bluuebunny/crossref_metadata_2025 huggingface dataset:
Rapid anisotropic motion of the Ca2+-transport ATPase of the rabbit skeletal muscle sarcoplasmic reticulum
Authors: Manuck Barbara A. , Sykes Brian D.
Published: December 2009

1H nuclear magnetic resonance techniques were used to study the binding of uridine 5′-triphosphate to the Ca2+-transport ATPase (EC 3.6.1.3) of sarcoplasmic reticulum vesicles from rabbit skeletal muscle. The nuclear spin relaxation times determined for the bound nucleotide are used to characterize the rotational motion of the ATPase to which the nucleotide is bound. The results, assuming an anisotropic model for the motion of the ATPase in the membrane, place a low upper limit on the rotational correlation time of the ATPase. This indicates that the motion of the ATPase in the membrane is quite rapid when compared, for example, with the motion found for other membrane-bound proteins such as rhodopsin.
Acyl specificity of CDPcholine:l,2-diacylglycerol cholinephosphotransferase in rat lung
Authors: Possmayer F. , Duwe G. , Hahn M. , Buchnea D.
Published: December 2009

Under optimal conditions, rat lung microsomal cholinephosphotransferase (EC 2.7.8.2) activity is markedly stimulated by exogenously added 1,2-sn-diacylglycerols containing an unsaturated fatty acid at the 2-position. Diacylglycerols containing long-chain saturated fatty acids at the 1- and 2-positions did not stimulate the incorporation of CDP[14C]choline above the incorporation observed with the endogenous diacylglycerols present in the microsomal preparation. 1-Oleoyl,2-palmitoyl-sn-glycerol was also ineffective in stimulating phosphatidylcholine synthesis. Diacylglycerols containing linoleate or linolenate at the 2-position were not as effective as 1-palmitoyl,2-oleoyl-sn-glycerol. Identical stimulations were observed with the latter diacylglycerol and 'natural' diacylglycerols prepared from egg-yolk phosphatidylcholine or pig liver phosphatidylcholine. Marked selectivities were observed with diacylglycerols containing two unsaturated fatty acids. Only minor amounts of 1,2-[14C]dipalmitoyl-sn-glycerol were incorporated into phosphatidylcholine, even when this radioactive diacylglycerol was dispersed with 'egg' diacylglycerols. When CDP[14C]choline was incorporated into rat lung microsomal lipids with endogenous diacylglycerols or diacylglycerols endogenously generated by phospholipase C (EC 3.1.4.3) (Bacillus cereus), little radioactivity was associated with the disaturated species of phosphatidylcholine.It has previously been suggested that cholinephosphotransferase catalyses the rate-limiting reaction in the biosynthesis of phosphatidylcholine by lung and that this enzyme is specifically induced in fetal lung by glucocorticoids. The present results indicate that these proposals are untenable and must be reevaluated. These experiments also suggest that dipalmitoyl phosphatidylcholine is not synthesized readily by the de novo pathway for lecithin synthesis and must be produced through reacylation or transesterification mechanisms.
The effect of proflavine on pyruvate kinase I of Escherichia coli B
Authors: McKellar Robin C. , Kushner Donn J.
Published: December 2009

Proflavine (PF) inhibited glucose use in sensitive but not resistant Escherichia coli B. Glucose transport (as measured by α-methylglucoside accumulation) was only partly inhibited by PF concentrations that completely blocked glucose use. Fructose 1,6-diphosphate-(FDP)-regulated pyruvate kinase (PK I) (EC 2.7.1.40), the only glycolytic enzyme affected by PF, was completely inhibited by a dye concentration of 0.8 mM. The inhibition curve for PF was sigmoidal, suggesting that PF was acting as an allosteric inhibitor. PF increased the K1/2 for phosphoenolpyruvate (PEP) and lowered the V; however, it had no effect on the Hill number for PEP. PF inhibition was partially reversed by FDP but not by cyclic AMP, AMP, ATP, fructose 6-phosphate, or dithiothreitol. Studies with a variety of acridines indicated that those substituted at the 3-position are the most effective inhibitors and also that hydrophobic interactions may be involved in PF inhibition of PK I. PK I for E. coli B/Pr was also strongly inhibited by PF, indicating that PF resistance does not lie at the level of this enzyme. Ribose-5-phosphate-regulated pyruvate kinase (EC 2.7.1.40) was much less sensitive than PK I to the inhibitory effects of PF. A role for PF as a molecular probe for PK I has been proposed.
Effect of metabolites on ε-N-hydroxylysine formation in cell-free extracts of Aerobacter aerogenes 62-1
Authors: Murray G. J. , Clark G. E. D. , Parniak M. A. , Viswanatha T.
Published: December 2009

The conversion of L-lysine to its corresponding ε-N-hydroxy derivative has been achieved for the first time by cell-free extracts of Aerobacter aerogenes 62-1. Partial fractionation by differential centrifugation (at 12 000 × g) revealed that both supernatant and pellet are essential for maximum enzymatic activity. The ω-N-hydroxylase (EC 1.14.99) was found to function optimally at pH 7–7.5 and exhibited an apparent Km of about 75 μM for L-lysine. L(+)-Lactate or DL-lactate and pyruvate greatly stimulate the ω-N-hydroxylase activity. The system is strongly inhibited by arsenite and sulfite.
Studies related to antitumor antibiotics. Part IX. Reactions of carzinophillin with DNA assayed by ethidium fluorescence
Authors: Lown J. William , Majumdar Krishna C.
Published: December 2009

