A method for the purification of milligram quantities of stable human phosphatidylcholine-cholesterol acyltransferase. A method for processing 3 litres of human plasma for the purification of phosphatidylcholine-cholesterol acyltransferase is described. The method involves (NH4)2SO4 fractionation, citric acid treatment, and DEAE-cellulose and hydroxyapatite chromatography. At this stage the enzyme preparation is purified approx. 8000-fold. This preparation appears to be free of lipoproteins as determined by immunoelectrophoresis against anti-human serum and is minimally contaminated with albumin (less than 30 mug/mg of enzyme protein) as determined by immunodiffusion. The activity of the enzyme was stable for 4 days, but most of its activity was lost after 20 days On electrophoresis on 5% polyacrylamide gel, a fast-moving band with enzyme activity and a slow-moving band with no enzyme activity was observed. A faint band of albumin was also present. Extracts of enzymically active bands cut from ten gels and then pooled and extracted with 0.15 M-NaC1/4mM-sodium phosphate, pH 7.0, showed a single band on re-electrophoresis on 5% polyacrylamide gel.


Molecular forms and activities of glycosidases in cultures of amniotic-fluid cells. Ion-exchange chromatography of gel filtration demonstrated the presence of different molecular forms of nine lysosomal enzymes in cultured amniotic-fluid cells. The patterns of molecular forms were similar to those known from skin fibroblasts and liver tissue. During cultivation total enzyme activities fluctuated with the number of passages, without any consistent trend of increase or decrease, and without correlation to the dominating cell type in the culture.


The determination of methionine in proteins by gas-liquid chromatography. Intact methionine residues in proteins were rapidly and precisely determined by measuring methyl thiocyanate released during the reaction with CNBr and separated by g.l.c. Conditions for the reaction and for chromatography on columns of Porapak P-S are described. The recovery of methyl thiocyanate from several methionine derivatives and analogues were examined. Carbamoylmethionine was adopted as a stable primary standard and ethyl thiocyanate as internal standard. The measured methionine content of several isolated proteins was close to the theoretical value indicated by previous work and the results for these and a range of food proteins agreed well with results obtained by ion-exchange chromatography after performic acid oxidation. Since CNBr does not react with methionine sulphoxide and a preliminary hydrolysis is not required, the method discriminates between methionine and any methionine sulphoxide that may be present. It could be useful in studies on the nutritional availability of methionine in processed foods.


Mode of interaction between beta-lactam antibiotics and the exocellular DD-carboxypeptidase--transpeptidase from Streptomyces R39. The exocellular DD-carboxypeptidase-transpeptidase of Streptomyces R39 is inhibited by beta-lactam antibiotics according to the same general scheme of reaction as the exocellular DD-carboxypeptidase-transpeptidase of Streptomyces R61. However, the values for the kinetic constants involved in the reaction are very different for the two enzymes and provide an explanation for the observation that the R39 enzyme is more sensitive to beta-lactam antibiotics than the R61 enzyme. Further, particular beta-lactams influence the kinetic constants to different extents depending on the source of the enzyme, so that a physical basis for the spectrum of antibiotic activity against particular enzyme systems is provided.


Isolation of (copper, zinc-) thioneins from pig liver. The copper-binding proteins with mol. wt. of approx. 12000 in the cytosol of pig liver have been identified as copper-thioneins. They contain variable amounts of zinc, with approx. 10% total metal in the protein. The molar cysteine/metal ratio is approx. 2:1.


The nature of the 5'-linked 5' nucleotide sequence at the 5' end of rabbit globin messenger ribonucleic acid. Poly(A)-containing messenger RNA isolated from rabbit reticulocytes as estimated by periodate oxidation and condensation with [3H]isoniazid has two oxidizable end groups per molecule of mol. wt. 220000. When the mRNA is subjected to stepwise degradation by beta-elimination, only one oxidizable end-group is found. This indicates that one of the 2',3' hydroxyl end-groups is linked through the normal 3'--5' phosphodiester bond, but that the other is linked in such a way that after stepwise degradation no new 2',3 hydroxyl group is revealed. This structure could be a 5'-linked 5'-phospho di- or tri-ester. On digestion with ribonuclease the isoniazid-labelled RNA produced oligonucleotide hydrazones consistent with a poly(A) sequence at the 3' end plus fragments that are not found after stepwise degradation. These fragments have a charge of --6 and --8 from pancreatic ribonuclease or --7 from ribonuclease T1 digestion. These charges are changed to --3.4 and --4.1 after pancreatic ribonuclease, ribonuclease T2 and alkaline phosphatase digestion. methyl-3H-labelled-poly(A)-containing RNA isolated from late erythroid cells contain a methyl-labelled fragment resistant to endonuclease and phosphodiesterase II digestion. After digestion with phosphodiesterase I this fragment produces methyl-3 H-labelled nucleotides with the electrophoretic mobility of pm7G and pAm. It is concluded that globin mRNA has the 5' sequences m7G(5')ppp'AmpYpGp ... and m7G(5')pppAmpApGpYp.


Kinetic and spectral properties of rabbit brain 4-aminobutyrate aminotransferase. Purification and 4-aminobutyrate-2-oxoglutarate aminotransferase (EC 2.6.1.19) from rabbit brain is described. The method was used as a routine to give between 5 and 10mg of pure enzyme from 750 g of rabbit brain. The enzyme is a dimer made up of subunits each with a mol. wt. of 58000. An absorption spectrum of the freshly prepared enzyme shows peaks at 415 and 330 nm. Treatment of the enzyme with the substrate 4-amino-butyrate or glutamate produces a decrease in the 415 nm and an increase in the 330 nm peak. This conversion, which is attributed to an aldimine into ketimine step in the reaction, is sufficiently slow when 4-aminobutyrate is the substrate to allow it to be followed by stopped-flow spectrophotometry. A first-order rate constant was determined for this step (12s-1) and compared with the turnover number for the enzyme derived by steady-state methods (9.5S-1). The first-order rate constant when glutamate was used as substrate was estimated to be approx. 30s-1.


Affinity purification and some molecular properties of human liver alkaline phosphatase. Alkaline phosphatase from human liver was purified to homogeneity. The purification procedure included solubilization with butanol, fractionation with acetone, and chromatography on concanavalin A-Sepharose, DEAE-cellulose, Sephadex G-200 and DEAE-Sephadex. Purity was established by standard and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The isoelectric point of the protein was determined to be 4.0. Sephadex-gel filtration gave a mol.wt. of 146000, although a higher value was obtained in the presence of 100mM-NaC1. The subunit mol.wt. 76700, was determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Neuraminidase treatment resulted in two enzyme-activity bands on isoelectric-focused gels with isoelectric points of 6.6 and 6.8. The desialylated enzyme gave only one protein band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with a subunit molecular weight indistinguishable from that of the non-neuraminidase-treated protein. The desialylated enzyme was more readily denatured by sodium dodecyl sulphate in the presence of mercaptoethanol than was the native enzyme.


Ligand-apomyoglobin interactions. Configurational adaptability of the haem-binding site. 1. The interaction of the haem-binding region of apomyoglobin with different ligands was examined by ultrafiltration, equilibrium dialysis and spectrophotometry, to study unspecific features of protein-ligand interactions such as they occur in, for example, serum albumin binding. 2. Apomyoglobin, in contrast with metmyoglobin, binds at pH 7, with a high affinity, one molecule of Bromophenol Blue, bilirubin and protoporphyrin IX, two molecules of n-dodecanoate and n-decyl sulphate and four molecules of n-dodecyl sulphate and n-tetradecyl sulphate. 3. The number of high-affinity sites and/or association constants for the alkyl sulphates are enhanced by an increase of hydrocarbon length, indicating hydrophobic interactions with the protein. 4. Measurements of the temperature-dependence of the association constants of the high-affinity sites imply that the binding processes are largely entropy-driven. 5. Binding studies in the presence of two ligands show that bilirubin plus Bromophenol Blue and dodecanoate plus Bromophenol Blue can be simultaneously bound by apomyoglobin, but with decreased affinities. By contrast, the apomyoglobin-protoporphyrin IX complex does not react with Bromophenol Blue. 6. Optical-rotatory-dispersion measurements show that the laevorotation of apomyoglobin is increased towards that of metmyglobin in the presence of haemin and protoporphyrin IX. Small changes in the optical-rotatory-dispersion spectrum of apomyoglobin are observed in the presence of the other ligands. 7. It is concluded that the binding sites on apomyoglobin probably do not pre-exist but appear to be moulded from predominantly non-polar amino acid residues by reaction with hydrophobic ligands. 8. Comparison with data in the literature indicates that apomyoglobin on a weight basis has a larger hydrophobic area avaialble for binding of ligands than has human serum albumin. On the other hand, the association constants of serum for the ligands used in this study are generally somewhat larger than those of apomyoglobin.


Abnormal carbohydrate composition of the major penetrating membrane protein of En(a-) human erythrocytes. The major penetrating membrane glycoprotein (band 3) was isolated from En(a-) and normal human erythrocytes. The two proteins differed only in carbohydrate composition. Band 3 from En(a-) erythrocytes contained greater amounts of galactose and N-acetyl-glucosamine. The loss of the sialoglycoprotein sialotetrasaccharides in the En(a-) cell is not compensated by the appearance of these units in band 3 of En(a-) erythrocytes.


Removal of adsorbed elastase from partially digested elastin. Bovine ligamentum nuchae elastin that had been partially digested by porcine pancreatic elastase and then thoroughly washed with 0.2 M-NaC1/0.05 M-sodium borate solution continued to release peptide fragments in solution. Complete removal of elastase from the elastin surface required extraction with dilute alkali, a procedure that does not irreversibly inactivate the enzyme or hydrolyse elastin.


Modification of brain deoxyribonucleic acid base content with maturation in normal and malnourished rats. The mol percentage of 5-hydroxymethylcytosine is 2.2 times greater in the adult than in 2-day-old rat brain DNA. The concentration of 5-hydroxymethylcytosine falls in corresponding liver DNA preparations. This normal increase in brain 5-hydroxymethylcytosine is abolished in rats placed on an 8%-protein diet 5 days after birth.


Dihydrofolate reductase and thymidylate synthetase activities in the liver of rabbit foetuses during the haemapoietic period. Dihydrofolate reductase activity did not change with variations of mitotic rate in foetal rabbit haemopoietic tissue, whereas the activity of thymidylate synthetase declined with decrease in cell divisions. Both the activities were normal in the brachydactylia strain, in which a failure in folate metabolism was previously assumed.


Inhibition of acetylcholinesterase by derivatives of 1,3,2-dioxaphosphorinane 2-oxide. Cholinesterases are inhibited by 2-fluoro-1,3,2-dioxaphosphorinane 2-oxide by a mechanism that involves a slow association step followed by a very slow phosphorylation step. No phosphorylation step was observed for the interaction between acetylcholinesterase and 2-S-[2'-(NN-diethylamino)ethyl]thio-1,3,2-dioxaphosphorinane 2-oxide.


