bluuebunny/crossref_metadata_2025 huggingface dataset:
Vitamin A and the biosynthesis of sulphated mucopolysaccharides. Experiments with rats and cultured neoplastic mast cells
Authors: Thomas D. B. , Pasternak C A
Published: August 2015

1. The uptake and incorporation of [35S]sulphate into mucopolysaccharides by colon and duodenum in vitro are unaffected by the vitamin A status of the animals. 2. Uptake and incorporation in vivo are unaffected at 4hr. after injection of [35S]sulphate, but at later times are decreased in some tissues of vitamin A-deficient animals. 3. The rate of removal of 35S from blood, its rate of appearance in urine, the plasma concentration of sulphate and the uronic acid content of several tissues are not significantly altered in vitamin A deficiency. 4. These results, and direct measurement of 35S in mucopolysaccharides at various times after injection of [35S]sulphate, suggest that the synthesis of mucopolysaccharides is unaffected but that their turnover is increased in vitamin A deficiency. 5. Neither the growth rate of, nor the incorporation of [35S]sulphate into heparin by, P815Y and HC cultured neoplastic mast cells is decreased when the horse serum necessary for growth is treated with ultraviolet light or is replaced by serum from vitamin A-deficient rats. 6. The addition of citral is no more toxic to growth rate or to incorporation of 35S than is the addition of vitamin A itself. 7. It is concluded that neoplastic mast cells in culture do not require vitamin A for growth or for the synthesis of heparin. 8. None of these results is compatible with the view that vitamin A or a derivative is directly involved in the biosynthesis of sulphated mucopolysaccharides.
Triglyceride synthesis by small-intestinal epithelium of the pig, sheep and chicken
Authors: Bickerstaffe R. , Annison E. F.
Published: August 2015

1. A comparative study was made of triglyceride synthesis by the intestinal epithelium of pigs, sheep and chickens. In pig and chicken tissue both the glycerol 3-phosphate and the monoglyceride pathway of triglyceride synthesis were operative, but the former pathway predominated in sheep tissue. 2. The fatty acid specificity of the glycerol 3-phosphate pathway was studied in pig and sheep total-homogenate preparations. Maximum incorporation was obtained with myristic acid and palmitic acid under optimum conditions for each fatty acid. Lauric acid, myristic acid, oleic acid, linoleic acid and linolenic acid were inhibitory at concentrations above their optimum, but octanoic acid, decanoic acid, palmitic acid and stearic acid did not show this effect. 3. Subcellular fractionation located the glycerol 3-phosphate and monoglyceride pathways of triglyceride synthesis in the microsomes in all instances. Phosphatidate phosphohydrolase was associated with both the microsomes and the particle-free supernatant. 4. Glycerol 1-mono-oleate was incorporated into triglycerides to a greater extent than glycerol 1-mono-palmitate or glycerol 1-monostearate by microsomal preparations from pig and chicken. 5. A lipase specific for monoglycerides was detected in the particle-free supernatant of all the species examined.
The controlling effect of carbohydrate in human, rabbit and bovine immunoglobulin G on proteolysis by papain
Authors: Payne R. B.
Published: August 2015

About two-thirds of the hexose of human and rabbit immunoglobulin G (IgG) was located in the Fc fragment and one-third in the ‘hinge’ region of the γ (heavy) polypeptide chain at the junction of the Fab and Fc fragments. In contrast, bovine IgG contained more hexose in the ‘hinge’ region than in the Fc fragment. The initial cleavage of susceptible IgG molecules into Fab and Fc fragments by papain under the conditions given by Porter (1959) had reached completion after digestion for 2hr., though bovine IgG was digested somewhat more slowly than human or rabbit IgG. The release of ‘hinge’ peptides from human and rabbit IgG had also reached completion by 2hr., but was slower from bovine IgG and continued for several hours longer. Since bovine IgG molecules contained on the average a greater amount of hexose in the ‘hinge’ region, carbohydrate on this part of the γ-chain may influence not only the initial rate of enzymic hydrolysis into Fab and Fc fragments, but also, and to a greater extent, the rate of further limited hydrolysis of the N-terminal regions of the Fc fragment. The presence of carbohydrate in the ‘hinge’ region does not appear to account for the resistance of some IgG molecules to papain digestion and of some Fc fragments to N-terminal degradation.
Metabolism of leucine in protein-caloriedeficient rats
Authors: McFarlane I. G. , von Holt C.
Published: August 2015

1. Protein-calorie-depleted rats exhibit a decreased ability to oxidize leucine as compared with control animals. 2. The block in degradation of l-leucine is due to decreased activities of l-leucine transaminase and d-amino acid oxidase associated with liver mitochondria. 3. Cytoplasmic l-leucine transaminase in liver is not affected by the protein depletion. 4. The specific activities of the mitochondrial enzymes can be increased by a single oral dose of 1g. of Bacto-Peptone (meat hydrolysate). The effect of Bacto-Peptone can be inhibited by prior administration of dosages of 10mg. of puromycin/100g. body wt., suggesting that the increased enzyme specific activities may be due to new synthesis of enzyme protein.
The specificity of purified porcine pancreatic elastase
Authors: Narayanan A. Sampath , Anwar R. A.
Published: August 2015

An electrophoretically homogeneous elastase preparation free from tryptic and chymotryptic activities was obtained by chromatography on DEAE-Sephadex and CM-cellulose. This preparation exhibits a narrower specificity towards peptide bonds than that observed by Naughton & Sanger (1961). With oxidized insulin B chain as substrate, the fastest breaks occur between alanine-14 and leucine-15 and between valine-18 and cysteic acid-19. The bond between glycine-23 and phenylalanine-24 is also efficiently hydrolysed. Other bonds hydrolysed are that between valine-12 and glutamic acid-13 and that between serine-9 and histidine-10. Oxidized insulin A chain is hydrolysed only at one of two points, between alanine-8 and serine-9 or between serine-12 and leucine-13, and the rate of hydrolysis is very low.
The composition of rat liver microsomes. The structural proteins of rat liver microsomes
Authors: Ward K. A. , Pollak J K
Published: August 2015

