Dehydroascorbic acid transport by GLUT4 in Xenopus oocytes and isolated rat adipocytes.

Dehydroascorbic acid (DHA), the first stable oxidation product of vitamin C, was transported by GLUT1 and GLUT3 in Xenopus laevis oocytes with transport rates similar to that of 2-deoxyglucose (2-DG), but due to inherent difficulties with GLUT4 expression in oocytes it was uncertain whether GLUT4 transported DHA (Rumsey, S. C. , Kwon, O., Xu, G. W., Burant, C. F., Simpson, I., and Levine, M. (1997) J. Biol. Chem. 272, 18982-18989). We therefore studied DHA and 2-DG transport in rat adipocytes, which express GLUT4. Without insulin, rat adipocytes transported 2-DG 2-3-fold faster than DHA. Preincubation with insulin (0.67 micrometer) increased transport of each substrate similarly: 7-10-fold for 2-DG and 6-8-fold for DHA. Because intracellular reduction of DHA in adipocytes was complete before and after insulin stimulation, increased transport of DHA was not explained by increased internal reduction of DHA to ascorbate. To determine apparent transport kinetics of GLUT4 for DHA, GLUT4 expression in Xenopus oocytes was reexamined. Preincubation of oocytes for >4 h with insulin (1 micrometer) augmented GLUT4 transport of 2-DG and DHA by up to 5-fold. Transport of both substrates was inhibited by cytochalasin B and displayed saturable kinetics. GLUT4 had a higher apparent transport affinity (K(m) of 0.98 versus 5.2 mm) and lower maximal transport rate (V(max) of 66 versus 880 pmol/oocyte/10 min) for DHA compared with 2-DG. The lower transport rate for DHA could not be explained by binding differences at the outer membrane face, as shown by inhibition with ethylidene glucose, or by transporter trans-activation and therefore was probably due to substrate-specific differences in transporter/substrate translocation or release. These novel data indicate that the insulin-sensitive transporter GLUT4 transports DHA in both rat adipocytes and Xenopus oocytes. Alterations of this mechanism in diabetes could have clinical implications for ascorbate utilization.

Cloning and functional characterization of the human sodium-dependent vitamin C transporters hSVCT1 and hSVCT2.

Two sodium-dependent vitamin C transporters, hSVCT1 and hSVCT2, were cloned from a human kidney cDNA library. hSVCT1 had a 1797 bp open reading frame encoding a 598 amino acid polypeptide. The 1953 bp open reading frame of hSVCT2 encoded a 650 amino acid polypeptide. Using a Xenopus laevis oocyte expression system, both transporters were functionally expressed. By Eadie-Hofstee transformation the apparent K(m) of hSVCT1 for ascorbate was 252.0 microM and of hSVCT2 for ascorbate was 21.3 microM. Both transporters were sodium-dependent and did not transport dehydroascorbic acid. Incubation of oocytes expressing either transporter with phorbol 12-myristate 13-acetate (PMA) inhibited ascorbate transport activity. Availability of the human transporter clones may facilitate new strategies for determining vitamin C intake.

Criteria and recommendations for vitamin C intake.

