Nucleo-cytoplasmic relationships of high-molecular-weight ribonucleic acid, including polyadenylated species, in the developing rat brain. The metabolism of high-molecular-weight RNA in the nuclear and cytoplasmic fractions of newborn and adult rat brain was investigated after the intracranial administration of [32P]Pi. In young brain, a considerable proportion of the newly synthesized radioactive RNA is transferred to the cytoplasm, in contrast with the adult brain, where there appears to be a high intranuclear turnover. Electrophoretic analysis of the newly synthesized RNA showed that processing of the rRNA precursor to yield the 28S and 18S rRNA may be more rapid in the adult than in the young, although most of the adult rRNA in the nucleus is not transferred to the cytoplasm. In young brain, processing is probably tightly coupled to transport of rRNA into the cytoplasm, so that 28S and 18S rRNA are not subjected to possible degradation within the nucleus. Polyadenylated RNA turns over in concert with high-molecular-weight RNA in the nuclei of the adult rat brain. In the cytoplasm the polyadenylated RNA has a higher turnover rate relative to rRNA. In the young brain the polyadenylated RNA is transferred to the cytoplasm along with rRNA, although polyadenylated RNA is transported into the cytoplasm at a faster rate. The nuclear and cytoplasmic polyadenylated RNA species of young brain are larger than their corresponding adult counterparts. These results suggest that there are considerable changes in the regulation of the nucleo-cytoplasmic relationship of rRNA and polyadenylated RNA during the transition of the brain from a developing replicative phase to an adult differentiated and non-dividing state.


The formation of alanine from amino acids in diaphragm muscle of the rat. Alanine production and pyruvate content of the isolated rat hemidiaphragm are increased by isoleucine or glutamate. These results support the hypothesis that amino acids are converted into pyruvate before oxidation and that some pyruvate is transaminated to alanine, which is released from the muscle.


Activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in brains of adult and 7-day-old rats. Rat brain contains 3-hydroxy-3-methylglutaryl-CoA reductase activity, but this enzyme is far more active in 7-day-old brain than in adult brain. This difference may partly explain why cholesterol biosynthesis is more rapid in growing than in adult rat brain.


Conformational analysis of deoxyribonucleic acid from PM2 bacteriophage. The effect of size on supercoil shape. Laser light-scattering studies of bacteriophage PM2 DNA showed the molecule to have mol.wt. 5.9 X 10(6) and root-mean -square radius 125 nm at an ionic strength of 0.2 mol/litre. Computer-generated curves compatible with these data were compared with the experimental interference curve for several structural models of the molecules. The data fit best to an asymmetric four-armed planar molecule in which all four arms emerge from or close to the one area of the molecule. This contrasts with the smaller DNA molecules investigated, which have shown a three-armed molecule, whose symmetry varies with primary structure.


Purification, characterization and identification of rat liver histidine-pyruvate aminotransferase isoenzymes. 1. Histidine-pyruvate aminotransferase (isoenzyme 1) was purified to homogeneity from the mitochondrial and supernatant fractions of rat liver, as judged by polyacrylamide-gel electrophoresis and isolectric focusing. Both enzyme preparations were remarkably similar in physical and enzymic properties. Isoenzyme 1 had pI8.0 and a pH optimum of 9.0. The enzyme was active with pyruvate as amino acceptor but not with 2-oxoglutarate, and utilized various aromatic amino acids as amino donors in the following order of activity: phenylalanine greater than tyrosine greater than histidine. Very little activity was found with tryptophan and 5-hydroxytryptophan. The apparent Km values were about 2.6mM for histidine and 2.7 mM for phenylalanine. Km values for pyruvate were about 5.2mM with phenylalanine as amino donor and 1.1mM with histidine. The aminotransferase activity of the enzyme towards phenylalanine was inhibited by the addition of histidine. The mol.wt. determined by gel filtration and sucrose-density-gradient centrifugation was approx. 70000. The mitochondrial and supernatant isoenzyme 1 activities increased approximately 25-fold and 3.2-fold respectively in rats repeatedly injected with glucagon for 2 days. 2. An additional histidine-pyruvate aminotransferase (isoenzyme 2) was partially purified from both the mitochondrial and supernatant fractions of rat liver. Nearly identical properties were observed with both preparations. Isoenzyme 2 had pI5.2 and a pH optimum of 9.3. The enzyme was specific for pyruvate and did not function with 2-oxoglutarate. The order of effectiveness of amino donors was tyrosine = phenylalanine greater than histidine greater than tryptophan greater than 5-hydroxytryptophan. The apparent Km values for histidine and phenylalanine were about 0.51 and 1.8 mM respectively. Km values for pyruvate were about 3.5mM with phenylalanine and 4.7mM with histidine as amino  donors. Histidine inhibited phenylalanine aminotransferase activity of the enzyme. Gel filtration and sucrose-density-gradient centrifugation yielded a mol.wt. of approx. 90000. Neither the mitochondrial nor the supernatant isoenzyme 2 activity was elevated by glucagon injection.


Interaction of human cathepsin D with the inhibitor pepstatin. 1. Because of the proposed role of cathepsin D in a variety of biological and pathological processes, the characteristics of inhibition by the potentially useful agent, pepstatin, were determined. 2. The beta and gamma forms of human cathepsin D, separated by isoelectric focusing, have identical specific extinction coefficients and specific activity in the degradation of haemoglobin. 3. Cathepsin D showed tight binding of 1 mol of pepstatin per 43000 g of protein, indicating that titration with the inhibitor represents a useful method for determination of absolute concentrations of the enzyme. 4. The titration curves were used to determine apparent dissociation constants (KD) for the binding of pepstatin and pepstatin methyl ester at pH3.5; values of approx. 5 X 10(-10)M were obtained. 5. Pepstatinyl-[3H]glycine was synthesized and shown to have a KD similar to that of pepstatin. Gel-chromatographic experiments showed that the binding of pepstatin and its derivatives is strongly pH-dependent. 6. The effect of pH on the KD for pepstatinyl-glycine was determined by equilibrium dialysis. As the pH was raised from 5.0 to 6.4, KD rose from 5 X 10(-10)M to 2 X 10(-6)M. 7. The catalytic activity of cathepsin D declines essentially to zero on going from pH5.0 to pH7.0, and we suggest that the binding site for substrate and pepstatin is abolished by a conformational change in the enzyme molecule. 8. The data indicate that, in biological experiments near neutral pH, large molar excesses of pepstatin over cathepsin D will be required for efficient inhibition.


Partial purification and characterization of collagen galactosyltransferase from chick embryos. Collagen galactosyltransferase was purified 50-150-fold from chick-embryo extract. The tissue homogenate was prepared in the presence of Triton X-100, since the addition of the detergent doubled the enzyme activity in the homogenate and the extract. Three species of the enzyme activity with different molecular weights were recovered on gel filtration, the mol.wts. being about 450000, 200000 and 50000. Collagen galactosyltransferase activity was strongly inhibited by p-mercuribenzoate, and stimulated by the addition of dithiothreitol to the incubation system. Studies on substrate requirements indicated that denatured citrate-soluble collagen is a more effective substrate than gelatinized insoluble collagen, as judged from their Km values. Experiments on three peptide fractions prepared from citrate-soluble collagen indicated that a fraction with an average mol.wt. of 500-600 contained peptides large enough to meet a minimun requirement for interaction with the enzyme. However, longer peptides were clearly better substrates. When native and heat-denatured citrate-soluble collagens were compared as substrates, practically no synthesis of galactosylhydroxylysine was found with native collagen. This finding suggests that the triple-helical conformation of collagen prevents the galactosylation of hydroxylysine residues.


Purification of the glycoprotein lectin from the broad bean (Vicia faba) and a comparison of its properties with lectins of similar specificity. 1. The lectin from the broad bean (Vicia faba) was purified by affinity chromatography by using 3-O-methylglucosamine covalently attached through the amino group to CH-Sepharose (an omega-hexanoic acid derivative of agarose). Its composition and the nature of its subunits were compared with concanavalin A and the lectins from pea and lentil. 2. Unlike the other three lectins, broad-bean lectin is a glycoprotein; a glycopeptide containing glucosamine and mannose was isolated from a proteolytic digest. 3. The mol.wt. is about 47500; the glycoprotein consists of two apprently identical subunits, held together by non-covalent forces. Fragments of the subunits, similar to those found in concanavalin A and soya-bean agglutinin, were found in active preparations. 4. Broad-bean lectin was compared with concanavalin A and the lectins from pea and lentil in an investigation of the inhibition of their action by a number of monosaccharides, methyl ethers of monosaccharides, disaccharides and glycopeptides. The most striking differences concern 3-O-substituted monosaccharides, which are strong inhibitors of the action of broad-bean, pea and lentil lectins but not of the action of concanavalin A. There is, however, no strong inhibition of the action of these lectins by 3-Olinked disaccharides.


2-O-methyl-D-glucuronic acid, a new hexuronic acid of biological origin. Acid hydrolysis of the extracellular polysaccharide of Porphyridium cruentum (a unicellular red alga) produced a mixture of aldobiuronic acids and free hexuronic acids. Fractionation of this mixture on an ion-exchange column yielded a hexuronic acid characterized as the title compound. Its identity was confirmed by chromatographic comparisons with the authentic compound, by reduction to the corresponding methylated aldose, by resistance to controlled lead tetra-acetate oxidation and by chemical-ionization mass spectrometry. Complete spectra have been deposited as Supplementary Publication SUP50062 (7 pages) with the British Library (Lending Division), Boston Spa, Wetherby, W. Yorkshire LS23 7BQ, U.K., from whom copies may be obtained under the terms given in Biochem. J. (1976) 153, 5.


Subunit composition and structure of subcomponent C1q of the first component of human complement. 1. Unreduced human subcomponent C1q was shown by electrophoresis on polyacrylamide gels run in the presence of sodium dodecyl sulphate to be composed of two types of non-covalently linked subunits of apparent mol.wts. 69 000 and 54 000. The ratio of the two subunits was markedly affected by the ionic strength of the applied sample. At a low ionic strength of applied sample, which gave the optimum value for the 54 000-apparent mol.wt. subunit, a ratio of 1.99:1.00 was obtained for the ratio of the 69 000-apparent mol.wt. subunit to the 5400-apparent-mol.wt. subunit. The amount of the 54 000-apparent-mol.wt. subunit detected in the expected position on the gel was found to be inversely proportional to increases in the ionic strength of the applled sample. 2. Human subcomponent C1q on reduction and alkylation, or oxidation, yields equimolar amounts of three chains designated A, B and C [Reid et al. (1972) Biochem. J. 130, 749-763]. The results obtained by Yonemasu & Stroud [(1972) Immunochemistry 9, 545-554], which showed that the 69 000-apparent-mol.wt. subunit was a disulphide-linked dimer of the A and B chains and that the 54 000-apparent-mol.wt. subunit was a disulphide-linked dimer of the C chain, were confirmed. 3. Gel filtration on Sephadex G-200 in 6.0M-guanidinium chloride showed that both types of unreduced subunit were eluted together as a single symmetrical peak of apparent mol.wt. 49 000-50 000 when globular proteins were used as markers. The molecular weights of the oxidized or reduced A, B and C chains have been shown previously to be very similar all being in the range 23 000-24 000 [Reid et al. (1972) Biochem. J. 130, 749-763; Reid (1974) Biochem. J. 141, 189-203]. 4. It is proposed that subcomponent C1q (mol.wt. 410000) is composed of nine non-covalently linked subunits, i.e. six A-B dimers and three C-C dimers. 5. A structure for subcomponent C1q is proposed and is based on the assumption that the collagen-like regions of 78 residues in each of the A, B and C chains are combined to form a triple-helical structure of the same type as is found in collagens.


