bluuebunny/crossref_metadata_2025 huggingface dataset:
A gas-liquid-chromatographic procedure for separating a wide range of metabolites occurring in urine or tissue extracts
Authors: Dalgliesh CE , Horning EC , Horning MG , Knox KL , Yarger K
Published: August 2015

1. A gas-liquid-chromatographic procedure is described which permits separation and identification on the same chromatogram of a wide range of substances occurring in urine or tissue extracts. The method uses hydrogen flame ionization, which detects organic compounds whether free or conjugated with no requirement for specific reactive groups. 2. For chromatography, carboxyl groups are quantitatively converted into methyl esters or trimethylsilyl esters. Phenolic, alcoholic and potential enolic groups are converted into trimethylsilyl ethers. Separations are carried out on a 6ft. column of either 10% F-60 (a polysiloxane) or 1% F-60, temperature programming at 2 degrees /min. being used over such part of the temperature range 30 degrees -260 degrees as is required. Propionyl derivatives of hydroxy compounds can also be used, but only on a non-quantitative basis. Derivatives and columns have been selected for optimum range of usefulness when large numbers of samples are examined by using automated gas chromatography. 3. The method is applicable to: fatty acids above butyric acid; di- and tri-carboxylic acids; hydroxy acids and keto acids; polyhydroxy and alicyclic compounds such as glycerol, inositol, quinic acid, shikimic acid, ascorbic acid and sugar alcohols; aromatic hydroxy and acidic compounds, both benzenoid and indolic; sesquiterpenes; steroids; glycine conjugates; mercapturic acids; glucuronides. It is not satisfactory for sulphate conjugates, iminazoles or polypeptides. 4. Methylene units provide an accurate and reproducible parameter for characterizing peak position. Methylene unit values are reported for a large variety of substances occurring in, or related to those occurring in, urine and tissue extracts. 5. The nature of derivatives was confirmed by combining gas chromatography with mass spectrometry. Combined gas chromatography-mass spectrometry gives a diagnostic tool of great power in the evaluation of metabolic patterns, and various uses are discussed.
Biosynthesis of intestinal mucins. Sialic acids of sheep colonic epithelial mucin
Authors: Kent P W , Draper P.
Published: August 2015

1. Sheep colonic mucin contains three types of sialic acids, separable from the macrostructure by mild acidic hydrolysis. These are composed chiefly of N-acetyl-and N-glycollyl-neuraminic acid in ratios between 1:1·2 and 1:3·5 for different preparations of the mucin. The third sialic acid appears to be a diacetylated neuraminic acid. 2. A particle-free enzyme preparation, obtained from sheep colonic mucosa by gentle homogenization and high-speed centrifugation, catalyses a series of reactions involving N-acylamino sugars and leading to the formation of sialic acids in vitro: (i) phosphorylation by ATP of d-glucosamine, N-acetyl-and N-glycollyl-d-glucosamine; (ii) conversion of N-acetylglucosamine 6-phosphate into N-acetyl-d-glucosamine 1-phosphate; (iii) formation of sialic acids from phosphoenolpyruvate and N-acetyl- or N-glycollyl-d-glucosamine; (iv) formation of N-acetylneuraminic acid from uridine diphospho-N-acetylglucosamine or from N-acetylmannosamine; (v) incorporation of l-[U−14C]serine into the mucin by whole mucosal preparations.
Degradation of insulin in vitro by liver and epididymal adipose tissue from obese–hyperglycaemic mice
Authors: Westman Sighild
Published: August 2015

The insulin-degrading activity of liver supernatants and epididymal adipose-tissue homogenates from genetically obese–hyperglycaemic mice (ob ob) and their lean litter mates was studied by measurement of radioactive trichloroacetic acid-soluble degradation products of the insulin molecule. Optimum assay conditions for the decomposition of the hormone were devised. The properties of the degrading activity suggested the presence of enzymic insulin destruction in both the liver and epididymal adipose tissue. There was no difference in insulin degradation in liver samples from obese and lean mice when the results were related to the protein content of the supernatants. The epididymal adipose-tissue homogenates from obese mice displayed about eightfold higher degrading activity per unit of protein than did homogenates from lean animals. The physiological significance of this finding is discussed in the light of the increased fat depots, hyperphagia, raised serum insulin concentrations and increased insulin tolerance previously recorded in this strain of mice.
The estimation of galactose, mannose and fucose in glycoproteins by radioisotope dilution
Authors: Graham E. R. B. , Neuberger A.
Published: August 2015

1. The principle of radioisotope dilution, as used previously for the estimation of mannose in egg albumin, was applied on a semi-micro scale to the estimation of fucose, mannose and galactose in some glycoproteins. The sugars were separated by partition chromatography on columns of Celite 545. 2. The release of mannose from egg albumin in 2n-hydrochloric acid at 100° after various times was determined by the radioisotope-dilution method and found to have a half-time of 7min. 3. The destruction of mannose in 2n-hydrochloric acid after 3hr. at 100° was found to be small if air was excluded. The destruction was slightly increased by the presence of lysozyme containing tryptophan in an amount equimolar with the mannose. The same amount of free tryptophan caused considerable loss of mannose. 4. Analytical values are reported for the non-amino sugar contents of egg albumin, rabbit γ-globulin and some samples of blood-group-specific substances. The values found were similar to the most reliable estimates published previously.
The assimilation of carbon by Chloropseudomonas ethylicum
Authors: Callely A G , Rigopoulos N. , Fuller R. C.
Published: August 2015