1. The structural-protein component of microsomal membranes was isolated by three separate methods. Analysis by polyacrylamide-gel electrophoresis indicated that the microsomal structural component is made up of a heterogeneous group of proteins. These proteins were further characterized by their phospholipid-binding capacity. The electrophoretic patterns of microsomal structural proteins were found to differ significantly from those of mitochondrial structural proteins. 2. The reticulosomal fraction was also characterized by electrophoresis with reference to total microsomal proteins, microsomal structural proteins and ribosomal proteins. The reticulosomes gave an electrophoretic pattern significantly different from those of the other three preparations examined. It is suggested that reticulosomes consist largely of enzymic proteins of the endoplasmic reticulum.
The stereospecific removal of a C-19 hydrogen atom in oestrogen biosynthesis
Authors: Skinner S. J. M. , Akhtar M
Published: August 2015

1. The synthesis of a number of 19-substituted androgens is described. 2. A method for the partially stereospecific introduction of a tritium label at C-19 in 19-hydroxyandrost-5-ene-3β,17β-diol was developed. The 19−3H-labelled triol produced by reduction of 19-oxoandrost-5-ene-3β,17β-diol with tritiated sodium borohydride is tentatively formulated as 19-hydroxy[(19-R)-19−3H]androst-5-ene-3β,17β-diol and the 19−3H-labelled triol produced by reduction of 19-oxo[19−3H]-androst-5-ene-3β,17β-diol with sodium borohydride as 19-hydroxy[(19-S)-19−3H]-androst-5-ene-3β,17β-diol. 3. In the conversion of the (19-R)-19−3H-labelled compound into oestrogen by a microsomal preparation from human term placenta more radioactivity was liberated in formic acid (61·6%) than in water (38·4%). In a parallel experiment with the (19-S)-19−3H-labelled compound the order of radioactivity was reversed: formic acid (23·4%), water (76·2%). 4. These observations are interpreted in terms of the removal of the 19-S-hydrogen atom in the conversion of a 19-hydroxy androgen into a 19-oxo androgen during oestrogen biosynthesis. 5. It is suggested that the removal of C-19 in oestrogen biosynthesis occurs compulsorily at the oxidation state of a 19-aldehyde with the liberation of formic acid.
Properties of heparan sulphate and chondroitin sulphate from young and old human aortae
Authors: Manley G. , Mullinger R. N. , Lloyd P. H.
Published: August 2015

1. Glycosaminoglycans were liberated from old and young human ascending aortae by digestion with papain. Heparan sulphate and chondroitin sulphate were separated by the different solubilities of their complexes with cetylpyridinium chloride in solutions of sodium chloride. Final fractionation was achieved by salt-gradient column chromatography on Dowex 1 (Cl−form). 2. Heparan sulphate from old aortae showed a slight, but consistent, increase in sulphation compared with heparan sulphate from young aortae. 3. The major amino acids associated with aortic heparan sulphate and chondroitin sulphate were serine, glycine, glutamic acid and aspartic acid. Heparan sulphate and chondroitin sulphate from old aortae contained about twice as much total amino acid as heparan sulphate and chondroitin sulphate from young aortae. Alkali hydrolysis resulted in the destruction of more serine in chondroitin sulphate from old, compared with young, aortae. 4. Molecular weights of glycosaminoglycans from old and young aortae were found to be similar, and in the region of 35000. 5. It is suggested that there is an increased degree of protein–glycosaminoglycan cross-linking in old aortae.
Immune reactions in polysaccharide media. Polysaccharide-enhanced precipitin reactions with antigens of various sizes
Authors: Hellsing Krister
Published: August 2015

The influence of the size of the antigen in the polysaccharide-enhanced precipitin reaction was investigated. The experiments were carried out by addition of homologous antigens of different molecular sizes (the monomer, dimer and trimer of serum albumin and the monomer and dimer of immunoglobulin G) to the same preparation of antibody in the absence and presence of dextran. Dextran decreased the solubility of the immune complexes to a larger extent when the antigen size increased. This is in accord with the view that the polysaccharide effect is due to steric exclusion.
A rat liver system that catalyses a pyridoxal phosphate-independent αβ-elimination
Authors: Tudball N. , Thomas J H , Fowler J. A.
Published: August 2015

1. Attempts were made to demonstrate the presence of pyridoxal phosphate in the rat liver system catalysing the αβ-elimination of l-serine O-sulphate. 2. Methods designed to resolve protein-bound cofactor, spectroscopic examination of a purified enzyme system and attempted reactivation of apo-(alanine aminotransferase) failed to demonstrate the presence of pyridoxal phosphate. 3. The activity of the αβ-eliminating system remained constant in vitamin B6-deficient animals even though the activities of other pyridoxal phosphate-dependent systems fell markedly. 4. The metabolism of l-serine O[35S]-sulphate in vivo appears to be normal in vitamin B6-deficient animals. 5. No incorporation of tritium into the αβ-eliminating system occurred after administration of tritiated pyridoxine to experimental animals.
Carbonic anhydrase isoenzymes in the erthrocytes and dorsolateral prostate of the rat
Authors: McIntosh J. E. A.
Published: August 2015

