bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification, properties and amino acid sequence of atypical cytochrome c from two protozoa, Euglena gracilis and Crithidia oncopelti
Authors: Pettigrew G W , Leaver J L , Meyer T E , Ryle A P
Published: August 2015

A basic cytochrome was isolated from the phytomastigophorean protozoan Euglena gracilis and a similar protein from the zoomastigophorean protozoan Crithidia oncopelti. In both cases chromatography on CM-cellulose in first the reduced and then the oxidized form proved to be an efficient means of purification. The two cytochromes can be classed in the cytochrome c family but they have certain atypical features. The alpha peak of the absorption spectrum is shifted towards the red and is asymmetrical. The pyridine ferrohaemochrome has an alpha-peak maximum intermediate between that of c-type cytochromes and proteins containing protohaem IX. The test for free vinyl groups was positive. The amino acid sequences of the two cytochromes were determined. Attention is drawn in the text to those parts of the evidence that are less satisfactory. Both sequences are homologous with the family of cytochrome c, but are unusual in having only one cysteine residue so that the haem is attached through only one thioether bond. Detailed evidence for the amino acid dequences of the two proteins has been deposited as Supplementary Publication SUP 50042 (70 pages) at the British Library (Lending Division) (formerly the National Lending Library for Science and Technology), Boston Spa, Wetherby, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1975) 145, 5.
The major human erythrocyte membrane protein. Evidence for an S-shaped structure which traverses the membrane twice and contains a duplicated set of sites.
Authors: Jenkins R E , Tanner J A
Published: August 2015

The structure of the major human erythrocyte membrane protein (protein E) was investigated by studying the products of proteolysis of the native protein in the membrane. The distribution and location of the tyrosine residues labelled by radioiodination by lactoperoxidase was determined. Proteolysis of the extracellular region of the protein by thermolysin released four tyrosine-containing peptides, all of which were also found to remain in the major fragment that is retained in the membrane. The presence of these duplicated sites in the extracellular region of the protein was confirmed by limited trypsin digestion of the intracellular region of the protein. Two groups of fragments were obtained. Both groups contained a set of the extracellular labelled sites, but they differed in containing distinct groups of intracellular sites, showing that the two sets of extracellular sites are linked by an intracellular region of the protein. The polypeptide chain thus traverses the membrane twice. An S-shaped model which is consistent with these data is proposed.
Association of xanthine oxidase with the bovine milk-fat-globule membrane. Nature of the enzyme-membrane association.
Authors: Briley M S , Eisenthal R
Published: August 2015

1. Xanthine oxidase (EC 1.2.3.2) was found to represent more than 8% of the intrinsic protein of the bovine milk-fat-globule membranes. 2. Less than 25% of the xanthine oxidase activity of the fat-globule membrane was solubilized with 0.1 M-sodium pyrophosphate buffer or 2M-NaCl. Of the particulate activity remaining 56% was solubilized with Triton X-100. 3. The xanthine oxidase activity solubilized with buffer, 2M-NaCl or Triton X-100 was not liberated as the free enzyme. Only tryptic digestion was found to release the free enzyme from the fat-globule membrane. Tryptic digestion also liberated free xanthine oxidase from those fractions solubilized by buffer or NaCl, but not from those fractions solubilized with Triton X-100 or by sonication. 4. The effect of membrane association on the catalytic properties of the enzyme could be mimicked by low pH or by the presence in the assay mixture of certain concentrations of 2-methyl-propan-2-ol, but not 1,4-dioxan, suggesting that hydrogen-bonding rather than low dielectric constant may be involved. 5. The origin of the milk-fat-globule membrane is discussed with reference to the intrinsic nature of the associated xanthine oxidase activity.
The effect of D-xylose, β-D-xylosides and β-D-galactosides on chondroitin sulphate biosynthesis in embryonic chicken cartilage
Authors: Robinson H C , Brett M J , Tralaggan P J , Lowther D A , Okayama M
Published: August 2015

The incorporation of [3H]acetate into chondroitin sulphate was used as a measure of the rate of synthesis of this polysaccharide in whole tibias and femurs of embryonic chicken cartilage in vitro. The incorporation is inhibited by puromycin and by cycloheximide, but the inhibition is relieved by the addition of D-xylose, xβ-D-xylosides and β-D-galactosides to the incubation medium. β-D-Xylosides can stimulate the incorporation to 300% of that of controls incubated in the absence of cycloheximide or puromycin, D-Xylose, β-D-xylosides and β-D-galactosides appear to act as artificial initiators of chondroitin sulphate synthesis and enable polysaccharide-chain synthesis to be studied as an event separate from the synthesis of intact proteoglycan.
Turnover of carnitine by rat tissues
Authors: Brooks D E , McIntosh J E
Published: August 2015

Radioactive carnitine, in the form of L-[methyl-3H]carnitine, was administered intravenously to male rats and the specific radioactivity of carnitine in blood plasma and 13 tissues was measured for 16 days. There was no evidence of metabolism of carnitine to other compounds. A compartmental analysis was made by comparing the variation with time of the specific radioactivity of each tissue with one of two models. Kidney, heart and epididymal fat were best represented as containing a single compartment of carnitine, whereas spleen, liver, lung, adrenal, prostate, seminal vesicle, pancreas, muscle, testis and brain were best represented in terms of two compartments each exchanging carnitine with blood plasma. Estimates were obtained of the turnover times of carnitine in the individual tissue compartments as well as the fluxes across each compartment boundary. Analysis of the variation in the specific radioactivity of carnitine in urine and blood plasma. Estimates were obtained of the turnover times of carnitine in the individual tissue compartments as well as the fluxes across each compartment boundary. Analysis of the variation in the specific radioactivity of carnitine in urine and blood plasma indicated an average total excretion rate of carnitine of 10.4mumol/day, of which about 3.2mumol was found in the urine.
Proportional activities of glycerol kinase and glycerol 3-phosphate dehydrogenase in rat hepatomas
Authors: Harding J W , Pyeritz E A , Morris H P , White H B
Published: August 2015

