bluuebunny/crossref_metadata_2025 huggingface dataset:
Factors controlling aminoacyl-transfer-ribonucleic acid synthesis in vitro by a plant system
Authors: Tao K. L. , Hall T. C.
Published: August 2015

1. Factors affecting aminoacyl-tRNA synthesis in vitro by cell-free preparations from bean leaves were investigated. 2. Evidence was obtained that optimum concentrations as well as correct ratios of Mg2+ and ATP are required for aminoacyl-tRNA synthesis in the bean-leaf system. 3. The results indicated that pH is a controlling factor having differential effects on the formation of individual aminoacyl-tRNA species. The possible micro-regulatory function of pH in protein synthesis in vivo is discussed with special reference to alanyl-tRNA formation. 4. Very low rates of alanine-stimulated pyrophosphate exchange were observed in the absence of tRNA. This observation is discussed relative to proposals about the mechanism of aminoacyl-tRNA synthesis.
Micro method for determination of reactive carbonyl groups in proteins and peptides, using 2,4-dinitrophenylhydrazine
Authors: Fields Robert , Dixon Henry B. F.
Published: August 2015

A method is described for determining carbonyl groups that is especially suitable for use with proteins and peptides. It involves the determination of the extinction at 370nm of a sample solution after adding 2,4-dinitrophenylhydrazine. The reaction of 2,4-dinitrophenylhydrazine with pyruvoylglycine and with transaminated ribonuclease T1 is presented; the isolation of protein hydrazones is discussed.
Nucleic acid enzymology of extremely halophilic bacteria. Gel-filtration and density-gradient-centrifugation studies of the molecular weights of Halobacterium cutirubrum polynucleotide phosphorylase and deoxyribonucleic acid- and ribonucleic acid-dependent ribonucleic acid polymerases
Authors: Louis B. Gregory , Peterkin Pearl I. , Fitt P. S.
Published: August 2015

1. Conditions have been established for the estimation of molecular weights of proteins by analytical gel filtration and sucrose-density-gradient centrifugation in 2.5m-potassium chloride–1m-sodium chloride; Halobacterium cutirubrum polynucleotide phosphorylase, DNA-dependent RNA polymerase and RNA-dependent RNA polymerase have been studied by these methods. 2. The RNA-dependent polymerase has also been studied by density-gradient centrifugation in the absence of salt. 3. All three proteins are of unusually low molecular weight compared with similar enzymes from non-halophilic bacteria.
Extent of propionate metabolism during absorption from the bovine ruminoreticulum
Authors: Weigand E , Young J W. , McGilliard A. D
Published: August 2015

1. Solutions containing acetate, [2-14C]propionate and butyrate were placed into the ruminoreticulum of calves to measure the extent to which propionate is metabolized by ruminoreticulum epithelium. In response to five different combinations of pH and total volatile fatty acid concentrations, propionate absorption rates ranged from 89 to 341mmol/h. 2. The extent of propionate conversion into lactate, calculated from both concentration and specific radioactivity in portal and arterial blood, averaged 4.9 (range 2.5–9.1)%. 3. Circulating glucose synthesized from propionate had a higher specific radioactivity than arterial lactate and was converted into lactate by gastrointestinal tissues. Thus conversion of propionate into lactate was overestimated but was corrected to average 2.3 (1.0–4.6)%. 4. The estimates of propionate conversion into lactate were negatively correlated with its rate of absorption.
Effects of testosterone on the metabolism of folate coenzymes in the rat
Authors: Rovinetti C. , Bovina C. , Tolomelli B. , Marchetti M.
Published: August 2015

1. The effects of castration and testosterone treatment on enzymic activities involved in folate coenzyme metabolism in the liver and in accessory sex organs of male adult rats were studied. 2. In the liver of castrated rats the concentration of 10-formyltetrahydrofolate (10-HCO-H4folate) synthetase and tetrahydrofolate (H4folate) dehydrogenase were significantly decreased whereas that of 5,10-methylenetetrahydrofolate dehydrogenase increased; the treatment with five doses of testosterone caused a return to normal values of these activities. 3. In the prostate of castrated rats a pronounced decrease in H4folate dehydrogenase, serine hydroxymethyltransferase and 10-HCO-H4folate synthetase activities was observed. The administration of testosterone restored the enzymic activities to normal values. 4. In the seminal vesicles of castrated rats only 10-HCO-H4folate synthetase was markedly depressed; testosterone treatment not only restored activity to normal values but raised it to higher than normal values. The slight changes observed in other enzymic activities also returned to normal values with the hormone treatment. 5. These results are discussed in relation to a possible control mechanism of folate metabolism by testosterone.
The immediate effects of insulin and fructose on the metabolism of the perfused liver. Changes in lipoprotein secretion, fatty acid oxidation and esterification, lipogenesis and carbohydrate metabolism
Authors: Topping D. L. , Mayes P. A.
Published: August 2015

