bluuebunny/crossref_metadata_2025 huggingface dataset:
The function of subcellular fractions in the oxidation of glutathione in rat liver homogenate
Authors: Jocelyn P. C.
Published: August 2015

1. The aerobic loss of GSH added to the supernatant fraction from rat liver is much increased by including the microsome fraction, which both inhibits the concurrent reduction of the GSSG formed and also augments the net oxidation rate. 2. Oxidation occurs with a mixture of dialysed supernatant and a protein-free filtrate; the latter is replaceable by hypoxanthine and the former by xanthine oxidase, whereas fractions lacking this enzyme give no oxidation. 3. In all these instances augmentation occurs with microsomes, with fractions having urate oxidase activity and with the purified enzyme; uric acid and microsomes alone also support the oxidation. 4. Evidence implicating additional protein factors is discussed. 5. It is suggested that GSH oxidation by homogenate is linked through glutathione peroxidase to the reaction of endogenous substrate with supernatant xanthine oxidase and of the uric acid formed with peroxisomal urate oxidase.
Mescaline-induced changes of brain-cortex ribosomes. Effect of mescaline on the hydrogen-bonded structure of ribonucleic acid of brain-cortex ribosomes
Authors: Datta R. K. , Ghosh J. J.
Published: August 2015

1. The action of mescaline sulphate on the hydrogen-bonded structure of the RNA constituent of ribosomes of goat brain-cortex slices was studied by using the hyperchromic effect of heating and formaldehyde reaction. 2. The ribosomal total RNA species of the mescaline-treated brain-cortex slices have a smaller proportion of hydrogen-bonded structure than the ribosomal RNA species of the untreated brain-cortex slices. 3. Mescaline also appears to have affected this lowering of hydrogen-bonded structure of the ribosomal 28S RNA of brain-cortex tissue.
Cerebral-cortex hexokinase. Comparison of properties of solubilized mitochondrial and cytoplasmic activities
Authors: Thompson M. F. , Bachelard H. S.
Published: August 2015

1. Cerebral-cortex mitochondria, after purification by using high-density sucrose solutions, were extracted with Triton X-100. The total hexokinase activity of the intact mitochondria was increased by 50–80% in the Triton extracts. 2. Triton X-100 was removed from mitochondrial extracts by a combination of ammonium sulphate fractionation and DEAE-cellulose chromatography. Mitochondrial hexokinase remained soluble after removal of extractant. 3. The behaviour of solubilized mitochondrial hexokinase was compared with soluble cytoplasmic hexokinase from the same samples of cerebral cortex on identical columns of DEAE-cellulose. Two peaks were eluted from each source of hexokinase. The distribution between hexokinase peaks was similar for the two sources. Peak I (approx. 80% of the total hexokinase) from each was eluted at identical concentrations of potassium chloride and slight differences were observed in the elution profiles for peak II. 4. The purified mitochondrial hexokinase showed the following kinetic properties: peak I, Km(ATP) 0.60mm, Km(glucose) 0.042mm; peak II, Km(ATP) 0.66mm, Km(glucose) 0.043mm. The purified cytoplasmic hexokinase Michaelis constants were: peak I, Km(ATP) 0.56mm, Km(glucose) 0.048mm; peak II, Km(ATP) 0.68mm, Km(glucose) 0.062mm. 5. Although no significant differences between mitochondrial and cytoplasmic hexokinases were noted in chromatographic behaviour or in the kinetic properties studied, the purified mitochondrial enzyme was activated slightly (approx. 20%) by Triton X-100, in contrast with the cytoplasmic enzyme, which was not affected. 6. The results, taken to indicate basic similarity between mitochondrial and cytoplasmic hexokinases, are discussed in relation to the role of the two sources of enzyme in the metabolism of the tissue.
Isoelectric focusing of human liver alkaline phosphatase
Authors: Latner A. L. , Parsons Mary E. , Skillen A. W.
Published: August 2015

1. Isoelectric focusing of human liver alkaline phosphatase in a sucrose density gradient with LKB Ampholine as carrier ampholytes is described. 2. Problems due to the chelating properties of the ampholytes and the pH gradient were examined. 3. A reactivation procedure to counter these effects was devised that can probably be used for other alkaline phosphatases. 4. The isoelectric point of human liver alkaline phosphatase was found to be pH3.9.
Dissociation of catalase. A correlation between changes in sedimentation and spectroscopic properties accompanying dissociation of bacterial catalase in alkaline solution
Authors: Jones Peter , Pain R. H. , Suggett A.
Published: August 2015

1. At high concentrations, in 10mm-phosphate buffer, pH7.0, the sedimentation coefficient of bacterial catalase varies with concentration according to: [Formula: see text] with S020,w=11.30S and ks=6.29×10−3ml mg−1. Sedimentation-equilibrium experiments yield a molecular weight of 240000. 2. Parallel studies of changes in sedimentation-velocity behaviour and in electronic spectra of bacterial catalase at pH>11 were made. Dissociation is indicated by the appearance of a slow-moving (2.9S) component in sedimentation patterns and this is accompanied by marked changes in absorption spectrum in the Soret region. Values of R=E406/E355 show a theoretically predictable near-linear dependence on α, the degree of dissociation calculated from ultracentrifuge data. 3. The Soret absorption of bacterial catalase subunits is much lower than that of the native enzyme, and it is suggested that dissociation produces an environmental constraint on the prosthetic group that results in distortion of the porphyrin ring.
Interaction of dehydroheliotridine, a metabolite of heliotridine-based pyrrolizidine alkaloids, with native and heat-denatured deoxyribonucleic acid in vitro
Authors: Black D. N. , Jago M. V.
Published: August 2015