The reactions of the antitumor antibiotic carzinophillin (CZ) with native DNAs and synthetic polynucleotides have been examined by an ethidium fluorescence assay. CZ rapidly produces covalent linkage of the complementary strands of a variety of DNAs without activation. This process is accompanied by extensive alkylation, as detected by reduced fluorescence due to destruction of potential intercalation sites for ethidium. These processes which occur without loss of purine or pyrimidine bases show a preference for bonding to guanine groups (but not at the N-7 position). Examination of the reversibility of the cross-links suggests they involve one 'permanent' link to guanine and a second weaker linkage, possibly to a cytosine residue. Both cross-linking and alkylation show strong pH dependence and are favored at lower pH, suggesting that reactive sites on the antibiotic are basic. The addition of intercalating agents to DNA before treatment with CZ inhibits the cross-linking.
Characteristics of potassium ion transport in pea cotyledon mitochondria isolated by a zonal technique
Authors: Hamman W. M. , Spencer Mary
Published: December 2009

Methodology was developed for measurement of K+ transport in pea cotyledon mitochondria isolated by zonal centrifugation.The valinomycin-mediated transport process required respiratory energy from Krebs cycle intermediates and was completely reversible in the absence of oxygen. The rate and extent of uptake were dependent on the presence of proton-conducting anions (with acetate plus phosphate being most effective), well-coupled mitochondria, and on the K+ concentration.
A kinetic analysis of the processive enzyme Lactobacillus plantarum exoribonuclease
Authors: Lam Kam-Fong , Logan David M.
Published: December 2009

Oligonucleotide chains consisting of adenosine residues and ranging from 1 to 70 residues in length have been tested as substrates or inhibitors with Lactobacillus plantarum exoribonuclease (EC 3.1.4.20). The kinetic constants V, Km, and Ki are all chain-length dependent. Ki decreases with increasing chain length to a minimum for oligonucleotides seven residues in length and then begins to increase slightly. Kinetic plots indicate that the oligonucleotides are almost all competitive inhibitors of poly A hydrolysis. However, the oligonucleotide (Ap)3A > p probably leads to mixed inhibition. The enzyme is unable to retain its processivity when it hydrolyzes short oligonucleotides such as (Ap)2A and (Ap)3A. It is proposed that L. plantarum exoribonuclease possesses seven binding sites for the polynucleotide. When the enzyme is bound to a long-chain-length substrate the complex is stabilized by a binding energy of about 8 Kcal/mol. After cleavage of the terminal nucleotide the remaining binding energy is still sufficient to maintain an enzyme–substrate complex. The shortened nucleotide chain is moved relative to the enzyme to re-form the seven-bond association by a gradient of energy of about 1.7 Kcal/mol for the change from six to seven bonds.
Use of 13C in biosynthetic studies. The labelling pattern in tenellin enriched from isotope-labelled acetate, methionine, and phenylalanine
Authors: Wright J. L. C. , Vining L. C. , McInnes A. G. , Smith D. G. , Walter J. A.
Published: December 2009

The biogenetic origin of the carbon atoms in tenellin has been established by adding 13C-enriched compounds to cultures of Beauveria bassiana, and determining the isotopic distribution in the metabolite by 13C nuclear magnetic resonance spectrometry. Tenellin is formed by condensation of an acetate-derived polyketide chain with a phenylpropanoid unit that may be phenylalanine. Alternate carbon atoms of the polyketide chain were labelled with sodium [1-13C]- and [2-13C]-acetate; sodium [1,2-13C]acetate was incorporated as intact two-carbon units, the presence of which in tenellin was apparent from coupling between adjacent 13C-enriched carbons. Substituent methyl groups of the polyketide-derived alkenyl chain were labelled with L-[Me-13C]methionine. The labelling patterns from DL-[carboxy-13C]phenylalanine and DL-[α-13C]phenylalanine indicated a rearrangement of the propanoid component at some stage in the synthesis. The mass spectrum of tenellin from cultures administered L-[15N]phenylalanine showed isotopic enrichment similar to that obtained with 13C- or 14C-labelled phenylalanine. During incorporation of L-[carboxy-14C, β-3H]phenylalanine 96% of the tritium label was lost, discounting the possibility of a 1,2-hydride shift during biosynthesis of the metabolite.
Regulation of selectivity of CDPcholine: 1,2-diacyl-sn-glycerol cholinephosphotransferase in rat liver microsomes towards different molecular species of 1,2-diacyl-sn -glycerols
Authors: Holub B. J.
Published: December 2009

The suitability of monoenoic, dienoic, tetraenoic, and hexaenoic molecular species of 1,2-diacyl-sn-glycerols as substrates for the CDPcholine: 1,2-diacyl-sn-glycerol cholinephosphotransferase (EC 2.7.8.2) was studied in rat liver microsomes. No statistically significant difference in the rates of phosphatidylcholine synthesis with the various diacylglycerols was found at 0.40 mM, although a moderate discrimination against hexaenoic species relative to monoenoic and dienoic species was observed at 0.25 mM. The addition of palmitoyl-CoA (7.5 μM) significantly enhanced cholinephosphotransferase activity when tetraenoic diacylglycerols were added at 0.25 or 0.40 mM. CDPethanolamine at 24.4 μM was found to inhibit the rates of phosphatidylcholine biosynthesis by 54 and 39% with hexaenoic and monoenoic 1,2-diacyl-sn-glycerols, respectively, whereas no significant effects were observed in the case of dienoic and tetraenoic species. These latter findings may partially explain why 1-saturated 2-docosahexaenoyl diacylglycerols are used to a greater extent for phosphatidylethanolamine than for phosphatidylcholine synthesis in rat liver in vivo. The present results also suggest that the selectivity of the cholinephosphotransferase for certain molecular species of 1,2-diacyl-sn-glycerols is a function of diacylglycerol concentration and may be mediated under physiological conditions by substrates for enzymes which compete for common diacylglycerol precursors.
Comparative study of rye and thymus histones: amino acid analysis and tryptic fingerprinting
Authors: Nadeau Paul , Pallotta Dominick , Lafontaine Jean-G.
Published: December 2009