Glucagon-like and insulin-like hormones of the insect neurosecretory system. Aqueous extracts of corpus cardiacum-corpus allatum complexes of the adult tobacco hornworm Manduca sexta produced both glycogenolysis and hypoglycaemia when injected into the larval form of the same species. Application of specific radioimmuno assays to similar extracts showed also that these gland complexes contain both glucagon-like and insulin-like peptides. Further, the partially purified immunoreactive peptides had the expected biological activities. The former decreased the glycogen content of the fatbody and the latter the circulating trehalose levels in recipient animals. These results suggest the existence of hormones in these invertebrates having both biological and structural similarities to vertebrate insulin and glucagon.


The inhibition in vivo of lipoprotein lipase (clearing-factor lipase) activity by triton WR-1339. 1. Lipoprotein lipase activity was measured in heart homogenates and in heparin-releasable and non-releasable fractions of isolated perfused rat hearts, after the intravenous injection of Triton WR-1339. 2. In homogenates of hearts from starved, rats, lipoprotein lipase activity was significantly inhibited (P less than 0.001) 2h after the injection of Triton. This inhibition was restricted exclusively to the heparin-releasable fraction. Maximum inhibition occurred 30 min after the injection and corresponded to about 60% of the lipoprotein lipase activity that could be released from the heart during 30 s perfusion with heparin. 3. Hearts of Triton-treated starved rats were unable to take up and utilize 14C-labelled chylomicron triacylglycerol fatty acids, even though about 40% of heparin-releasable activity remained in the hearts. 4. It is concluded that Triton selectively inhibits the functional lipoprotein lipase, i.e. the enzyme directly involved in the hydrolysis of circulating plasma triacylglycerols. 5. Lipoprotein lipase activities measured in homogenates of soleus muscle of starved rats and adipose tissue of fed rats were decreased by 25 and 39% respectively after Triton injection. It is concluded that, by analogy with the heart, these Triton-inhibitable activities correspond to the functional lipoprotein lipase.


Hydroxylation of lysine and glycosylation of hydroxylysine during collagen biosynthesis in isolated chick-embryo cartilage cells. Hydroxylation of lysine and glycosylation of hydroxylysine during collagen biosynthesis in isolated chick-embryo cartilage cells were studied by using continuous labelling and pulse-chase labelling experiments with [14C]lysine. Control experiments with [14C]proline indicated that in continuous labelling the hydroxylation of [14C]proline became linear with time after about 4 min and the secretion of collagen after about 35 min, as reported previously. In similar experiments with [14C]lysine the hydroxylation of [14C]lysine and the glycosylations of hydroxy[14C]lysine became linear at about 4 min, suggesting that these reactions were initiated while the polypeptide chains were growing on the ribosomes. Pulse-chase labelling experiments with [14C]lysine indicated that after a 5 min pulse-label the hydroxylation of [14C]lysine and the glycosylations of hydroxyl[14C]lysine continued during the chase period for about 20 min. The data suggest that these reactions are continued after the release of complete polypeptide chains into the cisternae of the endoplasmic reticulum, whereas the reactions are probably not continued after the formation of the triple helix and the movement of the molecules into the Golgi vacuoles.


Metabolism of L-(U-14C)valine, L-(U-14C)leucine, L-(U-14C)histidine and L-(U-14C) phenylalanine by the isolated perfused lactating guinea-pig mammary gland. 1. The incorporation of L-[U-14C]leucine, L[U-14C]histidine and L-[U-14C]phenylalanine into casein secreted during perfusion of isolated guinea-pig mammary glands was demonstrated. 2. The extent of incorporation of label into casein residues was consistent with their being derived from free amino acids of the perfusate plasma. 3. The mean transit time of the amino acids from perfusate into secreted casein was approx. 100 min. 4. Whereas radioactive histidine and phenylalanine were incorporated solely into milk protein, radioactivity from [U-14C]valine was also transferred to CO2 and to an unidentified plasma component, and from [U-14C]leucine to plasma glutamic acid. 5. Evidence from experiments with [U-14C]phenylalanine suggests that, as in rats, but in contrast with ruminant species, guinea-pig mammary tissue does not possess phenyl alanine hydroxylase activity. 6. The results are discussed in relation to the possible role of essential amino acid catabolism in the control of milk-protein synthesis.


A sensitive method for measuring protein turnover based on the measurement of 2-3H-labelled amino acids in protein. A method for measuring the rate of protein degradation is described. The method measures the change in 2-3H content of protein with time by racemization of the protein hydrolysate with acetic anhydride. The 3H on C-2 of amino acids is stable in proteins but becomes labile, owing to the action of transaminases, once the amino acids are released by proteolysis. The specific measurement of 2-3H in amino acids largely overcomes problems due to compartmentation and isotope recycling and evidence to support this claim is presented. Values for the half-life of Lemna minor (duckweed) protein determined by the new method are compared with values obtained by other methods.


Evidence for the lack of deoxyribonucleic acid dark-repair in Halobacterium cutirubrum. 1. Halobacterium cutirubrum does not perform dark-repair of DNA either after u.v. irradiation or during normal growth. 2. Cultures irradiated with u.v. are readily photoreactivated, but do not recover viability in the dark. 3. No increase in the rate of DNA synthesis is observed in the surviving cells after u.v. irradiation. 4. At early times during normal semiconservative replication, newly incorporated thymidine is found only in the hybrid DNA. 5. It is suggested that these bacteria may be useful in the study of DNA replication and photoreactivation.


Erythrocyte calcium metabolism. Calcium exchange in normal and sickle-cell-anaemia erythrocytes. Under exchange conditions (no net increase in calcium), erythrocytes incubated in isoosmotic phosphate-buffered saline have an exchangeable calcium pool comprising about 10% of the total erythrocyte calcium. This pool reaches exchange equilibrium, for either inward-directed or outward-directed transfer of the 45Ca-exchange label, with a half-time of about 20 min. The uptake of Ca2+ requires phosphate, even under hypo-osmotic conditions, where the calcium loading expected as the cells swell is obtained only when phosphate is present. The phosphate requirement is not due to Ca2+ transport as a phosphate salt. This exchangeable-calcium pool is also present in sickle-cell-anemia erythrocytes, and comprises a similar proportion of total cellular calcium.


Fatty acid synthesis in the perfused liver of adrenalectomized rats. 1. Fatty acid synthesis, measured in the perfused liver of fed adrenalectomized rats with 3H2O and 14C-labelled precursors, was less than in control sham-operated rats. 2. This defect was more extensive for synthesis of fatty acids incorporated into triacylglycerols than into phospholipids. 3. There was impairment in desaturation and export of newly synthesized fatty acid. 4. Fatty acid synthesis and desaturation were restored to normal rates 5h after treatment with cortisol in vivo. 5. Fatty acid synthesis was seasonally variable, being highest in the winter; the impairment after adrenalectomy was observed in all seasons. 6. In perfusions with oleate (0.7 mM), no further impairment in fatty acid synthesis was discerned in livers from adrenalectomized rats, in which the rate resembled that in control livers. 7. No defect in the incorporation of oleate into glycerides was discerned in livers from adrenalectomized rats. 8. Cortisol exerted no stimulatory effect on fatty acid synthesis when added to perfusion media. 9. The impairment in hepatic lipogenesis, demonstrable after adrenalectomy, shows that adrenal glucocorticoids promote hepatic capacity for fatty acid synthesis de novo, at least in intact non-diabetic rats. It is suggested that this effect is mediated by insulin, perhaps through direct action on the liver.


Lipogenesis from ketone bodies in rat brain. Evidence for conversion of acetoacetate into acetyl-coenzyme A in the cytosol. The metabolism of acetoacetate via a proposed cytosolic pathway in brain of 1-week-old rats was investigated. (-)-Hydroxycitrate, an inhibitor of ATP citrate lyase, markedly inhibited the incorporation of carbon from labelled glucose and 3-hydroxybutyrate into cerebral lipids, but had no effect on the incorporation of labelled acetate and acetoacetate into brain lipids. Similarly, n-butylmalonate and benzene-1,2,3-tricarboxylate inhibited the incorporation of labelled 3-hydroxybutyrate but not of acetoacetate into cerebral lipids. These inhibitors had no effect on the oxidation to 14CO2 of the labelled substrates used. (-)-Hydroxycitrate decreased the incorporation of 3H from 3H2O into cerebral lipids by slices metabolizing either glucose or 3-hydroxybutyrate, but not in the presence of acetoacetate. (-)-Hydroxycitrate also differentially inhibited the incorporation of [2-14C]-leucine and [U-14C]leucine into cerebral lipids. The data show that, although the acetyl moiety of acetyl-CoA generated in brain mitochondria is largely translocated as citrate from these organelles to the cytosol, a cytosolic pathway exists by which acetoacetate is converted directly into acetyl-COA in this cellular compartment.


Partial isolation and translation in vitro of messenger ribonucleic acid for phosphoenolpyruvate carboxykinase (guanosine triphosphate). 1. mRNA was extracted from the livers of starved rats and incubated in a heterologous cell-free protein-synthesizing system derived from rabbit reticulocytes. The presence of newly synthesized phosphoenolpyruvate carboxykinase (GTP) was detected by immunoprecipitation with a specific antibody to the enzyme and analysis by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 2. The synthesis of the enzyme was dependent on the addition of rat liver RNA, whereas RNA isolated from rat spleen was inactive. If ovalbumin and anti-ovalbumin were used to form the immunoprecipitates, no radioactivity that migrated as phosphoenolpyruvate carboxykinase was detected. 3. The optimal concentrations of magnesium acetate and KCl for phosphoenolpyruvate carboxykinase synthesis were determined. 4. When polyribosomal RNA was separated by sucrose-gradient centrifugation, phosphoenolpyruvate carboxykinase mRNA migrated between 20 and 26 S in keeping with the high mol. wt. of the protein (85 000). 5. The presence of poly(A) in phosphoenolpyruvate carboxykinase mRNA was suggested by retention of mRNA activity on oligo(dT)-cellulose columns. 6. It was concluded that the cell-free synthesis of phosphoenolpyruvate carboxykinase can serve as a bioassay for intracellular phosphoenolpyruvate carboxykinase mRNA.


Enzymic hydrolysis of myelin basic protein and other proteins in central nervous system and lymphoid tissues from normal and demyelinating rats. Proteolytic activity of central-nervous-system tissue of the normal rat was examined over the pH range 2-9 with casein, haemoglobin and myelin basic protein as substrates. With casein as a substrate, brain and spinal cord homogenates showed very similar activity profiles with increasing pH, with the main peaks of proteolytic activity at pH 3-4 and 5-6. When haemoglobin was used, one broad main peak of activity from pH 3 to 5 was demonstrated. There was no optimum pH, however, for proteolytic activity with myelin basic protein as a substrate, and considerable hydrolysis were observed from pH 3.5 up to pH8. Proteolytic activity at the various pH values was compared by using homogenates of spinal cords from rats with acute experimental allergic encephalomyelitis and those from rats injected with Freund's adjuvant alone. The profiles of activity were similar with peaks at pH 3.5 and 5.5 with casein as a substrate, but the specific activity was significantly higher at most pH values in the spinal-cord homogenates from rats with experimental allergic encephalomyelitis. Similarly the spinal-cord homogenates from these latter rats contained much more proteolytic activity toward myelin basic protein throughout the pH range than was present in the control spinal cords. Homogenates from lymph nodes of rats with experimental allergic encephalomyelitis and from those of the controls contained two to three times as much proteolytic activity as that of the central-nervous-system tissue and had a different proteolytic activity profile form that of the central-nervous system, with higher activity at the neutral than at acid pH. The results are discussed with regard to the probability that inflammatory cells such as lymphocytes may be the cause of the increased proteolytic activity in the central nervous system of animals with experimental allergic encephalomyelitis, and that enzymes from these cells possess the capability of digesting myelin basic protein.


