PMID: 2508629Sep 1, 1989Paper

Detection of GTP-binding proteins in purified derivatives of rough endoplasmic reticulum

The Biochemical Journal
J LanoixJ Paiement

Abstract

As a first step in determining the molecular mechanism of membrane fusion stimulated by GTP in rough endoplasmic reticulum (RER), we have looked for GTP-binding proteins. Rough microsomes from rat liver were treated for the release of ribosomes, and the membrane proteins were separated by SDS/polyacrylamide-gel electrophoresis. The polypeptides were then blotted on to nitrocellulose sheets and incubated with [alpha-32P]GTP [Bhullar & Haslam (1987) Biochem. J. 245, 617-620]. A doublet of polypeptides (23 and 24 kDa) was detected in the presence of 2 microM-MgCl2. Binding of [alpha-32P]GTP was blocked by 1-5 mM-EDTA, 10-10,000 nM-GTP or 10 microM-GDP. Either guanosine 5'-[gamma-thio]triphosphate or guanosine 5'-[beta gamma-imido]triphosphate at 100 nM completely inhibited binding, but ATP, CTP or UTP at 10 mciroM did not. Pretreatment of microsomes by mild trypsin treatment (0.5-10 micrograms of trypsin/ml, concentrations known not to affect microsomal permeability) led to inhibition of [alpha-32P]GTP binding, suggesting a cytosolic membrane orientation for the GTP-binding proteins. Two-dimensional gel-electrophoretic analysis revealed the 23 and 24 kDa [alpha-32P]GTP-binding proteins to have similar acid isoelectric points. [alp...Continue Reading

Citations

Jan 30, 1991·European Journal of Biochemistry·P J Van HaastertC Erneux
May 4, 2011·Progress in Histochemistry and Cytochemistry·Christine LavoieJacques Paiement
Oct 1, 1995·European Journal of Clinical Investigation·J SteinW F Caspary
Aug 30, 1991·Biochemical and Biophysical Research Communications·J Lanoix, J Paiement
Apr 1, 1990·Molecular Reproduction and Development·M J MironJ Paiement
Nov 1, 1992·Pharmacology & Therapeutics·L MissiaenR Casteels
Aug 10, 1992·Biochimica Et Biophysica Acta·K VerheydenG P Mannaerts
May 15, 1991·Biochemical and Biophysical Research Communications·J PaiementL Roy
Jan 15, 1991·Biochemical and Biophysical Research Communications·J M LenhardP D Stahl

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