1. When livers from fed rats were perfused with blood containing elevated concentrations of rat insulin or blood to which fructose was added, the oxidation of free fatty acids was depressed and their esterification was increased. 2. Raised concentrations of insulin or addition of fructose increased secretion of triglyceride in very-low-density lipoproteins, but only insulin caused more of the free fatty acids taken up by the liver to be incorporated into very-low-density lipoproteins. 3. When insulin and fructose were added together the combined effect on oxidation and esterification of free fatty acids and on secretion of very-low-density lipoproteins was equal to the sum of the effects of either alone. No statistically significant interaction between the effects of fructose and insulin was found for any of the parameters investigated. 4. Bovine insulin had similar effects, in most respects, to comparable studies with raised concentrations of rat insulin. 5. Lipogenesis was increased in the livers treated with fructose plus bovine insulin. 6. A significant proportion of the fatty acids in very-low-density lipoproteins were derived either from the liver triglyceride pool or from lipogenesis. This fraction was increased both by treatment with insulin or fructose, and was augmented further when both insulin and fructose were present together. 7. The uptake of fructose by the perfused liver was similar to that found in vivo. It was unaffected by the presence of insulin. 8. Addition of fructose to the perfused liver caused perfusate lactate concentrations to increase, as a result of diminished hepatic uptake of lactate. 9. The uptake of free fatty acids by the perfused liver was unaffected by the addition of either insulin or fructose. 10. The distribution among the various lipid classes in plasma lipoproteins of label arising from the hepatic uptake of [14C]oleate was unaltered by the addition of either fructose or insulin. 11. It is suggested that the effects described are due principally to control of the balance between esterification of fatty acids and lipolysis of the ensuing triglyceride, fructose enhancing esterification and insulin inhibiting lipolysis.
The biosynthesis in vitro of chondroitin sulphate in neonatal rat epiphysial cartilage
Authors: Handley C. J. , Phelps C. F.
Published: August 2015

1. A system is described, which was used to incubate neonatal rat epiphysial cartilage in vitro with [U-14C]glucose and [35S]sulphate. 2. The acid glycosaminoglycans of neonatal rat epiphyses were extracted and fractionated on cetylpyridinium chloride–cellulose columns. The major components were chondroitin 4-sulphate (65%), chondroitin 6-sulphate (15%), hyaluronic acid (4%) and keratan sulphate (2%). 3. The acid-soluble nucleotides and intermediates of glycosaminoglycan synthesis were separated on a Dowex 1 (formate) system. The tissue contents and cellular concentrations of these metabolites were determined. 4. The rates of synthesis of UDP-glucuronic acid and UDP-N-acetyl-hexosamine from [U-14C]glucose were found to be 0.79±0.16 and 3.2±0.08nmol/min per g wet wt. respectively. 5. The incorporation of [U-14C]glucose into the uronic acid and hexosamine moieties of the polymers was also measured and the turnover rates of the glycosaminoglycans were calculated. It was found that chondroitin sulphate was turning over in about 70h and hyaluronic acid in about 120h. 6. The relative rates of synthesis of the sulphated glycosaminoglycans were calculated from [35S]sulphate incorporation and were found to be in good agreement with those obtained from [U-14C]glucose labelling.
Studies on the mode of uptake of blood triglycerides by the mammary gland of the lactating goat. The uptake and incorporation into milk fat and mammary lymph of labelled glycerol, fatty acids and triglycerides
Authors: West C. E. , Bickerstaffe R. , Annison E. F. , Linzell J. L.
Published: August 2015

1. The mode of uptake of the precursors of milk fat by the mammary gland of the lactating goat has been examined by infusing radioactive fatty acids, glucerol or doubly labelled triglycerides into the mammary artery or jugular vein of animals surgically prepared to permit samples of arterial and venous blood to be withdrawn without disturbance to the animal. 2. Acetate was taken up by the mammary gland and incorporated into milk fat. The decrease in the specific radioactivity of blood acetate across the gland was evidence of acetate production, but there was no significant release of labelled lipid from the mammary gland. 3. When labelled long-chain fatty acids or glycerol were infused into the lactating goat, there was extensive transfer of radioactivity into milk in spite of the absence of net uptake of substrate by the mammary gland. The decrease in the specific radioactivity of each substrate across the mammary gland, however, showed that both fatty acids and glycerol were simultaneously taken up and released by mammary tissue. 4. The infusion of chylomicra and triglyceride emulsions labelled with3H and14C revealed that both glycerol and fatty acids were released during triglyceride uptake by mammary tissue. Changes in the3H/14C ratio during the transfer of triglyceride from blood into milk showed that at least 80% of the triglyceride was hydrolysed during uptake, but the potential re-utilization of both products of hydrolysis for triglyceride synthesis in mammary tissue implied that only a minimum value could be obtained from the change in the ratio. 5. The time-course of the transfer of3H and14C into milk and lymph were closely similar after the infusion of [2-3H]glycerol tri[1-14C]oleate or of a mixture of [2-3H]glycerol and [1-14C]oleate. 6. The results were consistent with the hypothesis that plasma triglycerides are extensively or completely hydrolysed during mammary uptake.
Changes in the protein–polysaccharides of pig articular cartilage during prenatal life, development and old age
Authors: Šimůnek Z. , Muir H.
Published: August 2015

Analysis of the knee-joint cartilage of pigs at five ages (namely foetuses from the second half of pregnancy and animals 10 weeks, 25 weeks, 3 years and 5 years old) showed that the composition approached that of adult cartilage by 25 weeks of age, the most marked differences being between foetal and 10 week-old cartilage. Protein–polysaccharides were extracted sequentially, first by brief low-speed homogenization with iso-osmotic sodium acetate, then by two extractions with 2m-CaCl2 for 24h with gentle agitation interspersed with brief low-speed homogenization and agitation for another 24h. About half of the protein–polysaccharides were removed from foetal cartilage by the first extraction and the remainder by the second. The proportion in the first extract declined sharply with the age of the animal, but that in the first CaCl2 extract was similar at all ages other than 10 weeks. The amount left in the residue increased approximately with the collagen content from about one-fifth at 10 weeks of age to one-third in adult and old cartilage. The proportion of medium-sized protein–polysaccharides in the extracts changed little with age after birth, but the glucosamine content increased about fivefold and the protein content almost doubled between 10 weeks and 5 years of age. Other analytical values changed little. These results cannot be explained solely by changes in the proportion of ‘link-glycoprotein’ in the protein–polysaccharides. Since major changes in most parameters had taken place by 25 weeks of age, the first weeks after birth may be a critical period for cartilage development in the pig.
Oestrogen and nuclear binding sites. Determination of specific sites by [3H]oestradiol exchange
Authors: Anderson J. , Clark J. H. , Peck E. J.
Published: August 2015