The activities of glycerol 3-phosphate dehydrogenase (EC 1.1.1.8), glycerol kinase (EC 2.7.1.30), lactate dehydrogenase (EC 1.1.1.27), “malic’ enzyme (L-malate-NADP+ oxidoreductase; EC 1.1.1.40) and the beta-oxoacyl-(acyl-carrier protein) reductase component of the fatty acid synthetase complex were measured in nine hepatoma lines (8 in rats, 1 in mouse) and in the livers of host animals. With the single exception of Morris hepatoma 16, which had unusually high glycerol 3-phosphate dehydrogenase activity, the activities of glycerol 3-phosphate dehydrogenase and glycerol kinase were highly correlated in normal livers and hepatomas (r = 0.97; P less than 0.01). The activities of these two enzymes were not strongly correlated with the activities of any of the other three enzymes. The primary function of hepatic glycerol 3-phosphate dehydrogenase appears to be in gluconeogenesis from glycerol.
The ribosomal ribonucleic acid of Agrobacterium tumefaciens
Authors: Schuch W , Loening U E
Published: August 2015

The 23S rRNA of Agrobacterium tumefaciens contains at least two nicks which result in the formation of RNA components with mol.wts. of 0.52 × 106 and 0.48 × 106. Thus under the usual conditions of extraction and analysis, no 23S rRNA was recovered from the bacterium. The experiments show that 23S rRNA is synthesized as a continuous chain, in which one or two nicks are formed almost immediately near the ends of the molecule and an additional nick in the middle at a later time.
The extraction and characterization of bovine epidermal α-keratin
Authors: Steinert P M
Published: August 2015

1. The α-fibrous protein (α-keratin) component of bovine epidermis has been extracted and characterized. 2. Prekeratin, a multichain unit of the epidermal tonofilaments, was shown to consist of six different polypeptide chains on polyacrylamide-gel systems containing sodium dodecyl sulphate or sodium decyl sulphate with discontinuous gel buffers, but only three chains were seen when a gel system containing sodium dodecyl sulphate with a continuous gel buffer was used. 3. Extraction of the ‘keratinized’ stratum corneum and the living part of the epidermis with urea buffers at pH 7.6 or 9.0 released 60% of the total dry weight of the tissues in the form of α-helical polypeptides. 4. The numbers, relative amounts and properties of the extracted polypeptides were the same as the subunits of prekeratin and thus are derived from the tonofilaments in situ. 5. The subunits of prekeratin and the polypeptides extracted from the living cell layers contained an average of six cysteine residues, but those from the stratum corneum contained an average of three intrachain disulphide bonds. 6. The polypeptide chains aggregated through non-covalent interactions in vitro into filaments that were similar to the tonofilaments. 7. Since the polypeptides could be released from the stratum corneum without breaking covalent bonds, it is concluded that such bonds do not cross-link the tonofilaments and non-fibrous keratohyalin. It is suggested that the tonofilaments and keratohyalin of bovine epidermis are associated by secondary bonding forces.
The effect of some amine oxides and disulphide compounds on the activity of acetylcholinesterase
Authors: Dawson R M
Published: August 2015

Three amine oxides, two aliphatic and one aromatic, and 2,2'-dithiodipyridine were found to be weak reversible inhibitors of acetylcholinesterase. 5,5'-Dithiobis-(2-nitro-benzoic acid) had no effect on the enzyme under the conditions of the Ellman spectrophotometric assay.
Glutamine and asparagine as nitrogen donors for reductant-dependent glutamate synthesis in pea roots
Authors: Miflin B J , Lea P J
Published: August 2015

Glutamine, in the presence of α-oxoglutarate, stimulates nicotinamide nucleotide oxidation by crude extracts of pea roots and leads to a reductant-dependent formation of glutamate. Commercially available asparagine also stimulates nicotinamide nucleotide oxidation in the presence of α-oxoglutarate, but the reaction causing the stimulation can occur in the absence of a reductant, is inhibited by transaminase inhibitors, and is additive to the glutamine reaction. The asparagine used was found to be contaminated with aspartate. Repurified asparagine, chromatographically free of aspartate, did not stimulate the rate of nicotinamide nucleotide oxidation, and it is probable that the original stimulation was due to aspartate contamination. It is concluded that pea-root glutamine (amide)-α-oxoglutarate aminotransferase (glutamate synthase), in common with the enzyme in leaves, is specific for glutamine as the N donor and α-oxoglutarate as the N acceptor. The significance of the enzyme in conjunction with glutamine synthetase in the assimilation of nitrate by roots is discussed.
Physicochemical properties of Planorbis corneus erythrocruorin
Authors: Wood E J , Mosby L J
Published: August 2015

The erythrocruorin from the snail Planorbis corneus had a sedimentation coefficient, s020,w, of 33.5 ± 0.31S, and a molecular weight of 1.65 × 10(6) ± 0.04 × 10(6) by high-speed sedimentation-equilibrium ultracentrifugation. The amino acid composition and absorption spectrum of the protein are reported. A very low number of half-cystine residues was found, corresponding to 0.4 residue per haem group. The haem content was 2.76 ± 0.22%, corresponding to a protein molecular weight of about 22300. Under both acid and alkaline conditions partial dissociation took place to yield mixtures of products that could not be identified. A subunit corresponding to that containing one haem group was not obtained under any of the dossociating conditions tried. Electron microscopy revealed a ring-shaped molecule about 12.2 ± 0.5 nm in diameter. The native erythrocruoerin bound O2 co-operatively, the intermediate value of h in Hill plots having values between 1.7 and 3.4 depending on the conditions.
Secondary alkylsulphatases in a strain of Comamonas terrigena
Authors: Fitzgerald J W , Dodgson K. S. , Matcham G. W. J.
Published: August 2015