1. Three forms of the zinc-containing enzyme carbonic anhydrase (EC 4.2.1.1) were isolated from the erythrocytes of the rat and two forms from the dorsolateral prostate of the rat. Several additional minor components were observed but not isolated. Separation of the isoenzymes was achieved by ion-exchange chromatography, polyacrylamide-gel electrophoresis and isoelectric focusing. 2. The general properties of the isolated isoenzymes, their molecular weights and their contents of zinc were closely similar. As catalysts of the hydration of carbon dioxide, however, they were distinctly different. The two most abundant isoenzymes of the erythrocytes, which were found in equal proportions, differed 70-fold in specific activity, whereas the isoenzymes of the dorsolateral prostate were similar to one another and resembled the high-activity component of the erythrocytes. The inhibition of the latter by acetazolamide (5-acetamido-1-thia-3,4-diazole-2-sulphonamide) was mainly competitive, whereas in identical conditions the low-activity erythrocyte component and the dorsolateral prostate isoenzymes were non-competitively inhibited. 3. The use of chloroform–ethanol to remove haemoglobin from the rat haemolysate was found (a) to bring about changes in the kinetic properties of the soluble isoenzymes and (b) to cause the appearance of an additional isoenzyme. 4. The actions were compared of the inhibitors acetazolamide, 1,1-dimethylaminonaphthalene-5-sulphonamide and ethoxzolamide (6-ethoxybenzothiazole-2-sulphonamide) on the hydrolysis of p-nitrophenyl acetate catalysed by the isoenzymes. 5. The low-activity erythrocyte isoenzyme was an efficient catalyst of the hydrolysis of β-naphthyl acetate whereas the high-activity forms were much less active towards this ester. Neither of the isoenzymes present in the dorsolateral prostate catalysed this reaction. 6. Carbonic anhydrase in the rat dorsolateral prostate accounts for no more than 5% of the unusually high content of zinc in this organ.
The induction of cross-links in soluble rat skin collagen in vitro
Authors: Bannister D. W.
Published: August 2015

1. Starch-gel electrophoresis was used to investigate the subunit composition of salt-soluble and acid-soluble rat skin collagen. 2. Cross-linkage of collagen subunits in vitro was performed (a) when in fibrillar form and (b) when in solution. In the former case the increase in number of cross-links appeared to be predominantly intermolecular and in the latter case predominantly intramolecular.
Structural requirements for active intestinal transport. Spatial and bonding requirements at C-3 of the sugar
Authors: Barnett J. E. G. , Ralph A. , Munday K A
Published: August 2015

Analogues of d-glucose modified at C-3, and in some cases at a second position, were prepared and tested for active accumulation by everted segments of hamster intestine. Their relative affinity for the sugar carrier was measured by tissue/medium ratio, Michaelis–Menten kinetics and competitive inhibition of d-galactose or methyl α-d-glucoside transport. d-Glucose and its 3-deoxy-3-fluoro, 3-chloro-3-deoxy and to a smaller extent its 3-bromo-3-deoxy derivatives, bound and were transported more strongly than 3-deoxy-d-glucose and other sugars not containing an electronegative atom in the gluco configuration at C-3. 3-Deoxy-d-galactose, 3,6-dideoxy-d-glucose and d-gulose, which have two alterations from the d-glucose structure, were not, or only very weakly, transported. The results are interpreted as indicating the presence of a hydrogen bond from the carrier to the hydroxyl group at C-3 of d-glucose. Spatial requirements are also discussed. New syntheses are reported for 3-chloro-3-deoxy- and 3-bromo-3-deoxy-d-glucose and 3,6-dideoxy-d-glucose.
Changes in the concentrations of hepatic metabolites on administration of dihydroxyacetone or glycerol to starved rats and their relationship to the control of ketogenesis
Authors: Williamson D. H. , Veloso Dulce , Ellington E. V. , Krebs H. A.
Published: August 2015

1. Glycerol and dihydroxyacetone, both antiketogenic and readily metabolized, but differing in their effects on the redox state of the hepatic NAD couples, were given to starved rats and the contents of metabolites were measured in freezeclamped liver and in the blood. The object was to study the effects of changes in the redox state and of the availability of oxidizable substrates on the rate of ketone-body formation. 2. Intramuscular administration of dihydroxyacetone, glycerol or glucose to starved rats decreased the concentrations of acetoacetate and 3-hydroxybutyrate in the blood by 70–80% within 60min., whereas there was no major change in the free fatty acid concentration. 3. Dihydroxyacetone, but not glucose or glycerol, caused an immediate and sustained twofold increase in the blood lactate concentration. 4. Dihydroxyacetone and glycerol caused a rapid fall in the hepatic concentrations of ketone bodies, dihydroxyacetone being more effective. 5. This decrease was not accompanied by significant changes in the concentrations of acetyl-CoA, long-chain acyl-CoA or free CoA. 6. The hepatic glycerophosphate concentration rose about 40-fold on administration of glycerol, whereas with dihydroxyacetone the increase was only about 50%. The large increase in glycerophosphate concentration after administration of glycerol was completely prevented by pretreatment of the rats with tri-iodothyronine. Triiodothyronine-treated rats showed the same decrease in ketone-body concentrations after administration of glycerol as the untreated rats. 7. Glycerol and dihydroxyacetone caused an increase in the hepatic lactate concentration; the pyruvate concentration rose only after injection of dihydroxyacetone. 8. Both compounds increased liver glycogen. 9. Calculation of the [free NAD+]/[free NADH] ratios indicated that dihydroxyacetone increased the ratio in cytoplasm and mitochondria, whereas glycerol caused a prompt fall in both compartments, followed at 10min. by a slight rise in the mitochondrial compartment. 10. Dihydroxyacetone did not alter the hepatic content of ATP. 11. The findings suggest that the main reason for the antiketogenic effect of glycerol and dihydroxyacetone was a consequence of their ready metabolism and the provision of an increased supply of C3 intermediates for conversion into oxaloacetate. Under the test conditions, neither the hepatic content of α-glycerophosphate nor the redox state of the NAD couples appeared to play a major role in the regulation of ketogenesis.
Mass-spectrometric determination of the amino acid sequences in peptides isolated from protein silk fibroin of Bombyx mori
Authors: Geddes A J , Graham G N , Morris H R , Lucas F. , Barber M. , Wolstenholme W. A.
Published: August 2015