1. The enzymes in ultrasonically prepared extracts of Chloropseudomonas ethylicum were studied to elucidate how this organism assimilates acetate and carbon dioxide and why it cannot grow with either of these two compounds alone. 2. Such extracts can (i) convert acetate and oxaloacetate into α-oxoglutarate, (ii) convert oxaloacetate into succinyl-CoA, (iii) convert phosphopyruvate into 3-phosphoglyceraldehyde and (iv) interconvert phosphopyruvate and pyruvate via oxaloacetate. 3. Pyruvate kinase, α-oxoglutarate dehydrogenase, ribulose diphosphate carboxylase, isocitrate lyase and malate synthase were not detected. 4. It is difficult to detect aconitate hydratase, fumarate hydratase and citrate synthase in extracts of the organism ultrasonically treated in tris buffer; to demonstrate these enzymes extracts should be prepared in phosphate buffer containing 2-mercaptoethanol. 5. Provided that this organism can synthesize pyruvate from acetate and carbon dioxide, the enzymes detected are sufficient to account for the nutritional requirements of this organism.
The influence of dietary protein on ascorbic acid metabolism in rats
Authors: Mukherjee Dipti , Kar N. C. , Sasmal N. , Chatterjee G. C.
Published: August 2015

1. d-Glucuronolactone reductase, l-gulonolactone oxidase, uronolactonase, dehydroascorbatase, l-gulonate dehydrogenase and l-gulonate decarboxylase have been measured in the tissues of rats fed on diets containing variable amounts of protein. Rats fed on a protein-free or a 2% casein diet for 15 days showed a marked decline in the activities of d-glucuronolactone reductase, l-gulonolactone oxidase, uronolactonase and dehydroascorbatase in the liver, and no change in l-gulonate dehydrogenase and l-gulonate decarboxylase activities in the kidney when compared with rats fed on diets containing 9%, 18% or 25% casein. Giving diets containing 60% or 88% casein to rats did not appreciably alter the activities of uronolactonase, dehydroascorbatase, l-gulonate dehydrogenase and l-gulonate decarboxylase, but inhibited considerably the activities of d-glucuronolactone reductase and l-gulonolactone oxidase in the liver, resulting in decreased synthesis of ascorbic acid. 2. Rats fed on a 25% casein diet showed maximal weight gain, higher tissue reserve of ascorbic acid and higher urinary excretion of both ascorbic acid and glucuronic acid when compared with rats fed on diets containing lower or higher amounts of protein.
Studies on alkaline phosphatase. Phosphorylation of calf intestinal alkaline phosphatase by 32P-labelled pyrophosphate
Authors: Fernley H N , Bisaz Sylvia
Published: August 2015

1. A purified preparation of alkaline phosphatase from calf-intestinal mucosa was phosphorylated by 32P-labelled PPi at a serine residue on the enzyme. Under the conditions employed, up to 0·15μm-labelled sites were obtained from 1μm-[32P]PPi. 2. The phosphorylated enzyme was labile, the rate of dephosphorylation being similar to the overall rate of substrate hydrolysis. 3. A stopped-flow technique was used to determine the number of phosphomonoesterase active sites, which agreed with the number of 32P-labelled sites. 4. It is concluded that calf-intestinal alkaline phosphatase is both a phosphomonoesterase and a pyrophosphatase.
The periodate oxidation of bovine bone sialoprotein, and some observations on its structure
Authors: Andrews A. T. De B. , Herring G. M. , Kent P. W.
Published: August 2015

1. Bovine bone sialoprotein (mol.wt. 23000) contains N-acetylneuraminic acid and N-glycollylneuraminic acid, fucose, galactose, mannose, N-acetylgalactosamine and N-acetylglucosamine residues in the form of a very small number, perhaps one, of highly branched oligosaccharide structures linked covalently to peptide. 2. Periodate oxidation of the sialoprotein results in quantitative destruction only of the sialic acid and fucose residue consistent with the earlier findings of their positions as terminal groups. 3. Terminal sialic acid residues are attached to galactopyranose residues by 2,3-linkages, and to some N-acetylgalactosamine residues (at C-6). 4. Sequential Smith degradation indicates that N-acetylgalactosamine residues may be present as points of branching (linked in C-1, C-3 and C-6) and N-acetylglucosamine residues are located in the inner part of the structure, adjacent to the carbohydrate–peptide bond(s). 5. Mannose residues appear to be linked in the 1,3-positions.
Hydrolysis of dehydroepiandrosterone sulphate by human placental steroid 3β-sulphatase in [18O]water
Authors: Logan Geoffrey G. , Warren James C.
Published: August 2015

1. Dehydroepiandrosterone sulphate was hydrolysed by human placental steroid 3β-sulphatase in H218O. Equimolar amounts of dehydroepiandrosterone and sodium sulphate were similarly incubated as controls. After incubation, unconjugated steroid was extracted with ether and sulphate precipitated as barium sulphate. Both were analysed for 18O-content by mass spectroscopy, the sulphate as carbon dioxide after initial pyrolysis with graphite. 2. In duplicate experiments, amounts of 18O equivalent to 67% and 72% respectively of the theoretical content calculated for rupture of the O–S bond were present in the sulphate. As the enzyme preparation used was a microsomal preparation containing unenriched endogenous sulphate and phosphate, and as no incorporation of isotope was found in the steroid, it is concluded that the placental enzyme effects hydrolysis by rupture of the O–S bond.
The combination of carbon monoxide–haem with apoperoxidase
Authors: Phelps Charles , Antonini Eraldo
Published: August 2015