N-terminal amino acid sequence of wheat proteins that lack phenylalanine and histidine residues. The 24 residues of the N-terminal CNBr peptide from a wheat albumin, that lacks phenylalanine and histidine, have been sequenced. Three of the assignments were made partly by analogy with two other proteins, as evidence is presented that all three proteins are probably identical in this region. Extra evidence for the sequences of the alpha-chymotryptic peptides derived from the N-terminal CNBr peptides of the three proteins, and also for their assembly, has been deposited as Supplementary Publication SUP 50063 (11 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem J. (1976) 153, 5. The nature of such evidence is stated in the text of this present communication.


High-performance liquid chromatography of phosphatidylcholine and sphingomyelin with detection in the region of 200 nm. A sensitive method for the separation of phosphatidylcholine and sphingomyelin by high-performance liquid chromatographic analysis is described. The elution of the phospholipids from a microparticulate (10 mum) silica-gel chromatographic column was monitored with an ultraviolet spectromonitor at 203 nm. Acetonitrile/methanol/water (65:21:14, by vol.) was used as the solvent. It was shown by using synthetic phosphatidylcholines of knowm fatty acid composition and of varying degree of unsaturation that the absorption at 203 nm was primarily due to the isolated double bonds and the response measured varied with the degree of unsaturation. Approx. 1 nmol of phosphatidylcholine, containing at least one double bond per molecule, can be detected. The amounts of phosphatidylcholine and sphingomyelin could be determined by high-performance liquid chromatography and ultraviolet absorption if the apparent extinction coefficient of the material analyzed was established. Alternatively, peaks were collected and the phospholipids were determined by the analysis of phosphorus. The analysis of phosphatidylcholine and sphingomyelin present in the lipid extracts from animal tissues, blood and amniotic fluids were made without interference from other phospholipids or ultraviolet-absorbing material. The method described here is complementary to the high-performance liquid chromatographic method described previously for the analysis of ethanolamine-containing phosphoglycerides and serine-containing phosphoglycerides [Jungalwala, Turel, Evans and McCluer (1975) Biochem. J. 145, 517-526].


Mouse haemoglobin Beta chains. Sequence data on embryonic y chain and genetic linkage of the Y-chain locus to the adult beta-chain locus Hbb. Embryonic haemoglobin y-chain types were determined by starch-gel electrophoresis for inbred Mus musculus strains Sec/ReJ and Au/SsJ (with Hbbs and Hbbp adult beta-chain alleles respectively) and for random-bred HA/ICR (Swiss) mice, which had been selected to be homozygous for the adult Hbbd beta-chain allele. The strain with the Hbbs allele had y1 embryonic beta chain and the strain with the Hbbd allele had y2. The strain with the Hbbp allele also had y2 chain. A breeding study showed that the maximum recombination frequency between the Hbb locus and the y-chain locus is 5.4% at the 95% confidence level. Embryonic y2 chain was sequenced for 39 positions from the N-terminus with an Edman-Begg sequenator. Some tryptic-peptide composition data were obtained on both y1 and y2 chains. No amino acid substitutions between y1 and y2 chains were found in the 40 positions for which there are common data on the two chains. y2 and adult beta chains are different in 12 of the first 21 positions, but show considerable similarity thereafter. It is suggested that homologous but unequal crossing-over between y-chain and adult beta-chain loci could have given rise to the adult beta-chain allele Hbbd, which produces structurally different major and minor beta chains from two closely linked genes.


Human lysosomal elastase. Catalytic and immunological properties. 1. The elastase of human spleen was shown to exhibit endopeptidase activity against azo-casein and elastin. 2. Activity against several synthetic substrates was detected, and benzyloxycarbonyl-L-alanine 2-naphthyl ester was found to be a good substrate for routine use. 3. The enzyme showed a broad pH optimum in the range of 8.2-9.2 against azo-casein and the synthetic substrate. 4. The effect of inhibitors on the spleen elastase showed it to be a serine proteinase with a specificity similar to that of porcine pancreatic elastase. 5. Specific antisera were raised against the enzyme, and it was shown to be immunologically identical with the lysosomal elastase of human neutrophil leucocytes.


The actin of muscle and fibroblasts.

The actin of muscle and fibroblasts. The isolation and quantification of an 18-residue peptide from the N-terminal region of chicken actin was used to quantify the amount of actin in acetone-dried powders of chicken breast muscle and chicken-embryo fibroblasts. Either isotope dilution or double labelling can be used for peptide quantification. About 17% of the protein of chicken breast muscle was estimated to be actin. However, only 0.25% of the protein of chicken-embryo fibroblasts was determined to be actin by quantification of this peptide. The actin content of fibroblasts may be low or the amino acid sequences of muscle and fibroblast actin may differ in the N-terminal region. The methodology used can be extended to examine whether other regions of muscle actin sequence are present in fibroblasts or other cell types.


The structures of the carbohydrate moieties of the alpha subunit of human chorionic gonadotrophin. The alpha subunit of human chorionic gonadotrophin was reduced with dithiothreitol followed by carboxymethylation with iodoacetic acid. The modified glycoprotein was hydrolysed with trypsin to give various peptides, the identities of which were established, and glycopeptides. The glycopeptides were separated by gel filtration and ion-exchange chromatography; they were subjected to component analysis and were found to represent the two carbohydrate moieties in the parent glycoprotein. Sequential removal with glycoside hydrolases of monosaccharide units from the glycopeptides demonstrated (1) that galactose, mannose, glucosamine (2-amino-2-deoxyglucose) and neuraminic acid (5-amino-3,5-dideoxy-glycero-galacto-2-nonulosonic acid) residues possess the D configurations, (2) that the glucosamine units are N-acetylated and (3) the order of the monosaccharide units in the chain, the neuraminic acid units being furthest from the peptide backbone of the subunit and substituting the D-galactose units. Methylation analysis of the glycopeptides by adaptation of the Hakomori technique demonstrated that: (4) D-galactose, D-mannose and N-acetylglucosamine (2-acetamido-2-deoxy-D-glucose) units exist in the pyranose forms; (5) the D-galactopyranose units are linked in the 1 and 6 positions; (6) the D-mannopyranose units exist in several forms, one in a terminal non-reducing position, one as 1,2-linked residues and some as 1,6-linked branch points; (7) the N-acetylglucosamine units are 1,6-linked. On the basis of the results of methylation and enzymic analysis, structures are proposed for the carbohydrate moieties and the assignments are compared with other data previously obtained by periodate-oxidation studies [Kennedy et al. (1974) Carbohydr. Res. 36, 369-377].


A comparison of the properties of renin isolated from pig and rat kidney. 1. On isoelectric focusing, renin from rat kidneys showed three activity peaks with pI values at pH 5.0, 5.2 and 5.4 after a purification procedure involving differential centrifugation, acidification, chromatography on Sephadex G-75 and dialysis. 2. The preparation (purified 140-fold) was compared with a crude kidney extract in the absence and presence of 3 M-urea by isoelectric focusing. The pattern of activity distribution was confirmed by these experiments and the content of isoenzymes in the three groups calculated. 3. Pig renin was prepared and compared with rat renin with regard to molecular weight, acid activation, behaviour on isoelectric focusing, immunogenicity and substrate affinity. 4. Extracts of rat kidney contained multiple forms of renin with mol.wt. between 39000and 42000, whereas active pig renin had an approximate mol.wt. of 40000. Acidification of rat renal extracts did not increase the activity of renin, indicating the absence of an inactive form of renin in rat kidneys, whereas pig renin was activated by this procedure. Pig renin has isoelectric points at pH 4.6, 4.8, 5.05 and 5.2, significantly lower than for rat renin. The isoenzymes from the two species had no antigenicity in common, as shown by crossed immunoelectrophoresis or rocket immunoelectrophoresis. 5. The Michaelis constants for pig and rat renin were in the same range, 1 X 10(-6) M, when rat renin substrate was used. The relative content of rat isoenzyme with pI in the pH ranges 4.9-5.1, 5.1-5.3 and 5.3-5.5 was approx. 20, 27 and 53% respectively. Purified pig renin prepared in two different ways had isoenzymes with pI in the pH regions 4.5-4.7, 4.7-4.9, 4.9-5.05 and 5.05-5.20 in the approximate proportions 14, 24, 28 and 29%.


The mechanism of folding of globular proteins. Suitability of a penicillinase from Staphylococcus Aureus as a model for refolding studies. 1. A homogeneous preparation of penicillinase (penicillin amido-beta-lactamhydrolase, EC 3.5.2.6) was isolated and purified from cultures of Staphylococcus aureus by a simple two-stage procedure. 2. The native protein contains 20-30% helix as determined by optical-rotatory-dispersion and circular-dichroism measurements. Some 54(+/-5)% of the 13 tyrosine residues are exposed to solvent molecules of diameter 0.44 and 0.94 nm. 3. Conditions that allow full recovery of enzymic activity and native conformation from the fully unfolded state in 4M-guanidinium chloride were defined. 4. Refolding of the protein was shown to be inhibited by intermolecular interaction, by small changes in ionization and by low concentrations (0.025 M) of phenol.


The mechanism of folding of globular proteins. Equilibria and kinetics of conformational transitions of penicillinase from Staphylococcus aureus involving a state of intermediate conformation. 1. The thermodynamically reversible unfolding and refolding of penicillinase between the native and fully unfolded states were followed by using guanidinium chloride as denaturant. 2. The equilibria, studied by optical rotation, u.v. absorption, viscosity and enzyme activity, show the presence of a state of intermediate conformation, termed state H, which is stable at 20 degrees C in 0.8 M-guanidinium chloride. 3. The physical properties of this state show that it is slightly expanded with an intrinsic viscosity of 8 ml-g-1, that the 13 tyrosine residues, which are distributed through the primary sequence, are maximally exposed to the solvent and that the helix content is the same as that of the native state. 4. The kinetics of the transition between the native state, state H and the fully unfolded state were followed by u.v. absorption and by optical rotation. They are interpreted as showing that state H lies on the folding pathway between the native and fully unfolded states. 5. The transition between the native state and state H exhibits monophasic unfolding kinetics and biphasic refolding kinetics. This indicates that there must be at least two intermediate states in this process, at least one of which lies on the folding pathway which may also involve cul-de-sac paths. 6. The results are discussed in terms of a mechanism involving rapid stabilization of nucleation regions in a moderately compact but internally solvated structure, with 'native format' [Anfinsen (1973) Science 181, 233-230] secondary structure stabilized by tertiary interaction. The final and rate-limiting step in refolding involves shuffling of these structural elements into the native state. 7. This model is discussed in relation to folding in vivo.