Dehydroheliotridine, a major pyrrolic metabolite of heliotridine-based pyrrolizidine alkaloids, interacted with both native and heat-denatured DNA in vitro under mildly acid conditions to form a soluble non-diffusible complex as shown by spectral data. The complex formed with heat-denatured DNA was more resistant than heat-denatured DNA alone to the combined action of deoxyribonuclease and phosphodiesterase. Analysis of the resistant fractions suggested the presence of at least one base not obtained from DNA alone.
Plasma protein catabolism by the perfused rat liver. The effect of alteration of albumin concentration and dietary protein depletion
Authors: Hoffenberg R. , Gordon A. H. , Black E. G. , Louis L. N.
Published: August 2015

1. The isolated perfused rat liver was used to study degradation rates of plasma albumin, transferrin and fibrinogen. 2. Constant fractional rates were observed for all three proteins even when the albumin concentration was drastically increased by the addition of large amounts to the perfusate pool. 3. Livers taken from rats deprived of dietary protein for 14–18 days showed greatly diminished fractional catabolic rates for albumin when perfused with blood from similarly deprived animals. 4. These rates could be restored to near-normal values by adding albumin or by perfusing with blood from normally fed rats. 5. These findings are consistent with the theory of pinocytosis as a step in the degradation of plasma proteins by hepatic parenchymal cells.
A study on the precursors of the acetyl moiety of acetylcholine in brain slices. Observations on the compartmentalization of the acetyl-coenzyme A pool
Authors: Nakamura R. , Cheng S.-C. , Naruse H.
Published: August 2015

1. A method was devised for the determination of the specific radioactivity of the acetyl moiety of acetylcholine synthesized from various 14C-labelled substrates. 2. The precursor for the acetyl moiety of acetylcholine was studied in slices of striatum and cerebral cortex from rat and guinea-pig brain. Incorporation of radioactivity into acetylcholine was determined after incubating the slices in the presence of [2-14C]acetate, [14C]bicarbonate, [1,5-14C]citrate, dl-[1- or 5-14C]glutamate or [1- or 2-14C]pyruvate. 3. After incubation for 1h, acetylcholine was accumulated significantly in both striatum slices (4.1nmol/mg of protein) and cerebral-cortex slices (0.57nmol/mg of protein) from the rat. Final concentrations were about 11 and 5 times respectively the initial values. 4. With slices from rat striatum, rat cerebral cortex and guinea-pig cerebral cortex, the specific radioactivity of acetylcholine derived from [2-14C]pyruvate was very high, reaching approx. 30, 20 and 6% respectively of the initial specific radioactivity of added pyruvate in the medium. With the striatum slices this high value was reached after incubation for 15min. Incorporation of radioactivity from [2-14C]acetate was only 1.25, 5.3 and 19.7% of that from [2-14C]pyruvate in rat striatum, rat cerebral-cortex and guinea-pig cerebral-cortex slices respectively. A small but definite incorporation was found from [5-14C]glutamate. No incorporation was found from the other substrates. The findings suggest that pyruvate is the most important precursor for the synthesis of the acetyl moiety of acetylcholine in brain slices. 5. The specific radioactivity of acetylcholine relative to that of citrate when [2-14C]pyruvate was used compared with that obtained when [2-14C]acetate was used. A marked difference was found in all slices, suggesting metabolic compartmentation of the acetyl-CoA pool.
The synthesis of bile acids in perfused rat liver subjected to chronic biliary drainage
Authors: Percy-Robb I. W. , Boyd G. S.
Published: August 2015

1. Isolated rat liver was perfused with heparinized whole blood under physiological pressure resulting in the secretion of bile at about the rate observed in vivo. 2. The preparation remained metabolically active for 4h and was apparently normal in function and microscopic appearance. 3. When the perfusate plasma and liver cholesterol pool was labelled by the introduction of [2-14C]mevalonic acid the specific radioactivity of the perfusate cholesterol increased. The biliary acids (cholic acid and chenodeoxycholic acid) were labelled and had the same specific radioactivity. 4. Livers removed from rats immediately after, and 40h after, the start of total biliary drainage, were perfused; increased excretion rates of both cholic acid and chenodeoxycholic acid were found when the liver donors had been subjected to biliary drainage. 5. The incorporation of [2-14C]mevalonic acid or rat lipoprotein labelled with [14C]cholesterol into bile acids was studied. 6. A dissociation between the mass of bile acid excreted and the rate of incorporation of 14C was found. This was attributed to the changing specific radioactivity of the cholesterol pool acting as the immediate bile acid precursor.
The isolation and composition of two phosphoproteins from hen's egg
Authors: Clark R. C.
Published: August 2015

1. Phosvitin extracted from domestic hen's-egg yolk was resolved on Sephadex G-100 into two phosphoprotein components. 2. The major component has a molecular weight of about 3.4×104 and alanine as an N-terminal residue. Glucosamine is present, but tyrosine is virtually absent. 3. The minor component has a molecular weight of about 2.8×104 and lysine as an N-terminal residue. Missing residues are glucosamine, methionine and leucine. Lysine, histidine, threonine, glycine, phenylalanine and tyrosine contents differ significantly from those of the major component. 4. Sephadex G-100 also removes small amounts of an impurity with a much higher molecular weight.
The pepsins from human gastric mucosal extracts
Authors: Etherington D. J. , Taylor W. H.
Published: August 2015

