bluuebunny/crossref_metadata_2025 huggingface dataset:
A temperature-jump study of the reaction between azurin and cytochrome c-551 from Pseudomonas aeruginosa (Short Communication)
Authors: Brunori Maurizio , Greenwood Colin , Wilson Michael T.
Published: August 2015

Temperature-jump studies on the electron-transfer reaction between azurin and cytochrome c-551 clearly reveal two chemical relaxations. The amplitudes of these relaxation processes have identical spectral distributions, but the relaxation times show different dependences on the reactant concentrations. These findings are discussed in terms of possible models.
Human placental cathepsin B1. Isolation and some physical properties
Authors: Swanson Arnold A. , Martin Bill J. , Spicer Sam S.
Published: August 2015

A reproducible procedure for the isolation, from human placenta, of a cathepsin B1 in a homogeneous state, demonstrated by electrophoretic, ultracentrifugal and enzymic criteria, was carried out. The pH optimum was near pH5.5. The placental enzyme catalysed the release of acid-soluble u.v.-dense products from haemoglobin and myoglobin. It was inhibited by heavy metals and several compounds which react with the thiol groups. The optimum temperature was between 37° and 42°C. The molecular weight of the enzyme was calculated to be 24250.
Thiol groups of normal immunoglobulin G
Authors: Buchwald Barbara M. , Connell G. E.
Published: August 2015

Normal human immunoglobulin G (IgG) was treated with radioactive N-ethylmaleimide in the absence of a reducing agent but in the presence of a denaturing solvent. The sites of reaction were determined by isolation of the radioactive peptides from thermolytic digests of the heavy and light chains. Six radioactive peptides were purified from the digest of the light chain and ten from that of the heavy chain. When sequenced, all the peptides were identical with peptides that would be predicted for the half-cystine residues involved in disulphide bonds. The specific radioactivity of the peptides indicated that the proportion of the half-cystine residues in the reduced form varied from 0.57 to 2.54%. These results indicate that the disulphide bonds of IgG are not completely oxidized. The estimate of 0.2mol of SH group/mol of IgG (Cecil & Stevenson, 1965; Luks & Connell, 1968) can be accounted for if 0.8% of every half-cystine residue of the intrachain bonds was in the reduced form. Variable cysteine residues as have been described in several myeloma proteins must occur extremely infrequently among the immunoglobulins.
Properties and substrate specificity of some reactions catalysed by a short-chain fatty acyl-coenzyme A synthetase from seeds of Pinus radiata
Authors: Young Owen A. , Anderson John W.
Published: August 2015

1. Crude extracts of seeds of Pinus radiata catalysed acetate-, propionate-, n-butyrate- and n-valerate-dependent PPi–ATP exchange in the presence of MgCl2, which was apparently due to a single enzyme. Propionate was the preferred substrate. Crude extracts did not catalyse medium-chain or long-chain fatty acid-dependent exchange. 2. Ungerminated dry seeds contained short-chain fatty acyl-CoA synthetase activity. The activity per seed was approximately constant for 11 days after imbibition and then declined. The enzyme was located only in the female gametophyte tissue. 3. The synthetase was purified 70-fold. 4. Some properties of the enzyme were studied by [32P]PPi–ATP exchange. Km values for acetate, propionate, n-butyrate and n-valerate were 4.7, 0.21, 0.33 and 2.1mm respectively. Competition experiments between acetate and propionate demonstrated that only one enzyme was involved and confirmed that the affinity of the enzyme for propionate was greater than that for acetate. CoA inhibited fatty acid-dependent PPi–ATP exchange. The enzyme catalysed fatty acid-dependent [32P]PPi–dATP exchange. 5. The enzyme also catalysed the fatty acyl-AMP-dependent synthesis of [32P]ATP from [32P]PPi. Apparent Km (acetyl-AMP) and apparent Km (propionyl-AMP) were 57μm and 7.5μm respectively. The reaction was inhibited by AMP and CoA. 6. Purified enzyme catalysed the synthesis of acetyl-CoA and propionyl-CoA. Apparent Km (acetate) and apparent Km (propionate) were 16mm and 7.5mm respectively. The rate of formation of acetyl-CoA was enhanced by pyrophosphatase. 7. It was concluded that fatty acyl adenylates are intermediates in the formation of the corresponding fatty acyl-CoA.
Curves of ligand binding. The use of hyperbolic functions for expressing titration curves
Authors: Dixon Henry B. F.
Published: August 2015

1. The dependences of the concentrations of the non-ligated, uni-ligated and bi-ligated forms of a molecule that binds two molecules of ligand are expressed as functions of the logarithm of free ligand concentration by means of hyperbolic functions. Expressions are also given for the saturation both of an individual site and of the molecule as a whole. This form of expression allows derivation of the following points. 2. The sharpness of bell-shaped curves of concentration of the uni-ligated form is analysed in terms of the heights of their points of inflexion; these can rise to 1/√2 of the curve. 3. A single group can exhibit a doubly sigmoid saturation curve if this group and another have comparable affinities for a ligand, and if ligand binding at one of them diminishes the affinity at the other. If the molecular pK values pK1 and pK2 for the first and second molecules of ligand are called pK*±logm, so that K*2=K1K2 and m2=K1/K2, then the doubly sigmoid curve can be represented by the sum of two independent one-site saturation curves, in general of unequal height, of pK values pK*±log½[m+√(m2-4)]. The error in such representation is small either if the mutual interaction between the groups (i.e. m) is large, or if the groups have very similar affinities for the ligand. 4. The sum of two one-site saturation curves, again of pK values of pK*±log½[m+√(m2-4)] but of equal heights, gives a precise value for the total saturation, provided that the binding of one molecule does not promote the binding of a second, i.e. providing that m≥2. Hence determinations of saturation cannot distinguish interacting and possibly identical sites from independent and different ones.
Substrate specificity of an α-amino acid ester hydrolase produced by Acetobacter turbidans A.T.C.C. 9325
Authors: Takahashi Takeshi , Yamazaki Yoshio , Kato Koichi
Published: August 2015

