Low molecular weight GTP-binding proteins in hepatocytes and an assessment of the role of p21ras 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 [alpha-32P]GTP. p21ras proteins could only be detected by immunological analysis. The amounts of p21ras proteins present in isolated hepatocytes and in a highly purified preparation of liver plasma membrane vesicles were 0.3 and 4 ng p21ras protein/micrograms membrane protein, respectively. In comparison with the total cell extract, the degree of enrichment of plasma membrane vesicles with p21ras was similar to that of 5'-nucleotidase. The p21ras proteins were tightly associated with the membrane. Treatment of [3H]choline-labelled plasma membranes with an excess concentration of the anti-p21ras antibody Y13-259 failed to inhibit either basal or guanosine 5'-[gamma-thio]triphosphate (GTP[S])-stimulated [3H]choline release. It is concluded that in hepatocytes (a) the majority of p21ras is bound to the plasma membrane and (b) p21ras is not directly involved in the activation by GTP[S] of phospholipase D.
Hurst
K M
KM
Department of Medical Biochemistry, School of Medicine, Flinders University, Adelaide, South Australia.
Chataway
T K
TK
Hughes
B P
BP
Barritt
G J
GJ
D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002457:Cell Extracts / Q000737:chemistry; D002458:Cell Fractionation; D002462:Cell Membrane / Q000737:chemistry; D002794:Choline / Q000378:metabolism; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D015689:Oncogene Protein p21(ras) / Q000737:chemistry* / Q000276:immunology*; D010739:Phospholipase D / Q000378:metabolism*; D051381:Rats
D000911:Antibodies, Monoclonal; D002457:Cell Extracts; D006160:Guanosine Triphosphate; D010739:Phospholipase D; D019204:GTP-Binding Proteins; D015689:Oncogene Protein p21(ras); D002794:Choline