Chromatographic characteristics and subcellular localization of synthase phosphatase, phosphorylase phosphatase and histone phosphatase in human polymorphonuclear leukocytes.
Synthase phosphatase, phosphorylase phosphatase and histone phosphatase activity in a leukocyte homogenate were found to have different sedimentation characteristics: both synthase phosphatase and phosphorylase phosphatase activity are associated with the microsomal fraction, while the majority of histone phosphatase activity (75-85%) was found in the cytosol. Synthase phosphatase, phosphorylase phosphatase and histone phosphatase activities accompanying the microsomal fraction are readily solubilized by 0.3% Triton X-100. When the solubilized microsomal enzymes were chromatographed on Sephadex G-200, the majority of synthase phosphatase, phosphorylase phosphatase and histone phosphatase activity migrated in single peaks corresponding to apparent molecular weights of 380 000, 250 000 and 68 000, respectively. A minor peak of 30 000, which had phosphatase activity against all three substrates was also obtained. Ethanol treatment resulted in solubilization and dissociation of the three phosphatase activities. It was found that although ethanol treatment resulted in a 4-fold increase of phosphorylase phosphatase activity, histone phosphatase activity was decreased (by 60%), while synthase phosphatase activity remained stable. Similar results were obtained when ethanol treatment was performed on the 17 000 X g supernatant. Chromatography of the ethanol-treated microsomes (or homogenate) on Sephadex G-200 showed that the phosphatase activity towards synthase D, phosphorylase a and phosphohistone coincided a Mr 30 000 species. Heat treatment of the Mr 30 000 peak resulted in dissociation of synthase phosphatase and phosphorylase phosphatase activity. Synthase phosphatase was inhibited by phosphorylase a in a kinetically non-competitive manner while histone phosphatase activity was not inhibited by synthase D (8.5 unit/ml) or by phosphorylase a (12 unit/ml).
D002451:Cell Compartmentation; D002850:Chromatography, Gel; D000431:Ethanol / Q000494:pharmacology; D006007:Glycogen-Synthase-D Phosphatase / Q000378:metabolism*; D006358:Hot Temperature; D006801:Humans; D008861:Microsomes / Q000201:enzymology; D008970:Molecular Weight; D009504:Neutrophils / Q000201:enzymology* / Q000648:ultrastructure; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010765:Phosphorylase Phosphatase / Q000378:metabolism*; D012995:Solubility; D013379:Substrate Specificity; D014357:Trypsin / Q000494:pharmacology
D000431:Ethanol; D010749:Phosphoprotein Phosphatases; D010765:Phosphorylase Phosphatase; D006007:Glycogen-Synthase-D Phosphatase; D014357:Trypsin