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Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Pure glutathione reductase from Saccharomyces cerevisiae catalyzed under anaerobic conditions the enzymatic reduction of GSSG using electrochemically reduced methyl viologen as electron donor. The new assay was completely dependent on the amount of active enzyme present, and involved the formation of 1 mol GSH per mole of reduced methyl viologen consumed. The enzyme followed a standard Michaelis-Menten kinetics; a Km = 230 microM for reduced methyl viologen and a turnover number of 969 mumol GSSG reduced per minute per micromole enzyme were determined. The enzymatic activity seemed to depend on the redox potential, showing half-maximal activity at -0.407 V. The enzyme was quite specific: the activity using reduced benzyl viologen as electron donor was just 1.5% of that obtained with reduced methyl viologen at the same concentration and potential. Glutathione reductase was totally inactivated after a brief anaerobic exposure with reduced methyl viologen in the absence of GSSG; a partial reactivation was observed following addition of glutathione disulfide. No inhibition of the methyl viologen-dependent activity was observed in the presence of 2',5'-ADP or 2'-P-5'-ADP-ribose, two NADP(H) analogs, at concentrations which drastically inhibited the NADPH-dependent activity, thus suggesting that the reduced viologen does not interact with the pyridine nucleotide-binding site.
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PMID:Electron transfer between reduced methyl viologen and oxidized glutathione: a new assay of Saccharomyces cerevisiae glutathione reductase. 353 78

Transient ATP synthesized by preparations enriched with plasmatic membranes of particles from the human placenta in the presence of insulin (4 micrograms/ml) and epidermal growth factor (1 microgram/ml) within 1 min after the addition of hormones at 30 degrees C, was isolated by means of chromatography on Dowex 1 X 8. ATP was synthesized in a medium containing Tris-HCl buffer, pH 7.5, ADP, Mg2+, and Pi during NADH-dependent oxidation in the presence of cytochrome C and oxygen. The amount of ATP was 10(-9) mole/mg protein/min. Quantitative assessment of ATP in lyophilized product was carried out by means of fluorimetry (excitation wavelength--360 nm; emission wavelength--460 nm) of NADH formed during coupled enzymatic reactions involving hexokinase and glucose-6-phosphate dehydrogenase. A possible biological role of peptide growth factor-stimulated formation of transient ATP in plasmatic membranes is discussed.
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PMID:[ATP generation by the plasma membranes of human placental cells as affected by insulin and epidermal growth factor]. 354 74

Creatine kinase from skeletal muscle (EC 2.7.3.2) was inactivated by means of imidazolides of AMP, ADP, ATP. Rates of the inactivation of the enzyme's M- and M'-subunits differ 50-100 fold and decrease in the presence of ADP and ATP. Differential spectrum of the native and modified enzymes corresponds to the spectrum of N,O-diacetyltyrosine. Kinetic curves of hydroxylamine-dependent destruction of N,O-diacetyltyrosine and of alteration of differential spectrum of the modified and native enzymes essentially coincide. The enzyme's inactivation appears to be caused mainly by the formation of a bond between nucleotide imidazolides activated carboxyl group of the active centre and OH-group of Tyr residue arranged in the close proximity. The stoichiometry of acyltyrosine formation is evaluated as 2.1 +/- 0.2 mole per mole of the functional dimer. Along with formation of ester bond between amino acid residues, a covalent attachment of 0.03-0.06 mole of [14C]nucleotides per mole of enzyme is observed. As the data of acid hydrolysis show, Im-ATP and Im-AMP block epsilon-amino group of Lys and guanidine group of Arg, respectively. Reasons of the multiple modification of creatine kinase by affinity reagents are discussed. The results obtained and literature data are summarised in the hypothetical scheme of disposition of various amino acid residues in the active centre of creatine kinase.
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PMID:[Creatine kinase from rabbit skeletal muscles: formation of O-acyltyrosine as a result of the activation of the carboxylic group of the enzyme active site by affinity reagents, nucleotide imidazolides]. 360 72