A partially purified preparation of an α-amino acid ester hydrolase was obtained from Acetobacter turbidans A.T.C.C. 9325, which catalyses synthesis of 7-(d-α-amino-α-phenylacetamido)-3-cephem-3-methyl-4- carboxylic acid (cephalexin) from methyl d-α-aminophenylacetate and 7-amino-3-deacetoxycephalosporanic acid. The enzyme preparation catalysed both cephalosprin synthesis from 7-amino-3-deacetoxycephalosporanic acid and suitable amino acid esters (e.g. methyl d-α-aminophenylacetate, l-cysteine methyl ester, glycine ethyl ester, d-alanine methyl ester, methyl dl-α-aminoiso-butyrate, l-serine methyl ester, d-leucine methyl ester, l-methionine methyl ester) and the hydrolysis of such esters. The substrate specificity of the enzyme preparation for the hydrolysis closely paralleled the acyl-donor specificity for cephalosporin synthesis, even to the reaction rates. Only α-amino acid derivatives could act as acyl donors. The hydrogen atom on the α-carbon atom was not always required by acyl donors. The hydrolysis rate was markedly diminished by adding 7-amino-3-deacetoxycephalosporanic acid to reaction mixtures, but no effect on the total reaction rate (the hydrolysis rate plus synthesis rate) was observed with various concentrations of 7-amino-3-deacetoxycephalosporanic acid. Both the hydrolytic and the synthetic activities of the enzyme preparation were inhibited by high concentrations of some acyl donors (e.g. methyl d-α-aminophenylacetate, ethyl d-α-aminophenylacetate). The enzyme preparation hydrolysed α-amino acid esters much more easily than α-amino acid derivatives with an acid–amide bond.
The effect of acetyl-coenzyme A on phosphate-activated glutaminase from pig kidney and brain
Authors: Kvamme Elling , Torgner Ingeborg Aasland
Published: August 2015

Phosphate-activated glutaminase (EC 3.5.1.2; l-glutamine amidohydrolase) purified from pig kidney and brain is activated by CoA and short-chain acyl-CoA derivatives. Acetyl-CoA is the most powerful activator (KA about 0.2mm). Acetyl-CoA is maximally effective in the absence of other activating anions such as phosphate and citrate, and at low glutamine concentrations. The negative co-operative substrate activation observed at pH7 becomes more pronounced in the presence of acetyl-CoA. Similarly to phosphate, acetyl-CoA produces at high protein concentrations a different type of activation, which is time-dependent, depends on protein concentration and is accompanied by an increase in the sedimentation coefficient. Acetyl-CoA, phosphate and citrate appear to have binding sites in common. No significant difference was observed between kidney and brain phosphate-activated glutaminase.
Regulation of muscle phosphorylase b kinase activity by inorganic phosphate and calcium ions
Authors: Sacktor Bertram , Wu Niou-Ching , Lescure Ono , Reed W. Douglas
Published: August 2015

The regulation of the activity of blowfly flight-muscle phosphorylase b kinase by Pi and Ca2+ was studied, and the actions of these effectors on the kinases from insect flight and rabbit leg muscles were compared. Preincubation of blowfly kinase with Pi increased activity severalfold. The effect was concentration-dependent, with an apparent Km of about 20mm, and time-dependent, requiring at least 10min for maximal activation. Neither ATP nor cyclic AMP was needed, suggesting that a protein kinase may not be involved. Maximal activation of the insect kinase required Mg2+ in addition to Pi. The apparent Km for Mg2+ was 3mm. Rabbit leg-muscle phosphorylase b kinase was slightly inhibited, rather than stimulated, by Pi, and was strongly inhibited by K+, Na+ and Li+. At physiological concentrations, Ca2+ activated the phosphorylase b kinases from both blowfly flight and rabbit leg muscles. However, the responses to Ca2+ of the enzymes from the two tissues were different. The mammalian kinase had virtually no activity in the absence of Ca2+, and showed a large increase in activity over a narrow range of Ca2+ concentrations. Flight-muscle kinase had appreciable activity in the absence of Ca2+, and had a smaller increase over a wide range of Ca2+ concentration. The concentrations of Ca2+ required for half-activation were 0.1 and 1μm for the blowfly and rabbit enzymes respectively. The pH–activity profiles of the non-activated, phosphate- and Ca2+-activated kinase revealed considerable enhancement of activity with little, if any, increase in the ratio of activities at pH6.8 to those at 8.2. These results are discussed in relation to the mechanism coupling contraction to glycogenolysis and to the biochemical distinction between asynchronous and synchronous types of muscle.
The purification of bovine cathepsin B1 and its mode of action on bovine collagens
Authors: Etherington D. J.
Published: August 2015