1. The pepsins and pepsinogens of the gastric mucosal extracts of two normal subjects, of seven patients with gastric adenocarcinoma and of two patients with duodenal ulcer have been investigated by agar-gel electrophoresis and by ion-exchange chromatography. 2. Of the eight zones of proteolytic activity that have previously been reported in normal human gastric juice, seven can be detected in activated fundic mucosal extracts. Of these seven, four can be attributed to discrete pepsins, numbered 1, 3a, 3 and 5. 3. Zone 7 results from the activity of one or more enzymes that are alkali-stable and are best referred to as gastric proteinases rather than as pepsins. Zone 7 is much more evident in mucosal extracts than in gastric juice. 4. Zones 4 and 6 may result respectively from the activity of a pepsin–inhibitor complex and of an unactivated zymogen. 5. It was not possible, by the chromatographic methods employed, to separate satisfactorily the individual pepsins from activated extracts or their precursors from unactivated extracts, so that the ascribing of a pepsin to a specific zymogen must be considered tentative. Even so, pepsin 3 appears to arise from at least two major precursors, if not from three, whereas pepsins 1 and 5 each arise from a single major precursor. 6. Pyloric mucosal extracts contain principally zone 5 but also zones 6 and 7. These zones in general behave similarly to the corresponding zones of fundic extracts, but pyloric pepsin 5 migrates slightly faster on agar-gel electrophoresis than does fundic pepsin 5 and is a different enzyme. Zones 1 to 4 are absent.
Catabolite repression of the lac operon. Effect of mutations in the lac promoter
Authors: Yudkin M. D.
Published: August 2015

1. Several lac diploid strains of Escherichia coli were constructed and tested to discover whether mutations in the lac promoter alleviate catabolite repression. 2. In each of these diploids the chromosome carries one of the promoter mutations, L8, L29 or L1; so that the rate of synthesis of the enzymes of the lac operon is only 2–6% of the fully induced wild-type. Each diploid harbours the episome F′lacM15 that specifies the synthesis of thiogalactoside transacetylase under the control of intact regulator, promoter and operator regions, but has a deletion in the structural gene for β-galactosidase. In each diploid more than 90% of the thiogalactoside transacetylase is synthesized from the episome, and 100% of the β-galactosidase is synthesized from the chromosome, and comparison of the extent of catabolite repression that the two enzymes suffered indicated whether the chromosomal promoter mutation relieves catabolite repression. 3. In the strains in which the promoter carries either of the point mutations L8 or L29 the enzymes were equally repressed, suggesting that neither L8 nor L29 affects catabolite repression. 4. In a diploid strain harbouring the same episome but carrying deletion L1 on the chromosome, synthesis of β-galactosidase suffered much less repression than that of thiogalactoside transacetylase. 5. In a diploid strain in which the chromosome carries L1 and also a second mutation that increases the rate of expression of lac to that permitted by L8 or L29, the synthesis of β-galactosidase again suffered much less repression than the synthesis of thiogalactoside transacetylase. 6. The effect of L1 (which deletes the boundary between the i gene and the lac promoter) is ascribed to its bringing the expression of lac under the control of the promoter of the i gene. 7. Even in strains carrying L1, some catabolite repression persists; this is not due to a trans effect from the episome since it occurs equally in a haploid strain with L1.
Early steps in the biosynthesis of mycobactins P and S
Authors: Tateson J. E.
Published: August 2015

1. Lysine is readily incorporated into mycobactins P and S. Incorporation is into the hydroxamic acid moieties only and is equal in the mycobactic acid and cobactin portions of the molecule. 2. 2-Amino-6-hydroxyaminohexanoic acid is not taken up by cells of Mycobacterium phlei and is not detectable in extracts of cells actively synthesizing mycobactin. 3. The most abundant material derived from lysine that can be detected in such cell extracts is an N6-acyl-lysine. Cells grown in the presence of iron contain markedly less of this material than do those grown under conditions of iron deficiency. 4. When added to growing cultures of M. phlei the N6-acyl-lysine is readily incorporated into mycobactin. 5. The hydroxy acid of cobactin P is derivable from propionate.
Formation of mercapturic acids in rats after the administration of aralkyl esters
Authors: Clapp J. J. , Young L.
Published: August 2015

1. Benzylmercapturic acid and hippuric acid were isolated from the urine of rats that had been injected subcutaneously with benzyl acetate. 2. 1-Menaphthylmercapturic acid and 1-naphthoic acid were isolated from the urine of rats after the subcutaneous injection of each of the following compounds: 1-menaphthyl alcohol and its acetate, propionate, butyrate and benzoate esters. 3. A quantitative method for determining 1-menaphthylmercapturic acid in urine was developed and used to measure the excretion of this compound in the urine of rats in the 4-day period after the subcutaneous injection of 1-menaphthyl alcohol and its acetate, propionate, butyrate and benzoate esters. 4. Chromatographic evidence was obtained for the presence of S-(1-menaphthyl)glutathione and S-(1-menaphthyl)-l-cysteine in bile collected from rats with cannulated bile ducts after the animals had been injected subcutaneously with each of the following compounds: S-(1-menaphthyl)glutathione, 1-menaphthyl acetate, propionate and butyrate. 5. Benzylmercapturic acid and 1-menaphthylmercapturic acid were isolated from the urine of rats that had been injected with sodium benzyl sulphate and sodium 1-menaphthyl sulphate respectively.
Glycine decarboxylase in Rhodopseudomonas spheroides and in rat liver mitochondria
Authors: Tait G. H.
Published: August 2015

