Purification and characterization from bovine brain cytosol of a protein that inhibits the dissociation of GDP from and the subsequent binding of GTP to smg p25A, a ras p21-like GTP-binding protein.

T Sasaki,A Kikuchi,S Araki,Y Hata,M Isomura,S Kuroda,Y Takai

doi:10.1016/s0021-9258(19)39980-6

Abstract

A novel regulatory protein for smg p25A, a ras p21-like GTP-binding protein, was purified to near homogeneity from bovine brain cytosol. This regulatory protein, designated here as smg p25A GDP dissociation inhibitor (GDI), inhibited the dissociation of GDP, but not of guanosine 5'-(3-O-thio)triphosphate (GTPgamma S), from smg p25A. smg p25A GDI also inhibited the binding of GTPgamma S to the GDP-bound form of smg p25A but not of that to the guanine nucleotide-free form. GDI did not stimulate the GTPase activity of smg p25A and by itself showed neither GTPgammaS-binding nor GTPase activity. GDI was inactive for other ras p21/ras p21-like GTP-binding proteins including c-Ha-ras p21, rhoB p20, and smg p21. The Mr value of GDI was estimated to be about 54,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, about 65,000 from the S value (4.5 S), and about 82,000 by gel filtration. The isoelectric point of GDI was about pH 5.6. The activities of GDI were killed by tryptic digestion or heat boiling. These results indicate that bovine brain cytosol contains a regulatory protein for smg p25A that inhibits the dissociation of GDP from and thereby the subsequent binding of GTP to this protein.

Highlights

Like GTP-binding protein, was purified to near homogeneity from bovine brain cytosol. This regulatory protein, designated here as smg p25A GDP dissociation inhibitor (GDI), inhibited the dissociation of GDP, but not of guanosine 5’-(3-0-thio)triphosphate (GTP-yS), from smg p25A. smg p25A GDI inhibited the binding of GTPrS to the GDP-bound form of smg p25A
Containing 0.3 M NaCl, smg p25A GDI was eluted by 800 ml of Buffer A containing 0.3 M NaCl
The sample was centrifuged at 20,000 x g for 20 min. smg p25A GDI was mostly recovered in the supernatant, while GTPase-activating proteins for c-Ha-ros ~21 and smg p21 were mostly recovered in the precipitate as described [14]

Summary

Introduction

Like GTP-binding protein, was purified to near homogeneity from bovine brain cytosol. This regulatory protein, designated here as smg p25A GDP dissociation inhibitor (GDI), inhibited the dissociation of GDP, but not of guanosine 5’-(3-0-thio)triphosphate (GTP-yS), from smg p25A. Ras p2 l-like GTP-binding proteins including c-Ha-r-us p2 1, rhoB ~20, and smg p2 1. The activities of GDI were killed by tryptic digestion or heat boiling. These results indicate that bovine brain cytosol contains a regulatory protein for smg p25A that inhibits the dissociation of GDP from and thereby the subsequent binding of GTP to this protein. Smg p25 family belongs to this superfamily and is composed of three highly homologous members, smg p25A, -B, and -C [3, 4]. smg p25A was first isolated from bovine brain membranes and characterized [3]. smg p25A is composed of 220 amino acids with a calculated M, of 24,954 [4]. smg p25A has Culture of Japan (1989), grants-in-aid for Abnormalities

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Conclusion