Amyloid A: amphipathic helixes and lipid binding. Polypeptide segments, composed of alpha helixes with specific surface topography termed amphipathic helixes, have been proposed as the basic lipid-associating domains of apolipoproteins from the plasma lipoproteins. A computer search for proteins having sequences that could form amphipathic helixes indicated that amyloid A, a pathologically occurring protein usually associated with "secondary" amyloidosis, also contained amphipathic helixes. In studies reported here, amyloid A is shown to associate spontaneously with phospholipid vesicles with the following results: (a) the formation of a protein-lipid complex isolated by equilibrium density gradient ultracentrifugation, (b) a 100% increase in alpha helicity as measured by circular dichroism, (c) a 9-nm shift in the fluorescence maximum due to the single tryptophan residue located in the amphipathic region, indicating the tryptophan is moving from a polar to a nonpolar environment, and (d) the formation of stacked disk-like protein-lipid complexes as visualized by negative stain electron microscopy. The temperature dependence of the circular dichroic spectrum of the amyloid A-phospholipid complex suggests that the complex is formed by insertion of protein between the fatty acyl chains of the lipid. These findings suggest that the amphipathic helix is an important structural unit in lipid-associating proteins and that this unit can be recognized on the basis of its amino acid sequence. In addition, these studies have implications for the origin and function of amyloid A protein.


Effects of pressure upon the fluorescence of the riboflavin binding protein and its flavin mononucleotide complex. The effect of pressure in the range of 10(-3)-10 kbars upon the ultraviolet fluorescence of the riboflavin binding protein and the fluorescences of its complex with flavin mononucleotide has been studied. The fluorescence spectrum of the isolated protein showed a reversible red shift of 12nm (1000 cm-1) at high pressure, indicating the reversible exposure of the tryptophan to solvent. From the pressure dependence of the visible fluorescence of the protein-flavin complex in the region of 1-4 kbars the volume change in dissociation of the protein-ligand complex was estimated to be +3.3ml/mol. A very sharp increase in fluorescence-up to 30-fold of the low-pressure value-takes place in the region 5-8 kbars. This increase is due to release of the flavin from the complex and is assigned to pressure denaturation of the protein. The midpoint, rho 1/2, of this transition was found at 6.5  kbars and the change in volume, delta, in the reaction (native-to-denatured) was calculated to be -74ml/mol. Addition of up to 30% methanol results in a progressive decrease both in delta and rho 1/2, in agreement with the concept that hydrophobic bonding stabilizes the native structure.


Purification and characterization of the agglutinins from the sponge Axinella polypoides and a study of their combining sites. The hemagglutinins from the sponge Axinella polypoides were isolated by affinity chromatography using Sepharose 4B as an absorbent and eluting with DGal. Further separation on DEAE-cellulose and preparative disc electrophoresis on polyacrylamide and agarose gave three fractions. The physicochemical properties and binding specificities of the two main agglutinins were studied. Homogeneity was tested by polyacrylamide electrophoresis and immunoelectrophoresis and by sedimentation analysis. In isoelectric focusing, agglutinin I (mol wt 21 000) showed two bands at pH 3.8 and 3.9. Agglutinin II (mol wt 15 000) showed one band at pH 3.9. Both agglutinins have a carbohydrate content of about 0.5%, are immunochemically unrelated, and differ in amino acid composition. Both precipitate A1, A2, B, Lea, and precursor I blood group substances but to different extents. Inhibition experiments revealed that both agglutinins are inhibited best by terminal nonreducing DGal glycosidically linked beta 1 leads to 6 or by p-nitrophenyl-betaDGal. DGal and DFuc are equally active but about 20 and 12 times less active with agglutinin I and agglutinin II, respectively. DGalNAc and LFuc were inactive even at much higher concentrations. Both agglutinins have similar specificities and react with the immunodominant determinants of blood group B and Lea but not with A and H substances; in A and H substances, reactivity is with side chains in which beta-linked DGal is unsubstituted at the nonreducing terminus. The Axinella polypoides lectins are compared with galactose-specific lectins of different origin and with the aggregation factor system is sponges.


Transcription of isolated mouse liver chromatin. Analysis of RNA transcription from isolated mouse liver chromatin has been undertaken by means of RNA-excess hybridizations with small amounts of radioactive DNA. This analysis indicates that mouse liver chromatin is a restricted template for the in vitro synthesis of RNA complements to repetitive DNA, but more RNA species are synthesized than are found in the RNA isolated from mouse liver nuclei. Extraction with 0.5 M NaC1 destroys the template restriction of isolated chromatin. RNA synthesized in vitro from DNA or chromatin templates by Escherichia coli RNA polymerase, as well as in vivo mouse liver nuclear RNA, were each hybridized to 125I-labeled DNA of high, intermediate, or low reiteration frequency. Chromatin-primed and nuclear RNA saturate a smaller portion of each DNA fraction than does DNA-primed RNA. However, chromatin-primed RNA  saturates more high and low reiteration frequency DNA than does nuclear RNA. Simultaneous hybridization of nuclear-and chromatin-primed RNA with 125I-labeled DNA indicates that chromatin-primed RNA contains all of the sequences present in nuclear RNA. Extraction of chromatin with 0.5 MNaC1 leads to removal of histone F1, as well as a wide variety of non-histone proteins. When used as a template for in vitro RNA synthesis, such salt-extracted chromatin produced RNAs that hybridize as large a portion of each DNA fraction  as does DNA-primed RNA.


Effect of histones and nonhistone chromosomal proteins on the transcription of histone genes from HeLaS3 cell DNA. To elucidate the manner in which histones and nonhistone chromosomal proteins interact to render histone genes transcribable in HeLa S3 cells, we have examined transcription of histone mRNA sequences from DNA, as well as from several DNA-chromosomal protein complexes. Histone mRNA sequences were assayed by hybridization to a 3H-labeled single-stranded DNA complementary to histone mRNAs. Our results indicate that DNA is an effective template for transcription of histone mRNA sequences and that histones by themselves inhibit transcription from DNA, including transcription of histone genes, in a dose-dependent, nonspecific manner. When complexed with DNA alone, nonhistone chromosomal proteins do not affect the transcription of histone mRNA sequences. However, when associated with DNA in the presence of histones, nonhistone chromosomal proteins are capable of selectively rendering histone genes transcribable. These results suggest a possible role for nonhistone chromosomal proteins in mediating the interactions of histones with DNA to render histone genes transcribable.


Activation of in vitro histone gene transcription from Hela S3 chromatin by S-phase nonhistone chromosomal proteins. Using a 3H-labeled single-stranded complementary DNA probe for detection of histone mRNA sequences (Thrall, CL., Park, WD., Rashba, HW., Stein, JL.,Mans, RJ., and Stein, GS.(1974), biochem. Biophys. Res. Commun. 61,1443) we have found that histone genes are transcribed in vitro from chromatin isolated from S-phase HeLa cells but not from chromatin isolated from G1-phase cells (Stein, G., Park W., Thrall, C., Mans, R., and Stein, J. (1975a), Nature (London) 2578 764; Stein, G., Park, W., Thrall, C., Mans, R., Steins, J.(1975b), Biochem. Biophys. Res. Commun. 63, 945). Utilizing the technique of chromatin reconstitution, we have recently demonstrated that it is the nonhistone chromosomal protein portion of the genome that is responsible for this  difference in in vitro histone gene expression (Stein et al., 1975a). In order to determine whether this is attributable to some component of the S-phase chromosomal proteins that promotes the transcriptin of histone genes, a component of the G1 phase chromosomal proteins that inhibits histone gene transcription, or both, in the present study chromatin from both G1 and S-phase cells was dissociated and then reconstituted in the presence of various chromosomal proteins. The results of this study confirm that it is the nonhistone chromosomal proteins that are responsible for the cell cycle stage specific differences in in vitro histone gene expression and further show that these differences can be accounted for by a component or components of the S-phase nonhistone chromosomal proteins that has the capacity, when reconstituted in the presences of G1 phase chromatin, to render the histone genes transcribable in a dose-dependent fashion.


Ethidium bromide as a probe of conformational heterogeneity of DNA in chromatin. The role of histone H1. The accessibility and the tertiary structure of the DNA inside chromatin were studied by using ethidium bromide (EB) as a fluorescent probe. The exclusion model of binding was refined by introductina a parameter alpha (0less than alpha less than 1) which measures the accessibility of the DNA and by taking into account when necessary the existence of two sets of binding sites. We were thus able to fit predicted and experimental isotherms and then to describe completely EB binding to native or partially histone depleted chromatin under various conditions. Itn native chromatin 95% of the DNA (alpha = 0.95) appears to be accessible to EB but two sets of sites are present. The first one corresponds to alpha = 0.13 and is characterized by an affinity constant which is higher by two orders of magnitude than that relative to pure DNA. The second set corresponds to alpha = 0.82 and the corresponding binding constant is only  three or four times lower than that of pure DNA. The sites with high affinity are still present after treatment with formaldehyde but disappear after removal of histon H1. By comparison with chromatin treated with deoxycholate of with artifical complexes between H1 and DNA, high affinity sites were found only when all of the histons are bound to DNA. An alpha value around 0.8 is still obtained in 1 M NaC1 treated chromatin, pointing to the fact that histones H3 and H4 are preventing 20% of the DNA to intercalate EB.


Removal of histone H1 exposes a fifty base pair DNA segment between nucleosomes. Micrococcal nuclease has been used to prepare chromatin from HeLa cells and to probe the structure of HeLa chromatin under various ionic conditions and after the removal of chromatin proteins by salt extraction. The results suggest that (1) HeLa chromatin DNA exists as 150-160 base pair beads interspersed with 40-50 base pair bridges;(2) the bead and bridge conformation exists at physiologic salt concentrations; and (3) removal of histone H1 renders the 40-50 base pair bridge, but not the 150-160 base pair bead, more nuclease susceptible.


Comparative orientation of the fluorene residue in native DNA modified by N-acetoxy-N-2-acetylaminofluorene and two 7-halogeno derivatives. Native calf thymus DNA was reacted with N-acetoxy-N-2-acetylaminofluorene (N-AcO-AAF) and its 7-fluoro and 7-iodo derivatives. Different ways of purification of the fluorene modified DNA samples were checked in order ot obtain a nucleic acid free from all noncovalently bound fluorene residues. The decrease in melting temperature in DNA samples modified by N-AcO-AAF(DNA-AAF) was carefully reinvestigated. From these experiments, we conclude that the melting temperature decrease is equal to 1.15 degree C per percent of modified bases, in DNA-AAF samples. Electric dichroism measurements on sonicated DNA samples modified by the different fluorene derivatives show the fluorene ring perpendicular to the helix axis in the case of the N-AcO-AAF and its fluoro derivative, and lying alone the phosphate-sugar backbone in the case of the iodo derivative. The results presented in this paper, along with those obtained earlier, led us to propose an "insertion-denaturation model" for the mode of binding of N-Aco-AAF and its fluoro derivative, and an "outside binding model" for the iodo derivative. Discrepancies with the data obtained by Chang et al.((1974) Biochemistry 13,2142-2148) concerning the melting temperature decrease and the electric dichroism results are observed and discussed.


