bluuebunny/crossref_metadata_2025 huggingface dataset:
P-cresol and 3,5-xylenol methylhydroxylases in Pseudomonas putida N.C.I.B. 9896
Authors: Keat M J , Hopper D J
Published: August 2015

Pseudomonas putida N.C.I.B. 9869, when grown on 3,5-xylenol, hydroxylates the methyl groups on 3,5-xylenol and on p-cresol by two different enzymes. 3,5-Xylenol methylhydroxylase, studied only in relatively crude extracts, requires NADH, is not active with p-cresol and is inhibited by cyanide, but not by CO. The p-cresol methylhydroxylase requires an electron acceptor and will act under anaerobic conditions. It was purified and is a flavocytochrome c of mol.wt. approx. 114,000 consisting of two subunits of equal size. The enzyme catalyses the hydroxylation of p-cresol (Km 16 micron) and the further oxidation of product, p-hydroxybenzyl alcohol (Km 27 micron) to p-hydroxybenzaldehyde. A different p-cresol methylhydroxylase of the flavocytochrome c type is induced by growth on p-cresol. It too was purified and has mol.wt. approx. 100,000, and again consisted of two equal-size subunits. The Km for p=cresol 3.6 micron and for p=hydroxybenzyl alcohol, 15 micron.
Resonance Raman-spectroscopic studies of the hapten features involved in the binding of 2,4-dinitrophenyl haptens by the mouse myeloma proteins MOPC 315 and MOPC 460
Authors: Kumar K , Phelps D J , Carey P R , Young N M
Published: August 2015

The binding of four dinitrophenyl haptens to the mouse myeloma proteins MOPC 315 IgA (immunoglobulin A) and MOPC 460IgA was studied by resonance Raman spectroscopy. Isotopic substitution with 15N and 2H was used to assign features in the resonance Raman spectra of the free haptens. Changes in each of these features on binding to the proteins could then be attributed to interactions of the proteins' binding sites with either the p-NO2 or the o-NO2/amine regions of the haptens. The interactions between a given hapten and MOPC 315 IgA are often quite distinct from those between the same hapten and MOPC 460 IgA. Moreover, for both antibodies the nature of the R side chain in a Dnp-NHR (Dnp, 2,4-dinitrophenyl) compound appears to modify the interactions between the Dnp chromophore and the protein. Thus, with the haptens studied, there is no unique set of contacts between the Dnp group and the binding site. The contacts expected between epsilon-2,4-dinitrophenyl-L-lysine and the site on MOPC 315 IgA, on the basis of a recent model for this site [Dwek, Wain-Hobson, Dower, Gettins, Sutton, Perkins & Givol (1977) Nature (London) 266, 31–37] were not detected. However, the contacts between this hapten and the site on MOPC 460 IgA were closer to those predicted by the model for MOPC 315 IgA.
The molybdenum centre of native xanthine oxidase. Evidence for proton transfer from substrates to the centre and for existence of an anion-binding site
Authors: Gutteridge S , Tanner S J , Bray R C
Published: August 2015

The observation by Bray & Knowles [Proc. R. Soc. London Ser. A (1968) 302, 351–353] of direct transfer, during the catalytic reaction, of hydrogen atoms from substrate molecules to the enzyme xanthine oxidase was reinvestigated. The experimental phenomenon and its basic interpretation were confirmed and extended. In the reduced functional enzyme, molybdenum(V) interacts with two enzyme-bound protons, which are exchangeable with solvent protons. One of these is coupled to the metal with AHav. 1.4mT and the other with AHav. 0.3mT. The molecule also contains a site for the binding of anions, presumably as ligands of molybdenum. This is shown by effects of nitrate ions on the e.p.r. spectra. The spectra of the nitrate and 1-methylxanthine complexes of the reduced enzyme are very similar to one another, and are designated Rapid type-1 spectra. It is concluded that, in the Michaelis complex, the substrate molecule occupies the anion site, probably being bound to molybdenum via the nitrogen in its 9-position. During the turnover process, hydrogen from the substrate C-8 position, after transfer to the enzyme, appears as the proton more strongly coupled to molybdenum. This proton then exchanges with solvent deuterium with a rate constant of 27s-1, at pH 8.2 and 12 degrees C. It has been confirmed that substrate molecules occupying the anion site do not interfere with observation of the transfer and exchange processes.
Isoelectric focusing of glutathione S-transferases from rat liver and kidney
Authors: Hales Barbara F. , Jaeger Valerie , Neims Allen H.
Published: August 2015

The glutathione S-transferases that were purified to homogeneity from liver cytosol have overlapping but distinct substrate specificities and different isoelectric points. This report explores the possibility of using preparative electrofocusing to compare the composition of the transferases in liver and kidney cytosol. Hepatic cytosol from adult male Sprague–Dawley rats was resolved by isoelectric focusing on Sephadex columns into five peaks of transferase activity, each with characteristic substrate specificity. The first four peaks of transferase activity (in order of decreasing basicity) are identified as transferases AA, B, A and C respectively, on the basis of substrate specificity, but the fifth peak (pI6.6) does not correspond to a previously described transferase. Isoelectric focusing of renal cytosol resolves only three major peaks of transferase activity, each with narrow substrate specificity. In the kidney, peak 1 (pI9.0) has most of the activity toward 1-chloro-2,4-dinitrobenzene, peak 2 (pI8.5) toward p-nitrobenzyl chloride, and peak 3 (pI7.0) toward trans-4-phenylbut-3-en-2-one. Renal transferase peak 1 (pI9.0) appears to correspond to transferase B on the basis of pI, substrate specificity and antigenicity. Kidney transferase peaks 2 (pI8.5) and 3 (pI7.0) do not correspond to previously described glutathione S-transferases, although kidney transferase peak 3 is similar to the transferase peak 5 from focused hepatic cytosol. Transferases A and C were not found in kidney cytosol, and transferase AA was detected in only one out of six replicates. Thus it is important to recognize the contribution of individual transferases to total transferase activity in that each transferase may be regulated independently.
Structure revision of disaccharidic conjugates of bilirubin-IX α in human bile and identification of phenylazo derivatives B4, B5, and B6 as 2-, 3- and 4-O-acylglucuronides
Authors: Compernolle F
Published: August 2015