The occurrence in a strain of Comamonas terrigena of secondary alkylsulphatase activity towards potassium decan-5-yl sulphate is reported. A number of cell-washing and osmotic-shock procedures for releasing bacterial exocytoplasmic enzymes were ineffective in releasing this activity. Primary alkylsulphatases are not present in the organism, nor can their formation be induced under a wide variety of experimental conditions tested.
The interaction of magnesium ions with teichoic acid
Authors: Lambert P A , Hancock I C , Baddiley J
Published: August 2015

The binding of Mg2+ to the wall teichoic acid of Lactobacillus buchneri N.C.I.B. 8007 was measured by equilibrium dialysis at controlled ionic concentration and pH. In an aqueous solution containing 10mM-NaCl at pH 5.0 one Mg2+ ion was bound for every two phosphate groups of the teichoic acid, with an apparent association constant, Kassoc. = 2.7 × 103 M-1. On lowering the pH below the pKa of the phosphate groups the amount of bound Mg2+ decreased concomitantly with decreasing ionization of the phosphate groups. Both the amount of Mg2+ bound to the teichoic acid and the apparent association constants were similar in the presence of 10 mM concentrations of NaCl or KCl but decreased markedly in the presence of 10 mM-CaCl2 because of competition between Ca2+ and Mg2+ for the binding sites. A similar effect was found when the concentration of NaCl was increased from 0 to 50 mM. The results are discussed in relation to the function of teichoic acid in the walls of Gram-positive bacteria.
A comparison of seven methods for fitting the Michaelis-Menten equation
Authors: Atkins G L , Nimmo I A
Published: August 2015

The Michaelis-Menten equation was fitted to simulated data containing different sorts of error by using the three linear transformations, and the methods of S. R. Cohen [Anal. Biochem. (1968) 22, 549-552], R. Eisenthal & A. Cornish-Bowden [Biochem. J. (1974) 139, 715-120], F. de M. Merino [Biochem. J.143, 93-95] and G. N. Wilkinson [Biochem. J. (1961) 80, 324-332). The best methods were those of Eisenthal & Cornish-Bowden (1974) and Wilkinson (1961).
The effects of ammonium, inorganic phosphate and potassium ions on the activity of phosphofructokinases from muscle and nervous tissues of vertebrates and invertebrates
Authors: Sugden P H , Newsholme E A
Published: August 2015

1. The effect of NH4+, Pi and K+ on phosphofructokinase from muscle and nervous tissues of a large number of animals was investigated. The activation of the enzyme from lobster abdominal muscle by NH4+ was increased synergistically by the presence of Pi or SO4(2-). In the absence of K+, NH4+ plus Pi markedly activated phosphofructokinase from all tissues studied. In the presence of 100 mM-K+, NH4+ plus Pi activated phosphofructokinase from nervous tissue and muscle of invertebrates and the enzyme from brain of vertebrates, but there was no effect of NH4+ plus Pi on the enzyme from the muscles of vertebrates. Nonetheless, NH4+ plus Pi increased the activity of vertebrate muscle phosphofructokinase in the presence of 50 mM-K+ at inhibitory concentrations of ATP, i.e. these ions de-inhibited the enzyme. In the absence of NH4+ plus Pi, K+ activated phosphofructokinase from vertebrate tissues at non-inhibitory ATP concentrations, but the effect was less marked with the enzyme from invertebrate tissues. Indeed, high concentrations of K+ (greater than 50 mM) caused inhibition of invertebrate tissue phosphofructokinase. Of the other alkali-metal ions tested, only Rb+ activated phosphofructokinase from lobster abdominal muscle and rat heart muscle. 2. The properties of lobster abdominal-muscle phosphofructokinase were studied in detail. This muscle was chosen as representative of invertebrate muscle because large quantities of tissue could be obtained from one animal and the enzyme was considerably more stable in tissue extracts than in extracts of insect flight muscle. In general, the properties of the enzyme from this tissue were similar to those of the enzyme from many other tissues: ATP concentrations above an optimum value inhibited the enzyme and this inhibition was decreased by raising the fructose 6-phosphate or the AMP concentration. In particular, NH4+ plus Pi activated the enzyme at noninhibitory concentrations of ATP and they also relieved ATP inhibition (see above). 3. It is suggested that increases in the concentration of NH4+ and Pi, under conditions of increased ATP utilization in certain muscles and/or nervous tissue, may play a part in the stimulation of glycolysis through the effects on phosphofructokinase (the effect may be a direct activation and/or a relief of ATP inhibition). Changes in the concentration of NH4+ and Pi are consistent with this theory in nervous tissue and the anaerobic type of muscles. The role of AMP deaminase in production of NH4+ from AMP in these tissues is discussed in relation to the control of glycolysis.
Sulphation of p-hydroxyphenylpyruvic acid and related compounds by the rat liver cytosol
Authors: Hext P M , Rose F A
Published: August 2015