Backbone conformational change in the A to B Transition of deoxyribonucleic acid. Infrared linear dichroism studies of A-and B-DNA films reveal six bands between 2800 and 3000 cm-1 which must arise from deoxyribose and thymine methyl CH stretching motions. The band at 2890 is perpendicularly polarized in A-DNA but parallel polarized in B-DNA. This band most probably originates in the C'(5)H2 symmetric stretch; the polarization flip is consistent with the structural change occurring at C'(5) during the A to B transition, according to models derived from x-ray work.


Accumulation and decay of messenger ribonucleic acid in mouse kidney. The stability of polyadenylated messenger ribonucleic acid(mRNA) from cytoplasmic structures sedimenting faster than 40S was analyzed in normal mouse kidney. Incorporation of radioactivity into poly(A)-containing and poly(A)-lacking cytoplasmic RNAs separated by oligo(dT)-cellulose chromatography was determined after sedimentation of RNA IN SODIUM DODECYL SULFATE CONTAINING  SUCROSE DENSITY GRADIENTS. Radioactivity accumulated in poly(A)-containing RNA during the first 6 h and then decayed exponentially. Beginning 8-12h after administering label, two components were evident in the decay curve of poly(A)-containing RNA; the short-lived component (approximately 57% of newly synthesized molecules) had an apparent half-life of 6h, and the second class (approximately 43% of new mRNA) was more stable, decaying with a 24-h half-life. These studies provide the basis for examining the regulation of mRNA stability during compensatory renal hypertrophy.


The allosteric effect of inositol hexasulfate on oxygen binding by hemoglobin. Myoinositol hexasulfate (IHS) is a powerful allosteric effector of oxygen binding by hemoglobin. It binds to deoxyhemoglobin at the same site as 2,3-diphosphoglycerate (DPG) and inositol hexaphosphate (IHP) with an affinity which is intermediate between that of the two phosphate esters. The binding constant calculated from the displacement of the oxygenation curve in the presence of low concentrations of IHS is 0.9X10(6) M. The value obtained directly from the number of protons bound as a function of IHS concentration is 1.0X10(6) M. The agreement between these two independent measurements provides an experimental verification of the empirical equation, relating the oxygen affinity to the binding constants, proposed previously (Benesch, R.E., Benesch, R., Renthal, R and Gratzer, W.B. (1971), Nature (London), New Biol. 2348 174).


Conformational aspects of the interaction of polyanions with liganded beta chains of human hemoglobin. The interaction of carbon monoxide beta chains with two allosteric effectors, namely inositol hexaphosphate and benzenehexacarboxylate, was studied. The sedimentation coefficient (s20,w) of the liganded beta chains was measured to be the same both in the presence and absence of the two effectors suggesting that the protein exists as a tetramer under the conditions of our titration and optical studies. The binding of benzenehexacarboxylate to the liganded beta chains was investigated by potentiometric titration in the pH range 6.7-8.0. The results at pH 7.4 showed a binding of 2 mol of benzenehexacarboxylate per tetramer, with an association constant of 1.26 X 10(4) 1. mol-1 at 20 degrees C. The Hill coefficient for the binding was determined to be 0.73. Similar experiments on the interaction of inositol hexaphosphate with the beta chains showed a binding of 2 mol of the effector per tetramer with identical Hill coefficient (0.737) and comparable association constants (0.88 X 10(4)1. mol-1). The value below unity of the Hill coefficient, found for the binding of the two effectors to the protein, probably reflected an anticooperativity produced by the different net electric charges of the free protein and the protein-effector complex. The difference in protons bound per mole of heme by the beta subunits in the presence and absence of benzenehexacarboxylate appeared consistent with the proposal that two groups per chain changed their pK from 6.6 to 7.4 upon the interaction. In the presence of benzenehexacarboxylate, the protonation of these groups appeared to be cooperative, suggesting a conformational change of the protein upon the binding. The absorption spectra of carbon monoxide beta chains in the Soret region was markedly altered by benzenehexacarboxylate and inositol hexaphosphate. The features in the difference spectra of the protein obtained with the two effectors were qualitatively identical and indicated changes in the heme environment produced by the interaction of the effectors with the beta chains. Concomitant changes in circular dichroism and optical rotatory dispersion of the liganded beta chains caused by the addition of the two effectors provided supporting evidence for the conformational change in the protein produced by the binding of the effectors.


Anomalous azide binding to metmanganomyoglobin. The reaction of metmanganomyoglobin (MnIIIMb) with azide presents a novel pattern with direct evidence for kinetic complexity. Although the kinetic analysis may not be unique, it appears that the azide complex cannot be fully formed, as judged by spectrophotometric changes, even with an infinitely great [N3-]. This is interpreted as resulting from an equilibrium between the final spectroscopically observable azide complex and an intermediate species whose spectrum is not substantially different from that of MnIIIMb itself. The two forms of the azide complex appear to exhibit roughly equal proportions at 3 degrees C. We propose that this intermediate is a weak Mn3+ -azide complex in which the metal ion remains out-of-plane toward the imidazole of the proximal histidine, but that the metal lies toward the anion in the "final" complex.


Reinvestigation of some physicochemical and chemical properties of human ceruloplasmin (ferroxidase). The original molecular weight determinations and hydrodynamic data on ceruloplasmin have later been contradicted by crystallographic and other investigations. In order to arrive at the correct copper stoichiometry of the protein we have undertaken a careful reinvestigation of some aspects of the physical chemistry and chemistry of ceruloplasmin with particular attention directed towards molecular weight and copper content. Human ceruloplasmin form I (the major form) was isoalted from fresh normal or retroplacental serum by a procedure designed to avoid proteolysis. The molecular weight of the protein, determined by meniscus depletion sedimentation equilibrium, was found to be 134 000+/-3 000. The sedimentation coefficient (7.25 S) and diffusion coefficient (4.46 10(-7) cm2/s) are consistent with this molecular weight. The frictional ratio calculated from these data, 1.41, and the observed intrinsic viscosity of 4.5 ml/g indicate that ceruloplasmin has a slightly more extended shape than a typical globular protein. This might at least partly be ascribed to its carbohydrate moiety. The amino acid composition, carbohydrate composition, and copper content of ceruloplasmin were determined. The copper content of several preparations varied between 6.0 and 6.6 atoms per mol. The copper stoichiometry of ceruloplasmin is discussed.


Protein kinase associated with tubulin: affinity chromatography and properties. Rat brain tubulin purified by colchicine-agarose affinity chromatography contains protein kinase activity. The kinase activity can be separated completely from tubulin by chromatography on casein columns and is not subsequently retained by colchicine affinity columns. Protein kinase activity associated with purified tubulin does not correlate with the total content of protein kinase activity in brain homogenates, since microtubules isolated from 48 000g fetal brain supernatants contain twice as much protein kinase activity than adult microtubules, although the total protein kinase activity is twice as high in the 48 000g adult supernatant. The protein kinase of tubulin preparations, while corresponding to a different molecule than tubulin, is probably not simply the result of contamination. These observations are interpreted in terms of specific associations between protein kinase and tubulin complexes. The protein kinase-tubulin association may be an important determinant in the regulation of tubulin function. Fetal tubulin polymerizes twice as well as adult tubulin in the absence of glycerol at the same tubulin concentration. The preferred substrate for the protein kinase either in vivo or in vitro (pH 7.4, 37 degrees C) is a specific high-molecular-weight protein, distinct from tubulin, which copurifies with tubulin through different kinds of isolation procedures (i.e., colchicine affinity chromatography and ammonium sulfate precipitation followed by diethylaminoethyl-cellulose chromatography). The tubulin-associated protein kinase is completely dependent on cyclic adenosine monophosphate (Km=10(-7)M), as demonstrated by the complete suppression of activity upon addition of the protein kinase modulator, a well-known specific inhibitor of cAMP-dependent protein kinases


Dental phosphoprotein-induced formation of hydroxylapatite during in vitro synthesis of amorphous calcium phosphate. (Ethylenedinitrilo)tetraacetic acid soluble phosphoproteins were isolated from rat incisor and bovine unerupted teeth. This material was examined for its effect on the stability of amorphous calcium phosphate in vitro. When the precipitation of amorphous calcium phosphate was attempted in the presence of small amounts of these phospho-proteins, an apatite-like mineral was observed to form, which was approximately 60% crystalline, as determined by infrared measurements. This apatite phase could not be induced by addition of phosphoprotein after the precipitation reaction. The organic phosphate bound to these phosphoproteins was shown to be directly responsible for the formation of the apatite phase, since removal of 60% of the covalently bound phosphate with alkaline phosphatase destroyed the protein's ability to induce hydroxylapatite formation. The properties of the dental phosphoproteins appear to be consistent with their possible involvement in the development of the mineral phase of dentine.


Evidence against an acyl-enzyme intermediate in the reaction catalyzed by clostridial phosphotransacetylase. Clostridial phosphotransacetylase catalyzes acyl group transfer between coenzyme A (CoA) and inorganic phosphate and also the arsenolysis of acetyl-coenzyme A (AcCoA) to yield acetate and CoA-SH. The enzyme mobility on sodium dodecyl sulfate electrophoresis corresponds to a molecular weight of 70 000. Kinetics of both forward and reverse reactions are of the ternary type as previously reported and product inhibition data are consistent with a random binding scheme. One essential sulfhydryl group per 70 000 daltons was inactivated in a pseudo-first-order process by either N-ethylmaleimide or 5,5'-dithiobis (nitrobenzoic acid). Reduction of the rate of this inactivation by 50% in the presence of AcCoA or acetyl phosphate concentrations near their kinetic K values demonstrates binding of these acyl donors in simple enzyme-substrate complexes. Moreover, pulse-chase experiments show these binary complexes to be functional and also show that they do not dissociate rapidly compared with their rates of catalytic turnover. Incubation of the enzyme with 14C-labeled acyl donors failed to produce labeled protein after passage through Sephadex. This was true despite efforts to mimic "substrate synergism" with desulfo-CoA or to compensate for unfavorable equilibria by means of CoA traps. Very slow isotope exchange reactions of 32Pi into acetyl phosphate and [3H]CoA into AcCoA were at first observed. As in the cases of several other enzymes recently reexamined, these were shown on careful inspection to be artifacts of contamination by second substrates. Attempts to detect exchange reactions between acetyl phosphate and Pi, even in the presence of the CoA analogue, desulfo-CoA, were also unsuccessful. Therefore, no evidence for an acyl-enzyme could be detected. Furthermore, our data allow us to develop arguments which, we believe, indicate that an acyl-enzyme intermediate is extremely improbable in the reaction catalyzed by phosphotransacetylase.