A method was developed for the determination of the number of specific oestradiol-binding sites in the nuclear fraction of oestrogen-sensitive tissues. The method is based on the exchange of [3H]oestradiol with non-labelled oestradiol that is bound to nuclear binding sites. The number of specific nuclear binding sites after the injection of 2.5μg of oestradiol, an amount sufficient to saturate all binding sites, is 1.6–1.7pmol per immature uterus. The number of sites occupied after an injection of physiological amounts of oestradiol (0.1μg) was 0.46pmol. The injection of oestradiol results in an increased number of nuclear binding sites in uterus and vagina, but has no effect on kidney or muscle. Injections of testosterone or progesterone failed to increase the number of uterine nuclear binding sites. This method permits an evaluation of the number of oestradiol-binding sites in the nuclear fraction of various tissues as a function of either endogenous oestradiol or non-labelled oestradiol administered by injection.
β-Fructofuranosidases from roots of dandelion (Taraxacum officinale Weber)
Authors: Rutherford P. P. , Deacon A. C.
Published: August 2015

1. Three β-fructofuranosidases were separated by chromatography on a DEAE-cellulose column from the soluble protein extracted from dandelion (Taraxacum officinale Weber) roots. 2. One enzyme, which acted on sucrose, was characterized as an invertase, with a Km of 2.00×10-2M and pH optimum of 7.5. 3. The other two enzymes are hydrolases (A and B), which act on the inulin series of oligosaccharides [general formula glucose-fructose-(fructose)n]. They both have a pH optimum of 4.0 and Km of 1.54×10-2M but differ in their chromatographic behaviour on DEAE-cellulose. Neither of the hydrolases is inhibited by sucrose. 4. The physiological role of these three hydrolytic enzymes is discussed.
The disulphide bridges of a mouse immunoglobulin G1 protein
Authors: Svasti J. , Milstein C.
Published: August 2015

[35S]Cystine-labelled immunoglobulin MOPC21 (IgG1) was prepared from myeloma cells in tissue culture. Carrier myeloma protein was added and the protein was digested with pepsin. The digest was fractionated on Sephadex G-50 into two fractions, further digested with trypsin and again fractionated on Sephadex. Disulphide-bridge peptides were purified by electrophoresis and chromatography and identified by radioautography. A peptide of 96 residues was isolated, which contains both the heavy–light interchain disulphide bridge and all the inter-heavy-chain disulphide bridges. Other peptides were isolated, accounting for all the intrachain disulphide bridges (which could be placed by homology with proteins of other species), except for the variable section of the light chain. Sequences describing this missing disulphide bridge were obtained from totally reduced and alkylated light chains. Peptides related to the interchain disulphide-bridge peptide were isolated from partially reduced and alkylated myeloma protein and from totally reduced heavy chain. The interchain disulphide-bridge peptide was placed at the C-terminal position of the F(ab′)2 fragment, prepared by digestion of the protein with pepsin at pH4.0. Sequences from the heavy-chain intrachain disulphide bridges of MOPC 21 immunoglobulin are compared with homologous sequences from mouse myeloma proteins of other subclasses and proteins of other species.
Effects of tetrodotoxin and anaesthetics on brain metabolism and transport during anoxia
Authors: Shankar R. , Quastel J. H.
Published: August 2015

1. Tetrodotoxin, at concentrations at which it abolishes generation of action potentials in the nervous system, enhances by about 300% the rate of anaerobic glycolysis of brain-cortex slices from adult rats, or from adult and infant guinea pigs. This occurs to a greater extent in Ca2+-deficient incubation media than in Ca2+-rich media. Tetrodotoxin has no accelerative effect on cerebral aerobic glycolysis. 2. Tetrodotoxin does not affect the rate of anaerobic glycolysis of 2-day-old rat brain-cortex slices, nor that of adult rat kidney medulla, nor that of an extract of an acetone-dried powder of brain. 3. Tetrodotoxin does not affect the rate of penetration of glucose into brain slices. 4. Its effect is not apparent if it is added 10min or later after the onset of anoxia. 5. Its effect diminishes as the concentration of K+ in the incubation medium is increased while that of Na+ is decreased. 6. Its salient effect, at the onset of anoxia, is to diminish influx of Na+ into, and efflux of K+ from, the brain slices. 7. Substances that promote cerebral influx of Na+, e.g. protoveratrine, sodium l-glutamate, diminish the accelerative action of tetrodotoxin. 8. It is concluded that tetrodotoxin exerts its effect on anaerobic glycolysis by suppressing, at the onset of anoxia, the generation of action potentials and thereby the accompanying influx of Na+ and efflux of K+. It is suggested that glycolytic stimulation occurs because a rate-limiting step, e.g. operation of pyruvate kinase, is stimulated by K+ and depressed by Na+. 9. Local anaesthetics behave in a manner similar to that of tetrodotoxin in enhancing cerebral anaerobic glycolysis. 10. Sodium Amytal has a marked effect at relatively high concentration. 11. Tetrodotoxin diminishes efflux of amino acids, particularly glutamate and aspartate, at the onset of anoxia.
Polyprenols of Aspergillus niger. Their characterization, biosynthesis and subcellular distribution
Authors: Barr R. M. , Hemming F. W.
Published: August 2015