1. Glycine decarboxylase and glycine–bicarbonate exchange activities were detected in extracts of Rhodopseudomonas spheroides and in rat liver mitochondria and their properties were studied. 2. The glycine decarboxylase activity from both sources is stimulated when glyoxylate is added to the assay system. 3. Several proteins participate in these reactions and a heat-stable low-molecular-weight protein was purified from both sources. 4. These enzyme activities increase markedly when R. spheroides is grown in the presence of glycine, glyoxylate, glycollate, oxalate or serine. 5. All the enzymes required to catalyse the conversion of glycine into acetyl-CoA via serine and pyruvate were detected in extracts of R. spheroides; of these glycine decarboxylase has the lowest activity. 6. The increase in the activity of glycine decarboxylase on illumination of R. spheroides in a medium containing glycine, and the greater increase when ATP is also present in the medium, probably accounts for the increased incorporation of the methylene carbon atom of glycine into fatty acids found previously under these conditions (Gajdos, Gajdos-Török, Gorchein, Neuberger & Tait, 1968). 7. The results are compared with those obtained by other workers on the glycine decarboxylase and glycine–bicarbonate exchange activities in other systems.
Autolysis of cell walls of Bacillus stearothermophilus B65 and the chemical structure of the peptidoglycan
Authors: Grant W. D. , Wicken A. J.
Published: August 2015

1. The cell walls of Bacillus stearothermophilus B65 contain glucosamine, muramic acid, alanine, α∈-diaminopimelic acid (Dap), glutamic acid, aspartic acid, glycine, and serine in the molecular proportions 0.60:0.64:2.30:0.85:1.00:0.11:0.13:0.31. 2. Both d- and l-alanine are present, but glutamic acid and diaminopimelic acid are present only as the d- and meso-isomers respectively. 3. The peptide fragments Ala-Dap, Dap-Ala, and Dap-Ala-Dap have been isolated from a partial acid hydrolysate of the cell walls. 4. The major products of autolysis of the cell wall were d-alanine, a peptide mixture, peptidoglycan material and a peptidoglycan–teichoic acid complex. 5. Separation of the peptide mixture into ten major peptides was achieved by DEAE-Sephadex and paper chromatography, and paper electrophoresis. 6. The structures of these peptides have been determined and they fall into four groups, the individual members of each group differing only in number or position of carboxamide substituents. 7. The structures are I, a tripeptide l-Ala–d-Glu-meso-Dap; II, a pentapeptide made up by the tripeptide (I) linked through the ∈-amino group of its diaminopimelic acid residue to the carboxyterminal of the dipeptide meso-Dap-d-Ala; III, a heptapeptide made up by a similar linkage between the tripeptide (I) and the tetrapeptide l-Ala-d-Glu-meso-Dap-d-Ala; IV, a possible undecapeptide made up by a further tetrapeptide similarly linked to the heptapeptide (III) structure. 8. The structure of the peptidoglycan and the actions of the autolytic enzymes are discussed in terms of these peptide structures.
Chemical modification of a cerebral sodium-plus-potassium ion-stimulated adenosine triphosphatase preparation
Authors: Pull I.
Published: August 2015

1. A particulate Na++K+-stimulated adenosine triphosphatase preparation obtained by treatment of bovine cerebral microsomes with a sodium iodide reagent has been further treated with acid anhydrides likely to convert amino groups into acidic derivatives. 2. The extent of acylation of amino groups was determined by reaction of the remaining amino groups with 2,4,6-trinitrobenzenesulphonic acid. The unmodified preparation contains about 1.2 μequiv. of amino groups/mg of protein of which only about 0.5 μequiv. are accounted for by protein amino groups. Kinetics of the trinitrobenzenesulphonic acid reaction with the unmodified preparation are complex and are altered by ATP or ouabain. 3. The compounds examined cause loss of Na++K+-stimulated adenosine triphosphatase activity when relatively few amino groups are modified but ATP was found to afford partial protection against inactivation by methylmaleic anhydride. Na++K+-stimulated adenosine triphosphatase activity is partly restored to the dimethylmaleylated preparation by hydrolysis of the dimethylmaleyl–amide bonds but not if more than about 20% of the amino groups have been acylated. 4. Supernatants obtained by high-speed centrifugation of the dimethylmaleylated preparation contained up to 45% of the total protein with less than 10% of the total phospholipid. Methylmaleyl and benzenetricarboxylyl derivatives of the enzyme preparation behaved similarly but tetrafluorosuccinylated material was almost entirely deposited by centrifugation.
Enzyme-catalysed reactions between some 2-substituted 5-nitrofuran derivatives and glutathione
Authors: Boyland E. , Speyer B. E.
Published: August 2015

1. A glutathione transferase present in rat and human liver supernatant catalyses the reaction of some 2-substituted 5-nitrofuran derivatives with GSH, with formation of a conjugate and release of the nitro group as inorganic nitrite. Some of the substrates undergo the same reaction at a slower rate in the absence of enzyme. Nitrofuran derivatives commonly used as drugs, and five other drugs containing nitro groups, did not react. 2. Substrate activity in the nitrofuran derivatives showed an approximate correlation with the lability of the nitro group to alkali. 3. Optimum pH values ranging from 6.6 to 9.0 were found for the enzymic reaction with various derivatives, the values being influenced by alkali-lability and pK values of the compounds. 4. Tenfold purification of rat liver glutathione S-aryl-transferase resulted in an equal purification of the activities that catalyse the reaction of two of the nitrofuran derivatives with GSH.
Isolation of 2-amino-1-naphthyl hydrogen sulphate with cetylpyridinium bromide from metabolic and chemical oxidations of 2-naphthylamine
Authors: Manson D.
Published: August 2015

1. 2-Amino-1-naphthyl hydrogen sulphate can be rapidly isolated from the urine of dogs dosed with 2-naphthylamine by precipitation with cetylpyridinium bromide. 2. No evidence was obtained for the presence of 2-naphthylhydroxylamine-O-sulphonic acid, noteworthy as a possible source of the carcinogens, 2-naphthylhydroxylamine and 2-amino-1-naphthol. 3. Treatment of neutral persulphate oxidations of 2-naphthylamine with the reagent gave only the cetylpyridinium salt of 2-amino-1-naphthyl hydrogen sulphate.
Free and membrane-bound polyribosomes in normal and Rauscher-virus-infected mouse spleen cells
Authors: Burghouts J. Th. M. , Stols A. L. H. , Bloemendal H.
Published: August 2015