Identification of the amatoxin-binding subunit of RNA polymerase B by affinity labeling experiments. Subunit B3-the true amatoxin receptor protein of multiple RNA polymerase B. Crude calf thymus DNA-dependent RNA polymerase, RNA polymerase B (ribonucleoside triphosphate: RNA nucleotidyltransferase, EC 2.7.7.6), was incubated with the tritium labeled, potent inhibitor [3H]amanin, in order to form the enzymatically inactive [3H]amanin-polymerase complex ([3H]A-P complex). Subsequent purification procedures for the [3H]A-P complex were based on radioactive assays. Phosphocellulose chromatography separated two radioactive components: PCI, the previously reported amatoxin binding protein, ABP (Brodner and Wieland, 1976), and PC II, the [3H]A-P complex. Sodium dodecyl sulfate gel electrophoresis of the complex showed the presence of a new heavy band very close to subunit B 1 and a decreased intensity of subunit band B 3. These were the only differences noted in the subunit structure of RNA polymerase B. [3H]Amanin was covalently coupled to the enzyme, affinity labeling, by a water-soluble carbodiimide and the resultant conjugate submitted to sodium dodecyl sulfate gel electrophoresis. The profile of radioactivity showed one main peak (greater than 2000 cpm) coinciding with the 550-nm absorption peak of subunit B 3 on a stained parallel gel. Since no other protein band contains any significant radioactivity, the binding site for [3H]amanin and most probably for all amatoxins is localized on the B 3 subunit SB 3.


Chemical characterization and subunit structure of human N-acetylhexosaminidases A and B. Human hexosaminidases A and B were purified from placentae, using two stages of affinity chromatography, to a high degree of purity. Each enzyme was purified 5000-6000-fold, and isolated in 25-40% yield. Enzyme preparations appeared homogeneous in the analytical ultracentrifuge and by acrylamide gel electrophoresis. Hexosaminidase A contained 1.65 residues of sialic acid per molecule, whereas no sialic acid was present in hexosaminidase B. The molecular weights of the A and B isozymes as determined by gel filtration and sedimentation equilibrium are 100 000 and 108 000, respectively. In 5 M guanidine-HCl each of the enzymes yielded a 50 000-dalton species, which can further be dissociated into 25 000-dalton polypeptide chains by reduction and alkylation. The hexosaminidase B yielded one type of polypeptide chain, denoted beta, whereas the product from hexosaminidase A could be separated by ion-exchange chromatography into two species of chains, denoted alpha and beta, in equal amounts. The amino acid compositions of the separated alpha and beta chains were determined, and were found to correlate well with those of the intact enzymes. These findings enable the construction of a plausible model for the molecular structure of both enzymes. According to this model hexosaminidase A is composed of two subunits alpha2 and beta2, in which the two polypeptide chains are linked by a disulfide bridge. The structure of hexosaminidase B is, in parallel, beta2beta2. The suggested model is discussed in view of the accumulated information about the interrelationships between hexosaminidase A and B and the genetic metabolic disorders with which they are involved.


Synergistic activation of adenylate cyclase by guanylyl imidophosphate and epinephrine. A kinetic analysis of the synergistic activation of turkey erythrocyte adenylate cyclase by 1-catecholamines and guanylyl imidodiphosphate (Gpp(NH)p) is described. We have found that the role of the catecholamine hormone is to facilitate the activation of the enzyme by the guanyl nucleotide according to the following mechanism: R-E+G=R-EG R-EG+H=HR-EG leads to HR-E''G where R is the receptor, E the enzyme, G the guanyl nucleotide effector, and H the hormone. The binding steps are fast and reversible but the conversion of the inactive enzyme E to its active stable form (E'') occurs with a rate constant of k=0.7 min-1. This step is essentially irreversible in the presence of high Gpp(NH)p concentrations. In the absence of beta-agonist (1-catecholamine) and at low free Mg2+ concentrations, the activation of the enzyme is insignificant. At high Mg2+ concentration the conversion of E to E'' occurs slowly in the absence of hormone, probably by another pathway. Thus, the presence of a guanyl nucleotide at the allosteric site is obligatory but not sufficient to induce the conversion of the inactive enzyme to its active form. The process of enzyme activation requires both Gpp(NH)p and hormone and under these conditions is essentially irreversible. The permanently active enzyme is stable in the absence of hormone and Gpp(NH)p and its high catalytic activity is stable for many hours. However, hormone and ATP induce a conversion of the high activity to the low activity form. Thus, it seems that both the process of enzyme activation by Gpp(NH)p and its reversal are hormone dependent. Both processes are blocked by the beta-blocker propranolol.


Affinity labeling of bovine colostrum galactosyltransferase with a uridine 5'-diphosphate derivative. The dialdehyde produced by the periodate cleavage of the ribose moiety of uridine 5'-diphosphate (UDP) has been used as an affinity label for the UDP-galactose/UDP binding site of galactosyltransferase from bovine colostrum. This derivative causes progressive inactivation of galactosyltransferase at a rate dependent on its concentration, and under certain conditions is a competitive inhibitor with respect to UDP-galactose. The substrate UDP-galactose protects the enzyme from inactivation. The inactivation is also dependent on Mn2+ concentration in a range that implies that the binding of Mn2+ at site I is a prerequisite for the binding of the UDP derivative. The inactivation can be progressively reversed by nitrogenous bases, or stabilized by KBH4 reduction, which is consistent with the hypothesis that a Schiff base has formed with a lysine residue. Galactosyltransferase was inactivated with a [3H]UDP derivative and the predominant labeled peptide, from thermolysin digestion, isolated and characterized as: Ser-Gly-Lys-UDP.


A class of chromatin particles associated with nonhistone proteins. Unfixed nucleoproteins may be banded isopycnically in metrizamide (2(3-acetamido-5-N-methylacetamido-2,4,6-triiodobenzamido)-2-deoxy-D-glucose) according to the protein/nucleic acid ratio. Unsheared or lightly sheared chromatin bands sharply (p=1.2 g/ml); it has a protein/DNA ratio of 1.4. Chromatin sheared by sonication to approximately 350 base pairs of DNA contains two components with protein/nucleic acid ratios of approximately 1.3 (p=1,185 g/ml) and 2 (p=1.245 g/ml). When chromatin is digested exhaustively with staphylococcal nuclease, two density components are found, one with a protein/DNA ratio of 1.5 (p=1.21 g/ml), the other with a protein/DNA ratio of 2 (p=1.24 g/ml). In both instances the denser particle (p=1.24 g/ml) contains nearly all the nonhistone proteins, while both dense and light fractions contain histones in similar amounts. The base sequence complexity of DNA from the fractions is not distinguishable from that of total DNA and there is no evidence of any concentration of sequences complementary to polysomal polyadenylated RNA molecules.


Characterization of the association of specific proteins with messenger ribonucleic acid. Isolation of messenger ribonucleic acid (mRNA)-protein particles from cytosol or dissociated polyribosomes yielded complexes in which several proteins were consistently associated with mRNA. Some of the mRNA-associated proteins appeared to have a high affinity for mRNA since they remained complexed to mRNA during centrifugation in CsCl gradients. Quantitation of RNA and protein in polyribosomal mRNPs suggested that each molecule of mRNA bound a molecule of each of the two major proteins of 78 000 and 52 000 apparent molecular weights and/or one or more of several minor proteins found in mRNPs. Of the several mRNP proteins, only the protein of 78 000 apparent molecular weight appeared to form a stable complex with the polyriboadenylic acid [poly(A)]-tract of mRNA, suggesting that the remaining mRNA-associated proteins bind to other regions which may be common to many or all mRNAs. Binding of [3H]poly(A)-rich RNA to mRNP proteins was effectively inhibited by unlabeled poly(A)-rich RNA or the homopolymer polyriboguanylic acid [poly(G)], but not by poly(A) or other natural or synthetic mRNAs. The properties of non-poly(A)-dependent binding of mRNA by mRNP protein were similar to those of mRNA binding by the guanosine triphosphate dependent Met-tRNAfMet-binding protein.


In vitro translation of globin: effect of proteins purified by affinity chromatography on polyadenylate-Sepharose. By means of affinity chromatography on poly(adenylic acid) (poly(A))-fixed Sepharose, protein fractions having strong affinity to poly(A) were prepared from postribosomal supernatants of rabbit reticulocyte and rat liver. These fractions contained several proteins similar by electrophoretic analysis to rabbit globin messenger ribonucleoprotein. Protein fractions from both sources were shown to form ribonucleoprotein complexes with rabbit globin mRNA, and these complexes sedimented at the same rate as native globin messenger ribonucleoprotein. Binding of the proteins to RNA was not highly specific, since not only poly(A) but also other polynucleotides as poly(C) or poly(U) were bound to these proteins. Ribosomal RNAs, tRNA, or DNAs did not bind the proteins. In order to ascertain the function of the poly(A)-Sepharose purified proteins, their effects on translation of globin mRNA was studied in vitro. Addition of rabbit reticulocyte protein to globin mRNA resulted in no more than a slight stimulation of both alpha- and beta-chain synthesis. Poly(A)-Sepharose purified protein from rat liver, however, caused a marked preferential reduction of alpha-chain synthesis. These results showed that at least some proteins in the poly(A)-Sepharose purified proteins affect the translation of globin. This inference suggested a possibility that protein moiety in globin mRNP might be involved in control of globin synthesis.


The effect of various treatments in vitro and in vivo on the binding of 125I-labeled anti-rat serum albumin Fab' to rat tissue polyribosomes. A quantitative assay was developed to permit estimation of the relative amounts of albumin-synthesizing polyribosomes in rat liver. The polyribosomes synthesizing albumin were identified by their capacity to bind anti-RSA Fab' radiolabeled with 125I. The anti-RSA Fab'-binding sites occur on the nascent peptide chains attached to liver polyribosomes. These binding sites can be saturated by preincubation of the polyribosomes with large quantities of unlabeled anti-RSA Fab'. The iodinated antibody did not react with polyribosomes isolated from a tissue which does not synthesize rat serum albumin. Pretreatment of hepatic polyribosomes with bovine pancreatic ribonuclease resulted in a 42% enhancement of binding of anti-RSA Fab'. Pretreatment of these polyribosomes with detergents or various levels of Mg2+ did not significantly affect the specific binding of the iodinated antibody. Anti-RSA Fab' associated preferentially with membrane-bound polyribosomes when compared with free polyribosomes following their isolation from animals maintained either on a 90% or a 0% protein diet fed ad libitum. Binding of anti-RSA Fab' to each A260 unit of membrane-bound polyribosomes is from 2.4 to 27 times greater than to each A260 unit of free polyribosomes. However, each A260 unit of free polyribosomes was found to associate with 1.8 times more anti-RSA Fab' when compared with the "loosely bound" subclass of membrane-bound polyribosomes. Each A260 unit of the "tightly bound" subclass of membrane-bound polyribosomes reacted with 4.3 times as much antibody as compared with free polyribosomes. Polyribosomes isolated from the livers of rats sacrificed 6 h after treatment with actinomycin D showed a 42% reduction in their capacity to bind anti-RSA Fab'. Polyribosomes from rats sacrificed 2 h after treatment with actinomycin D showed no reduction in binding capacity. Free polyribosomes from three Morris hepatomas were capable of binding anti-RSA Fab' whereas the antibody would not associate with the membrane-bound polyribosomes of the same hepatomas. Thus the binding of 125I-labeled Fab' antibody molecules to polyribosomes is a useful technique for the subcellular localization of polyribosomes synthesizing specific proteins and for the estimation of the relative proportions of such polyribosomes.


