The genomic DNA sequence of the widely studied yeast Saccharomyces cerevisiae, which is a model organism for eukaryotic cells, contains three NAD kinase homologues, namely, Utr1p, Pos5p and Yel041wp [1][2][3]. NAD kinase (EC 2.7.1.23) catalyses NAD phosphorylation by using phosphoryl donors (ATP or inorganic polyphosphate [poly(P)]), constituting the last step of the NADP biosynthetic pathway [4,5] ATP-NAD kinase phosphorylates NAD to produce NADP by using ATP, whereas ATP-NADH kinase phosphorylates both NAD and NADH. Three NAD kinase homologues, namely, ATP-NAD kinase (Utr1p), ATP-NADH kinase (Pos5p) and function-unknown Yel041wp (Yef1p), are found in the yeast Saccharomyces cerevisiae. In this study, Yef1p was identified as an ATP-NADH kinase. The ATP-NADH kinase activity of Utr1p was also confirmed. Thus, the three NAD kinase homologues were biochemically identified as ATP-NADH kinases. The phenotypic analysis of the single, double and triple mutants, which was unexpectedly found to be viable, for UTR1, YEF1 and POS5 demonstrated the critical contribution of Pos5p to mitochondrial function and survival at 37°C and the critical contribution of Utr1p to growth in low iron medium. The contributions of the other two enzymes were also demonstrated; however, these were observed only in the absence of the critical contributor, which was supported by complementation for some pos5 phenotypes by the overexpression of UTR1 and YEF1. The viability of the triple mutant suggested that a 'novel' enzyme, whose primary structure is different from those of all known NAD and NADH kinases, probably catalyses the formation of cytosolic NADP in S. cerevisiae. Finally, we found that LEU2 of Candida glabrata, encoding b-isopropylmalate dehydrogenase and being used to construct the triple mutant, complemented some pos5 phenotypes; however, overexpression of LEU2 of S. cerevisiae did not. The complementation was putatively attributed to an ability of Leu2p of C. glabrata to use NADP as a coenzyme and to supply NADPH.Abbreviations CgLEU2, LEU2 of yeast Candida glabrata; FOA, 5-fluoroorotic acid; GFP, green fluorescent protein; KNDE, 10 mM potassium phosphate, pH 7.0, containing 0.1 mM NAD, 0.5 mM dithiothreitol and 1.0 mM EDTA; poly(P), inorganic polyphosphate; ScLEU2, LEU2 of yeast Saccharomyces cerevisiae; SD, synthetic dextrose; SG, synthetic glycerol; SD+FOA+Ura, synthetic dextrose ⁄ 5-fluoroorotic acid ⁄ uracil; WT, wild type; YPD, yeast extract ⁄ peptone ⁄ dextrose; YPG, yeast extract ⁄ peptone ⁄ glycerol.

Transformation is an indispensable method for the manipulation of Saccharomyces cerevisiae cell. The spf1 cell, in which the gene encoding an endoplasmic reticulum-located P-type ATPase is deleted, has been known to show the high-transformation phenotype. In this study, fluorescent microscopic observation of transformation process of S. cerevisiae using plasmid DNA labelled with fluorescent DNA probe, YOYO-1, suggested that the spf1 cell absorbed more plasmid DNA on cellular surface than did the wild-type cell and the unwashed cell did more plasmid DNA than the washed cell. The amounts of the absorbed DNA correlated with the transformation efficiency (number of transformants per μg plasmid DNA) and frequency (transformation efficiency per viable cell number). The high-transformation phenotype of spf1 cell and the effect of heat shock, which effectively induces the transformation of intact cell, disappeared upon cell wall digestion. Electron microscopic observation of the transformation process using negatively charged Nanogold as a mimic of plasmid DNA supported the result obtained using YOYO-1 and implied that plasmid DNA enters into cell together with membrane structure. These data strongly suggest that during the transformation of intact cell, plasmid DNA is initially absorbed on the cell wall, passes through the cell wall with the aid of heat shock, reaches to the membrane, and enters into the cell together with the membrane structure and that the capacity of the cell wall to absorb DNA is at least one of the determinants of transformation efficiency and frequency.

Intact cells of the yeast Saccharomyces cerevisiae were transformed with exogenous DNA by incubating the cells with plasmid in the presence of polyethylene glycol (PEG), which has been shown to be required, although the underlying has not been elucidated. In this study, we found that incubation of the S. cerevisiae cells with PEG was not only required for the PEG-dependent transformation but also enhanced transformation, suggesting that PEG might cause an intracellular response. To understand the response, microarray and metabolome analyses were conducted. We found that incubation of the cells without PEG caused up-regulation of several genes, including those which are involved in carbon source metabolisms, e.g. fatty acid metabolism, yielding acetyl-CoA and those involved in stress-response. Contrary to this, incubation of the cells with PEG gave no transcriptional change. These microarray data were supported by the results of metabolome analysis for anionic metabolites, implying that the physical effect of PEG on cell membrane, rather than the effect of PEG itself on the intracellular response, could cause high transformation in the PEG-dependent transformation.

The Zimmerman reaction was found to be adaptable for the assay of prostaglandin synthetase, which is capable of catalyzing the conversion of arachidonic acid into 1 lcr-l5a-dihydroxy-9-oxo-5-cis,l3-truns-prostadienoic acid. The bovine seminal vesicle microsomes possess active prostaglandin synthetase activity when a heat-labile, nondialyzable inhibitor, present in the supernatant fraction was re-moved. Various parameters affecting the rate of this biosynthetic reaction have been quantitatively defined. To achieve maximal rate, both GSH and a cofactor must be present. The following compounds were found to be suitable cofactors: p-aminophenol, hydroquinone, L-norepinephrine, L-epinephrine, serotonin, and 5-hydroxyindolacetic acid. The p H optimum of this reaction depends on the specific coenzyme used.

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