A polyprenol complex of Aspergillus niger was shown, by using spectrometric methods, to consist of a family of exo-methylene-hexahydroprenols that contain between 18 and 24 isoprene residues per molecule. Each prenol contains two trans residues, three saturated residues (α, ω and ψ) and an exo-methylene substituent on the carbon atom β to the isopropyl group in each ω-residue. The ubiquinone complex consisted of 90% ubiquinone-9, 9% ubiquinone-8 and 1% ubiquinone-10. The amount of polyprenol complex present reached a maximum of 1.7mg/culture bottle after 9–10 days of growth, coincident with the maximum weight of mycelium. The amount of ergosterol (10mg/culture bottle) and ubiquinone (1mg/culture bottle) reached a peak at 8 days. By the 13th day of growth the yield of ergosterol had fallen by 20% and that of ubiquinone by 85%. A study of the incorporation of [2-14C]mevalonate over different time-intervals confirmed that there was a slow turnover of prenol, a more rapid turnover of ergosterol and a very rapid turnover of ubiquinone. At any one time each member of the prenol complex had essentially the same specific radioactivity as other members of the complex. A similar conclusion was made about the ubiquinone mixture. Just over half of the polyprenol present was esterified to fatty acids. Subcellular fractionation studies indicated that the unesterified prenol is associated primarily with a mitochondrial fraction, whereas the ester is more widely distributed.
The role of intracellular pH in the regulation of cation exchanges in yeast
Authors: Ryan J. P. , Ryan H.
Published: August 2015

1. When yeast oxidizes propan-2-ol in the presence of KCl no uptake of K+occurs. 2. When propionate is added to suspensions containing propan-2-ol, or if the suspensions are bubbled with CO2, a considerable uptake of K+occurs. 3. Maximum K+uptake occurs at a propionate concentration of 2mm. 4. The addition of 20mm-propionate to the suspension lowers the intracellular pH of the yeast from a resting value in the region of 6.2 to approx. 5.6. 5. When K+uptake is measured in the presence of 20mm-propionate, progressive changes in the rate of K+uptake and intracellular pH occur. The optimum rate of K+uptake occurs at an intracellular pH of 5.70. 6. The effect of both intra- and extra-cellular pH on K+–K+exchange was studied and an optimum rate was found at an extracellular pH of 5.35, the corresponding intracellular pH being 6.44. 7. When a Na+-loaded yeast oxidizes propan-2-ol in the presence of KCl, a steady efflux of Na+and influx of K+occurs. The addition of 10mm-propionate to the suspension markedly inhibited the Na+efflux but only slightly decreased the K+influx. 8. The effect of both extra- and intra-cellular pH on Na+efflux was studied with propan-2-ol and with glucose. The results can be best interpreted in terms of intracellular pH changes, and an optimum was obtained at approx. pH6.40.
Dietary protein intake and skeletal-muscle protein metabolism in rats. Studies with salt-washed ribosomes and transfer factors
Authors: Alexis S. D. , Basta S. , Young Vernon R.
Published: August 2015

1. Aspects of skeletal muscle protein synthesis in vitro were studied in young rats given a low-protein diet for up to 10 days and during re-feeding with an adequate diet. 2. Partially purified muscle transfer factors (transferases I and II), crude and purified (NH4Cl-washed) ribosomes and a pH5 enzyme fraction were prepared for this purpose. 3. A marked decrease in the capacity of crude ribosomes to carry out cell-free polypeptide synthesis occurred within 4 days of feeding the low-protein diet. 4. The capacity of salt-washed ribosomes to promote amino acid polymerization, in the presence of added transfer factors and aminoacyl-tRNA, was only slightly decreased by the dietary treatment. 5. However, the capacity of salt-washed ribosomes to bind 14C-labelled aminoacyl-tRNA was decreased by feeding the low-protein diet. 6. The capacity of the pH5 enzyme fraction to promote amino acid incorporation in a complete cell-free system was decreased within 2 days of feeding the low-protein diet. There is no evidence that the change is associated with aminoacyl-tRNA synthetase or binding enzyme activities of the pH5 fractions. 7. These changes are discussed in relation to the diminished rate of protein synthesis in the intact muscle cell when rats are given a low-protein diet.
Preparation and properties of nexuses and lipid-enriched vesicles from mouse liver plasma membranes
Authors: Evans W. H. , Gurd J. W.
Published: August 2015

Extraction of mouse liver plasma membranes with 4% (w/v) N-laurylsarcosinate–tris buffer, pH7.8, solubilized 80–90% of the protein and 60% of the 5′-nucleotidase activity. The membrane residue remaining after extraction was resolved on sucrose gradients into two fractions: a vesicular membrane fraction and a fraction characterized by the presence of large numbers of nexuses in an amorphous background. The vesicular fraction had a phospholipid/protein weight ratio of 7:1, it contained most of the plasma-membrane glycolipids, and polyacrylamide-gel electrophoresis indicated the presence of only five to eight proteins, including two or three glycoproteins. The 5′-nucleotidase and leucine naphthylamidase specific activities were 23- and 6-fold higher respectively than in the plasma membranes. Electron microscopy of thin sections and negatively stained preparations indicated that the nexuses present in the second fraction closely resembled gap junctions present in tissue sections and isolated plasma membranes. The nexus fraction contained a distinctive protein pattern, and of the 20 proteins present about four were identified as glycoproteins by Schiff–periodate staining. Examination of the lipid composition of the fractions by t.l.c. showed that in the nexus fraction, phospholipids and glycolipids were present in small amounts compared with triglycerides and cholesterol. Amino sugar analyses confirmed the t.l.c. results and amino acid analysis showed the fractions to have characteristic protein compositions. A ‘reconstituted’ membranous fraction prepared by dialysis against MgCl2 of membrane components soluble in N-laurylsarcosinate–tris buffers, pH7.8, lacked the trilaminar image characteristic of the two other membrane fractions isolated and was devoid of enzyme activities. The results indicate that proteins and glycoproteins play an important role in the structural maintenance of the nexuses isolated from the liver by the present procedure.
The effects of halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) on glycolysis and biosynthetic processes of the isolated perfused rat liver
Authors: Biebuyck J. F. , Lund Patricia , Krebs H. A.
Published: August 2015