Recommendations for vitamin C intake are under revision by the Food and Nutrition Board of the National Academy of Sciences. Since 1989 when the last recommended dietary allowance (RDA) of 60 mg was published, extensive biochemical, molecular, epidemiologic, and clinical data have become available. New recommendations can be based on the following 9 criteria: dietary availability, steady-state concentrations in plasma in relationship to dose, steady-state concentrations in tissues in relationship to dose, bioavailability, urine excretion, adverse effects, biochemical and molecular function in relationship to vitamin concentration, direct beneficial effects and epidemiologic observations in relationship to dose, and prevention of deficiency. We applied these criteria to the Food and Nutrition Board's new guidelines, the Dietary Reference Intakes, which include 4 reference values. The estimated average requirement (EAR) is the amount of nutrient estimated to meet the requirement of half the healthy individuals in a life-stage and gender group. Based on an EAR of 100 mg/d of vitamin C, the RDA is proposed to be 120 mg/d. If the EAR cannot be determined, an adequate intake (AI) amount is recommended instead of an RDA. The AI was estimated to be either 200 mg/d from 5 servings of fruits and vegetables or 100 mg/d of vitamin C to prevent deficiency with a margin of safety. The final classification, the tolerable upper intake level, is the highest daily level of nutrient intake that does not pose risk or adverse health effects to almost all individuals in the population. This amount is proposed to be less than 1 g of vitamin C daily. Physicians can tell patients that 5 servings of fruits and vegetables per day may be beneficial in preventing cancer and providing sufficient vitamin C intake for healthy people, and that 1 g or more of vitamin C may have adverse consequences in some people.

Menaquinone (vitamin K2) biosynthesis: overexpression, purification, and characterization of a new isochorismate synthase from Escherichia coli.

The first committed step in the biosynthesis of menaquinone (vitamin K2) is the conversion of chorismate to isochorismate, which is mediated by an isochorismate synthase encoded by the menF gene. This isochorismate synthase (MenF) is distinct from the entC-encoded isochorismate synthase (EntC) involved in enterobactin biosynthesis. MenF has been overexpressed under the influence of the T7 promoter and purified to homogeneity. The purified protein was found to have a molecular mass of 98 kDa as determined by gel filtration column chromatography on Sephacryl S-200. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a molecular mass of 48 kDa. Thus, the enzyme is a homodimer. The purified enzyme showed a pH optimum of 7.5 to 8.0 and a temperature optimum of 37 degrees C. The enzyme carries out the irreversible conversion of chorismate to isochorismate in the presence of Mg2+. The enzyme was found to have a Km of 195 +/- 23 microM and a k(cat) of 80 min(-1). In the presence of 30 mM beta-mercaptoethanol (BME), the k(cat) increased to 176 min(-1). The reducing agents BME and dithiothreitol stimulated the enzymatic activity more than twofold. Treatment of the enzyme with the cysteine-specific modifying reagent N-ethylmaleimide (NEM) resulted in the complete loss of activity. Preincubation of the enzyme with the substrate, chorismate, before NEM treatment resulted in complete protection of the enzyme from inactivation.

A new isochorismate synthase specifically involved in menaquinone (vitamin K2) biosynthesis encoded by the menF gene.

A new gene (menF) encoding an isochorismate synthase specifically involved in menaquinone (vitamin K2) biosynthesis has been cloned and sequenced. Overexpression of the encoded polypeptide under the influence of a T7 promoter showed an increase in specific activity of 2200-fold. Treatment with protamine sulfate resulted in another 3.5-fold increase in specific activity (7700-fold compared to the parent strain). The relative molecular mass of the overexpressed protein was M(r) 49 000, which is in full agreement with the DNA sequence predicted molecular mass of 48 777 Da. Purified enzyme converted chorismate to isochorismate with the product of the reaction shown to be isochorismate by its thermal conversion to salicylic acid. The fluorescence spectrum generated by the formed salicylic acid was identical to that of authentic salicylic acid. The 5' end of the flanking menD gene has also be redefined.

Dimethyl sulfoxide reductase is not required for trimethylamine N-oxide reduction in Escherichia coli.

Deletion mutants of Escherichia coli lacking dimethyl sulfoxide (DMSO) reductase activity and consequently unable to utilize DMSO as an electron acceptor for anaerobic growth have been isolated. These mutants retained the ability to use trimethylamine N-oxide (TMAO) as an electron acceptor and the TMAO reductase activity was found to be unaltered. Heating the cell-free extract of the wild-type strain at 70 degrees C for 15 min selectively inactivated the DMSO reductase activity while the TMAO reductase activity remained unchanged for at least 1 h.