Several peptides were isolated from the protein silk fibroin of Bombyx mori by means of ion-exchange chromatography of a chymotryptic digest. The sequences of three of the peptides, Gly-Ala-Gly-Tyr, Gly-Val-Gly-Tyr and Gly-Ala-Gly-Ala-Gly-Ala-Gly-Tyr, were known from previous chemical work, but the sequence of the fourth, Gly-Ala-Gly-Val-Gly-Ala-Gly-Tyr, was previously only partially known. The necessary volatility for mass-spectrometric examination of the peptides was achieved by permethylation of the N-acetyl-peptide methyl ester derivatives. From the mass spectra it was possible to confirm the known sequences and to establish that of the partially known one. In one instance it was possible to deduce from the same mass spectrum the sequence of a main peptide component and that of a small amount of contaminating peptide. These results demonstrate for the first time the use of mass spectrometry in the determination of the amino acid sequences in peptides from a protein hydrolysate.
Studies on the structure of hyaluronic acid. Characterization of the product formed when hyaluronic acid is treated with ascorbic acid
Authors: Swann David A.
Published: August 2015

Physical and chemical methods were used to characterize hyaluronic acid before (fraction HAIIBI) and after (fraction HA-AA) treatment with ascorbic acid. Fraction HA-AA was recovered with an almost quantitative yield and was shown to be chemically identical with fraction HAIIBI by all the methods used. These two materials, however, differed markedly in their molecular sizes and degree of polydispersity. By using sedimentation, diffusion and sedimentation-equilibrium analyses, weight-average molecular weights of about 1·2×106 and 6·5×104 respectively were obtained for fractions HAIIBI and HA-AA. It is concluded from these results that hyaluronic acid has a molecular weight of about 65000 and that the polysaccharide chain of this molecule is not depolymerized by ascorbic acid. It is further proposed that hyaluronic acid molecules in the matrix of connective tissues are present either in an aggregated form or as subunits of heterogeneous macromolecules, and that it is the linkages responsible for the organization of these structures which are broken by ascorbic acid.
Characterization of protein–polysaccharides of articular cartilage from mature and immature pigs
Authors: Brandt Kenneth D. , Muir Helen
Published: August 2015

Protein–polysaccharides of femoral articular cartilage from pigs of ages 9 months and 5 weeks were compared after extraction at pH6·8 with iso-osmotic sodium acetate followed by 0·63m-calcium acetate. The cartilage from the younger animals had a higher moisture content and contained considerably larger amounts of protein–polysaccharide, but less than half as much collagen/g. dry weight, than cartilage from the older pigs. There was notably less keratan sulphate in the fractions from the less mature animals. After gel filtration on 6% agarose, elution profiles of the calcium acetate extracts were similar to those of the sodium acetate extracts of the same tissue. Chemical analyses, however, showed that in both age-groups the extraction procedure had achieved a sequential solubilization of protein–polysaccharides in that the initial extracts contained a higher proportion of keratan sulphate than those that were extracted subsequently. Both extracts from the older animals contained up to 25% of a relatively small protein–polysaccharide that was retarded on 6% agarose and that had a lower protein content and less keratan sulphate than the larger protein–polysaccharides. In contrast, in extracts from the less mature cartilage only about 5% of the protein–polysaccharides were small enough to be retarded by 6% agarose, suggesting that the small components may not be precursors of the larger. The average length of chondroitin sulphate chains, as calculated from the analytical data, was the same in the smaller protein–polysaccharides as in the larger.
Strain differences in the activities of rat liver enzymes
Authors: Eggleston L. V. , Krebs H. A.
Published: August 2015

1. The activities of four enzymes of glycolysis were assayed in the liver of five different strains of rats (four ‘Wistar-derived’, one Sprague–Dawley) kept on three different but very similar diets. 2. Major strain differences were found for the activities of pyruvate kinase (3-fold), α-glycerophosphate dehydrogenase (5-fold), glyceraldehyde phosphate dehydrogenase (3-fold) and triokinase (1·5-fold). 3. Although the initial activities of pyruvate kinase differed greatly the percentage responses to starvation or a diet high in soluble carbohydrate were of the same order in two strains. 4. The importance of considering strain differences is emphasized when making comparisons of measurements carried out in different laboratories.
Incorporation of [2−14C,(5R)-5−3H1]mevalonic acid into cholesterol by a rat liver homogenate and into β-sitosterol and 28-isofucosterol by Larix decidua leaves
Authors: Goad L J , Gibbons G. F. , Bolger Loretta M. , Rees H H , Goodwin T W
Published: August 2015