1. With reference to the post-operative dysfunction of the liver observed after halothane anaesthesia, the effects of the anaesthetic on some metabolic functions were studied in the isolated perfused rat liver. Oxygen uptake, glycolysis, gluconeogenesis and urea synthesis were affected by halothane at a concentration (2.5% of the gas phase) within the range used in clinical anaesthesia. 2. At this concentration of halothane uptake of oxygen was inhibited in livers from both fed and starved rats. 3. In livers from fed rats there was a 16-fold increase in lactate production. This was accompanied by a fivefold decrease in the tissue content of 2-oxoglutarate and a more than twofold decrease in citrate. The calculated [free NAD+]/[free NADH] ratio in both cytoplasm and mitochondria was lower in the halothane-exposed livers than in controls. 4. In livers of starved rats the rate of gluconeogenesis from lactate was decreased by halothane to 30% of the control rate. 5. Halothane inhibited gluconeogenesis from alanine and propionate to the same extent as from lactate, whereas glucose formation from dihydroxyacetone, glycerol, fructose and sorbitol was relatively unaffected. 6. During gluconeogenesis from 10mm-lactate the tissue content of ATP was decreased by 50%, glutamate by 50% and 2-oxoglutarate was decreased eightfold in the halothane-exposed livers. 7. Halothane decreased urea synthesis in the presence of 10mm-NH4Cl and 2mm-ornithine to 15% of the control rate. 8. The inhibitions of gluconeogenesis and urea synthesis were completely abolished within 15min of withdrawal of the anaesthetic. 9. The stimulation of uptake of oxygen brought about by the addition of lactate or precursors of urea was abolished by halothane. 10. Effects on gluconeogenesis similar to those of halothane occurred in livers exposed to the anaesthetic methoxyflurane, although normal rates were not restored on withdrawal of the drug. Other anaesthetic agents tested (ketamine–HCl and trichloroethylene) decreased gluconeogenesis to 66% of the control rate. 11. The inhibitory effects of halothane are consistent with an interference at the stage of the NADH dehydrogenase of the electron-transport chain.
The formation of ω-cyclohexyl-fatty acids from shikimate in an acidophilic thermophilic bacillus. A new biosynthetic pathway
Authors: De Rosa M. , Gambacorta A. , Minale L. , Bu'lock J. D.
Published: August 2015

Labelled acetate, phenylalanine and shikimic acid were fed to Bacillus acidocaldarius. A high proportion of the 14C incorporated from acetate and shikimate was recovered in methyl esters from the cell lipids, but such recovery of 14C from phenylalanine was low. Only the 14C from shikimate was selectively incorporated into 11-cyclohexylundecanoate and 13-cyclohexyltridecanoate. Degradation of these cyclohexyl-fatty acids showed that shikimate was incorporated as an intact C7 unit.
An adenosine triphosphate-dependent stabilization of proteolytic activity in heterolysosomes. Evidence for a proton pump
Authors: Mego John L. , Farb R. M. , Barnes Judith
Published: August 2015

1. Homogenization of mouse kidneys or livers in 0.25m-sucrose buffered with tris–acetate, pH7.3, resulted in a decreased rate of proteolysis within isolated heterolysosomes containing injected 125I-labelled albumin when these particles were incubated at 35°C. Proteolysis in mouse kidney or liver heterolysosomes isolated from homogenates made in 0.25m-sucrose buffered at pH7.3 was stimulated by pH5 buffer or by additions of ATP. 2. A greater inhibition of proteolysis was produced by including bicarbonate or pH8 borate buffers in the incubation media, and this inhibition was also reversed by ATP. 3. Other nucleoside triphosphates were not as effective as ATP, but GTP and ITP were more effective than CTP or UTP. ADP, AMP, or adenosine 3′:5′-cyclic monophosphate were completely without effect. 4. Although ATP prevented some heterolysosome breakage in media containing bicarbonate, the primary effect appeared to be to promote proteolytic activity. 5. These observations are consistent with the presence of a proton pump in the heterolysosome membrane, which functions to maintain intralysosomal pH in alkaline media.
The isolation of pure cholinergic synaptic vesicles from the electric organs of elasmobranch fish of the family Torpedinidae
Authors: Whittaker V. P. , Essman W. B. , Dowe G. H. C.
Published: August 2015

1. Zonal centrifuging permitted the separation, on the milligram scale and in a form largely free from contamination by soluble cytoplasmic protein or membrane fragments derived from other structures, of synaptic vesicles from the purely cholinergic terminals of the electric organ of Torpedo. Up to 100g of tissue could be processed in a single run. 2. As much as 46% of the bound acetylcholine from the original tissue preparation was recovered as a single peak of density equivalent to 0.38m-sucrose–0.21m-NaCl and with a concentration of up to 680nmol of acetylcholine/mg of protein. 3. The limiting concentration of acetylcholine in isolated vesicles when allowance had been made for non-vesicular protein appeared to be about 600nmol/mg of protein. 4. Vesicle counts by a ‘bead-tagging’ procedure indicated an acetylcholine content of about 360μmol/ml of vesicles; thus the vesicle protein content would be about 60% (w/v). 5. Calculations showed that the core of the vesicle, accounting for about 55% of the vesicle volume, could be largely filled with acetylcholine and protein.
Protein fluorescence of lactate dehydrogenase
Authors: Holbrook J. J.
Published: August 2015