The calcium conductance of the inner membrane of rat liver mitochondria and the determination of the calcium electrochemical gradient. 1. A method is described for establishing steady-state conditions of calcium transport across the inner membrane of rat liver mitochondria and for determining the current of Ca2+ flowing across the membrane, together with the Ca2+ electrochemical gradient across the native Ca2+ carrier. These parameters were used to quantify the apparent Ca2+ conductance of the native carrier. 2. At 23 degrees C and pH7.0, the apparent Ca2+ conductance of the carrier is close to 1 nmol of Ca2+-min-1-mg of protein-1 mV-1. Proton extrusion by the respiratory chain, rather than the Ca2+ carrier itself, may often be rate-limiting in studies of initial rates of Ca2+ uptake. 3. Under parallel conditions, the endogenous H+ conductance of the membrane is 0.3 nmol of H+-min-1-mg of protein-1-mV-1. 4. Ruthenium Red and La3+ both strongly inhibit the Ca2+ conductance of the carrier, but are without effect on the H+ conductance of the membrane. 5. The apparent Ca2+ conductance of the carrier shows a sigmoidal dependence on the activity of Ca2+ in the medium. At 23 degrees C and pH7.2, half-maximum conductance is obtained at a Ca2+ activity of 4.7 muM. 6. The apparent Ca2+ conductance and the H+ conductance of the inner membrane increase fourfold from 23 degrees to 38 degrees C. The apparent Arrhenius activation energy for Ca2+ transport is 69kJ/mol. The H+ electrochemical gradient maintained in the absence of Ca2+ transport does not vary significantly with temperature. 7. The apparent Ca2+ conductance increases fivefold on increasing the pH of the medium from 6.8 to 8.0. The H+ conductance of the membrane does not vary significantly with pH over this range. 8. Mg2+ has no effect on the apparent Ca2+ conductance when added at concentration up to 1 mM. 9. Results are compared with classical methods of studying Ca2+ transport across the mitochondrial inner membrane.


A sparing effect of increased plasma fatty acids on muscle and liver glycogen content in the exercising rat. Increasing plasma free fatty acids decreased the degree of glycogen depletion, and increased the citrate concentration, in slow-red (soleus) and fast-red (deep portion of vastus lateralis) muscle during exercise (approx. 50% depletion of glycogen, as against 75% in control animals). There was no effect in fast-white muscle (superficial portion of vastus lateralis). Glycogen concentration in the liver decreased by 83% in controls, but only by 23% in animals with increased free fatty acids during exercise. The decreased glycogen depletion may be partly explained by the findings that (a) plasma-insulin concentration was two- to three-fold higher in animals with increased plasma free fatty acids and (b) the exercise-induced increase in plasma glucagon was lessened by increased free fatty acids. Blood glucose was higher in the animals with increased free fatty acids after the exercise. The rats with increased plasma free fatty acids utilized approx. 50% as much carbohydrate as did the controls during the exercise.


Measurement of protein turnover in rat liver. Analysis of the complex curve for decay of label in a mixture of proteins. The curve for decay of 14C in rat liver protein labelled by injection of NaH14CO3 was analysed to obtain the average turnover rate of mixed liver protein. Three different methods of analysis were used. (1) Unlike decay curves from homogeneous proteins, the curve did not fit a single exponential, but a good fit was obtained with three exponentials. By assuming that the mixture contained three major components with different turnover rates, the calculated value for the average turnover rate (k) was close to 40% per day. (2) k was also calculated from the area under the decay curve, a method which makes no assumptions about the number of proteins in the mixture. This method also gave a value close to 40% per day. (3) It was shown empirically, both by simulation of decay of label in model mixtures of protein and with the decay curve measured in vivo, that k can be calculated from the time taken for the specific radioactivity to fall to 10% of its maximum value. This is an advantage, since the other two methods require the decay curve to be measured over a much longer period of time.


Effect of phenobarbitone on the nucleocytoplasmic transport of ribonucleic acid in vitro. The transport of nucleic acids from the nucleus to the cytoplasm is a potential site for modification of normal cellular processes by drugs and hormones. In this study the effect of phenobarbitone on nucleocytoplasmic transport of ribosomes was measured in an assay system in vitro. The transport of radioactive ribosomes from isolated rat hepatic nuclei to unlabelled post-microsomal supernatant was measured in rats treated with 80 mg of phenobarbitone/kg body wt. or saline 3h before death. With either treatment, transport was linear with time, and dependent on temperature and the presence of ATP. However, phenobarbitone treatment increased transport of ribonucleoproteins over saline-treated animals nearly twofold. The effect of phenobarbitone was mediated through the cytosol, but was not the result of altered stability of the RNA transported to the cytosol. Cycloheximide (5 mg/kg body wt.) given 3.5 h before death inhibited the stimulation of transport by phenobarbitone. The data indicate that phenobarbitone increased the transport of RNA by stimulating the synthesis of cytosol factors that regulate transport of RNA from the nucleus.


Gluconeogenesis in isolated intact lamb liver cells. Effects of glucagon and butyrate. 1. Isolated lamb liver cells were prepared from 24-h-starved animals by venous perfusion of the excised caudate lobe with buffer containing collagenase. On the basis of Trypan-Blue exclusion, rate of O2 uptake, adenine nucleotide content and retention of constitutive enzymes, these cells were judged to be intact. 2. Isolated caudate-lobe liver cells showed rates of gluconeogenesis from 10 mM-propionate and 10 mM-lactate that compared favourably with rates determined in isolated median-lobe cells and with rates determined with the isolated perfused lamb liver. 3. The gluconeogenic potential of substrates tested depended on the lamb's age. Cells prepared from suckling lambs (up to 20 days of age and essentially non-ruminant) showed highest rates from galactose, serine and alanine; those prepared from post-weaned lambs (older than 30 days of age and ruminant) showed highest rates from propionate, lactate and fructose. 4. Gluconeogenic rates from endogeneous precursors, 10 mM-propionate and 10mM-galactose, were linear for 1 h and were both stimulated by 1 muM-glucagon. Provided the endogenous rate of gluconeogenesis remained unchanged after substrate addition, glucagon caused a net stimulation of gluconeogenesis from each of these substrates. 5. Gluconeogenic capacity and glucagon sensitivity were examined in cells maintained in substrate-free oxygenated buffer at 37 degrees, 22 degrees and * degrees C. Even under the best of the three conditions of storage that were tested (i.e. at 22 degrees C in gelatin-containing buffer) deterioration of the lamb cells proceeded rapidly, and loss of glucagon responsiveness preceeded the loss of ability to convert precursor into glucose. 6. n-Butyric acid, 2-methylpropanoic acid and 3-methylbutanoic acid at concentrations comparable with those found in lamb portal-vein blood each stimulated gluconeogenesis from 10mM-galactose or 10mM-propionate; gluconeogenesis from galactose was stimulated to the greater extent. 7. The regulatory effects of glucagon and sodium butyrate on lamb liver-cell gluconeogenesis and glycogenolysis were compared. Glucagon (1 muM) and 2mM-butyrate accelerated the rate of glucose formation of liver cells of 24h-starved animals from lactate+pyruvate or fructose. Insulin (20nM) decreased both gluconeogenesis and the efficacy of 1 muM-glucagon. For lactate+pyruvate as substrate, the stimulatory effect of butyrate was additive to that of 1muM-glucagon and for both lactate+pyruvate and fructose the stimulatory effect of butyrate was not influenced by 20nM-insulin. In contrast with glucagon, which stimulated the rate of glycogenolysis in cells prepared from fed lambs, butyrate (0.1-20mM) had no effect. 8. It is concluded that glucagon and butyrate stimulate lamb liver-cell gluconeogenesis by different mechanisms.


Inhibition by botulinum toxin of depolarization-evoked release of (14C)acetylcholine from synaptosomes in vitro. 1. Cerebral-cortex synaptosomes were shown to synthesize (14C)acetylcholine after incubation with (14C)choline, and 25mM-KCl released (14C)acetylcholine (but not (14C)choline) into the medium by a Ca2+-dependent and Mg2+-sensitive process. 2. The K+-stimulated release of (14C)acetylcholine was inhibited by more than 80% after preincubation of the synaptosomes with 10(5) mouse lethal doses of botulinum toxin/ml. (14C)choline uptake, (14C)acetylcholine synthesis, intrasynaptosomal K+ and occluded lactate dehydrogenase were unaffected by the toxin. It also failed to prevent the K+-stimulated release of (3H)noradrenaline and (14C)glycine from synaptosomes. 3. Fractionation of hypo-osmotically shocked synaptosomes revealed that more than 75% of the radioactive acetylcholine was in the cytoplasmic compartment, although the vesicle pellet contained more total acetylcholine than the cytoplasmic pool. Consequently the specific radioactivity of acetylcholine in the cytoplasmic pool was almost 5 times that of the vesicles. This distribution was unaffected by preincubation with botulinum toxin. It is concluded that the toxin acts directly on the release of acetylcholine, rather than influencing its storage. 4. After K+-stimulation, toxin-inhibited synaptosomes contained increased amounts of total acetylcholine, which suggests that its rate of synthesis is controlled by depolarization rather than release.


Stimulation of peptide elongation by thyroxine. This study suggests that thyroxine stimulates peptide elongation in a cell-free rat liver polyribosome system. The thyroxine effect persists in the presence of sufficient aurintricarboxylic acid to prevent polyuridylic acid-stimulated peptide initiation. In addition, thyroxine stimulates elongation of pre-existing polyphenylalanine chains providing conclusive evidence that the effect does not depend on peptide initiation. Thyroxine does not stimulate release of nascent peptides from ribosomes into the supernatant phase of the reaction mixture. Therefore in this protein-synthesis system the thyroxine effect is expected to occur at one or more of the reactions of peptide chain elongation, which include aminoacyl-tRNA binding, peptide bond synthesis and translocation.


A new diagnostic test for pancreatic disorders by examination of parotid saliva. The parotid saliva test was performed in 146 subjects, including 48 patients with pancreatic disorders, 82 with nonpancreatic disorders and 16 healthy persons. The following results were obtained: 1. The salivary output as well as the maximum bicarbonate concentration and amylase content in the parotid saliva of patients with pancreatic disorders were significantly less than those of patients with nonpancreatic disorders. 2. An abnormal saliva test was found in 83.3% of the patients with the pancreatic disorders. 3. Comparison was made of the parotid saliva test with the pancreozyminsecretin test in regard to diagnostic reliability in 44 subjects, including 22 with pancreatic disorders and 22 with nonpancreatic disorders. The data indicated that, in this series, an abnormal parotid saliva test was 88.6% accurate in diagnosing pancreatic disorders, whereas positive pancreozymin-secretin was only 65.9%.