Active fragments obtained by cyanogen bromide cleavage of ovomucoid. Cleavage of the two methionine residues in the glycoprotein trypsin inhibitor ovomucoid, variant O1, with CNBr resulted in two fragments whose mol.wts. were approx. 16 600 (fragment LS) and 11 000 (fragment M). Both fragments formed precipitates with antisera to ovomucoid. Fragment LS retained 56% of the trypsin-inhibitory activity of ovomucoid, but fragment M did not inhibit. After reduction and alkylation, the molecular weight of fragment M was unchanged, but fragment LS could be resolved into two segments of peptide chain with mol.wts. of approx. 12000 (fragment L) and 4700 (fragment S). Each of these peptides contained carbohydrate. Marked heterogeneity was observed in the hexose and hexosamine contents of fragment L. This may account for much of the heterogeneity in neutral carbohydrate occurring in ovomucoid preparations. It was found that fragment M was located at the N-terminal end, fragment S was in the centre and fragment L made up the C-terminal portion of the molecule.


Bilirubin conjugates in bile of man, rat and dog. Semi-quantitative analysis of bile composition by thin-layer chromatography. 1. Conjugated bile pigments, separated in two fractions by semi-quantitative t.l.c. performed on silicic acid with phenol/water as the developing solvent, were treated with diazotized ethyl anthranilate. Resulting dipyrrylazo derivatives were analysed by quantitative t.l.c. 2. The tentative structure elucidation of tetrapyrrolic bilirubin conjugates and semi-quantitative evaluation of rat bile, post-obstructive human bile and dog bile composition is presented. 3. Homogeneous and mixed hexuronic acid diesters of bilirubin containing glucuronic acid constitute 51% of the total conjugates in normal rat bile, 45% of those in human post-obstructive bile and 38% of those in obstructed rat biles. 4. Monoconjugated bilirubin amounts to 33% of total conjugated bile pigments in normal rat bile, and 17 and 14% in post-obstructive hepatic human bile and gall-bladder bile of dog respectively. After loading with unconjugated bilirubin a greater amount of monoconjugates (56%) occur in the rat bile, whereas bilirubin diglucuronide excretion is decreased (34%). 5. In gall-bladder bile of normal dog, 40% of glucose-containing diconjugates, 32% of homogeneous and/or mixed hexuronic acid (mainly glucuronic acid) diesters of bilirubin and 14% of xylose-containing diconjugates are estimated. 6. Increased amounts of bilirubin conjugates, including some with unidentified uronic acid groups, were observed in cholestatic rat biles and quantities of conjugates with glucuronic acid were decreased.


Separation of rat muscle aminopeptidases. By means of chromatography on DEAE-Sephadex, two arylamidases (hydrolysing L-arginine 2-naphthylamide) and three dipeptidyl peptidases (hydrolysing dipeptide 2-naphthylamides) were distinguished in extracts of rat muscle. However, the arylamidase from the larger peak also hydrolysed the dipeptide 2-naphthylamides. Glycyl-L-arginine amide, an alternative substrate for dipeptidyl peptidase I, was not hydrolysed by arylamidase. L-Leucine amide was hydrolysed by an enzyme, presumed to be leucine aminopeptidase, from a separate peak, but was also hydrolysed by arylamidase. Arylamidase, dipeptidyl peptidase III and most of the leucine aminopeptidase could be extracted from the muscle with a neutral salt solution, but dipeptidyl peptidase I was extracted only in the presence of Triton X-100; dipeptidyl peptidase II showed an intermediate extraction behaviour.


Preparation of the 3-monosulphates of cholic acid, chenodeoxycholic acid and deoxycholic acid. 1. The 3-sulphates of cholic, chenodeoxycholic and deoxycholic acids were prepared as crystalline disodium salts. 2. The method described shows that it is possible to prepare specific sulphate esters of polyhydroxy bile acids and to remove protecting acyl groups without removing the sulphate. 3. A study of bile acid sulphate solvolysis showed that none of the usual methods give the original bile acid in major yield in a single step. 4. An understanding of the preparation, properties and methods of solvolysis of bile acid sulphates is basic for investigations of cholestasis and liver disease.


Synthesis and separation by thin-layer chromatography of bilirubin-IX isomers. Their identification as tetrapyrroles and dipyrrolic ethyl anthranilate azo derivatives. Procedures for the synthesis, separation and determination of structure of the bilirubin-IX isomers are described. 1. The four biliverdin-IX isomers were prepared by oxidative cleavage of haemin and were separated as their dimethyl esters. The individual esters were reduced with NaBH4, and the bilirubin esters obtained were subjected to alkaline hydrolysis yielding the corresponding bilirubin-IX isomers. 2. The bilirubin-IX isomers were structurally characterized (a) at the tetrapyrrolic stage by mass spectrometry of their trimethylsilyl derivatives and (b) by formation and structural analysis of their dipyrrolic ethyl anthranilate azo derivatives. 3. The absorption spectrum of bilirubin-IX alpha differed strikingly from the spectra of the other isomers. The presence of a pronounced shoulder around 453 nm in the spectrum of bilirubin-IXbeta allows easy differentiation from bilirubin-IXdelta. Methylation of the carboxyl groups largely eliminates the spectral differences between the IXalpha- and non-alpha isomers. 4. The bilirubin-IX isomers are conveniently separated by t.l.c. Detection and unequivocal identification is possible on a micro-scale by (a) t.l.c. with respect to reference compounds and (b) subsequent formation and t.l.c. of the more stable ethyl anthranilate azopigments. 5. Pronounced differences in polarity, i.e. solvent distribution, between the bilirubin-IX isomers indicate that a re-evaluation of conclusions reached previously with regard to the presence in, or absence from, biological fluids of some isomers and their relative amounts is needed.


The effect of some analogues of disulfiram on the aldehyde dehydrogenases of sheep liver. The effect of some close structural analogues of disulfiram on the activity of the cytoplasmic and mitochondrial aldehyde dehydrogenases of sheep liver was studied. Several thiuram disulphides are equally potent inhibitors of the cytoplasmic enzyme, but in all cases the mitochondrial enzyme is much less strongly affected. Tetramethylthiuram monosulphide decreases the activity of the cytoplasmic enzyme in a process apparently involving a covalent interaction.


The computation of hyperbolic dependences in enzyme kinetics. A computer program aimed at analysing results following Michaelis-Menten kinetics can be used unmodified in the treatment of other kinetic results provided that the kinetic equations in these cases can be written in the form of the Michaelis-Menten equation. A list is presented of the parameters to be set instead of substrate concentration and reaction rate, and of constants replacing Km and V, if such a program is applied in analysing enzyme inhibitions, activations and pH-dependences.


The use of ninhydrin as a reagent for the reversible modification of arginine residues in proteins. A simple technique was developed for the specific reversible modification of guanidino groups in proteins involving reaction with ninhydrin. The extent of the reaction is easily determined non-destructively by spectrophotometric analysis. The reagent can also be used for the titration of sterically unhindered thiol groups in proteins.


Preferential inhibition by poly(adenylic acid) of minus-strand synthesis by bacteriophage Qbeta ribonucleic acid replicase. Among the homopolymers examined, poly(A) was found to inhibit preferentially the synthesis of the minus strand by bacteriophage Qbeta RNA replicase in the presence of host factor. A specific interaction of poly(A) with the host factor is suggested to be a principal cause for the observed preferential inhibition by poly(A) of the host-factor-requiring Qbeta RNA replicase reaction.


Induction of peripheral blood lymphocyte transformation by autologous synovial fluid lymphocytes and synovial fluid. Synovial fluid lymphocytes and synovial fluid from most patients with rheumatoid arthritis induced blastogenesis of autologous peripheral blood lymphocytes. In vitro transfer of the mitogenic activity of the synovial fluid to peripheral blood lymphocytes was not accomplished, but diminished response of the peripheral blood lymphocytes to phytohemagglutinin stimulation after incubation in synovial fluid was demonstrated. These findings suggest that a similar blastogenic response in vivo may induce the lymphoid hyperplasia regularly observed in rheumatoid arthritis.


Cellular hypersensitivity in rheumatoid arthritis, ankylosing spondylitis, and anterior nongranulomatous uveitis. The humoral and cellular responses to normal human ocular and joint tissue antigens were studied in rheumatoid arthritis, ankylosing spondylitis, and anterior non-granulomatous uveitis. No free-circulating autoantibodies could be detected by hemagglutination, immunodiffusion, and immunofluorescence. Uvea-retina, synovial membrane, and articular cartilage antigen preparations inhibited the migration of leukocytes from patients with rheumatoid arthritis. In ankylosing spondylitis inhibition could be induced only by synovial membrane antigens. Patients with anterior nongranulomatous uveitis were hypersensitive to uvea-retina antigens alone.


Periarticular uptake of 99mtechnetium diphosphonate in psoriatics: correlation with cutaneous activity. The periarticular uptake of 99mtechnetium-labeled diphosphonate (99mTcDp) was compared in 12 patients hospitalized for psoriasis and in 12 hospitalized for other dermatoses not associated with arthropathy. The 12 patients with psoriasis had recent onset disease of less than 5 years duration; neither group had historical or clinical evidence of arthritis. All psoriatics had markedly abnormal scans with symmetrically increased periarticular uptake about the imaged joints. None of the controls had similar findings. In 4 patients scanned with 99mtechnetium-pertechnetate within 24 hours of their 99mTcDP scan, no evidence of inflammatory synovitis was found. Three of these patients were serially imaged with 99mTcDP at intervals of 2 weeks to 3 months after their initial study, when obvious clinical improvement in their psoriasis was apparent. Improvement in the radionuclide joint images was demonstrated in some of the patients, but none reverted to normal during the study period. In light of recent evidence for the preferential binding of 99mTcDP to immature collagen, it is suggested that psoriasis may represent a generalized, but uncharacterized, collagen disorder present in bone as well as skin, linking the cutaneous disease with the potential for arthropathy.


Articular manifestations of hypertrophic pulmonary osteoarthropathy in bronchogenic carcinoma. Eight patients with bronchogenic carcinoma presented with painful joint effusions as part of the syndrome of hypertrophic osteoarthropathy. Elevated sedimentation rates and symptomatic relief with aspirin were common, but synovial fluids all had leukocyte counts less than 500/mm3. Synovial microvascular injury was found in all patients, and electron-dense deposits in synovial vessel walls were identified in 5 cases. These vascular deposits suggest a role for circulating materials in pathogenesis.