1. Static titrations reveal an exact stoicheiometry between various haem derivatives and apoperoxidase prepared from one isoenzyme of the horseradish enzyme. 2. Carbon monoxide–protohaem reacts rapidly with apoperoxidase and the kinetics can be accounted for by a mechanism already applied to the reaction of carbon monoxide–haem derivatives with apomyoglobin and apohaemoglobin. 3. According to this mechanism a complex is formed first whose combination and dissociation velocity constants are 5×108m−1sec.−1 and 103sec.−1 at pH9·1 and 20°. The complex is converted into carbon monoxide–haemoprotein in a first-order process with a rate constant of 235sec.−1 for peroxidase and 364sec.−1 for myoglobin at pH9·1 and 20°. 4. The effects of pH and temperature were examined. The activation energy for the process of complex-isomerization is about 13kcal./mole. 5. The similarity in the kinetics of the reactions of carbon monoxide–haem with apoperoxidase and with apomyoglobin suggests structural similarities at the haem-binding sites of the two proteins.
Iodination of insulin in aqueous and organic solvents
Authors: Massaglia A. , Rosa U. , Rialdi G. , Rossi C. A.
Published: August 2015

1. The iodination of insulin was studied under various experimental conditions in aqueous media and in some organic solvents, by measuring separately the uptake of iodine by the four tyrosyl groups and the relative amounts of monoiodotyrosine and di-iodotyrosine that are formed. In aqueous media from pH1 to pH9 the iodination occurs predominantly on the tyrosyl groups of the A chain. Some organic solvents increase the iodine uptake of the B-chain tyrosyl groups. Their efficacy in promoting iodination of Tyr-B-16 and Tyr-B-26 is in the order: ethylene glycol and propylene glycol≃methanol and ethanol>dioxan>8m-urea. 2. It is suggested that each of the four tyrosyl groups in insulin has a different environment: Tyr-A-14 is fully exposed to the solvent; Tyr-A-19 is sterically influenced by the environmental structure, possibly by the vicinity of a disulphide interchain bond; Tyr-B-16 is embedded into a non-polar area whose stability is virtually independent of the molecular conformation; Tyr-B-26 is probably in a situation similar to Tyr-B-16 with the difference that its non-polar environment depends on the preservation of the native structure.
Substrate specificity and kinetic properties of α-galactosidases from Vicia faba
Authors: Dey P. M. , Pridham J. B.
Published: August 2015

1. The hydrolysis of a variety of galactosides and other glycosides by α-galactosidases I and II of Vicia faba was studied. 2. The effect of temperature on kinetic parameters was also examined. 3. Both enzymes are inhibited by excess of substrate (p-nitrophenyl α-d-galactoside); with enzyme I this is competitive and is caused by the galactosyl moiety. 4. Enzyme I is inhibited by oligosaccharides possessing terminal non-reducing galactose residues and to a smaller extent by l-arabinose and d-fucose. 5. The effect of pH on Km and Vmax. values suggests that carboxyl and imidazole groups are involved in the catalytic activity of enzyme I. 6. Photo-oxidation experiments with enzyme I also suggest that an imidazole group is present at the active site.
An accurate method for measurement of aldosterone production
Authors: Thomas J. P. , Oake R. J.
Published: August 2015

A method is described for isolation of the acid-hydrolysable metabolite of aldosterone in sufficient purity to measure accurately the daily production rate. Values obtained with six hospital patients were 84–131μg./day on a daily intake of 100m-equiv. of Na+ and 227–464μg./day on a daily intake of 10m-equiv. of Na+. Corresponding values for aldosterone excretion were also recorded, but these are a poor index of production rate since they represent from 1·6 to 9·8% of the total daily output of aldosterone.
Studies on the preparation of water-insoluble derivatives of rennin and chymotrypsin and their use in the hydrolysis of casein and the clotting of milk
Authors: Green Margaret L. , Crutchfield G.
Published: August 2015

1. Enzymically active insoluble derivatives of chymotrypsin and rennin were prepared by coupling each enzyme to agarose as described by Porath, Axén & Ernback (1967) and rennin to aminoethylcellulose by the method of Habeeb (1967). 2. Agarose–chymotrypsin was stable over the range pH2–9, but agarose–rennin released active enzyme into solution at above pH2 and aminoethylcellulose–rennin was similarly unstable at certain pH values. 3. Each derivative appeared to catalyse the clotting of milk at 30°, but this was probably entirely due to enzyme released into solution from the carrier. 4. The presence of a competitive inhibitor of chymotrypsin during its coupling to agarose had no effect on the activity or stability of the resulting derivative. 5. The characteristics of agarose and cellulose render them not entirely suitable for use in a continuous system with milk.
Metabolism of vitamin D. A new cholecalciferol metabolite, involving loss of hydrogen at C-1, in chick intestinal nuclei
Authors: Lawson D. E. M. , Wilson P. W. , Kodicek E.
Published: August 2015