Lichen verrucosus et reticularis of Kaposi (porokeratosis striata of Nékam): a manifestation of acquired adult toxoplasmosis. Three young adults are described with features of lichen verrucosus et reticularis of Kaposi (porokeratosis of Nékam). Serological evidence of toxoplasmosis (active or recent infection) was found in all three. Marked improvement on appropriate treatment occurred in the one patient who presented evidence of systemic toxoplasmosis.


DNA synthesis and mitosis in uninvolved epidermis of persistent palmoplantar pustulosis. Mitotic and DNA synthesizing cell counts have been performed in uninvolved epidermis of twenty-one patients with persistent palmoplantar pustulosis (PPP). There was no difference in mitotic counts and DNA synthesis in PPP compared with normal epidermis, but both were significantly lower than those found in the clinically uninvolved epidermis of patients with psoriasis.


Glucose-6-phosphate dehydrogenase in lichen planus skin. The apparent Michaelis constant (Km) for glucose-6-phosphate of the enzyme glucose-6-phosphate dehydrogenase has been measured in extracts prepared from biopsies of normal human skin and from both affected and apparently normal skin of patients with lichen planus. No differences of Km were found and starch gel electrophoresis of extracts from lichen planus lesions and normal controls showed similar patterns when stained for glucose-6-phosphate dehydrogenase activity. These results do not support the view that lichen planus is an inborn error of metabolism in which the structure of glucose-6-phosphate dehydrogenase of skin is affected.


The significance of the local sweat response in assessing the progress of leprosy. Serial observations on cutaneous sensation and on sweat response in hypopigmented flat patches of twenty-nine patients with tuberculoid and five with dimorphous leprosy on dapsone therapy were studied over 2 years. Sensation was determined by routine methods, while sweat stimulation was done by intradermal injection of 0-1 ml (0.025 mg) of carbachol (carbaminoylcholine). There was significant improvement in sensation and considerable augmentation of sweat response in the final as compared to the initial tests. The difference in sweat response was statistically significant, suggesting the value of this simple procedure in assessing the progress of leprosy.


Regression of a hamster melanoma with intralesional Corynebacterium granulosum. The present study was undertaken to compare the antitumour effects of intralesionally administered Corynebacterium granulosum, Corynebacterium parvum, BCG and saline on a hamster melanoma. Forty-eight golden hamsters were inoculated subcutaneously with Forthers melanotic melanoma no. 1. Twenty-four animals bore palpable tumours which were excised. No non-excised group animal showed primary tumour regression. In the excised group, the tumours generally recurred at the excision site in the C. parvum, BCG and saline-treated animals. By contrast, the incidence of recurrence was significantly reduced by intralesional injections of C. granulosum.


Accidental kidney biopsies in psoriasis. Eight accidental kidney biopsies in psoriatics were obtained together with 429 liver biopsies. None of the biopsies showed signs of renal damage. All patients had severe psoriasis, six had been treated with methotrexate for 1-2-6 years, receiving total dosages from 660 to 1757 g.


The treatment of hypermelanosis with 4-isopropylcatechol. Over the past 6 years sixty-eight patients have been treated with 1% or 3% 4-isopropylcatechol (4-IPC). Fifty-four had melasma, the others a variety of disorders of pigmentation. Two-thirds of the patients were significantly improved. Twenty patients had skin irritation due to 4-IPC and four developed an allergic contact dermatitis. One patient developed confetti-like areas of depigmentation in the 4-IPC-treated areas. Light and electron microscopic studies showed that in the 4-IPC-treated areas there was a loss and damage of the melanocytes, but the keratinocytes and Langerhans cells were unaffected. Melanosomal complexes containing many melanosomes were frequently found in the keratinocytes of the 4-IPC-treated negro skin. 4-Isopropylcatechol is a potent depigmenting agent and is of use in the topical therapy of selected patients with hypermelanosis. However, like other substituted phenols and hydroquinone it is irritant to the skin and should be used with caution.


Hodgkin's disease and pemphigus.

Hodgkin's disease and pemphigus. The second case of pemphigus associated with Hodgkin's disease is reported. Circulating pemphigus auto-antibodies were detected in this patient and cell-mediated (T cell) immunity was markedly depressed. It is postulated that this T-cell immunodepression predisposed the patient to the development of pemphigus.


Rates of production of methane in the rumen and large intestine of sheep. 1. An isotope tracer method for estimating methane production in sheep is described. 2. The technique was used to estimate methane produced in both the upper and lower digestive tract and to determine the routes by which it was excreted. 3. Four Merino ewes given lucerne chaff (33 g every hour) were used. 4. Total methane production rate was 21 +/- 1-1 (SE) ml/min; production in the rumen accounted for 87 +/- 1-2% of the total production; 95 +/- 1-4% of the methane produced in the rumen was excreted by eructation. 5. Of the methane produced in the lower digestive tract, 89 +/- 2-3% was excreted through the lungs and 11% through the anus.


Studies on the appearance of a hepatic copper-binding protein in normal and zinc-deficient rats. 1. A study has been made by gel-filtration techniques of the soluble copper- and zinc-binding proteins in rat liver after both intraperitoneal injection of Cu and dietary Cu supplementation. 2. Liver Cu and Zn concentrations increased after injection of Cu, both metals accumulating in the cytosol, mainly in a fraction with an apparent molecular weight of about 12 000. 3. When Zn-deficient rats were injected with Cu, there was little change in liver Zn concentration and the occurence of Cu in the low-molecular-weight form (about 12 000) was more transient. At most periods after injection, Cu accumulated mainly in a fraction with a molecular weight greater than 65 000. 4. When the rats were Cu-loaded by dietary supplementation, virtually no Cu or Zn was found in the low-molecular-weight form in Zn-deficient rats, although they were found in the Zn-supplemented animals. 5. The results suggest that Zn is essential for the accumulation of Cu in this form, but not for Cu to stimulate production of the metal-binding protein by a process requiring active protein protein synthesis.


Diets and living conditions of Asian boys in Coventry with and without signs of rickets. 1. The diets and living conditions of nin Asian boys with biochemical, and in most instances also radiological, signs of rickets were compared with those of nine other boys who appeared to be normal. The groups were matched according to age, religion, place of father's origin and boy's own place of birth. 2. There were no outstanding differences between the diets of the boys with, and of those without signs of rickets. All had adequate to high calcium intakes. Most of the boys had low intakes of vitamin D, and those withsigns of rickets generally had lower intakes than the normal boys. The food tables used for making the calculations of vitamin D intakes report the amount of the vitamin in the lipid fraction of milk. If it proves to be true that most of the vitamin D activity of milk is in the aqueous fraction, the boys would have been getting considerably more vitamin D than the results suggested, and their average intake may have been about 3-5mug/d. 3. It was not possible to make any quantitative measure of the exposure of the boys to sunlight. All Asian boys studied appeared to have their bodies more completely covered than British or West Indian boys. 4. The problem of why nine boys had signs of rickets and nine had none has not been solved. It is suggested that those with signs of rickets may have had higher requirements for vitamin D than others. When the intake of vitamin D is low and exposure to sunlight is minimal, those with high requirements will be the ones to develop signs of rickets.


The site of magnesium absorption from the ruminant stomach. 1. A low-magnesium diet was fed to four sheep, each of which had been surgically prepared with a rumen fistula, a tube into the cranial one-third of the omasum, a tube to the cranial one-third of the abomasum and a re-entrant duodenal cannula. Mg, as gluconate or acate or acetate, was continuously infused for 12-14 d in turn into (1) the caudal duodenal cannula, (2) the abomasum, (3) the omasum, (4) the rumen. A continuous infusion of the chromium-ethylenediaminetetraacetic acid complex (CrEDTA) was maintained to the  rumen. The abomasal effluent which flowed through the cranial duodenal cannula was continually sampled and the flow of Mg calculated from the concentrations of Mg and CrEDTA. Blood and rumen fluid samples were taken and urine and faeces collected during each period of Mg infusion. 2. The Mg infused to either the abomasum or omasum was completely recovered at the duodenum, indicating a lack of net absorption of Mg from these stomach compartments. In contrast, 13-7-18-7 mmol (36-61%) of the Mg infused to the rumen was not recovered at the duodenum which suggested that a substantial net absorption of the infused Mg occurred from the reticulo-rumen. Absorption of Mg caudal to the pylorus was not related to the site of Mg infusion and averaged 3-28 +/- 0-56 (SEM) mmol/d. 3. Compared with the intraruminal infusion, the post-ruminal infusion of Mg was associated with decreased plasma and rumen fluid Mg concentrations, decreased urinary Mg exretion, decreased Mg balance and increased faecal Mg excretion. 4. It is concluded that no significant absorption of Mg occurs from either the omasum or abomasum in sheep and that the reticulo-rumen is the principal site of Mg absorption before the pylorus. Absorption of Mg post-ruminally is insufficient to maintain normal Mg status in the animal.


Effect of feeding a carbohydrate-free diet on the growth and metabolism of preruminant kids. 1. Experiments were done using 8-d-old kids to determine the metabolic effect of feeding a carbohydrate-free diet, and the effects of supplementation of this diet with a small amount of glycerol, sodium propionate or glucose. 2. The experimental (carbohydrate-deficient) diets permitted growth nearly equal to that with the control diet (cow's milk). The kids given the experimental diets generally had lower levels of blood glucose than those given the control diet. 3. With all experimental diets there were increases in the concentrations of plasma lipid and total liver lipid and a decrease in the concentration of liver glycogen; supplementation of the carbohydrate-free diet with glycerol, sodium propionate or glucose had no additional effect on these values. 4. The ingestion of cow's milk produced hyperglycaemia 2 h after feeding, while in kids given the carbohydrate-free diet there was no increase in blood glucose level. The concentration of plasma free fatty acids in the kids given the carbohydrate-free diet was higher than that in control animals 24 h after feeding, suggesting that the kids given the experimental diet preferentially utilize free fatty acids as an energy source.


The measurement of production rates of volatile fatty acids in the caecum of the conscious rabbit. 1. The decrease in specific radioactivity of individual volatile fatty acids (VFA) after a single injection of tracer was monitored. The results obtained indicated the occurrence of a first-order process. 2. Regression analysis indicated the high flux of VFA through the caecal pool, equivalent to 30% of the maintenance energy requirement of the animal. 3. Interconversion of VFA was monitored, and results indicated substantial synthesis of butyric acid from acetic acid. 4. Results were obtained from animals on two dietary regimens, and these were compared with results reported for the other species.