Genu amoris.

Genu amoris. Intractable pain in a patient with chondromalacia patellae was found to be related to an unusual type of repetitive trauma.


Hypertrophic osteoarthropathy and rheumatoid arthritis: simultaneous occurrence in association with diffuse interstitial fibrosis. A patient is described who was treated with high-dose prednisone in an attempt to halt progressive respiratory insufficiency associated with diffuse interstitial fibrosis. On cessation of steroid therapy the patient was noted to have radiologic manifestations of hypertrophic osteoarthropathy (HOA) as well as clinical and laboratory features of rheumatoid arthritis (RA). Subsequently a diffuse vasculitis developed with bowel perforation and sepsis leading to death.


Serum lysozyme as a marker of host resistance. II. Patients with malignant melanoma, hypernephroma or breast carcinoma. Serum lysozyme activity was measured in groups of untreated patients with malignant melanoma, hyperneophroma and breast carcinoma. Significant elevation of serum levels of the enzyme was confined to patients with localized disease. In the presence of metastatic disease such elevation was not detected. The rise in serum lysozyme activity was not due to renal damage or any infective process and in the case of malignant melanoma was shown to be associated with infiltration of the tumour mass by macrophages. In vitro studies demonstrated that the macrophages resident in a tumour mass are responsible for relasing lysozyme in large amounts. It is proposed that the elevation of serum lysozyme in these cases may be an indicator of macrophage-mediated host resistance and that the measurement of macrophage products such as lysozyme in the extracellular fluid may under well defined conditions provide useful clinical information concerning host reactions.


In vitro production of a transfer factor specific for transitional-cell carcinoma of the bladder. Human dialysable Transfer Factor (TFd) extracted from lymphocytes of patients with transitional cell carcinoma of bladder (TCCB) was replicated in culture by lymphoblastoid cell lines. The effectiveness of two such TFdLs produced in vitro in transferring sensitivity to TCCB was assessed in the lymphocyte migration test (LMT) using formalin-treated TCCB cells as antigen. The results, showed that one TFdL transferred sensitivity in 5/14 cases and the other in 12/15, not only to leucocytes of healthy individuals but also to leucocytes of TCCB patients. Preliminary results showing an in vivo transfer of sensitivity are discussed.


The multipotential carcinogenic action of N-ethyl-N-nitrosourea administered neonatally to mice. Newborn A, C57BL, DBAf and IF mice were injected s.c. with a range of doses of N-ethyl-N-nitrosourea (ENU). A high proportion of treated mice developed tumours, particularly hepatomata, pulmonary adenomata and carcinomata, and malignant lymphomata of thymus and spleen. Liver tumours occurred most frequently in C57BL and DBAf mice, lung tumours in A mice, and lymphomata in A and DBAf mice. A small proportion of C57BL, DBAf and IF mice developed tumours of the nervous system. The results are discussed with reference to the ready induction of nervous system tumours in similarly treated rats, and their relevance for human cancer.


Inducers of Friend leukaemic cell differentiation in vitro--effects of in vivo administration. Studies were conducted of the in vivo therapeutic potential of compounds which induce the differentiation of Friend leukaemia cells (FLC) in vitro. DBA2/J mice were inoculated with Friend leukaemia cells grown in tissue culture and at various times thereafter were treated with either N-methylacetamide, dimethylacetamide, or tetramethylurea. While survival was only occasionally prolonged, in every study these agents significantly inhibited leukaemia cell proliferation in the spleen and to a lesser extent in the marrow. These agents had no effect on the rate of proliferation of FLC growing subcutaneously nor on the proliferation of myeloid leukaemia in RFMS mice. These studies indicate that the administration of inducing agents to mice bearing Friend leukaemia can alter the proliferation characteristics of the leukaemia cells and hence suggest that these agents may have therapeutic potential.


Ovarian failure following abdominal irradiation in childhood. Ovarian function was studied in 18 female patients treated for abdominal tumours during childhood. All received abdominal radiotherapy as part of their treatment and were studied between 1 and 26 years after irradiation. The serum gonadotrophins and oestradiol levels were consistent with ovarian failure in each case but there was a disproportionate elevation in serum follicle stimulating hormone (FSH) when compared to serum luteinizing hormone (LH) in 16. In 2 patients, the radiotherapeutic field extended downwards only as far as the sacral promontory. However, these 2 girls show similar evidence of ovarian failure to that in the other 16.


Purification and crystallization of human carboxypeptidase A. Human carboxypeptidase A has been isolated from activated pancreatic juice by means of affinity chromatography employing the competitive inhibitor benzylsuccinic acid as an affinity ligand. The structural and functional features of the human and bovine enzymes are quite analogous. The molecular weights of human and bovine carboxypeptidases A are virtually identical, their amino acid compositions are similar, both contain 1 g-atom of zinc/mole, and the activities of both are restored by addition of zinc to the apoenzyme. The inhibition of human carboxypeptidase by chelating agent is reversed by either dilution or addition of a metal such as Cu2+. When other metals are substituted for the native zinc, peptidase activity of the human metallocarboxypeptidases follows the order: cobalt greater than nickel greater than manganese greater than cadmium, while the sequence for esterase activities is: manganese greater than cobalt = cadmium greater than nickel. The latter sequence differs from that observed for the bovine enzyme. Human carboxypeptidase A crystallizes after dialysis at low ionic strength. Hydrolysis of the dipeptide carbobenzoxyglycyl-L-phenylalanine and of the ester benzoylglycyl-L-alpha-hydroxy-beta-phenyllactate exhibits kinetic anomalies, but that of their longer homologues does not. Chemical modifications with tyrosine reagents alters esterase and peptidase activities. The affinity chromatographic method here described should greatly facilitate future studies of this enzyme from human and other sources.


Creatine biosynthesis during embryonic development. False feedback suppression of liver amidinotransferase by N-acetimidoylsarcosine and 1-carboxymethyl-2-iminoimidazolidine (cyclocreatine). The level of arginine:glycine amidinotransferase in liver of the developing chick embryo is partially suppressed following injection of arginine into the yolk, and the level can be completely suppressed following injection of guanidinoacetate or creatine (Walker, J.B. (1963), Proc. Soc. Exp. Biol. Med. 112, 245; Walker, J.B., and Wang, S.-H. (1964), Biochim, Biophys. Acta 81, 435). In this investigation structural requirements for the physiological suppressor were examined by testing certain analogues of creatine and its biosynthetic precursors for their ability to suppress liver amidinotransferase levels in developing chick embryos and growing chicks. The creatine analogues, N-acetimidoylsarcosine and 1-carboxymethyl-2-iminoimidazolidine (cyclocreatine), were found to suppress liver amidinotransferase levels of both developing embryos and growing chicks. Compounds ineffective as suppressors included: the arginine analogue, N5-acetimidoylornithine; the guanidinoacetate analogue, N-acetimidoylglycine; and the creatine analogue, 1-carboxymethyl-2-iminohexahydropyrimidine. Our findings suggest that (i) arginine and guanidinoacetate must be converted to creatine before serving as a suppressor, and (ii) creatine, not phosphocreatine, is most closely related to the physiological suppressor of amidinotransferase.


Voltage-induced formation of alamethicin pores in lecithin bilayer vesicles. The interaction of alamethicin with lecithin bilayer vesicles in the presence of a transmembrane potential difference has been studied by proton magnetic resonance spectroscopy. Asymmetrical vesicles with alamethicin trapped within the intravesicular compartment were prepared and the formation of ion channels, were then triggered by imposing a suitable potassium ion gradient across the bilayer membrane in the presence of valinomycin. These alamethicin channels were found to permit the outward passage of europium ions into the extravesicular medium when the transmembrane potential is more positive for the inner compartment.


A beta-coupled gauche kink description of the lipid bilayer phase transition. A statistical-mechanical model for a lipid bilayer was built around the assumption that beta-coupled gauche kinks are the only nonstraight hydrocarbon chain configurations allowed. Packing of these chains was considered and expressions for the energy and entropy are developed based on those considerations. A solution was obtained which was not exact but was still accurate enough to be useful. In addition, an intermolecular potential was formulated which includes the attractive and repulsive forces between the hydrocarbon chains, the polar group repulsions, and hydrophobic forces. Combining the solution to the kinking model and the intermolecular potential, we obtain the total free energy of the lipid bilayer, which exhibits a first-order phase transition at a characteristic temperature. The theoretical heat of transition indicates that kinks alone can provide enough disorder for the transition to occur. Other physical quantities can be calculated, including order parameters. The order parameters calculated here give a picture of chains which are rigid near the polar group and more fluid near the methyl group.


Carboxypeptidase inhibitor from potatoes. Interaction with derivatives of carboxypeptidase A. The mechanism of action of a carboxypeptidase inhibitor from potatoes has been probed by studying its interaction with derivatives of carboxypeptidase A containing modified residues at the active site. Arsanilazocarboxypeptidase A, a derivative containing a chromophore attached to tyrosine 248, exhibits a circular dichroism spectrum which is sensitive to the presence of ligands at the active site (Kagan, H.M., and Vallee, B.L. (1969), Biochemistry 8, 4223). Since the spectral change attending binding of the carboxypeptidase inhibitor to arsanilazocarboxypeptidase A is similar to that produced by small substrates and inhibitors, the enzyme-inhibitor interaction also involves the enzyme active site. Catalytic activity is not required for inhibitor binding. Complexes of the inhibitor with apocarboxypeptidase A anc carboxypeptidase A which was inactivated by treatment with the affinity label, N-bromoacetyl-N-methyl-L-phenylalanine, are demonstrated by gel filtration experiments. Morever, competitive binding studies reveal that the latter derivative, in which the binding pocket is presumably blocked by reagent, binds inhibitor nearly as strongly as does the native enzyme, and differences in free energy of association being only 0.4 kcal/mol of a total binding energy of - 11 kcal/mol. A model is proposed to account for both the tight binding of inhibitor to the N-bromoacetyl-N-methyl-L-phenylalanine derivative and the involvement of the active site of arsanilazocarboxypeptidase A. It is suggested that the inhibitor fits into a shallow depression at the active site of the enzyme but does not penetrate into the binding pocket.


Electrooptical properties of reduced protein-sodium dodecyl sulfate complexes. Two independent electrooptical properties, the specific Kerr constants and the electric birefringence relaxation times, of the saturated sodium dodecyl sulfate complexes of a series of reduced polypeptides of known molecular weight are reported. Both the Kerr constants and the relaxation times are unique functions of the molecular weight of the polypeptide chain. The specific Kerr constants depend upon the square of the polypeptide molecular weight. The relaxation times of the complexes, which are proportional to the rotational diffusion constants, are dependent on the molecular weight to approximately the first power. The latter finding is inconsistent with the compact prolate ellipsoid model for sodium dodecyl sulfate-protein complexes proposed by Reynolds and Tanford ((1970) J. Biol. Chem. 245, 5161) in which the cross section is constant and the length depends on linearly on molecular weight; for this model the relaxation times would depend on approximately the 2.5 power of the molecular weight in the range of sizes investigated. Combination of the present results with other properties in the literature rules out a number of other models characterized by compactness and near inflexibility. No firm conclusions can be drawn as to the suitability of the free-draining flexible model of Shirahama, Tsujii, and Takagi ((1974) J. Biochem. (Tokyo) 75, 309).


