EFFECTS OF GELSOLIN ON HUMAN PLATELET CYTOSOLIC PHOSPHOINOSITIDE-PHOSPHOLIPASE-C ISOZYMES

The effective resolution of human platelet cytosolic phosphoinositide- phospholipase C (PLC) revealed five distinct activity peaks by Q-Sepha rose and heparin-Sepharose column chromatographies when assayed using phosphatidylinositol (PI) and phosphatidylinositol 4,5-bisphosphate (P IP2). The results of Western blotting analysis with various antibodies against PLC isozymes showed that peak-Ia (PLC-delta-type), peak-Ib (P LC-gamma(1) type), and peak-IIc (PLC-beta-type) and two unidentified a ctivity peaks (PLC-IIa and PLC-IIb) were present in human platelet cyt osol. A protein with guanosine 5'-3-O-(thio)triphosphate-binding activ ity was coeluted with the PLC-IIa and was purified to homogeneity. It exhibited 86- and 42-kDa polypeptide bands upon sodium dodecyl sulfate -polyacrylamide gel electrophoresis, which were identified as gelsolin and actin by immunostaining, respectively. Large amounts of gelsolin/ actin (1:1) complex "gelsolin complex" were detected in the PLC-delta and PLC-gamma(1) fractions. The PLC-gamma(1) and the gelsolin complex were coimmunoprecipitated by the antibody raised against PLC-gamma(1). Furthermore, the partially purified bovine brain PLC-gamma(1) fractio n also was found to be associated with the gelsolin complex and the as sociation was released by the addition of 1% sodium cholate.This findi ng has prompted us to examine effects of the gelsolin complex and the free gelsolin on activities of the above PLC isoforms from platelet cy tosol. The gelsolin complex did not affect the PIP2 hydrolyzing activi ties of all PLC isoforms. In contrast, the purified gelsolin inhibited distinctly PIP2 hydrolyses by PLC-Ia (delta), PLC-Ib (gamma(1)), and PLC-IIa (unidentified), whereas the inhibitory effects for PLC-IIb (un identified) and PLC-IIc (beta) were moderate. The inhibitory effect of gelsolin on PIP2-hydrolysis by PLC-gamma(1) was diminished by a large amount of PIP2 substrate. These results suggested that the inhibition of PLC by gelsolin is due to sequestration of substrate PIP2 by its c ompetitive binding.