Characterization of multiple acid phosphatases in bovine liver cytosol and lysosome. Inactivation of cytosolic enzymes by disulfides and its redox regulation by thioltransferase.
Cytosolic and lysosomal acid phosphatases have the ability to hydrolyze orthophosphoric monoesters below pH 5-6. However, it is thought they may have different intracellular roles. To clarify their properties, substrate specificity, inhibitor sensitivity and the modulation of enzyme by redox conditions were determined using bovine liver enzymes. DEAE-cellulose chromatography following (NH4)2SO4 fractionation revealed three forms of cytosolic acid phosphatases as in the KCl gradient (0-500 mM). After Sephadex G-75 gel filtration, the enzymes appeared as single bands on SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Their activities for D-erythrose 4-phosphate co-purified with p-nitrophenylphosphatase activities in all steps. In contrast the lysosomal enzyme was purified by Octyl-Sepharose column chromatography after n-butanol treatment, (NH4)2SO4 fractionation, Bio gel P-200 gel filtration and DE-52 chromatography. The relative molecular masses (M(r)) determined by SDS-PAGE indicated that M(r) of the cytosolic enzymes (16,000) was less that of lysosomal enzyme (160,000). The cytosolic enzymes were active against sugar phosphates and were inhibited by 1 mM Cu2+. In addition, the cytosolic enzymes were inactivated by 5 mM oxidized glutathione and protected by 10 mM reduced glutathione (in the presence or absence of thioltransferase), suggesting that sensitive cysteinyl residue(s) existed. The lysosomal enzyme was active against various substrates and was strongly inhibited by 1 mM Cu2+ and 2 mM fluoride. The results presented here suggest that cytosolic enzymes have different properties from those of lysosomal enzyme with respect to substrates, inhibitors and regulation of activity.
Terada
T
T
Faculty of Pharmaceutical Sciences, Osaka University, Japan.
D000135:Acid Phosphatase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D003600:Cytosol / Q000201:enzymology*; D004220:Disulfides / Q000378:metabolism; D054477:Glutaredoxins; D005978:Glutathione / Q000378:metabolism; D019803:Glutathione Disulfide / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism*; D008099:Liver / Q000201:enzymology*; D008247:Lysosomes / Q000201:enzymology*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism; D011490:Protein Disulfide Reductase (Glutathione)
D004220:Disulfides; D054477:Glutaredoxins; D007527:Isoenzymes; D010088:Oxidoreductases; D011490:Protein Disulfide Reductase (Glutathione); D000135:Acid Phosphatase; D005978:Glutathione; D019803:Glutathione Disulfide