NADP(+)-dependent D-arabinose dehydrogenase shows a limited contribution to erythroascorbic acid biosynthesis and oxidative stress resistance in Saccharomyces cerevisiae.

The molecular aspects and physiological significance of NADP(+)-dependent D-arabinose dehydrogenase (ARA), which is thought to function in the biosynthesis of an analog of ascorbic acid, D-erythroascorbic acid in yeasts, were examined. A large subunit of ARA, Ara1p produced in E. coli, was purified as a homodimer, some of which was degraded at the N-terminus. It showed sufficient ARA activity. Degradation of Ara1p occurs naturally in yeast cells, and the small subunit of ARA previously thought as is, in fact, a naturally occuring degradation product of Ara1p. A deficient mutant of ARA1 lost almost all NADP(+)-ARA activity, but intracellular D-erythroascorbic acid was only halved. This mutant showed increased susceptibility to H(2)O(2) and diamide but not to menadione or tert-butylhydroperoxide. Feeding D-arabinose to mutant cells led to increases in intracellular D-erythroascorbic acid, suggesting the presence of another ARA isozyme. The deficient mutant of ARA1 recovered resistance to H(2)O(2) with feeding of D-arabinose. Our results suggest that the direct contributions of Ara1p both to D-erythroascorbic acid biosynthesis and to oxidative stress resistance are quite limited.

Heterologous expression of dehydroascorbate reductase from rice and its application to determination of dehydroascorbate concentrations.

We constructed an expression vector for rice dehydroascorbate reductase (DHAR) (EC 1.8.5.4) with a polyhistidine tag at the amino terminus and introduced the vector into several strains of Escherichia coli. On conventional induction treatment with isopropylthiol-beta-D-galactoside, E. coli harboring rice DHAR cDNA produced doublet polypeptides of about 27 kDa. Induction duration or growth temperature did not affect the ratio of these polypeptides. Only the larger polypeptide, corresponding to full-length recombinant DHAR, was produced in E. coli supplemented with tRNAs for several minor codons. Most of the enzymatic characteristics of the recombinant DHAR were similar to those of the native one, although the recombinant protein showed increased heat susceptibility. Using recombinant DHAR, we developed a method for simple and precise determination of dehydroascorbate concentrations in tissue extracts by spectrophotometry, and we successfully applied the method to several fruit juices and vegetables.

Gender-related differences in mouse hepatic ethanol metabolism.

The effect of an acute oral load of 2 g ethanol/kg body weight was studied in a group of male and female 10-wk-old C3H/HeNCrj (C3H/He) mice to investigate gender change throughout differences of the hepatic ethanol metabolism of mice. The following parameters were measured in the serum from 0 h to 3 h after the start of the experiment: ethanol, acetaldehyde, and acetate. Their concentrations in the serum in female mice tended to show lower levels than in male mice. In female mice, the concentration of ethanol at 1 h and the concentration of acetate at 1 h, 2 h, and 3 h after ethanol administration showed significantly lower levels than in male mice. Ten-week-old male and female C3H/He mice were subcutaneously injected 50 microg/kg body weight beta-estradiol and 1.45 mmol/kg body weight testosterone propionate (testosterone) in olive oil, respectively, and changes in the activity of enzymes related to the hepatic ethanol metabolism of mice were examined at 24 h after the administration of sex hormones. The activity of the cytosolic alcohol dehydrogenase (ADH) and microsomal aniline hydroxylase (ANH) and the low Km, high Km and total aldehyde dehydrogenase (AlDH) activities in the mitochondrial, the cytosolic, and the microsomal fraction of the liver were higher. Moreover, the density of the band of CYP2E1 in the microsome in female mice was stronger than in male mice, and in the microsomal fraction of the liver, the total content of cytochrome P-450 (CYP) and ethoxyresorufin O-dealkylase (EROD) activity in male mice showed significantly higher values than in female mice. The density of the band of CYP2E1 and the three activities of AlDH in the hepatic mitochondrial fraction of male mice increased significantly under treatment with beta-estradiol. The three activities of AlDH of the cytosolic fraction of the liver in female mice significantly decreased under treatment with testosterone. The present findings suggested that in C3H/He mice livers, the rate of ethanol metabolism is faster in females than in males, and the enzymes related to ethanol metabolism are controlled by testosterone or beta-estradiol. It is suggested that ethanol and its metabolite disappear faster from the serum of female mice than from the serum of male mice because the activities of hepatic enzymes related to ethanol metabolism are higher in female mice than in male mice. C3H/He mice, hepatic ethanol metabolism, gender different, ADH, AlDH