Amino acid metabolism in the piglet: influence of level of protein and of methionine in the diet on tissue uptake and in vivo oxidation. 1. 14C-labelled lysine, glutamic acid and methionine were each administered as single infusions through catheters in the jugular veins of pigs between 28 and 42 d old, receiving either a low-protein (LP) (140 g protein/kg), or a high-protein (HP) (200 g protein/kg) diet. The pigs received 40 g diet/kg body-weight per d. 2. L-[U-14C]lysine and L-[U-14C]glutamic acid were both rapidly removed from the plasma; 3 min after completion of the infusion less than 10% of the activity from the [14C]-lysine, and between 10 and 20% of the activity from the [14C]glutamic acid remained in the plasma. 3. In pigs killed 15 min after infusion of either [14C]lysine or [14C]glutamic acid 26% of the activity from the dose of lysine was in the liver of those receiving the LP diet, whereas the corresponding value was only 16% for those which had received the HP diet. The uptake of activity from [14C]glutamic acid by the liver was unaffected by the level of protein in the diet. More activity from [14C]lysine than from [14C]glutamic acid was recovered in the spleen, heart and brain. 4. The recovery of the activity from [14C]lysine as 14CO2 in the 6 h after the infusion of the dose was lower for the pigs on the LP diet (2-7%) than for those on the HP diet (4-8%). The corresponding values for [14C]glutamic acid infusion were 34 and 52% respectively. 5. Supplementing the LP diet with five successive increments of 1-5 g DL-methionine/kg resulted in an increase from 8 to 17% dose in the recovery of activity as 14CO2 from a dose of L-[I-14C]methionine, whereas with the same supplementation of the HP diet the recovery increased from 8 to 24% dose.


Nalpha-acetylfusarinines: isolation, characterization, and properties. The isolation, purification, and structural elucidation of a new group of naturally occurring hydroxamic acids are described. These compounds are produced by an unidentified species of Penicillium when grown on iron deficient medium. They are closely related structurally to the fusarinines, whose basic structure contains Ndelta-(cis-5-hydroxy-3-methylpent-2-enoyl)-Ndelta-hydroxy-L-ornithine, but the alpha-amino groups in the new compounds are protected by Nalpha acetyl groups. The most complex of the compounds is N,N',N"-triacetylfusarinine C, or N,N'N"-triacetylfusigen, a cyclic triester composed of three molecules on Nalpha-acetylfusarinine. The trimer has growth factor activity for Arthrobacter JG-9 about equal to fusarinine C itself. The monohydroxamate, Nalpha-acetylfusarinine, is more active than the trimer. The greater chemical stability of these compounds compared to the unacetylated fusarinines reconciles the utilization of otherwise unstable amino acid esters as iron transport agents.


Fungal ornithine esterases: relationship to iron transport. Extracts of Fusarium roseum (ATCC 12822) contain an enzyme which hydrolyzes the ornithine ester bonds of fusarinine C, a cyclic trihydroxamic acid produced by this organism. The methyl ester of Ndelta-dinitrophenyl-L-ornithine is also a substrate for the enzyme, and an assay was devised using this substrate. The enzyme exhibits a sharp maximum of activity at pH 7.5 and is extremely temperature sensitive. It is strongly inhibited by HgCl2 and p-chloromercuribenzoate, and it is competitively inhibited by Ndelta-dinitrophenyl-D-ornithine methyl ester (Ki = 0.3mM). Methyl esters of glycine, L-alanine, dinitrophenyl-L-alanine, dinitrophenyl-beta-alanine, and Ndelta-dinitrophenyl-Nalpha-acetyl-L-ornithine are not substrates, although Nepsilon-dinitrophenyl-L-lysine methyl ester is as effective as the ornithine derivative. Nonspecific lipases do not hydrolyze ornithine esters, nor does trypsin. The three ester bonds of fusarinine C are progressively hydrolyzed by the enzyme to eventually yield the monomer, fusarinine. The ferric chelate of fusarinine C is not hydrolyzed. An enzyme from Penicillium sp. was isolated with identical properties toward Nbeta-dinitro-phenyl-L-ornithine methyl ester as substrate. It also hydrolyzes N,N',N"-triacetylfusarinine C, a cyclic trihydroxamate containing Nalpha-acetylornithine ester bonds, which is produced by this organism. This substrate is hydrolyzed to Nalpha-acetylfusarine. In contrast to the Fusarium enzyme, this enzyme is fully active toward the ferric trihydroxamate chelate. However, replacement of iron by aluminum leads to a completely inactive substrate. Production of the enzyme is severely suppressed by iron in the growth medium. It is proposed that these specific ornithylesterases provide a mechanism of cellular iron release by hydrolysis of the ferric ionophores, and that  an iron-exchange step occurs prior to, and is a prerequisite for, hydrolysis of the ester bonds.


Methylation of adenosine in strongly alkaline medium: Preparation and Properties of o'-methyl derivatives of adenosine and N6-methyladenosine. In strongly alkaline aqueous medium, 9-substituted adenines, including adenine nucleosides, are relatively resistant to alkylation of the ring nitrogens and the exocyclic amino group. This fact was utilized to obtain the various possible Omicron'-methyl derivatives of adenosine by dimethyl sulfate treatment of the latter in alkaline medium, followed by separation of the products on a Dowex OH-column. In strongly alkaline aqueous dimethyl sulfoxide, several derivatives additionally methylated on the amino group were obtained, including N6,O2'-dimethyladenosine, a component located recently at the 5' terminus of animal cell and viral mRNAs. The latter was also prepared by diazomethane methylation of N6-methyladenosine in the presence of SnCl2. Alkylation in alkaline medium possesses the advantage that the products are not limited to those involving etherification of cis hydroxyls and is applicable to adenine nucleosides with sugar components other than ribose. The properties of the various O'-methylderivatives, including proton magnetic resonance data, are presented in detail.


Biosynthesis of bacterial menaquinones: the membrane-associated 1,4-dihydroxy-2-naphthoate octaprenyltransferase of Escherichia coli. It has been postulated that 1,4-dihydroxy-2-naphthoic acid is the naphthalenic intermediate in the biosynthesis of menaquinone (vitamin K2) in Escherichia coli to which the octaprenyl side chain is attached to from demethylmenaquinone. In the present work the presence of enzyme, 1,4-dihydroxy-2-naphthoate octaprenyltransferase, which catalyzes the conversion of 1,4-dihydroxy-2-naphthoate to demethylmenaquinone was demonstrated in cell extracts of E. coli. Demethylmenaquinone-9 was formed when the naphthoate was incubated with cell extracts and the synthetic substrate, solanesyl pyrophosphate, in the presence of Triton X-100. Solanesyl monophosphate could not substitute for the pyrophosphate in the reaction. The prenylation of of 1,4-dihydroxy-2-naphthoate was also studied in a strain of E. coli which accumulates octaprenyl pyrophosphate, the natural precursor of the menaquinone side chain. The octaprenyltransferase was shown to be membrane bound and to require magnesium ions for optimal activity. A menA-mutant of E. coli was found to lack the octaprenyltransferase activity, suggesting that the menA gene is the structural gene for this enzyme. However, this strain had normal levels of 4-hydroxybenzoate octaprenyltransferase, the enzyme catalyzing the analogous prenylation reaction in ubiquinone biosynthesis, providing additional evidence that the two octaprenyltransferases are quite distinct.


Microviscosity of the hydrocarbon region of the bovine retinal rod outer segment disk membrane determined by fluorescent probe measurements. The microviscosity of the hydrocarbon region of the bovine retinal rod outer segment disk membrane was determined by measuring the anisotropy of fluorescence from the  probe 1,6-diphenyl-1,3,5-hexatriene. The microviscosity ranged from 1.4 P at 40 degreeC to 15 P at O degree C, and no phase transition was observed in this temperature range. Bleaching of rhodopsin in the disk membrane produced no change in the microviscosity within the limits of error of our measurement (not equal to 5%). The presence of retinal in the disk membrane caused string quenching of the probe fluorescence. Removal of retinal from bleached disks produced a threefold increase in total fluorescence intensity and only a 10% drop in fluorescnece anisotropy, suggesting that large lateral gradients in microviscosity do not exist in the disk membrane. Bilayers prepared from extracted disk lipids had a microviscosity which was about one-fourth that of the intact disk membrane, demonstrating that rhodopsin hinders the mobility of the hydrocarbon chains of the disk phospholipids or egg phosphatidylcholine had identical microviscosities despite the much higher degree of unsaturation of the disk phospholipids.


Adduct of tyrosine and the oncogen 3-acetoxyxanthine. A major product of the reaction of L-tyrosine (I) with the activated oncogen 3-acetoxyxanthine (II) at neutral pH has been characterized as 8-xanthinyl-4'-O-tyrosine (III). A similar product is formed with tyrosylglycine. Another major product was the hydantoin of tyrosine. 8-Xanthinyl-4'-O-tyrosine is hydrolyzed in acid to yield tyrosine and uric acid but is stable in base. The synthesis of 8-phenoxyxanthine is also described. A metabolite identical with III in several chromatographic systems was found in the urine of two rats after administration of radioactive 3-hydroxyxanthine. This represented about 0.05% of the urinary radioactivity.


Rate of tritium labeling of specific purines in relation to nucleic acid and particularly transfer RNA conformation. The kinetics of the incorporation of tritium into the C-8 positions of purine units in nucleic acids has been studied. The polymers investigated include poly(A), poly(A): poly (U) duplex, a double-stranded viral RNA, tRNA, and DNA. In the random coil state, the kinetics of incorporation of tritium into the purine sites of the polymers are identical with those for the corresponding purine mononucleotides. When the nucleic acids are in their native conformations, however, the purine labeling rates are reduced below that expected for the free mononucleotides. The magnitude of the effect is remarkably dependent upon the particular nucleic acid. For example, at 37 degrees C the purines in double-stranded DNA label at a rate two- to threefold slower than the corresponding mononucleotides, but in a double-stranded viral RNA, a 30- to 40-fold effect is found. The data suggest a strong influence of microscopic helix structure on the rate of tritium incorporation. First-order rate constants for the exchange of tritium into specific purine sites in yeast tRNAPhe were also determined. This was done by partially labeling the nucleic acid in tritiated water, and subsequently removing free and loosely bound tritium. Under conditions where exchange-out does not occur, the nucleic acid was digested with specific nucleases; chromatographic separation then enabled specific activities of purines from specific sites to be obtained. The rate constants for these sites show a large variation. They are markedly reduced for those residues occurring in cloverleaf helical sections and, in certain cases, for those known from crystallographic data to be involved in tertiary interactions. As examples of bases that can participate in tertiary interactions, the crystal structures show A14 and G15 in special base-pairing arrangements. Both purines (A14 and G15) occur in single-stranded sections of the cloverleaf; both show markedly reduced C-8 hydrogen-exchange rates. On the other hand, rate constants for bases and regions known to be on the outside of the moleculesuch as the anticodon loop and the 3' terminusāre perturbed the least. In one instance, a base in the dihydrouridine loop believed to be involved in tertiary interactions, according to crystallographic studies, incorporates tritium as if it were relatively unperburbed by the tRNA structure. The structural interactions of this base may be partially or completely broken at 37 degrees C in solution.