1. Free and membrane-bound polyribosomes and ribosomal monomers were isolated from normal and Rauscher-virus-infected mouse spleens by means of discontinuous sucrose density gradients. 2. The addition of ribonuclease inhibitor from rat liver was essential to protect these polyribosomes from degradation. To separate the smooth and rough membranes from ribosomal monomers an additional centrifugation step through a continuous sucrose density gradient was necessary. 3. After infection a marked increase in rRNA from both membrane-bound and free polyribosomes was observed. Treatment of the membrane-bound polyribosomes with sodium deoxycholate yielded only 80S particles even when ribonuclease inhibitor was added. 4. A striking feature of the infected spleen was the occurrence of large polyribosomes. Up to 40 monomers per polyribosome could be counted on electron micrographs.
Studies on the incorporation of [U-14C]glucose and [35S]sulphate into the acid glycosaminoglycans of neonatal rat skin
Authors: Hardingham T. E. , Phelps C. F.
Published: August 2015

1. The incorporation of [35S]sulphate in vivo into the acid-soluble intermediates extracted from young rat skin showed three sulphated hexosamine-containing components. 2. The rates of synthesis of these components were determined in vivo by measuring the incorporation of radioactivity from [U-14C]glucose into their isolated hexosamine moieties. 3. The incorporation of radioactivity from [U-14C]glucose into the isolated hexosamine and uronic acid moieties of the acid glycosaminoglycans was also measured. These results, combined with those obtained on the intermediary pathways of hexosamine and uronic acid biosynthesis previously determined in this tissue, indicated that the acid-soluble sulphated hexosamine-containing components were not precursors of the sulphated hexosamine found in the acid glycosaminoglycans. 4. The rates of synthesis of the acid glycosaminoglycan fractions were calculated from the incorporation of radioactivity from [U-14C]glucose into the hexosamine moiety. The sulphated components containing principally dermatan sulphate, chondroitin 6-sulphate and in smaller amounts, chondroitin 4-sulphate, heparan sulphate and heparin appeared to be turning over about twice as rapidly as hyaluronic acid and about four times as rapidly as the small keratan sulphate fraction. The relative rates of synthesis of the sulphated glycosaminoglycans were calculated from the incorporation of [35S]sulphate and were in agreement with those from 14C-labelling studies.
Preparation and characterization of the plasma membrane of pig lymphocytes
Authors: Allan D. , Crumpton M. J.
Published: August 2015

Lymphocyte plasma membrane was isolated from minced pig mesenteric lymph node by differential centrifugation and by centrifuging through a sucrose density gradient. The yield of membrane was approx. 0.1% (dry wt. relative to wet wt. of lymph node). The purified material had a sucrose density of 1.14g/cm3 and consisted mainly of smooth vesicles. The membrane fraction contained, apart from protein and lipid, 59μg of carbohydrate, 11μg of sialic acid and 28μg of RNA/mg of protein; no DNA was detected. The cholesterol/phospholipid molar ratio was 1.01. Specific activities (μmol of product/h per mg of protein) of 5′-nucleotidase, succinate dehydrogenase, acid phosphatase and glucose 6-phosphatase were 10.1, 0, 0.51 and 0.30 respectively. The membrane vesicles were aggregated by an antiserum against pig lymphocytes and adsorbed the agglutinins to whole lymphocytes present in the antiserum; the membrane fraction was 28 times as effective as whole cells (on a dry wt. basis) in removing the lympho-agglutinins. Antisera against the membrane fraction agglutinated whole lymphocytes. It is concluded that the preparation represents the plasma membrane of small lymphocytes. The plasma membrane of pig thymocytes was isolated by using the same procedure. Its properties were similar to those of the lymphocyte plasma membrane.
The control of ribonucleic acid synthesis in bacteria. Steady-state content of messenger ribonucleic acid in Escherichia coli M.R.E. 600
Authors: Gray W. J. H. , Midgley J. E. M.
Published: August 2015

1. The technique of DNA–RNA hybridization was used to follow changes in the amount and average lifetime of unstable messenger RNA in Escherichia coli M.R.E. 600 over a wide range of different growth conditions. The method of analysis was based on the kinetics of incorporation of exogenous labelled nucleic acid bases into the RNA of steadily growing cultures, as described by Bolton & McCarthy (1962). 2. The ratio of the average lifetime of messenger RNA to the mean generation time of E. coli cultures was constant over the temperature range 25–45°C in a given medium, but the constant varied with the nature of the growth medium. For cultures growing in sodium lactate–salts or glucose–salts media the ratio was 0.046±0.005 and in enriched broth it was 0.087±0.009. Measurements of the amounts of transfer RNA, ribosomal RNA and messenger RNA were also made. The results confirmed earlier reports that the ratio of the amount of messenger RNA to the amount of ribosomes in the cells is virtually constant. On the other hand, the ratio of the amount of transfer RNA to the amount of ribosomal RNA decreased with increasing growth rate at a given temperature. 3. In cultures at temperatures higher than necessary for optimum rates of growth the average lifetime of messenger RNA lengthened in harmony with the increased time required for cell division. It seems that suboptimum growth rates at higher temperatures cannot be explained simply as a combination of increased rates of synthesis and breakdown of messenger RNA with a grossly decreased efficiency of translation. The absolute rate of messenger RNA synthesis was lowered, and its amount in the cells was typical of all other cultures grown at lower temperatures in the same medium. 4. The rate of entry of exogenous labelled uracil into unstable messenger RNA and stable ribosomal RNA was constant in all media at all temperatures in the approximate ratio 1:2. In media supporting a lower rate of growth, e.g. lactate–salts or glucose–salts media, the messenger RNA fraction constituted 2.2±0.3% of the total cellular RNA. In enriched broth 3.6±0.3% of the total RNA was messenger.
Isolation and properties of conjugated bilirubin from bile
Authors: Ostrow J. Donald , Murphy Nancy H.
Published: August 2015