Aniline azopigments B4, B5 and B6, derived from conjugates of bilirubin-IX alpha in human bile, and previously characterized as disaccharidic esters [Kuenzle (1970) Biochem. J. 119, 387-394 and 411-435], were analysed by using t.l.c. and mass spectrometry. The compounds were identified as partially separated mixtures of 2-, 3- and 4-O-acylglucuronide positional isomers. The 1-O-acylglucuronide was not detected in the mixtures and was the only compound hydrolysed with beta-glucuronidase. Further scrutiny of structural assignments made by Kuenzle [(1970) Biochem. J. 119, 411-435] led to identification of the lactone and hexuronic acid derivatives that were obtained from azopigment B5 along with glucuronolactone and glucuronic acid. A branched-chain structure, i.e. 3-C-hydroxy-methyl-D-riburonic acid, was assigned previously, but the derivatives have now been identified as various incompletely silylated forms of glucuronolactone and glucuronic acid. Several trimethylsilyl derivatives glucuronolactone were isolated and characterized by n.m.r. and mass spectrometry.
Evidence for a reciprocal relationship between lipogenesis and ketogenesis in hepatocytes from fed virgin and lactating rats
Authors: Benito M , Williamson D H
Published: August 2015

Lipogenesis is increased in hepatocytes from fed lactating rats compared with virgin rats. Inhibition of lipogenesis with 5-(tetradecyloxy)-2-furoic acid resulted in increased ketogenesis from endogenous substrate, but not from oleate. Dihydroxyacetone increased ketogenesis from endogenous substrate, but not from oleate. Dihydroxyacetone increased lipogenesis and esterification of [1–14C]oleate and decreased ketogenesis; these changes were reversed by the inhibitor. The reciprocal relationship between lipogenesis and ketogenesis in hepatocytes from fed rats may be due to alterations in [malonyl-CoA] [McGarry, Mannaerts & Foster (1977) J. Clin. Invest. 60, 265–270; Cook, King & Veech (1978) J. Biol. Chem. 253, 2529–2531], but this mechanism is not considered to be sufficient to explain the increased ketogenesis in starvation completely.
A method for measuring relative changes in guanosine 3′:5′-cyclic monophosphate in mouse neuroblastoma cells on muscarinic cholinergic stimulation
Authors: Strange P G
Published: August 2015

A convenient, inexpensive assay was developed for measuring relative changes in cyclic GMP in whole mouse neuroblastoma cells (clone NIE 115) based on labelling the cellular GTP pool with [8(-3)H]guanine. The time course of cell labelling and the distribution of radioactivity among possible products were studied; GTP is the only major labelled species. Radioactive cyclic GMP produced from the radioactive GTP on cell stimulation is isolated by column chromatography nad its identity has been rigorously established by paper chromatography and ion-exchange chromatography. The assay was used to study the time course of the cyclic GMP changes that occur after stimulation of neuroblastoma cells with carbamoylcholine and the dependence of the cyclic GMP changes on the carbamoylcholine concentration. The assay gives results comparable with those obtained by using a radioimmunoassay for cyclic GMP and should be applicable to other whole-cell and tissue-slice systems.
Enhancement by double-stranded polyribonucleotides of production by cultured mouse peritoneal macrophages of differentiation-stimulating factor(s) for mouse myeloid leukaemic cells
Authors: Tomida M , Takenaga K , Yamamoto Y , Hozumi M
Published: August 2015

Mouse peritoneal macrophages release a factor(s) that stimulates differentiation of a mouse myeloid leukaemic cell line into mature granulocytes and macrophages. Treatment of the macrophages with the synthetic double-stranded polyribonucleotides poly(I).poly(C) and poly(A).poly(U) resulted in enhanced release of the factor into the culture medium. The effect was maximal after treatment with polyribonucleotides for 1 h, and the optimal dose of poly(I).poly(C) was 50 microgram/ml. The single-stranded polyribonucleotides poly(I) and poly(C) at the same concentration were far less effective. The differentiation-stimulating factor was detected not only in the cultured medium but also in the cell lysate. Exposure of macrophages to poly(I).poly(C) enhanced the total activity of the factor in both the culture medium and the cell lysate. The effect of this compound was blocked by the presence of cycloheximide. These results suggest that double-stranded polyribonucleotides enhance production of the differentiation-stimulating factor by peritoneal macrophages.
The use of rat liver nucleoplasm for the characterization of heterogeneous nuclear ribonucleic acid synthesis in vitro
Authors: Beebee T J C
Published: August 2015

1. A nucleoplasmic fraction rich in endogenous RNA polymerase II activity was isolated from rat liver nuclei and conditions were determined under which elongation of RNA molecules initiated in vivo continued at maximal rates in vitro. 2. Elongation rates in vitro were calculated to be about 0.25 nucleotide/s and there were about 7 × 10(3) RNA molecules in the process of being elongated by form-II RNA polymerase per original nucleus. 3. Evidence was obtained suggesting that transcription-dependent release of RNA polymerase II molecules from the template occurred during the incubations in vitro. 4. The nascent RNA was tightly associated with protein and banded as ribonucleoprotein in caesium salt gradients. 5. RNA molecules labelled in vitro were up to 13000 nucleotides in length, but consisted of long unlabelled chains transcribed in vivo with only short labelled sequences added in vitro, and without significant polyadenylation. 6. Hybridization of transcripts in the presence of a vast excess of DNA demonstrated that both form-II RNA polymerase and another enzyme, resistant to low alpha-amanitin concentrations, were synthesizing RNA molecules complementary to both reiterated and unique DNA sequences in the genome.
Sources of ammonia for mammalian urea synthesis
Authors: Krebs H A , Hems R , Lund P , Halliday D , Read W W
Published: August 2015