Amino acid composition and tryptic fingerprints of rye (Secale cereale) H1, H2B (PHI), and H2A (PHII) histones indicate the presence of major differences between these and the corresponding calf or rabbit fractions. In addition to variations for other amino acids, fraction H1 from rye contains twice as much arginine as the corresponding animal fraction; the plant H2B (PHI) and H2A (PHII) histones show lysine to arginine ratios greater than those of their animal counterparts. The tryptic maps of the same proteins appear to differ between plants and animals by the number and the general pattern of the peptides, as well as by the quantity and distribution of the arginine-containing peptides. Such results suggest the presence of differences in the primary structure of the calf and rye lysine-rich and moderately lysine-rich histones. Furthermore, the possibility is ruled out that each of these plant histones consists of an animal-like protein with an additional segment of 20–30 amino acid residues. On the other hand, the rye and calf arginine-rich fractions H3 and H4 show similar amino acid compositions and tryptic peptides maps.
Biosynthesis of gibberellins in Gibberella fujikuroi. Gibberellin A47
Authors: McInnes A. G. , Smith D. G. , Durley R. C. , Pharis R. P. , Arsenault G. P. , MacMillan J. , Gaskin P. , Vining L. C.
Published: December 2009

Two minor radioactive products in cultures of G. fujikuroi strain ACC 917 supplemented with labeled gibberellin A4 have been identified as the 2α-hydroxy derivative, gibberellin A47, and the hydrate, gibberellin A2. In addition, evidence was obtained by combined gas chromatography – mass spectrometry for the formation of 3-O-acetylgibberellin A1 3-O-acetylgibberellin A3, and gibberellin A20 by the culture. The time course of gibberellin synthesis in defined and complex media, as well as changes in the relative amounts of radioactive gibberellins formed when [14C]gibberellin A4 was added to cultures at different ages, suggest that the composition of the gibberellin mixture produced is determined by the balance between synthesis and decay of key enzymes in the branching pathway.
Human pituitary thyrotropin. Isolation and characterization of the hormone and its subunits
Authors: Sairam M. R. , Li Choh Hao
Published: December 2009

Procedures have been described for the isolation of highly purified thyrotropin from frozen or acetone-preserved glands or from side fractions of somatotropin isolation and for the separation of its α and β subunits. The products have been characterized by terminal residue analyses, amino acid composition, carbohydrate content, disc electrophoresis, ultracentrifugation, and biological activity.
A new method for the fractionation of human plasma high density lipoprotein
Authors: Rubenstein Bernard , Evans Susan , Steiner George
Published: December 2009

We have devised a new method for the fractionation of human plasma high density lipoprotein (HDL). The HDL was chromatographed on DEAE-agarose columns using a continuous gradient of 0.06–0.15 M NaCl. The elution pattern obtained showed three phases, each with differing peptide composition. Examination of the three subfractions showed that each contained both apo A-I and apo A-II, but indifferent proportions. Subfraction 1 contained noapoC-II orC-III-1 and only a trace of apo C-III-2, subfraction 2 contained apo C-II and C-III-1 but no C-III-2, while subfraction 3 contained considerable apo C-III-2 with only traces of apo C-II or C-III-1.
Evidence for 16β-hydroxylation of estrogens by guinea pig liver slices
Authors: Hobkirk R. , Nilsen Mona
Published: December 2009

In an earlier communication, we conclusively proved that estrone-3-sulfate could be hydroxylated in the 16α position by guinea pig liver slices. Disulfates of 16α-hydroxyestrone and estriol were identified. Suggestive evidence for 6-hydroxylated metabolites was also mentioned. A careful reinvestigation has now shown that the steroid disulfate fraction is composed of 16α- and 16β-hydroxylated steroids. 16β-Hydroxyestrone in particular is an important quantitative metabolite of estradiol-17β. It is concluded that no firm evidence is available for the formation of 6-hydroxysteroids in the tissue system under consideration.
Competitive inhibition of catechol O-methyltransferase by RO-4-4602
Authors: Buu Nguyen T. , Kuchel Otto , Parent Marie T.
Published: December 2009

RO-4-4602, a peripheral L-DOPA decarboxylase (EC 4.1.1.28) inhibitor was shown to also inhibit the activity of catechol O-methyltransferase (COMT) (EC 2.1.1.6). Kinetic study indicated that the inhibition is of a competitive nature. The apparent Ki values of 7 × 106, 5.3 × 10−6, and 9 × 10−1 M calculated from Line weaver–Burke plots when dopamine, norepinephrine, and epinephrine, respectively, were used as substrate, showed that this drug is the most potent inhibitor of COMT yet known.
Metabolism of monoamines and diamines in hyperthyroid and hypothyroid rats
Authors: Sourkes T. L. , Missala K. , Bastomsky C. H. , Fang T. Y.
Published: December 2009

The in vivo rates of catabolism of 14C-labelled pentylamine, ethylamine, putrescine, and cadaverine were studied in thyroidectomized rats and others made hyperthyroid by the daily administration of 0.2 mg of L-thyroxine per kilogram for 20–21 days. Hyperthyroid rats metabolized the monoamines at an accelerated rate; thyroidectomized animals oxidized pentylamine at a reduced rate. There was no effect of hypophysectomy on the rate of pentylamine oxidation. The in vitro monoamine oxidase (MAO) activity of liver was reduced in hyperthyroid rats and unchanged in those thyroidectomized; MAO activity in skeletal muscle was increased in the hyperthyroid rats and decreased in the hypothyroid rats. Because of the large mass of skeletal muscle compared with liver, it is considered that the changes in muscle MAO could play an important role in determining the rate of oxidation of pentylamine in vivo. The oxidation of the two diamines tested was not significantly affected by thyroidectomy; the rates were increased in the hyperthyroid rats, but the increase was significant only for cadaverine.
Partial characterization of a soluble ATPase from pea cotyledon mitochondria
Authors: Grubmeyer Charles , Duncan Ian , Spencer Mary
Published: December 2009