1. A comparison was made of the nature and intestinal intracellular distribution of the metabolites formed in vitamin D-deficient chicks from [4−14C]cholecalciferol and [1−3H]cholecalciferol. 2. The simultaneous administration of the two radioactive substances showed the presence in blood, liver, intestine, kidney and bone of cholecalciferol, its ester, 25-hydroxycholecalciferol and a further metabolite of cholecalciferol more polar than 25-hydroxycholecalciferol. The 3H/14C ratios in these four radioactive components were the same as that of the dosed material (4·7:1) with the exception of the most polar material. The 3H/14C ratio was lower in the fourth, most polar, metabolite (0·4:1–1·8:1) in all tissues examined, with the exception of blood. 3. In the chick intestine the polar metabolite accounted for almost 70% of the radioactivity in this tissue after a dose of 0·5μg. of [4−14C,1−3H]cholecalciferol. This polar metabolite from the intestine also had the lowest 3H/14C ratio of all the tissues. It appears that in the chick intestine the polar metabolite reaches a maximum concentration of 1ng./g. of tissue, above which it cannot be increased irrespective of the dose of the vitamin. 4. The intestinal intracellular organelle with the highest concentration of 14C radioactivity is the nucleus, and this radioactivity is almost entirely due to the polar metabolite with the lowered 3H/14C ratio, in this case <0·2:1. It appears to be further localized in the chromatin of the nuclei. However, about half of the polar metabolite in the intestine is extranuclear. 5. Double-labelled 25-hydroxycholecalciferol was prepared and after its administration to vitamin D-deficient chicks the polar metabolite with the lowered 3H/14C ratio was detected in liver, kidney, intestine, bone, muscle and heart. 6. None of the polar metabolite with the lowered 3H/14C ratio was detected 16hr. after dosing with either the double-labelled vitamin or the double-labelled 25-hydroxycholecalciferol in blood and adipose tissue of vitamin D-deficient chicks, nor in the intestine, liver and kidney of supplemented birds. 7. The reasons for this loss of 3H relative to 14C are discussed in relation to possible chemical structures of this new polar metabolite.
An automatic apparatus for the study of enzyme kinetics
Authors: Illingworth J. A. , Tipton K F
Published: August 2015

A continuous-flow apparatus is described for automatically plotting substrate saturation curves, and is suitable for use with a variety of enzymes. A linear concentration gradient of the variable substrate is combined with a fixed proportion of the other substrates and the enzyme, and after passing through a reaction coil the product concentrations are measured spectrophotometrically. Use of a 4cm. flow cell and modified spectrophotometer permits accurate measurement of NADH concentration in the region of 0·1μm. Precise control over reaction times and substrate concentration is achieved by using power-driven syringes with an integral mixer. Specimen results are given for yeast alcohol dehydrogenase.
The effects of unsaturated fatty acids on hepatic microsomal drug metabolism and cytochrome P-450
Authors: Di Augustine Richard P. , Fouts James R.
Published: August 2015

1. The effects of unsaturated fatty acids on drug-metabolizing enzymes in vitro were measured by using rat and rabbit hepatic 9000g supernatant fractions. 2. Unsaturated fatty acids inhibited the hepatic microsomal metabolism of ‘type I’ drugs with inhibition increasing with unsaturation: arachidonic acid>linolenic acid>linoleic acid>oleic acid. Inhibition was independent of lipid peroxidation. Linoleic acid competitively inhibited the microsomal O-demethylation of p-nitroanisole and the N-demethylation of (+)-benzphetamine. 3. The hepatic microsomal metabolism of ‘type II’ substrates, aniline and (−)-amphetamine, was not affected by unsaturated fatty acids. 4. The rate of reduction of p-nitrobenzoic acid and Neoprontosil was accelerated by unsaturated fatty acids. 5. Linoleic acid up to 3·5mm did not decelerate the generation of NADPH by rat liver soluble fraction, nor the activity of NADPH–cytochrome c reductase of rat liver microsomes. Hepatic microsomal NADPH oxidase activity was slightly enhanced by added linoleic acid. 6. No measurable disappearance of exogenously added linoleic acid occurred when this fatty acid was incubated with rat liver microsomes and an NADPH source. 7. The unsaturated fatty acids used in this study produced type I spectra when added to rat liver microsomes, and affected several microsomal enzyme activities in a manner characteristic of type I ligands.
Distribution, purification and properties of 1-aspartamido-β-N-acetylglucosamine amidohydrolase
Authors: Conchie J. , Strachan I.
Published: August 2015

1. The activity of the enzyme that splits 2-acetamido-1-l-β-aspartamido-1,2-dideoxy-β-d- glucose (1-aspartamido-β-N-acetylglucosamine) was measured in tissues from different mammalian species. 2. The enzyme from an aqueous extract of rat liver was purified 150-fold in 56% yield. 3. Optimum activity for the hydrolysis of 1-aspartamido-β-N-acetylglucosamine was at pH7, and ammonia and N-acetylglucosamine were liberated in equimolar amounts. At pH8·5, 1-amino-N-acetylglucosamine was the only sugar produced after short periods of incubation. On prolonged incubation there was spontaneous liberation of ammonia from this compound. 4. It is concluded that the enzyme is an amidase.
Penicillins and other acylamino compounds synthesized by the cell-bound penicillin acylase of Escherichia coli
Authors: Cole M.
Published: August 2015

1. The penicillin acylase of Eschericha coli N.C.I.B. 8743 is a reversible enzyme. Reaction rates for the two directions have been determined. 2. Measurements of the rates of enzymic synthesis of penicillins from 6-aminopenicillanic acid and various carboxylic acids revealed that p-hydroxyphenylacetic acid was the best substrate, followed by phenylacetic, 2-thienylacetic, substituted phenylacetic, 3-hexenoic and n-hexanoic acids. 3. The rate of synthesis of penicillin improved when amides or N-acylglycines were used; α-aminobenzylpenicillin and phenoxymethylpenicillin were only synthesized when using these more energy-rich compounds. 4. Phenyl-acetylglycine was the best substrate for the synthesis of benzylpenicillin compared with other derivatives of phenylacetic acid. 5. The enzyme was specific for acyl-l-amino acids, benzylpenicillin being synthesized from phenylacetyl-l-α-aminophenylacetic acid but not from phenylacetyl-d-α-aminophenylacetic acid. 6. α-Phenoxyethylpenicillin was synthesized from 6-aminopenicillanic acid and α-phenoxypropionylthioglycollic acid non-enzymically, but the rate was faster in the presence of the enzyme. 7. The E. coli acylase catalysed the acylation of hydroxylamine by acids or amides to give hydroxamic acids, the phenylacetyl group being the most suitable acyl group. The enzyme also catalysed other acyl-group transfers.
Regulation of synthesis of glutamate dehydrogenase and glutamine synthetase in micro-organisms
Authors: Pateman J. A.
Published: August 2015