The initial rate of incorporation of [15N]alanine into the 6-amino group of the adenine nucleotides in rat hepatocytes was about one-eighteenth of the rate of incorporation into urea. Thus the purine nucleotide cycle cannot provide most of the ammonia needed in urea synthesis for the carbamoyl phosphate synthase reaction (EC 2.7.2.5). On the other hand, contrary to the view expressed by McGivan & Chappell [(1975) FEBS Lett. 52, 1–7], the experiments support the view that hepatic glutamate dehydrogenase can supply the required ammonia.
An alternative hypothesis of cellular transport of lysosomal enzymes in fibroblasts. Effect of inhibitors of lysosomal enzyme endocytosis on intra- and extra-cellular lysosomal enzyme activities
Authors: Von Figura Kurt , Weber Ernst
Published: August 2015

Recapture of lysosomal enzymes secreted by fibroblasts was inhibited by growing the cells in the presence of either free or immobilized antibodies against lysosomal enzymes or in the presence of phosphorylated carbohydrates known to interact with the cell-surface receptors for lysosomal enzymes. The following results were obtained. 1. Conditions that prevent recapture of released lysosomal enzymes increase the rate of extracellular accumulation of these enzymes up to twice that of controls. 2. Growing cells for 12 days in the presence of 0.5mm-mannose 6-phosphate, which decreases β-N-acetylglucosaminidase endocytosis to less than 10% of that of controls, has no effect on the intracellular activity of this and four other lysosomal enzymes. 3. Growing cells for 4 days in the presence of 50mm-mannose 6-phosphate, which is a 1000-fold higher concentration than that required for 50% inhibition of lysosomal enzyme endocytosis, leads to a 4-fold increase in extracellular β-N-acetylglucosaminidase accumulation and a decrease in intracellular enzyme. These results give evidence that, in fibroblasts, transfer of lysosomal enzymes into lysosomes does not require secretion before a receptor-mediated recapture [Hickman & Neufeld (1972) Biochem. Biophys. Res. Commun.49, 992–999]. We propose that (a) lysosomal enzymes are present in a receptor-bound form in those vesicles that fuse with the cell membrane, (b) the major part of the lysosomal enzyme cycles via the cell surface in a receptor-bound form and (c) only a minor part of the lysosomal enzyme is released into the extracellular space during its life cycle.
Occurrence of an endo-1,3-β-glucanase in culture fluids of the yeast Candida utilis. Purification and characterization of the enzyme activity
Authors: Villa T G , Notario V , Villanueva J R
Published: August 2015

The endo-1,3-beta-glucanase (EC 3.2.1.6) secreted into the culture medium by cells of Candida utilis was isolated and purified to homogeneity on polyacrylamide-gel electrophoresis and in ultracentrifugation studies (s20,w = 1.97S). The purified enzyme represented only 0.001% of the total 1,3-beta-glucanase activity, the remainder being due to an exo-1,3-beta-glucanase enzyme, and behaved as an acidic glycoprotein (pI 3.3) in isoelectric-focusing experiments. The mol.wt. was estimated to be 21 000 by gel filtration and polyacrylamide-gel electrophoresis. Studies on the hydrolysis of different substrates showed that the enzyme was only able to break down (1 leads to 3)-beta-linkages, by an endo-splitting mechanism. Glucono-delta-lactone, D-glucoronolactone and heavy metal ions such as Hg2+ were inhibitors of the enzyme activity. The function of this endo-beta-glucanase in C. utilis is discussed.
The biosynthesis of methylcitrate
Authors: Weidman S W , Drysdale G R
Published: August 2015

Citrate synthase catalyses the formation in vitro of two diastereoisomers of methylcitrate from propionyl-CoA and oxaloacetate. The proportion of diastereoisomers produced is temperature-sensitive. In the presence of oxaloacetate and a thiol trap, the enzyme catalyses the exchange of both alpha-protons of propionyl-CoA with 2H2O. The pro-S-proton at the alpha-carbon atom is exchanged 15 times faster with 2H2O than is the pro-R-proton. The exchanges are over 1000 and 100 times faster respectively than Vmax. These results are interpreted in the light of recent reports that 2R,3S- and 2S,3S-methylcitrates are produced by the enzyme-catalysed condensation and also excreted by patients with propionic acidaemia.
Purification and properties of four species of lysyl oxidase from bovine aorta
Authors: Kagan Herbert M. , Sullivan Kathleen A. , Olsson Theodore A. , Cronlund Anne L.
Published: August 2015