1. Cathepsin B1 was isolated from bovine spleen by autolysis, (NH4)2SO4 fractionation and chromatography on Amberlite IRC-50. Two isoenzyme forms were purified to homogeneity by chromatography on CM-cellulose and DEAE-Sephadex. 2. A collagenolytic cathepsin was separated from cathepsin B1 during purification. The remaining collagenolytic activity of the purified cathepsin-B1 isoenzymes was no greater than 0.3 unit/unit of cathepsin B1 compared with about 5.0 unit/unit of cathepsin B1 in the autolysed spleen extracts. 3. The cathepsin B1 isoenzymes lowered the viscosity of gelatin at 37°C. Optimum activity was at pH4–5. 4. At 28°C the interchain cross-links in native tropocollagen were cleaved most effectively at pH4–5. Insoluble tendon collagen was digested at pH3.5 and 28°C to yield mainly α-chain components, with the loss of a short N-terminal sequence. 5. Electron-microscope studies of collagen fibrils showed that cathepsin B1 caused longitudinal splitting and dissociation of the protofilaments. The effect was not general but occurred at selected sites. 6. The isoenzymes of cathepsin B1 cleaved the telopeptide region of calf skin tropocollagen between the lysine-derived cross-link and the triple helix. The CB1 peptide fragments obtained from enzyme-degraded α1 chains were hydrolysed at Gly12-Ile13 and Ser14-Val15. The residual α2 CB1 peptides were hydrolysed at Ala8-Asp9 and Asp9-Phe10.
The enzymic and non-enzymic degradation of colneleic acid, an unsaturated fatty acid ether intermediate in the lipoxygenase pathway of linoleic acid oxidation in potato (Solanum tuberosum) tubers
Authors: Galliard T. , Wardale D. A. , Matthew J. A.
Published: August 2015

Colneleic acid is an unsaturated ether fatty acid derived from linoleic acid via a lipoxygenase-mediated enzyme pathway. It is degraded (a) by an enzyme in potato tubers which is heat-labile and non-dialysable and (b) by a model system containing catalytic amounts of Fe2+ ions. Both enzyme- and Fe2+-catalysed systems have similar properties with respect to pH optima (pH5.0–5.5), oxygen requirement (0.6–0.7 mol of O2 consumed/mol of ether degraded), inhibitors and reaction products. An unstable product breaks down to C8 and C9 carbonyl fragments. Both systems are inhibited by low concentrations of antioxidants (e.g. 5μm-butylated hydroxytoluene) and some chelating agents (e.g. 5μm-diethyldithio-carbamate). The model system is strongly inhibited by metal ions, particularly Cu2+ and Fe3+, at 20μm. Hydrogen peroxide and haemoproteins do not substitute for the enzyme or Fe2+ ions but the non-haem iron protein, ferredoxin, does catalyse the degradation.
Oxidation of formate by peroxisomes and mitochondria from spinach leaves
Authors: Halliwell Barry
Published: August 2015

1. Spinach (Spinacia oleracea L.) leaf extracts catalyse the oxidation of formate to CO2. 2. Two enzymic systems are responsible for this oxidation, the peroxidatic action of catalase (EC 1.11.1.6) and NAD-dependent formate dehydrogenase (EC 1.2.1.2). 3. Formate dehydrogenase is mainly, if not exclusively, located in the mitochondria. This enzyme has a pH optimum of 6–6.5 and a Km for formate of 1.7mm in the presence of 1 mm-NAD+. 4. Peroxidatic action of catalase is presumed to take place in peroxisomes, since these seem to be the subcellular site of catalase. Formate oxidation at pH5 by chloroplast and mitochondrial fractions is due to their ability to generate H2O2 and the presence of contaminating catalase. 5. During photorespiration, peroxidatic oxidation of formate by catalase can occur over a wide range of pH values, but the rate of this reaction is probably controlled by the concentration of formate present, to an extent dependent on the pH.
Fate of corticotrophins in an isolated adrenal-cell bioassay and decrease of peptide breakdown by cell purification
Authors: Bennett Hugh P. J. , Bullock Gillian , Lowry P. J. , McMartin Colin , Peters Judith
Published: August 2015

1. The fate of corticotrophins in a trypsin-dispersed rat adrenal-cell assay system was investigated with a view to establishing whether differences in the rate of inactivation might contribute to potency differences observed between analogues. 2. Corticotrophin-(1–24)-tetracosapeptide and to a lesser extent synthetic 1–39 corticotrophins were found to be inactivated during incubation with cell suspension. 3. Peptide fragments were isolated by using [[3H2]Tyr23]corticotrophin-(1–24)- tetracosapeptide as a marker. The fragments indicate a peptidase with a predominantly tryptic specificity. 4. The peptidase is present in the extracellular fluid and is released from cells when they are damaged. 5. Cells were fractionated on an albumin gradient. Cells from the zona fasciculata and the zona intermedia or reticularis were present in fractions which produced fluorogenic steroids in response to corticotrophin. 6. Purification of the cells by centrifugation through albumin decreased degradation by peptidases, so that if the assay is carried out with a dilute suspension of purified cells peptide breakdown should not affect the observed potencies of adrenocorticotrophin analogues. 7. No binding of [[3H2]Tyr23]corticotrophin-(1–24)- tetracosapeptide to cells could be detected at low concentrations of the peptide. This indicated that less than 120 receptors/cell are occupied during stimulation by a dose that would elicit approx. 80% of the maximal response.
The development of cytochromes during the cell cycle of a glucose-repressed fission yeast, Schizosaccharomyces pombe 972h−
Authors: Poole Robert K. , Lloyd David , Chance Britton
Published: August 2015

1. Spectrophotometric analysis of intact cells of Schizosaccharomyces pombe, harvested from exponentially growing cultures during the phase of glucose repression, revealed the presence of cytochromes a+a3, c and at least two species of cytochrome b. 2. An absorption maximum at 554nm at 77°K, previously attributed to cytochrome c1, has been identified as a b-type cytochrome. 3. CO-difference spectra reveal the presence of cytochromes P-420 and P-450 in addition to cytochrome a3. 4. The cell cycle was analysed by separation of cells into classes representing successive stages in the cell cycle by isopycnic zonal centrifugation. 5. Cytochromes c548, b554 and b560 each exhibited a single broad maximum of synthesis during the cell cycle. 6. Amounts of cytochromes a+a3 and b563 (tentatively identified as cytochrome bT by its reaction on pulsing anaerobic cell suspensions with O2) oscillated in phase, and showed two maxima during the cycle; the second maximum of cytochromes a+a3 was coincident with a maximum of activity of enzymically active cytochrome c oxidase. 7. The amount of cytochrome P-420 decreased during the first three-quarters of the cell-cycle, whereas that of cytochrome P-450 increased during this period. 8. The discrepancy between spectrophotometric and enzymic assay of cytochrome c oxidase, the changing ratio of cytochrome a3/cytochrome a and the relationship between changes in cellular content of cytochromes and previous observations on respiratory oscillations during the cell cycle are discussed.
Oxidative phosphorylation in Escherichia coli K12. An uncoupled mutant with altered membrane structure
Authors: Cox G. B. , Gibson F. , McCann L.
Published: August 2015

