VITAMINS

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The singlet oxygen (^1O_2) deactivating rate constants (k_s) of α-tocopherol (α-Toc) and carotenoids such as β-carotene, astaxanthin and canthaxanthin and their inhibiting activities for ^1O_2-dependentlipid peroxidation (IC_50' that is, the concentration required for 50% inhibition of lipid peroxidation) were investigated in liposomes and compared to those in EtOH solution. Liposomes were prepared from dimyristoylphosphatidylcholine to measure the k_s and egg yolk phosphatidylcholine to measure the IC_50 values. ^1O_2 was generated by photoirradiation using three photosensitizers, Rose bengal and methylene blue, which generate ^1O_2 at the membrane surface or in bulk water, and 2-(1-pyrene) dodecanoic acid, which generates ^1O_2 in the membrane hydrophobic inner region. The k_s and IC_50 values were determined in membranes considering the following two points: (1) antioxidants exist in membranes and their active moieties are distributed in different regions of membranes at different concentrations; (2) dynamics of ^1O_2 in membranes (generation site of ^1O_2 depending on the localization of photosensitizer in membranes, and solubility of ^1O_2 which differs in the site of membranes). In conclusion, the following three factors are experimentally confirmed to be important for the consideration of ^1O_2 deactivating activities of α-Toc and carotenoids in membranes:(1) the concentration of antioxidants (EtOH solution < membrane), especially the local concentration of their active moieties in membranes (in the case of OH-group of α-Toc, 0%, 50-60%, and 40-50% at polar zone, hydrogen belt, and hydrophobic core, respectively, of the membranes); (2) the mobility of antioxidants (EtOH solution > membrane), the mobility of membrane phospholipid (liquid crystalline state > gel state), the mobility of antioxidants in membranes (antioxidants located at one half of the bilayer membrane such as α-tocopherol > antioxidants located across the bilayer such as β-carotene; antioxidants interactive with polar head group of lipid in membrane such as astaxanthin < antioxidants nointeractive with polar head group of lipid in membrane such as β-carotene), and the local mobility of their active moieties (in case of OH-group of α-Toc, membrane surface < membrane inner region); (3) the dielectric constant (micropolarity) at the domains in membranes, that is higher in membrane surface than in membrane inner region, where the active moieties of antioxidants deactivate ^1O_2 (in case of α-Toc, reactivity is high in the solution with high dielectric constant).

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In order to obtain information on NAD biosynthetic and metabolic pathways of Euglena, we investigated the effects of NAD precursors on growth and various enzyme activities in Euglena gracilis Z. Among the NAD precursors, dihydroxyacetone phosphate had no effect on growth of Euglena gracilis Z cultured heterotrophically at a concentration of 5 mM, while NAD content increased about 1.3 fold. We found that quinolinic acid was biosynthesized from aspartic acid (Asp) and dihydroxyacetone phosphate in Euglena gracilis Z, suggesting that NAD may be biosynthesized via Asp-pathway. Among enzymes related to NAD biosynthesis and metabolism, nicotinamidase, nicotinate phosphoribosyltransferase, nicotinic acid mononucleotide adenyltransferase, and NAD synthetase activities were all found in Euglena gracilis Z. In contrast, activities of quinolinate phosphoribosyltransferase, nicotinamide mononucleotide adenyltransferase, nicotinamide phosphoribosyltransferase, nicotinate methyltransferase, and nicotinamide methyltransferase were barely or little detected. These results suggest that NAD may be biosynthesized in a salvage pathway via nicotinic acid. Nicotinate phosphoribosyltransferase was inhibited by nicotinic acid mononucleotide and nicotinic acid adenine dinucleotide, whereas nicotinamidase by nicotinamide mononucleotide, NAD, and N^1-methylnicotinamide. We suggest that Euglena had its own unique regulatory mechanism in the biosynthesis and metabolism of NAD.

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