A new mammalian DNA polymerase with 3' to 5' exonuclease activity: DNA polymerase delta. A new species of DNA polymerase has been purified more than 10 000-fold from the cytoplasm of erythroid hyperplastic bone marrow. This DNA polymerase, in contrast to previously described eukaryotic DNA polymerases, is associated with a very active 3' to 5' exonuclease activity. Similar to the 3' to 5' exonuclease activity associated with prokaryotic DNA polymerases, this enzyme catalyzes the removal of 3'-terminal nucleotides from DNA, as well as a template-dependent conversion of deoxyribonucleoside triphosphates to monophosphates. The exonuclease activity is not separable from the DNA polymerase activity by chromatography on DEAE-Sephadex or hydroxylapatite, and upon sucrose density gradient centrifugation the two activities cosediment at 7 S or at 11 S depending on the ionic strength. Both exonuclease and polymerase activities have identical rates of heat inactivation and both are equally sensitive to hemin and Rifamycin AF/013, inhibitors of DNA synthesis that act by binding to DNA polymerase and causing its dissociation from its template/primer. These results are consistent with the coexistence of two enzyme activities in a single protein.


Circular dichroic studies of the DNA and RNA of nucleoli. Circular dichroism (CD) in the 240-300-nm region was used to study the conformation of DNA and RNA complexed with proteins in isolated nucleoli form HeLa cells. Deoxyribonuclease or ribonuclease digestion was employed to obtain (1) the individual CD spectra of nucleolar DNA or RNA in complex form with proteins, or in free form; and (2) the experimental CD baseline correction to exclude contributions from nonnucleic acid sources such as light scattering  artifacts and proteins. The CD spectrum of nucleolar DNA in DNA-protein complexes was highly reduced in ellipticity in comparison with protein-free DNA. It showed a positive peak at 283 nm with a molar ellipticity [theta]283 = 1200 deg cm2 dmol-1 and a crossover at 262 nm. Addition of sodium dodecylsulfate shifted the peak to 276 nm with [theta]276 8000 deg cm2 dmol-1 and a crossover at 254 nm. The CD spectrum of nucleolar RNA in RNA-protein complexes was also reduced in comparison with protein-free RNA, showing a peak at 269 nm ([theta]269 = 6900 deg cm2 dmol-1), and a crossover at 250 nm. Addition of sodium dodecyl sulfate shifted the peak to 265 nm with [theta]265 = 18 000 deg cm2 dmol-1 and a crossover at 246 nm. The low ellipticity of both nucleolar DNA and RNA when complexed with proteins was increased by treatment with sodium chloride, urea, or heparin. This suggests that some ionic, hydrophobic, and hydrogen bondings are involved in the nucleic acid-protein interaction in nucleolar chromatin similar to that observed in nuclear chromatin.


NH2-terminal extensions on skin collagen from sheep with a genetic defect in conversion of procollagen into collagen. A modified form of procollagen was extracted with 10 M urea from the skin of lambs with dermatosparaxis, a disease which is produced by a genetic defect in the conversion of procollagen to collagen. The extracts contained little if any alpha1 and alpha2 chains of normal type I collagen, and instead they contained the larger polypeptides palpha1 and palpha2 together with high polymers. palpha1 was purified by ion-exchange chromatography and gel filtration. The polypeptide was shown to be related to alpha1 by its chromatographic behavior, its amino acid composition, and the peptides obtained after cleavage with cyanogen bromide. The molecular weight of palpha1 by gel filtration was 112 300 +/- 6300. After digestion of palpha1 with bacterial collagenase, a fragment of about 100 amino acid residues was obtained which was similar in amino acid composition and antigenic activity to a comparable fragment previously obtained from the NH2-terminal region of palpha1 chains from dermatosparaxic cattle. However, after cleavage of palpha1 with cyanogen bromide, a larger NH2-terminal fragment of about 160 amino acid residues was obtained. The larger cyanogen bromide fragment contained 8 residues of hydroxyproline, 12 residues of proline, and 19 residues of glycine not found in the NH2-terminal fragment isolated after digestion with bacterial collagenase. The results indicated that, in addition to containing amino acid sequences similar to those found in globular proteins, the peptide extensions on the NH2-terminal end of the palpha1 chain of procollagen also contain amino acid sequences similar to those found in the triple-helical region of the collagen molecule. The molecular weight of palpha2 by gel filtration was 102 400 +/- 6800. No additional peptide fragment was recovered after digestion of palpha2 with bacterial collagenase.


Utilisation of exogenous fatty acids by rat epididymal fat cells in vitro. When isolated rat fat cells are incubated with radioactively labelled palmitate the extracellular fatty acid pool is diluted by fatty acids of endogenous origin. Using fat cell concentration as an experimental variable, a simple graphical procedure has been devised to correct for the difficulty such precursor dilution causes in measurement of oxidation and esterification of exogenous fatty acids. After application of such corrections it was calculated that extracellular palmitate was utilised for oxidation with a composite Km of approx. 7 mu M. The composite Km for palmitate esterification was 29 mu M (no other substrates present, albumin included 1t 10 mg/ml).


Skin surface lipids of the guinea pig.

Skin surface lipids of the guinea pig. Skin surface lipids of the guinea pig were found to contain sterol esters (33%), wax diesters (diacyl alkanediols) (24%), glycerol ether diesters (28%), free fatty alcohols (6%) and free sterols (9%). The sterol esters and diacyl alkanediols contained saturated fatty acids (40 and 67%, respectively) having straight and singly-branched chains and mono-unsaturated acids (60 and 33%,respectively) derived predominantly by delta 9-desaturation of C15 and C16 straight-chain saturated fatty-acid precursors. The 1-O-alkylglycerols and fatty acids from the glycerol ether diesters were both entirely saturated series containing straight, branched and multi-branched chains. Both the free and the esterified sterols consisted principally of cholesterol with a small proportion of lathosterol.


Lipids of Nitrobacter and effects of cultural conditions on fatty acid composition. The nitrite-oxidizing autotroph, Nitrobacter was studied with respect to fatty acid composition and lipids. One fatty acid, shown to be cis-11-actadecenoic acid (cis-vaccenic) accounted for almost 96% of the total fatty acids of the extractable lipids of Nitrobacter agilis, Nitrobacter winogradskyi and each of several isolates from Minnesota and Moroccan soils studied. The cis-vaccenic acid was high in all organisms, ranging from 85 to 95% when grown at 27degreesC in the log growth phase, the other major acid was plamitic (16 : 1). All converted ces-vaccenic acid to a 19-carbon cyclopropanecarboxylic acid upon entering the stationary growth phase. The 11-carbon cyclopropanecarboxylic acid was not degraded when stationary phase cells were reinoculated into fresh medium. In N. agilis the levels of cis-vaccenic acid ranged from 86.8% when grown at 33degreesC to 95.6% when grown at 19degreesC. Addition of acetate or casein hydrolyzate to the inorganic medium had virtually no effect on the fatty acid composition of N. agilis, while propionate effected both qualitative and quantitative changes. In all organisms phosphatidylcholine made up a large portion of the extractable lipids. The distribution was phosphatidylcholine, 54%; phosphatidylethanolamine, 23%; phosphatidylglycerol, 10%; and neutral lipids, 11% for N. agilis.


Studies on nucleotide diphosphate diacylglycerol specificity of acidic phospholipid biosynthesis in rat liver subcellular fractions. (1)Cytidine diphosphate diacylglycerol, uridine diphosphate diacylglycerol, adenosine diphosphate diacylglycerol and guanosine diphosphate diacylglycerol were synthesized chemically and their purity assessed.  The acticity of these compounds in acidic phospholipid synthesis was examined in rat liver mitochondria and microsomes.  (2) Phosphatidylglycerol synthesis in rat liver mitochondria exhibited considerable activity with CDP diacylglycerol (v 7.0 nmol mg-1 h-1).  UDPdiacylglycerol (v 5.4) and ADP diacylglyc erol (v 4.2).  GDP diacylglycerol activity was detectable but very low.  (3) Diphophatidylglycerol formation in mitochondria and phosphatidylinositolsythesis in microsomes exhibited considerable more specificity for CDP diacylglycerol.  However, at high concentrations, measurable diphosphaticylglycerol and phophatidylinositol synthesis was observed with the other liponucleotides.  (4) Although considerable phosphatidylglycerol formation was observed with UDPdiacaylglycerol and ADP diacylglycerol, it is unlikelky that these compounds are of physiologic importance, at least in rat liver, since CTP:phophatidic acid cytidyltransferase in microsomes and mitochondria was shown to be specific for cytidine triphosphate.  The lack of specificity of  phosphatiidylglycerol synthesis for CDP diacylglycerol is currently unexplained but may be of some importance in other tissues or in other organisms.


Sphingolipid base metabolism. Sphinganine-1-phosphate lyase: identification of ethanolamine 1-phosphate as product. Ethanolamine 1-phosphate has been characterized as a product of the action of rat liver microsomal sphinganine 1-phosphate lyase on erythro-sphinganine 1-phosphate. The product was characterized by various forms of chromatography, gas-liquid chromatography-mass spectral analysis of appropriate derivatives, and by conversion to ethanolamine. The results of detailed studies of the mass spectral fragmentation of the tetra-trimethylsilyl derivative of ethanolamine 1-phosphate are also reported.


Effect of glutathione peroxidase activity on lipid peroxidation in biological membranes. Results are presented indicating that, although glutathione peroxidase activity inhibits lipid peroxidation in membranes, it does not appear to do so by reducing membrane lipid peroxides to lipid alcohols, as has been shown by others to be the case for free fatty acid peroxides in solution. Lipid peroxidation was studied in an enzymic system (microsomal NADPH oxidase) and in a non-enzymic system (mitochondria plus ascorbate). A study of the fatty acids in the phospholipids of microsomes and mitochondria demonstrated that detectable amounts of hydroxy fatty acids were not formed in the membranes when the latter were incubated in the presence of the glutathione peroxidase system even under conditions known to have generated significant levels of lipid peroxides in the membrane. Fatty acid analyses of the microsomal and mitochondrial particles indicated that glutathione peroxidase activity inhibited loss of polyunsaturated fatty acids when these organelles were exposed to peroxidizing conditions. If glutathione peroxidase activity were inhibiting the formation of malondialdehyde (a product of lipid peroxidation) by converting peroxide groups to alcohols, the loss of the constitutive polyunsaturated fatty acids in the membrane should not have been appreciably affected by addition of the peroxidase system. The protective effect cannot be due to quenching of an autocatalytic type of lipid peroxidation (at least in the microsomal system) since it has been established that the microsomal enzyme system (NADPH oxidase) catalyzes a continuous attack on microsomal polyunsaturated fatty acyl groups during the reaction and that the peroxidative process is not autocatalytic in nature. It appears, therefore, that glutathione peroxidase activity must exert its effect on this system by preventing free radical attack on the polyunsaturated membrane lipids in the first place. A possible mechanism for the interruption of a free radical attack on the lipids is proposed.


The mechanism for nonspecific lipase from rat pancreas. Purified nonspecific lipase from rat pancreas appears to mediate the followin reaction sequence: (2) HE +nB in equilibrium HEBn; (2) HEBn + S inequilibrium HEBnS; (3) HEBnS IN EQUILIBRIUM EBnS' + ROH; (4) EBnS' + h2o vector HEBn + R'COO- + H+, where E equils enzyme, B equils bile salt, and S equils R'COOR. Evidence is presented for the occurrence, sequence and reversibility or irreversibility of the above four reactions. Also discussed are the activation energies for both a "good" and a "poor" substrate, the correlation between V and subsitituent function (o-) for a series of substituted benzoic acid esters, and the effects of steric hindrance. Studies of the incorporation of label from H2(18)O further detail the hydrolytic mechanism, and the effects of the physical state in which substrate is presented to the enzyme (micelles, emulsion, crude bulk phase, etc.) are discussed.


