Phospholipids are major components of cellular membranes that participate in a range of cellular processes. Phosphatidic acid (PA) is a key molecule in the phospholipid biosynthetic pathway. In Saccharomyces cerevisiae, SLC1 has been identified as the gene encoding lysophosphatidic acid acyltransferase, which catalyzes PA synthesis. However, despite the importance of PA, disruption of SLC1 does not affect cell viability (Nagiec, M. M., Wells, G. B., Lester, R. L., and Dickson, R. C. (1993) J. Biol. Chem. 268, 22156 -22163). We originally aimed to identify the acetyl-CoA:lyso platelet-activating factor acetyltransferase (lysoPAF AT) gene in yeast. Screening of a complete set of yeast deletion clones (4741 homozygous diploid clones) revealed a single mutant strain, YOR175c, with a defect in lysoPAF AT activity. YOR175c has been predicted to be a member of the membrane-bound O-acyltransferase superfamily, and we designated the gene LPT1. An Lpt1-green fluorescent protein fusion protein localized at the endoplasmic reticulum. Other than lysoPAF AT activity, Lpt1 catalyzed acyltransferase activity with a wide variety of lysophospholipids as acceptors, including lysophosphatidic acid, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylglycerol, lysophosphatidylinositol, and lysophosphatidylserine. A liquid chromatography-mass spectrometry analysis indicated that lysophosphatidylcholine and lysophosphatidylethanolamine accumulated in the ⌬lpt1 mutant strain. Although the ⌬lpt1 mutant strain did not show other detectable defects, the ⌬lpt1 ⌬slc1 double mutant strain had a synthetic lethal phenotype. These results indicate that, in concert with Slc1, Lpt1 plays a central role in PA biosynthesis, which is essential for cell viability.

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The antioxidant activities of several extracts from Susabinori (Porphyra yezoensis) were measured by the ferric thiocyanate method and the thiobarbituric acid method. The methanol, acetone, ethyl acetate, and hexane extracts, and the chloroform-soluble and water-soluble fractions from the chloroform-methanol extract exhibited higher activities than α-tocopherol. The hot water extract showed little activity. Thinlayer chromatography analysis of the active extracts suggested the existence of several antioxidants. The activity of the chloroform soluble fraction was due to chlorophyll analogs. A strong antioxidant was isolated from the methanol extract, accompanied by several amino acids such as leucine and phenylalanine. This compound was identified as usujilene, a kind of mycosporine-glycine like amino acid.Paper no. J8916 in JAOCS 76, 649-653 (May 1999).Seaweeds, such as laver and kelp, are commonly eaten in Japan and their high dietary fiber and vitamin content have stimulated their evaluation as health foods. In addition, seaweeds contain high levels of polyunsaturated fatty acids (1). Although polyunsaturated fatty acids are very easily oxidized, seaweeds are known to resist oxidation during storage. Fujimoto et al.(2) reported the antioxidant effect of 21 species of marine algae and found that the phospholipid fraction in arame (Eisenia bucyclis) showed efficient activities. Tutour (3) described that brown algae had strong antioxidant activity and that they synergistically enhanced the effect of vitamin E. Furthermore, Nishibori et al. (4) identified pheophytin a, one of the chlorophyll analogs, as a major antioxidant in Aonori (Enteromorpha sp.). In this paper, we describe the antioxidant effect of several extracts from Susabinori (Porphyra yezoensis), one of laver, and the isolation of antioxidants from the methanol extract. EXPERIMENTAL PROCEDURESMaterials and extraction. Cultured P. yezoensis was purchased from Akashiura Fisherman's Union (Hyogo, Japan) in February 1995. A part (10 g each) of the ground freeze-dried material was extracted independently with 200 mL of n-hexane, ethyl acetate, acetone, chloroform/methanol (2:1), methanol, and hot water (90°C) under stirring. Each extract was filtered and concentrated in vacuo. Water was added to the chloroform/methanol extract and then partitioned with chloroform to afford a chloroform-soluble fraction and an aqueous fraction. Antioxidative assay. (i) Ferric thiocyanate method (FTC). The method of Kikuzaki et al. (5) was slightly modified. To a mixture of 2 mg of the fractions of the Susabinori extract, α-tocopherol or isolated compounds in 2 mL of 99.5% ethanol in a vial (φ = 35, h = 80 mm), 2.05 mL of 2.51% linoleic acid in 99.5% ethanol, 4 ml of a 0.05 M phosphate buffer (pH 7.0), and 1.95 mL of water were added. The solution including linoleic acid without any antioxidant or extract was used as control. The vial was placed in an oven at 40°C in the dark. At each test time, to 0.1 mL of this sample solution were added 9.7 mL of 75% ethanol and 0.1 mL of 3...

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To examine the antioxidative compounds of nonprotein amino acids in a red alga, Porphyra yezoensis, an ethanol extract of the cultured thalli was fractionated with Dowex columns. The basic fraction V showed strong antioxidative capacities with ferric thiocyanate and TBARS measurements. In this basic fraction, histidine, 3-methylhistidine, carnosine, and anserine were detected beside ornithine, lysine, and arginine by amino acid analysis. The occurrence of carnosine and anserine suggests that the histidine-related compounds also contribute to the antioxidative reactions in P. yezoensis.

y-Glutamyltranspeptidase was purified ca. 15,200-fold from cell-free extracts of Proteus mirabiis to electrophoretic homogeneity and then crystallized. The enzyme has an estimated molecular weight of 80,000 and consists of two different subunits with molecular weights of ca. 47,000 and 28,000. The purified enzyme catalyzed hydrolysis and transpeptidation of various y-glutamyl compounds, including the oxidized and reduced forms of glutathione, y-glutamyl compounds of L-phenylalanine, L-tyrosine, L-histidine, L-aaminobutyrate, L-leucine, and p-nitroaniline. Glycylglycine, L-phenylalanine, L-methionine, L-histidine, Ltryptophan, and L-isoleucine were good acceptors of the y-glutamyl moiety in the transpeptidation reaction. Km values for-y-glutamyl compounds were on the order of 10-4 to 10-5 M, and those for acceptor peptides and amino acids were on the order of 10-2 to 10-3 M. The enzyme was inhibited by L-serine plus borate and 6diazo-5-oxo-L-norleucine, which are inhibitors of y-glutamyltranspeptidases isolated from mammals. Various amino acids alone were found to inhibit the transpeptidation competitively with a y-glutamyl donor. Kinetic analysis suggested that the reaction sequence of substrate binding and product release proceeds according to a ping pong bi bi mechanism.

Glutathione leaked from cells of Proteus mirabilis grown in medium containing an inhibitor of-yglutamyltranspeptidase. In medium containing 100 mM L-serine and borate, up to 300 ,uM glutathione accumulated. L-Serine in the medium was consumed during the logarithmic phase of growth,-y-glutamyltranspeptidase activity was restored, and glutathione decreased in the medium. In the presence of 2 mM 6diazo-5-oxo-L-norleucine, cells increased normally,-y-glutamyltranspeptidase was inhibited completely, and the maximum concentration of glutathione which accumulated in the medium was 20 ,uM. The glutathione content of cells rose before leakage began. Glutathione leaked from intact cells of other bacteria, although to a lesser extent than was seen with P. mirabilis.