Biochemical properties and excretion behavior of repressible acid phosphatases with altered subunit composition.

Yeast repressible acid phosphatase (rAP) is the oligomeric extracellular enzyme encoded by the three structural genes PH05 (p60), PHO10 (p58) and PHO11 (p56). We examined the ability of acid phosphatases formed by various subunit combinations to be excreted into the medium. Plasmids with repressible acid phosphatase structural genes under control of the yeast glyceraldehyde-phosphate dehydrogenase (GAP) promoter were constructed to obtain constitutive expression of acid phosphatase, and yeast strains with disruptions in PHO5, PHO10 and PHO11, respectively, were used to generate mutants expressing single genes or specific gene combinations. EndoF treatment of acid phosphatases, produced by these strains, followed by SDS-electrophoresis in combination with densitometry techniques revealed that the ratio p60/(p56 + p58) among structural polypeptides in extracellular enzyme is constant and equals to 6.0. A study of acid phosphatases formed by single type subunits was undertaken. Expression products of PHO5, PHO10 and PHO11 genes were isolated from the culture medium. The specific activities of the enzymes were found to be 33, 2 and 2 mM x mg-1 x min-1, respectively. The values of Mr estimated by HPLC chromatography for the enzymes encoded for by the genes PHO5, PHO10 and PHO11 and SDS-polyacrilamide gel electrophoresis data suggested an oligomeric organisation of the enzymes. Isoelectric focusing in polyacrylamide gel with immobilised pH gradient followed by activity staining yielded numerous sharp bands of homopolymeric acid phosphatases forms being different in their pI. The kinetic characterisation of the enzymes revealed differences in Km values, sensitivity to temperature inactivation, inhibition by orthophosphate and the effect of pH on the enzyme activity.

[Analysis of the quaternary structure of secreted repressible acid phosphatase from the yeast Saccharomyces cerevisiae]. / Analiz chetvertichnoi struktury sekretiruemoi repressibel'noi kisloi fosfatazy drozhzhei Saccharomyces cerevisiae.

The structural organization of extracellular repressible acid phosphatase from S. cerevisiae has been studied. The existence of multiple acid phosphatase forms with isoelectric points at pH 4.1-4.8 has been confirmed by isoelectrofocusing. The molecular masses of three acid phosphatase isoforms (56, 57-59, and 60 kDa) obtained after enzymatic deglycosylation correlate with the data obtained previously during the analysis of translation products in cell-free systems. Electron microscopic studies revealed that the acid phosphatase molecule has a square shape and is made up of four identical subunits with molecular masses of about 125 kDa.

[Possible existence of alternative secretion routes of acid phosphatase in the yeast Saccharomyces cerevisiae]. / O vozmozhnosti sushchestvovaniia razlichnykh putei sekretsii kisloi fosfatazy u drozhzhei Saccharomyces cerevisiae.

The secretion of different acid phosphatase forms into the growth medium was studied in the yeast Saccharomyces cerevisiae. The secretion of constitutive acid phosphatase highly correlated with the process of bud formation. This exoenzyme might be involved in the construction of the bud cell wall. Analysis of the rate, at which repressible acid phosphatase was excreted into the growth medium, as well as a negative correlation between its secretion and the process of bud formation were indicative of an additional route via which the repressible form of acid phosphatase was secreted. It is possible that yeast cells contain vesicles similar to "safe" vesicles in higher eukaryotic cells.