1. Incubation of a rat liver homogenate with 3R-[2−14C,(5R)-5−3H1]mevalonic acid gave cholesterol with 3H/14C atomic ratio 6:5. 2. Conversion of the labelled cholesterol into 3β-acetoxy-6-nitrocholest-5-ene or cholest-4-ene-3,6-dione resulted in the loss of one tritium atom from C-6. 3. These results show that during cholesterol biosynthesis the 6α-hydrogen atom of a precursor sterol is eliminated during formation of the C-5–C-6 double bond. 4. Incorporation of 3R-[2−14C,(5R)-5−3H1]mevalonic acid into the sterols of larch (Larix decidua) leaves gave labelled cycloartenol and β-sitosterol with 3H/14C atomic ratios 6:6 and 6:5 respectively. 5. One tritium atom was lost from C-6 on conversion of the labelled β-sitosterol into either 3β-acetoxy-6-nitrostigmast-5-ene or stigmast-4-ene-3,6-dione, demonstrating that formation of the C-5–C-6 double bond of phytosterols also involves the elimination of the 6α-hydrogen atom of a precursor sterol. 6. The 3R-[2−14C,(5R)-5−3H1]mevalonic acid was also incorporated by larch (L. decidua) leaves into a sterol that co-chromatographed with 28-isofucosterol. Confirmation that the radioactivity was associated with 28-isofucosterol was obtained by co-crystallization with carrier 28-isofucosterol and ozonolysis of the acetate to give radioactively labelled 24-oxocholesteryl acetate. 7. The significance of these results to phytosterol biosynthesis is discussed.
Effects of added adenine nucleotides on renal carbohydrate metabolism
Authors: Weidemann M. J. , Hems D. A. , Krebs H. A.
Published: August 2015

1. The regulatory effects that adenine nucleotides are known to exert on enzymes of glycolysis and gluconeogenesis were demonstrated to operate in kidney-cortex slices and in the isolated perfused rat kidney by the addition of exogenous ATP, ADP and AMP to the incubation or perfusion media. 2. Both preparations rapidly converted added ATP into ADP and AMP, and ADP into AMP; added AMP was rapidly dephosphorylated. AMP formed from ATP was dephosphorylated at a lower rate than was added AMP, especially when the initial ATP concentration was high (10mm). Deamination of added AMP occurred more slowly than dephosphorylation of AMP. 3. Gluconeogenesis from lactate or propionate by rat kidney-cortex slices, and from lactate by the isolated perfused rat kidney, was inhibited by the addition of adenine nucleotides to the incubation or perfusion media. In contrast, oxygen consumption and the utilization of propionate or lactate by slices were not significantly affected by added ATP or AMP. 4. The extent and rapidity of onset of the inhibition of renal gluconeogenesis were proportional to the AMP concentration in the medium and the tissue, and were not due to the production of acid or Pi or the formation of complexes with Mg2+ ions. 5. Glucose uptake by kidney-cortex slices was stimulated 30–50% by added ATP, but the extra glucose removed was not oxidized to carbon dioxide and did not all appear as lactate. Glucose uptake, but not lactate production, by the isolated perfused kidney was also stimulated by the addition of ATP or AMP. 6. In the presence of either glucose or lactate, ATP and AMP greatly increased the concentrations of C3 phosphorylated intermediates and fructose 1,6-diphosphate in the kidney. There was a simultaneous rise in the concentration of malate and fall in the concentration of α-oxoglutarate. 7. The effects of added adenine nucleotides on renal carbohydrate metabolism seem to be mainly due to an increased concentration of intracellular AMP, which inhibits fructose diphosphatase and deinhibits phosphofructokinase. This conclusion is supported by the accumulation of intermediates of the glycolytic pathway between fructose diphosphate and pyruvate. 8. ATP or ADP (10mm) added to the medium perfusing an isolated rat kidney temporarily increased the renal vascular resistance, greatly diminishing the flow rate of perfusion medium for a period of several minutes.
Differential stability of 28s and 18s rat liver ribosomal ribonucleic acids
Authors: Venkov P. V. , Hadjiolov A. A.
Published: August 2015

Rat liver ribosomal RNA (rRNA) free from nuclease contaminants was isolated by a modification of the phenol technique. The 28s and 18s rRNA species were separated by preparative agar-gel electrophoresis. The two rRNA species were heated at different temperatures under various conditions and the amount of undegraded rRNA was determined by analytical agar-gel electrophoresis. The 18s rRNA remained unaltered after heating for up to 10min. at 90° in water, acetate buffer, pH5·0, or phosphate buffer, pH7·0. Under similar or milder conditions 28s rRNA was partially degraded, giving rise to a well-delimited 6s peak and a heterogeneous material located in the zone between 28s and 6s. The dependence of degradation of 28s rRNA on the temperature and the ionic strength of the medium was studied. The greatest extent of degradation of 28s rRNA was observed on heating at 90° in water. It is suggested that the instability of rat liver 28s rRNA is due to two factors: the presence of hidden breaks in the polymer chain and a higher susceptibility of some phosphodiester bonds to thermal hydrolysis.
Developmental and regional variations in ribonucleic acid synthesis on cerebral chromatin
Authors: Bondy S. C. , Roberts Sidney
Published: August 2015

1. Chromatin was prepared from purified nuclei isolated from liver and cerebral regions of the rat. 2. The capacity of these preparations to promote RNA synthesis in the presence of bacterial RNA polymerase was determined. 3. The rate of RNA synthesis on chromatin was normally 12–21% of the rate observed with native DNA, but was markedly stimulated on addition of 200mm-ammonium sulphate. 4. At physiological concentrations (80μg./ml.), the brain-specific S-100 protein inhibited RNA synthesis on DNA and chromatin. 5. Cerebral chromatin from foetal and newborn animals was more active in RNA synthesis than were the analogous preparations from liver. 6. Cerebellar chromatin maintained a high rate of RNA synthesis during brain maturation. In contrast, RNA synthesis on chromatin from other brain regions and liver declined with age of the rat. 7. RNA synthesized on chromatin stimulated amino acid incorporation in an Escherichia coli ribosomal system and hybridized with homologous DNA. 8. RNA synthesized on chromatin from adult cortex or hindbrain hybridized with DNA to a greater extent than that synthesized on cerebellar chromatin. 9. The proportion of RNA formed on cerebral-cortical chromatin that hybridized with DNA increased with age of the rat. 10. The results indicate that the total amount and the types of RNA synthesized on cerebral chromatin vary regionally and during development.
Quantitative determination of deoxyribonucleic acid in rat brain
Authors: Penn N. W. , Suwalski R.
Published: August 2015