Cytosol preparations of rat liver and kidney were examined for their ability to transfer sulphate from adenosine 3′-phosphate 5′-sulphatophosphate to p-hydroxyphenylpyruvic acid. Little activity towards this substrate was observed, and the main product detected in the reaction mixtures was identified as p-hydroxybenzyl alcohol O-sulphate. This was not formed from p-hydroxybenzaldehyde, a spontaneous oxidation product of p-hydroxyphenylpyruvic acid, by sulphation followed by a rapid enzyme-catalysed reduction of the intermediate phydroxybenzaldehyde O-sulphate. This product was formed mainly by this sequence of reactions, but the reverse, reduction followed by sulphation, also appeared possible. p-Hydroxybenzyl alcohol itself was very readily sulphated by both preparations, and the liver also produced a sulpho-conjugate of homogentisic acid. These observations are important in calculating the turnover of L-tyrosine O-sulphate in the mammalian system, and establish that p-hydroxyphenylpyruvic acid O-sulphate is an end product of its metabolism, rather than an intermediate in its synthesis by reversed transamination.
Rate-limiting factors in the oxidation of ethanol by isolated rat liver cells
Authors: Meijer A J , van Woerkom G M , Williamson J R , Tager J M
Published: August 2015

The oxidation of ethanol by isolated liver cells from starved rats is limited by the rate of removal of reducing equivalents generated in the cytosol by alcohol dehydrogenase. Evidence is presented suggesting that, in these cells, transfer of reducing equivalents from the cytosol to the mitochondria is regulated by the intracellular concentrations of the intermediates of the malate-aspartate and glycerol 3-phosphate cycles, as well as by flux through the respiratory chain. In liver cells isolated from fed rats, the availability of substrate increased the cell content of intermediates of the hydrogen-transfer cycles, and enhanced ethanol uptake. Under these conditions, ethanol consumption is limited by the availability of ADP for oxidative phosphorylation.
Progesterone synthesis by pig corpus luteum tissue during superfusion
Authors: Watson John , Wrigglesworth Pauline M.
Published: August 2015

A modified superfusion technique is described with which it was demonstrated that the action of gonadotrophin on progesterone secretion by pig corpus luteum tissue is twofold, in that it first stimulates the rapid release of progesterone (either performed or partially synthesized), which is followed by prolonged synthesis of the steroid de novo from acetate.
An 18O double-labelling study of haemoglobin catabolism in the rat
Authors: Brown S B , King R F G J
Published: August 2015

The degradation of haemoglobin to bilirubin in the rat was investigated by 18O labelling of the molecular oxygen consumed by the animal. The oxygen atoms incorporated into bilirubin were derived from two different oxygen molecules. Implications of this finding for the mechanism of haem catabolism in vivo are discussed; both verdohaem and a dioxygen-bridged compound appear to be excluded at intermediates.
Adenosine 3′:5′-cyclic monophosphate-dependent and plasma-membrane-associated protein kinase(s) from bovine corpus luteum. Properties of associated enzyme and phosphorylation of specific plasma-membrane proteins
Authors: Azhar S , Menon K M
Published: August 2015

Plasma-membrane fractions FI and FII isolated from bovine corpus luteum by discontinuous sucrose-density-gradient centrifugation, at sucrose-density interfaces of 1.14/1.16 and 1.16/1.18 respectively, contained membrane-associated protein kinases that phosphorylated both the structural proteins of membranes as well as exogenously added protein substrates. Both fractions were characterized with respect to endogenous and exogenous protein substrate specificity, pH-dependence, effect of bivalent metal ions and sensitivity toward cyclic nucleotides. These membrane-associated kinases showed an optimum pH of 6.0 and had an absolute requirement for bivalent metal ions such as Mg2+, Mn2+, or Co2+ that cannot be replaced by Ca2+. Both the activities were stimulated two- to four-fold by cyclic AMP in vitro with an apparent Km of 83 and 50 nM for fractions FI and FII respectively. Other cyclic 3′:5′-nucleotides were effective only at higher concentrations, but even the most effective, cyclic IMP, showed a stimulation nearly an order of magnitude lower than that of cyclic AMP. In contrast, stimulation by cyclic dTMP and cyclic dAMP was very weak. Cyclic AMP showed no significant effect on the apparent Km value of both enzymes for histone and MgCl2 but it somewhat decreased the Km value for ATP. Nucleoside triphosphates like GTP, CTP and UTP inhibited the transfer of [32P]Pi from [γ-32P]ATP into mixed histone catalysed by membrane-associated kinases either in the presence or in the absence of cyclic AMP. In addition to protein kinases, these membrane fractions also possessed cyclic AMP-binding activities. The apparent association constant (Kalpha) for cyclic AMP binding was 1.0 × 10(10) and 2.6 × 10(10) M for FI and FII membrane fractions respectively.
The reversible delipidation of a solubilized sodium-plus-potassium ion-dependent adenosine triphosphatase from the salt gland of the spiny dogfish
Authors: Ottolenghi P
Published: August 2015

A microsomal fraction rich in Na+, K+-ATPase (sodium-plus-potassium ion-dependent adenosine triphosphatase) and the corresponding K+-dependent p-nitrophenyl phosphatase from the rectal salt gland of the spiny dogfish was solubilized by treatment with deoxycholate at high ionic strength. On gel filtration through Sepharose 6B, the ATPase apoenzyme could be separated, in apparently soluble form, from the tissue-fraction phospholipids and was almost free of enzymic activity (2% of the p-nitrophenyl phosphatase activity and 0.2% of the ATPase activity being recovered). On mixing the apoenzyme with an activator consisting of cooked ox brain, a large proportion of the original enzymic activity was obtained. Specific activities of the re-activated enzyme were somewhat higher than in the material before gel filtration: values of 1300-1450 mumol and 250-290 mumol/h per mg of protein were obtained for the hydrolysis of ATP and of p-nitrophenyl phosphate respectively. The activity was inhibitible by ouabain.
The transport of 25-hydroxycholecalciferol in a New World monkey
Authors: Hay A W
Published: August 2015