1. A new mutant strain (AN228) of Escherichia coli K12, unable to couple phosphorylation to electron transport, has been isolated. The mutant allele (unc-405), in strain AN228, was found to map near the uncA and uncB genes at about minute 74 on the E. coli genome. 2. A transductant strain (AN285) carrying the unc-405 allele is similar to the uncA and uncB mutants described previously in that it is unable to grow on succinate, gives a low aerobic yield on limiting concentrations of glucose, has a normal rate of electron transport, is unable to couple phosphorylation to electron transport, and lacks ATP-dependent transhydrogenase activity. 3. Strain AN285 (unc-405) is similar to an uncA mutant, but different from an uncB mutant, in that it is unable to grow anaerobically in a glucose–mineral-salts medium, and membrane preparations do not have Mg2+-stimulated adenosine triphosphatase activity. 4. Strain AN285 (unc-405) does not form an aggregate analogous to the membrane-bound Mg2+-stimulated adenosine triphosphatase aggregate found in normal cells. In this respect it differs from strain AN249 (uncA−), which forms an inactive membrane-bound Mg2+-stimulated adenosine triphosphatase aggregate.
Microbial metabolism of amino alcohols. Aminoacetone metabolism via 1-aminopropan-2-ol in Pseudomonas sp. N.C.I.B. 8858
Authors: Faulkner Anne , Turner John M.
Published: August 2015

1. Pseudomonas sp. N.C.I.B. 8858 grew well on d- and l-1-aminopropan-2-ol and on aminoacetone. 2. Cell-free extracts possessed high activities of inducibly formed l-1-aminopropan-2-ol-NAD+ oxidoreductase, amino alcohol–ATP phosphotransferase, dl-1-aminopropan-2-ol O-phosphate phospho-lyase and aldehyde–NAD+ oxidoreductase, but no 1-aminopropan-2-ol racemase or d-1-aminopropan-2-ol-NAD+ oxidoreductase. 3. The amino alcohol kinase (activated by ADP) was non-stereospecific towards 1-aminopropan-2-ol and was one-third as active with ethanolamine. The phospho-lyase was active with l- and d-1-aminopropan-2-ol O-phosphate, but ethanolamine O-phosphate was only one-tenth as active as its higher homologues. The purified aldehyde dehydrogenase was active with propionaldehyde, acetaldehyde and also with methylglyoxal. The previously observed 2-oxo aldehyde dehydrogenase activity was considered to be due to the broadly specific aldehyde dehydrogenase. 4. Mutants of Pseudomonas sp. N.C.I.B. 8858 deficient in 1-aminopropan-2-ol kinase, 1-aminopropan-2-ol O-phosphate phospho-lyase, aldehyde dehydrogenase or an enzyme involved in propionate metabolism were incapable of growth on aminoacetone or 1-aminopropan-2-ol as carbon source, although all except the kinase- or phospho-lyasedeficient mutants could use these compounds and ethanolamine as nitrogen sources. The aldehyde dehydrogenase-deficient mutants produced copious amounts of propionaldehyde and acetaldehyde during growth on the corresponding amino alcohols. 5. The path of aminoacetone metabolism in Pseudomonas sp. N.C.I.B. 8858 was concluded to involve l-1-aminopropan-2-ol, the O-phosphate ester of this compound, propionaldehyde and propionate as obligatory intermediates. d-1-Aminopropan-2-ol was metabolized by the same route as the l-isomer, gratuitously inducing formation of the stereospecific l-1-aminopropan-2-ol dehydrogenase. 6. Extracts of the pseudomonad grown with ethanolamine as the nitrogen source were devoid of 1-aminopropan-2-ol dehydrogenase, the kinase and the phospho-lyase, but exhibited cobamide coenzyme-dependent deaminase activity. Mutants deficient in kinase or phospho-lyase (deaminating) grew well on ethanolamine as the nitrogen source. Ethanolamine deaminase was inactive with, but inhibited by, 1-aminopropan-2-ol.
Isolation and structure of nocobactin NA, a lipid-soluble iron-binding compound from Nocardia asteroides
Authors: Ratledge Colin , Snow G. Alan
Published: August 2015

Nocobactin NA, a lipid-soluble iron-chelating product with an unusual and characteristic u.v.-absorption spectrum, was isolated from Nocardia asteroides grown under conditions of iron deficiency. Its structure was determined by physical methods and by synthesis of one of its degradation products. Nocobactin NA was obtained as a homologous mixture of compounds with side chains of differing length, and resembles mycobactin M in structure except that it has an oxazole ring in place of an oxazoline ring, and the side chains in the cobactin fragment are considerably shorter.
The purification and amino acid sequence of cytochrome c-552 from Euglena gracilis
Authors: Pettigrew G. W.
Published: August 2015

