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Biomedical subjects

C H Reynolds

Publications and source records attributed to C H Reynolds.

At least 37 records · Page 2Linked to original sources
PubMed1988

Inactivation of soybean lipoxygenase by lipoxygenase inhibitors in the presence of 15-hydroperoxyeicosatetraenoic acid.

Soybean lipoxygenase is rapidly inactivated when incubated with arachidonic acid and any of several lipoxygenase inhibitors, including NDGA, the aminopyrazolines BW 755C and BW 540C, and the acetohydroxamic acid derivatives BW A4C and BW A137C. Little or no inactivation was found when the enzyme was incubated with substrate or with inhibitors alone. 15-HPETE was as effective as arachidonic acid in promoting inactivation, but linoleic acid and 13-HPOD were much less effective. The UV absorption at 235 nm, due to the conjugated diene in 15-HPETE or 13-HPOD, was rapidly destroyed in the presence of soybean lipoxygenase and inhibitor in a presumed pseudoperoxidase reaction. The products of the reaction between linoleic acid, BW A137C and soybean lipoxygenase have been partially characterized. A derivative of arachidonic acid is postulated to be the inactivating agent.

4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyraz↗
PubMed1988

Heparin cofactor II: cDNA sequence, chromosome localization, restriction fragment length polymorphism, and expression in Escherichia coli.

Heparin cofactor II (HCII) is an inhibitor of thrombin in plasma that is activated by dermatan sulfate or heparin. An apparently full-length cDNA for HCII was isolated from a human liver lambda gt11 cDNA library. The cDNA consisted of 2215 base pairs (bp), including an open-reading frame of 1525 bp, a stop codon, a 3'-noncoding region of 654 bp, and a poly(A) tail. The deduced amino acid sequence contained a signal peptide of 19 amino acid residues and a mature protein of 480 amino acids. The sequence of HCII demonstrated homology with antithrombin III and other members of the alpha 1-antitrypsin superfamily. Blot hybridization of an HCII probe to DNA isolated from sorted human chromosomes indicated that the HCII gene is located on chromosome 22. Twenty human leukocyte DNA samples were digested with EcoRI, PstI, HindIII, KpnI, or BamHI, and Southern blots of the digests were probed with HCII cDNA fragments. A restriction fragment length polymorphism was identified with BamHI. A slightly truncated form of the cDNA, coding for Met-Ala instead of the N-terminal 18 amino acids of mature HCII, was cloned into the vector pKK233-2 and expressed in Escherichia coli. The resultant protein of apparent molecular weight 54,000 was identified on an immunoblot with 125I-labeled anti-HCII antibodies. The recombinant HCII formed a complex with 125I-thrombin in a reaction that required the presence of heparin or dermatan sulfate.

Amino Acid Sequence↗
PubMed1988

5-Lipoxygenase and leukotriene synthesis: effects of calcium ions and of inhibitors.

5-Lipoxygenase and leukotriene (LT) A4 synthase, the first two enzymes in the pathway converting arachidonic acid to leukotrienes, can be co-purified. The Ca2+-activated conversion of arachidonic acid and of 5-hydroperoxyeicosatetraenoic acid (5-HPETE) to LTB4 have been compared, using cytosol from human leucocytes. The two activities showed identical responses to a number of inhibitors, suggesting that the two catalytic activities may involve the same active centre. The effects of Ca2+ ions were further investigated. With 5-HPETE as substrate, substantial synthesis of LTB4 was given in the absence of Ca2+, and the inhibitor sensitivity of this component was quite different from that of the Ca2+-activated component. This Ca2+-independent synthase activity was, however, very low in saponin-permeabilised washed leucocytes and it may therefore be not significant physiologically. With arachidonic acid as substrate at pH 7, the activity was highly Ca2+-dependent at a low substrate concentration (6.6 microM), but at a high concentration (132 microM) substantial activity was observed without Ca2+. This was also found when 5-lipoxygenase was assayed in cytosol from RBL cells. At pH 8-8.5, however, Ca2+ was required at both high and low concentrations of arachidonic acid. This suggests that Ca2+ is required for 5-lipoxygenase activity on arachidonate ions in solution but possibly not on protonated arachidonic acid or micelles.

Arachidonate 5-Lipoxygenase↗
PubMed1988

Selective inhibition of arachidonate 5-lipoxygenase by novel acetohydroxamic acids: biochemical assessment in vitro and ex vivo.

