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D B McCormick

Publications and source records attributed to D B McCormick.

At least 73 records · Page 4Linked to original sources
PubMed1983

Correlation of H2O2 production and liver catalase during riboflavin deficiency and repletion in mammals.

A substantial decrease in liver peroxisomal catalase was found during riboflavin deficiency in rats. This decrease is greater than that found among other hemoproteins and seems to follow decrease in flavin-dependent peroxisomal oxidases. This is not due to a general depression of peroxisomal enzymes, since Cu-dependent urate oxidase activity was not changed. Furthermore, the level of catalase activity as well as flavin-dependent oxidases was restored by riboflavin repletion. These results suggest that hydrogen peroxide, the substrate for catalase produced by several flavoprotein oxidases, induces catalase in mammals as has been indicated for certain bacteria.

Animals↗
PubMed1983

Kinetic properties of pyridoxamine (pyridoxine)-5'-phosphate oxidase from rabbit liver.

The kinetic properties of pyridoxamine (pyridoxine)-5'-phosphate oxidase have been studied using the physiological substrates pyridoxine 5'-phosphate (PNP) and pyridoxamine 5'-phosphate (PMP) at 25 degrees C and pH 8.0. Under steady-state conditions with different concentrations of PNP and O2, a series of parallel lines and competitive substrate inhibition with a KI of 50 microM are seen in double reciprocal plots. This is suggestive of a binary complex mechanism. Secondary plots yield a turnover number of 42 min-1 and Km values for both PNP (8.2 microM) and O2 (182 microM). A large deuterium isotope effect, VH/VD of 6.5, was observed with [4',4'-2H]PNP. In analogous studies using PMP, a turnover number of 6.2 min-1 and respective Km values for PMP and O2 of 3.6 and 85 microM were calculated. No significant substrate inhibition and a small deuterium isotope effect (VH/VD = 1.1) were observed with PMP. Anaerobic stopped flow data showed that the enzyme-bound flavin was reduced at a rate similar to catalytic turnover with PNP as a substrate, whereas with PMP, the rate of enzyme reduction is 500-fold faster than turnover. Stopped flow kinetic data also showed the reduced enzyme to react with O2 at rates at least 10(2)-10(3) faster than turnover. These results indicate that enzyme reduction is rate-limiting when the alcohol form (PNP) is the substrate, but in the case of the amine (PMP), the rate-limiting step in catalysis occurs subsequent to reduction. With PMP as substrate, release of product from the complex with reduced enzyme is 15-fold slower than turnover, and thus, it is suggested that oxygen reacts with the complex. The pH dependence of the deuterium isotope effect and the Km for PMP showed substantial change in the pH range between 6.0 and 7.5, whereas little or no pH dependence was observed for PNP. These data show that the kinetic mechanism of pyridoxamine (pyridoxine)-5'-phosphate oxidase can function via either a binary or ternary complex mechanism, depending upon the nature of the substrate.

Animals↗
PubMed1983

Effect of riboflavin status on hepatic activities of flavin-metabolizing enzymes in rats.

The effect of riboflavin status on hepatic activities of the relatively substrate-specific flavokinase and FAD synthetase and the relatively nonspecific FMN phosphatase and FAD pyrophosphatase was investigated in weanling, male Sprague-Dawley rats fed 0, 5, 15, 30 and 60 micrograms riboflavin/15 g diet for 1, 3 and 5 weeks. Flavokinase activity was determined by using [14C]riboflavin and ATP as substrates and measuring product [14C]FMN after incubation and separation by high performance liquid chromatography. Similarly, FAD synthetase activity was determined by using [3H]ATP and FMN and quantitating [3H]FAD formed. Flavokinase activities among all groups were similar after only 1 week of feeding experimental diets; by 3 weeks, activities were depressed to about 60% of normal in animals that received suboptimal riboflavin; by 5 weeks, activity of rats fed riboflavin-free diet was further decreased to about 40% of normal. FAD synthetase activities were unaffected by riboflavin status at 1 and 3 weeks; however, at 5 weeks, activities were moderately decreased to 85, 65 and 52% of normal with rats which had received 15, 5 and 0 microgram riboflavin/15 g diet, respectively. FMN phosphatase and FAD pyrophosphatase activities decreased with age, but were not influenced by riboflavin status at any period. Overall results indicate the effect of increasing severity of riboflavin deficiency is greater with flavokinase, which is physiologically rate-limiting in the biosynthesis of flavocoenzymes, than with FAD synthetase.