Lysyl oxidase of bovine aorta was resolved into four enzymically active species by elution from DEAE-cellulose with a salt gradient in 6m-urea, consistent with purification results obtained with enzyme of other tissues [Stassen (1976) Biochim. Biophys. Acta438, 49–60]. In the present study, each of the four peaks of activity was purified to apparent homogeneity by subsequent chromatography on gel-filtration media in 6m-urea. Each enzyme is eluted as a species with mol.wt. approx. 30000 under these conditions, although lysyl oxidase polymerizes to a series of multimers with molecular weights ranging up to 1000000 in the absence of urea. The apparent subunit molecular weight of each enzyme species determined by electrophoresis in sodium dodecyl sulphate and 8m-urea is approx. 32000–33000. The amino acid compositions of the purified forms of lysyl oxidase are similar to each other, although sufficient differences exist to conclude that each is a unique molecular species. Incorporation of α-toluenesulphonyl fluoride into the purification scheme does not alter the resolution of enzyme into four species, suggesting that proteolysis during isolation is not the basis of the heterogeneity. The similar sensitivities of each form of enzyme to chelating agents and to semicarbazide and isoniazid indicate that each requires the participation of a metal ion, presumably Cu2+, and of a carbonyl compound for enzyme function. The present study describes a method for the purification of multiple species of lysyl oxidase and reveals that significant chemical differences exist between the different enzyme forms.
Interaction of dinitrophenyl groups bound to bovine serum albumin with univalent fragments of anti-dinitrophenyl antibody
Authors: Knight C. Graham , Green N. Michael
Published: August 2015

Two lysine residues of bovine serum albumin reacted with 1-fluoro-2,4-dinitrobenzene with apparent second-order rate constants approx. 500-times greater than those observed in similar reactions with low-molecular-weight lysine derivatives. A series of dinitrophenyl (Dnp)-bovine serum albumins were prepared and their ability to bind univalent fragments of anti-Dnp antibody was measured by fluorescence-quenching titrations. Compared with the Dnp group of the free hapten, 6-N-Dnp-aminohexanoate, the majority of the protein-bound Dnp groups were unavailable to the antibody at pH8.0. When the same Dnp-albumins were titrated at pH3.0 the availability of the Dnp groups increased approx. 3-fold. Dnp-albumins were treated with pepsin at pH3.0 and Dnp-containing fragments isolated by chromatography on DE-52 DEAE-cellulose. Fluorescence-quenching titrations showed that the Dnp groups on the fragments behaved like the free hapten with respect to quenching efficiency, although with an increased dissociation constant. The association between the Dnp-albumins and the antibody was measured also by difference-spectral titrations at high protein concentrations. Antibody binding was increased under these conditions, but the Dnp group of mono-Dnp-albumin remained unavailable to antibody. We propose that the reactive lysine residues are located in clefts between the globular sub-domains of the single polypeptide chain. Dnp groups attached to these lysine residues are fully exposed to the solvent, but binding of the macromolecular probe, anti-Dnp antibody, is sterically hindered by the adjacent surface of the albumin molecule.
A necessary modification to the preparation of papain from any high-quality latex of Carica papaya and evidence for the structural integrity of the enzyme produced by traditional methods
Authors: Baines Baldev S. , Brocklehurst Keith
Published: August 2015

A method of preparation of papain (EC 3.4.22.2) from relatively soluble types of latex of Carica papaya, including spray-dried latex produced by a controlled and relatively mild process, was devised. Spray-dried latex dissolves easily in water up to 350mg/ml at 22°C, which corresponds to approx. 230mg of protein/ml. When the usual method of preparation of crystalline papain contaminated only by its oxidation products, developed by Kimmel & Smith [J. Biol. Chem. (1954) 207, 515–531], is applied to spray-dried latex, the result is a preparation of papain heavily contaminated by chymopapains A and B (EC 3.4.22.6), and to a lesser extent by papaya peptidase A. This applies also to other types of papaya-latex currently commercially available, which, though less soluble than spray-dried latex, are more soluble than the types of latex available when the method of Kimmel & Smith (1954) was developed. This contamination is avoided by adjusting the concentration of the initial latex extract to 65mg of protein/ml (or less) before salt fractionation. For spray-dried latex this corresponds to 100mg of latex/ml. Papain isolated from spray-dried latex was characterized by using 2,2′-dipyridyl disulphide and 4-chloro-7-nitrobenzofurazan as thiol-specific reactivity probes and α-N-benzoyl-l-arginine ethyl ester as substrate. Papain isolated from this source appears to have the same catalytic-centre characteristics as papain isolated previously from latex produced by harsher methods. The catalysis of the hydrolysis of α-N-benzoyl-l-arginine ethyl ester by the mixture of thiol proteinases extracted from spray-dried latex by application of the method of Kimmel & Smith (1954) appears to obey Michaelis–Menten kinetics. The presence of the other enzymes results in an increase in the value of Km and a decrease in the catalytic-centre activity (kcat.) relative to the values for the catalysis by papain.
Enzyme-induced inactivation of transminases by acetylenic and vinyl analogues of 4-aminobutyrate
Authors: John R A , Jones E D , Fowler L J
Published: August 2015

The reactions of two analogues of 4-aminobutyrate, namely 4-aminohex-5-ynoate and 4-aminohex-5-enoate, with three transaminases were studied. Three pure enzymes were used, aminobutyrate transaminase (EC 2.6.1.19), ornithine transaminase (EC 2.6.1.13) and aspartate transaminase (EC 2.6.1.1), and the course of the reactions was studied by observing changes in the absorption spectrum of the bound coenzyme and by observing loss of activity. All of the enzymes were inactivated by either inhibitor, but amino-hexenoate showed a marked specificity for aminobutyrate transaminase. Aminohexynoate was most potent towards ornithine transaminase, and with this enzyme transamination of the inhibitor is an important factor in protecting the enzyme. Most of the reactions could be analysed as first order, with the observed rate constant showing a hyperbolic dependence on inhibitor concentration.
Lipid and steroid hydroperoxides as substrates for the non-selenium-dependent glutathione peroxidase
Authors: Shreve M R , Morrissey P G , O'Brien P J
Published: August 2015