A partially purified soluble ATPase (ATP phosphohydrolase, EC 3.6.1.3) from pea cotyledon mitochondria was characterized. Inhibition patterns with azide, NaF, and cold, and a stimulation by 2,4-dinitrophenol were typical of F1-ATPases from mammalian mitochondria. The enzyme hydrolysed GTP, ITP, and ATP, but not CTP, UTP, ADP, or IDP. ATPase and ITPase activities were strongly inhibited by ADP and to a lesser extent by IDP. Distinctive properties of the pea mitochondrial enzyme were activation by high concentrations of CaCl2 and stimulation by NaCl.
Role of orthophosphate concentration in the regulation of ribose phosphate synthesis and purine metabolism in Ehrlich ascites tumor cells
Authors: Barankiewicz Jerzy , Battell Mary L. , Henderson J. Frank
Published: December 2009

Concentrations of intracellular orthophosphate were determined in Ehrlich ascites tumor cells incubated with glucose, inosine, or uridine in media of different orthophosphate concentration. The effects of orthophosphate concentration on the accumulation of lactate and of phosphoribosyl pyrophosphate and on concentrations of ribose 1-phosphate and ribose 5-phosphate in tumor cells incubated with glucose were also determined. Both the phosphorolysis of inosine and the rate of catabolism of ATP in cells incubated with 2-deoxyglucose were also influenced by the orthophosphate concentration of the medium.
Identification of specific phosphoproteins in nuclease-digested chromatin subunits
Authors: Chan P. K. , Liew C. C.
Published: December 2009

Rat liver chromatin subunits (nucleosomes), which were isolated from nuclease-digested chromatin either treated or not treated with 0.5% sodium deoxycholate and 5% Triton X-100, were found to be similar with regard to sedimentation at 11 S and appeared as spherical particles with a diameter of about 100 Å (1 Å = 0.1 nm) in electron micrographs. The ratio of histone to nonhistone chromatin proteins (NHCP) in these subunits was 1:0.1 in the absence of detergents and 1:0.25 in the presence of detergents.After ultracentrifugation of the nuclease-digested chromatin in a 5–30% sucrose density gradient two groups of phenol-soluble NHCP were identified, one being released in the top of the gradient (fA) and the other still bound to the chromatin subunits after the nuclease digestion (fB). Two specific phosphoproteins were found in fA, one (A4) with molecular weight (MW) 39 000, pI 5.3–6.0, and the other (A5) with MW 31 000, pI 5.1–5.8. These proteins were not present in fB. Another protein in fB (B2) was highly phosphorylated, with MW 68 000, pI 6.5–8.2, and this was not found in fA.These phosphoproteins were further characterized and found to contain phosphoserine. The presence of specific phosphorylated protein(s) in fB suggests that the interaction of phosphoproteins with DNA in the eukaryotic genome is more than a random process.
Resolution of rat renin substrates by isoelectric focusing
Authors: Faiers A. A. , Loh A. Y. , Osmond D. H.
Published: December 2009

Pooled plasmas from normal or binephrectomized rats and perfusates of isolated livers were used as sources of renin substrate for isoelectric focusing. After desalting, preliminary fractionation (plasma only), and concentration, the preparations were focused in a pH 3–10 gradient on 20-cm glass plates layered with Sephadex slurry. The pH 4–6 region, containing all the substrate, was scraped from this plate and refocused in a pH 4–6 gradient. Substrate content of 1-cm strips of slurry from half of the plate was determined by both radioimmunoassay and bioassay of angiotensin resulting from incubation with added renin. Corresponding strips from the other half of the plate were incubated without renin as a control for any preformed angiotensin. The asymmetry and broad distribution (pH 4–5) of substrate from different sources suggested the existence of more than one form. Higher resolution achieved by using the high substrate concentration of postnephrectomy plasma and 0.5-cm strips of slurry on 20-cm or 40-cm plates revealed peaks and shoulders of substrate activity. Our data suggest that multiple forms of substrate are synthesized by the liver and circulate in plasma. Postnephrectomy rat plasma appears to contain relatively more substrate(s) with higher isoelectric points than in normal plasma, possibly an accumulation of forms ordinarily degraded by endogenous renal renin.
Incorporation in vivo of [32P]orthophosphate and [Me-3H]choline into rough microsomal, Golgi, and plasma membranes of rat liver
Authors: Chang Patricia L. , Riordan John R. , Moscarello Mario A. , Sturgess Jennifer M.
Published: December 2009