1. The chemically novel acetohydroxamic acids, BW A4C, BW A137C and BW A797C, are potent inhibitors of the synthesis of leukotriene B4 (LTB4) from arachidonic acid by human leucocyte homogenates: the concentrations required for 50% inhibition (IC50) were 0.1 microM, 0.8 microM and 0.5 microM respectively. Inhibition was less at higher concentrations of arachidonic acid. 2. These compounds also inhibited the synthesis of [14C]-5-HETE from [14C]-arachidonic acid and the calcium-dependent synthesis of LTB4 from 5-HPETE. This, therefore, suggests that they inhibit 5-lipoxygenase and LTA4 synthase. 3. Concentrations of acetohydroxamic acids required to inhibit metabolism of arachidonic acid by cyclo-oxygenase, 12-lipoxygenase and 15-lipoxygenase were 10 to 100 times higher than those required to inhibit 5-lipoxygenase. 4. The compounds were potent inhibitors of LTB4 synthesis induced by the ionophore, A23187, in human intact leucocytes. This inhibition was reversed by washing the cells. They were also potent, selective inhibitors of LTB4 synthesis induced by A23187 in whole rat blood: binding to rat plasma proteins did not greatly reduce the effectiveness of the compounds. 5. The effects of the acetohydroxamic acids, administered either intravenously or orally to rats, on the synthesis of LTB4, and thromboxane B2 (TXB2) in A23187-stimulated blood ex vivo was studied. The three compounds caused dose-dependent inhibition of the synthesis of LTB4 but not TXB2. Inhibition of LTB4 synthesis persisted for up to 6 h after a single oral dose of 50 mg kg-1. 6. The plasma concentrations of unchanged compound determined by h.p.l.c. correlated with the inhibition of LTB4 synthesis ex vivo.

Animals↗
PubMed1986

Activation of human erythrocyte 2,3-bisphosphoglycerate phosphatase at physiological concentrations of substrate.

In human erythrocytes the reactions of the 2,3-bisphosphoglycerate shunt are catalyzed primarily by one protein, 2,3-bisphosphoglycerate synthase-phosphatase. At low concentrations of 2,3-bisphosphoglycerate the phosphatase is activated by several anions including inorganic phosphate and sulfite, and the phosphate activation is inhibited by low concentrations of 3-phosphoglycerate [Z. B. Rose and J. Liebowitz (1970) J. Biol. Chem. 245, 3232-3241]. Phosphate and sulfite also activate at high but physiological concentrations of 2,3-bisphosphoglycerate (5 mM), but the inhibition by 3-phosphoglycerate is much weaker. The basal activity (without added phosphate or sulfite) was also found to be higher and to be 3-phosphoglycerate sensitive; this is attributed to activation either by 2,3-bisphosphoglycerate itself or by a contaminant in it. These results allow previous observations of 2,3-bisphosphoglycerate hydrolysis in intact erythrocytes to be reconciled with the properties of the purified enzyme under near-physiological conditions.

2,3-Diphosphoglycerate↗
PubMed1984

The role of phosphoenolpyruvate in insulin secretion: the effect of L-phenylalanine.

Incubation of rat islets with phenylalanine increased the tissue content of phosophoenolpyruvate, both in the presence and in the absence of glucose. At the same time, L-phenylalanine neither stimulated nor inhibited insulin release. It is unlikely that insulin secretion is tightly coupled to the availability of phosphoenolpyruvate in rat islets.

Alanine↗
PubMed1984

Kinetics of inhibition of angiotensin converting enzyme by captopril and by enalapril diacid.

Using a continuous spectrophotometric assay, inhibition of angiotensin converting enzyme by captopril and by the active diacid derivative of enalapril was reinvestigated. The onset of inhibition was comparatively slow, but the inhibition achieved was stronger than previous estimates: approximate Ki-values were 0.3 nM for captopril and 0.06 nM for enalapril diacid. The rate-constants for association and dissociation of these enzyme-inhibitor complexes were estimated, and half-times of approximately 12 min for the captopril complex and 60 min for the enalapril diacid complex were calculated. The rate of dissociation of the captopril-enzyme complex was measured directly by reacting the thiol group in free captopril with 5,5'-dithiobis(2-nitrobenzoic acid) and observing the reactivation of the enzyme; a half-time of approximately 30 min was obtained. Therefore the release of these inhibitors from the enzyme may be slow enough to affect the duration of their hypotensive action.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1983

Citrate utilization by Escherichia coli: plasmid- and chromosome-encoded systems.