1. There is a non-linear decrease in the protein fluorescence (F) of lactate dehydrogenase with the increase in the fraction (α) of the coenzyme-binding sites occupied with NADH. 2. By a curve-fitting procedure it is shown that the fluorescence intensity can be represented by the equation F=[1−α(1−x)]n where n is the number of identical and indistinguishable coenzyme-binding sites per protein molecule and x=Fs1/n (Fs is the protein fluorescence at α=1). This equation implies that the relative protein fluorescence of molecules bearing j ligands form the geometric series xj. 3. Non-linear quenching of protein fluorescence for this enzyme is probably due to radiationless transfer of energy from the protein molecule to the bound NADH and should also be observed when other potential acceptors of protein fluorescence are bound at unique sites. 4. The intercept with Fs of an initial tangent to a curve of protein fluorescence against α will be at a value of α equal to (Kd+[E0])· (1−xn)/n·(1−x) and not at a value equal to the sum of the dissociation constant (Kd) and the concentration of identical ligand-binding sites ([E0]). 5. A use of non-linear protein fluorescence quenching to investigate the state of aggregation of a protein is discussed.
The regulation of glyceride synthesis in isolated white-fat cells. The effects of palmitate and lipolytic agents
Authors: Saggerson E. D
Published: August 2015

1. 0.5mm-Palmitate stimulated incorporation of [U-14C]glucose into glyceride glycerol and fatty acids in normal fat cells in a manner dependent upon the glucose concentration. 2. In the presence of insulin the incorporation of 5mm-glucose into glyceride fatty acids was increased by concentrations of palmitate, adrenaline and 6-N-2′-O-dibutyryladenosine 3′:5′-cyclic monophosphate up to 0.5mm, 0.5μm and 0.5mm respectively. Higher concentrations of these agents produced progressive decreases in the rate of glucose incorporation into fatty acids. 3. The effects of palmitate and lipolytic agents upon the measured parameters of glucose utilization were similar, suggesting that the effects of lipolytic agents are mediated through increased concentrations of free fatty acids. 4. In fat cells from 24h-starved rats, maximal stimulation of glucose incorporation into fatty acids was achieved with 0.25mm-palmitate. Higher concentrations of palmitate were inhibitory. In fat cells from 72h-starved rats, palmitate only stimulated glucose incorporation into fatty acids at high concentrations of palmitate (1mm and above). 5. The ability of fat cells to incorporate glucose into glyceride glycerol in the presence of palmitate decreased with increasing periods of starvation. 6. It is suggested that low concentrations of free fatty acids stimulate fatty acid synthesis from glucose by increasing the utilization of ATP and cytoplasmic NADH for esterification of these free fatty acids. When esterification of free fatty acids does not keep pace with their provision, inhibition of fatty acid synthesis occurs. Provision of free fatty acids far in excess of the esterification capacity of the cells leads to uncoupling of oxidative phosphorylation and a secondary stimulation of fatty acid synthesis from glucose.
Preparation and characterization of the lateral and basal plasma membranes of the rat intestinal epithelial cell
Authors: Douglas A. P. , Kerley R. , Isselbacher K. J.
Published: August 2015

A technique is described for the isolation of a plasma-membrane fraction from the rat intestinal epithelial cell which is distinct from the microvillus membrane of that cell. The isolated fraction contains only about 0.2% of the sucrase activity in the original homogenate and negligible quantities of nuclear and mitochondrial membrane markers. It contains 12% of the total Na+,K+-dependent adenosine triphosphatase and 7% of the alkaline phosphatase, with significant increments in specific activity of these enzymes. Multiple membrane preparations were highly reproducible with respect to the specific activities of the markers studied. The small intestine of one rat yields material containing about 1.3mg of protein. In addition an assay is described suitable for determining 5′-nucleotidase in the small intestine.
Specific inhibition of mitochondrial fatty acid oxidation by 2-bromopalmitate and its co-enzyme A and carnitine esters
Authors: Chase J. F. A. , Tubbs P. K.
Published: August 2015

1. The CoA and carnitine esters of 2-bromopalmitate are extremely powerful and specific inhibitors of mitochondrial fatty acid oxidation. 2. 2-Bromopalmitoyl-CoA, added as such or formed from 2-bromopalmitate, inhibits the carnitine-dependent oxidation of palmitate or palmitoyl-CoA, but not the oxidation of palmitoylcarnitine, by intact liver mitochondria. 3. 2-Bromopalmitoylcarnitine inhibits the oxidation of palmitoylcarnitine as well as that of palmitate or palmitoyl-CoA. It has no effect on succinate oxidation, but inhibits that of pyruvate, 2-oxoglutarate or hexanoate; however, the oxidation of these substrates (but not of palmitate, palmitoyl-CoA or palmitoyl-carnitine) is restored by carnitine. 4. In damaged mitochondria, added 2-bromopalmitoyl-CoA does inhibit palmitoylcarnitine oxidation; pyruvate oxidation is unaffected by the inhibitor alone, but is impaired if palmitoylcarnitine is subsequently added. 5. The findings have been interpreted as follows. 2-Bromopalmitoyl-CoA inactivates (in a carnitine-dependent manner) a pool of carnitine palmitoyltransferase which is accessible to external acyl-CoA. This results in inhibition of palmitate or palmitoyl-CoA oxidation. A second pool of carnitine palmitoyltransferase, inaccessible to added acyl-CoA in intact mitochondria, can generate bromopalmitoyl-CoA within the matrix from external 2-bromopalmitoylcarnitine; this reaction is reversible. Such internal 2-bromopalmitoyl-CoA inactivates long-chain β-oxidation (as does added 2-bromopalmitoyl-CoA if the mitochondria are damaged) and its formation also sequesters intramitochondrial CoA. Since this CoA is shared by pyruvate and 2-oxoglutarate dehydrogenases, the oxidation of their substrates is depressed by 2-bromopalmitoylcarnitine, unless free carnitine is available to act as a ‘sink’ for long-chain acyl groups. 6. These effects are compared with those reported for other inhibitors of fatty acid oxidation.
Inhibition of the thrombin–fibrinogen reaction by a macromolecular factor from rat liver
Authors: Flengsrud Ragnar , Østerud Bjarne , Prydz Hans
Published: August 2015