Purification and chemical characterization of salt-extractable glycoproteins from porcine mitral valve. Four major glycoproteins were extracted by dilute salt solution from procine mitral valvular tissue. Two of these major glycoproteins, procine valve glycoprotein I and porcine valve glycoprotein III, were isolated and purified by fractionation of salt extract with ammonium sulfate followed by column chromatography on DEAE-cellulose. The purified glycoproteins appeared to be homogeneous by polyacrylamide disc electrophoresis in several buffer systems, and by Sephadex filtration. The porcine valve glycoprotein I  has a molecular weight of approximately 120000. Isoelectric focusing yielded a single band, pI = 5.8. The glycoprotein contained large amounts of acidic amino acids, and amide nitrogen. The carbohydrate moiety was composed of fucose, mannose, galactose, glucose, glucosamine, and galactosamine in the molar ratio of 5:10:15:12:7:2 per mole of glycoprotein. The second major glycoprotein, porcine valve glycoprotein III, has an approximate molecular weight of 72000. This glycoprotein gives two bands upon analytical isoelectric focusing with isoelectric points of pI = 4.1 and 4.3. Porcine valve glycoprotein III contained large amounts of acidic amino acids and low amounts of amide nitrogen. Its carbohydrate moiety was composed of glucose, galactose, mannose, fucose, glucosamine, and sialic acid in the ratio of 3:3:2:1:4:1 mol/mole of glycoprotein. This glycoprotein was similar to a glycoprotein preparation isolated from porcine aortic intima by P.V. Wagh and B.I. Roberts (1972), Biochemistry 11, 4222.


Complete primary structure of the major component myoglobin of California gray whale (Eschrichtius gibbosus). The complete primary structure of the major component myoglobin from the California gray whale, Eschrichtius gibbosus, was determined by specific cleavage of the protein to obtain large peptides for degradation by the automatic sequenator. Cleavage at the two methionine residues of the apomyoglobin with cyanogen bromide and at the three arginine residues of the methyl acetimidated protein with trypsin resulted in three and four easily separable peptides, respectively, which when sequenced accounted for 85% of the primary structure. The remainder of the covalent structure was obtained by further digestion of the central cyanogen bromide peptide with trypsin and S. aureus strain V8 protease. This protein differs from that of the sperm whale, Physeter catodon, at 12 positions, from that of the common porpoise, Phocoena phocoena, and the Black Sea dolphin, Delphinus delphis, at 14 positions, and from that of the Amazon River dolphin,  Inia geoffrensis, at 7 positions. All substitutions observed in this sequence fit easily into the tertiary structure of sperm whale myoglobin.


Transfer free energies and average static accessibilities for ribonuclease A in guanidinium hydrochloride and urea solutions. Isopiestic vapor pressure measurements have been used to obtain free energies of transfer of ribonuclease A from dilute buffer to solutions of either urea or guanidine hydrochloride (GdnHCl) over a wide cosolute concentration range. The free energies of transfer vary monotonically from 0 to -8 kcal/mol in 8 M urea and to -18 kcal/mol in 6 M GdnHCl. These values are not large in relation to free energies of transfer of constituent groups of the protein from water to cosolute solutions of the same concentration. The assumption is made that the magnitude of the free energy of transfer of the protein is governed by the average static accessibility of the constituent groups to the solution. The free energies of transfer to different cosolute concentrations of a hypothetical 100% accessible ribonuclease A were determined using literature values of the free energies of transfer of constituent groups and the amino acid composition. The ratio of the experimentally determined free energy of transfer to the free energy of transfer of the 100% accessible protein gave 11% accessible surface area for the native protein in 1 M GdnHCl or 2 M urea. Additional considerations led to a value of 36% for the accessible surface area of the denatured protein in 6 M GdnHCl or 8 M urea.


Chromatin-bound protease: (3H)diisopropyl fluorophosphate labeling patterns of chromatin. The nuclei and chromatin of rat liver contain three major proteins reacting with diisopropyl fluorophosphate (DFP). The molecular  weights of the three proteins determined by gel filtration in the presence of sodium dodecyl sulfate and sodium dodecyl sulfate-polyacrylamide gel electrophoresis are 70000, 60000, and 25000. The chromatin isolated from whole liver, instead of nuclei, contains an additional DFP-binding protein whose molecular weight is 100000 in the presence of sodium dodecyl sulfate and beta-mercaptoethanol. The small molecular weight DFP-binding protein can be fractionated from chromatin by 0.25 N HC1 and was found to be a protease which is active in the most commonly used solution for chromatin dissociation, that is, 2-3 M NaCl-5 M urea. This enzyme appears to be the major DFP-binding chromatin-bound protease in the chromatin of most rat tissues. The acid-soluble protease is converted from a 25000-dalton form to a 20000-dalton form during 0.25 N HC1 acid extraction from chromatin, which retains proteolytic activity.


Proteolytic fragmentation of Helix pomatia alpha-hemocyanin: structural domains in the polypeptide chain. alpha-Hemocyanin from the Roman snail Helix pomatia is composed of polypeptide chains with a molecular weight of 360000 +/- 30000. The cylindrically shaped hemocyanin molecule contains 20 of these large chains. The polypeptide chain has been split into components with molecular weights of: 210000, 154000, 147000, 112000, 120000, 98000, 55000, and 50000, by gentle proteolysis with enzymes of different specificities. Most of the fragments have molecular weights which are about 50000 or a multiple of 50000. Departure from these values, as found in the 112000 and 120000 fragments, is probably caused by the high carbohydrates content of these components. A mixture of these fragments has the same oxygen binding properties as the nondigested protein. Subtilisin converts the hemocyanin polypeptide chain, under appropriate conditions, almost completely into fragments of 50000 and 55000 daltons with conservation of the oxygen binding properties of the nondigested protein. We conclude from these studies that the polypeptide chain of Helix pomatia alpha-hemocyanin is folded into about seven compact tertiary structures, which are covalently interconnected. This chain of structural domains has been visualized. (Siezen and Van Bruggen (1974), J. Mol. Biol. 90, 77-89) by electron microscopy, which shows 1/20 hemocyanin molecules to be flexible structures consisting of 7-8 apparently spherical units of 55-60 A diameter.


Conformation and unfolding thermodynamics of epidermal growth factor and derivatives. Mouse submaxillary epidermal growth factor (EGF) is a 53-residue single chain peptide hormone of known amino acid sequence which contains three disulfides, five tyrosines, and two tryptophans. Circular dichroic (CD) spectra have been obtained and resolved for EGF, several well-characterized chemical and enzymic derivatives, and related low molecular weight model compounds. Assignments have been made to most of the resolved bands; these include the peptide, aromatic, and disulfide chromophores. From a comparison of the rotational strength of the 213-nm resolved CD band in native EGF with that of standard proteins, EGF is estimated to contain about 22% beta structure and no alpha helicity. A derivative of EGF lacking the five carboxyl-terminal residues (prepared by limited trypsin digestion) and the cyanogen bromide derivative, in which there is a single main-chain cleavage at residue 21, have spectra properties indicative of approximately 10 and 12% beta structure, respectively. The near-ultraviolet CD spectra of the derivatives are similar to, albeit not identical with, that of EGF. The rotational strengths characteristic of the side-chain chromophores in EGF and these derivatives are several-fold higher than the corresponding values in low molecular weight model compounds. Thus, it appears that EGF and these modified forms contain a stable (and similar) tertiary structure. In contrast, the S-aminoethylated derivative of EGF exhibits a drastically altered CD spectrum relative to EGF indicating a different conformation(s). Equilibrium studies on the guanidinium hydrochloride (GdmCl) mediated reversible unfolding of EGF showed that the transition midpoint is quite high (i.e., 6.89 M GdmCl at 25.0 degrees C), thus, indicating considerable stability. From these data a rough estimate of 16 kcal/mol can be made for the unfolding free energy (delta G degrees) of EGF in the absence of denaturant. Interestingly, EGF exhibits greater stability characteristics than several proteins two to four times its size. The cyanogen bromide derivative of EGF exhibited greatly reduced stability characteristics, e.g., the transition midpoint occurred at 4.19 M GdmCl (25.0 degrees C) and delta G degrees was estimated to be approximately 4 kcal/mol. Thus, a single main-chain cleavage reduced the stability of EGF by about 70%. Thermal transitions of EGF and the cyanogen bromide derivative in the presence of concentrated GdmCl are characterized by a relatively high enthalpy of about 25 kcal/mol at 40 degrees C and a low (probably zero) heat capacity. From these thermodynamic parameters one can calculate that the large reduction in delta G degrees due to scission of the single peptide bond between residues 21 and 22 can be attributed almost completely to a change in entropy; e.g., at 40 degrees C the apparent entropy of unfolding of EGF is 20.4 cal mol-1 deg-1 while that of the cyanogen bromide derivative is 66.4 cal mol-1 deg-1.


DNA polyintercalating drugs. Proton magnetic resonance studies of a new acridine dimer. Conformations and interactions with mono- and dinucleotides. The conformation in aqueous solution of one acridine dimer which is able to bisintercalate in DNA (1, 14-bis(5-methoxy-6-chloro-9-acridinyl)-1,5,10,14-tetraazatetradecane tetrahydrochloride) (AcDi) and its interactions with mono- and dinucleotides have been investigated by fast Fourier transform proton magnetic resonance spectroscopy. Variations in chemical shifts of the most distingusihable protons of the acridine dimer with temperature bring evidence of a folded in equilibrium unfolded fast conformational equilibrium in the temperature range of 4-85 degrees C. Equilibrium parameters were estimated. The folded conformation has been deduced from iso-shielding curves of the acridine ring. In the complex between AcDi and 3'- or 5"AMP, and adenine ring is intercalated between the two acridine rings to give a sandwich-like complex. Studies of the interaction with adenylyl(3' leads to 5') adenosine (ApA) show two different complexes in equilibrium with the 3' or 5' moiety of ApA intercalated in the acridine dimer. These conclusions are derived from comparative studies with 2-methoxy-6-chloro-9-(3-dimethylaminopropylamino)acridine dihydrochloride which is the corresponding acridine monomer-(AcMo). In that case the self-association constant was determined. A model of the AcMo-5'-AMP complex was deduced from the analysis of the chemical shifts of the adenine protons. In this model, the N10+-D bond of the acridine points toward the negatively charged phosphate of the nucleotide.