Cytochrome c-552 from Euglena gracilis was purified and the amino acid sequence determined. The protein is a single peptide chain of 87 residues with the haem prosthetic group bound through two thioether linkages to two cysteine residues near the amino-terminal region. The amino acid sequence shows some similarities to mitochondrial cytochrome c and to two prokaryote c-type cytochromes. The sequence, taken with the known characteristics of cytochrome c-552, indicates that it is an f-type cytochrome. The possible functional and evolutionary significance of these features in common is discussed. Detailed evidence for the amino acid sequence of Euglena cytochrome f has been deposited as Supplementary Publication SUP 50027 at the British Library, Lending Division (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7QB, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973) 131, 5.
Adrenocorticotrophic and melanocyte-stimulating peptides in the human pituitary
Authors: Scott Alexander P. , Lowry Philip J.
Published: August 2015

The adrenocorticotrophic and melanocyte-stimulating peptides of the human pituitary were investigated by means of radioimmunoassay, bioassay and physicochemical procedures. Substantial amounts of adrenocorticotrophin and a peptide resembling β-lipotrophin were identified in pituitary extracts, but α-melanocyte-stimulating hormone, β-melanocyte-stimulating hormone and corticotrophin-like intermediate lobe peptide, which have been identified in the pars intermedia of pituitaries from other vertebrates, were not found. The absence of β-melanocyte-stimulating hormone appears to contradict previous chemical and radioimmunological studies. Our results suggest, however, that it is not a natural pituitary peptide but an artefact formed by enzymic degradation of β-lipotrophin during extraction.
The preparation and properties of pyruvate kinase from yeast
Authors: Fell David A. , Liddle Peter F. , Peacocke Arthur R. , Dwek Raymond A.
Published: August 2015

A new method is described for the preparation of pyruvate kinase from yeast. This eliminates proteolysis during the preparation. The molecular weight of yeast pyruvate kinase is 215000, and it is composed of four subunits. Such properties of the enzyme as its extinction coefficient, cold-lability, thiol-group reactivity and binding of Mn2+ ions are compared with those previously reported for yeast pyruvate kinase prepared by different methods. The specific activity is significantly higher than previously observed, but otherwise the enzyme is similar, apart from its molecular weight and Mn2+-binding characteristics, to preparations from Saccharomyces cerevisiae obtained in this laboratory (e.g. Fell et al., 1972, and references therein) and that of C. H. Suelter (e.g. Kuczenski & Suelter, 1971, and references therein), and is different from the enzyme isolated from Saccharomyces carlsbergensis by B. Hess and his co-workers (e.g. Wieker & Hess, 1972, and references therein).
Triterpenoid carotenoids and related lipids. The triterpenoid carotenes of Streptococcus faecium UNH 564P
Authors: Taylor Richard F. , Davies Brian H.
Published: August 2015

1. The occurrence of a novel series of triterpenoid carotenes in Streptococcus faecium UNH 564P is reported. 2. This series, which comprises the C30 analogues of phytoene, phytofluene, ζ-carotene, 7,8,11,12-tetrahydrolycopene and neurosporene, appears to be analogous to the C40 Porter–Lincoln series and a pathway of triterpenoid carotene dehydrogenation is proposed to account for the formation of these compounds. 3. Two cis isomers of the C30 analogue of neurosporene are described. 4. An appropriate system of nomenclature for these novel compounds is proposed.
Triterpenoid carotenoids and related lipids. Triterpenoid monohydroxy- and monoglucosyloxy-carotenoids from Streptococcus faecium UNH 564P
Authors: Taylor Richard F. , Davies Brian H.
Published: August 2015

1. The characterization of two novel triterpenoid xanthophylls occurring in Streptococcus faecium UNH 564P is described. 2. Both are derived formally, and probably biosynthetically, from the C30 analogue of neurosporene and have been identified as 4-hydroxy-4,4′-diaponeurosporene and its glucoside, 4-d-glucopyranosyloxy-4,4′-diaponeurosporene. 3. Problems associated with the use of specific glucosidases in defining the anomerism of carotenoid glucosides are discussed.
A spectrophotometric assay of β-lactamase action on penicillins
Authors: Waley Stephen G.
Published: August 2015

A new method for measuring the enzymic hydrolysis of the β-lactam ring in penicillins is described. The change in extinction in the u.v. region is determined. The method is sensitive (50μm-benzylpenicillin can be used) and convenient.
The occurrence of two types of deoxyribonucleic acid polymerase activity in the main classes of nuclei obtained from the brains of infant and adult rats
Authors: Stambolova M. A. , Cox D. , Mathias A. P.
Published: August 2015

1. The influence of exogenous or activated DNA template on the DNA polymerase activity in the different types of intact nuclei from rat brain tissue was determined. The different amounts or physical state of the DNA template did not produce significant differences in the relative distribution of the DNA polymerase activity between the separate groups of nuclei. 2. The DNA polymerase activities, fractionated by sucrose gradient centrifugation into enzyme A and enzyme B, were found to be present in the extracts of all types of rat brain nuclei. The distribution of these two activities in the ‘particulate’ and ‘soluble’ fractions of the separate groups of nuclei from 10-day-old and adult rats was studied. The findings are related to the DNA-synthetic activity in vivo of the intact nuclei and the possible biological functions of the DNA polymerase activities are discussed.
Some inhibitory effects of (−)-emetine on growth of Ehrlich ascites carcinoma
Authors: Johnson Randall K. , Jondorf W. Robert
Published: August 2015

(−)-Emetine has little or no effect on O2 consumption of Ehrlich ascites-cell suspensions or on the viability of transplanted Ehrlich ascites-tumour cells exposed to, or incubated with, the drug in vitro before inoculation into new-host mice. (−)-Emetine administered as a single injection to mice bearing Ehrlich ascites-tumour cells slows the growth rate of the tumour. The subcutaneous or intraperitoneal injection of the drug depresses protein and DNA synthesis of the tumour cells in vivo in a reversible manner. Mice bearing ascitic sarcoma 180 or Ehrlich ascites-tumour cells when given a course of treatment with (−)-emetine or (−)-O-methyltubulosine have a substantially lower tumour load than untreated controls and correspondingly longer survival times.
Utilization of gluconate by Escherichia coli. Uptake of d-gluconate by a mutant impaired in gluconate kinase activity and by membrane vesicles derived therefrom
Authors: Pouysségur J. M. , Faik Pelin , Kornberg H. L.
Published: August 2015