Albumin is responsible for the transport of 25-hydroxycholecalciferol in the Capuchin monkey. This was confirmed by gel filtration, analytical polyacrylamide-disc-gel electrophoresis, polyacrylamide-gel isoelectric focusing and a competitive protein-binding assay. Association constants of the serum transport proteins of a New and an Old World monkey towards 25-hydroxycholecalciferol were calculated; the transport protein in the New World monkey has a lower affinity for the vitamin D metabolite than the transport protein in the Old World primate.
The amino acid sequence of Staphylococcus aureus penicillinase
Authors: Ambler R P
Published: August 2015

The amino acid sequence of the penicillinase (penicillin amido-β-lactamhydrolase, EC 3.5.2.6) from Staphylococcus aureus strain PC1 was determined. The protein consists of a single polypeptide chain of 257 residues, and the sequence was determined by characterization of tryptic, chymotryptic, peptic and CNBr peptides, with some additional evidence from thermolysin and S. aureus proteinase peptides. A mistake in the preliminary report of the sequence is corrected; residues 113-116 are now thought to be -Lys-Lys-Val-Lys- rather than -Lys-Val-Lys-Lys-. Detailed evidence for the amino acid sequence has been deposited as Supplementary Publication SUP 50056 (91 pages) at the British Library (Lending Division), Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1975) 145, 5.
A genetic marker in the variable region of rabbit immunoglobulin heavy chain
Authors: Mole L R
Published: August 2015

A previous study [Mole et al. (1971) Biochem. J. 124, 301-318] showed several differences in sequence between the variable (V) regions of rabbit immunoglobulin Aa1 and Aa3 heavy chains. The inheritance of one such difference has been followed in a family of 38 rabbits by a radioautographic peptide-‘map’ technique and is shown to segregate in a Mendelian fashion. This clearly demonstrates the presence of a genetic marker in the rabbit heavy-chain V region, although the finding that Aa2 and Aa3 heavy chains have identity of sequence in the region studied obscures the relationship of this genetic marker to the a locus.
Purification and characterization of kynurenine-2-oxoglutarate aminotransferase from the liver, brain and small intestine of rats
Authors: Noguchi T , Minatogawa Y , Okuno E , Nakatani M , Morimoto M , Kido R
Published: August 2015

1. Kynurenine-2-oxoglutarate aminotransferase (isoenzyme 1) was purified to homogeneity from the liver, brain and small intestine of rats by the same procedure. The three enzyme preparations had nearly identical pH optima, substrate specificities and molecular weights. Isoenzyme 1 was active with 2-oxoglutarate but not with pyruvate as amino acceptor, and utilized a wide range of amino acids as amino donors. Amino acids were effective in the following order to activity: L-aspartate greater than L-tyrosine greater than L-phenylalanine greater than L-tryptophan greater than 5-hydroxy-L-tryptophan greater than L-kynurenine. The molecular weight was approximately 88 000 as determined by sucrose-density-gradient centrifugation. The pH optimum was between 8.0 and 8.5. On the basis of substrate specificity, substrate inhibition, subcellular distribution and polyacrylamide-disc-gel electrophoresis, it is suggested that liver, brain and small intestinal kynurenine-2-oxoglutarate aminotransferase (isoenzyme 1) is identical with mitochondrial tyrosine-2-oxoglutarate aminotransferase and also with mitochondrial aspartate-2-oxoglutarate aminotransferase. 2. An additional kynurenine-2-oxoglutarate aminotransferase (isoenzyme 2) was purified from the liver. This enzyme was specific for 2-oxoglutarate and L-kynurenine. Sucrose-density-gradient centrifugation gave a molecular weight of approximately 100 000. The pH optimum was between 6.0 and 6.5. This enzyme was not detected in the brain or small intestine.
Short Communications. Evidence for an active dimer of Escherichia coli β-galactosidase
Authors: Kaneshiro C M , Enns C A , Hahn M G , Peterson J S , Reithel F J
Published: August 2015

β-Galactosidase (EC 3.2.1.23), prepared from strains ML 308 and K12 3300 of Escherichia coli, dissociated into an inactive monomer in the presence of Ag+. When such a monomer preparation is treated with excess of thiol an enzymically active dimer is formed in addition to an active tetramer. It is suggested that Ag+ may be of value in studies on other multimeric proteins as a mild dissociating agent.
Physical studies on phosphonium phosphatidylcholine. A unique [31P]phosphorus nuclear-magnetic-resonance probe for model and biological membranes
Authors: Sim E , Cullis P R , Richards R E
Published: August 2015

1. Distearoyl phosphatidylcholine and the phosphonium analogue, in which the nitrogen atom is replaced by phosphorus, show similar gel-liquid crystalline transition temperatures as detected by differential scanning calorimetry. 2. The temperature-dependence of the 31P n.m.r. (nuclear-magnetic-resonance) linewidths of the phosphate resonances of sonicated vesicles of distearoyl phosphatidylcholine and the phosphonium analogue are similar. Below the phase-transition temperature the linewidths decrease as the temperature is raised. Above the phase-transition temperature the phosphate resonances are relatively temperature-independent. The phosphonium 31P n.m.r. signal exhibits the same pattern of temperature-dependence. 3. The 31P n.m.r. phosphonium resonance is sensitive to the paramagnetic shift reagent, K3Fe(CN)6. Use of K3Fe(CN)6, together with Nd(NO3)3, enabled the determination of the trans-bilayer distribution of egg-yolk phosphatidylcholine and its phosphonium analogue in co-sonicated vesicles. Both are distributed comparably across the bilayer of the vesicles. 4. The phosphonium 31P n.m.r. signal is much sharper than the corresponding phosphate resonance in both sonicated and unsonicated dispersions of the phosphatidylcholine analogue. 5. The properties of the phosphonium analogue of phosphatidylcholine are discussed in terms of its suitability as a probe of membrane structure.
Interchain disulphide bridges of mouse immunoglobulin M
Authors: Milstein C P , Richardson N E , Dieverson E V , Feinstein A
Published: August 2015