1. A procedure is given for spectrophotometric analysis of rat brain DNA after its resolution into component bases. Amounts of tissue in the range 50–100mg. can be used. 2. The amount of DNA obtained by the present method is 80% greater than that reported for rat brain by a previous procedure specific for DNA thymine. Identity of the material is established by the base ratios of purines and pyrimidines. The features responsible for the higher yield are the presence of dioxan during alkaline hydrolysis of tissue, the determination of the optimum concentration of potassium hydroxide in this step and omission of organic washes of the initial acid-precipitated residues. 3. The requirement for dioxan during alkaline hydrolysis suggests a possible association of brain DNA with lipid. The concentration of potassium hydroxide that gives maximum yield is 0·1m, indicating that there may be internucleotide linkages in this DNA that are more sensitive to alkali than those of liver or thymus DNA. 4. This procedure gives low yields of DNA from liver. It is not suitable for analysis of the DNA from this tissue.
Studies on the metabolism of steroids in the foetus. Biosynthesis of 6α-hydroxytestosterone in the human foetal liver
Authors: Lisboa B. P. , Gustafsson J.-Å.
Published: August 2015

After incubation of testosterone with 105000g microsomes of human foetal liver, 6α-hydroxytestosterone was isolated and identified by t.l.c. and g.l.c.–mass spectrometry. This is the first example of 6α-hydroxylation of C19 steroids in the human liver, and the finding is discussed in relation to earlier reports of 6-oxygenated C19 and C18 steroids in pregnant women.
Purification and properties of d-4-deoxy-5-oxoglucarate hydro-lyase (decarboxylating)
Authors: Jeffcoat R. , Hassall H. , Dagley S.
Published: August 2015

1. An enzyme extracted from Pseudomonas acidovorans was purified and shown to catalyse the simultaneous dehydration and decarboxylation of d-4-deoxy-5-oxoglucarate. It is proposed to name the enzyme d-4-deoxy-5-oxoglucarate hydro-lyase (decarboxylating), trivial name ‘deoxyoxoglucarate dehydratase’. 2. No added cofactors were required, and the enzyme was inactivated when incubated with its substrate in the presence of sodium borohydride. Under these conditions the substrate and enzyme appeared to be bound covalently. 3. The action of the enzyme is readily explained if it is assumed that d-4-deoxy-5-oxoglucarate forms a Schiff base with a lysine residue in the enzyme.
Transport of reduced nicotinamide–adenine dinucleotide into mitochondria of rat white adipose tissue
Authors: Robinson B. H. , Halperin M. L.
Published: August 2015

Mitochondria from rat white adipose tissue were prepared, exhibiting good respiratory control and P/O ratios. They would not oxidize NADH unless NNN′N′-tetramethyl-p-phenylenediamine was added as a carrier of reducing equivalents. These mitochondria were found to oxidize neither l-glycerol 3-phosphate nor l-glutamate plus l-malate at significant rates. The activity of aspartate aminotransferase in these mitochondria was found to be low compared with that found in rat liver mitochondria. As a consequence of this, the adipose-tissue mitochondria exhibited very low rates of cytoplasmic NADH oxidation in a reconstituted Borst (1962) cycle compared with liver mitochondria.
The mechanism of action of serine transhydroxymethylase
Authors: Jordan P. M. , Akhtar M.
Published: August 2015

1. The preparation of stereospecifically tritiated glycines and the determination of their absolute configurations by the use of d-amino acid oxidase are described. 2. The reaction catalysed by serine transhydroxymethylase, which results in the conversion of glycine into serine, has been separated into at least four partial reactions. It is suggested that the first event in this conversion is the formation of a Schiff base intermediate of glycine and pyridoxal phosphate. The next important step involves the removal of the 2S-hydrogen atom of glycine to give a carbanion intermediate. Experiments pertinent to the mechanism of conversion of this carbanion intermediate into serine are described. 3. The enzyme preparation catalysing the conversion of glycine into serine also participates in the conversion of glycine into threonine and allothreonine. In both these conversions, glycine → serine and glycine → threonine, the 2S-hydrogen atom of glycine is eliminated and the 2R-hydrogen atom of glycine is retained. 4. In the light of these experiments the mechanism of action of serine transhydroxymethylase is discussed. It is suggested that methylenetetrahydrofolate is the carrier of formaldehyde, from which formaldehyde may be liberated at the active site of the enzyme, thus allowing the overall reaction to take place.
Studies on heterogeneity of human gastric zymogens
Authors: Turner Michael D. , Mangla Jagdish C. , Samloff I. Michael , Miller Leon L. , Segal Harry L.
Published: August 2015

The peptic zymogens of human gastric mucosa have been studied in the past by three different techniques: phosphate-gradient chromatography on a column of DEAE-cellulose, chloride-gradient chromatography on DEAE-cellulose and agar-gel electrophoresis. These techniques have given different results, each suggesting the presence of a different number of pepsin zymogens in human gastric mucosa. In the present experiments gastric mucosal homogenates were subjected to analysis by all three techniques. The two major zymogen peaks obtained from phosphate-gradient chromatography were found to be composed of the three fractions found on chloride-gradient chromatography; these fractions in turn were shown to be heterogeneous on agar-gel electrophoresis. The latter technique demonstrated the presence of seven separable enzymically active components in human gastric mucosal homogenates. The relationships of the components separated by the three techniques are discussed.
The metabolic fate of amphetamine in man and other species
Authors: Dring L. G. , Smith R. L. , Williams R. T.
Published: August 2015