1. From Escherichia coli strain K2.1.5c.8.9, which is devoid of 6-phosphogluconate dehydrogenase (gnd) and 6-phosphogluconate dehydratase (edd) activities, a mutant R6 was isolated that was tolerant to gluconate though still edd-, gnd-. 2. Measurements of the fate of labelled gluconate, of the conversion of gluconate into 6-phosphogluconate, and of the induction of gluconate kinase by the two organisms show that, although both inducibly form a gluconate-transport system, strain R6 is impaired in its ability to convert the gluconate thus taken up into 6-phosphogluconate; it was therefore used for study of the kinetics and energetics of gluconate uptake. 3. Suspensions of strain R6 induced for gluconate uptake took up this substrate via a ‘high affinity’ transport process, with Km about 10μm and Vmax. about 25nmol/min per mg dry mass; a ‘low affinity’ system demonstrated to occur in certain E. coli mutants was not induced under the conditions used in this work. 4. The uptake of gluconate was inhibited by lack of oxygen and by inhibitors of electron transport; such inhibitors also promoted the efflux of gluconate taken up. 5. Membrane vesicles prepared from strain R6 also manifested these properties when incubated with suitable electron donors, at rates similar to those observed with whole cells. 6. The results indicate that the active transport of gluconate into the cells is the rate-limiting step in gluconate utilization by E. coli, and that the mechanism of this process can be validly studied with membrane vesicles.
Glycogen metabolism in the liver of the foetal rat
Authors: Devos Pierre , Hers Henri-Géry
Published: August 2015

1. The glycogen present in the liver of rat foetuses was labelled by injecting a trace amount of [6-3H]glucose into the mother at 19.5 days of gestation. The radioactivity incorporated in the glycogen 4h after the administration of the label was still present 38h later. A large proportion of this radioactivity was on the outer chains of the polysaccharide. These results indicate that there is normally almost no glycogen degradation in the foetal liver. In contrast, glycogen breakdown occurs very rapidly in the livers of foetuses whose mother is anaesthetized. 2. Glycogen synthetase is present in the liver at day 16 of gestation at a concentration as high as 30% of that in the adult, but essentially as an inactive (b) enzyme. The appearance of synthetase phosphatase between days 18 and 19 corresponds to that of synthetase a and to the beginning of glycogen synthesis. From day 19 to 21.5 the amount of synthetase a present in the foetal liver is just sufficient to account for the actual rate of glycogen deposition. 3. The content of total phosphorylase in the foetal liver increases continuously from day 16 to birth. However, a precise measurement of the a and b forms of the enzyme in the liver of non-anaesthetized foetuses is not possible. Taking the rate of glycogenolysis as an appropriate index of phosphorylase activity, we conclude that this enzyme is almost entirely in the inactive form in the foetal liver under normal conditions. 4. The accumulation of glycogen in the liver during late pregnancy may therefore be explained by a relatively slow rate of synthesis and a nearly total absence of degradation.
Ionophore-mediated coupling between ion fluxes and amino acid absorption in mouse ascites-tumour cells. Restoration of the physiological gradients of methionine by valinomycin in the absence of adenosine triphosphate
Authors: Reid M. , Gibb L. E. , Eddy A. A.
Published: August 2015

1. Preparations of mouse ascites-tumour cells depleted of ATP and Na+ ions accumulated l-methionine, in the presence of cyanide and deoxyglucose, from a 1mm solution containing 80mequiv. of Na+/l and about 5mequiv. of K+/l. Valinomycin increased, from about 4 to 16, the maximum value of the ratio of the cellular to extracellular concentrations of methionine formed under these conditions without markedly affecting the distributions of Na+ and of K+. Similar observations were made with 2-aminoisobutyrate, glycine and l-leucine. Increasing the extracellular concentration of K+ progressively decreased the accumulation of methionine in the presence of valinomycin. Over the physiological range of ionic gradients, the system behaved as though the absorption of methionine with Na+ was closely coupled to the electrogenic efflux of K+ through the ionophore. The process was insensitive to ouabain and so the sodium pump was probably not involved. 2. The amount of methionine accumulated during energy metabolism was similar to the optimal accumulation in the presence of valinomycin when ATP was lacking. It was also similarly affected by increasing the methionine concentration. 3. A mixture of nigericin and tetrachlorosalicylanilide mimicked the action of valinomycin. The anilide derivative inhibited the absorption of 2-aminoisobutyrate in the presence of valinomycin, but not in its absence. 4. Gramicidin inhibited methionine absorption and caused the preparations to absorb Na+ and lose K+. 5. The observations appear to verify the principle underlying the gradient hypothesis by showing that the tumour cells can efficiently couple the electrochemical gradient of Na+ to the amino acid gradient.
The biochemistry of virus-induced cell fusion. Changes in membrane integrity
Authors: Pasternak Charles A. , Micklem Kingsley J.
Published: August 2015

