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Biomedical subjects

J H Harrison

Publications and source records attributed to J H Harrison.

At least 73 records · Page 4Linked to original sources
PubMed1984

Selenium content and glutathione peroxidase activity in tissues of the dairy cow after short-term feeding.

The effect of selenium and vitamin E on concentrations of selenium and selenium-dependent glutathione peroxidase in tissues of dairy cows was studied. Selenium (5 mg/day) and vitamin E (2 g/day) were supplemented for 10 days in a 2 X 2 factorial arrangement. Selenium supplementation increased content of selenium in whole blood, plasma, ovary, and liver and increased activity of selenium-dependent glutathione peroxidase in liver. Activity of selenium-dependent glutathione peroxidase in follicular fluid was closely correlated with selenium-dependent glutathione peroxidase in plasma. Significant activity of selenium-dependent glutathione peroxidase was detected in luteal tissue of the ovary. Relationships were linear between content of selenium and selenium-dependent glutathione peroxidase in ovary, uterus, and adrenal tissues.

Administration, Oral↗
PubMed1983

Rapid and sensitive method for the microassay of nitrosobenzene plus phenylhydroxylamine in blood.

An assay method has been developed for the determination of the combined concentration of nitrosobenzene plus phenylhydroxylamine (as nitrosobenzene) in small volumes of blood. The initial step in the procedure consisted of the simultaneous oxidation of phenylhydroxylamine to nitrosobenzene and of ferrous hemoglobin to methemoglobin by ferricyanide. Nitrosobenzene in the ferricyanide-treated blood samples was then extracted into ethyl acetate, and separated and quantitated by reversed-phase high-performance liquid chromatography with electrochemical detection. The sensitivity limit for nitrosobenzene in blood was in the pmol/ml concentration range, less than 100 microliter of blood was required for assay, and the procedure was convenient for routine multisample use. In comparison with previous assays, this method was more sensitive, had a lower coefficient of variation, and required 25-40 fold smaller blood sample volumes. The method was combined with the orbital sinus bleeding technique in order to follow the nitrosobenzene time course in vivo using small serial blood samples from rats treated with intraperitoneal injections of phenylhydroxylamine or aniline.

Animals↗
PubMed1982

Regulation of mitochondrial malate dehydrogenase. Evidence for an allosteric citrate-binding site.

The effect of citrate on the structure and function of porcine heart mitochondrial malate dehydrogenase (EC 1.1.1.37) has been characterized. The native dimeric form of this enzyme is specifically activated by citrate in the NAD+ leads to NADH direction and inhibited by citrate in the NADH leads to NAD+ direction. It is proposed that citrate is bound at a regulatory site that is distinct from the catalytic site of the enzyme. In binding to this regulatory site, citrate greatly reduces the binding of NADH as determined by fluorescence titration and "Hummel-Dreyer"-type experiments, but does not diminish the binding of NAD+. As would be expected for an effector altering the equilibrium between two conformational forms of an enzyme, citrate favorably perturbs the equilibrium for the reaction in the direction of NAD+ reduction. Using [14C]citrate, the stoichiometry of citrate binding to mitochondrial malate dehydrogenase has been determined to be two equivalent sites per dimer, with a dissociation constant of 12.5 microM. In detailed kinetic studies, it has also been observed that activation by citrate abolishes (masks) the enzymatic activation induced by high concentrations of the substrate, L-malate. In addition, Hummel-Dreyer-type experiments indicate that less than a stoichiometric amount of NADH is bound to the enzyme under conditions of malate activation. These data are consistent with a previously suggested second "substrate" binding site proposed to explain the enzymatic activation observed at high concentrations of the substrate, L-malate (Telegdi, M., Wolfe, D. V., and Wolfe, R. G. (1973) J. Biol. Chem. 248, 6484-6489). This allosteric site may exist only on the enzyme conformation capable of binding NAD+.

Allosteric Regulation↗
PubMed1982

Active subunits in hybrid-modified malate dehydrogenase.