To study membrane biogenesis and to test the validity of the endomembrane flow hypothesis, incorporation of 32P and [Me-3H]choline in vivo into membranes of the rat liver was followed. Rough microsomal, Golgi-rich, and plasma membrane fractions were monitored with marker enzyme assays and shown with morphometric analysis to contain 82% rough microsomes, at least 70% Golgi complexes, and 88% plasma membranes, respectively. Membrane subfractions from the rough microsomal and Golgi-rich fractions were prepared by sonic disruption.At 5 to 30 min after 32P injection, the specific radioactivity of phosphatidylcholine was higher in the rough microsomal membranes than in the Golgi membranes. From 1 to 3 h, the specific activity of phosphatidylcholine in Golgi membranes became higher and reached the maximum at about 3 h. Although the plasma membrane had the lowest specific radioactivity throughout 0.25–3 h, it increased rapidly thereafter to attain the highest specific activity at 5 h. Both rough microsomal and plasma membranes reached their maxima at 5 h.The specific radioactivity of [32P]phosphatidylethanolamine in the three membrane fractions was similar to that of [32P]phosphatidylcholine except from 5 to 30 min, when the specific radioactivity of phosphatidylethanolamine in the Golgi membranes was similar to the rough microsomal membranes.At 15 min to 5 h after [Me-3H]choline injection, more than 90% of the radioactivity in all the membranes was acid-precipitable. The specific radioactivities of the acid-precipitated membranes, expressed as dpm per milligram protein, reached the maximum at 3 h. After [Me-3H]choline injection, the specific radioactivity of phosphatidylcholine separated from the lipid extract of the acid-precipitated membranes (dpm per micromole phosphorus) did not differ significantly in the three membrane fractions. The results indicated rapid incorporation of choline into membrane phosphatidylcholine by the rough endoplasmic reticulum, Golgi, and plasma membranes simultaneously.The data with both 32P and [Me-3H]choline precursors did not support the endomembrane flow hypothesis. The Golgi complexes apparently synthesized phosphatidylethanolamine and incorporated choline into phosphatidylcholine as well as the endoplasmic reticulum. The results are discussed with relevance to current hypotheses on the biogenesis and transfer of membrane phospholipids.
Altered amounts of hemoglobin synthesis in livers of dystrophic hamsters
Authors: Wrogemann K. , Quilliam N. M. , Nylen E. G. , Johnson M. D. , Clancy Jr. J. F.
Published: December 2009

In liver supernatants similar amounts of leucine are incorporated into proteins of normal and dystrophic hamsters. However, using a dual labeling technique, a protein fraction is detected which shows different levels of leucine incorporation between normal and dystrophic animals in an age-dependent fashion. The major protein in this fraction comigrates with hemoglobin or its subunits under various conditions of polyacrylamide gel electrophoresis. In animals younger than 3 days there is more synthesis of this protein fraction in dystrophic animals, while at 6 days there is considerably less synthesis of this protein. These changes are paralleled by differences in the number of erythroid foci in histological sections of the livers. There is no evidence for structurally altered hemoglobin in dystrophic hamsters. The significance of this finding and its possible relation to the dystrophy process are not known at present.
Simultaneous separation of halophilic proteins and nucleic acids after adsorption onto agarose gels
Authors: Pater Alan , Pater Mary M.
Published: December 2009

In dilute solutions, proteins and nucleic acids from the extremely halophilic bacterium Halobacterium cutirubrum remain soluble in 2.38 M ammonium sulphate and bind firmly to unsubstituted agarose gel columns. These macromolecules are clearly separated during elution with a decreasing salt concentration gradient.
Formiminotransferase–cyclodeaminase from porcine liver. A sulfhydryl essential for the deaminase activity of the bifunctional enzyme
Authors: Drury Elizabeth J. , MacKenzie Robert E.
Published: December 2009

Reaction of the bifunctional enzyme formiminoglutamate:tetrahydrofolate formimino-transferase (EC 2.1.2.5) – formiminotetrahydrofolate cyclodeaminase (EC 4.3.1.4) with the sulfhydryl reagent 5,5′-dithiobis (2-nitrobenzoic acid) selectively inactivates the cyclodeaminase. Loss of activity correlates with the modification of two sulfhydryl groups per subunit. The inhibitor folic acid reduces the rates of inactivation and sulfhydryl modification, and protection experiments demonstrate that only one of the two sulfhydryls modified is important for enzyme activity. The results indicate the presence of a cyclodeaminase site on each polypeptide, assuming one sulfhydryl per site, in agreement with a quaternary structure containing identical polypeptides. Modification does not cause dissociation of the enzyme and is reversible with dithiothreitol.
The role of divalent cations in the activation of the NADP+-specific isocitrate dehydrogenase from Pisum sativum L.
Authors: Maloney Robert J. , Dennis David T.
Published: December 2009

A divalent cation electrode was used to measure the stability constants (association constants) for the magnesium and manganese complexes of the substrates for the NADP+-specific isocitrate dehydrogenase (EC 1.1.1.42) from pea stems. At an ionic strength of 26.5 mM and at pH 7.4 the stability constants for the Mg2+–isocitrate and Mg2+–NADP+ complexes were 0.85 ± 0.2 and 0.43 ± 0.04 mM−1 respectively and for the Mn2+–isocitrate and Mn2+–NADP+ complexes they were 1.25 ± 0.07 and 0.75 ± 0.09 mM−1 respectively. At the same ionic strength but at pH 6.0 the Mg2+–NADPH and Mn2+–NADPH complexes had stability constants of 0.95 ± 0.23 and 1.79 ± 0.34 mM−1 respectively. Oxalosuccinate and α-ketoglutarate do not form measureable complexes under these conditions. Saturation kinetics of the enzyme with respect to isocitrate and metal ions are consistent with the metal–isocitrate complex being the substrate for the enzyme. NADP+ binds to the enzyme in the free form. Saturation kinetics of NADPH and Mn2+ indicate that the metal–NADPH complex is the substrate in the reverse reaction. In contrast the pig heart enzyme appears to bind free NADPH and Mn2+. A scheme for the reaction mechanism is presented and the difference between the reversibility of the NAD+ and NADP+ enzyme is discussed in relation to the stability of the NADH and NADPH metal complexes.
Purine metabolism in Saccharomyces cerevisiae
Authors: Burridge Philip W. , Woods Robin A. , Henderson J. Frank
Published: December 2009