The reduction of linoleic acid hydroperoxide catalysed by rat liver cytosol was previously shown to be catalysed by a selenium-dependent glutathione peroxidase. In contrast, the enzyme responsible in guinea-pig liver cytosol is not selenium-dependent and appears to be a glutathione transferase.
A steady-state-kinetic model for formaldehyde dehydrogenase from human liver. A mechanism involving NAD+ and the hemimercaptal adduct of glutathione and formaldehyde as substrates and free glutathione as an allosteric activator of the enzyme
Authors: Uotila L , Mannervik B
Published: August 2015

The steady-state kinetics of formaldehyde dehydrogenase from human liver have been explored. Non-linearities were obtained in v-versus-v[S] plots. It was necessary and sufficient to consider two reactants of the equilibrium mixture of formaldehyde, glutathione and their hemimercaptal adduct for a complete description of the kinetics. A random sequential reaction scheme is proposed in which adduct and beta-NAD+ are the substrates. In addition, glutathione can bind to an allosteric regulatory site and only the glutathione-containing enzyme is considered productive. Various alternative reaction models were examined but no simple alterative was superior to the model chosen. The discrimination was largely based on results of non-linear regression analysis. Several S-substituted glutathione derivatives were tested as activators or inhibitors of the enzyme, but all were without effect. Thio-NAD+, nicotinamide–hypoxanthine dinucleotide and 3-acetylpyridine-adenine dinucleotide could substitute for beta-NAD+ as the nucleotide substrate. alpha-NAD+ and ADP-ribose were competitive inhibitors with respect to beta-NAD+ and non-competitive with glutathione and the adduct. When used simultaneously, the inhibitors were linear competitive versus each other, indicating a single nucleotide-binding site or, if more than one, non-co-operative binding sites.
Liver monoamine oxidase in the obese–hyperglycaemic (ob/ob) mouse
Authors: Tong J H , Limson-Zamora M , D'Iorio A , Bégin-Heick N
Published: August 2015

1. The specific activity of monoamine oxidase was found to be greater in liver mitochondria from ob/ob mice than from lean mice. The activities of marker enzymes were similar in both tissues. 2. Experiments with various substrates (5-hydroxytryptamine, benzylamine and tyramine) and inhibitors (clorgyline and deprenyl) indicated that, unlike rat liver mitochondria, mouse liver mitochondria contain a predominance of the B-form of monoamine oxidase. 3. The Km values for lean and ob/ob mice were the same for any given substrate and were in the increasing order 5-hydroxytryptamine less than tyramine less than benzylamine. Vmax. was approximately 50% greater in obese than in lean mice. 4. Extraction of liver mitochondria with acetone/water or acetone/water/NH3 to remove lipids decreased the enzyme activity relatively more in obese- than in lean-mice preparations, but residual activity was the same in both preparations.
A rapid and sensitive method for measuring ribonucleic acid in ribosomal preparations
Authors: Mackie C , Bryant T H , Mowbray J
Published: August 2015

By using the fluorescence enhancement of ethidium bromide when it binds to RNA, a very rapid, simple and sensitive assay for the concentration of ribosomal RNA in complex mixtures has been devised.
Distinction between progestin- and glucocorticoid-binding sites in mammary glands. Apparent lack of cytoplasmic progesterone receptors in lactating mammary glands
Authors: Shyamala G , McBlain W A
Published: August 2015

The cytosol fraction of the lactating mammary glands of mice does not appear to contain detectable amounts of progesterone receptors. Mixing experiments indicate that the absence of receptors is not due to interference by other factors in the cytosol. However, in the cytosol of mammary glands, there is specific binding or progestins to certain low-affinity sites which have characteristics of specific glucocorticoid-binding sites.
Effects of carbon tetrachloride on isolated rat hepatocytes. Inhibition of protein and lipoprotein secretion
Authors: Gravela E , Albano E , Dianzani M U , Poli G , Slater T F
Published: August 2015

The effects of carbon tetrachloride on protein and lipoprotein secretion, and on lipid peroxidation, have been investigated in isolated rat hepatocytes. It was found that although the free-radical scavenger promethazine completely suppressed the increased peroxidation produced by carbon tetrachloride, it had no effect on the inhibitory action of carbon tetrachloride on lipoprotein secretion. In consequence, the latter effect of carbon tetrachloride does not appear to be mediated through a peroxidative stage.
Regulation of mammalian protein synthesis in vivo. Simulated transport of nuclear ribonucleoprotein complexes to the cytoplasm after cycloheximide treatment
Authors: Ch'ih J J , Duhl D M , Faulkner L S , Devlin T M
Published: August 2015

By studies in vivo with purified nuclei from rat liver, it was shown that a non-lethal dose of cycloheximide causes a decrease in the content of total nuclear ribonucleoprotein complexes by 2h after treatment. Analysis of the complex by sucrose-density-gradient centrifugation substantiated this observation for the faster-sedimenting complex, but showed an increase in the content of a smaller complex. Radioisotope incorporation studies showed that the overall decrease in nuclear ribonucleoprotein content was not due to a decreased synthesis, but rather to an increased transport to the cytoplasm. The results of a double-radioisotope technique support the conclusion that, during the inhibitory phase of protein synthesis brough on by cycloheximdie, gene transcription continues and the gene product is transported to the cytoplasm for subsequent translation.
Selective cleavage of variant surface glycoproteins from Trypanosoma brucei
Authors: Johnson J G , Cross G A
Published: August 2015