1. Phospholipids prelabelled with [14C]acetate, [32P]phosphate, [3H]- or [14C]-choline or [3H]inositol are not significantly degraded during fusion of Lettrée cells mediated by Sendai virus, nor are carbohydrates prelabelled with [3H]fucose, [14C]galactose or [3H]glucosamine. Less than 1nmol of lysophosphatidylcholine/107 cells is formed during fusion. Diethyl p-nitrophenyl phosphate, which inhibits phospholipase A by more than 95% has no effect on fusion. It is concluded that none of the events leading to cell fusion is accompanied by significant turnover of phospholipids or other membrane components. 2. Intracellular K+ leaks out during virally mediated cell fusion; the loss is not as extensive as that of intracellularly accumulated choline or deoxyglucose. Movement of Ca2+ into or out of cells could not be detected. 3. At concentrations of Lettrée cells insufficient to be agglutinated by virus, intracellularly accumulated choline and deoxyglucose leak out. Agglutination caused by concanavalin A does not result in leakage of intracellular metabolites. 4. P815Y cells, which agglutinate but do not fuse in the presence of virus, show leakage of intracellularly accumulated metabolites. The extent of leakage does not alter during the G1 and S periods of the cell cycle. 5. Leakage is inhibited by Ca2+, but is unaffected by EDTA. 6. It is concluded that the interaction of Sendai virus with mammalian cells causes a weakening of membrane integrity so that intracellular metabolites leak out. Such destabilization may facilitate viral entry and is therefore an interesting system for further biochemical studies.
Inhibition by local anaesthetics of adenine nucleotide translocation in rat liver mitochondria
Authors: Spencer Terry L. , Bygrave Fyfe L.
Published: August 2015

1. The mechanism of adenine nucleotide translocation in mitochondria isolated from rat liver was further examined by using the local anaesthetics procaine, butacaine, nupercaine and tetracaine as perturbators of lipid–protein interactions. Each of these compounds inhibited translocation of ADP and of ATP; butacaine was the most effective with 50% inhibition occurring at 30μm for 200μm-ATP and at 10μm for 200μm-ADP. The degree of inhibition by butacaine of both adenine nucleotides was dependent on the concentration of adenine nucleotide present; with low concentrations of adenine nucleotide, low concentrations of butacaine-stimulated translocation, but at high concentrations (greater than 50μm) low concentrations of butacaine inhibited translocation. Butacaine increased the affinity of the translocase for ATP to a value which approached that of ADP. 2. Higher concentrations of nupercaine and of tetracaine were required to inhibit translocation of both nucleotides; 50% inhibition of ATP translocation occurred at concentrations of 0.5mm and 0.8mm of these compounds respectively. The pattern of inhibition of ADP translocation by nupercaine and tetracaine was more complex than that of ATP; at very low concentrations (less than 250μm) inhibition ensued, followed by a return to almost original rates at 1mm. At higher concentrations inhibition of ADP translocation resulted. 3. That portion of ATP translocation stimulated by Ca2+ was preferentially inhibited by each of the local anaesthetics tested. In contrast, inhibition by the anaesthetics of ADP translocation was prevented by low concentrations of Ca2+. 4. The data provide further support for our hypothesis that lipid–protein interactions are important determinants in the activity of the adenine nucleotide translocase in mitochondria.
Changes in phylloquinone epoxidase activity related to prothrombin synthesis and microsomal clotting activity in the rat
Authors: Willingham Allan K. , Matschiner John T.
Published: August 2015

The oxidation of phylloquinone to the 2,3-epoxide (by phylloquinone epoxidase) was studied in liver from control and warfarin-resistant rats. The reaction requires microsomal fraction, soluble protein, a heat-stable soluble factor and O2. It is not inhibited by CO or CN−. Epoxidase activity was stimulated if plasma prothrombin was lowered either by anticoagulants or the absence of vitamin K. The activity of the enzyme rapidly returned to normal values after the administration of vitamin K to hypoprothrombinaemic rats. These differences in the activity of the enzyme occur in the microsomal fraction and not the cytosol. A thrombin-generating polypeptide that accumulates in microsomal fraction of hypothrombinaemic rats correlated directly with epoxidase activity. These data support the view that enzymic interconversion of phylloquinone and its 2,3-epoxide participates in the biological activity of vitamin K.
The mechanism of intestinal absorption of phosphatidylcholine in rats
Authors: Parthasarathy Sampath , Subbaiah Papasani V. , Ganguly Jagannath
Published: August 2015

1. The mechanism of absorption of phosphatidylcholine was studied in rats by injecting into the intestine phosphatidylcholine specifically labelled either in the fatty acid or in the glycerol moiety or with 32P, when considerable amounts of 1-acyl-lysophosphatidylcholine were found in the intestinal lumen. 2-([14C]Acyl)phosphatidylcholine gave markedly more radioactive unesterified fatty acids in the lumen, compared with the 1-([14C]acyl) derivative. Some of the radioactivity from either the fatty acid or the glycerol moiety of the injected phosphatidylcholine appeared in the mucosal triacylglycerols. 2. Injection of 32P-labelled phosphatidylcholine or 32P-labelled lysophosphatidylcholine led to the appearance of radioactive glycerylphosphorylcholine, glycerophosphate and Pi in the mucosa. 3. Rat mucosa was found to contain a highly active glycerylphosphorylcholine diesterase. 4. It was concluded that the dietary phosphatidylcholine is hydrolysed in the intestinal lumen by the pancreatic phospholipase A to 1-acylglycerylphosphorylcholine, which on entering the mucosal cell is partly reacylated to phosphatidylcholine, and the rest is further hydrolysed to glycerylphosphorylcholine, glycerophosphate, glycerol and Pi. The fatty acids and glycerophosphate are then reassembled to give triacylglycerols via the Kennedy (1961) pathway.
The metabolism of phenolic acids in the rat
Authors: Ranganathan S. , Ramasarma T.
Published: August 2015

Some of the enzyme systems in the formation of p-hydroxybenzoate from tyrosine have been studied in the rat liver in vitro. The conversion of p-hydroxycinnamate into p-hydroxybenzoate, which was found in rat liver mitochondria showed a number of differences when compared with the β-oxidation of fatty acids. Studies with p-hydroxy[U-14C]cinnamate indicated that 14CO2 was released during the formation of p-hydroxybenzoate. The formation of p-hydroxycinnamate from tyrosine of p-hydroxyphenyl-lactate could not be demonstrated in vitro. The interconversion of p-hydroxycinnamate and p-hydroxyphenylpropionate was demonstrated in rat liver mitochondria.
Membrane glycopeptides from old and young human erythrocytes (Short Communication)
Authors: Balduini Cesare , Balduini Carlo Luigi , Ascari Edoardo
Published: August 2015