Citrate utilization plasmids have previously been identified in atypical Escherichia coli isolates. A different citrate-utilizing (Cit+) variant of E. coli K-12 arose as a consequence of two chromosomal mutations (B. G. Hall, J. Bacteriol. 151:269-273, 1982). The processes controlling the transport of citrate in both a Cit+ chromosomal mutant and a Cit+ plasmid system were studied. Both systems were found to be inducible in growth experiments. In transport assays with whole cells, citrate-grown cells accumulated [1,5-14C]citrate at two to three times the rate of uninduced cells. Only the Vmax was affected by induction, and the Km for whole cells remained at 67 microM citrate for the chromosomal strain and 120 microM citrate for the plasmid-conferred system. There was no detectable accumulation of radioactivity with [6-14C]citrate, because of rapid metabolism and the release of 14CO2. Energy-dependent citrate transport was found with membrane vesicles obtained from both the chromosome-conferred and the plasmid Cit+ systems. The vesicle systems were inhibited by valinomycin and carbonyl cyanide m-chloro-phenylhydrazone but not by nigericin and monensin. In contrast to whole cells, the vesicle systems were resistant to Hg2+ and showed identical kinetics with [1,5-14C]citrate and [6-14C]citrate. H+ appeared to be important for citrate transport in whole cells and membranes. Monovalent cations such as Na+ and K+, divalent cations such as Mg2+ and Mn2+, and anions such as PO4(3-), SO4(2-), and NO3- were not required. The two systems differed in inhibition by citrate analogs.

Aconitic Acid↗
PubMed1982

Inhibition of calmodulin-activated cyclic nucleotide phosphodiesterase: multiple binding-sites for tricyclic drugs on calmodulin.

A cyclic nucleotide phosphodiesterase from guinea-pig heart is activated by calmodulin in the presence of calcium ions. Activation was measured over a range of calmodulin concentrations, and is antagonised by several tricyclic psychotropic drugs including trifluoperazine, imipramine, chlorpromazine and amitriptyline. When the concentration of amitriptyline was increased, its apparent inhibition constant for binding to calmodulin decreased. This was due in part to binding of amitriptyline to glass surfaces; but after correction for this the discrepancy was still significant. It is proposed that this is due to two sites on calmodulin for amitriptyline, with binding to either site being sufficient to prevent calmodulin from activating phosphodiesterase.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1982

Simulations of the roles of multiple cyclic nucleotide phosphodiesterases.

1. Simulations were performed using a model for cellular cyclic AMP metabolism involving a hormone-activated adenylate cyclase and two cyclic nucleotide phosphodiesterases with different Michaelis constants. 2. The response curves of cyclic AMP concentration as a function of hormone concentration were affected by regulating the phosphodiesterases. The maximum velocity of the high-affinity phosphodiesterase (V1) was important in determining the position of the response curve; when v1 was less than the maximal activity of adenylate cyclase (Vc), sigmoid response curves were readily produced. The maximum attainable concentration of cyclic AMP was determined primarily by V1 when Vc less than V1, and primarily by the activity of the low-affinity enzyme when Vc greater than V1 (V2 much greater than Vc in all cases). 3. The glucagon-stimulated adenylate cyclase and insulin-stimulated phosphodiesterase of the rat liver plasma membrane were simulated using experimentally determined values for the enzyme-kinetic parameters, and a considerable potential for regulation of the system by insulin was demonstrated. 4. Other possible functions for the regulation of phosphodiesterases are considered, in particular the value of increasing the speed of response to decreases in hormone concentration.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1980

Activation and inactivation of phosphoenolpyruvate carboxykinase by ferrous ions.

Phosphoenolpyruvate carboxykinase from rat liver cytosol is activated by Fe2+ ions in either direction of catalysis. Preincubation of the purified enzyme with Fe2+ ions causes a time-dependent irreversible loss of activity; this is not seen with unpurified enzyme. Purified enzyme can be protected from inactivation by Fe2+ ions by partially purified protein fractions from liver (ferroactivator fractions). The possible role of ferroactivator and Fe2+ ions in regulating phosphoenolpyruvate carboxykinase is discussed.

Animals↗
PubMed1980

Determination of the stability constants of Mn2+ and Mg2+ complexes of the components of the NADP-linked isocitrate dehydrogenase reaction by electron spin resonance.