Two conformationally distinct regions were revealed by tryptic cleavage of six undenatured variant surface glycoproteins purified from clones of Trypanosoma brucei. Within 5 min, the native glycoproteins (65,000 mol.wt.) were cleaved, yielding a large N-terminal fragment (48,000-55,000 mol.wt. depending on the variant) together with one or more C-terminal fragments. After 30-60 min incubation, further breakdown of the large fragment occurred in some variants. The ultimate large product (40,000-52,000 mol.wt.) was very resistant to further degradation by trypsin (in the absence of denaturation). The distinction between N-terminal and C-terminal domains may be significant in relation to the organization and function of these glycoproteins on the trypanosome surface.
Biosynthesis of a 7-α-methoxycephalosporin. Incorporation of molecular oxygen
Authors: O'Sullivan J , Aplin R T , Stevens C M , Abraham E P
Published: August 2015

A 7-alpha-methoxycephalosporin containing a carbamoyloxymethyl substituent at C-3 (cephamycin C) has been isolated from the extracellular fluid of an aqueous suspension of Streptomyces clavuligerus shaken in the presence of 18O2. The cephalosporin has been converted into its N-acetyl dimethyl ester and the distribution of 18O in the latter determined by chemical-ionization mass spectrometry. The results indicate that the oxygen atom of the methoxy group, as well as that linked to the exocyclic methylene group at C-3, is derived from molecular O2.
Solubilization of human erythrocyte membrane glycoproteins by triton X-100
Authors: Pratt R S , Cook G M
Published: August 2015

1. The enzymic removal of sialic acid residues from the glycoproteins of the human erythrocyte decreases the solubilization of membrane glycoprotein by Triton X-100. 2. The solubilization of asialoglycoprotein by Triton X-100 may be restored by the addition of borate. 3. Use of this non-ionic detergent in the presence of borate, as a general procedure for the mild solubilization of membrane glycoproteins deficient in sialic acid residues, is discussed.
The relationship of detergent-solubilization plasma-membrane components of rabbit alveolar macrophages to an isolated inhibitor of phagocytosis
Authors: Pratt R S , Cook G M
Published: August 2015

1. A plasma-membrane fraction prepared from rabbit alveolar macrophages by hyposmotic borate lysis is described. 2. Rabbit lung lavages, containing a glycoprotein inhibitor of phagocytosis, may be fractionated by preparative isoelectric focusing in the presence of Triton X-100. 3. Chemical analysis indicates that the glycoproteins of the lung lavage contain sialic acid, fucose, mannose, galactose, hexosamine and appreciable quantities of glucose. 4. The relationship of macrophage membrane glycoproteins, solubilized with Triton X-100 in the presence of borate, to the lung lavage glycoproteins is demonstrated immunoelectrophoretically.
Identification of histidine residues that act as zinc ligands in β-lactamase II by differential tritium exchange
Authors: Baldwin G S , Waley S G , Abraham E P
Published: August 2015

1. Four histidine-containing peptides have been isolated from a tryptic digest of the Zn2+-requiring beta-lactamase II from Bacillus cereus. One of these peptides probably contains two histidine residues. 2. The presence of one equivalent of Zn2+ substantially decreases the rate of exchange of the C-2 proton in at least two and probably three of the histidine residues of these peptides for solvent 3H. 3. It is concluded that peptides containing at least two of the three histidine residues acting as Zn2+ ligands at the tighter Zn2+-binding site of beta-lactamase II have been identified.
The formation and distribution of bilirubin monoglucuronide and diglucuronide in rat liver slices
Authors: Campbell M T , Dutton G J
Published: August 2015

1. Bilirubin conjugation in rat liver slices was reassessed by using analysis of ethyl anthranilate azopigments to estimate separately the formation of bilirubin mono- and di-glucuronides. 2. Conjugation in slices resembles the situation in vivo more closely than does microsomal conjugation, in that diglucuronide is formed in appreciable quantity. 3. Both bilirubin mono- and di-glucuronides were present in slices in approximately equal amounts, but the monoglucuronide was the major product found in the incubation medium. 4. These results are discussed in relation to recent theories on the relationship between bilirubin mono- and di-glucuronide formation in vivo.
Analysis of the ratio of α- to β-globin and globin messenger ribonucleic acid content of fractionated rabbit erythroid bone-marrow cells
Authors: Mezl V A , Kawasaki E S , Hunt J A
Published: August 2015

The ratio of alpha- to beta-globin mRNA was measured by hybridization of a constant amount of highly purified alpha- or beta-globin cDNA (complementary DNA) with increasing amounts of RNA in the range up to 20% cDNA hybridization, where an essentially linear reaction is obtained. Statistical analysis indicates that the ratio of alpha- to beta-globin can be measured within a maximal error of +/- 0.3 and in most cases is better than +/- 0.15. Under these conditions there is no significant deviation from the ratio of 1.3 in the alpha- to beta-globin mRNA ratio of RNA isolated from erythroid cells rich in pronormoblasts through to reticulocytes. If the ratio of alpha- to beta-globin mRNA exceeded 1.7 or was less than 0.9 in pronormoblasts, it would be detected in these experiments. The overall globin mRNA content increases to a maximal value in the fractions rich in basophilic normoblasts of 30,000–50,000 molecules/cell. However, the accuracy of these determinations is not as great as for the ratio determinations, and no significant deviations were seen except in the cells rich in pronormoblasts, which contained less globin mRNA than the later stages.
Interactions of rat α-foetoprotein with bilirubin
Authors: Versée V , Barel A O
Published: August 2015

The binding of bilirubin to rat-alpha-foetoprotein has been measured by changes in the absorption spectrum and by the appearance of two intense circular-dichroism bands. Furthermore binding of this bile pigment has been demonstrated by a decrease of its enzymic oxidation rate and by competition experiments with bovine serum albumin-agarose. The binding parameters have been determined as follows: n = 1.0 mol bound/mol of protein and Ka = 2.9 × 10(6) M-1. Competition of oestradiol for the bilirubin-binding site has not been established.
NADH- and NADPH-dependent formation of superoxide anions by bovine heart submitochondrial particles and NADH–ubiquinone reductase preparation
Authors: Takeshige Koichiro , Minakami Shigeki
Published: August 2015