Hormonal and nutritional effects on the binding of 125I-labeled anti-serine dehydratase Fab' to rat tissue polysomes. With 125I-labeled Fab' specific for rat liver serine dehydratase it has been possible to localize polyribosomes synthesizing the enzyme under several different environmental conditions. Evidence is presented to show that, following the administration of amino acids in vivo, the relative synthetic capabilities of free and membrane-bound polyribosomes synthesizing serine dehydratase vary with time. Early during the period of induction of the enzyme by administration of amino acids or by feeding a high protein diet the majority of the newly synthesized enzyme is derived from membrane-bound polyribosomes. Later in the induction process an increasing proportion of the enzyme is synthesized by the free polyribosomes. Subcellular localization studies clearly show that serine dehydratase is synthesized by both subclasses of hepatic membrane-bound polyribosomes, the loose and tight membrane-bound polyribosomes, as well as by the free polyribosomes. It was found that the membrane-bound polyribosomes are the preferential sites of synthesis of the majority of serine dehydratase molecules in the Morris hepatomas 5123C and 7800. It is concluded that the synthesis of the enzyme, serine dehydratase, in rat liver is not discretely compartmentalized in either class of free or membrane-bound polyribosomes. Rather, the relative proportions of the serine dehydratase synthesizing polyribosomes within these two classes of polyribosomes can vary depending on the metabolic and physiologic state of the liver cell.


Preparation of inside-out vesicles of pig lymphocyte plasma membrane. Between 30 and 50% of pig lymphocyte plasma membrane vesicles were not bound by concanavalin A (Con A)-Sepharose. Various results suggest that the Con A-unretarded fraction represents "inside-out" membrane vesicles. First, an alternative cell surface ligand, anti-lymphocytic serum, gave a similar fractionation to Con A. Second, lack of binding by Con A was not due to lack of carbohydrate or to masking of carbohydrate by extraneous protein, because the unfractionated membrane and the unretarded fraction had similar carbohydrate and polypeptide compositions. Third although the carbohydrate of the unretarded membrane vesicles was accessible to 125I-labelled Con A and to release by soluble trypsin, it was not accessible to ferritin-Con A or trypsin-Sepharose. Fourth, antisera against the external surface of the Con A-unretarded vesicles strongly agglutinated the unretarded membrane, but caused negligible agglutination of whole lymphocytes. When attached to Sepharose these antisera bound all of the Con A-unretarded fraction, but failed to bind the membrane that adhered to Con A-Sepharose.


High sensitivity amino acid sequence determination. Application to proteins eluted from polyacrylamide gels. An automatic solid-phase procedure is described for determining the amino-terminal amino acid sequence of very small quantities of proteins. The sample is covalently attached to an inert support so that mechanical and physical losses during sequencing are eliminated. High sensitivity is achieved by using an initial coupling with high specific activity phenyl [35S]isothiocyanate followed by a longer reaction with the unlabeled reagent. The radioactive phenylthiohydantoins are identified by autoradiography after two-dimensional thin-layer chromatography. Unlabeled phenylthiohydantoin-amino acids are added to each fraction to assist in the identification and to act as carriers, hence reducing absorptive and extractive losses of the small quantities of sample. The method may be used on proteins eluted from polyacrylamide gels containing sodium dodecyl sulfate without removal of the detergent. Sequences of up to 20 residues have been obtained on quantities of protein ranging from 2.5 to 70 pmol. Results from proteins of hitherto unknown sequence are included.


Conformational properties of dinucleoside monophosphates in solution: dipurines and dipyrimidines. In order to obtain information about the conformational features in a polyribonucleotide at the nearest neighbor level, detailed nuclear magnetic resonance studies of the dinucleoside monophosphates ApA, ApG, GpA, UpU, CpC, UpC, and CpU were undertaken. Proton spectra were recorded at 100, 220, 270, or 300 MHz for D2O solutions, 0.01-0.03 M, pD 7.4 at 20+/-2 degrees C. Spectra of ApA, ApG, UpU, and UpC were also recorded in the temperature range of 70-90 degrees C. Unambiguous signal assignments of all proton resonances were made with the aid of selectively deuterated dimers. Complete, accurate sets of nuclear magnetic resonance (NMR) parameters were derived for each nucleotidyl unit by simulation-iteration methods. A complete set of chemical shift and coupling constant data was also obtained for all the constituent monomeric units at a concentration and ionization state comparable to that of the dimers. Conformational properties were evaluated quantitatively for most of the bonds in the dinucleoside monophosphates using procedures developed in earlier studies. All of the dimers have a flexible conformational framework in aqueous solution. While flexibility is allowed and alternate conformations are accessible, these molecules nevertheless attempt to achieve conformational identity by showing preferences--sometimes overwhelming preferences--for certain orientations. Thus the ribose rings exist as equilibrium mixtures of C2'-endo in equilibrium C3'-endo conformers with a bias for the C3'-endo pucker in most cases. The C4'-C5' bonds of both nucleotidyl units show significant preference (70-85%) for a gg conformation. Similarly, the dominant conformer (80-90%) about C5'-O5' is g'g'. Even though an unambiguous determination of the orientation about C3'-O3' cannot be made, there is suggestive evidence that the orientation of the 3' phosphate group is coupled to the ribose conformational equilibrium and it is likely that a 3Eg- in equilibrium 2Eg+ equilibrium exists with a bias for the 3Eg- coupled conformation in which the H3'-C3'-O3'-P dihedral angle is about 34-38 degrees. The individual nucleotidyl units in the dimers differ in several key ways from corresponding monomer conformations. Specifically, the ribose equilibrium C2'-endo in equilibrium C3'-endo shifts in favor of C3'-endo upon dimerization, the only exception being UpU. The C4'-C5' and C5'-O5' bonding network in the dimer forms a stable conformational unit and no correlation exists in the dimers between the conformational preference of this fragment and ribose conformer population. The temperature data for the dimers and dimerization data clearly indicate that the transition C2'-endo leads to C3'-endo is directly related to XCN changes brought about by dimerization and stacking...


Effect of 1-beta-D-arabinofuranosylcytosine triphosphate on DNA synthesis in isolated HeLa cell nuclei. 1-beta-D-Arabinofuranosylcytosine 5'-triphosphate (ara-CTP) was found to be a competitive inhibitor of DNA synthesis in nuclei isolated from synchronized S-phase HeLa S3 cells. The inhibition was reversed by dCTP. The main inhibitory effect was to reduce the rate of elongation of the primary DNA pieces from ara-CTP-treated nuclei after a 60-s pulse was about 115-120 nucleotides vs. 215-220 in control nuclei. A reduction in the frequency of initiation of primary DNA pieces probably also occurred. A slight inhibition of the transfer of labeled material from the primary pieces into high-molecular weight DNA was noted and probably arose from the fact that the reduced rate of growth of the primary pieces caused the critical size for ligation to be reached later. Synchronized HeLa S3 cells in suspension were treated with 1-beta-D-arabinofuranosylcytosine for 2 h in S-phase to give a 99% inhibition of DNA synthesis prior to isolation of nuclei. Neither the rate nor the extent of [3H]TTP incorporation into DNA decreased when such nuclei were incubated in the absence of ara-CTP, but the rate of ligation of primary pieces was reduced.


Ligand interactions with the acetylcholine receptor from Torpedo californica. Extensions of the allosteric model for cooperativity to half-of-site activity. The solubilized acetylcholine receptor from Torpedo californica showed positive cooperativity in acetylcholine binding with a dissociation constant of 1.2 X 10(-8) M. Blockade of acetylcholine binding by nicotine was competitive; blockade by d-tubocurarine appeared to result from an allosteric interaction that altered half of the acetylcholine binding sites to a lower affinity form; decamethonium blockade displayed properties of competitive and allosteric inhibition suggesting less specificity for decamethonium binding than seen with either nicotine or d-tubocurarine. The d-tubocurarine inhibition data were evaluated by several possible models involving either differential competitive inhibition or allosteric inhibiton. The data were best described by the allosteric model.


Cholinergic sites in skeletal muscle. II. Interaction of an agonist and two antagonists with the acetylcholine site. The equilibrium interactions of alpha-bungarotoxin, d-tubocurarine, and carbamylcholine with junctional and extrajunctional skeletal muscle acetylcholine receptors were examined. d-Tubocurarine is a competitive inhibitor of the bindings of alpha-bungarotoxin to the acetylcholine receptor. No substantive difference was observed in the association of d-tubocurarine with the junctional and extrajunctional receptors. In contrast, the carbamylcholine inhibition of toxin binding is not competitive. The data indicate that either the single set of alpha-bungarotoxin and d-tubocurarine bindings sites contains two subsets of carbamylcholine sites or that the carbamylcholine binds in a cooperative manner to a single set of sites. In addition, the affinity of carbamylcholine for extrajunctional receptors may be higher than the affinity for junctional receptors.


Binding of chloromethyl ketone substrate analogues to crystalline papain. Papain (EC 3.4.22.2) is a proteolytic enzyme, the three-dimensional structure of which has been determined by x-ray diffraction at 2.8 A resolution (Drenth, J., Jansonius, J.N., Koekoek, R., Swen, H. M., and Wothers, B.G. (1968), Nature (London) 218, 929-932). The active site is a groove on the molecular surface in which the essential sulfhydryl group of cysteine-25 is situated next to the imidazole ring of histidine-159. The main object of this study was to determine by the difference-Fourier technique the binding mode for the substrate in the groove in order to explain the substrate specificity of the enzyme (P2 should have a hydrophobic side chain (Berger and Schechter, 1970) and to contribute to an elucidation of the catalytic mechanism. To this end, three chloromethyl ketone substrate analogues were reacted with the enzyme by covalent attachment to the sulfur atom of cysteine-25. The products crystallized isomorphously with the parent structure that is not the native, active enzyme but a mixture of oxidized papain (probably papain-SO2-) and papain with an extra cysteine attached to cysteine-25. Although this made the interpretation of the difference electron density maps less easy, it provided us with a clear picture of the way in which the acyl part of the substrate binds in the active site groove. The carbonyl oxygen of the P1 residue is near two potential hydrogen-bond donating groups, the backbone NH of cysteine-25 and the NH2 of glutamine-19. Valine residues 133 and 157 are responsible for the preference of papain in its substrate splitting. By removing the methylene group that covalently attaches the inhibitor molecules to the sulfur atom of cysteine-25 we obtained acceptable models for the acyl-enzyme structure and for the tetrahedral intermediate. The carbonyl oxygen of the P1 residue, carrying a formal negative charge in the tetrahedral intermediate, is stabilized by formation of two hydrogen bonds with the backbone NH of cysteine-25 and the NH2 group of glutamine-19. This situation resembles that suggested for the proteolytic serine enzymes (Henderson, R., Wright, C. S., Hess, G. P., and Blow, D. M. (1971), Cold Spring Harbor Symp. Quant. Biol. 36, 63-70; Robertus, J. D., Kraut, J., Alden, R. A., and Birktoft, J. J. (1972b), Biochemistry 11, 4293-4303). The nitrogen atom of the scissile peptide bond was found close to the imidazole ring of histidine-159, suggesting a role for this ring in protonating the N atom of the leaving group (Lowe, 1970). This proton transfer would be facilitated by a 30 degrees rotation of the ring around the C beta-Cgamma bond from an in-plane position with the sulfur atom to an in-plane position with the N atom. The possibility of this rotation is derived from a difference electron-density map for fully oxidizied papain vs. the parent protein.