Mouse IgM (immunoglobulin M) was selectively and partially reduced and treated with iodo[2-14C]acetate to label the interchain disulphide bridges. The carboxymethylation was studied in some detail. The labelled peptides were purified, sequenced and positioned by homology with human IgM. Only peptides originating from three interchain disulphide bridges were labelled, in contrast with the four labelled bridges obtained in human IgM under the same conditions. These peptides are homologous to human bridge peptides forming the heavy-light bridge and two inter-heavy bridges, one present in the Cμ2 region and the other in the C-terminal region. The inter-heavy bridge in the Cμ2 region was alone cleaved and radioactively labelled in selectively reduced IgM held together as a pentamer by non-covalen interactions. The same bridge was the only one to be totally cleaved in subunits released after more extensive, though still selective, reduction. In the light of these results a possible arrangement of the disulphide bridges of the mouse IgM pentamer is proposed.
Purification of the Thy-1 molecule from rat brain
Authors: Barclay A N , Letarte-Muirhead M , Williams A F
Published: August 2015

The present paper describes the characterization of the Thy-1 molecule from rat brain. The molecule was recognized by its antigens, which could be solubilized from brain membrane with deoxycholate. In the solubilized form the Thy-1 antigens were associated with a homogenous component with the following hydrodynamic properties: s20,w=2.2s,v=0.72ml/g and Stokes radius=3.0nm. The mol.wt. of the deoxycholateantigen complex was estimated to be 27000; these values are not significantly different from those obtained thymocyte Thy-1. Brain Thy-1 was further purified by affinity chromatography with lentil lectin coupled to Sepharose 4B, and more than 80% of the antigen was bound. The material eluted with methyl α-D-glucopyranoside was then filtered on a column of Sephadex G-200, and only one glycoprotein was found in the antigenically active fraction. On sodium dodecyl sulphate-polyacrylamide-gel electrophoresis the glycoprotein was very similar to the Thy-1 from thymocytes that binds to lentil lectin. Its apparent mol.wt. on 12.5% acrylamide gels was 24000, and it electrophoresed as a symmetrical band. Brain Thy-1 was antigenically indistinguishable from thymocyte Thy-1 when analysed with rabbit antisera raised against brain or thymocyte Thy-1.
Palmitate activation and esterification in microsomal fractions of rat liver
Authors: Lloyd-Davies K A , Brindley D N
Published: August 2015

1. Palmitoyl-CoA synthetase activity in the microsomal fraction of rat liver was measured directly by palmitoyl-CoA production, and indirectly by converting the palmitoyl-CoA into palmitoylcarnitine under optimum conditions. Even in the latter system, palmitoyl-CoA accumulated. The rate of palmitoyl-CoA hydrolysis and the inhibition of palmitoyl-CoA synthetase by palmitoyl-CoA were each estimated to be less than 10% of the maximum rate of palmitoyl-CoA production. The concentration of palmitoyl-CoA present in the assay systems used for measuring palmitate esterification to glycerol phosphate and the activity of palmitoyl-CoA synthetase by using the carnitine-linked determination were measured. These concentrations were not altered by the addition of glycerol phosphate, or of carnitine plus carnitine palmitoyltransferase. The relationship between the activity of palmitoyl-CoA synthetase and the rate of glycerolipid synthesis was investigated. The latter activity was measured by using palmitoyl-CoA generated from palmitate, palmitoyl-AMP or palmitoylcarnitine. It is concluded that, at optimum substrate concentrations, the activity of glycerol phosphate acyltransferase is rate-limiting in the synthesis of phosphatidate by rat liver microsomal fractions. The implications of these results in the measurement of palmitoyl-CoA synthetase and in the control of glycerolipid synthesis are discussed.
The activity of phosphoenolpyruvate carboxykinase in rat tissues. Assay techniques and effects of dietary and hormonal changes
Authors: Pogson Christopher I. , Smith Stephen A.
Published: August 2015

1. Phosphoenolpyruvate carboxykinase was assayed by three methods: (i) incorporation of H14CO3- into oxaloacetate: (ii) conversion of oxaloacetate into phosphoenolpyruvate, subsequently assayed enzymically; and (iii) transfer of 32P from [γ-32P]GTP to oxaloacetate. 2. Enzyme activity is increased in liver and epididymal adipose tissue in alloxan-diabetes and starvation, and in kidney in starved, acidotic and steroid-treated animals. 3. The ratios of the ‘back’ to the ‘forward’ reactions in liver, kidney and epididymal adipose tissue are different and characteristic of each tissue; they differ markedly from values reported for the purified mitochondrial enzyme. 4. The ratio of the ‘back’ to ‘forward’ reaction in any one tissue is constant in adrenalectomized, diabetic, acidotic and steroid-treated animals. 5. In starved animals, the ratio is increased in liver and kidney, but decreased in epididymal adipose tissue. 6. Administration of l-tryptophan results in an acute (1h) increase in activity measured in the ‘forward’ direction alone in liver and epididymal adipose tissue, but not in kidney.
Short-chain fatty acid synthesis in brain. Subcellular localization and changes during development
Authors: Reijnierse G L , Veldstra H , Van der Ber C J
Published: August 2015