Glycopeptides were extracted by papain digestion from old and young human erythrocyte membranes and fractionated on DEAE-Sephadex A-25. Chemical characterization of the unfractionated samples and of the main peak eluted from the column indicates that glycoproteins of the erythrocyte membrane undergo significant decreases in sialic acid and galactosamine content with aging.
Comparison of the amino acid sequences of the variable regions of light chains derived from two homogeneous rabbit anti-pneumococcal antibodies
Authors: Jaton Jean-Claude
Published: August 2015

The amino acid sequence of the N-terminal 139 residues of the L (light) chain derived from a homogeneous rabbit antibody to type III pneumococci was determined. This L chain, designated BS-5, exhibits a greater degree of homology with the basic sequence of human κ chains of subgroup I (72%) than with subgroups II and III. L-chain BS-5 differs from another L chain (BS-1), also derived from an antibody to type III pneumococci (Jaton, 1974), by eight amino acid residues, even though the chains are identical within the N-terminal 30 residues. Six of these eight substitutions are located within the three hypervariable sections of the variable half: Asn/Ser in position 31, Glu/Ala in position 55, Asx/Thr, Thr/Gly, Thr/Gly and Val/Tyr in positions 92, 94, 96 and 97 respectively. The two anti-pneumococcal L chains BS-1 and BS-5 are much more similar to each other than to an anti-azobenzoate L chain (Appella et al., 1973), from which they differ by 30 and 29 residues respectively. Of these interchanges 13–15 are confined to the three hypervariable sections, and 11 occur within the N-terminal 27 positions. The three chains have an identical sequence from residue 98 to residue 139, except for a possible inversion of two residues in positions 130–131 of the anti-azobenzoate chain.
Changes in the number of binding sites for ribonucleic acid polymerase in chromatin of WI-38 fibroblasts stimulated to proliferate
Authors: Hill Bridget T. , Baserga Renato
Published: August 2015

1. When WI-38 human diploid fibroblasts form confluent monolayers, DNA synthesis and cell division almost completely cease. A change of medium causes these density-inhibited cells to proliferate and within 1h after the application of the stimulus there is an increase in template activity of the chromatin isolated from stimulated cells. 2. The number of binding sites for Escherichia coli RNA polymerase was determined on chromatin from WI-38 cells by two different methods, i.e. incorporation of [3H]UTP into RNA in the absence of reinitiation, and incorporation of [γ-32P]GTP into chain termini. 3. Both methods indicate that the capacity of chromatin to bind E. coli RNA polymerase is increased in WI-38 cells stimulated to proliferate. 4. The increase in the number of binding sites for E. coli RNA polymerase parallels the increase in chromatin template activity and suggests that the latter reflects an increase in the number of initiation sites, rather than an increase in the rate of transcription.
Molecular heterogeneity of mouse duodenal alkaline phosphatase. Association of lipids and peptides
Authors: Nayudu P. R. V. , Hercus Fraser B.
Published: August 2015

Polyacrylamide-gel electrophoresis and Bio-Gel P-300 molecular-sieve chromatography of mouse duodenal alkaline phosphatase demonstrates its molecular heterogeneity, which, in a kinetic sense, is manifest also in the differential relative velocities of the heterogeneous forms of the enzyme with two substrates, phenylphosphate and β-glycerophosphate. Different treatments that eliminate most of the electrophoretic and chromatographic variability of the enzyme also decrease the velocities with both substrates so that the molar ratio of hydrolysis of one substrate relative to the other is also altered to a low but stable value. Concomitant with these changes, lipids and peptides are dissociated from the enzyme. The lipids are tentatively identified as a sterol and phospholipids. The peptides have an average composition of four to six amino acids and appear to be strongly electropositive. The conditions of dissociation suggest that their binding to the enzyme is non-covalent and predominantly based on hydrophobic and ionic bonding. The concept of lipid and peptide association would suggest prima facie differential molecular weights as a factor in the observed electrophoretic and chromatographic heterogeneity. However, the molecular forms of the enzyme with differences in elution volume equivalent to more than one-half the void volume of the Bio-Gel P-300 column, or even enzyme fractions dissociated from the lipids and peptides compared with undissociated portions, do not show any differences in sedimentation on sucrose-density-gradient centrifugation. This may be because the alterations in molecular weight owing to binding of small molecules are too small to be detected by this method. Alternatively, since lipids are involved, the binding may alter the partial specific volume in such a way that the buoyant density is not significantly altered.
Purification of human placental alkaline phosphatase. Salt effects in affinity chromatography
Authors: Doellgast George J. , Fishman William H.
Published: August 2015

Human placental alkaline phosphatase was chromatographed on Sepharose derivatives of d- and l-phenylalanine, l-leucine, glycine, aniline and p-aminobenzoic acid in high concentrations of (NH4)2SO4. Retention on these columns was greatest at the highest concentrations of (NH4)2SO4. By using decreasing concentrations and changing the types of salts, elution was effected from each of the columns. The (NH4)2SO4-mediated retention appeared to be related to the hydrophobic character of the substituted Sepharose, rather than to any specific binding site of the enzyme. It is suggested that this provides a way of controlling hydrophobic affinity chromatography. By use of chromatography on l-phenylalanine–Sepharose and of DEAE-Sephadex chromatography in the presence of Triton X-100 detergent, a preparation of highly purified (1000-fold) human placental alkaline phosphatase was obtained in 22% yield.