Inactivation of porcine heart mitochondrial malate dehydrogenase (L-malate: NAD+ oxidoreductase, EC 1.1.1.37) by selective modification of an active center histidine residue with the reagent iodoacetamide has been further investigated to examine the existence of and the enzymatic activity of a hybrid (half)-modified dimer. The loss of enzymatic activity during iodo(1-14C) acetamide modification is linear with 14C incorporation. Enzyme was modified to various extents and the reaction was quenched. Microzonal electrophoresis was performed to separate native dimeric enzyme, hybrid-modified enzyme, and doubly modified enzyme. The distribution of each species was quantitated by scanning densitometry. The distribution generated throughout the time course of inactivation indicates that both subunits are modified independently and at the same rate. It is apparent that the hybrid-modified dimer contributes one-half of the enzymatic activity of a native dimer in the standard assay. Kinetic studies were performed and the results indicate that there is no apparent change in kinetic parameters between a subunit of the native dimer and the active subunit in the hybrid-modified dimer. Dissociation and reassociation of a mixture of native enzyme and doubly-iodoacetamide-modified enzyme indicates that there is no preferential association of a modified subunit with another modified subunit, or of a native subunit with another native subunit, but rather, association is random with respect to native and iodoacetamide-modified subunits.

Animals↗
PubMed1981

The N-ethylmaleimide-sensitive cysteine residue in the pH-dependent subunit interactions of malate dehydrogenase.

The specific chemical modification by N-ethylmaleimide of a cysteine residue at pH 5.0 in porcine heart mitochondrial malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37) has been shown to result in an enzymatically inactive, monomeric product, which does not reassociate at pH 7.5 to yield the native dimer. In this report, an investigation of proton release and uptake upon NADH binding to the native enzyme and to the N-ethylmaleimide-modified enzyme has implicated the above cysteine residue as being directly linked to the pH-dependent subunit dissociation of mitochondrial malate dehydrogenase. The results are consistent with the view that the modified cysteine residue is not located at the subunit interaction site, although it is probably near this site. A recent study from this laboratory has demonstrated that the monomeric enzyme obtained at pH 5.0 exists in a conformation which is enzymatically inactive and which has an enhanced intrinsic protein fluorescence. Interpretation of protein fluorescence data has suggested that the N-ethylmaleimide modification results in inactivation of the enzyme by preventing the pH-induced conformational change to the active dimer. However, NADH is able to induce reassociation of the N-ethylmaleimide-modified enzyme at pH 7.5 but not at pH 5.0. This reassociation at pH 7.5 is accompanied by a significant regain of enzymatic activity, indicating that NADH binding is able to partially overcome the negative effect of the cysteine modification on the pH-dependent subunit reassociation of mitochondrial malate dehydrogenase.

Animals↗
PubMed1981

Subunit interactions in mitochondrial malate dehydrogenase. Kinetics and mechanism of reassociation.

The pH-dependent dissociation of porcine heart mitochondrial malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37) has been more extensively characterized. The native, dimeric form of the enzyme (Mr = 70,000) which exists at pH 7.5 has previously been shown to dissociate into its constituent subunits (Mr = 35,000) at pH 5.0 (Bleile, D. M., Schulz, R. A., Gregory, E. M., and Harrison, J. H. (1977) J. Biol. Chem. 252, 755-758). The dissociation is accompanied by a concomitant decrease in enzymatic specific activity and an increase in intrinsic protein fluorescence. By using the characteristics of specific activity and intrinsic protein fluorescence as probes of dimerization, the kinetics of subunit reassociation was investigated. In order to facilitate reassociation, a pH jump method was utilized in which enzyme at pH 5.0 was diluted into a large excess of pH 7.5 buffer. The regain of enzymatic specific activity and the decrease in protein fluorescence were observed to follow first order kinetics. The rate constant in both cases was dependent upon the protein concentration, and in all cases, full recovery of either enzymatic activity or native protein fluorescence was obtained. The Arrhenius activation energy for the reassociation of the subunits was found to be approximately 20 kcal/mol, an observation which is consistent with a refolding process whose rate-limiting step may be the cis/trans-isomerization about one or more proline imino bonds. A model for subunit reassociation which is consistent with the kinetic data is proposed.

Animals↗
PubMed1981

The immobilization of mitochondrial malate dehydrogenase on Sepharose beads and the demonstration of catalytically active subunits.