Animals↗
PubMed1983

Uptake of riboflavin by isolated rat liver cells.

The effect of riboflavin status on uptake was investigated in hepatocytes isolated from control, riboflavin-sufficient and riboflavin-deficient rats. The uptake exhibited biphasic characteristics with an initial rapid phase [13.2 +/- 1.8 pmol/(10(6) cells X minute)] for the first couple of minutes followed by a second slower phase which continued for over an hour. The accumulation of riboflavin at near equilibrium conditions was 2.5- and 5.2-fold greater than external concentration in control and riboflavin-deficient cells, respectively. An apparent Km of 12 +/- 1.3 microM and Vmax of 82.3 +/- 9.1 pmol/(10(6) cells X minute) were obtained for control and riboflavin-sufficient rats while a similar Km but higher Vmax were obtained with deficient animals. Correspondence of the Km to that of flavokinase for riboflavin suggested the possibility that uptake of the vitamin may occur via metabolic trapping, i.e., phosphorylation. As substantiation of this, the rate of uptake was decreased by lumiflavin and 2'-hydroxyethylflavin, which are competitive inhibitors, and by 7,8-dichloroflavin, a substrate for flavokinase. Furthermore, the uptake was found to be temperature-dependent and studies with carbonylcyanide-p-trifluoro-methoxyphenylhydrazone (FCCP) and ethionine indicated a requirement for ATP. These results showed that overall, entry of riboflavin into hepatocytes occurs predominantly by a facilitated diffusion process followed by rapid trapping by flavokinase-catalyzed phosphorylation to FMN.

Adenosine Triphosphate↗
PubMed1983

Purification and general characterization of FAD synthetase from rat liver.

Flavin adenine dinucleotide synthetase (ATP:FMN adenylyltransferase, EC 2.7.7.2) has been enriched more extensively than previously from fresh rat liver. For this, 10% homogenates in sucrose-phosphate buffer were treated with 0.1% Tween-20 prior to high-speed centrifugation to obtain soluble proteins. Those precipitated by 40% saturation with ammonium sulfate were subjected to stepwise addition of calcium phosphate gel to remove pyrophosphatase, and the remaining synthetase was further enriched by passage through a tricalcium phosphate column. An apparent yield of greater than 70% and purification over 70-fold was achieved from the high-speed supernatant fraction. The synthetase activity in solution at 4 degrees was largely lost within a week unless protected by thiols which could partly restore inactivated enzyme. The pH optimum for synthetase activity is near 7.7 when assayed with suitable concentrations of FMN, ATP, and Mg2+. Purified enzyme could be separated into lower (140,000) and higher (325,000) molecular weight components when subjected to molecular sieving on a Sephadex G-200 column.

Animals↗
PubMed1982

Distribution and properties of flavokinase in the developing chick embryo.

1. Localization of flavokinase was studied in the nonembryonic portions of the chicken egg, in embryos at various stages of development, and in the adult hen. 2. Embryonic biosynthesis of flavin nomonucleotide (FMN) increases with increasing protein synthesis. 3. In the embryo, higher activity of flavokinase was observed in intestine, heart, liver, and yolk sac. 4. Lower activity was observed in allantoic membrane and fluid. 5. No significant activity was measured in yolk. 6. Both specific and total flavokinase activities in organs of the more aerobic and metabolically active adult chicken are considerably greater than those of embryo organs. 7. Flavokinase from embryonic liver was enriched by affinity chromatography by using N-10-flavinyl agarose. 8. The partially purified enzyme has properties generally similar to other animal flavokinases.

Animals↗
PubMed1982

Factors affecting riboflavin requirements of oral contraceptive users and nonusers.