1. The stability constants (Ks) of Mn2+ and Mg2+ complexes of isocitrate, 2-oxoglutarate, NADP and NADPH have been estimated by using electron spin resonance to measure free Mn2+ in ligand--metal-ion solutions. 2. The values of Ks for the Mn2+ complexes at 25 degrees C, in triethanolamine buffer containing NaCl, pH 7.0 and ionic strength 0.15 M, are 497 M-1 for isocitrate, 39 M-1 for 2-oxoglutarate, 467 M-1 for NADP and 943 M-1 for NADPH. 3. For the Mg2+ complexes under the same conditions, the Ks values are 357 M-1, 25 M-1, 133 M-1 and 179 M-1 respectively. The large difference between the stabilities of the isocitrate and 2-oxoglutarate complexes is thus largely responsible for the observed variation of the apparent equilibrium constant of the NADP-linked isocitrate dehydrogenase reaction with magnesium ion concentration. 4. NADP-linked isocitrate dehydrogenase from bovine heart mitochondria binds Mn2+, and the stability constant of the complex is about 2.2 x 10(4) M-1. The formation of this complex may explain the inhibition of the enzyme-catalysed reaction observed with Mn2+ concentrations greater than 0.2 mM in initial rate measurements.

Drug Stability↗
PubMed1979

Factors affecting coenzyme binding and subunit interactions in glyceraldehyde-3-phosphate dehydrogenase.

There is no evidence, at pH 9.4, of negative cooperativity in the binding of NAD+ or NADH to rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phorphorylating), EC 1.2.1.12) nor in the binding of acetyl pyridine adenine dinucleotide at pH 7.6 and ph 9.4. The binding of NAD+ to carboxymethylated enzyme at pH 7.6 and pH 9.4 also occurs without cooperativity. The possible implications of these findings for the involvement of ionising groups in the enzyme in the subunit interactions responsible for negative cooperativity, previously reported for coenzyme binding at pH 7.4--8.6, are discussed.

Alkylation↗
PubMed1978

Equilibrium binding of coenzymes and substrates to nicotinamide-adenine dinucleotide phosphate-linked isocitrate dehydrogenase from bovine heart mitochondria.

1. The stoicheiometries and affinities of ligand binding to isocitrate dehydrogenase were studied at pH 7.0, mainly by measuring changes in NADPH and protein fluorescence. 2. The affinity of the enzyme for NADPH is about 100-fold greater than it is for NADP+ in various buffer/salt solutions, and the affinities for both coenzymes are decreased by Mg2+, phosphate and increase in ionic strength. 3. The maximum binding capacity of the dimeric enzyme for NADPH, from coenzyme fluorescence and protein-fluorescence measurements, and also for NADP+, by ultrafiltration, is 2 mol/mol of enzyme. Protein-fluorescence titrations of the enzyme with NADP+ are apparently inconsistent with this conclusion, indicating that the increase in protein fluorescence caused by NADP+ binding is not proportional to fractional saturation of the binding sites. 4. Changes in protein fluorescence caused by changes in ionic strength and by the binding of substrates, Mg2+ or NADP+ (but not NADPH) are relatively slow, suggesting conformation changes. 5. In the presence of Mg2+, the enzyme binds isocitrate very strongly, and 2-oxoglutarate rather weakly. 6. Evidence is presented for the formation of an abortive complex of enzyme-Mg2+-isocitrate-NADPH in which isocitrate and NADPH are bound much more weakly than in their complexes with enzyme and Mg2+ alone. 7. The results are discussed in relation to the interpretation of the kinetic properties of the enzyme and its behaviour in the mitochondrion.

Animals↗
PubMed1978

Transient kinetics of nicotinamide-adenine dinucleotide phosphate-linked isocitrate dehydrogenase from bovine heart mitochondria.

Pre-steady-state studies of the isocitrate dehydrogenase reaction show that the rate constant for the hydride-transfer step is above 990s-1, and that both subunits of the enzyme are simulataneously active. After the fast formation of NADPH in amounts equivalent to the enzyme subunit concentration, the rate of NADPH formation is equal to the steady-state rate if the enzyme has been preincubated with isocitrate and Mg2+. If the enzyme has been preincubated with NADP+ and Mg2+, in 0.05 M-triethanolamine chloride buffer, pH 7.0, with the addition of 0.1 M-NaCl, the amount of NADPH formed in the fast phase is only 60% of the enzyme subunit concentration, and the turnover rate is at first lower than the steady-state rate. In 0.05 M-triethanolamine chloride buffer, pH 7.0, if the enzyme is preincubated with NADP+ or NADPH, the turnover rate increases 3-fold to reach the steady-state rate after about 5 s. Preincubation of the enzyme with isocitrate and Mg2+ abolishes this lag phase, the steady-state rate being reached at once. It is suggested that the enzyme exists in at least two conformational forms with different activities, and that the lag phase represents the transition (k = 0.4s-1) from a form with low activity to the fully active enzyme, induced by the binding of isocitrate and Mg2+.

Animals↗