1. Aspergillus nidulans, Neurospora crassa and Escherichia coli were grown on media containing a range of concentrations of nitrate, or ammonia, or urea, or l-glutamate, or l-glutamine as the sole source of nitrogen and the glutamate dehydrogenate and glutamine synthetase of the cells measured. 2. Aspergillus, Neurospora and Escherichia coli cells, grown on l-glutamate or on high concentrations of ammonia or on high concentrations of urea, possessed low glutamate dehydrogenase activity compared with cells grown on other nitrogen sources. 3. Aspergillus, Neurospora and Escherichia coli cells grown on l-glutamate possessed high glutamine synthetase activity compared with cells grown on other nitrogen sources. 4. The hypothesis is proposed that in Aspergillus, Neurospora and Escherichia colil-glutamate represses the synthesis of glutamate dehydrogenase and l-glutamine represses the synthesis of glutamine synthetase. 5. A comparison of the glutamine-synthesizing activity and the γ-glutamyltransferase activity of glutamine synthetase in Aspergillus and Neurospora gave no indication that these fungi produce different forms of glutamine synthetase when grown on ammonia or l-glutamate as nitrogen sources.
The configuration of 2,6-diamino-3-hydroxypimelic acid in microbial cell walls
Authors: Perkins H R
Published: August 2015

β-Hydroxydiaminopimelic acid, together with some diaminopimelic acid, occurs in the cell-wall mucopeptide of certain Actinomycetales. These components were converted into their di-DNP derivatives and separated by chromatography. Hence the relative proportions present in the cell walls of a number of species were measured. The problem of acid-induced inversion of configuration was studied. Of the diaminohydroxypimelic acids isomer B (see Scheme 2; amino groups meso, hydroxy group threo to its neighbouring amino group) always predominated but a small proportion of isomer D (amino groups l, hydroxy group erythro) also occurred. The configuration of the diaminohydroxypimelic acids was determined by periodate oxidation to glutamic γ-semialdehyde, which underwent spontaneous ring-closure. Reduction with sodium borohydride produced optically active proline, the configuration of which was determined by direct measurement of the optical rotation of DNP-proline. Un-cross-linked diaminohydroxypimelic acid in the cell wall was oxidized with periodate in the presence of ammonia. Since the remaining amino group was bound in peptide linkage, ring-closure was prevented and borohydride reduction of the aldehyde–ammonia presumed to be present resulted in the formation of ornithine. The quantity of ornithine was used as a measure of the degree of cross-linking.
Separation of the bovine colostrum M-1 glycoprotein into two components
Authors: Bezkorovainy Anatoly , Grohlich Dietmar
Published: August 2015

1. Two glycoproteins were isolated from the M-1 acid glycoprotein fraction of bovine colostrum. 2. The lighter glycoprotein had a molecular weight of 7200, contained about 28·4% of carbohydrate, and had an absorption maximum at 275nm. The heavier glycoprotein had a molecular weight of 12000, contained 39·0% of carbohydrate, and had no absorption maxima in the 240–300nm. range of the spectrum. 3. The carbohydrate moiety of both glycoproteins was removable from the polypeptide moiety under the conditions of the β-elimination reaction. 4. Periodate oxidation experiments showed that sialic acid was linked to galactose in both proteins.
Rates of efflux and intracellular concentrations of potassium, sodium and chloride ions in isolated fat-cells from the rat
Authors: Perry M. C. , Hales C. N.
Published: August 2015

1. The metabolism of K+, Na+ and Cl− has been investigated in isolated fat-cells prepared from the epididymal adipose tissue of rats. 2. Methods are described for measuring the intracellular water space, the rates of loss of intracellular 42K+, 22Na+ and 36Cl− and the intracellular concentrations of K+, Na+ and Cl− in isolated fat-cells. 3. The intracellular water space, measured as the [3H]water space minus the [carboxylic acid−14C]inulin space, was 3·93±0·38μl./100mg. cell dry wt. 4. The first-order rate constants for radioisotope effluxes from isolated fat-cells were 0·029min.−1 for 42K+, 0·245min.−1 for 22Na+ and 0·158min.−1 for 36Cl−. 5. The intracellular concentrations of K+, Na+ and Cl− were 146m-equiv./l., 18·6±2·9m-equiv./l. and 43±2·4m-equiv./l. respectively. 6. The total intracellular K+ content of isolated fat-cells was determined by atomic-absorption spectrophotometry to confirm the value obtained from the radioisotope-efflux data. 7. The ion effluxes from isolated fat-cells were: K+, 1·5pmoles/cm.2/sec., Na+, 1·6pmoles/cm.2/sec., and Cl−, 2·4pmoles/cm.2/sec. 8. The membrane potential of isolated fat-cells calculated from the Cl− distribution ratio was −28·7mv.
Isolation of human β1A-globulin by modern techniques
Authors: Charlwood P. A.
Published: August 2015

1. Human β1A-globulin was isolated from serum by precipitation with ammonium sulphate, gel filtration and electrophoresis in polyacrylamide gel. 2. The product was found by ultracentrifugation, analytical electrophoresis in polyacrylamide gel and two-dimensional immunoelectrophoresis to be of satisfactory quality for further study. 3. The amino acid composition of β1A-globulin was determined. 4. In ordinary dilute buffers near neutrality, β1A-globulin had S020,w 6·42s and M 131 000, but some reversible aggregation occurred at lower pH. In neutral 6m-guanidine hydrochloride the molecular weight was not measurably different from that in dilute buffer.
Schiff bases formed from retinal and phosphatidylethanolamine, phosphatidylserine, ethanolamine or serine
Authors: Plack P. A. , Pritchard D. J.
Published: August 2015