1. Both NADH and NADPH supported the oxidation of adrenaline to adrenochrome in bovine heart submitochondrial particles. The reaction was completely inhibited in the presence of superoxide dismutase, suggesting that superoxide anions (O2−) are responsible for the oxidation. The optimal pH of the reaction with NADPH was at pH7.5, whereas that with NADH was at pH9.0. The reaction was inhibited by treatment of the preparation with p-hydroxymercuribenzoate and stimulated by treatment with rotenone. Antimycin A and cyanide stimulated the reaction to the same extent as rotenone. The NADPH-dependent reaction was inhibited by inorganic salts at high concentrations, whereas the NADH-dependent reaction was stimulated. 2. Production of O2− by NADH–ubiquinone reductase preparation (Complex I) with NADH or NADPH as an electron donor was assayed by measuring the formation of adrenochrome or the reduction of acetylated cytochrome c which does not react with the respiratory-chain components. p-Hydroxymercuribenzoate inhibited the reaction and rotenone stimulated the reaction. The effects of pH and inorganic salts at high concentrations on the NADH- and NADPH-dependent reactions of Complex I were essentially similar to those on the reactions of submitochondrial particles. 3. These findings suggest that a region between a mercurialsensitive site and the rotenone-sensitive site of the respiratory-chain NADH dehydrogenase is largely responsible for the NADH- and NADPH-dependent O2− production by the mitochondrial inner membranes.
Subcellular localization and ionic properties of acetyl-coenzyme A synthetase in the electric organ of Torpedo marmorata
Authors: Morot-Gaudry Y , Diebler M F
Published: August 2015

Acetyl-CoA synthetase activity was shown to be present in pure cholinergic synaptosomes from electric organ of Torpedo marmorata. After osmotic disruption of synaptosomes a substantial part of the activity was recovered in the soluble fraction. The effects of varying pH and increasing K+ concentrations on the synaptosomal enzyme activity were shown to differ from those observed with the mitochondrial enzyme. Whereas this latter enzyme showed optimal activity above pH 8.5, and a maximal activation in the presence of 120 mM-K+, the synaptosomal enzyme exhibited an optimal activity at pH 7.9 and a moderate K+ stimulatory effect with an optimal concentration of 30 mM.
The role of insulin in the regulation of stearic acid desaturase activity in liver and adipose tissue from obese–hyperglycaemic (ob/ob) and lean mice
Authors: Enser M
Published: August 2015

The relationship between the hyperinsulinaemia of obese–hyperglycaemic (ob/ob) mice and their high activity of stearic acid delta 9-desaturase compared with lean mice has been investigated. The concentrations of plasma insulin in obese mice were decreased by 71, 88 and 96% after treatment either with alloxan or food restriction to maintain the same weight as lean mice, or treatment of the weight restricted mice with alloxan followed by feeding ad libitum. The concentration of plasma insulin produced by the latter treatment was the same as in normal lean mice. After treatment the hepatic desaturase activities were 24, 68 and 19% less respectively on a cell basis than in livers from untreated obese mice, and the total epididymal fat-pad activities were lower by 16, 62 and 57%. These results suggest that hyperinsulinaemia is not essential for the increased hepatic desaturase, controlling the hepatic desaturase activity, but even this may be subject to overriding regulation by the concentration of esterified linoleic acid in the liver lipids, which was negatively correlated (r = 0.91, P less than 0.001) with desaturase activity.
Effects of temperature, metabolic inhibitors and some other factors on fluid-phase and adsorptive pinocytosis by rat peritoneal macrophages
Authors: Pratten M K , Lloyd J B
Published: August 2015

Low temperature, NaF and 2,4-dinitrophenol could each abolish the pinocytic uptake of 125I-labelled poly(vinylpyrrolidone) or colloidal [198Au]gold by rat peritoneal macrophages cultured in vitro. Cytochalasin B caused only partial inhibition, even at 10 microgram/ml, and colchicine (10 or 25 microgram/ml) inhibited uptake of colloidal [198Au]gold much more than that of 125I-labelled poly(vinylpyrrolidone). Dibutyryl cyclic AMP and ouabain were without effect on uptake of 125I-labelled poly(vinylpyrrolidone), and slight stimulation was seen with ATP and theophylline. Uptake of 125I-labelled poly(vinylpyrrolidone) was abolished by EGTA (5mM), but restored by adding CaCl2 (5mM). The results appear not to support the conventional criteria for the division of pinocytic phenomena into macropinocytosis, requiring a metabolic energy supply and cytoskeletal components, and micropinocytosis, requiring neither.
A potassium ion diffusion potential causes adrenaline uptake in chromaffin-granule ‘ghosts’
Authors: Njus D , Radda G K
Published: August 2015

Membrane vesicles (‘ghosts’) formed from bovine chromaffin granules accumulate adrenaline in response to a diffusion potential produced by adding K+ in the presence of valinomycin. This uptake occurs as a short (2–5 min) burst because of the transient nature of the diffusion potential. The potential-driven uptake is optimal at pH approximately 7.2, is inhibited by reserpine, and has an initial rate comparable with that of ATP-driven uptake. These results show that ATP-dependent adrenaline uptake may occur at least partly in response to the membrane potential generated by an electrogenic proton-translocating adenosine triphosphatase found in chromaffin-granule membranes.
The application of microcalorimetry to the assessment of metabolic efficiency in isolated rat hepatocytes
Authors: Jarrett Ivan G. , Clark Dallas G. , Filsell Owen H. , Harvey John W. , Clark Michael G.
Published: August 2015