Studies on the intramolecular and intermolecular kinetic isotope effects in pyruvate carboxylase catalysis. A deuterium kinetic isotope effect of 2.1 was observed when [2H3]pyruvate was used as the substrate for pyruvate carboxylase. The effect is on Vmax/Km alone and disappears at infinite substrate concentration. This is interpreted to mean that the slowest step in the overall catalysis is in the half-reaction involving the carboxylation of enzymebiotin by ATP and HCO3-. A tritium intramolecular isotope effect of 4.8 and an intermolecular effect of 1.2 were also observed. The former was interpreted as the isotope effect on the "effective kcat", while the latter the one on V max/Km. With these data, the rate constant for binding of pyruvate was estimated to be 4.5 X 10(6) M-1 min-1, and the deuterium kinetic isotope effect on the catalytic step to be 3.1. Relative values for various rate constants were also obtained. Fluoropyruvate was also shown to be a substrate, reacting six times slower. A deuterium kinetic isotope effect of 1.5 was observed, which remained even at infinite substrate concentration. This is interpreted to mean that the slowest step in the overall catalysis is now the carboxylation of fluoropyruvate.


Poly(2-aminoadenylic acid): interaction with poly(uridylic acid). Poly(2-aminoadenylic acid) forms both double and triple helices with poly(uridylic acid) [poly(U)]. The 2-amino group forms a third hydrogen bond, elevating the 2 leads to 1 transition temperature by 33 degrees C. The third strand, however, has about the same stability as poly(A)-2poly(U), as measured by Tm 3 leads to 2. This selective stabilization of the two-stranded helix results in a much greater resolution of the differnt thermal transitions than that observed in analogous polynucleotide systems. In contrast to other A, U systems 3 leads to 1 and 2 leads to 3 transitions are not observed under any conditions, and the triple helix always undergoes a 3 leads to 2 transition even at very high ionic strength. A 1:1 mixture of poly(2NH2A) and poly(U) exhibits no transient formation of 1:2 complex, unlike similar mixtures of poly(A) with poly(U) and poly(T). This difference is evidently due to a more rapid displacement reaction: [poly(2NH2A) + poly(2NH2A)-2poly(U) leads to 2 poly(2NH2A)-poly(U)] With poly(2NH2A) than with poly(A). We describe a method for establishing the combining ratios of polynucleotide complexes which used a computer to calculate the angles of intersection of mixing curves as explicit and continuous functions of the wavelength. The wavelength dispersions of the angles of intersection determine optimum wavelengths for establishing stoichiometry and can also provide reliable negative evidence that presumably plausible complexes are not formed. Analogous computer procedures have been developed to determine wavelengths which are selective for the formation of both 1:1 and 1:2 complexes. Infrared spectra of the 1:1 and 1:2 complexes resemble those of other A, U homoribopolynucleotide helices in having two and three strong bands, respectively, in the region of carbonyl stretching vibrations. CD spectra of the two complexes are unusual in having negative first extrema of moderate intensity. We attribute these extrema to intrastrand interactions of strong, well-resolved transitions at 278 nm (B2u) of the 2-aminoadenine residues. The CD spectra are correlated with those of other polynucleotide helices.


Temperature dependence of the 31P chemical shifts of nucleic acids. A prode of phosphate ester torsional conformations. The temperature dependence of the 31P chemical shifts of the ribodinucleoside monophosphates, ApA, GpC, CpC, UpU, and ApU, of the deoxyribonucleic acids, d-ApT, TpT, d-ApA, and d-pApT, and of the homopolyribonucleic acids poly(G), poly(U), poly(A) is shown to provide information on the helix-coli transition in nucleic acids. The base stacked, helical structure with a gauche,gauche phosphate ester torsional conformation is 0.2-0.6 ppm upfield from the random coil conformation. In contrast, the 31P chemical shifts of dimethyl and diethyl phosphate do not change significantly with temperature. These results support our earlier hypothesis that 31P shifts are sensitive probes of torsional conformations with phosphate esters in gauche,gauche conformations having 31P shifts upfield from nongauche conformations.


Isolation and purification of hen oviduct protein synthesis initiation factors A2A and A2B. Two initiation factors, IF-A2A and IF-A2B, required for protein synthesis in a fractionated system have been isolated from hen oviduct. These factors were obtained from a 0.5 M KCl extraction of a nuclear-microsomal fraction of the oviduct. The crude extract inhibited protein synthesis, but, following DEAE-cellulose chromatography, activity was detected. Sephadex G-200 chromatography separated the activity into two active frations, A2A and A2B. These factors have been characterized with respect to their activities in polyphenylalanine polymerization at a low Mg2+ concentration, AUG- and GTP-dependent Met-tRNAf binding to 40S ribosomal subunits, the hydrolysis of GTP, and natural messenger ribonucleic acid (mRNA) dependent protein synthesis. In all of these systems A2A and A2B were able to substitute for rabbit reticulocyte M2A and M2B. The molecular weights of A2A and A2B have been estimated by gel filtration chromatography to be 280 000 and 23 000, respectively. Sedimentation analysis on sucrose gradients showed A2A to have a sedimentation coefficient of 5.2 S. Combining these data, the molecular weight of A2A was calculated to be 125 000. These values are similar to those for corresponding reticulocyte proteins. Finally, in the presence of added ovalbumin mRNA, A2A and A2B stimulated protein synthesis on non-salt-whashed hen oviduct and rabbit reticulocyte polysomes. Moreover, A2A- and A2B-dependent synthesis of ovalbumin was shown to occur on reticulocyte polysomes programmed with ovalbumin mRNA. This supports the conclusion that these factors are initiation factors for protein synthesis and not ribosomal structural proteins.


Digestion of RNA of chromatin and nuclear ribonucleoprotein by staphylococcal nuclease. Following a 1-h [3H]uridine pulse of cells of a human colon carcinoma line, 15% of the radioactivity in heterogeneous nuclear RNA associated with both chromatin and nuclear ribonucleoprotein was not digested to acid soluble fragments during a 2-h incubation with staphylococcal nuclease (EC 3.1.4.7). These [3H]uridine-labeled oligonucleotides were approximately 26 nucleotides in length. An RNA containing structure which sedimented no faster than 2 S could be isolated from the digests. Major and mino peptide species, of molecular weights 40 000 and 66 000, respectively, were associated with this structure isolated from either chromatin or nuclear ribonucleoprotein. The results demonstrate that some protein of nuclear ribonucleoprotein is complexed with the transcript while it is still associated with chromatin.


Use of gel chromatography for the determination of the Stokes radii of proteins in the presence and absence of detergents. A reexamination. In the course of a routine investigation of the complex between the erythrocyte membrane protein spectrin and sodium dodecyl sulfate, we observed a large discrepancy between the true Stokes radius (178 A, measured by hydrodynamic methods)  and the apparent value derived from gel chromatography (107 A). In attempting to resolve this discrepancy, we have experiments that indicate that all large asymmetric particles may be subject to a similar discrepancy; e.g., native fibrinogne has a true Strokes radius of 108 A, whereas the value derived by column chromatography after calibration with globular proteins is only 71 A. The simplest interpretation is that end-on insertion of asymmetric particles into the gel pores contributes to their retardation. The phenomenon clearly limits the usefulness of gel chromatography as a quantitative measure of the hydrodynamic Stokes radius. Incidental data obtained in the course of this work indicate that spherical viruses may have weak chemical affinity for the porous gel. Chromatography of large proteins in the presence of detergents produced no effects ascribable to absorption of the detergents, but the results suggest a need for further  study of possible interaction between detergents and small gel pores.


Bovine milk acid phosphatase. III. Purification and characterisation of the enzyme. The acid phosphatase of bovine milk has been purified by ion-exchange chromatography on Amberlite CG-50 resin followed by Sephadex G-100 gel filtration and further purification steps using CM-Sephadex C-50, cellulose phosphate and affinity chromatography on alphas1-casein-Sepharose. These gave an apparently homogeneous enzyme with a specific activity of about 30 I.U./mg which possessed a molecular weight of about 42000 and E1%1cm = 9.4 at 280 nm. Electrophoresis under dissociating conditions, gel filtration in a 6.6 M urea buffer and ultracentrifugal analysis in the presence of 6 M guanidine HCl and 0.1 M 2-mercaptoethanol all gave molecular weight values close to 42000 indicating that the molecule was probably composed of a single polypeptide chain. The acid phosphatase was a glycoprotein with 2 mol/mol of galactose, 2 mol/mol of mannose and 4 mol/mol of glucosamine. Amino acid analysis showed that the enzyme was devoid of methionine.


Experimental allergic aspermatogenic orchitis. IV. Chemical properties of sperm glycoproteins isolated from guinea pig testes. Of four glycoproteins isolated from guinea pig testes, two were aspermatogenic (types I and IV) and two (types II and III) were inactive. The glycoproteins were rich in carbohydrate, varying from 41.5% to 49.5% carbohydrate by weight. Each glycoprotein had a unique amino acid composition, but in general low levels of tyrosine, tryptophan, and basic amino acids were found along with relatively high contents of serine, threonine, glutamic acid, and proline. Types I and IV glycoproteins were remarkably stable; their aspermatogenic activity was not affected by urea, trypsin, or heating at 100 degrees C in water or in 1 M HCl for 15 min. Carbohydrate analysis revealed little difference in the monosaccharide compositions of types I and IV glycoproteins, except that only the type I contained sialic acid. In contrast, types II and III glycoproteins lacked sialicacid and fucose and contained much less mannose. Both N-acetylglucosamine and N-acetylgalactosamine were present in all four glycoproteins, and they dominated in the types II and III. Fucose and at least 20-25% of the galactose appeared to occupy terminal positions in type IV glycoprotein as shown by their release after 15 min hydrolysis in 1 M HCl. All of the glycoproteins contained a relatively high percentage of galactose by weight, from 12.6 to 19.3%. The molecular weights of the glycoproteins were estimated by sodium dodecyl sulfate gel electrophoresis to be 47000, 105000 and 18000 respectively for the types I, II, and IV; type III glycoprotein showed two major bands, with molecular weights of 41500 and 22800. All the above molecular weight values are probably overestimated because of high carbohydrate content. The molecular weight of type IV glycoprotein was found to be 13000 by ultracentrifugation; a corrected value of 29000 was calculated for type I glycoprotein.