1. Conditions were established for the reaction of retinal with phosphatidylethanolamine, phosphatidylserine, ethanolamine and serine in chloroform, ethanol or ethanol–water solutions to form retinylidene compounds, or Schiff bases. 2. The Schiff bases were reduced to retinyl compounds with sodium borohydride. 3. Absorption maxima and molar extinction coefficients were determined for the various retinylidene and retinyl compounds and for the corresponding coloured products formed by their reaction with antimony trichloride.
The pentose phosphate pathway of glucose metabolism. Enzyme profiles and transient and steady-state content of intermediates of alternative pathways of glucose metabolism in Krebs ascites cells
Authors: Gumaa K. A. , McLean Patricia
Published: August 2015

1. The pentose phosphate pathway in Krebs ascites cells was investigated for regulatory reactions. For comparison, the glycolytic pathway was studied simultaneously. 2. Activities of the pentose phosphate pathway enzymes were low in contrast with those of the enzymes of glycolysis. The Km values of glucose 6-phosphate dehydrogenase for both substrate and cofactor were about four times the reported upper limit for the enzyme from normal tissues. Fructose 1,6-diphosphate and NADPH competitively inhibited 6-phosphogluconate dehydrogenase. 3. About 28% of the hexokinase activity was in the particulate fraction of the cells. The soluble enzyme was inhibited by fructose 1,6-diphosphate and ribose 5-phosphate, but not by 3-phosphoglycerate. The behaviour of the partially purified soluble enzyme in vitro in a system simulating the concentrations of ATP, glucose 6-phosphate and Pi found in vivo is reported. 4. Kinetics of metabolite accumulation during the transient state after the addition of glucose to the cells indicated two phases of glucose phosphorylation, an initial rapid phase followed abruptly by a slow phase extending into the steady state. 5. Of the pentose phosphate pathway intermediates, accumulation of 6-phosphogluconate, sedoheptulose 7-phosphate and fructose 6-phosphate paralleled the accumulation of glucose 6-phosphate. Erythrose 4-phosphate reached the steady-state concentration by 2min., whereas the pentose phosphates accumulated linearly. 6. The mass-action ratios of the pentose phosphate pathway reactions were calculated. The transketolase reaction was at equilibrium by 30sec. and then progressively shifted away from equilibrium towards the steady-state ratio. The glucose 6-phosphate dehydrogenase was far from equilibrium at all times. 7. Investigation of the flux of [14C]glucose carbon confirmed the existence of an operative pentose phosphate pathway in ascites cells, contributing 1% of the total flux in control cells and 10% in cells treated with phenazine methosulphate. 8. The pentose phosphate formed by way of the direct oxidative route and estimated from the 14CO2 yields represented 20% of the total accumulated pentose phosphate, the other 80% being formed by the non-oxidative reactions of the pentose phosphate pathway. 9. The pentose phosphate pathway appears to function as two separate pathways, both operating towards pentose phosphate formation. Control of the two pathways is discussed.
Examination of an equilibrium interpretation of deoxyribonucleic acid–ribonucleic acid hybridization data
Authors: Bishop John O.
Published: August 2015

1. When a constant amount of denatured DNA is annealed for a constant time with a series of different RNA concentrations, it is often observed that the reciprocal of the amount of RNA hybridized is linearly proportional to the reciprocal of the RNA concentration. This may be explained by assuming that an equilibrium is set up between free RNA and DNA on the one hand and DNA–RNA hybrid on the other. The hybridization of Escherichia coli DNA and ribosomal RNA was used to test this proposition. Rate constants were estimated from the initial rates of the forward and back reactions and compared with direct estimates of the dissociation constant. 2. The rate constants of the forward and back reactions were estimated to be 1.82mlμg-1h-1 (160lmol-1s-1) and 0.023h-1 (6.4×10-6s-1) respectively, giving a ratio k2/k1=0.013μgml-1. After 24h annealing the dissociation constant was estimated to be 0.114μgml-1, and by extrapolation to infinite time, 0.047μgml-1. 3. It is concluded that (a) equilibrium greatly favours the hybrid complex, (b) equilibrium is not established in 24h, (c) the equilibria that were directly estimated are incompatible either with the measured rates of the forward and back reactions or with the simple formulation of the reaction that was adopted, and finally (d) for these reasons the equilibrium interpretation of the linear reciprocal relationship is unsatisfactory.
Specific alkylation of a histidine residue in carnitine acetyltransferase by bromoacetyl-l-carnitine
Authors: Chase J. F. A. , Tubbs P. K.
Published: August 2015

Incubation of carnitine acetyltransferase with low concentrations of bromoacetyl-l-carnitine causes a rapid and irreversible loss of enzyme activity; one mol of inhibitor can inactivate one mol of enzyme. Bromoacetyl-d-carnitine, iodoacetate or iodoacetamide are ineffective. l-Carnitine protects the transferase from bromoacetyl-l-carnitine. Investigation shows that the enzyme first reversibly binds bromoacetyl-l-carnitine with an affinity similar to that shown for the normal substrate acetyl-l-carnitine; this binding is followed by an alkylation reaction, forming the carnitine ester of a monocarboxymethyl-protein, which is catalytically inactive. The carnitine is released at an appreciable rate by spontaneous hydrolysis, and the resulting carboxymethyl-enzyme is also inactive. Total acid hydrolysis of enzyme after treatment with 2-[14C]bromoacetyl-l-carnitine yields N-3-carboxy[14C]methylhistidine as the only labelled amino acid. These findings, taken in conjunction with previous work, suggest that the single active centre of carnitine acetyltransferase contains a histidine residue.
Molecular weights and metabolism of rat brain proteins
Authors: Vrba R. , Cannon Wendy
Published: August 2015