The synthesis, interconversion, and catabolism of purine bases, ribonucleosides, and ribonucleotides in wild-type Saccharomyces cerevisiae were studied by measuring the conversion of radioactive adenine, hypoxanthine, guanine, and glycine into acid-soluble purine bases, ribonucleosides, and ribonucleotides, and into nucleic acid adenine and guanine. The pathway(s) by which adenine is converted to inosinate is (are) uncertain. Guanine is extensively deaminated to xanthine. In addition, some guanine is converted to inosinate and adenine nucleotides. Inosinate formed either from hypoxanthine or de novo is readily converted to adenine and guanine nucleotides.
Insulin-like effects of fluoroacetate on lipolysis and lipogenesis in adipose tissue
Authors: Taylor Wayne M. , D'Costa Mario , Angel Aubie , Halperin Mitchell L.
Published: December 2009

Hormone-stimulated lipolysis in adipose tissue was inhibited by fluoroacetate and there was a concomitant decrease in both the basal and hormone-stimulated cyclic AMP levels. Adenylate cyclase (EC 4.6.1.1) activity in membrane preparations was inhibited by fluoroacetate. There was no influence of fluoroacetate on the low Km cyclic AMP phosphodiesterase (EC 3.1.4.17) activity.The rate of glucose conversion to fatty acids was increased when adipose tissue was incubated in the presence of fluoroacetate. The outputs of pyruvate and lactate into the incubation medium were decreased at this time, suggesting decreased tissue pyruvate levels and a site of activation of lipogenesis distal to pyruvate formation. Pyruvate dehydrogenase (EC 1.2.4.1) activity was increased twofold in adipose tissue incubated in the presence of fluoroacetate. This was attributed to a fluoroacetate-induced inhibition of pyruvate dehydrogenase kinase, the enzyme responsible for inactivating the pyruvate dehydrogenase complex. Glucose transport was increased to a small but significant degree by fluoroacetate. In addition, both the tissue content of citrate and its release into the incubation medium were increased, suggesting that fluoroacetate resulted in an inhibition of aconitase (EC 4.2.1.3). The tissue ATP content was unchanged.Because the antilipolytic and lipogenic effects of fluoroacetate parallel those of insulin, they may share a common mechanism.
The reactivity to N-ethyl maleimide of the subunits of cytochrome oxidase
Authors: McGeer A. , Lavers B. , Williams G. R.
Published: December 2009

Beef heart cytochrome oxidase (EC 1.9.3.1) prepared in this laboratory consistently presents 10 Coomassie blue staining zones on SDS–polyacrylamide gel electrophoresis. At pH 7.0 only two of these polypeptides (III and VIa) are labelled by radioactive N-ethyl maleimide (NEM). The labelling of VIa is variable and correlates with the activity of particular oxidase preparations. When cytochrome oxidase is isolated from alkylated membranes, either mitochondria or electron transport particles, polypeptide VIa is found not to be labelled; polypeptide III is more strongly labelled than when isolated oxidase is alkylated, and label now appears in polypeptide I which is not alkylated upon treatment of isolated oxidase with NEM.
Inhibition of rat liver CDPethanolamine: 1,2-diacylglycerol ethanolamine-phosphotransferase activity by ATP and pantothenic acid derivatives
Authors: Liteplo Robert G. , Sribney Michael
Published: December 2009

The properties of rat liver microsomal CDPethanolamine: 1,2-diacylglycerol ethanolaminephosphotransferase (ethanolaminephosphotransferase; EC 2.7.8.1) are studied with respect to metal ion and substrate concentration. The enzyme requires magnesium (20 mM) or manganese (1 mM) ions for optimum activity. Manganese ions are better activators of ethanolaminephosphotransferase activity than are magnesium ions. Calcium (1 mM) inhibits the magnesium-activated ethanolaminephosphotransferase by 86% and the manganese-activated enzyme by 57%. The Km for CDPethanolamine is 2.4 and 0.7 × 10−4 M, in the presence of magnesium or manganese ions, respectively. ATP plus CoA or ATP plus pantetheine significantly inhibit the manganese-activated ethanolaminephosphotransferase, while the magnesium-activated enzyme is inhibited by ATP plus pantetheine and slightly stimulated by ATP plus CoA. In the presence of either metal ion, ATP itself inhibits enzyme activity, while CoA or pantetheine when added alone have no effect. Evidence is presented indicating that the inhibition of ethanolaminephosphotransferase activity by ATP plus CoA or ATP plus pantetheine is not due to the formation of acyl-CoA or acyl-S-pantetheine esters. At the present time, however, the true mechanism of inhibition is unknown. The results indicate that the cellular levels of ATP, CoA, pantetheine, magnesium, manganese, and calcium ions may all play a role in the regulation of phosphatidylethanolamine biosynthesis.
Discrete electrophoretic products of mitochondrial protein synthesis in the Chinese hamster ovary cell line
Authors: Yatscoff R. W. , Freeman K. B.
Published: December 2009

Mitochondrial proteins labelled with [35S]methionine for 1 h in whole Chinese hamster ovary (CHO) cells in the presence of cycloheximide or emetine, known inhibitors of cytosolic protein synthesis, have been enumerated and characterized by their electrophoretic mobility in sodium dodecyl sulfate slab gel electrophoresis. Ten distinct electrophoretic bands were observed. The components were relatively stable during a 2 h postlabelling period. The same 10 bands were also seen with the CHO cell line tsH1, labelled at 40 °C, a temperature at which cytosolic but not mitochondrial protein synthesis is inhibited in this cell line, and with isolated mitochondria labelled in the presence of cycloheximide. An 11th band was present when [3H]leucine but not [35S] methionine was used for labelling. The width of the major band suggested that it consists of two components making a total of at least 12 proteins synthesized in mitochondria. The molecular weights of these mitochondrial proteins ranged from 5000 to 50000 and there was a sixfold difference in the relative molar amounts synthesized in a 1-h period in the presence of [3H]leucine or [3SS] methionine.No differences in number or electrophoretic mobility of the mitochondrially synthesized proteins were found among the seven CHO cell lines examined. These results suggest the stability of the mitochondrial genome in the CHO cell line.
Proteolytic digestion and labelling studies of the organization of the proteins in the outer membrane of Escherichia coli
Authors: Reithmeier Reinhart A. F. , Bragg Philip D.
Published: December 2009

