Hirosuke Fukuda

Immobilization of glycolysis system of yeasts and production of cytidine diphosphate choline

Glycolysis system of yeast was successfully immobilized into a derivative of polyethylene glycol hydroxyethylacrylate. The immobilized system could produce ATP and then phosphorylate nucleotides (CMP). The CTP thus formed was effectively converted to CDP-choline in the same system (Fig.2).

Transfer of mitochondria into protoplasts of saccharomyces cerevisiae by mini-protoplast fusion

Protoplast fusion has become a valuable method in the study of yeast genetics and biochemistry. Intraspecific, interspecific and intergeneric fusion of yeast protoplasts has been accomplished [l-8]. Isolated mitochondria have also been transferred into yeast protoplasts [9,10], although with very low efficiency of transformation (-10V8). To raise this efficiency, the mini-protoplast method was devised, by which mitochondria contained in mini-protoplasts (protoplasts lacking nuclei) were transferred into protoplasts of a respiratory-deficient (p”) strain to yield respiratory-sufficient cells (A. Ma&z, personal communication). Mini-protoplasts can be obtained by treating log-phase cells with lytic enzyme [ 111. In the log phase, yeast cells are dividing, and the migration of mitochondria into buds or daughter cells precedes that of the nuclei. We have conducted intraspecific mitochondrial transfer experiments as the first step in investigating through intergeneric transfer the inhibitory effect of mitochondria on expression of the hexokinase isozyme gene in Hansenula jadinii, which we found in the analysis of fermentative production of CDPcholine [12-141.

Some properties of an immobilized glycolysis system of yeast in fermentative phosphorylation of nucleotides

SummaryImmobilized dried yeast cells, which contain glycolytic and some other emzymes, required NAD but not ATP for the (e.g. choline kinase, pyrophosphorylase) fermentative production of CDP-choline, when washed and reused. The immobilized system was more resistant to heat than dried cells, which had previously been used for the same purpose. However, when too many cells were immobilized, leakage of the enzymes from the resin lattice was observed during repeated use. To prevent this leakage, the ratio of cells to resin should be considered.

Transfer of mitochondria of Hansenula wingei into protoplasts of Saccharomyces cerevisiae by mini-protoplast fusion

Intraspecific transfer of mitochondria has been reported with isolated mitochondria, although the efficiency of the transformation was very low (-1 O-“) [ 11. mini-protoplasts containing mitochondria, but not nuclei, intraspecific transfer of mitochondria was also achieved in [2]. This study presents data on the intergeneric transfer of mitochondria from Hansen&a wingei into protoplasts of Saccharomyces cerevisiae using the miniprotoplast method. Mini-protoplasts were prepared by treating log-phase cells of H. wingei with a lytic enzyme (zymolyase). In these dividing cells, migration of mitochondria into buds or daughter cells precedes that of the nuclei. After separation, the mini-protoplasts were fused with the normal protoplasts prepared from a respiration-deficient (p”) strain of S. cerevisiae. Seven fusants were isolated and two of them were shown to be haploid cells of S. cerevisiae to which only mitochondria, but not nuclei had been transferred from H. wingei.

Effect of acriflavine on the hexokinase isozyme pattern of a yeast, Hansenula jadinii

Dried cells of a yeast, Hansenula jadinii, that had been cultured aerobically with acriflavine, contained three hexokinase isozymes and metabolized glucose at 0.6 M to produce ATP to phosphorylate nucleotides in the presence of a high concentration of phosphate. Dried cells cultured aerobically without acriflavine contained two hexokinase isozymes and could not metabolize glucose under the same conditions. Two of the isozymes of the yeast cultured with acriflavine were similar to isozymes of the yeast cultured without acriflavine. However, the third isozyme was resistant to a high phosphate concentration and caused regeneration of ATP through glycolysis and phosphorylation of nucleotides.

Controlling mechanism of ATP regeneration by glycolytic pathway in a yeast: Hansenula jadinii

SummaryThe regulatory mechanism of ATP regeneration by the glycolytic pathway in Hansenula jadinii cells was investigated by analyzing the initial stage of CDP-choline fermentation. As a result, the “on-off” of ATP regeneration was found to be determined by the ATP concentration overcoming the inhibitory effect of phosphate buffer on hexokinase activity. The concentration of ATP at the initial stage of fermentation was greatly influenced by the kinds and amounts of glycogen in cells. Based on these results, the regulatory mechanism of ATP regeneration by the glycolytic pathway is discussed in detail.

Action of 5-(2-thienyl)valeric acid as a biotin antagonist.

5-(2-Thienyl)valeric acid (TVA), a biotin analogue which can be easily prepared through chemical process, inhibited the growth of a biotin synthesizing Rhodotorula glutinis. The growth inhibition was reversed by the addition of biotin. Among biotin intermediates, dethiobiotin and 7,8-diaminopelargonic acid reversed the inhibition by TVA, while 7-keto-8-amino-pelargonic acid and pimelic acid did not. From these results, it was concluded that TVA is a biotin antagonist which probably acts as an inhibitor of biotin biosynthesis.