1. A simple, rapid solvent partition method is described for isolation of conjugated bilirubin, free of unconjugated bilirubin, bile salts, phospholipids and cholesterol, from rat bile. Yields are 40–58%. The product is a phosphate-buffered solution containing approx. 0.4mg of bilirubin/ml, principally as mono- and di-glucuronide conjugates. The method may be modified for isolation of conjugates from human bile with 15–22% yield, and for preparation of unconjugated bilirubin from rat or human bile with yields of 55–62%. 2. The conjugated pigment has red–brown fluorescence and an absorption maximum at 450nm with ∈mM 59.8cm−1. Diazotization by the Malloy–Evelyn method gives a direct Van den Bergh reaction (in water) 12% greater than the total reaction (in methanol), with ∈total 28.4×103lmol−1cm−1 at 550nm. After desalting by elution from Sephadex LH-20 in 50% (v/v) ethanol, the product gave water-soluble mustard-yellow crystalline needles. Such desalted conjugates were precipitated by Pb2+ but not by Ba2+, Ca2+ or Zn2+. 3. At pH7.0 and 37°C the conjugated bilirubin was oxidized at a rate of 1%/h without hydrolysis, whereas 84% was hydrolysed by β-glucuronidase or aqueous alkali. 4. Mono- and di-glucuronides were separated by elution from Sephadex LH-20 in 95% (v/v) ethanol or by extraction with chloroform at pH3.2–3.4. The monoconjugated bilirubin did not become labelled during incubation with unconjugated [14C]bilirubin, and chromatographed as a single spot without dissociating into unconjugated bilirubin and diglucuronide as would be expected of a complex. 5. After intravenous injection of mono- or di-conjugated [14C]bilirubin into normal or Gunn rats, 79–91% was excreted in bile and 2–7% in urine over 2h. In these experiments injected diglucuronide was not hydrolysed whereas 30–41% of injected monoglucuronide was converted into diglucuronide by the normal but not by the Gunn rats. The evidence favours the existence of a true bilirubin mono-glucuronide that is not a complex.
Protein–polysaccharide of chicken cartilage and chondrocyte cell cultures
Authors: Shulman Herbert J. , Meyer Karl
Published: August 2015

The nature of the matrix produced by embryonic chicken chondrocytes in cell culture was studied, and compared with adult and embryonic chicken cartilage. Adult chicken cartilage contains a protein–polysaccharide easily extracted with EDTA–sodium chloride at 4°C. Purification of this macromolecule on Bio-Gel P-300 and Bio-Gel A-50m yielded a progressively more homogeneous species in the ultracentrifuge. It contained mostly chondroitin 4-sulphate, some chondroitin 6-sulphate, and keratan sulphate. Embryonic chicken cartilage was previously shown to contain mostly chondroitin 4-sulphate, some chondroitin 6-sulphate and essentially no keratan sulphate. The matrix produced in chondrocyte cell cultures was shown to contain a protein–polysaccharide with alkali-labile linkages of chondroitin 4-sulphate to the protein core. A fraction was isolated from the matrix with many properties of keratan sulphate.
Biosynthesis of glycosaminoglycans in bovine cornea. The effect of uridine diphosphate xylose
Authors: Balduini C. , Brovelli A. , Castellani A. A.
Published: August 2015

1. The role of UDP-xylose in the regulation of corneal glycosaminoglycan biosynthesis was investigated. Bovine corneas were incubated with [U-14C]-glucose in the presence and in the absence of the nucleotide, and the radioactivity of chondroitin, chondroitin sulphate and keratan sulphate, as well as of their monosaccharide constituents, was determined. 2. A decrease in the rate of biosynthesis of chondroitin and chondroitin sulphate and an increase in that of keratan sulphate were observed in the samples incubated with UDP-xylose. 3. The UDP-glucuronic acid isolated after the incubation in the presence of UDP-xylose showed a noticeable decrease in the amount of radioactivity incorporated; this result suggests that UDP-xylose inhibits the UDP-glucose dehydrogenase, causing an accumulation of UDP-glucose and consequently an increase in the formation of UDP-galactose and keratan sulphate. 4. Galactose and galactosamine isolated from the polysaccharides showed variations in the amount of radioactivity incorporated in accordance with those observed for the macromolecules; this fact confirms that in the system we used in vitro a real biosynthesis of the polysaccharide chain took place and that the regulatory effect of UDP-xylose was active at the monosaccharide level.
Interactions between potassium ions and glycine transport in the yeast Saccharomyces carlsbergensis
Authors: Eddy A. A. , Indge K. J. , Backen K. , Nowacki J. A.
Published: August 2015

A study has been made of the effects of both varying the pH and extracellular [K+] on the initial rate of uptake of glycine (v) by a strain of Saccharomyces carlsbergensis that concentrated the amino acid, with respect to the extracellular phase, by up to 1400 times. When no other substrate than glycine was provided and [glycine] was relatively small (≤0.2mm) (1) v increased fivefold when the pH was lowered from 7 to 4.5; (2) v fell by up to about 80% as [K+] rose, K+ behaving as a non-competitive inhibitor of the system, with Ki 0.33mequiv./l at pH7; (3) the absorption of glycine caused up to about 2 or 3 equiv. of K+ to leave the yeast cells. These three phenomena were each less evident when glucose was present. An analogy is drawn between the respective interactions of H+ and K+ with the yeast system and the well recognized effects of Na+ and K+ on amino acid transport in certain mammalian systems.
Molecular aspects of the regulation of rat kidney alkaline phosphatase
Authors: Melani F. , Farnararo M. , Chiarugi V. P.
Published: August 2015