The arrangement of the proteins in the outer membrane of Escherichia coli was examined by treating intact cells and isolated membrane preparations with fluorescamine and with pronase. Intact wild-type cells, or those of a mutant in which the core region of the lipopolysaccharide was absent, were equally resistant to pronase treatment. The protein components of isolated outer membrane preparations varied in their rate of digestion and labelling with fluorescamine. The N-terminal portion of protein B was removed by pronase to yield a fragment (protein Bp) still embedded in the membrane. Protein Bp was not significantly enriched in nonpolar amino acids, suggesting that protein B may not be held in the membrane primarily by hydrophobic interactions. This was confirmed by reconstitution experiments in which protein B could be reassociated with itself, without lipopolysaccharide or phospholipid, in the presence of a divalent cation such that pronase digestion of the reassociated material gave protein Bp.
Cinétiques de pénétration transcutanée et de fixation du 3-propyl éther, 17-méthyl éther oestradiol et de l'oestradiol au niveau de différents tissus chez la ratte
Authors: Tresca Jean-Pierre , Ponsard Geneviève , Jayle Max-Fernand
Published: December 2009

The transcutaneous penetration of 3-propyl ether, 17-methyl ether oestradiol (POM) occurs by a diffusion phenomenon and does not seem to be modulated by a cutaneous receptor as it is the case for oestradiol.After transcutaneous administration of POM and oestradiol, a comparison of the kinetics of uptake on the uterus and of uterotrophic effects, as well as an analysis of radioactivity taken up by a partition method between petroleum ether and sodium hydroxide, indicates that a cleavage of both ether groups of POM occurs leading to oestradiol. It is likely that this de-etherification takes place in the liver after a period of quiescence.The lipophilic nature of POM allows an obvious uptake by the aorta and a very significant uptake by the adipose tissue. The etherification of the alcohol functions of oestradiol allows an adequate protection of the hormone against hepatic catabolism. This may explain, along with the release of metabolites taken up by the adipose tissue, that POM is bound to a greater extent than oestradiol by various tissues.
In vivo studies on the conversion of m-tyrosine to 3,4-dihydroxyphenylalanine in the rat
Authors: Tong J. H. , Smyth R. G. , Benoiton N. L. , D'Iorio A.
Published: December 2009

The question whether m-tyrosine can give rise to catechols in vivo has been investigated using labelled precursor. DL-[2-l4C]m-tyrosine (38 μCi/mmol (1 Ci = 37 GBq)) was synthesized from [2-14C]glycine. Radioactive catechols in rat brain, liver, and kidneys were examined 15 min after intraperitoneal administration of DL-[2-14C]m-tyrosine (100 mg/kg). The kidney was the only organ which showed demonstrable amounts of radioactive catechols, and about 14% of the catechols formed was identified as 3,4-dihydroxyphenylalanine (dopa), 22% as 3,4-dihydroxyphenylacetic acid, and 56% as dopamine. However, when the animals were pretreated with dopa decarboxylase inhibitor, labelled catechols were also observed in liver and brain, and dopa accounted for over 95% of the catechols formed in all three organs examined. Thus it is clear that m-tyrosine can be hydroxylated in vivo. Results from experiments using [2-14C]m-tyrosine enantiomers and specific enzyme inhibitors suggest that phenylalanine hydroxylase could be the enzyme catalyzing this reaction.
Solubilization and partial purification of particulate catechol-O-methyltransferase from rat liver
Authors: Tong J. H. , D'Iorio A.
Published: December 2009

Particulate catechol-O-methyltransferase (COMT) from rat liver has been solubilized by acetone treatment and partially purified. Results from the present study demonstrate that the solubilized, partially purified enzyme is similar to the cytosol COMT with respect to molecular weight, pH profile, sensitivity toward inhibitors, Mg2+ requirement, and substrate affinities. However, a comparison of the crude particulate COMT and the solubilized enzyme shows that there is a significant difference in their affinity for catechol substrates. This finding suggests that membrane protein and (or) lipid components may play an important role in catecholamine metabolism. The relationship of particulate COMT to [3H]norepinephrine binding was investigated. No correlation between the COMT and [3H]norepinephrine binding activities was observed in vitro.
Optimum pH and ionic strength for the assay of cytochrome c oxidase from pea cotyledon mitochondria
Authors: Bomhoff Gerrit H. , Spencer Mary
Published: December 2009

Cytochrome c oxidase (EC 1.9.3.1) has been solubilized by use of the nonionic detergents Triton X-114 and Triton X-100, from pea cotyledon mitochondria. Optimum assay conditions were determined for the oxidation of ferrocytochrome c in air. The results indicate that the plant cytochrome c oxidase resembles mammalian preparations in its sensitivity towards ionic strength and pH of the assay buffer.
Analyses of relative protein content and distribution of histones in euchromatic and heterochromatic fractions
Authors: Paul Izhak J. , Duerksen Jacob D.
Published: December 2009

