Low molecular weight GTP-binding proteins in hepatocytes and an assessment of the role of p21(ras) proteins in the activation of phospholipase D

A GTP-binding protein with an apparent molecular weight of 25 kDa was detected in hepatocyte extracts using SDS-PAGE and [α-³²P]GTP. p21(ras) proteins could only be detected by immunological analysis. The amounts of p21(ras) proteins present in isolated hepatocytes and in a highly purified preparation of liver plasma membrane vesicles were 0.3 and 4 ng p21(ras) protein/μg membrane protein, respectively. In comparison with the total cell extract, the degree of enrichment of plasma membrane vesicles with p21(ras) was similar to that of 5'-nucleotidase. The p21(ras) proteins were tightly associated with the membrane. Treatment of [³H]choline-labelled plasma membranes with an excess concentration of the anti-p21(ras) antibody Y13-259 failed to inhibit either basal or guanosine 5'[γ-thio]triphosphate (GTP[S])-stimulated [³H]choline release. It is concluded that in hepatocytes (a) the majority of p21(ras) is bound to the plasma membrane and (b) p21(ras) is not directly involved in the activation by GTP[S] of phospholipase D.