1. Rats were injected with [U-14C]glucose and after various intervals extracts of whole brain proteins (and in some cases proteins from liver, blood and heart) were prepared by high-speed centrifugation of homogenates in 0.9% sodium chloride or 0.5% sodium deoxycholate. 2. The extracts were subjected to gel filtration on columns of Sephadex G-200 equilibrated with 0.9% sodium chloride or 0.5% sodium deoxycholate. 3. Extracts prepared with both solvents displayed on gel filtration a continuous range of proteins of approximate molecular weights ranging from less than 2×104 to more than 8×105. 4. The relative amount of the large proteins (mol.wt.>8×105) was conspicuously higher in brain and liver than in blood. 5. At 15min after the injection of [U-14C]glucose the smaller protein molecules (mol.wt.<2×104) were significantly radioactive, whereas no 14C could be detected in the larger (mol.wt.>2×104) protein molecules. The labelling of all protein samples was similar within 4h after injection of [U-14C]glucose. Fractionation of brain proteins into distinctly different groups by the methods used in the present work yielded protein samples with a specific radioactivity comparable with that of total brain protein. 6. No evidence could be obtained by the methods used in the present and previous work to indicate the presence of a significant amount of ‘metabolically inert protein’ in the brain. 7. It is concluded that: (a) most or all of the brain proteins are in a dynamic state of equilibrium between continuous catabolism and anabolism; (b) the continuous conversion of glucose into protein is an important part of the maintenance of this equilibrium and of the homoeostasis of brain proteins in vivo.
Human immunoglobulin subclasses. Partial amino acid sequence of the constant region of a γ4 chain
Authors: Pink J. R. L. , Buttery S. H. , De Vries G. M. , Milstein C.
Published: August 2015

The heavy chain of a human myeloma protein (Vin) belonging to the γ4 subclass was subjected to tryptic digestion after reduction and carboxymethylation. Cyanogen bromide fragments were also prepared and all 19 tryptic peptides that account for one of them (the Fc-like fragment) were studied. Selected peptic peptides were isolated and provided evidence for the order of 15 of the tryptic peptides. In addition the sequence of two large peptic peptides derived from two sections of the molecule including all the interchain bridges is presented. Comparison with published data on other chains allows us to propose a sequence of γ4 chains that extends from just before the presumed starting point of the invariable region (at about residue 113) to the C-terminal end of the chain (approx. residue 446), except for a section of about 50 residues. The results of the comparison suggest that the immunoglobulin subclasses have a recent independent evolutionary origin in different species. Implications for complement fixation and for the evolutionary origin of antibody diversity are also discussed.
Ribonucleic acid synthesis and transport in animal cells at 27°C
Authors: Horisberger Michel , Amos Harold
Published: August 2015

The failure to induce glutamine synthetase in retinal cells with hydrocortisone at 27°C (37°C is the normal temperature for induction) led to a study of some aspects of RNA synthesis at 27°C by using the method of selective labelling with radioactive precursors, sucrose-density-gradient centrifugation and radioautography. The low-temperature treatment not only decreases the rate of RNA synthesis but also interferes with the general pattern of RNA maturation and distribution. It has been found that nuclear–cytoplasmic exchange is severely altered at low temperature and may be an explanation of the failure to induce the enzyme. When the temperature is raised from 27°C to 37°C, the incorporation of exogenous radioactive uridine into the RNA is distorted by a pool-dilution effect that results in an ‘over-shoot’ phenomenon. The kinetics of labelled uridine equilibration into the UMP pool accounts for the difficulty encountered by many investigators in ‘chase’ efforts with unlabelled nucleosides.
Biosynthesis of N-(α-hydroxyethyl)-lysergamide, a metabolite of Claviceps paspali Stevens & Hall
Authors: Castagnoli N. , Corbett K. , Chain E. B. , Thomas R.
Published: August 2015

1. The biosynthetic origin of the amide substituent of N-(α-hydroxyethyl)lysergamide has been studied. 2. [1-14C]Acetate, [14C]formate, [2-14C]mevalonic acid lactone, [2-14C]indole, dl-[3-14C]tryptophan, dl-[3-14C]serine, dl-[2-14C]alanine and [2-14C]pyruvate were efficiently incorporated into the alkaloid, but not dl-[1-14C]alanine or [1-14C]pyruvate. 3. Only the dl-[2-14C]alanine- and [2-14C]pyruvate-derived alkaloid contained appreciable radioactivity in the amide substituent. 4. l-[15N]Alanine-derived alkaloid was shown to be specifically labelled in the amide nitrogen. However, l-[14C,15N]alanine was found to be incorporated into the methylcarbinolamide substituent with an appreciable increase in the 15N/14C ratio, suggesting that alanine is not the direct precursor of this moiety.
Hepatic drug-metabolizing enzyme activity in rats pretreated with (−)-emetine or (±)-2,3-dehydroemetine
Authors: Miller H. H. , Johnson R. K. , Donahue J. D. , Jondorf W. R.
Published: August 2015