Comparative analysis of the nuclear basic proteins in rat, human, guinea pig, mouse and rabbit spermatozoa. Cysteine-rich protamines (Arg = 47-61%, Cys = 8-16%) were isolated from the sperm of an individual guinea pig, human and rabbit and from pooled samples of mouse and rat sperm. Appreciable concentrations of histones were not found in the sperm nuclei of these species. In addition to the protamines, a substance of relatively low molecular weight, which reacted with the Lowry reagent, appeared in crude acid-soluble extracts of sperm nucleoprotein. This unidentified contaminent was resolved from the protamines by chromatography on Bio-Rex 70. Heterogeneity of human and mouse protamines was revealed by electrophoresis at pH 2.7, in the presence of 2.5 M urea, and confirmed by amino acid analysis, which also suggested the presence of 2 or more species of protamine in the rabbit. By contrast, the guinea pig and rat preparations displayed nearly stoichiometric ratios of amino acid residues, approaching homogeneity by this criterion. The functional consequences of crosslinks between cysteine residues of these proteins and the possible species-specific significance of their differing percentages of histidine are discussed. Potentially analogous functions are suggested for phosphorylated serine and threonine, and for ionized cysteine and tyrosine, within the protamines of developing spermatids. Their amino acid compositions indicate that the protamines of eutherian mammals are coded by a C.G-rich genome which has been unusually susceptible to genetic drift. An especially high rate of G leads to A transitions seems to have occurred in the human protamine genes.


The effect of ligand size and stereochemistry on the reactivity of the alpha and beta chains within hemoglobin. The reactions of human hemoglobin A with methyl, ethyl, n-propyl, n-butyl, iso-butyl, sec-butyl, and tert-butyl isocyanide were examined in the presence and absence of inositol hexaphosphate. As the size and bulk of the aliphatic side-chain increases, the relative association rates and affinities of the beta-chains for isonitriles increase compared to those of the alpha chains. This result indicates that the beta heme pocket within hemoglobin is more open and accessible to ligand molecules than the alpha heme pocket.


Dependence of magneto-optical rotatory dispersion and magnetic circular dichroism of deoxy- and methemoglobin on their quaternary structure. Methods of magnetic optical activity, magneto-optical rotatory dispersion (MORD) and magnetic circular dichroism (MCD), were shown to be sensitivie to the quaternary structure of deoxyhemoglobin. The isolated alpha and beta chains, the monomeric hemoglobins (leghemoglobin, fraction III of Chironomus thummi thummi hemoglobin) and hemoglobin in the R state (hemoglobin digested with carboxypeptidases A and B) exhibit in the visible region two MORD minima of equal intensities. In native tetrameric hemoglobins studied (human, horse, porcine, feline, carp, tortoise, frog) the ratio of the intensities of these MORD minima is about 2. The MORD data for deoxy-des Arg-N-ethylsuccinimide-hemoglobin indicate that in solution there is a mixture of the T and R states, the equilibrium between the states being shifted toward the R structure. The identity of the MORD curves for deoxy-bis(N-maleimidoethyl)-ester-hemoglobin and for native deoxyhemoglobin indicates that deoxy-bis(N-maleimidoethyl)ester-hemoglobin has the T structure in solution. Comparison of the MORD curves exhibited by a native methemoglobin, a native metmyoglobin and the modified hemoglobins in the met form in the absence and presence of organic and inorganic phosphates reveals no direct correlation between the MORD changes and methemoglobin quaternary structure.


Biochemical studies on human ceruloplasmin. Ceruloplasmin from nephrotic urine, ascites fluid and plasma has been partially characterized. All ceruloplasmin preparations were found to be comprised of two light and two heavy polypeptide subunits. Characterization of the purified subunits indicated that the alpha chain had a mol. wt. of 16000 and had N-terminal valine while the beta chain had a mol. wt. of 59000 and had N-terminal lysine. All carbohydrate resided in the beta subunit. Incomplete cleavage of the 5-methionine residues of the alpha chain enabled a preliminary ordering of the CNBr fragments. Automated sequence analysis of the alpha chain was carried out and the sequence determined was Val-Phe-Asx-Pro-Arg-Arg-Lys-Leu-Glx-Phe-Ala-Leu-Leu-Phe-Leu-Val-Phe-Asx-Glx-Asx-Glx.


The heterogeneity of the protamines from human spermatozoa. Nuclear basic protein from ejaculated human spermatozoa were labelled with iodo[14C1]acetic acid and fractionated by ion-exchange chromatography into several pools (named A-K). Gel electrophoresis indicated that the minor protamine components, were present in pool D and that, of the major protamine components, component 1 (pools E, F, G, H) was well separated from the unresolved mixture of component 2 and component 3 (pools I, J, K). Pools G and J were free of other contaminants. Pools D, G, and J produced different radioactive peptides on digestion with trypsin and with thermolysin, and also had quite distinct amino acid compositions. This suggests that the heterogeneity of human protamines is caused by differences in amino acid sequence. Major component 1 also seems to be heterogenous, since it was found in two distinct peaks (pools E and G), but post-translational modification as a cause of the two types of component 1 has not been ruled out. Although all the human protamine components are similar to other mammalian protamines in containing half-cysteine and tyrosine, they also have unique common features such as high histidine and high glutamic acid contents.


DNA-binding proteins in Yoshida ascites tumor fluid. DNA-binding proteins were isolated from Yoshida ascites tumor fluid by chromatography on DNA-cellulose. This fraction represents 1-2% of the total ascites protein. Most of the DNA-binding proteins will bind to phosphocellulose as well. The proteins migrate by agarose gel electrophoresis at pH 8.6 as alpha and beta globulins. Quantitative immunoelectrophoresis revealed the presence of 12-18 proteins. SDS-polyacrylamide electrophoresis indicated molecular weights ranging from 3-10(4) to 10(6). Seven of the proteins were identified by specific immunoprecipitation as beta1-Eglobulin, beta2-glycoprotein I, fibrinogen split product E (fibrinogen E), coagulation factor XIII (factor XIII), alpha2-macroglobulin, IgG and IgM. Alpha1-antichymotrypsin might also be represented. In nuclear extracts of the tumor cells only factor XIII was present. With the exception of fibrinogen E and P5 all recognized DNA-binding proteins are present in normal rat plasma. With increasing tumor age the concentration of fibrinogen E, factor XIII, P5 and IgM increased both in ascites fluid and in plasma, while the concentration of other DNA-binding-proteins decreased or remained constant. Evidence is presented that the DNA- and phosphocellulose binding ascites protein fraction inhibit tumor cell growth. No inhibition was induced by corresponding protein fractions isolated from normal rat plasma.


Effects of organic and inorganic phosphate ions on the charge-transfer band of ferric myoglobin. (1) Splitting of the charge-transfer band positioned at 630 nm of metmyoglobin disappeared on lowering the pH from 7.5 to 4.5 in the presence of Pi and PPi at the temperature of liquid N2. (2) The observed apparent pK value of the disappearance of the splitting depended upon the species of phosphate ions. (3) In the case of Pi, the low-temperature absorption spectra suggested that at least two phosphate ions bound to one myoglobin molecule.


Activities of nucleoprotein particles derived from rat liver ribosome. 80-S ribosomes and 60-S subunits from rat liver were treated at increasing KC1 concentrations giving protein-deficient ribosomal particles whose components were analyzed and their activity tested. Most of the activities assayed stand treatment up to KC1 concentrations of around 0.6 M; peptidyl transferase, measured by the fragment reaction, however was 50% inhibited by 0.5 M KC1 in 60-S subunits but not in 80-S ribosomes. Three proteins, L21, L26 and L31, might be implicated in this loss of activity. 60-S subunits forming part of the 80 S ribosome are more resistant to the salt treatment and the pattern of proteins released by the treatment differs from the one obtained from free 60-S subunits, implying perhaps a change of conformation of this subunit upon association to form 80-S couples. According to their resistance to release by KC1 the proteins of the large sub-unit can be divided into three groups: (1) easily removed, including proteins: L1, L11, L17 and L25 in 80-s subunits and in addition, L5, L8, L9, L13, L20, L22, L26, L29, L31 and L32/33 in 60-S subunits; (2) proteins resistant to release by high salt concentrations in 80-S ribosomes as well as in 60-S subunits, namely proteins L3, L14, L27, L36, L40, L41, X1 and X2; (3) the rest of the proteins which are released in a more or less continuous way throughout the treatment. 5 S RNA is not released by KC1 treatment at the concentrations used. The binding sites for the antibiotics trichodermin and anisomycin are affected in a different way by the salt treatment, indicating that they are structurally different.


Preformed poly(A)-containing RNA in zoospores of Blastocladiella emersonii. Discharged zoospores of Blastocladiella emersonii contain poly(A)-containing RNA, which is performed during the initial stages of zoospore differentiation in the sporangium and is stable in vivo for at least 4 h. The average molecular weight of this RNA fraction is estimation to be 5.5-10(5) or 1600 nucleotides. The average length of the poly(A) sequence is approx. 100 nucleotides Poly(A)-containing RNA does not appear to accumulate in a gamma-particle fraction previously suggested to be involved in the storage of mRNA.


Effects of estrogen and progesterone on transcription, chromatin and ovalbumin gene expression in the chick oviduct. The effects of estrogen, progesterone and estrogen + progesterone combined on nuclear transcriptional processes in oviducts of immature chicks, previously withdrawn from estrogen, are reported. The responses to the steroids of the endogenous nuclear RNA polymerase activities, both nucleolar (I) and nucleoplasmic (II), the chromatin compositions and template capacities, and the appearance of ovalbumin messenger RNA (mRNA) are compared. When immature chicks (previously treated at 14 days with estrogen) are withdrawn from estrogen treatment, there is a gradual reduction in both polymerase activities. Diurnal variations in polymerase II activties in the oviduct of withdrawn chicks required that subsequent experiments include time-matched controls. The hormones alter RNA polymerase II and II activities in vivo as assayed in isolated nuclei. Progesterone represses the polymerase I and II activities, while estrogen alone and estrogen + progesterone enhance both polymerase activities immediately after injection. Diethylstillbestrol, a synthetic estrogen, causes changes similar to those of estrogen. The effects of these steroids on the polymerases are detected within 15 min of hormone injection. Changes in the capacities of chromatins to serve as template for RNA synthesis in general correlated with changes in polymerase II activities. Interestingly, in the case of estrogen treatment, the acidic chromatin protein (but not histone) levels fluctuate positively with the template capacities of the chromatin. An antagonism between estrogen and progesterone is observed in the responses of both RNA polymerases I and II activities as well as in the chromatin template capacity. Levels of messenger RNA coding for ovalbumin, as detected by hybridization with labeled complementary DNA, increase in oviducts of withdrawn chicks within 2--3 of the injection of estrogen, progesterone or estrogen + progesterone. This rapid accumulation of ovalbumin mRNA is not accompanied in each case by a similar increase in polymerase II activity or chromatin template capacity.


