Purification of a glycosyl-phosphatidylinositol-specific phospholipase D from human plasma.
Mammalian plasma contains a phospholipase D, which is specific for the glycosyl-phosphatidylinositol anchor found on many eukaryotic cell surface proteins (Davitz, M. A., Hereld, D., Shak, S., Krakow, J., Englund, P. T., and Nussenzweig, V. (1987) Science 238, 81-84; Low, M. G., and Prasad, A. R. S. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 980-984; Cardoso de Almeida, M. L., Turner, M. J., Stambuk, B. V., and Schenkman, S. (1988) Biochem. Biophys. Res. Commun. 150, 476-482). We have purified this phospholipase D to homogeneity by a four-step procedure involving a Mono Q and phenyl-5PW columns, followed by wheat germ lectin affinity chromatography and finally another Mono Q column. A 4,500-fold purification was achieved with a 5% yield. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the homogeneous enzyme has a Mr of 110,000 and appears to consist of a single polypeptide chain. It exhibits identical substrate specificity as compared with the crude preparation, is active over a broad pH range (4.0-8.5), inhibited by the thiol-blocking agent p-chloromercuriphenylsulfonic acid and by 1,10-phenanthroline, and is partially heat-labile.
Department of Pathology, New York University School of Medicine, New York 10016.
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Schenkman
D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D006801:Humans; D008970:Molecular Weight; D010716:Phosphatidylinositols; D010739:Phospholipase D / Q000097:blood* / Q000302:isolation & purification; D010740:Phospholipases / Q000097:blood*; D013379:Substrate Specificity
D010716:Phosphatidylinositols; D010740:Phospholipases; D010739:Phospholipase D