The mechanisms by which phosphate regulates the activity of alkaline phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1) in rat kidney were investigated. Measurements of incorporation of [14C]leucine into kidney alkaline phosphatase in rats fed on complete or phosphate-free diet provide evidence of a twofold increase in the rate of synthesis of the enzyme in diet-treated animals. Cycloheximide experiments indicated that control and diet-adapted enzyme decreases in activity according to first-order kinetics with a calculated half-life of 10.3 and 6.5h after complete and phosphate-free diet administration respectively. Basal and diet-adapted enzymes exhibit similar Km values for several phosphomonoesters and an identical degree of inhibition is produced by cysteine. In addition, the enzyme from both sources is the same with regard to heat inactivation at 45, 56 or 64°C, to the profile of elution from Sephadex and to electrophoretic properties on polyacrylamide gel. A failure of rat kidney alkaline phosphatase to respond to cortisol (hydrocortisone) was also observed.
Complex-formation between cytochrome c and cytochrome c peroxidase. Kinetic studies
Authors: Nicholls Peter , Mochan Eugene
Published: August 2015

1. The kinetics of ferrocytochrome c peroxidation by yeast peroxidase are described. Kinetic differences between the older and more recent preparations of the enzyme most probably arise from differences in intrinsic turnover rates. 2. The time-courses of cytochrome c peroxidation by the enzyme follow essentially first-order kinetics in phosphate buffer. Deviations from first-order kinetics occur in acetate buffer, and are due to a higher enzymic turnover rate in this medium accompanied by a greater tendency to autocatalytic peroxidation of cytochrome c. 3. The kinetics of ferrocytochrome c peroxidation by yeast peroxidase are interpreted in terms of a mechanism postulating formation of reversible complexes between the peroxidase and both reduced and oxidized cytochrome c. Formation of these complexes is inhibited at high ionic strengths and by polycations. 4. Oxidized cytochrome c can act as a competitive inhibitor of ferrocytochrome c peroxidation by peroxidase. The Ki for ferricytochrome c is approximately equal to the Km for ferrocytochrome c and thus probably accounts for the observed apparent first-order kinetics even at saturating concentrations of ferrocytochrome c. 5. The results are discussed in terms of a possible analogy between the oxidations of cytochrome c catalysed by yeast peroxidase and by mammalian cytochrome oxidase.
Temperature and the regulation of enzyme activity in poikilotherms. Regulatory properties of fructose diphosphatase from muscle of the Alaskan king-crab
Authors: Behrisch Hans W.
Published: August 2015

1. The properties of fructose diphosphatase from skeletal muscle of the Alaskan king-crab (Paralithodes camtschatica) were examined over the physiological temperature range of the animal. 2. King-crab muscle fructose diphosphatase is first activated by Na+ and NH4+ and is then partially inhibited by these cations at concentrations higher than 10mm at 0°, 8° and 15°C. Enzyme activity is stimulated by K+ at 0°C, but is curtailed at 8°C and 15°C, an effect that could render rate independent of temperature. 3. Affinity for substrate increases with decreasing temperature; below the temperature of acclimatization, Km for fructose 1,6-diphosphate increases, resulting in a complex U-shaped temperature–Km curve. 4. King-crab muscle fructose diphosphatase is inhibited by low concentrations of AMP. As with enzymes of other poikilotherms, inhibition by AMP is sensitive to temperature; the enzyme is least sensitive to inhibition by AMP near the temperature of acclimatization. 5. The affinity of fructose diphosphatase for fructose 1,6-diphosphate is enhanced by phosphoenolpyruvate, and this activation is temperature-sensitive; 0.5mm-phosphoenolpyruvate causes a sevenfold decrease in Km for fructose 1,6-diphosphate at 15°C but a 25-fold decrease at 0°C. 6. Phosphoenolpyruvate appears to decrease the affinity of king-crab muscle fructose diphosphatase for AMP at low temperature, whereas at the higher temperature it appears to enhance inhibition by AMP. Phosphoenolpyruvate was not observed to cause a reversal of inhibition of fructose diphosphatase activity by AMP. The identification of phosphoenolpyruvate as an activator of a rate-limiting step in gluconeogenesis permits the suggestion of a coupling of the controlling mechanisms of several steps in the glycolytic and gluconeogenic chains. 7. These findings suggest mechanisms for the maintenance and regulation of control of fructose diphosphatase activity in king-crab skeletal muscle at low temperature and under conditions that favour concomitant activity of phosphofructokinase.
N-Carbamoyl-2-(p-hydroxyphenyl)glycine from leaves of broad bean (Vicia faba L.)
Authors: Eagles J. , Laird W. M. , Matai S. , Self R. , Synge R. L. M. , Drake A. F.
Published: August 2015