1. The supernatant obtained by centrifugation of a rat liver homogenate at 100000g for 1h contained a heat-labile macromolecular inhibitor of the thrombin–fibrinogen reaction. 2. The inhibitor was purified to electrophoretic homogeneity by repeated preparative polyacrylamide disc electrophoresis. Inhibition was observed with purified inhibitor equivalent to about 1μg of protein/ml. 3. The inhibitor had a pI of 3.50–3.75, a molecular weight (from sodium dodecyl sulphate–polyacrylamide-gel electrophoresis) of 72000±3000 and was inactivated by p-hydroxymercuribenzoate or 5,5′-dithiobis-(2-nitrobenzoic acid). 4. Kinetic studies revealed a non-competitive inhibition, with the inhibitor probably acting on the thrombin–fibrinogen complex.
A β-d-fructofuranosidase from Claviceps purpurea
Authors: Dickerson A. G.
Published: August 2015

Evidence suggests that sucrose is the main carbon source for growth of Claviceps spp. in the parasitic condition. The sucrose acts as substrate for an active β-fructofuranosidase, produced by the fungus, which in the first instance converts the disaccharide into glucose and an oligofructoside. In this way, 50% of the glucose, supplied as sucrose, is made available to the parasite for assimilation. Subsequent action of the enzyme on both sucrose and the oligofructoside leads to the release of more glucose and the formation of additional oligosaccharides. The structures of the main oligosaccharides formed have been elucidated and the interactions of each compound studied. In experiments with purified enzyme in vitro the interaction of the oligosaccharides is rapid but in culture they are assimilated only slowly; in each case some free fructose is liberated. Free fructose is not assimilated in the presence of glucose and, further, inhibits growth at concentrations which might be expected to occur in the parasitic condition. A dual role has been suggested for the enzyme, with sucrose as substrate, in which glucose is made available to the growing parasite, while at the same time transfer of the fructose to form oligosaccharides prevents it from accumulating at inhibitory concentrations. Ultimately, when glucose becomes limiting, the fungus will adapt to fructose assimilation.
A modified procedure for fractionating histones
Authors: Oliver Denis , Sommer K. R. , Panyim Sakol , Spiker Steven , Chalkley Roger
Published: August 2015

A method is described, which is capable of fractionating histones obtained from any animal source into five major groups. Although the method is based on procedures initially developed by E. W. Johns (1964), involving differential solubility in solutions of acids and of ethanol, it gives a cleaner separation and possesses the considerable advantage that the starting material is whole histone rather than a nucleoprotein preparation.
Glycogen synthesis in the perfused liver of the starved rat
Authors: Hems D. A. , Whitton P. D. , Taylor E. A.
Published: August 2015

1. In the isolated perfused liver from 48h-starved rats, glycogen synthesis was followed by sequential sampling of the two major lobes. 2. The fastest observed rates of glycogen deposition (0.68–0.82μmol of glucose/min per g fresh liver) were obtained in the left lateral lobe, when glucose in the medium was 25–30mm and when gluconeogenic substrates were present (pyruvate, glycerol and serine: each initially 5mm). In this situation there was no net disappearance of glucose from the perfusion medium, although 14C from [U-14C]glucose was incorporated into glycogen. There was no requirement for added hormones. 3. In the absence of gluconeogenic precursors, glycogen synthesis from glucose (30mm) was 0–0.4μmol/min per g. 4. When livers were perfused with gluconeogenic precursors alone, no glycogen was deposited. The total amount of glucose formed was similar to the amount converted into glycogen when 30mm-glucose was also present. 5. The time-course, maximal rates and glucose dependence of hepatic glycogen deposition in the perfused liver resembled those found in vivo in 48h-starved rats, during infusion of glucose. 6. In the perfused liver, added insulin or sodium oleate did not significantly affect glycogen synthesis in optimum conditions. In suboptimum conditions (i.e. glucose less than 25mm, or with gluconeogenic precursors absent) insulin caused a moderate acceleration of glycogen deposition. 7. These results suggest that on re-feeding after starvation in the rat, hepatic glycogen deposition could be initially the result of continued gluconeogenesis, even after the ingestion of glucose. This conclusion is discussed, particularly in connexion with the role of hepatic glucokinase, and the involvement of the liver in the glucose intolerance of starvation.
Studies on the biosynthesis in vivo and excretion of 16-unsaturated C19 steroids in the boar
Authors: Saat Y. A. , Gower D. B. , Harrison F. A. , Heap R. B.
Published: August 2015