Porcine heart mitochondrial malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37) has been immobilized by covalent attachment to CNBr-activated Sepharose 4B-Cl gel. The gel was activated with low levels of CNBr to produce a low density of linkage sites and, hence, to facilitate linkage of the enzyme through a single subunit. Matrix-bound mitochondrial malate dehydrogenase was found to possess 50-65% of the native mitochondrial malate dehydrogenase specific activity when assayed in the NAD+ leads to NADH direction but only 5-15% of the native enzyme specific activity when assayed in the NADH leads to NAD+ direction. MB-dimeric mitochondrial malate dehydrogenase was dissociated to MB-monomer by exposure to pH 5.0 buffer. The MB-monomer was found to be catalytically active, possessing only a slightly decreased specific activity when compared to MB-dimer. The reconstitution of Mb-monomer to MB-dimer was accomplished by adding dissociated mitochondrial malate dehydrogenase, which exists at pH 5.0, to MB-monomer and adjusting to pH 7.5. The kinetic parameters, pH activity profile, and stability toward heat denaturation for MB-mitochondrial malate dehydrogenase (monomer and dimer) were determined and compared to native mitochondrial malate dehydrogenase. MB-mitochondrial malate dehydrogenase exhibited enhanced stability and similar pH activity profiles when compared to native mitochondrial malate dehydrogenase. Immobilization of mitochondrial malate dehydrogenase altered the enzyme's kinetic parameters in such a manner as to increase the values of Km for the substrates and decrease the values of Vmax.

Animals↗
PubMed1980

Mini-pump: method of diabetic control during minor surgery under general anaesthesia.

A simple method for maintaining diabetic control during and after minor surgery requiring a general anaesthetic was studied in 20 insulin-treated diabetics. Long-acting insulin was omitted on the night before the operation, and a mini-pump delivering insulin at a rate of 0.5 units/h was strapped to the arm early on the morning of the operation regardless of the time of operation. Insulin was infused at this rate throughout the day, the usual evening dose of insulin given and followed by supper, and the pump then stopped. In all 20 patients studied blood glucose concentrations fell steadily throughout the day. Hypoglycaemia did not occur. The mini-pump is simple to use and with the same standard insulin dose for all patients maintains satisfactory blood glucose concentrations throughout the day of operation.

Adolescent↗
PubMed1977

Investigation of the relation of the pH-dependent dissociation of malate dehydrogenase to modification of the enzyme by N-ethylmaleimide.

The pH-dependent dissociation of porcine heart mitochondrial malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37) has been further characterized using the technique of sedimentation velocity ultracentrifugation. The increased rate and specificity of the inactivation of mitochondrial malate dehydrogenase by the sulfhydryl reagent N-ethylmaleimide has been correlated with the pH-dependent dissociation of the enzyme. Data obtained using NAD+ and its component parts to reassociate the enzyme and also to protect the enzyme from inactivation by N-ethylmaleimide suggest that the sulfhydryl residues being modified by N-ethylmaleimide are inaccessible when the enzyme is in its dimeric form. A dissociation curve for the pH-dependent dissociation suggests that a limited number of residues are being protonated concomitant with dissociation of the enzyme. An apparent pKa of 5.3 has been determined for this phenomenon. Studies using enzyme modified by the sulfhydryl reagent N-ethylmaleimide indicate that selective modification of essential sulfhydryl residues alters the proper binding of NADH.

Animals↗
PubMed1977

Investigation of the subunit interactions in malate dehydrogenase.

The dissociations of porcine heart mitochondrial, bovine heart mitochondrial, and porcine heart cytoplasmic malate dehydrogenase dimers (L-malate: NAD+oxidoreductase, EC 1.1.1.37) have been examined by Sephadex G-100 gel filtration chromatography and sedimentation velocity ultracentrifugation. The porcine mitochondrial enzyme was found to chromatograph as subunits when applied to a gel filtration column at a concentration of .02 muM or less at pH 7.0. The presence of coenzymes shifted the dissociation equilibrium at low enzyme concentrations in favor of dimer formation. Monomer formation was also favored when procine mitochondrial enzyme was incubated at pH 5.0 even at concentrations as high as 120 muM. This shift in equilibrium has been correlated with the increased rate and specificity of sulfhydryl residue modification with N-ethylmaleimide at pH 5.0 (Gregory, E.M., Yost, F.J.,Jr., Rohrbach, M.S., and Harrison, J.H. (1971)J. Biol. Chem. 246, 5491-5497). Bovine mitochondrial enzyme did not exhibit a concentration-dependent disociation under the conditions examined. However, at pH5.0 monomer formation was favored, and correlations could again be drawn with sulfhydryl residue modification (Gregory, E.M. (1975)J.Biol. Chem. 250, 5470-5474). In both mitochondrial enzymes, coenzyme binding was found capable of overcoming the effects of pH on the dissociation equilibrium, and dimer formation was favored. Unlike either of the above mentioned enzymes, porcine cytoplasmic malate dehydrogenase did not dissociate into its monomeric form under any conditions investigated.

Animals↗