Acetyl-CoA synthase (EC 6.2.1.1), Propionyl-CoA synthase (EC 6.2.1.-) and butyryl-CoA synthase (EC 6.2.1.2) were measured in subcellular fractions prepared by primary and density-gradient fractionation from adult rat brain by a method resulting in recoveries close to 100%. Most of the activity of the three enzymes was recovered in the crude mitochondrial fraction. On subfractionation of this crude mitochondrial fraction with continuous sucrose density gradients, most of the activity of the three enzymes was found at a higher density than NAD+-isocitrate dehydrogenase and at about the same density as glutamate dehydrogenase, confirming earlier reported data for acetyl-CoA synthase. The finding that propionyl-CoA synthase and butyryl-CoA synthase had about the same distribution in the gradients as acetyl-CoA synthase adds support to the hypothesis that mitochondria involved in the metabolism of these short-chain fatty acids (all three of which have been shown to result in a rapid and high labelling of glutamine in vivo) form a distinct subpopulation of the total mitochondrial population. The three synthase activities were found to differ from each other in their rate of change and their subcellular localization during rat brain development. This, in combination with the observation that in gradients of adult brain preparations the three activities did not completely overlap, suggests that the three synthase activities are not present in the same proportion to each other in the same subpopulation (s) of mitochondria in the brain.
The pentose cycle and insulin release in isolated mouse pancreatic islets during starvation
Authors: Hedeskov C J , Capito K
Published: August 2015

When islets from mice were incubated with 16.7 mM-glucose, previous starvation for 48 h decreased the rate of insulin release by approx. 50% and glucose utilization was decreased by approx. 35%. The maximally extractable activity of glucose 6-phosphate dehydrogenase was diminished by 28% after starvation. The formation of 14CO2 from both [1-14C]glucose was, however, higher than the rate of oxidation of [6-14C]-glucose in islets from both fed and starved mice. The fraction of glucose utilized that was oxidized (specific 14CO2 yield) ranged from one-fifth to one-third and was higher in islets from starved mice with both [1-14C]glucose and [6-14C]glucose as substrate. The contribution of pentose-cycle oxidation to total glucose metabolism was small (3% in the fed state and 4% in the starved state). The absolute rates of glucose carbon metabolism via the pentose-cycle oxidation to total glucose metabolism was small (3% in the fed state and 4% in the starved state). The absolute rates of glucose carbon metabolism via the pentose cycle and the turnover of NADPH in this pathway were identical in islets from fed and starved animals. After incubation at 16.7 mM-glucose for 30 min the contents of glucose (6-phosphate and 6-phosphogluconate were both unchanged by starvation. It is concluded that there is no correlation between the decreased sensitivity of the insulin secretory mechanism during starvation and the metabolism of glucose via the pentose cycle, the islet content of glucose 6-phosphate or 6-phosphogluconate.
A neutral collagenase from human gastric mucosa
Authors: Woolley D E , Tucker J S , Green G , Evanson J M
Published: August 2015

Biopsy specimens of human gastric mucosa, maintained in culture for 7 days in the absence of serum, released a collagen-degrading enzyme into the medium. The yield of active enzyme reached a maximum after 2-3 days, and viable tissue, capable of protein synthesis, was essential for its production. 2. At 25 degrees C the enzyme attacked undenatured collagen in solution, resulting in a 55% loss of specific viscosity and producing the two products TCA and TCB characteristic of neutral-collagenase action. 3. Electron microscopy of segment-long-spacing crystallites of these reaction products showed the exact cleavage locus of the collagen molecules to be between bands 43 and 44 (I-43). The larger TCA and smaller TCB products were fragments representing 77 and 23% respectively of the length of the collagen molecule. 4. Optimal enzyme activity was observed over the pH range 7.5-8.5 and a mol.wt. of approx. 38000 was derived from gel-filtration studies. 5. The enzyme was shown to be inhibited by the human serum proteins ²2-macroglobulin and a smaller component of mol.wt. approx. 40000; α1-anti-trypsin was not inhibitory. 6. EDTA, 1, 10-phenanthroline, cysteine and dithiothreitol all inhibited collagenase activity. 7. The gastric enzyme has properties similar to other well characterized collagenases, but differences exist with respect to its molecular size and the site of attack on the collagen molecule.
Mobility of sodium dodecyl sulphate - protein complexes
Authors: Dunker A K , Kenyon A J
Published: August 2015

Reduced and unreduced lysozyme aggregates formed by formaldehyde cross-linking comprise a set of model compounds for studying the effects of protein conformation on the electrophoretic mobilities of sodium dodecyl sulphate-protein complexes. The reduced aggregates were indistinguisable from normal proteins, but the unreduced aggregates migrated anomalously fast by about 14%. Contrary to expectations, plots of logarithm Rf versus Kr (retardation coefficient) failed to reveal an unusual conformation for the unreduced aggregates. Thus the anomalous mobility caused by several intramolecular disulphide bonds escaped detection by the above two diagnostic plots. Also included in this paper is a discussion of the implications of these results with regard to current models for sodium dodecyl sulphate-protein complexes.
A new subunit of horse liver alcohol dehydrogenase and subunit composition of the polymorphic form
Authors: Pietruszko R , Ryzewski C N
Published: August 2015