1. The fate of [14C]amphetamine in man, rhesus monkey, greyhound, rat, rabbit, mouse and guinea pig has been studied. 2. In three men receiving orally 5mg each (about 0.07mg/kg), about 90% of the 14C was excreted in the urine in 3–4 days. About 60–65% of the 14C was excreted in 1 day, 30% as unchanged drug, 21% as total benzoic acid and 3% as 4-hydroxyamphetamine. 3. In two rhesus monkeys (dose 0.66mg/kg), the metabolites excreted in 24h were similar to those in man except that there was little 4-hydroxyamphetamine. 4. In greyhounds receiving 5mg/kg intraperitoneally the metabolites were similar in amount to those in man. 5. Rabbits receiving 10mg/kg orally differed from all other species. They excreted little unchanged amphetamine (4% of dose) and 4-hydroxyamphetamine (6%). They excreted in 24h mainly benzoic acid (total 25%), an acid-labile precursor of 1-phenylpropan-2-one (benzyl methyl ketone) (22%) and conjugated 1-phenylpropan-2-ol (benzylmethylcarbinol) (7%). 6. Rats receiving 10mg/kg orally also differed from other species. The main metabolite (60% of dose) was conjugated 4-hydroxyamphetamine. Minor metabolites were amphetamine (13%), N-acetylamphetamine (2%), norephedrine (0.3%) and 4-hydroxynorephedrine (0.3%). 7. The guinea pig receiving 5mg/kg excreted only benzoic acid and its conjugates (62%) and amphetamine (22%). 8. The mouse receiving 10mg/kg excreted amphetamine (33%), 4-hydroxyamphetamine (14%) and benzoic acid and its conjugates (31%). 9. Experiments on the precursor of 1-phenylpropan-2-one occurring in rabbit urine suggest that it might be the enol sulphate of the ketone. A very small amount of the ketone (1–3%) was also found in human and greyhound urine after acid hydrolysis.
Characterization of rapidly labelled rat liver ribonucleic acid showing high affinity for columns of methylated albumin on kieselguhr
Authors: Kunz W. , Niessing J. , Schnieders B. , Sekeris C. E.
Published: August 2015

Most of the rapidly labelled RNA from rat liver submitted to column chromatography on methylated albumin on kieselguhr remains tightly bound to the column and can only be recovered by elution with m-ammonia. The tightly bound RNA is composed mainly of DNA-like RNA. The binding capacity is dependent not only on base composition but also on molecular size: the heavier RNA molecules show a greater affinity to the column than do the lower-molecular-weight components. Rapidly labelled mouse liver and Saccharomyces cerevisiae RNA show similar behaviour to rat liver RNA on columns of methylated albumin on kieselguhr.
The subunits of thyroglobulin
Authors: Charlwood P. A. , Pitt-Rivers Rosalind , Schwartz H. L.
Published: August 2015

1. Pig thyroglobulin was reduced with dithiothreitol in the presence of sodium dodecyl sulphate, 8m-urea or 6m-guanidinium chloride. 2. The molecular weights of the reduction products were about 165000. 3. Two stable subunits with molecular weights as low as 35000 and 20000 were separated by Sephadex chromatography after prolonged exposure of the reduction products to sodium dodecyl sulphate at pH8.7. Although a hydrolytic reaction is probably implicated, the nature of the chemical linkages broken was not established.
The effect of substrate concentration and pH on the enzymic sulphation of l-tyrosyl derivatives
Authors: Mattock P. , Barford D. J. , Basford J. M. , Jones J. G.
Published: August 2015

1. The kinetics of the enzymic transfer of sulphate from adenosine 3′-phosphate 5′[35S]-sulphatophosphate to derivatives of l-tyrosine were investigated with a partially purified enzyme preparation from rat liver. 2. At pH7.5 and 37°C the Km values for l-tyrosine methyl ester and adenosine 3′-phosphate 5′[35S]-sulphatophosphate are 0.3mm and 8nm respectively. The Km value for either substrate is independent of the concentration of the other. The available data are consistent with the sulphation reaction proceeding according to a rapid-equilibrium random Bi Bi mechanism. 3. From the effect of pH on the Km and Vmax. values for l-tyrosine methyl ester, tyramine and N-acetyl-l-tyrosine ethyl ester it is concluded that the enzyme is specific for substrate molecules with a free and unprotonated amino group and an un-ionized hydroxyl group. 4. The only ionizing group that can be positively attributed to the enzyme appears to influence the binding of adenosine 3′-phosphate 5′[35S]-sulphatophosphate and has an apparent pK value of approx. 9.5. It is suggested that this group may be an essential thiol. 5. The enzyme is inhibited by iodoacetamide at pH7.5 and 30°C and this inhibition is prevented by the presence of adenosine 3′-phosphate 5′[35S]-sulphatophosphate but not by l-tyrosine methyl ester.
The isolation of three neurophysins from porcine posterior pituitary lobes
Authors: Uttenthal L. O. , Hope D. B.
Published: August 2015

1. Three neurophysins, proteins that bind the polypeptide hormones oxytocin and vasopressin, have been isolated from acetone-dried porcine posterior pituitary lobes. The proteins have been named porcine neurophysins-I, -II and -III in order of their electrophoretic mobilities at pH8.1. 2. Electrophoretic comparison of the purified proteins, which are homogeneous on starch-gel electrophoresis, with the soluble proteins of fresh porcine posterior pituitary lobes extracted in 0.1m-HCl and in buffer pH8.1 suggests that the isolated proteins are native to the fresh tissue. 3. Neurophysins-I and -II are present in similar amounts in the tissue, whereas neurophysin-III is present only in small quantities. Acetone-dried tissue also contains traces of other hormone-binding neurophysin components. 4. All the neurophysins can bind both oxytocin and [8-lysine]-vasopressin. 5. The apparent molecular weights of the neurophysins increase with increasing protein concentration as measured by equilibrium sedimentation in the ultracentrifuge. 6. Neurophysins-I and -III are of similar molecular dimensions, contain one residue of methionine per molecule and lack histidine. The minimum molecular weight of neurophysin-I obtained by amino acid analysis is 9360. Neurophysin-II is of larger molecular dimensions than neurophysins-I and -III and can be separated from these by gel filtration on Sephadex G-75. It contains no histidine or methionine, and its minimum molecular weight has been estimated as 14020 by amino acid analysis. 7. Each of the three neurophysins possesses N-terminal alanine. 8. The possible biological significance of the existence of several neurophysins within one species is discussed.
The amino acid sequence of Phaseolus aureus L. (mung-bean) cytochrome c
Authors: Thompson E. W. , Laycock M. V. , Ramshaw J. A. M. , Boulter D.
Published: August 2015