The nucleotide analogue 5'-p-fluorosulfonylbenzoyladenosine (FSBA) reacts irreversibly with rat liver cytosolic 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase kinase, causing a rapid loss of the AMP activation capacity and a slower inactivation of the catalytic activity. The rate constant for loss of AMP activation is about 10 times higher (kappa 1 = 0.112 min-1) than the rate constant of inactivation (kappa 2 = 0.0106 min-1). There is a good correspondence between the time-dependent inactivation of reductase kinase and the time-dependent incorporation of 5'-p-sulfonylbenzoyl[14C]adenosine ([14C]SBA). An average of 1.65 mol of reagent/mol of enzyme subunit is bound when reductase kinase is completely inactivated. The time-dependent incorporation is consistent with the postulate that covalent reaction of 1 mol of SBA/mol of subunit causes complete loss of AMP activation, whereas reaction of another mole of SBA/mol of subunit would lead to total inactivation. Protection against inactivation by the reagent is provided by the addition of Mg2+, AMP, Mg-ATP, or Mg-AMP to the incubation mixtures. In contrast, addition of ATP, 2'-AMP, or 3'-AMP has no effect on the rate constants. Mg-ATP protects preferentially the catalytic site against inactivation, whereas Mg-AMP at low concentration protects preferentially the allosteric site. Mg-ADP affords less protection than Mg-AMP to the allosteric site when both nucleotides are present at a concentration of 50 microM with 7.5 mM Mg2+. Experiments done with [14C]FSBA in the presence of some protectants have shown that a close correlation exists between the pattern of protection observed and the binding of [14C]SBA. The postulate is that there exists a catalytic site and an allosteric site in the reductase kinase subunit and that Mg-AMP is the main allosteric activator of the enzyme.
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PMID:Affinity labeling of the catalytic and AMP allosteric sites of 3-hydroxy-3-methylglutaryl-coenzyme A reductase kinase by 5'-p-fluorosulfonylbenzoyladenosine. 365 27

Yeast phosphoglycerate kinase is irreversibly inactivated upon incubation with 5'-[p-(fluorosulfonyl)-benzoyl]-1-N6-ethenoadenosine (5'-FSB epsilon A), an analogue to the nucleotide substrate. Marked protection against inactivation occurs with MgATP, ATP, MgADP, ADP, and 3-phosphoglycerate, suggesting that a part of the catalytic center is modified. The time dependence of the inactivation is characterized by a nonlinear kinetic profile. Curve fitting of various models for ligand binding to the enzyme suggested a two-site model. Modification of one of the sites appears to protect the catalytically essential site from modification. Stoichiometric studies show that the relationship between moles of 5'-FSB epsilon A incorporated per mole of enzyme and the residual enzymatic activity also shows nonlinear behavior. An extrapolated value of 1.5 mol of bound label/mol of enzyme corresponds to complete inactivation. The apparent overall pseudo first-order rate constant for the reaction between phosphoglycerate kinase and 5'-FSB epsilon A, as well as the separate rate constants for the modification, exhibit saturation behavior with respect to the concentration of 5'-FSB epsilon A, indicative of a rapid reversible binding of the reagent to the enzyme prior to modification.
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PMID:Affinity labeling of phosphoglycerate kinase by 5'-[p-(fluorosulfonyl)benzoyl]-1,N6-ethenoadenosine. 366 85