The most cathodal (on starch-gel electrophoresis), steroid-active band of horse liver alcohol dehydrogenase, whose catalytic properties were shown to be dependent on the livers used as a starting material [Pietruszko (1974) Biochem. Biophys. Res. Commun. 60, 687-694], has been prepared from A-type and S-type horse livers by identical methods. Results presented here show that different isoenzymes are present in these preparations.
The extraction and purification of a cysteine transfer ribonucleic acid from baker's yeast
Authors: Holness N J , Atfield G
Published: August 2015

1. A modification of the RPC 1 system of A.D. Kelmers, G.D. Novelli & M.P. Stulberg (1965) (J. Biol. Chem. 240, 3979-3983) is described in which the support medium is a Celite of narrow range particle size treated with dichlorodimethylsilane. 2. By using this system an apparently pure preparation of tRNA Cys was isolated from baker's yeast tRNA. 3. This preparation accepted at least 60% of the theoretical quantity of [3-14C]cysteine in a conventional assay and failed to accept isoleucine, phenylalanine, proline, serine or tyrosine. 4. A theoretical countercurrent-distribution curve calculated by assuming a distribution coefficient K of 2.03 was in excellent agreement with the profiles of E260 and cysteine-acceptor ability after 537 transfers in the 1.85 M-phosphate/formamide/propan-2-ol system of C.M. Connelly & B.P. Doctor (1965) (J. Biol. Chem. 241, 715-719). 5. Chromatography of tRNA Cys on Bio-Gel P100 polyacrylamide beads afforded two components one of which was far less efficient than the other in accepting cysteine. The base compositions of the two were similar.
Hydroxylation of p-coumaric acid by horseradish peroxidase. The role of superoxide and hydroxyl radicals
Authors: Halliwell B , Ahluwalia S
Published: August 2015

1. In the presence of dihydroxyfumarate, horseradish peroxidase catalyses the conversion of p-coumaric acid into caffeic acid at pH 6. This hydroxylation is completely inhibited by superoxide dismutase. 2. Dihydroxyfumarate cannot be replaced by ascorbate H2O2, NADH, cysteine or sulphite. Peroxidase can be replaced by high (10 mM) concentrations of FeSO4, but this reaction is almost unaffected by superoxide dismutase. 3. Hydroxylation by the peroxidase/dihydroxyfumarate system is completely inhibited by low concentrations of Mn2+ or Cu2+. It is proposed that this is due to the ability of these metal ions to react with the superoxide radical O2–. 4. Hydroxylation is partially inhibited by mannitol, Tris or ethanol and completely inhibited by formate. This seems to be due to the ability of these reagents to react with the hydroxyl radical -OH. 5. It is concluded that O2– is generated during the oxidation of dihydroxyfumarate by peroxidase and reacts with H2O2 to produce hydroxyl radicals, which then convert p-coumaric acid into caffeic acid.
Isolation of S-adenosyl-3-thiopropylamine from the eye of the sea catfish (Arius felis)
Authors: Ito S , Nicol J A C
Published: August 2015

A method is reported for the separation of S-adenosyl-3-methylthiopropylamine and other basic compounds in the eye of the sea catfish (Arius felis) by ion-exchange chromatography on CM-Sephadex. One of the basic compounds was isolated in crystalline form and was shown to be S-adenosyl-3-thiopropylamine by chemical and spectroscopic characterizations and by comparison with a synthetic sample.
Inhibition of glycolytic enzymes by 2-phosphotartronate
Authors: Thomas M K , Spring T G
Published: August 2015

2-Phosphotartronate has been synthesized by permanganate oxidation of glycerol 2-phosphate and has been tested as an inhibitor of five glycolytic enzymes that bind phosphoglycerate or phosphoglycollate. Competitive inhibition of rabbit muscle phosphoglycerate mutase, enolase and pyruvate kinase was observed. Triose phosphate isomerase and 3-phosphoglycerate kinase were not inhibited.
Lipid biosynthesis in liver slices of the foetal guinea pig
Authors: Jones C T , Ashton I K
Published: August 2015

Lipid synthesis as measured by the incorporation of acetate or 3H2O into slices of foetal liver, is much higher than in slices of adult liver and shows a peak at about two-thirds of gestation. At this time the synthesis from glucose was low and reached a peak 10 days later. The changes in the activity of ATP citrate lyase, which mirrored acetate incorporation, and the effect of glucose and pyruvate on acetate corporation into lipid suggests that some of the lipid synthesis occurs via intramitochondrial acetyl-CoA production from acetate. Despite this, lipid synthesis was not inhibited by (-)-hydroxycitrate. The low rate of synthesis from glucose at two-thirds of gestation is ascribed to the low activity of pyruvate carboxylase at this time and a role for a phosphoenolpyruvate carboxykinase in providing oxaloacetate for lipogenesis is proposed. The activity of fatty acid synthetase broadly agreed with the changes in lipid synthesis, whereas the activity of acetyl-CoA carboxylase was barely sufficient to account for the rates of lipid synthesis in vivo. Acetate and short-chain fatty acids are likely to be the major precursors for lipid synthesis in vivo.
Membrane attachment of folded chromosome of Escherichia coli
Authors: Dworsky P
Published: August 2015

By fractionation of the envelope part of membrane-associated folded chromosomes it is shown that only the outer membrane is bound to the DNA. Experiments with the M-band technique suggest the presence of attachment points for the membrane also in membrane-released folded chromosomes.
Effect of vitamin A nutritional status on the growth of oestrogen-primed chick oviduct
Authors: Joshi P S , Murthy S K , Ganguly J
Published: August 2015

Growth and development of the oestrogen-primed oviduct of immature chicks in terms of weight, length, total protein, total RNA, total DNA and total phospholipids are markedly arrested on deprivation of vitamin A; supplementation with retinyl acetate reversed the effect of the deficiency almost fully, whereas retinoic acid was only partly effective.