1. dl-2-(p-Hydroxyphenyl)glycine was resolved through the bromocamphorsulphonate to give its d-isomer. The N-carbamoyl derivatives of these amino acids were synthesized. Circular-dichroism studies on these and related compounds, reported in a deposited Annex, helped to establish the optical configuration. 2. N-Carbamoyl-dl-2-(p-hydroxyphenyl)glycine was isolated from broad-bean leaves. It amounted to about 0.1% of the leaf dry matter. Racemization may or may not have occurred during the isolation. There were indications of the same compound in chicory and in savoy cabbage. Under weakly acidic conditions it was converted gradually into 5-(p-hydroxyphenyl)hydantoin. Both these compounds yielded 2-(p-hydroxyphenyl)glycine on acid hydrolysis. 3. The occurrence is discussed of 2-phenylglycine derivatives in Nature and of N-carbamoyl-amino acids and hydantoins in plants. 4. Gradient elution from anion-exchange resin with acetic acid, besides proving useful for the present work, gave useful separations of pyrrolidonecarboxylic acid and of some N-acetyl-amino acids. 5. Supplementary material (Annex 1: details of experimental work other than ultraviolet and circular-dichroism spectra; Annex 2: ultraviolet absorption and circular dichroism of d-2-phenylglycine and some related compounds) has been deposited as Supplementary Publication SUP 50003 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. (1971), 121, 7.
The biosynthesis of valine from isobutyrate by Peptostreptococcus elsdenii and Bacteroides ruminicola
Authors: Allison Milton J. , Peel J. L.
Published: August 2015

1. Growing cultures of Peptostreptococcus elsdenii and Bacteroides ruminicola incorporate 14C from [1-14C]isobutyrate into the valine of cell protein. With P. elsdenii some of the 14C is also incorporated into leucine. 2. Crude cell-free extracts of both organisms in the presence of glutamine, carbon dioxide and suitable sources of energy and electrons incorporate 14C from [1-14C]isobutyrate into valine but not into leucine. 3. With extracts of P. elsdenii treated with DEAE-cellulose the reaction is dependent on ATP, CoA, thiamin pyrophosphate, molecular hydrogen and a low-potential electron carrier (ferredoxin, flavodoxin or benzyl viologen). 4. The same extracts incorporate 14C from NaH14CO3 into valine in the presence of isobutyrate plus ATP, CoA, glutamine and ferredoxin; isobutyryl-CoA or isobutyryl phosphate plus CoA will replace the isobutyrate plus CoA and ATP. With acetyl phosphate in place of isobutyryl phosphate, 14C is incorporated into alanine. With isovalerate or 2-methylbutyrate in place of isobutyrate, 14C is incorporated into leucine and isoleucine respectively. 5. When carrier 2-oxoisovalerate is added to the carboxylating system 14C from [1-14C]isobutyrate passes into the oxo acid fraction. 6. It is concluded that these two organisms form valine from isobutyrate by the sequence isobutyrate→isobutyryl-CoA→2-oxoisovalerate→valine and that the reductive carboxylation of isobutyrate is catalysed by a system similar to the pyruvate synthetase of clostridia and photosynthetic bacteria.
The amino acid sequence of sesame (Sesamum indicum L.) and castor (Ricinus communis L.) cytochrome c
Authors: Thompson E. W. , Richardson M. , Boulter D.
Published: August 2015

The amino acid sequences of sesame (Sesamum indicum L.) and castor (Ricinus communis L.) cytochrome c were determined by using 1.5μmol of protein from each species. Both molecules consist of a single chain of 111 amino acid residues and are homologous with other mitochondrial cytochrome c molecules. Both have an N-acetylated ‘tail’ of eight amino acids and two ∈-N-trimethyl-lysine residues, as also reported for wheat germ (Delange, Glazer & Smith, 1969) and mung-bean cytochrome c (Thompson, Laycock, Ramshaw & Boulter, 1970). Two different preparations of castor cytochrome c differed by one residue. This was glutamic acid for glutamine in position 100. The results for sesame and castor cytochrome c led to a re-examination and subsequent correction to the N-terminal region of the mung-bean cytochrome c sequence, as given by Thompson et al. (1970).
Properties of yeast grown anaerobically in media limiting in potassium
Authors: Bartley W. , Broomhead Valerie
Published: August 2015

1. Saccharomyces cerevisiae was grown anaerobically in media with different concentrations of K+ down to less than 1mm. Below 3.2mm the K+ concentration limited the growth rate and yield. 2. Yeast extract was essential for maximum growth. The yield of cells when the medium contained 0.83mm-K+ was only 30% of the yield with 90mm-K+. 3. At the end of anaerobic growth the cells grown in 0.83mm-K+ had a higher concentration of oxidative enzymes than cells grown in 90mm-K+. 4. The cells grown anaerobically in 0.83mm-K+ could adapt to aerobic conditions if K+ was present in the adaptation medium, but not otherwise. 5. The enzyme pattern of the yeast grown aerobically in 0.83mm-K+ was very similar to the anaerobically grown cells and did not change markedly after the glucose was consumed.
The arrangement of ribosomes in ribosome tetramers from hypothermic chick embryos
Authors: Carey N. H. , Read Gail S.
Published: August 2015

1. Ribosomes and the tetramer arrangement peculiar to the tissues of chick embryos exposed to low temperatures were separated by sucrose-density-gradient centrifugation, and the effects of variation of the concentrations of Mg2+, Ca2+ and K+ studied. 2. Lowering of the Mg2+ concentration from standard buffer conditions caused a reversible dissociation of tetramers into monomers and of these into subunits. 3. Ca2+ replaced Mg2+ in causing the re-formation of tetramers and monomers from subunits after dissociation in low Mg2+ concentrations. 4. Ca2+ also caused an almost complete conversion of monomers into dimers in the presence of Mg2+. 5. The effect of Ca2+ on the formation of dimers was abolished by pretreatment of the ribosomes with ribonuclease, but the re-formation of tetramers was unaffected. 6. Increase of the K+ concentration from that of the standard buffer caused dissociation of monomers and dimers into subunits. 7. Raised K+ concentration also caused a stepwise alteration of the tetramer from a particle with a sedimentation coefficient of 197S, which constitutes the bulk of the tetramer at low K+ concentrations, first to a 184S peak and finally to material with a sedimentation coefficient of about 155S. 8. The implications of these results on hypotheses of the arrangement of the individual monomers in the tetramer are discussed and a new model for the structure is proposed.