Detection of the sites of alkylation in DNA and polynucleotides by laser Raman spectroscopy. A laser Raman study of the alkylation of calf thymus DNA, poly(dG)-poly(dC) and poly(dA)-(dT) has been made using two water soluble alkylating agents: an antitumor drug, the difunctional methyl nitrogen mustard (HN2), which froms interstrand cross-links, and the dimethyl nitrogen half mustard (HN1). When an excess of the alkylating agent was used, the observed Raman frequencies due to the guanine ring modes in DNA and poly(dG)-poly(dC) changed virtually quantitatively to those of 7-methylguanosine (7-Me-Guo) showing that essentially all of the guanine bases were alkylated in the N-7 position. Furthermore, this alkylated DNA formed a stable double helical complex at neutral pH in which the alkylated guanine residues are in the keto form. No changes in the Raman bands of any of the other bases were observed in alkylated DNA. The DNA double helix, completely alkylated in at the N-7 position of guanine, melts about 35 degrees C below that of the native DNA. Upon melting, the alkylated guanine changes from the keto to the zwitterionic form.


Effect of cross-linking on the secondary structure of DNA I. Cross-linking by photodimerization. An investigation has been made into the effect produced by photo-induced pyrimidine cross-links upon the secondary structure of DNA. We have studied the effect of uv irradiation upon the B in equilibrium A transition in DNA brought about by a change of solvent from 70 to 80% ethanol. Circular dichroism (CD) was used to monitor the conformational changes. However, we first showed by means of laser Raman spectroscopy that CD is a reliable monitor of the conformational change, even though the DNA is aggregated in 80% alcohol solutions. It is suggested that this aggregation stabilizes the A form through lateral interaction between the helices. The uv irradiation experiments show that pyrimidine-dimer cross-links induced into the B-form DNA will lock it irreversibly into that conformation and prevent it from going to the A form in 80% EtOH solution. The A-form DNA can tolerate a few cross-links but converts cooperatively to the B form if a larger number of cross-links is introduced. Irradiation-induced pyrimidine cross-links create locally denatured regions in B-form DNA. Upon continued irradiation, the entire DNA moelcule becomes denatured.


Regulation of ribosomal RNA synthesis and processing during inhibition of protein synthesis by 1,3-bis(2-chloroethyl)-1-nitrosourea. The antineoplastic agent BCNU (1,3-bis(2-chloroethyl)-1nitrosourea) at a concentration of 25 mug/ml inhibits initiation of protein synthesis in HeLa cells. At this low concentration of the drug, the rate of synthesis of 45S ribosomal precursor RNA (pre-rRNA) is selectively inhibited without a marked inhibition of nucleoplasmic RNA. The inhibitory effects of the drug are readily reversible upon removal of BCNU from the growth medium. Pulse-chase analysis of the labeled nucleolar RNA in sucrose-gradients and acrylamide gels indicated that the 45S pre-rRNA synthesized before the addition of BCNU matures normally in the presence of the inhibitor. However, the processing of precursor RNA molecules synthesized following the addition of the drug is inhibited when incubation is continued on in the presence of 25 mug/ml BCNU. Since the formation of mature ribosomes is blocked by BCNU, the data would suggest that the effectiveness of the drug as a potent cell growth inhibitor may result from its inhibition of ribosome formation induced by inhibition of protein synthesis.


Folding and interaction of subunits at the antibody combining site. The Fv fragment derived from mouse myeloma protein 315 possessing anti-dinitrophenyl (DNP) activity, is composed of two subunits, the peptide chain VL and VH. In 8 M urea there is a complete dissociation of VL and VH and an approximately twofold increase in the fluorescence emission of Fv with a characteristic red shift of 11 nm. Upon dilution of Fv from 8 M urea into neutral buffer full regain of activity was observed, concomitant with regain of native fluorescence spectrum. The decrease in fluorescence upon dilution from 8 M urea was used to follow the renaturation process of Fv. At relatively high protein concentration (2.5 x 10(-6) M) two steps were observed during renaturation: a fast one, which is completed in less than 30 s, and a slower step, which proceeds for approximately 20 min. The fast process represents the refolding and association of VL and VH to form an active FV, whereas the slow step is attributed to the formation of "incorrect" associates between VL and VH which slowly reshuffle to the thermodynamically stable active FV. Indeed, at low protein concentration (1.5 x 10(-8 M) only the fast step is observed and renaturation is completed in less than 30s. The presence of hapten does not affect the rate of renaturation of FV. Reoxidation of FV completely reduced in 8 M urea was also found to yield a fully active Fv. Since either VL or VH have only one intrachain disulfide bond, reoxidation was performed at high protein concentration (3 mg/ml) in 8 M urea followed by dilution into neutral buffer. This demonstrates that variable domains not only exist in immunoglobulin structure but can also fold correctly independent of the rest of the peptide chains.


Proteins of the turkey erythrocyte membrane. A new and simplified method is described for preparation of turkey erythrocyte membranes which are essentially devoid of supernatant or nuclear contamination, but retain catecholamine-sensitive adenylate cyclase activity. These membranes have been solubilized in sodium dodecyl sulfate and analyzed by polyacrylamide gel electrophoresis and the major protein components identified. The turkey erythrocyte membranes exhibit a protein profile very similar to that of the human erythrocyte membrane, but contain a protein component of apparent molecular weight of 50000 which is not present in the human membranes. Three surface glycoprotein components of the turkey erythrocyte membranes (apparent molecular weights of 90000, 41000, and 26000) have been identified by periodic acid-Schiff staining of polyacrylamide gels and by cell surface 125I labeling using lactoperoxidase followed by polyacrylamide gel electrophoresis. After deoxycholate solubilization of membranes prepared from iodinated cells, glycoprotein with molecular weights of 90000 and 41000 bind to an infinity column of concanavalin A-Sepharose 4B and elute upon application of methyl alpha-Dmannopyrannoside. The lowest molecular weight glycoprotein component, however does not bind to the insolubilized concanavalin A.


Importance of the flux of phosphate across the inner membrane of kidney mitochondria for the activation of glutaminase and the transport of glutamine. The effect of mersalyl, an inhibitor of phosphate transport across the inner mitochondrial membrane, was investigated on the uncoupled respiration of pig kidney mitochondria in the presence of glutamine as substrate and on the activity of the phosphate-dependent glutaminase in the intact organelles. In addition, the submitochondrial location of the enzyme was reinvestigated. (1) It was found that mersalyl completely inhibits uncoupled respiration of the mitochondria in the presence of glutamine as substrate, whereas respiration with glutamate was not affected. The same amount of mersalyl which inhibits coupled oxidation of glutamine also inhibits coupled oxidation of glutamate and some other substrates. (2) Mersalyl strongly inhibited the activation of glutaminase in intact mitochondria only in the presence of inhibitiors of electron transport or of an uncoupler. The addition of a detergent prevented or fully released the inhibition. The effect of mersalyl was observed even when the mitochondria were pre-incubated with phosphate or incubated in the phosphate-free medium. If mersalyl and carbonyl cyanide m-chlorophenylhydrazone (CCCP) were added 3 min after pre-incubation with phosphate the same intramitochondrial concentration of the anion as in control experiments was found, whereas the activity of glutaminase was severely inhibited. These findings suggest that the activation of the enzyme by phosphate in intact nonenergized mitochondria occurs only if the activator moves across the inner mitochondrial membrane. (3) Mersalyl (plus CCCP) markedly decreased [14C]glutamine- and [32P]-phosphate-permeable mitochondrial spaces. A close correlation between the decrease of phosphate and glutamine permeable spaces and the inhibition of glutaminase activity was found. (4) If the activation energy of the enzyme was determined with frozen mitochondrial preparations, a discontinuity or break in the Arrhenius plot was observed, whereas the presence of a detergent completely abolished the break. Digitonin or ultrasonic treatment of the mitochondria followed by separation of the membrane and the soluble fraction revealed that glutaminase is a membrane-bound enzyme. On the basis of these findings it is concluded that there is an association between the transport on one side and the transport of glutamine and glutaminase activity on the other. It is possible that the movement of phosphate across the membrane activates the enzyme which facilitates diffusion of glutamine down a concentration gradient. However, the existance of a specific glutamine-phosphate carrier is not ruled out.


Energetics and chronology of phototransients in the light response of the purple membrane of Halobacterium halobium. Arrhenius parameters for formation and decay of phototransients in suspensions of purple membrane fragments in H2O and 2H2O have been determined in the temperature range 0-60 degree C. Kinetic isotope effects are found which show that proton transfer steps are involved in both formation and decay of the two longest-lived transients absorbing at 410 nm and 660 nm, respectively. The results also suggest that these transients do not occupy a single pathway in the spontaneous deexcitation of bacteriorhodopsin within the purple membrane. Purple membrane undergoes a phase transition at 25-30 degrees C in both H2O and 2H2O.


Photoelectrolysis using chlorophyll electrodes. By the conversion of sunlight to chemical energy, photoelectrolysis was carried out using two different chlorophyll-redox compound lining electrodes. These electrodes were prepared by covering platinum plates with chlorophyll and naphthoquinone or anthrahydroquinone and a conducting adhesive. These electrodes exhibit a photoexciting property. The potential was found to shift to a less noble state when the system of the chlorophyll-naphthoquinone electrode was inserted into NAD solution with illumination. On the other hand, the photoexcitation of the system of the chlorophyll-anthrahydroquinone electrode inserted into ferrocyanide solution made the potential more noble. (If the potential in the dark is in the dark is in the positive region of the scheme and the potential moves in the positive direction when the light is turned on, it can be said to be more positive region, it can be said to become less noble, but it is not suitable to say that it becomes more negative.) To make the potential difference between two electodes as big as possible, various factors such as intensity of illumination, molar ratio of chlorophyll to naphthoquinone or anthrahydroquinone and concentration of redox compound in electrolyte were examined. A cell was set up by combining the system of the chlorophyll-naphthoquinone electrode in NAD solution with that of the chlorophyll-anthrahydroquinone electode in ferrocyanide solution, and photoelectrolysis was carried out by closing the external circuit with illumination. The photovoltage between the two electrodes was 0.25 V and the current density was 8 muA/cm2. It was found that NAD was reduced to form NADH at the chlorophyll-naphthoquinone electrode and ferrocyanide was oxidized to form ferricyanide at the chlorophyll-anthrahydroquinone electrode.


Anomalies in the kinetics of photosynthetic oxygen emission in sequences of flashes revealed by matrix analysis. Effects of carbonyl cyanide m-chlorophenylhydrazone and variation in time parameters. The model of Kok et al. (Kok, B., Forbush, B. and McGloin, M. (1970) Photochem. Photobiol. 11, 457-475) is considered the best kinetic explanation of the damped oscillations of O2 evolution induced in higher plants by a sequence of brief saturating flashes. Matrix analysis applied to this model shows that the parameters involved (distribution of S states at zero time, probabilities of transition between states induced by a flash) cannot be completely known from the O2 yield sequence, Yn. However, four quantities, with limited content of information, are readily derived from data, without additional assumptions. They are sigma1, sigma2 and sigma3, three quasisymmetrical functions of the transition coefficients, and Y, a weighed average of four consecutive Yn values. The extent of misses and double hits and their variations can be qualitatively ascertained by inspection of the relative values of sigma1, sigma2and sigma3. In a regular sequence (stricly obeying Kok's model), all four quantities should be constant along the time axis. It is shown that actual sequences are seldom regular, in particular in the following conditions: (1) variable flashing frequency, (2) addition of carbonylcyanide m-chlorophenylhydrazone, (3) incomplete deactivation, (4) change of flashing frequency at steady state. In order to account for these anomalies, it is proposed to modify Kok's model by introducing, in parallel to the four state storage entity (S states), a side carrier C, which can reversibly exchange a positive charge with it. In the new model, the transition coefficients are essentially time varying, thus producing a nonregular behaviour of Yn sequences.