1. Pretreatment of female rats with (−)-emetine or (±)-2,3-dehydroemetine (at 18μmol/kg body wt. for 24h) prolongs the hexobarbital-induced sleeping-time of the treated animals. 2. This effect is not observed on pretreating animals with other compounds closely related to (−)-emetine, such as (−)-isoemetine or (+)-O-methylpsychotrine. 3. Liver microsomal drug-metabolizing enzyme activity in vitro as measured by N-demethylation of aminopyrine and azo-reduction of Neoprontosil is inhibited in rats pretreated with (−)-emetine or with (±)-2,3-dehydroemetine. 4. These inhibitory effects on drug metabolism in vitro are not observed in corresponding experiments involving pretreatment of rats with (−)-isoemetine or (+)-O-methylpsychotrine. 5. Co-administration of emetine or 2,3-dehydroemetine and sodium phenobarbital or 1,1-dichloro-2-o-chlorophenyl-2-p-chlorophenylethane to rats abolishes or greatly diminishes the stimulation of drug-metabolizing enzyme activity in vitro usually obtained by the administration of phenobarbital or 1,1-dichloro-2-o-chlorophenyl-2-p-chlorophenylethane alone. 6. Further, in rats pretreated with sodium phenobarbital and subsequently injected with emetine or 2,3-dehydroemetine the pre-stimulated drug-metabolizing enzyme activity in vitro is diminished. 7. The inhibitory effects on drug-metabolizing enzyme activity after pretreatment with (−)-emetine or (±)-2,3-dehydroemetine do not appear to be related to NADPH generation.
Purification and properties of threonine aldolase from Clostridium pasterianum
Authors: Dainty R. H.
Published: August 2015

1. Threonine aldolase was purified about 200-fold in 10% yield from Clostridium pasteurianum and its properties were examined. The final preparation gave three bands after ionophoresis on polyacrylamide gel. 2. The purified enzyme was shown to produce glycine and acetaldehyde in stoicheiometric amounts from threonine. The reverse reaction was demonstrated qualitatively. 3. The enzyme has a broad pH optimum at 6.5–7.0. 4. The enzyme is highly specific for l-threonine. 5. The enzyme is completely inhibited by 1mm concentrations of hydroxylamine and semicarbazide. Activity is decreased to 20% of the original by treatment with cysteine plus mercaptoethanol; most of the loss is regained on incubation with pyridoxal phosphate. It is concluded that pyridoxal phosphate is a prosthetic group. 6. The relationship between velocity and substrate concentration is atypical but indicates a Km value of 0.42mm. 7. The enzyme was demonstrated in several other strictly anaerobic bacteria.
Micro-analysis of pure deoxyribonucleic acid-dependent ribonucleic acid polymerase from Escherichia coli. Action of heparin and rifampicin on structure and function
Authors: Neuhoff Volker , Schill Wolf-Bernhard , Sternbach Hans
Published: August 2015

By using micro disc electrophoresis and micro-diffusion techniques, the interaction of pure DNA-dependent RNA polymerase (EC 2.7.7.6) from Escherichia coli with the template, the substrates and the inhibitors heparin and rifampicin was investigated. The following findings were obtained: (1) heparin converts the 24S and 18S particles of the polymerase into the 13S form; (2) heparin inhibits RNA synthesis by dissociating the enzyme–template complex; (3) rifampicin does not affect the attachment of heparin to the enzyme; (4) the substrates ATP and UTP are bound by enzyme loaded with rifampicin; (5) rifampicin is bound by an enzyme–template complex to the same extent as by an RNA-synthesizing enzyme–template complex. From this it is concluded that the mechanism of the inhibition of RNA synthesis by rifampicin is radically different from that by heparin. As a working hypothesis to explain the inhibitory mechanism of rifampicin, it is assumed that it becomes very firmly attached to a position close to the synthesizing site and only blocks this when no synthesis is in progress.
Computer simulation of the non-steady-state radioactive labelling of a system of metabolite pools with constant rates of influx and efflux
Authors: England P. J.
Published: August 2015

The derivation is given of the general case of a differential equation describing the labelling of a single metabolite pool that is subjected to constant rates of influx of radioactive material when the pool size is itself changing at constant rate. A programme, written in ALGOL, is briefly described that solves a set of these differential equations, and that can be used to simulate the radioactive labelling of a system of metabolite pools. A copy of the programme, together with examples of input and output, has been deposited as Supplementary Publication no. SUP 50001 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1970), 116, 7.
Rates of aminoacyl-transfer-ribonucleic acid synthesis in vivo and in vitro by bean leaves
Authors: Hall T. C. , Tao K. L.
Published: August 2015

1. A procedure for measuring rates of aminoacyl-tRNA synthesis in vitro and in intact leaves is presented. 2. Leaf discs showed rates close to those of intact leaves. 3. Cell-free preparations showed similar rates when assayed by pyrophosphate exchange, but actual aminoacyl-tRNA formation rates appeared to be much lower. Evidence is presented that dilution of supplied labelled amino acids was a major factor causing the low apparent rates. 4. Attempts to strip endogenous amino acids from plant tRNA resulted in low acceptor capability of the tRNA.
Effect of sedimentation through sucrose solutions on the protein-synthesizing ability of rat liver microsomes
Authors: Leslie Ruth A. , Mansbridge J. N.
Published: August 2015

1. Centrifugation of the postmitochondrial supernatant of rat liver through 1.0m-sucrose produces particles that have 85–95% less incorporating ability in a cell-free protein-synthesizing system than either ribosomes or microsomes. 2. The incorporation of [14C]phenylalanine into protein by particles prepared by sedimentation through 1.0m-sucrose is stimulated about 20-fold by addition of poly U. 3. The content of rapidly labelled RNA of microsomes is decreased during centrifugation through 1.0m-sucrose. 4. It is suggested that degradation of mRNA occurs during the formation of the pellet in the centrifuge tube as a result of a membrane-bound alkaline ribonuclease, after removal of the ribonuclease inhibitor of the soluble fraction.