Physical and Functional Association of Cytosolic Inositol-phospholipid-Specific Phospholipase C of Calf Thymocytes with a GTP-Binding Protein

Abstract

A soluble inositolphospholipid-specific phospholipase C (PI-phospholipase C) has been purified 5, 800-fold from the cytosolic fraction of calf thymocytes. The puri-fication was achieved by sequential column chromatographies on DEAE-Sepharose CL-6B, heparin-Sepharose CL-6B, Sephacryl S-300, Mono S, and Superose 12, followed by column chromatography on Sephadex G-100 in the presence of 1% sodium cholate. The enzyme thus purified was found to be homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The mo-lecular weight of the enzyme was estimated to be 68 kDa by SDS-PAGE. The enzyme is specific for inositol phospholipids. Phosphatidylinositol and phospha-tidylinositol 4, 5-bisphosphate (PIP₂) were hydrolyzed, but phosphatidylcholine and phosphatidylethanolamine were not affected by the enzyme. GTPγS-binding activity was detected in the enzyme fractions after all the purification steps, but not in the final enzyme preparation. The PI-phospholipase C and GTPγS-binding activities in the partially purified enzyme preparation could be separated by the column chro-matography on Sephadex G-100 only in the presence of 1 % sodium cholate. Thus, the soluble PI-phospholipase C has affinity to a GTP-binding protein. SDS-PAGE of the GTP-binding fractions eluted from the Sephadex G-100 column gave three visible bands of 54, 41, and 27 kDa. The 54 kDa polypeptide was specifically ADP-ribosylated by pertussis toxin. Furthermore, it was found that GTP and GTPγS (10μM and 1 mM) could enhance the PIP₂ hydrolysis activity of the partially purified enzyme in the presence of 3 mM EGTA, but the purified enzyme after separation from the GTP-binding activity was not affected by GTP and GTPγS. The soluble PI-phospholipase C of calf thymocytes may be not only physically but also functionally associated with a GTP-binding protein.