Studies on the rate-limiting reaction of photosynthetic oxygen evolution in spinach chloroplasts. The modulated oxygen polarography has been used to study the rate-determining steps of photosynthetic oxygen evolution in spinach chloroplasts. The rate constant, k, of the reaction has a value of 218 +/- 10 (S.E.) s-1 at 23 degrees C and an activation energy of 7 +/- 2 (S.E.) kcal-mol-1. A kinetic isotope experiment indicated that this step is probably not the water-splitting reaction. These findings resemble previous results with the unicellular alga Chlorella (Sinclair, J. and Arnason, T (1974) Biochim. Biophys. Acta 368, 393-400). In other experiments we changed the pH, O2 concentration and osmolarity of the medium, and treated the chloroplasts with 1 mM NH4Cl without detecting any significant change in k. These results suggest that the step is irreversible. However, a significantly lower value of k, 110 +/- 20 (S.E.) s-1 was obtained when all salts except 1 mM MgCl2 were removed from the medium bathing the chloroplasts.


Exciton interaction among chlorophyll molecules in bacteriochlorophyllaproteins and bacteriochlorophyllareaction center complexes from green bacteria. Absorption and CD spectra of bacteriochlorophyll a proteins and bacteriochlorophyll a reaction center complexes from two strains of Chlorobium limicola were recorded at 77 degrees K. Visual inspection showed that the Qy-band of chlorophyll in either protein was split into at least five components. Analysis of the spectra in terms of asymmetric Gaussian component pairs by means of computer program GAMET showed that six components are necessary to fit the spectra from strain 2K. These six components are ascribed to an exciton interaction between the seven bacteriochlorophyll a molecules in each subunit. The clear difference between the exciton splitting in the two bacteriochlorophyll a proteins shows that the arrangement of the chlorophyll molecules in each subunit must be slightly different. The spectra for the bacteriochlorophyll a reaction center complexes have a component at 834 nm (absorption) and 832 nm (CD) which does not appear in the spectra of the bacteriochlorophyll a proteins. The new component is ascribed to a reaction center complex which is combined with bacteriochlorophyll a proteins to form the bacteriochlorophyll a reaction center complex. The complete absorption (or CD) spectrum for a given bacteriochlorophyll a reaction center complex can be described to a first approximation in terms of the absorption (or CD) spectrum for the corresponding bacteriochlorophyll a protein plus the new component ascribed to the reaction center complex.


Correlation of reaction-center chlorophyll (P-700) oxidation and bound iron-sulfur protein photoreduction in chloroplast photosystem I at low temperatures. The extent of P-700 photooxidation at 18 degrees K has been followed in three different chloroplast preparations (unfractionated chloroplasts and two preparations enriched in Photosystem I). More than 90% of P-700+ formation in all preparations was eliminated by the addition of sodium dithionite at pH 10. Photoreduction of a bound chloroplast iron-sulfur protein was also decreased by at least 90% under similar conditions. Electron paramagnetic resonance spectra of the chloroplast preparations in the presence of dithionite showed chemical reduction of bound iron-sulfur protein under conditions where primary photochemistry is eliminated. These results indicate that P-700 photooxidation is concomitant with photoreduction of a bound iron-sulfur protein and that this iron-sulfur protein functions as the primary electron acceptor of Photosystem I.


Unusual C24, C25, C26 and C27 polyunsaturated fatty acids of the marine sponge Microciona prolifera. (1) Complete characterization of the fatty acids of the marine sponge Microciona prolifera, including double bond positional isomers, has identified 95 different acids in amounts of 0.1% or more. Trace amounts of 23 other acids were found. (2) 48% of the fatty acids present have C24--C28 chain lengths. These are all saturates, monoenes, dienes and trienes; the tetraene, pentaene and hexaene acids possess the usual C18--C22 carbon chains. The numerous C24--C28 acids present apparently originate within the sponge itself, indicating a highly active chain elongation system. (3) A new family of C24, C25, C26 and C27 polyunsaturated acids with isolated double bonds has been discovered. All contain delta 5,9 unsaturation. Specific acids identified were 5,9-24:2; 5,9-25:2; 5,9-26:2; 5,9,17-26:3; 5,9,19-26:3; 5,9,19-27:3 and 5,9,20-27:3. Biosynthetic pathways for such acids are proposed, based on intermediates found in our fatty acid analyses.


Influence of valine deprivation and its reversal on fatty acid metabolism in HeLa cells. The effects that amino acid starvation and re-supplementation have on fatty acid metabolism in HeLa cells have been studied using radio gas chromatographic techniques. Deprivation of valine for 13.5 h caused fatty acid de novo biosynthesis, elongation and desaturation to cease. This effect was reversed within 5 h by adding valine back to the culture. During deprivation accumulation of triacylglycerol occurred. The return of valine to the culture caused compositional changes in the triacylglycerols and phosphatidylcholines.


Contribution of intermuscular fat to lipogenesis from dietary glucose carbon in mice. We assessed the contribution of various tissues to the synthesis of fat from glucose carbon in mice during rapid lipogenic activation induced by a glucose test meal. Nibbling and gorging mice were maintained on a 58% glucose, fat-free diet. The mice were fasted 22 h and refed 5-10 muCi [U-14C]glucose (120 mg/20 g body weight) either by gastric intubation or as a test meal (58% glucose diet). The muscular carcass in both nibblers an gorgers contained more than 75% of the total radioactivity in the fatty acids derived from glucose; liver and epididymal fat pad accounted for only a small percentage. Mort than half the carcass activity was in the "muscular" tissue as neutral lipid acids. We could isolate a discrete fat body in the popliteal region of the leg muscle ("muscle fat", "intermuscular fat"). The popliteal fat converted glucose carbon to fatty acids an order of magnitude faster, per unit weight, than the epididymal fat pad or skeletal muscle. The fatty acid moiety of the triacylglycerols had the major portion of the label in the popliteal fat 2 and 6 h after ingestion of the glucose test meals. The diacylglycerol pool was active at 2 h and its activity faded at 6 h implicating its intermediary role in lipid metabolism similar to published findings in epididymal fat pad. These results indicate that fat cells associated with the muscular carcass may play a major role in the de novo synthesis of fat from dietary carbohydrate in mice.


Net changes in intermuscular fat before and during rapid lipogenic activation in mice. We have attempted to measure net changes in lipid content in a discrete "intermuscular" fat pad during rapid lipogenic activation that occurs after a previously fasted mouse nibbles a glucose-rich test meal for several minutes. The popliteal fat pad was chosen for the study since it has been shown to be about an order of magnitude more active than the epididymal fat pad in the synthesis of fatty acids from glucose carbon in fasted-refed mice. We found a highly reproducible net loss in the popliteal fat pad's weight and lipid content during fasting. Net deposition of lipid occurred when 24-h fasted mice were allowed to eat a fat-free, 58% glucose diet for several minutes. In two out of three experiments lipid repletion was complete after one brief period of nibbling. Significant decreases in the net amounts of each majority fatty acid, 16:0, 16: 1, 18:1 and 18:2, were found to occur in the popliteal fat pad during a 24-h fast. After nibbling their test meal for several minutes, previously fasted mice restored their major essential fatty acid, linoleic acid, to the original fed level within 2 h, even though total lipid repletion was incomplete. Highly significant net increases in each major non-essential fatty acid were also found after brief ingestion of the test meal; however, in one experiment (incomplete repletion) only about half of the depleted fatty acids was restored. When two successive glucose test meals were eaten (2-h interval), popliteal fat converted glucose carbon to fatty acids more than twice as fast after the second feeding as after the first. However, no significant additional increment either in tissue weight or in total lipid fatty acids was found after the second test meal. Based on these findings, the possible significance of intermuscular fat in the utilization and deposition of fat is discussed.


Is fatty liver induction a general feature of the administration of foreign sulfhydryl compounds? The intraperitoneal administration of 2-mercaptoethanol or 2-mercaptoacetate (40 muM/100 g body weight) to the rat induces a fatty liver, a marked and early increase of free fatty acids and a decrease of triacylglycerol and phospholipids in the blood. These changes are accompanied by a decrease of the ketone body level and the beta-hydroxybutyrate/acetoacetate ratio in the liver. Under the same experimental conditions, however, administration of 2-mercaptopropionate fails to induce a fatty liver and does not modify the hepatic ketone body level or the blood triacylglycerol and free fatty acid levels. These results led us to conclude that fatty liver induction is not a general feature of foreign thiols, and suggest that increased peripheral fat mobilization as well as decreased hepatic lipoprotein synthesis and/or release are responsible for the 2-mercaptoethanol- and 2-mercaptoacetate-induced fatty liver.


Effects of feeding ethyl-dihomo-gamma-linolenate on prostaglandin biosynthesis and platelet aggregation in the rabbit. 1. The ethyl ester of dihomo-gamma-linolenic acid (20:3omega6) (1 g/kg/day) was fed to rabbits for 25 days. Plasma lipids and platelet aggregation were analyzed on day 1, 11, 16, 21 and 26. 2. All plasma lipid classes were greatly enriched with 20:3omega6. Arachidonic acid levels were elevated to a smaller extent. The different platelet phospholipid fractions analyzed were also highly enriched with 20:3omega6, whereas the arachidonic acid content in platelet phospholipids was significantly lower than in control animals. 3. The excretion of 7 alpha-hydroxy-5,11-diketotetranorprostane-1,16-dioic acid, the major urinary metabolite of prostaglandin E1 and E2 was increased 4.6 fold by the treatment. 4. Platelet aggregation in response to ADP, collagen and arachidonic acid did not differ at any time betweeen 20:3omega6 treated rabbits and controls. 5. It is concluded that prostaglandin E biosynthesis can be increased by enriching the prostaglandin precursor pool. Platelet aggregation in vitro is not altered by feeding ethyl 20:3omega6.


Lipid transport across the intestinal epithelial cell. Effect of colchicine. Rats injected with colchicine (0.5 mg/100 g of body weight) 1 h before ingestion of a margarine emulsion (1 g in 2 ml of saline) do not show the rise in plasma triacylglycerol concentration found in controls during the subsequent hours. The effect of colchicine is more dramatic when the experiment is performed after prior administration of Triton WR-1339, a substance known to inhibit the catabolism of lipoproteins. Colchicine-treated rats also showed a five-fold increase in the content of triacylglycerol in proximal jejunum, when compared to controls. These results are consistent with the idea that colchicine interferes with the intracellular phase of fat absorption, suggesting that the microtubular-microfilamentous system could be involved in the release of chylomicrons from the intestinal cell into the circulation.