Interaction of concanavalin A with membrane-bound and solubilized lipoprotein lipase of rat heart. Concanavalin A was used to study the configuration of lipoprotein lipase at the surface of capillary endothelium. Incubation of heart homogenates with increasing concentrations of concanavalin A for 5-60 min resulted in inhibition of up to 50% of enzyme activity. The inhibition was related to the concentration of lectin and the time of incubation and was fully reversible by postincubation with alpha-methyl-D-mannoside. Rat hearts were perfused for 5-60 min and lipoprotein lipase activity determined in postheparin perfusates and in the perfused heart. When the lectin was introduced into the perfusate a significant reduction of heparin-releasable enzyme was found after 30 min of perfusion. The missing enzyme could be recovered by postperfusion with alpha-methyl-D-mannoside, but not by addition of the sugar to the perfusate withdrawn from the apparatus. These results suggested binding of lectin to the surface-located enzyme and support for such a binding was obtained by the finding of release of labeled lectin into the perfusate by heparin. Perfusion of hearts with concanavalin A for 60 min resulted also in a fall in nonreleasable lipoprotein lipase. The mechanism of this fall is not due to impairment of enzyme synthesis, as leucine incorporation into protein was not reduced. Since neither perfusion nor postincubation with alpha-methyl-D-mannoside restored enzyme activity, the fall was most probably due to irreversible inhibition. It is concluded that mannose residues of lipoprotein lipase in heart homogenates and at the endothelial surface of heart capillaries are available to interact with a specific lectin. Such an interaction renders the enzyme less releasable by heparin during perfusion and causes a significant inhibition of enzyme activity in homogenates.


Lipoprotein lipase and uptake of chylomicron triacylglycerol and cholesterol by perfused rat mammary tissue. The role of lipoprotein lipase in the uptake of chylomicron triacylglycerol and cholesterol from blood was studied in perfused inguinal-abdominal mammary tissue of rats lactating 10-15 days.  Lipoprotein lipase activity in the tissue was reduced, from 0.47 to 0.10 units/g, by removing the anterior pituitary gland from lactating rats 2 days before the experiment. Perfused mammary tissue of normal lactating rats took up 12% of the chylomicron triacylglycerol infused, whereas the tissue of hypophysectomized lactating rats took up less than 1%. About two-thirds of the triacylglycerol taken up was retained as glyceride, and the rest was hydrolyzed and released to the perfusing fluid as fatty acids and glycerol. Autoradiographic studies of perfused tissues of normal lactating rats showed that both the acyl and glycerol moieties derived from chylomicron triacylglycerol were incorporated into milk lipid droplets. Perfused mammary tissue of normal lactating rats also took up 15% of the chylomicron cholesterol infused, whereas the tissue of hypophysectomized lactating rats took up less than 1%. The findings demonstrate that chylomicron cholesterol is taken up with triacylglycerol by lactating mammary tissue, and that uptake of both lipids is markedly suppressed when lipoprotein lipase activity is low, as in tissue of hypophysectomized rats. It is proposed that uptake of triacylglycerol from chylomicrons by mammary tissue requires the action of lipoprotein lipase, while uptake of cholesterol is dependent on reduction of the triacylglycerol core, resulting from action of the enzyme on the core and uptake of lipolytic products by the tissue.


Hydrolysis of chylomicron phosphatidylcholine in vitro by lipoprotein lipase, phospholipase A2 and phospholipase C. The effects of lipoprotein lipase, phospholipase A2 and phospholipase C on chylomicron phosphatidylcholine and triacylglycerol were studied with rat lymph chylomicrons containing phosphatidylcholine labeled with [14C]oleic acid. Lipoprotein lipase purified from bovine milk readily hydrolyzed chylomicron phosphatidylcholine to lysophosphatidylcholine and fatty acid, and triacylglycerol to monoacylglycerol, fatty acid and glycerol. The rates of hydrolysis of phosphatidylcholine and triacylglycerol increased with enzyme concentration, and both decreased when fatty-acid binding sites on albumin in the incubation medium were limited. The proportion and amount of phosphatidylcholine hydrolyzed was always less than that of triacylglycerol. Analyses of hydrolytic products showed that lipoprotein lipase cleaved the 1-acyl ester bond of phosphatidylcholine. The findings indicate that lipoprotein lipase can account for some of the phospholipase A1 activity found in postheparin plasma. Phospholipase A2 and phospholipase C hydrolyzed chylomicron phosphatidylcholine, greater than 92% in 10 min, but not triacylglycerol. The resultant phosphatidylcholine-deficient chylomicrons, which could be concentrated by ultra-centrifugation and resuspended in incubation medium, were readily depleted of triacylglycerol when incubated with lipoprotein lipase. The findings indicate that phosphatidylcholine can be removed from the surface film of chylomicrons without disrupting the particles or blocking the action of lipoprotein lipase on the core triacylglycerol.


Studies on the glycolipids of sheep thymus and of normal and concanavalin A-stimulated sheep peripheral lymphocytes. The neutral glycolipids and gangliosides of sheep thymus and of sheep peripheral lymphocytes were compared. The patterns of both of these major classes of glycolipids were more complex in thymus than in the lymphocytes. The incorporation of radioactivity from D-[1-14C]galactose into the individual glycolipids of control and concanavalin A-stimulated sheep peripheral lymphocytes was also studied. A marked enhancement of incorporation into trihexosylceramide and an alteration of the pattern of incorporation into gangliosides were noted in the mitogen-treated cells. The results suggest that significant alterations of glycolipid composition and metabolism may occur during at least certain stages of lymphocyte differentiation.


Specific reductive metabolism of steroid sulphates in rat liver. The reductive metabolism of steroid sulphates and corresponding unconjugated steroids has been investigated in male and female rat liver. Enzyme activities studied were 5alpha-reductase, 5beta-reductase and 3alpha-, 3beta- and 20beta-hydroxy-steroid oxidoreductases in microsomal and soluble cellular fractions. With the exception of microsomal 3alpha-reductase all enzymes were equally or more active using sulphoconjugated substrates than using corresponding unconjugated substrates. 20beta-reduction of corticosterone and deoxycorticosterone occurred only with the 21-sulphurylated derivatives. This sulphate-specific 20beta-reductase, which is only present in male rat liver, probably participates in the formation of the sex-specific 20beta-reduced corticosterone metabolites present in bile from male but not from female rats. Sulphoconjugated deoxycorticosterone and corticosterone were also better substrates for microsomal 5alpha-reductase and sulphoconjugated 5alpha-dihydrotestosterone for microsomal 3beta-hydroxysteroid oxidoreductase than the corresponding unconjugated analogues. In conclusion, these results indicate that steroid sulphates generally are good substrates for hepatic reducing enzymes; in many cases even better substrates than the unconjugated analogues.


On the formation of thromboxane B2 and 12l-hydroxy-5,8,10,14-eicosatetraenoic acid (12 ho-20:4) in tissues from the guinea pig. Homogenates of tissues from the guinea pig were incubated at 37 degrees C for 30 min and the amounts of thromboxane B2 and 12L-hydroxy-5,8,10,14-eicostetraenoic acid (12ho-2-:4) formed from endogenous arachidonic acid were determined by multiple-ion analysis. Lung and spleen homogenates produced 7146 +/- 500 ng/g and 3046 +/- 542 ng/g, respectively, of thromboxane B2 and 15 122 +/- 3536 ng/g and 29 516 +/- 1966 ng/g, respectively, of 12ho-20:4. The amounts of both compounds in homogenates of kidney, stomach, liver, heart and whole blood were at least 10-times lower than those found in lung and spleen. 5,8,11,14-Eicostetraynoic acid and nordihydroguaiaretic acid inhibited 12ho-20:4 formation in spleen homogenates whereas indomethacin and arachidonic acid stimulated it.


Purification and properties of the RNA polymerase of an extremely thermophilic bacterium: Thermus aquaticus T2. The RNA polymerase of the extremely thermophilic bacterium Thermus aquaticus T 2 has been purified to homogeneity. The apparent molecular weights of the subunits of the enzyme are : 165 000, 130000, 92 000 and 44 000. The in vitro temperature optimum of enzyme activity is around 65 degrees C. The enzyme has a preference towards the homologous template and is strongly inhibited by KCI. Rifampicin inhibits the enzyme only to 50% even at very high concentrations. Heparin inhibits it completely, but only at higher molar excess than in the case of the Escherichia coli enzyme. The enzyme can form heparin-resistant complexes at elevated temperatures on the homologous template, but not on E. coli DNA.


Dissociation and reconstitution of the DNA replicase system of HeLa cell nuclei. The DNA replication system of S-phase HeLa nuclei has been dissociated by cautious extraction at 0 degrees C with 0.25 M NaCl. Replicase activity has been reestablished by recombination of the fractions and reduction of the salt concentration. The reconstituted system, like the starting nuclei, depended on ATP, 4dNTP, MgCl2, the proper ionic strength and the soluble cytoplasmic protein fraction. The activity of the nuclear extract showed a cell cycle dependency and was elevated in the nuclei of cells at the G1 leads to S boundary. In the presence of Mg2+ the major activity of the nuclear extract precipitated during dialysis to reduce the salt concentration; this precipitate exhibited DNA polymerase alpha activity. Chromatography of the active extracts over phosphocellulose separated the replicase supporting factors into three fractions. The major activity eluted in the fraction containing the DNA polymerase alpha activity; the other two active fractions were devoid of polymerase activity. The fraction containing DNA polymerase alpha from the nuclear extracts supported DNA replicase activity in salt-extracted nuclei whereas an equivalent level of DNA polymerase alpha from the cytoplasm was not effective. The data suggest that the DNA polymerase alpha of the salt extracts of S-phase nuclei is either different than the cytoplasmic enzyme or is associated with some essential replicase-supporting factor.


Arthropod ribosomes. Integrity of ribosomal ribonucleic acids from aphids and water fleas. The 28 S aphid rRNA differs from those of the other insects in two points: (1) it lacks the primary nick; (2) it is larger by 0.2 - 106 daltons. The 28 S rRNA from water fleas also, like that from crayfish, is larger than those from comon insects by 0.2 - 106 daltons. These crustacean 28-S rRNAs were shown to contain the primary nick, which is probably not located in the central point. The 18-S rRNAs from aphids and water fleas had molecular weights significantly larger than 0.7 - 106, the common vaalue for the eukaryotic 18 S rRNA. It was was suggested that the 3'-terminal base sequences of these RNAs are different from the common sequences of these RNAs are different from the common sequence proposed for the 18 S rRNAA of eukaryotes. These exceptional characteristics of the rRNAs from the parthenogenetic animals may provide a probe for general functions of the rRNA in the eukaryotic ribosomes.