1. In one experiment [7α-3H]pregnenolone was infused continuously for 12min into the left spermatic artery of a sexually mature boar and blood was collected during this period by continuous drainage from the spermatic vein. After infusion, the testis was removed and immediately cooled to −196°C. 2. From both the testicular tissue and the spermatic venous plasma, 3H-labelled 16-unsaturated C19 steroids were isolated and characterized and their radiochemical purity was established. 5α-Androst-16-en-3α- and 3β-ol occurred mainly as sulphate conjugates and to a lesser extent as free steroids. Only traces of these alcohols occurred as glucosiduronate conjugates. 5α-Androst-16-en-3-one was found in the free (ether-extractable) fraction. 3. The isotope concentration of each of the 3H-labelled 16-unsaturated C19 steroids in testicular tissue was different from that in spermatic venous plasma. 4. The ratios of tritiated 5α-androst-16-en-3α- and 3β-ol (free steroids) to their respective sulphate conjugates in the testicular tissue were less than the ratios of the same compounds in the spermatic venous plasma. The possibility that the sulphates are partially hydrolysed by testicular sulphatases before secretion is discussed. 5. In a second experiment, a continuous close-arterial infusion of [7α-3H]pregnenolone into the left testis was performed over a 200min period and all the urine that accumulated during the infusion was collected for analysis. 6. No 3H-labelled 16-unsaturated C19 steroids were detected in the urine as free steroids. Only a trace of 5α-androst-16-en-3α-ol was detected conjugated as glucosiduronate, whereas the corresponding 3β-alcohol occurred mainly as glucosiduronate and to a lesser extent as sulphate. 7. The absence of 5α-androst-16-en-3β-ol glucosiduronate in the spermatic venous blood and its presence in considerable amount in the urine may be attributed to hepatic glucuronyl transferase activity.
The purification and characterization of wheat-germ agglutinin
Authors: LeVine Dreania , Kaplan Michael J. , Greenaway Peter J.
Published: August 2015

The purification of wheat-germ agglutinin by precipitation with ammonium sulphate and by chromatography on Sephadex G-75, Sepharose–ovomucoid and CM-cellulose is described. This procedure gave agglutinin preparations which were homogeneous on polyacrylamide gels under a variety of conditions. Purified wheat-germ agglutinin formed colourless solutions and was relatively insoluble at neutral pH; maximum solubility in 1mm-tris–HCl buffer, pH7.4, was approx. 1mg/ml. The agglutinin was a glycoprotein containing a single polypeptide chain with an approximate molecular weight of 23000. The N-terminus of the oxidized agglutinin was cysteic acid and the C-terminus was glycine. The amino acid composition showed that the protein was extremely rich in cysteine and cystine; there were 15–17 free SH groups/mol. The absorption maximum for the protein was at 272nm and the molar extinction coefficient at 280nm was 1.09×105 litre·mol-1·cm-1. Equilibrium dialysis indicated that there was only one binding site per molecule for N-acetylglucosamine.
Interactions of insulin, corticosterone and prolactin in promoting milk-fat synthesis by mammary explants from pregnant rabbits
Authors: Forsyth Isabel A. , Strong Christopher R. , Dils Raymond
Published: August 2015

1. The rate of fatty acid synthesis by mammary explants from rabbits pregnant for 16 days or from rabbits pseudopregnant for 11 days was stimulated up to 15-fold by culturing for 2–4 days with prolactin. This treatment initiated the predominant synthesis of C8:0 and C10:0 fatty acids, which are characteristic of rabbit milk. 2. Inclusion of insulin in the culture medium increased the rate of synthesis of these medium-chain fatty acids. By contrast the inclusion of corticosterone led to the predominant synthesis of long-chain fatty acids. When explants were cultured for 2–4 days with insulin, corticosterone and prolactin, the rate of fatty acid synthesis increased up to 42-fold, but both medium- and long-chain fatty acids were synthesized. 3. These results show that the stimulus to mammary-gland lipogenesis and the initiation of synthesis of medium-chain fatty acids observed between days 16 and 23 of pregnancy in the rabbit can be simulated in vitro by prolactin alone. 4. When mammary explants from rabbits pregnant for 23 days were cultured for 2 days with insulin, corticosterone and prolactin, the rate of fatty acid synthesis increased fivefold, but there was a preferential synthesis of long-chain fatty acids. Culture with prolactin alone had little effect on the rate or pattern of fatty acids synthesized. 5. The results are compared with findings in vivo on the control of lipogenesis in the rabbit mammary gland, and are contrasted with the known effects of hormones in vitro on the mammary gland of the mid-pregnant mouse.
Metabolism of myricetin and related compounds in the rat. Metabolite formation in vivo and by the intestinal microflora in vitro
Authors: Griffiths L. A. , Smith G. E.
Published: August 2015

1. The metabolism of a group of polyphenols related in structure to myricetin (3,5,7,3′,4′,5′-hexahydroxyflavone), including myricetin, myricitrin, 3,4,5-trihydroxyphenylacetic acid, delphinidin, robinetin, tricetin, tricin, malvin and 5,7-dihydroxy-3′,4′,5′-trimethoxyflavone, has been studied both in vivo after oral administration to the rat and in vitro in cultures of micro-organisms derived from the intestine of the rat. 2. It was shown that the rat intestinal microflora are able to degrade compounds of this group to the ring-fission products observed in urine after oral administration of the specific flavonoid. 3. All flavones and flavonols possessing free 5- and 7-hydroxyl groups in the A ring and a free 4′-hydroxyl group in the B ring gave rise to ring-fission products that included 3′,5′-dihydroxyphenylacyl derivatives. 4. The metabolites 3,5-dihydroxyphenylacetic acid, 3-hydroxyphenylacetic acid, 3,5-dihydroxyphenylpropionic acid and 3-hydroxyphenylpropionic acid were isolated and identified by chromatographic and spectral methods. 5. On anaerobic incubation in a thioglycollate medium it was shown that intestinal micro-organisms can effect cleavage of glycosidic bonds, ring fission of certain flavonoid molecules showing 3′,4′,5′-trihydroxyphenyl substitution and dehydroxylation of certain flavonoid metabolites. 6. The urinary excretion of the metabolites 3,5-dihydroxyphenylacetic acid and 3-hydroxyphenylacetic acid was completely abolished when neomycin-treated rats were used.