Riboflavin depletion has been identified in women on oral contraceptives (OC) but change in riboflavin nutriture has not been consistently demonstrated in all OC user groups studied. Discrepant findings in reports have been attributed to differences of pill formulation or riboflavin intake. Aims of this study were to compare the riboflavin requirements of healthy OC users and nonusers on diets prepared in a metabolic unit. A single daily menu and meal pattern was used. The basic diet providing riboflavin at a level of 0.6 mg/1000 kcal was used in the period of acclimation and period 1. In periods 2 and 3, the riboflavin content of the diet was increased to 0.8 and 1.0 mg/1000 kcal, respectively. The riboflavin status of subjects was monitored by erythrocyte glutathione reductase assay and urinary riboflavin excretion. Eight women on OC and 10 nonusers participated. Erythrocyte glutathione reductase assay values and urinary riboflavin excretion showed intersubject and interperiod differences but no significant group differences (OC versus non-OC) in erythrocyte glutathione reductase values or in urinary riboflavin per g creatinine. It was concluded that when dietary intake is controlled, OC do not significantly influence riboflavin status. Riboflavin needs were related to energy requirements of the subjects.

Adult↗
PubMed1980

Spectroscopic studies of complexes between pyridoxamine (pyridoxine)-5'-phosphate oxidase and pyridoxyl 5'-phosphate compounds differing at position 4'.

The absorbance spectrum of pyridoxamine (pyridoxine)-5'-phosphate oxidase (EC 1.4.3.5) is altered upon the binding of pyridoxal 5'-phosphate or analogs with different substituents at position 4'. The absorbance difference spectra are similar for complexes of the oxidase and pyridoxal 5'-phosphate, 4'-desoxypyridoxine 5'-phosphate, and 4-ethynyl-4-deformylpyridoxal 5'-phosphate; hence, these perturb the flavoprotein absorbance by similar interactions primarily involving the pyridoxyl 5'-phosphate moiety, and not specifically the 4-formyl group or other relatively small and uncharged functions at this position. A different type of spectral perturbation is caused by analogs with larger substituents at position 4' (i.e. 4'-methoxypyridoxine 5'-phosphate, 4-methyl-vinyl-4-deformylpyridoxal 5'-phosphate, and pyridoxal 5'-phosphate oxime and hydrazone). These analogs impose bulky groups in a region of the active site that critically influences the environment of the flavin, and, thus, may reflect positioning of this portion of the substrates close to the flavin ring, as is required for their redox interaction.

Animals↗
PubMed1980

Affinity chromatographic purification and properties of flavokinase (ATP:riboflavin 5'-phosphotransferase) from rat liver.

Flavokinase (ATP:riboflavin 5'-phosphotransferase, EC 2.7.1.26) has been purified to apparent homogeneity from rat liver by affinity chromatography using flavinyl agarose beads (agarose-OCH2CONH(CH2)2NHCO(CH2)/N10-7,8-dimethylisoalloxazine). The specific activity of the pure enzyme is 9,900 units (nmol of FMN formed/h at 37 degrees C)/mg of protein, and reflects a one-step, 7000-fold purification. Flavokinase thus obtained, unlike previous preparations from mammalian sources, is free from contaminating phosphatase and FAD synthase. The purified enzyme rapidly loses activity upon storage but is stabilized by riboflavin and thiol-protecting reagents. The apparent molecular weight, estimated by gel filtration on Sephadex G-100 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is 28,000 +/- 1,000. Flavokinase phosphorylates and/or is inhibited by a large number of riboflavin analogs; however, the physiologically important 8 alpha-(amino acid)riboflavins are poorly accommodated. The strongly preferred phosphate donors are ATP and dATP. Both Zn2+ and Mg2+, as well as several other divalent cations, activate flavokinase, but Zn2+ yields greatest activity (1.8 times that with Mg2+). The pH optimum for activity with either Zn2+ or Mg2+ is approximately 9.3; at pH 7.0, the activity is 40% of that at the pH optimum.

Animals↗