Protein synthesis in dystrophic muscle. Activity of the pH 5 supernatant fraction of muscle in dystrophic mice. The pH 5 supernatant fractions prepared from homogenates of tissues of normal and dystrophic mice were used to study the incorporation of [14C]phenylalanyl-tRNA into peptide. The incorpoation was markedly reduced using the muscle pH 5 supernatant fraction from dystrophic animals but no reduction was seen with brain, liver or heart preparations from dystrophic mice. The lower incorporation with dystrophic muscle pH 5 supernatant was not due to altered activity of ribonuclease, elongation factors, proteolytic enzymes, GTP or sulfhydryl reagents, but was attributable to the presence of activity that was inhibitory to protein synthesis.


Analysis of the human genome by complementary RNA/DNA gradient hybridization and relaxes cesium sulfate-silver ion density centrifugation. 3H-Labeled complementary RNA (cRNA) transcribed from total nuclear human DNA was hybridized to homologous DNA and the hybrids formed localized in CsC1 density gradients. Radioactive peaks indicative of cRNA/DNA hybrids identified ten density components. The experimental conditions used indicated that these components correspond to DNA rich in repeated sequences. In addition, cRNAs transcribed from five Cot fractions (31% of total DNA; Cot less than 100) were hybridized to total DNA in CsC1 gradients. The hybrids sedimented at similar densities to those seen in total cRNA but showed a differential distribution along the five fractions. To isolate native double-stranded DNA components containing the various families of repeated sequences observed, nuclear DNA was fractionated using relaxes Cs2SO4/Ag+ density gradient centrifugation. These fractions revealed the presence of seven major components a densities of 1.715, 1.711, 1.708, 1.705, 1.702, 1.700 and 1.698 g/cm3 and three minor components at densities of 1.696, 1.693 and 1.687 g/cm3. The density components corresponded to those observed by cRNA/DNA hybridization, thus suggesting that the bulk of the human genome is made up of ten density-biased components containing sequences of varying degrees of repetitiveness.


Temperature-induced perturbations in the circular dichroic spectrum of the synthetic polymer poly[d(G-C)]. The effect of increasing temperature on the double-helical synthetic DNA polymer poly[d(G-C)] with alternating base sequence, was studied by ultraviolet absorbance and circular dichroic techniques. While a cooperative hyperchromic effect was noticed in the ultraviolet absorbance spectrum above a temperature of 70 degrees C, an inversion of the circular dichroic spectrum was observed at a similar temperature range. On cooling to room temperature, the spectrum was reversed to the initial one. These observations, first reported here, are tentatively attributed to a conformational change of the polymer, from the right-handed double helix to the left-handed single strands, prior to complete denaturation.


Raman spectroscopic detection and examination of the interaction of amino acids, polypeptides and proteins with the phophatidylcholine lamellar structure. Raman spectral peaks in the vicinity of 1100 and 2900 cm-1 for phosphatidylcholine were found to be sensitive to interactions with amino acids, polypeptides and plasma proteins. The amino acids L-luecine, L-isoleucine, L-tryptophan, L-arginine HCl, L-histidine HCl, L-threonine and L-aspartic acid decreased the dipalmitoyl phosphatidylcholine Raman intensity ratio  I1064/I1089 indicating an increase in the gauche hydrocarbon chain character of the lipid. The increase in the lipid approx. 2930 cm-1 peak intensity in relation to the approx. 2850 and approx. 2890 cm-1 peaks upon the addition of the amino acids L-arginine HCl, L-histidine-HCl and L-lysine-HCl to the lipid dispersion indicates that the lipid hydrocarbon chain environment becomes more polar in their presence. The lipid-alamethecin and lipid-valinomycin interactions produced a decrease in the lipid Raman intensity ratio I1064/I1089 again indicating an increase in the gauche hydrocarbon chain character of dicyristoyl phosphatidylcholine while producing no change in this ratio for dipalmitoyl phosphatidylcholine. Human fibrinogen and bovin serum albumin were found to increase the I2890/I2850 dimyristoyl phosphatidylcholine Raman intensity ratio while decreasing the I2850/I2930 dimyristoyl phosphatidylcholine Raman intensity ratio indicating that the lipid underwent a conformational change and that the hydrocarbon chain environment was more polar in the presence of albumin or fibrinogen.


31P NMR studies of unsonicated aqueous dispersions of neutral and acidic phospholipids. Effects of phase transitions, p2H and divalent cations on the motion in the phosphate region of the polar headgroup. 1. The 129 MHz (non-proton decoupled) and 36.4 MHz (proton decoupled) 31P NMR spectra arising from unsonicated aqueous dispersions of well defined species of phospholipid have been investigated. The phospholipids employed (and the parameters varied) include phosphatidylcholine (temperature), phosphatidylethanolamine (temperature), phosphatidic acid (temperature and p2H) and phosphatidylglycerol (temperature, p2H and Ca2+ (or Mg2+)) concentration. 2. At p2H = 7 the 31 P MNR spectra arising from saturated species of phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol become progressively broader as the temperature is reduced below the phase transition, demonstrating reduced motion in the phosphate region of the polar headgroup. 3. In the liquid crystalline state at p2H = 7 the molecular dipolar order parameters obtained for saturated species of phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol and very similar, and are independent of the acyl chain length for species derived from lauric and myristic acid. Thus the motion in the methylene-phosphate-methylene region is similar for these different liquid crystaline phospholipid species. 4. The 31 P NMR spectra of aqueous dispersions of 14:0/14:0 phosphatidic acid display anomalous temperature and p2H dependences. The effective chemical shift anistropy (delta v CSA EFF) at 5 degrees C varies from 71 ppm at p2H = 8.5 to 38 ppm at p2H = 2.5. Further, the motion in the phosphate region is relatively insensitive to the gel or liquid crystalline nature of the hydrocarbon chains. 5. The addition of 40 mol% Ca2+ (or Mg2+) to saturated species of phosphatidylglycerol causes an increase of approx. 20 degrees C in the hydrocarbon phase transition temperature as indicated by 31 P NMR. Equimolar concentrations of Ca2+ increase the transition temperature by approx. 70 degrees C, and no 31P NMR signal could be observed for the very condensed precipitate formed below this temperature. In the liquid crystalline state the motion in the phosphate region of the polar headgroup is not significantly affected by the presence of Ca+ or Mg2+. 6. The 31P NMR spectra obtained from 18 : 1c/18 : 1c phosphatidylethanolamine are consistent with a phase transition from a lamellar to an hexagonal (HII) phase in the region 10-15 degrees C. 7. The observed narrowing of the 31 P NMR spectra of aqueous dispersions of phospholipids as the temperature is raised toward the hydrocarbon transition temperature is discussed in terms of the "pretransition" observed in calorimetric studies.


Kinetics and stability of alamethicin conducting channels in lipid bilayers. It is already well-established that conduction in lipid bilayers containing alamethicin arises from the presence of complexes in which there are several molecules of the polypeptide. It is with the nature of these complexes that this paper is primarily concerned. While it is clear that increasing alamethicin concentration and increasing potential across the membrane favour their formation, the nature of the reactions involved has not yet been elucidated. Attempts have therefore been made to clarify the sequence of events leading to the establishment of a complex in its conducting state. It has been concluded that the most likely mechanism involves, initially, a non-field-dependent aggregation of the alamethicin, in the plane of the membrane, into non-conducting oligomers. These then appear to undergo movement normal to the membrane (which is field dependent) to form the conducting species. Temperature studies have shown that the various conducting states of the oligomer have effectively equal enthalpies, and that the activation energies for transitions between these states are all approx. 1.2kcal/mol. The corresponding rate constants are very sensitive to the lipid composition of the membrane and a variety of different systems has been examined in order to clarify the origins of this effect. The only conclusion from this part of the work is that lipid fluidity might be involved.


Studies on 6C3HED murine ascites tumor cell receptors for mannosyl-binding lectins. We have investigated the receptor site activity present on 6C3HED tumor cells for concanavalin A, fava, lentil and pea lectins. The binding of the tritiated lectins to the tumor cells was inhibited by methyl-alpha-D-mannoside but not by D-galactose. The number of binding sites for the lectins was 3.5-10(6)/cell for concanavalin A, 3.3-10(6)/cell for fava, 3.6-10(6)/cell for lentil and 4.8-10(6)/cell for pea. The apparent association constants were 3.6 and 1.3 muM-1 for concanavalin A, 3.9 muM-1 for fava, 4.2 muM-1 for lentil and 4.6 and 0.6 muM-1 for pea. Competitive inhibition studies showed that lentil was a good inhibitor of pea binding; concanavalin A was a poor inhibitor of pea binding; and fava was a better inhibitor than concanavalin A but not as good as lentil. Reciprocal inhibition experiments indicated that concanavalin A and pea may bind to different receptors as well as to common receptors. This was also indicated by the observation that trypsin or protease treatment of the cells decreased the binding of pea lectin by 20-40 percent whereas concanavalin A binding was unaffected.


A simple model for calcium induced exocytosis. We have developed a simple model showing how the presence or obsence of Ca2+ can determine whether an uncurved or curved membrane surface is favored energetically. The model shows why fusion of vesicles with the presynaptic membrane is favored in the presence of calcium and why the budding off of vesicles is favored in the absence of calcium inside of the presynaptic membrane. The model accurately predicts the radius of a synaptic vesicle using known properties of lipids and suggests consequences of temperature change, varied stimulation rate and addition of calcium by artificial means on rates of transmitter release.