Feeding natural fats varying in contents of palmitate (16:0), stearate (18:0), oleate (18:1), and linoleate (18:2) to rabbits resulted in modulation of platelet phospholipid fatty acyl composition. Rabbits were fed high fat semipurified diets containing 2% corn oil (CO) + 18% CO, cocoa butter (CB) or milkfat (M) for periods of up to 300 d. Platelet phospholipid linoleate contents corresponded to diet levels with 18:2 highest in CO-fed rabbits and following the sequence CO greater than CB greater than M. Stearate was highest in CB-fed rabbits, corresponding to high 18:0 levels in CB, but palmitate levels were not affected by diet. Both CB and M-fed rabbits were higher than CO-fed rabbits in oleate. Though CO is highest in 18:2, the accepted 20:4 precursor, arachidonate was highest in M-fed rabbits. Adding cholesterol (0.2%) to the diets did not affect platelet phospholipid fatty acyl composition except to elevate 20:4 in M-fed rabbits. CO-fed rabbits showed uniquely high levels of tetracosadienoate (24:2). Fatty acyl composition data were essentially constant between 200 and 300 d on diet. Phospholipid fatty acyl unsaturation was apparently homeostatically controlled as mole percent unsaturate to saturate ratios were independent of diet. The observed homeostasis resulted in minimal diet influences on platelet membrane fluidity and ADP or collagen stimulated platelet aggregation. Platelet fluidity, determined by fluorescence polarization, was a function of oleate and linoleate contents of the cells. Cholesterol feeding generally lowered platelet fluidity and altered the dependence of fluidity on fatty acyl composition.
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PMID:Influence of saturated and unsaturated fats on platelet fatty acids in cholesterol-fed rabbits. 382 74

Important advances have been made in recent years in the study of the structure of pyruvate kinase: the amino acid sequence of the enzymes from chicken muscle and yeast have been established and the three-dimensional structure of the cat muscle enzyme has been determined at 0.26 nm resolution. Work in our laboratory has shown that dialdehyde-ADP (oADP) can be used as an affinity label of rabbit muscle pyruvate kinase: if the enzyme is incubated with cold oADP in the presence of high ADP concentrations, dialyzed and then incubated with 14C-oADP, the enzyme inactivates and one mole of radioactive oADP incorporates per mole of enzyme subunit. A labeled peptide with a molecular weight of about 5900 has been purified from a tryptic digest of the modified enzyme. The first 26 residues of the peptide have been sequenced and this sequence is identical to a region in the chicken muscle enzyme and a peptide isolated from the bovine muscle enzyme specifically labeled with trinitrobenzenesulfonate. High homology is also found with a region of the yeast enzyme. All this suggests that the isolated peptide is part of the active site; the modified amino acid, probably a lysine, seems to be located in one of the alfa helices of domain A of the enzyme, according to the x-ray data.
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PMID:Pyruvate kinase: studies on affinity labeling and active-site structure using the rabbit muscle enzyme. 383 41

The N-terminal formic acid fragment (FA1) of the N-[3H]ethylmaleimide-labeled and carboxymethylated bovine mitochondrial phosphate transport protein (PTPN*CM) has been purified and completely sequenced: NH2-Ala-Val-Glu-Glu-Gln-Tyr-Ser-Cys-Asp-Tyr10-Gly-Ser-Gly-Arg-Phe- Phe-Ile-Leu-Cys- Gly20-Leu-Gly-Gly-Ile-Ile-Ser-Cys-Gly-Thr-Thr30-His-Thr -Ala-Leu-Val-Pro-Leu-Asp- -Leu-Val40-Lys-Cys(N-[3H]ethylmaleimide)-Arg-Met-Gln-Val-Asp- COOH. By thermolysin digestion of FA1 and high-performance liquid chromatography isolation of the radioactive subfragment Leu39-Arg43, the sole N-ethylmaleimide-binding residue has been identified as Cys42. FA1 contains a high mole percentage of cysteine (8.5%) and shows silver staining anomaly. Its sequence reveals significant homology in the triplicated gene regions (Pro27,132,229) of the mitochondrial ADP/ATP carrier from beef heart and Neurospora crassa. The hydropathic profile suggests that FA1 contains a transmembrane segment (Phe15-Val40) with only one basic (His31) and one acidic (Asp38) residue. The presence of the phosphate transport protein gene among nuclear genes is suggested from a lack of significant homology between the reverse-translated FA1 (mitochondrial codons) and the bovine mitochondrial genome. The inhibitory action of N-ethylmaleimide on the phosphate transport mechanism is discussed.
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PMID:Sequence of the N-terminal formic acid fragment and location of the N-ethylmaleimide-binding site of the phosphate transport protein from beef heart mitochondria. 406 97