Molecular orbital calculations on the conformation of phosphodiesters. An extended correlation between the geometry and the conformation of the phosphate group. An extension of our previous correlation (C.R. Acad. Sci. Paris, Ser. D (1973) 277, 2257; Biochim. Biophys. Acta (1974) 340, 299) between the geometry of the phosphate group and the conformation of phosphodiesters established by PCILO computations, shows that the probability of gauche-trans or trans-gauche conformations should become appreciable for low values (100-101) of the 33'-P-O5', angle and may even become predominant when the C-O-P angles have also a low value (congruent to 117 degrees). The results agree satisfactorily with available X-ray crystallographic data.


Studies on adenine and adenosine metabolism by intact human erythrocytes using high performance liquid chromatography. Adenine and adenosine metabolism has been studied in intact human erythrocytes in vitro using high performance liquid chromatography, isotopic labeling and electrophoresis. Their metabolism to nucleotides was controlled by phosphoribose diphosphate synthesis which was phosphate dependent. Adenosine formed hypoxanthine or IMP depending upon Pi concentration, but adenosine kinase and deaminase activities were not affected by P levels. Free [14C]adenine and [14C]hypoxanthine were found in cellular extracts. Rapid interconversions occurred to give a distribution for ATP : ADP : AMP of 10 : 1 : 0.1. Marked decomposition of ATP to ADP and AMP occurred during incubations in plasma and Earle's media in air on nitrogen, but ATP levels remained stable in phosphate buffers and in the presence of oxygen. At physiological Pi (1 mM) adenosine kinase activity grossly exceeded adenine phosphoribosyltransferase activity. The latter was approximately 7 fold that of hypoxanthine phosphoribosyltransferase activity. These differences decreased with increasing Pi levels. No significant increase in corresponding nucleotides was obtained by incubation with high levels (0.5 mM) of adenine, guanine or guanosine at physiological Ii, ATP increased by 10% independently of the substrate employed and significant amounts of IMP and GTP were formed adenosine and guanosine, respectively. The existence of a bound intracellular pool of ATP is suggested.


Interaction between dermatan sulphate chains. I. Affinity chromatography of copolymeric galactosaminioglycans on dermatan sulphate-substituted agarose. (1) Binding of copolymeric as well as homopolymeric galactosaminoblycan to dermatan sulphate-substituted gels has been demonstrated. Materials bound in the presence of 0.15 M NaC1 was eluted with either 1 M urea, 0.5 M guanidine - HC1 or 0.5 M NaC1. Homopolymeric galactosaminoglycans were also displaced by 0.5 M sodium acetate. The interaction was not dependent on divalent cations. (2) Dermatan sulphate has been fractionated into aggregating and nonaggregating species by gel chromatography in the presence of 0.5 M sodium acetate. In the presence of 3.1 M sodium acetate or 0.5 M guanidine - HC1 no aggregation was observed. (3) Crosslinks formed during periodate oxidation at physiological ionic strength have been ascribed to chain-chain interaction. (4) Chondroitin 4-sulphate, heparan sulphate and heparin also showed interaction with gels substituted with copolymeric galactosaminoglycans, while chondroitin 6-sulphate, hyaluronate and keratan sulphate did not. (5) Binding of copolymeric galactosaminoglycan chains to dermatan sulphate- or chondroitin sulphate-substituted gels was most pronounced when the copolymeric chains similar proportions of L-iduronic and D-glucuronic acid.


Desulphation of heparin by mice and guinea pig leukocytes. Leukocytes from mice and guinea pigs were tested for their sulphate-splitting activity on heparin. Mouse macrophages showed the highest degrading activity while mouse neutrophils and lymphocytes showed only a week degrading activity. Mouse macrophages maintained in tissue culture were also found to degrade heparin, the amount of sulphate released increasing with time up to 96 h. Spleen extracts were found to neutralize the anticoagulatory activity of heparin.


Intracellular localization of arginase in chick kidney. Although chickens are uricotelic and do not have significant urea-ornithine cycle in any tissue, the kidneys contain a high concentration of arginase which apparently functions to regulate degradation of dietary arginine. A series of investigations has been made to determine the intracellular localization of this arginase in chicken kidney. Tissue fractionation using sucrose density gradients and differential centrifugation showed as association of arginase activity with certain marker enzymes and with fractions identified as mitochondria by electron microscopy. This is consistent with the localization of the arginase in the mitochondrial matrix of chicken kidney cells. Such a finding has significance in understanding the regulation of arginine degradation in chickens.


The metabolism of l-[3-3h]lactate by isolated hamster liver cells. The rate of tritium removal from L-[3-3H]lactate by hamster liver cells is faster than the analytical rate of lactate utilization, or the rate of 14C disappearance from 6-[U-14C, 3-3H]Llactate decrease. However, addition of low concentrations (0.1 to 1.0 mM) of L-cycloserine, a glutamate pyruvate transaminase inhibitor, nearly equalizes the rates of isotope utilization from L-[3-3H]lactate and L-[U-14C]lactate. The results suggest a very limited rate of recycling of phosphoenolpyruvate back to pyruvate during gluconeogenesis from lactate in fasted hamster liver cells.


Occurrence of free creatine, phosphocreatine and creatine phosphokinase in adipose tissue. We evaluated brown and white adipose tissues for the presence of creatine, phosphocreatine and creatine phosphokinase activity. In rats 3.6 and 0.4 mumol of total creatine were found per g wet weight of brown and white adipose tissues, respectively. We were able to identify creatine by thin-layer chromatography after a pulse label of [14C]creatine had been given in vivo. Free creatine and phosphocreatine were shown to occur by column chromatography. Of the total creatine of brown adipose tissue, approximately one third to one half were attributable to phosphocreatine. The activity of creatine phosphokinase was demonstrated in both white and brown adipose tissue, the values of the latter prevailing over those of the former by a factor of 200, if based on wet weight, or 50, if expressed as specific enzyme activity. The labeling of total creatine in vivo proceeded much faster in adipose tissue than in skeletal muscle. The results strongly suggest that the energy metabolism of adipose tissue is closely dependent on the presence of creatine. The specific activities of free creatine and phosphocreatine of brown adipose tissue differed strikingly as long as 24 h after radioactive creatine was injected; this difference points to a metabolic or structural compartmentation of creatine.


Immunological properties of nerve growth factors. Antisera were raised against nerve growth factors isolated from mouse salivary gland and five venoms representative of the three main families of poisonous snakes. Immunochemical cross-reactivity was assessed from the ability of the antisera to inhibit the biological activities of the heterologous antigens. The high and low molecular weight forms of the salivary gland factors were found to be immunologically identical but distinct from the snake venom factors. The snake venom factors, on the other hand, were immunologically closely related to each other but not identical.


On the sol-gel transition insolutions of kappa-carrageenan. The disorder-order transition, which takes place at the gelpoint of k-carrageenan solutions was monitored by optical rotation and light scattering measurements. The coincidence of both sets of experimental data affords good evidence that the sol-gel transition is accompaned by a conformational change. Transition temperatures were observed to be linearly dependent on the logarthm of the salt concentration and this result is explained by the formation of double helices. Heats of gelation were measured by differential scanning calorimetry. It was found that the enthalpy increases with ionic strength, which was ascribed to the occurrence of a secondary process in which double helices are assembled into larger aggregates.


Effect of the CO-2-bicarbonate buffer system on the water and ion contents of rat renal cortical slices. (1) Partial replacement of medium chloride by bicarbonate, with pHmaintained constant by addition of CO2, lead to a greater rate of swelling when rat renal corti cal slices were leached in nitrogenated medium at 0--2 degree C. This greater rate of swelling was still present when 1 mM iodoacetate was added to the leaching media. (2) Rat renal cortical slices incubated in oxygenated bicarbonate medium at 25 degree C maintained a slightly greater tissue water content than did slices incubated in phosphate-buffered media. Inclusion of malate in the incubation medium also led to a greater steady-state water content when slices were incubated at 25 degree C in oxygenated media. These results suggest that that particular cell volume which is maintained by metabolism and the double Donnan system is determined at least in part by the nature of the extracellular anions. (3) The rates of net sodium extrusion and of net potassium uptake were not significantly altered by presence of CI2 and bicarbonate in the incubation medium.


Iron dynamics in the ruminant.

Iron dynamics in the ruminant. We have compared the plasma clearance rate of radioactive iron in cows both as ferric chloride and as iron specifically bound to transferrin. We have also repeated the transfusion experiment of Dern et al. (Dern, R.J., Monti A. and Glynn, M.F. (1963) J. Lab. Clin. Med. 61,280-291) using goats. The results show that neither non-specificity bound iron (Bates, F.W. and Schlabach, M.R. (1973) J. Biol. Chem. 248, 3228-3232) nor the iron bound to the two different sites in transferrin (Awai, M., Chipman, B. and Brown, E.B. (1975) J. Lab. Clin. Med. 85,769-784) can be identified as distinguishable iron pools by this technique.


Formation in vitro of ascorbic acid 2-sulfate. The sulfation of ascorbic acid by an ascorbic acid sulphotransferase was investigated using rat liver and colon homogenates. When Na2 35 SO4 or 3'-phosphoadenylyl [35S]sulfate (P-Ado-P-35S) and ascorbic acid were used as substrates, chromatographic behavior of the reaction products on thin-layer cellulose suggested that ascorbic acid 2-[35S]sulfate was formed. With Na2 35SO4 as the source of radioactive sulfate in the assay system, ATP was found to be an obligatory cofactor. Incorporation of [35S]sulfate frofrom Na2 35SO4 into ascorbic acid 2-[35S]sulfate was also decreased when ATP sulfurylase inhibitors were added to the system. P-Ado-O35S alone in the assay without ATP was an extemely effective sulfating agent. In addition, liver and colon homogenates from vitamin A deficient and sufficient rats were used in one of the studies. Vitamin A deficiency appeared to have little effect on ascorbic acid 2-sulfate formation.


The actions of avenaciolide and ethanol on glucose metabolism and on related enzyme activities in the isolated perfused rat liver. 1. The metabolic response of livers to perfusion with ethanol with and without avenaciolide, has been followed by measuring the perfusate levels of glucose, lactate, pyruvate, beta-hydroxybutyrate, ethanol, amino acids, urea and lipid. 2. Analysis of the perfused livers showed changes in the activities of some of the key enzymes of glycolysis, gluconeogenesis and lipogenesis. Ethanol perfusion decreased the levels of phosphofructokinase, glucokinase and cytosolic isocitrate dehydrogenase, while avenaciolide lowered pyruvate carboxylase and phosphoenolpyruvate carboxykinase as well as glucokinase. Isocitrate dehydrogenase and phosphofructokinase were unchanged, but the ionophore increased the level of fructose-1,6-diphosphatase. Ethanol plus avenaciolide showed the same pattern as ethanol alone, together with the decrease in phosphoenolpyruvate carboxykinase found with avenaciolide. 3. Neither ethanol nor avenaciolide had any effect on kexokinase, pyruvate kinase or acetyl-CoA carboxylase. There were small changes in glucose-6-phosphatase and malic enzyme, and a tendency for citrate lyase levels to decline in avenaciolide perfusions.