The metabolism of the phosphonium analogue of choline in vitro and in vivo, and its detection in phospholipids by 31P-NMR. 1. The phosphonium analogues of choline, phosphorylcholine, CDPcholine and phosphatidylcholine were synthesized chemically and characterized by 1H-NMR and 31P-NMR; in 1,2-distearoyl-DL-glycero-3-phosphorylphosphocholine, the 31P-NMR chemical shift of phosphonium relative to phosphate was--28.2 ppm. 2. A comparison was made of the rates of reaction of choline kinase, cholinephosphate cytidyltransferase, cholinephosphotransferase and phospholipase C on natural and phosphonium substrates. Enzyme reaction rates were similar for all but the cytidyltransferase, which exhibited a 3-fold preference for the normal substrate. 3. Weanling rats were maintained for 6 weeks on a diet in which choline was fully replaced by phospho[1,2-14C2]choline mixed with a trace of [Me-3H] choline. Incorporation of phosphocholine into liver lipids was detectable by 31P-NMR even in crude tissue homogenates. Choline-based phospholipids of liver, kidney, lung and brain were extracted, and phosphocholine incorporation calculated from 31P-NMR peak area ratios. The phosphatidylcholine analogues were separated by preparative thin-layer chromatography. Incorporation of phosphocholine ranged from 33% in lung phosphatidylcholine to 6% in kidney sphingomyelin. Variations in 14C/3H ratio between feed and phospholipid extracts indicated preferences for exogenous choline over phosphocholine varying from 1.3: 1 in brain to 3.2: 1 in liver. The results indicated that phosphocholine is a potentially useful 31P-NMR probe for the study of membrane lipids.


Formation of bile acids in man. Metabolism of 7alpha-hydroxy-4-cholesten-3-one in normal subjects with an intact enterohepatic circulation. The formation of bile acids in man is thought to involve a series of reactions in which the initial steps are the same for both cholic acid and chenodeoxycholic acid. The point of bifurcation of the pathway is postulated to occur after the formation of 7alpha-hydroxy-4-cholesten-3-one. To test the hypothesis that the entire synthesis of both bile acids proceeds through this intermediate we studied the metabolism of labeled 7alpha-hydroxy-4-cholesten-3-one in eight normal subjects with an intact enterohepatic circulation. If all the production of cholic acid and chenodeoxycholic acid takes place via 7alpha-hydroxy-4-cholesten-3-one, the areas under the specific decay curves of cholic acid and chenodeoxycholic acid should be identical following a single injection of this labeled intermediate. However, in 6 of the 8 subjects studied the area under the cholic acid specific activity decay curve was significantly less than the area under the chenodeoxycholic acid specific activity decay curve. These results that the production of cholic acid in man may not always involve the intermediate 7alpha-hydroxy-4-cholesten-3-one.


The fluorescence decay of human serum albumin and its subfractions. Human serum albumin does not decay monoexponentially although it contains a single tryptophan residue per molecule. The molecular population is thus heterogeneous with respect to the tryptophan emission. The separated monomeric and dimeric molecules of this protein, as well as various fractions isolated by the procedures of Foster and his coworkers, exhibit deviations from monoexponential decay which are comparable to those of the unfractionated protein; thus, the heterogeneity in molecular population of human serum albumin persists in the various fractions. By comparing the fluorescence decay data of this protein in the presence of thyroxine with the corresponding quenching data it was found that the fluorescence of the protein does not respond uniformly to the binding for all protein molecules. Qualitatively similar behavior was found for bovine serum albumin. In view of the above, binding studies followed by fluorescence should be viewed as averages over a heterogeneous population of the molecules of the serum albumin.


Energetics of the deformation of a peptide unit. Semi-empirical molecular orbital and ab initio study of N-methyl acetamide and N-acetyl-L-alanine N-methyl amide. The problem of non-planarity of peptide unit has been investigated using N-methyl acetamide as a theoretical model. A semi-empirical molecular orbital method: Iterative Extended Hückel Theory viz. IEHT/2 (Adams S. (1974) Doctoral dissertation, State University of New York at Buffalo, U.S.A.) and non-empirical abinitio method with minimal basis set, STO-3G (Hehre, W.J., Stewart, R.F. and Pople, J.A. (1969) J. Chem. Phys. 51, 2657-2664) were used to probe the energetics of the distortion of a planar peptide unit. Distortion of one of the peptide units in a dipeptide, N-acetyl-L-alanine N-methyl amide has also been investigated using abinitio method. The studies amply demonstrate the possibility of the existence of a non-planar peptide unit. Distortion of about 10-15 degrees is predicted to bring about very small loss in energy. The results are substantiated by results from experimental studies.


The hemoglobin system of the primitive fish, Amia calva: isolation and functional characterization of the individual hemoglobin components. Blood from the primitive holostean fish, the bowfin, Amia calva, contains 2 mo of ATP per mol of hemoglobin. The hemolysates contain at least five tetrameric hemoglobin components which differ in their oxygen affinities and their response to cofactors such as ATP. The binding of oxygen by each chromatographically isolated component, including a cathodal component, is influenced by pH and organic phosphates; there is no significant differentiation of function or structure as seen in trout and certain other fish hemolysates. Kinetic analyses of ligand binding indicate that the Bohr and Root effects of Amia calva hemoglobins are best explained by changes in both the "on" and "off" constants. At low pH, the increase in the "off" constant is smaller than for most other Root hemoglobins. The hemoglobin system of Amina calva is functionally undifferentiated and may be representative of the ancestral condition in teleosts.


The presence of N-terminal pyroglutamyl residues in hog thyroglobulin. A method was devised to isolate N-terminal peptide fragments from the polypeptide chains constituting thyroglobulin even in the case when the terminal amino groups are naturally blocked, for instance, acylated. Reduced and carboxymethylated hog thyroglobulin was first acetylated and digested with thermolysin. The blocked N-terminal peptide fragments were separated from the unblocked N-terminal fragments by column chromatography on Dowex 50, then on Dowex 1 after dinitrophenylation, and finally fractionated into ten fractions by paper chromatography after gel filtration on Sephadex G-10. Structural analyses by enzymic or partial acid hydrolysis of these peptide fractions failed to detect N-terminal acetyl amino acid. Instead, pyroglutamyl peptides including pyroglutamylleucine were found. By the same method, acetylated lysine and glycine were identified for chicken lysozyme and horse myoglobin, respectively. The use of thermolysin because of its unique specificity, and the possible relevance of the present result to the previous data on the N-terminal analysis of thyroglobulin are discussed.


Purification of human blood clotting factor X by Blue Dextran agarose affinity chromatography. Blue Dextran 2000 coupled covalently to agarose has been used as an affinity column for the rapid separation of human blood clotting Factor X. Factor X has been isolated with approximately 2000-fold purification from human citrated plasma and shows single-component behavior by sodium dodecyl sulfate gel electrophoresis. Affinity columns prepared with Blue Dextran chromophore (Cibacron blue) derivatized to agarose or Sepharose gave negative results. These studies have shown that Blue Dextran agarose possesses unique biospecific and nonbiospecific properties, both of which are essential to achieve resolution of Factor X from other vitamin K-dependent blood clotting factors.


The use of omicron-phthaldialdehyde in the detection of proteins and peptides. A highly sensitive method utilizing omicron-phthaldialdehyde for an automated fluorescent assay of proteins and peptides is described in this paper. As an example of the utility of omicron-phthaldialdehyde, the separation of bovine serum albumin monomer from bovine serum albumin oligomers is discussed.


The stoichiometry of the components of arthropod thin filaments. Limulus thin filaments confer calcium sensitivity on calcium-independent myosins and contain in addition to actin and tropomyosin, three troponin components. The molar ratio of actin:tropomyosin:troponin sub-unit T (TN-T): troponin sub-unit C (TN-C) is approximately 7:1:1:1, as in vertebrates, but twice the amount of the troponin sub-unit I (TN-I) may be present. Arthropod troponin binds approximately 1 mole Ca/mol troponin, a significantly smaller amount than bound by vertebrate troponin.


Amidated carboxyl groups in elastin.

Amidated carboxyl groups in elastin. Dicarboxylic amino acids constitute the most numerous residues of insoluble elastin in which are potentially ionizable in the physiological range of pH. These residues are essential in facilitating productive electrostatic interaction between elastase and elastin. The present study has investigated the possibility that the glutamic and aspartic acid residues of elastin are amidated. Acid-labile amide-bound ammonia of elastin was quantitated after hydrolysis of the insoluble protein with 2 M HC1 by incubating aliquots of microdistilled hydrolysates with glutamate dehydrogenase, excess alpha-ketoglutarate, and reduced nicotinamide adenine dinucleotide and measuring the resultant decrease in A340 due to oxidation of the dinucleotide cofactor. It was found that ligament elastin purified by repeated autoclaving contains approximately 2.29 mumol of acid-labile amide nitrogen per 10 mg of protein, a value equivalent to approximately 70% of the total number of dicarboxylic amino acid residues. Independent analysis of the amide content was obtained by amino acid analysis of an esterified and reduced elastin sample in which the free dicarboxylic amino acid residues had been converted to the corresponding alcohol derivatives. This analysis indicated that autoclaved ligament elastin contains approximately 18 glutamine, 3 asparagine, 4 glutamic acid and 5 aspartic acid residues per 1000 residues, in good agreement with the analysis of total acid-labile ammonia. The esterified and reduced elastin derivative was nearly inert as an elastase substrate, consistent with a lack of free dicarboxylic amino acid residues. However, addition of sodium dodecyl sulfate to this elastin derivative restores enzyme-substrate charge complementarity, and the elastin-ligand complex was readily hydrolyzed by elastase at the fully stimulated rate, emphasizing the control such ligands can exert in elastolysis. The amide bonds of elastin were found to be significantly more resistant to hydrolysis by 0.1 M NaOH at 98 degrees C than were those of lysozyme or free amidated amino acids. The finding that most of dicarboxylic amino acid residues of elastin exist at neutral amides further emphasizes the apolar character of elastin and has bearing upon the metabolic susceptibility, ligand-binding ability and structural aspects of this connective tissue protein.


A variant of prothrombin induced in cattle by prolonged administration of warfarin. Cows on long term Warfarin therapy produce a form of prothrombin which, although it binds to barium citrate, has a low biological activity. Activation experiments on this form of prothrombin show that it is only slowly converted to thrombin in the presence of Ca2+, although the thrombin produced has normal activity. Further experiments show that the Fragment 1 region of the molecule has a reduced calcium binding capacity. The results indicate the existance of a partially carboxylated form of prothrombin.