The amino acid sequence of Phaseolus aureus L. (mung-bean) cytochrome c has been determined. The molecule consists of a single polypeptide chain of 111 amino acid residues and is homologous with other mitochondrial cytochromes c. Comparison with the amino acid sequence of wheat-germ cytochrome c (Stevens, Glazer & Smith, 1967) shows 14 differences. On alignment with mammalian cytochromes c, mung-bean cytochrome c has an N-acetylated ‘tail’ of eight amino acid residues similar to that found in wheat-germ cytochrome c. Of the 22 positions in wheat-germ cytochrome c that contain amino acid residues unique to these positions, 20 were found to contain the same ones in mung-bean cytochrome c. The ∈-N-trimethyl-lysine residues reported for wheat-germ cytochrome c (Delange, Glazer & Smith, 1969) in positions 72 and 86 were also found in these positions in mung-bean cytochrome c. The sequence was determined from 3μmol, by using chymotryptic and tryptic peptides which were analysed by the ‘dansyl’–Edman method (Gray & Hartley, 1963a), with confirmation by amino acid analysis.
The biosynthesis of 4-hydroxycoumarin and dicoumarol by Aspergillus fumigatus Fresenius
Authors: Bye A. , King H. K.
Published: August 2015

A strain of Aspergillus fumigatus Fresenius, isolated from spoiled hay, converts melilotic acid (o-hydroxyphenylpropionic acid) and o-coumaric acid into 4-hydroxycoumarin and dicoumarol. The sequence is shown to be melilotic acid (I) [Formula: see text] coumaric acid (IV) [Formula: see text] β-hydroxymelilotic acid (II) [Formula: see text] β-oxomelilotic acid (III) [Formula: see text] 4-hydroxycoumarin (VI), on the basis of (1) studies on the formation of postulated intermediates, (2) experiments with isotopically labelled materials and (3) sequential enzyme induction. In the presence of semicarbazide, o-coumaraldehyde is formed from o-coumaric acid: there is no evidence, however, that this lies on the normal metabolic pathway.
Steroid metabolism in the cat. Biliary and urinary excretion of metabolites of [4-14C]oestradiol
Authors: Karim M. F. , Taylor W.
Published: August 2015

1. [4-14C]Oestradiol was administered to seven male, seven female and two castrated male cats as a single intravenous injection. Bile and urine were collected for 6h. 2. The radioactivity was excreted mainly in the bile of all animals (53–60%); only approx. 1% of the dose appeared in the urine. 3. Bile and urine samples were hydrolysed successively by β-glucuronidase, cold acid and hot acid. There were significant differences (P<0.005) between the percentage of the dose present in the bile fractions hydrolysed by β-glucuronidase (male, 9.0±1.7%; female, 18.6±1.45%) and by cold acid (male, 18.9±1.44%; female 12.1±1.02%). The excretion of radioactivity in these fractions by the castrated male cats was closer to that of female cats. 4. Approx. 20–27% of the dose could not be extracted from aqueous solution (pH10.5) by ethyl acetate–ether after hydrolysis.
The reactivity of thiol groups and the subunit structure of aldolase
Authors: Anderson P. J. , Perham R. N.
Published: August 2015

1. Seven unique carboxymethylcysteine-containing peptides have been isolated from tryptic digests of rabbit muscle aldolase carboxymethylated with iodo[2-14C]acetic acid in 8m-urea. These peptides have been characterized by amino acid and end-group analysis and their location within the cyanogen bromide cleavage fragments of the enzyme has been determined. 2. Reaction of native aldolase with 5,5′-dithiobis-(2-nitrobenzoic acid), iodoacetamide and N-ethylmaleimide showed that a total of three cysteine residues per subunit of mol.wt. 40000 were reactive towards these reagents, and that the modification of these residues was accompanied by loss in enzymic activity. Chemical analysis of the modified enzymes demonstrated that the same three thiol groups are involved in the reaction with all these reagents but that the observed reactivity of a given thiol group varies with the reagent used. 3. One reactive thiol group per subunit could be protected when the modification of the enzyme was carried out in the presence of substrate, fructose 1,6-diphosphate, under which conditions enzymic activity was retained. This thiol group has been identified chemically and is possibly at or near the active site. Limiting the exposure of the native enzyme to iodoacetamide also served to restrict alkylation to two thiol groups and left the enzymic activity unimpaired. The thiol group left unmodified is the same as that protected by substrate during more rigorous alkylation, although it is now more reactive towards 5,5′-dithiobis-(2-nitrobenzoic acid) than in the native enzyme. 4. Conversely, prolonged incubation of the enzyme with fructose 1,6-diphosphate, which was subsequently removed by dialysis, caused an irreversible fall in enzymic activity and in thiol group reactivity measured with 5,5′-dithiobis-(2-nitrobenzoic acid). 5. It is concluded that the aldolase tetramer contains at least 28 cysteine residues. Each subunit appears to be identical with respect to number, location and reactivity of thiol groups.