1. Heat output by suspensions of isolated rat hepatocytes was determined by using a modified batch-type microcalorimeter. 2. The ratio of O2 uptake (determined polarographically) to heat output was used to assess the metabolic efficiency of isolated hepatocytes. 3. Cells from starved or fed rats incubated in either bicarbonate-buffered physiological saline containing gelatin, or bicarbonate-buffered physiological saline containing amino acids, serum albumin and glucose showed no significant difference with respect to the ratio of O2 uptake to heat output. 4. For liver cells from 24h-starved rats, the addition of 10mm-dihydroxyacetone and 2.5mm-fructose significantly decreased the ratio of O2 uptake to heat output from 1.94±0.05 in the controls to 1.52±0.04 and 1.54±0.01μmol/J respectively. 5. Glucagon (1μm), which slightly increased both O2 uptake and heat output, did not significantly alter the ratio. 6. The addition of extracellular 10mm-NH4Cl and urease to provide an energetically wasteful cycle by ensuring hydrolysis of newly synthesized urea, lowered the ratio of O2 uptake to heat output from 1.81±0.08 to 1.47±0.06μmol/J, indicating a reduced metabolic efficiency. 7. Metabolic efficiency in rats of different dietary regimen, age and genetically based obesity was also assessed. No differences in the ratio of O2 uptake to heat output were found between liver cell suspensions prepared from rats maintained on colony diet and high-fat diet or sucrose-rich diet nor between animals ranging from 38 to 179 days of age. Comparison of the ratio of liver cell O2 uptake to heat output between homozygote Zucker fa/fa obese rats and their lean littermates showed no significant difference. 8. It is concluded that the ratio of O2 uptake to heat output for isolated hepatocytes is relatively constant unless perturbed by conditions that markedly enhance substrate cycling.
Comparison of human alkaline phosphatase isoenzymes. Structural evidence for three protein classes
Authors: McKenna M J , Hamilton T A , Sussman H H
Published: August 2015

The structural relationships among human alkaline phosphatase isoenzymes from placenta, bone, kidney, liver and intestine were investigated by using three criteria. 1. Immunochemical characterization by using monospecific antisera prepared against either the placental isoenzyme or the liver isoenzyme distinguishes two antigenic groups: bone, kidney and liver isoenzymes cross-react with anti-(liver isoenzyme) serum, and the intestinal and placental isoenzymes cross-react with the anti-(placental isoenzyme) antiserum. 2. High-resolution two-dimensional electrophoresis of the 32P-labelled denatured subunits of each enzyme distinguishes three groups of alkaline phosphatase: (a) the liver, bone and kidney isoenzymes, each with a unique isoelectric point in the native form, can be converted into a single form by treatment with neuraminidase; (b) the placental isoenzyme, whose position also shifts after removal of sialic acid; and (c) the intestinal isoenzyme, which is distinct from all other phosphatases and is unaffected by neuraminidase digestion. 3. Finally, we compare the primary structure of each enzyme by partial proteolytic-peptide ‘mapping’ in dodecyl sulphate/polyacrylamide gels. These results confirm the primary structural identity of liver and kidney isoenzymes and the non-identity of the placental and intestinal forms. These data provide direct experimental support for the existence of at least three alkaline phosphatase genes.
Neuraminidase inhibition by chemically sulphated glycopeptides
Authors: Mian N , Anderson C E , Kent P W
Published: August 2015

Chemically sulphated glycopeptides (derived from pig duodenal mucosa) inhibited Clostridium perfringens neuraminidase (EC 3.2.1.18) activity in a pH-dependent manner. Analysis of inhibition kinetics data indicated that, although the enzyme inhibition could not be categorized into any of the classical types of inhibition, it could be interpreted as a function of the size and shape of the substrates used. The enzyme activity was inhibited by 86% and 40% when tested with bovine submaxillary-gland mucin (mol. wt. 4 x 10(5)-40 x 10(5) and N-acetylneuraminyl-lactose (mol. wt. 633) as substrates respectively. Presence of sulphated glycopeptide did not affect the binding of N-acetylneuraminic acid (mol. wt. 309), a competitive inhibitor of Vibrio cholerae neuraminidase, to the enzyme active site. The enzyme inhibition was thus considered to be due to steric hindrance as a consequence of the non-specific interactions between the enzyme molecule and polyanionic sulphated glycopeptide affecting the differential accessibility of the substrate molecules to the enzyme active site. The enzyme-inhibitor interaction could be suppressed by rapid and many-fold dilution of the reaction mixture, by concurrent addition of the inactive enzyme or by partial removal of the sulphate esters from the sulphated glycopeptide molecule by the action of Helix pomatia arylsulphatase (EC 3.1.6.1).
Characterization of an oxygen-stable nitrogenase complex isolated from Azotobacter chroococcum
Authors: Robson R L
Published: August 2015

In crude cell-free extracts of Azotobacter chroococcum, nitrogenase was much less sensitive to irreversible inactivation by O2 than was the purified enzyme. When nitrogenase was partially purified by anaerobic discontinuous sucrose-density-gradient centrifugation, O2-tolerance was retained. This preparation was considerably enriched in four polypeptides, three of which were derived from the Mo-Fe(molybdenum-iron) protein and Fe (iron) protein of nitrogenase. The fourth was purified to homogeneity and shown to be an iron-sulphur protein (mol.wt. 14000) probably containing a 2Fe–2S centre. When this protein was added to purified nitrogenase, the enzyme was rendered O2-tolerant, through stabilization was Mg2+-dependent. The isolated O2-tolerant nitrogenase was an equimolar stoicheiometric complex between the MO–Fe, Fe and protective proteins. It is likely that the formation of this complex in vivo is the mechanism of ‘conformational protection’ in this organism.