Role of calcium in exocrine pancreatic secretion. III. Comparison of calcium and magnesium movements in rabbit pancreas. 1. Calcium and magnesium movements in the isolated rabbit pancreas and in rabbit pancreas fragments are compared in a qualitative and quantitative way. 2. At the basal secretion rate calcium and magnesium are present in the secreted fluid in concentrations of about 30% of their concentrations in the bathing medium. 3. Addition of 10(-6) M carbachol to the bathing medium results in enzyme secretion accompanied by calcium and magnesium release, in divalent cation-free medium as well as in a complete medium. 4. The secretion of each divalent cation is the sum of two components: an extracellular flux and a flux of protein-associated cations, the so-called secretory flux. 5. The extracellular flux is proportional to the concentration of the divalent cation in the bathing medium. The secretory flux is not dependent on the presence of the divalent cation in the bathing medium, but is proportional to the amount of protein secreted. About 25 nmol of each cation is secreted per mg protein. 6. Ca2+ and Mg2+ can be nearly completely separated from the digestive enzymes by gel filtration. They equilibrate completely with their radioactive isotopes added to the sample just before elution, indicating that the cations are rapidly exchangeable after secretion. 7. Efflux studies on rabbit pancreas fragments, pre-loaded with 45Ca2+, show a carbachol-stimulated 45Ca2+ efflux (the stimulatory flux) in addition to a release of amylase. Fragments pre-loaded with 28Mg2+ do not show carbachol stimulation of the tracer efflux. 8. These studies indicate that calcium and magnesium behave quite similarly with respect to the extracellular and secretory fluxes. The absence of a stimulatory flux for magnesium, suggests that the increase of the cytoplasmic calcium concentration plays a specific role in the stimulus-secretion coupling of pancreatic enzyme secretion.


Hydrophobic affinity partition of spinach chloroplasts in aqueous two-phase systems. The surface properties of spinach chloroplasts, both of intact chloroplasts with surrounding envelope and broken chloroplasts consisting of the inner lamellar system, have been studied by partitioning them between two aqueous phases, especially using counter-current distribution technique. The two-phase system consists of poly(ethyleneglycol), dextran and water. The two polymers are enriched in opposite phases and by binding deoxycholate or palmitate to one of the polymers the affinity of chloroplasts for the corresponding phase is strongly enhanced. The partition of the two classes of chloroplasts, however, is not affected to the same degree and the affinity of the chloroplast envelope for deoxycholate and palmitate is stronger than that of the lamellar system. This has been correlated to the chemical composition of the two types of membranes. By studying the effect of salts on the partition it has been found that the lamellar system bears a larger number of negative charges as compared to the envelope of the intact chloroplast.


An x-ray study of the condensed and separated states of sciatic nerve myelin. Low-angle X-ray diffraction patterns of peripheral nerve myelin after modification by either rehydration in various solutions or by chemical treatment have been recorded. These X-ray patterns and the previously reported modified nerve myelin patterns demonstrate that nerve myelin has at least five different states: the normal state, condensed state I and II and separated state I and II. There are two membranes per unit cell in the normal state and in states II whereas there is one membrane per unit cell in states I. Under certain conditions normal nerve can go reversibly into either of states II. With continued treatment the nerve myelin structure moves irreversibly from state II to state I and, once in state I, the nerve myelin layers cannot return to the normal state. Our results demonstrate that there is a reversible transformation between condensed state I and separated state I. Fourier profiles of nerve myelin in the normal state, condensed state I and separated state I are presented.


Outside-inside distributions and sizes of mixed phosphatidylcholine-cholesterol vesicles. (1) The effect of the incorporation of cholesterol upon the distribution of various molecular species of phosphatidylcholine across the bilayers of mixed sonicated liposomes (vesicles) has been studied with 31P-MNR. (2) The outside-inside ratio of both saturated and unsaturated phosphatidylcholine species was not much affected by the incorporation of up to 30 mol% cholesterol. Above 30 mol% cholesterol the outside-inside ratio strongly increased for phosphatidylcholines with cis unsaturated fatty acid chains. In contrast the outside-inside ratio of trans unsaturated and fully saturated phophatidylcholine species was either not affected or decreased by the incorporation of more than 30 mol% cholesterol. (3) a simple relationship between the size of the vesicle and the linewidth of the 31P-NMR resonance is described. From the measured linewidths the sizes of the various cholesterol containing vesicles have been obtained. It is found that incorporation of 0-30 mol% cholesterol does not significantly affect the size of the vesicle whereas above 30 mol% cholesterol does not significantly affect the size of the vesicle whereas above 30 mol% cholesterol the size of all phosphatidylcholine vesicles sharply increases. The increase in size is the largest for the more saturated phosphatidylcholine species. (4) From the outside-inside ratio and the size of the vesicle the composition of the outer and inner layer of the mixed vesicles could be obtained. Below 30 mol% cholesterol the composition of outer and inner layer is nearly identical. Above 30 mol% cholesterol the distribution of lipid across the bilayer of all visicles becomes assymetric with a disporportionately larger amount of cholesterol present in the inside monolayer.


Saccharides on teratocarcinoma cell plasma membranes. Their investigation with radioactively labelled lectins. We have studied the interaction of five lectins differing in their sugar specificity, with the surface of clonal cell lines derived from transplantable murine teratocarcinoma. The results show that the differentiation from primitive embryonal carcinoma cells into parietal yolk sac cells is accompanied by changes in cell surface saccharides. These changes consist of a marked decrease in the total number of binding sites for the L-fucose-specific lectin of Lotus tetragonolobus and a large increase in the total number of binding sites for wax bean agglutinin. It is suggested that these differences can be used as markers in the study of this early embryonic differentiation. No agglutination of primitive embryonal carcinoma cells or of parietal yolk sac cells by low concentrations (10mug/ml) of concanavalin A, soybean agglutinin or the fucose binding proteins was observed.


Alteration of birefringence signals from squid giant axons by intracellular perfusion with protease solution. The optical signal, arising from a transient birefringence change associated with excitation, was recorded from a squid giant axon together with the membrane potential change, and the effect of removal of the axoplasm on the optical signal was examined. In an unperfused axon, repetitive stimulation at a frequency of about 100 Hz produced two kinds of optical response. The initial response had a brief, spike-like time course and was elicited by each stimulating pulse. The delayed response had a slow time course and the sign of decreased light intensity, and summated with repetitive stimulation. Most of the axoplasm was removed from interior of the axon by intracellular perfusion with solutions containing pronase at a concentration of 0.1 mg/ml. The delayed response could selectively be eliminated by perfusion with a pronase-containing solution for 2-8 min. The result was interpreted as showing that the delayed birefringence signal originates from axoplasm when its gel structure was transiently disturbed by an increased Ca2+ influx associated with excitation. When perfusion was further continued the duration of the action potential started increasing and often a prominent after-depolarization appeared. At this stage the initial optical response was again followed by a large slow signal with the sign of increased light intensity. This reversed delayed response was tentatively assumed to originate from the membrane with some remaining axoplasm, but its cause is still not understood.


Physicochemical studies of taste reception. III. Interpretation of the water response in taste reception. The model membrane composed of a Millipore filter paper and the total lipids from bovine tongue epithelium or phosphatidylcholine from egg yolk simulated well the water response of a living taste cell, The water response observed with the model membrane adapted to various salt solutions was interpreted in terms of changes in electric potential at the membrane-solution interface, i.e. the water response was attributed to the e.m.f. change produced by diffusion of the electrolytes dissolved in (or adsorbed on) the membrane surface into the bulk solution. The water response of the frog tongue was also investigated by measuring the neural response of the glossopharyngeal nerve. The results obtained were consistent with the mechanism proposed in the present paper. The response of the frog to Ca2+ was examined under the condition where the water response was suppressed, and it was concluded that the water response of the frog is different from the response to Ca2+.


An optical study of the exchange  kinetics of membrane bound molecules. The kinetics of molecular exchange between lipid bilayers are studied using a special fluorescence technique. Pyrene and pyrene decanoic acid are chosen as typical examples of an apolar and amphiphilic molecule. Their property of forming dimers in the excited state (excimer) is exploited. The time dependencies of monomer and excimer intensities after rapid mixing of vesicles doped with fluorescent probe with undoped ones are studied by stopped-flow technique. The transient curves reveal the information on the exchange kinetics. A theoretical analysis shows that the molecular exchange follows a first order kinetics. Surprisingly short half life-times tex for this exchange process are obtained (for dipalmitoyl phosphatidylcholine tex=3.3 s for T=23 degrees C, tex=0.5 s for T=68 degrees C). Multilamellar systems (onion like structure) show much slower exchange rates. The exchange rates are nearly equal for polar and unpolar molecules. Addition of cholesterol has a strong reducing effect on this rate. Charging of dipalmitoyl phosphatidylcholine vesicle surfaces by the addition of (a) EuCl3 to the aqueous phase and (b) dipalmitoyl phosphatidic acid to the lipid phase reduces the exchange rate by about an order of magnitude above the phase transition. In a separate experiment it is shown that the lipid exchange or fusion for two different lipids is a much slower process compared to the label exchange. In fact vesicles kept below the phase transition temperature Ttr for both lipids, do not fuse even after 70 h. Noticeable fusion occurs after 10 h when the mixture stays above Ttr. Experiment shows that the fusion of pure lipid vesicles is not very much affected by the presence of a charged lipid. Change in concentration of the monovalent ions in the aqueous solution by two orders of magnitude does not have an appreciable effect on the exchange rate of phospholipids.


The effect of hashish compounds on pphospholipid phase transition. The interaction of hashish compounds, delta 1-tetrahydrocannabinol and cannabidiol, with dipalmitoyl phosphatidylcholine was investigated using differential scanning calorimetry. Both drugs affect the transition of dipalmitoyl phosphatidylcholine from the gel to liquid crystalline state, decreasing both the melting temperature and the enthalpy of melting. At a drug to dipalmitoyl phosphatidylcholine ratio of approx. 1:5, two peaks appear in the transition profile, suggesting a phase separation in the drug dipalmitoyl phosphatidylcholine mixture.


Semlike Forest virus membrane proteins. Preparation and characterization of spike complexes soluble in detergent-free medium. After Triton X-100 delipidation and subsequent Triton X-100 removal in a sucrose gradient the membrane protein spikes of Semliki Forest virus remained soluble in aqueous buffers. It was shown they were present as octameric complexes with a molecular weight of 95-10(4) and that they contain less than 4% lipid and detergent by weight. In electron microscopy after negative staining they appeared as "rosette"-shaped particles. Part of the protein could also be found associated in ordered paracrystalline arrays.


Isolation and characterization of three unusual membrane glycopeptides present in rat intestinal endoplasmic reticula. Three glycopeptides have been isolated from the mucosal homogenates of the rat small intestine without using proteolysis. These glycopeptides appear to be localized exclusively in the membranes of the endoplasmic reticula. Although they have similar molecular weights of about 2550 and have similar amino acid compositions, they differ in the carbohydrate constituents. The major glycopeptide has 2 mol glucose per polypeptide chain while the two other glycopeptides contain 1 mol fucose, mannose and galactose with either 1 or 2 mol glucose. No hexosamine or sialic acid was detected in any of the glycopeptides. An unusual physical property was found associated with these glycopeptides. Below pH 6.5 they formed a precipitate which prevented them from  diffusing through a dialysis membrane and allowed them to be rapidly purified following solubilization from the membrane. These glycopeptides appear to represent a new group of heretofore uncharacterized membrane constitutents which may play a role in some function specific for the endoplasmic reticula.