When whole urinary bladders of the toad were incubated with adenosine 3',5'-monophosphate-(3)H (cyclic AMP-(3)H) > 95% of the radioactivity could be extracted from the tissue with trichloroacetic acid (TCA). The TCA-soluble radioactivity was separable by cation-exchange (Dowex-50) chromatography into residual cyclic AMP-(3)H, 5'-AMP-(3)H, adenosine diphosphate (ADP)-(3)H and inosine-(3)H. Thus, neither substantial tight binding of cyclic AMP to TCA-precipitable cell constituents nor any novel metabolite of cyclic AMP were found. On exposure of cyclic AMP-(3)H to a crude homogenate of the epithelial cells scraped from the mucosal face of the bladder, the principal metabolite was inosine-(3)H, whereas 5'-AMP-(3)H was either absent or present in undetectible amounts. However, when the homogenate included added 5'-AMP (2 x 10(-2) mole/liter), substantial quantities of 5'-AMP-(3)H were recovered. Metabolism of cyclic AMP-(3)H by homogenates of the epithelial cell scrapings from the bladder was strongly stimulated by alkalinization over the range in pH of 6-9. Theophylline inhibited metabolism of cyclic AMP only to a limit of 50%, the inhibition being limited to the OH(-)-stimulated component. These results suggest the possibility that a second pathway for metabolism of cyclic AMP may exist. If such is the case, its relationship, if any, to the ultimate biological effects of cyclic AMP within cells will be studied.
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PMID:Metabolism of adenosine 3',5'-monophosphate by epithelial cells of the toad bladder. 430 52

The relation of cyclic 3',5'-adenosine monophosphate to platelet function has been studied by investigating the influence of this compound and of its N(6)-2'-0-dibutyryl derivative on platelet aggregation and other aspects of platelet behavior after demonstration of adenyl cyclase activity in disrupted platelets. Dibutyryl cyclic AMP inhibited platelet aggregation induced by ADP, epinephrine, collagen, and thrombin. Cyclic AMP was also inhibitory but was less effective. The platelet "release reaction" was also inhibited; specifically, there was inhibition of the induction of platelet factor 3 activity and of the release of labeled 5-hydroxytryptamine. Platelet swelling produced by ADP was not inhibited. The action of dibutyryl cyclic AMP did not result from contamination with 5'-AMP, nor was it attributable to production of 5'-AMP by plasma enzymes. Dibutyryl cyclic AMP was degraded to 2'-O-monobutyryl cyclic AMP and to cyclic AMP in plasma, but plasma exhibited no cyclic nucleotide phosphodiesterase activity, and the production of 5'-AMP did not occur. The in vitro effects of dibutyryl cyclic AMP were associated with uptake of the compound by platelets. Adenyl cyclase activity of platelet homogenates was demonstrated with production of 9.27 x 10(-11) (+/-2.62 x 10(-11)) mole cyclic AMP per min per 10(10) platelets. The activity was increased by NaF and by prostaglandin PGE(1) and was decreased by epinephrine. The effect of epinephrine was blocked by phentolamine but not by propanolol. Adenyl cyclase activity was also inhibited by collagen, 5-hydroxytryptamine, and thrombin. ADP, dibutyryl cyclic AMP, and cyclic AMP did not alter adenyl cyclase activity. These observations are consistent with the hypothesis that platelet aggregation is favored by a decrease in platelet cyclic AMP and inhibited by an increase in cyclic AMP.
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PMID:Cyclic 3',5'-adenosine monophosphate in human blood platelets. II. Effect of N6-2'-o-dibutyryl cyclic 3',5'-adenosine monophosphate on platelet function. 432 65


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