Euchromatic and heterochromatic fractions obtained by autodigestion of mouse TLT (taper liver tumour) hepatoma chromatin (Paul, I. J. &Duerksen, J. D. (1976) Arch. Biochem. Biophys. 174, 491–505) were analyzed for relative protein content and histone content. With one exception, all fractions had the same DNA to protein ratio. Similarly, the total histone to DNA ratio was also constant in all fractions. In addition, the relative contents of the major histones, H1, (H2A + H2B + H3), and H4, were also constant in all fractions.
Quinonoid dihydropterin reductase from beef liver
Authors: Korri Karl K. , Chippel Diana , Chauvin Michael M. , Tirpak Anne , Scrimgeour K. G.
Published: December 2009

Quinonoid dihydropterin reductase has been purified from beef liver. This enzyme has been shown to be indistinguishable from the reductase of sheep liver in molecular weight, subunit composition, and terminal residues. Both beef and sheep liver reductases possess acyl isoleucine as the N-terminal residue. Use of improved isolation techniques, including general ligand affinity chromatography, has yielded enzyme preparations of much higher specific activity than previously reported. Affinity chromatography experiments also suggest that the enzymic reaction proceeds by a compulsory ordered mechanism.
Synthesis of molecular classes of cytidine diphosphate diglyceride by rat liver in vivo and in vitro
Authors: Thompson W. , MacDonald G.
Published: December 2009

Cytidine diphosphate (CDP) diglyceride isolated from pooled rat liver was shown like phosphatidyl inositol to contain stearate and arachidonate as the major fatty acids. On the other hand palmitate and oleate were the predominant fatty acids of total liver phosphatidic acid.Labelling of molecular classes of phosphatidic acid, CDP-diglyceride, and phosphatidyl inositol was examined in liver at various time intervals after either intraportal or intrajugular injections of [3H]glycerol. In general the major flux of radioactivity was through the oligoenoic classes [Δ1 and Δ2] of phosphatidic acid. In contrast the labelling of oligoenoic classes of CDP-diglyceride was less and of polyenoic classes [Formula: see text] significantly enhanced, raising the possibility that there may be some discrimination in selection of species of phosphatidic acid for liponucleotide synthesis. After intrajugular injections of isotope the monoenoic classes of phosphatidyl inositol were most highly labelled but between 5 and 240 min there was a progressive decrease of radioactivity in this class and increase in the tetraenoic species, presumably as a result of deacylation and reacylation reactions. The backflow of radioactivity from phosphatidyl inositol was deemed not a likely explanation for the observed labelling pattern of CDP-diglyceride.The conversion of sonicated dispersions of [3H]phosphatidic acid, labelled in the glycerol moiety, to CDP-diglyceride by rat liver microsomes was examined. There was slightly less label in the oligoenoic classes and slightly more in trienoic and polyenoic (> Δ4) classes of CDP-diglyceride as compared with phosphatidic acid. These differences were much smaller in vitro than those observed in vivo. The in vitro data provide no convincing evidence for a high enough selectivity to explain the differences in arachidonate content between phosphatidic acid and the liponucleotide.
The relative utilization of different molecular species of unsaturated 1,2-diacylglycerols by the acyl-CoA: 1,2-diacyl-sn-glycerol acyltransferase in rat liver microsomes
Authors: Holub B.J. , Piekarski J.
Published: December 2009

The possible selectivity of the acyl-CoA:1,2-diacyl-sn-glycerol acyltransferase in rat liver microsomes towards different molecular species of 1,2-diacyl-sn-glycerols was studied. Triacylglycerol synthesis was determined by monitoring the entry of radioactive palmitate from [1-14C]palmitoyl-CoA into triacylglycerol in the presence of various diacylglycerols. The diacylglycerol acyltransferase did not exhibit any selectivity against hexaenoic precursors under the assay conditions used (0.25 mM diacylglycerol in medium) and the rates of triacylglycerol synthesis observed with the various diacylglycerols were not significantly different when the data were analyzed statistically. The results suggest that the extremely low level of docosahexaenoic acid in triacylglycerols cannot be attributed to a discrimination of the diacylglycerol acyltransferase against 1-saturated 2-docosahexaenoyl precursors.
Breakdown of mucin and plant polysaccharides in the human colon
Authors: Vercellotti J. R. , Salyers A. A. , Bullard W. S. , Wilkins T. D.
Published: December 2009

To obtain an estimate of the extent to which complex carbohydrates are degraded by bacteria in the human colon, aqueous extracts of colon contents from four human subjects were separated into high and low molecular weight fractions by chromatography on a Sephadex G-100 column. The composition of these fractions was compared with the composition of similar fractions from ileal contents, i.e., from material entering the colon. In all four subjects, high molecular weight carbohydrate concentrations were lower in the colon than in the ileum, indicating that breakdown of complex carbohydrate occurs in the colon. The high molecular weight carbohydrate fraction contained sugars characteristic of plant polysaccharides (arabinose, xylose, mannose, rhamnose) as well as sugars characteristic of mucin (fucose, hexosamines, sialic acids). Concentrations of most of these sugars were uniformly lower in the colon than in the ileum. Since high molecular weight protein concentrations were lower in the colon than in the ileum of two of the four subjects tested, some degradation of protein may also occur in the colon.
Synthesis of pppGpp by ribosomes from an Escherichia coli spoT mutant and the metabolic relationship between pppGpp and ppGpp
Authors: Leung Kwok-Luen , Yamazaki Hiroshi
Published: December 2009

Both ribosomes and a cell-free extract (S-30) prepared from an Escherichia coli spoT mutant catalyzed the synthesis of guanosine pentaphosphate (pppGpp) and guanosine tetraphosphate (ppGpp) as efficiently as did ribosomes and S-30 from a spoT+ strain. In both cases, the level of pppGpp reached its maximum before ppGpp maximally accumulated. pppGpp added to the ribosome system was rapidly converted to ppGpp. These results indicate that the spoT+ gene product may not have a direct role in the synthesis of pppGpp and that pppGpp is a precursor of ppGpp.