Open AccessGTP‐blot Analysis of Small GTP‐binding Proteins
Author(s) -
Klinz FranzJosef
Publication year - 1994
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1994.00099.x
Subject(s) - gtp' , recombinant dna , western blot , biochemistry , cytosol , g protein , blot , gtpase , microbiology and biotechnology , chemistry , binding protein , gtp binding protein regulators , biology , receptor , enzyme , gene
Recombinant c‐Ha‐ras, ralA and rap2, but not rap1A or rap1B proteins retained their ability to bind [α‐ 32 P] GTP after SDS/PAGE and transfer to nitrocellulose. Recombinant c‐Ha‐ras missing the C‐terminal 23 amino acid residues failed to bind [α‐ 32 P] GTP after the blot, and the ability of recombinant ralA missing the C‐terminal 28 amino acid residues to bind [α‐ 32 P] GTP was decreased manyfold. The presence of nonionic detergents of the polyoxyethylene type such as Tween 20, Triton X‐100, Nonidet P40 or Lubrol PX in the incubation buffer was necessary to induce renaturation of blotted recombinant c‐Ha‐ras protein, whereas other types of detergents were ineffective. We propose that detergents of the polyoxyethylene type induce the refolding of some types of blotted small GTP‐binding proteins and that the C‐terminus is involved in the refolding process. Membranes from NIH3T3 fibroblasts overexpressing c‐Ha‐ras protein showed much weaker binding of [α‐ 32 P] GTP as expected from the level of ras immunoreactivity. Treatment of fibroblasts with lovastatin, an inhibitor of hydroxymethylglutaryl‐coenzyme A reductase, caused the accumulation of the unfarnesylated form of c‐Ha‐ras in the cytosol. Examination of [α‐ 32 P] GTP‐binding and immunoreactivity for cytosolic and membrane‐bound c‐Ha‐ras revealed that binding of [α‐ 32 P] GTP to unprocessed c‐Ha‐ras was increased about threefold compared to the same amount of processed c‐Ha‐ras. Our results demonstrate that detection and quantification of small GTP‐binding proteins in eukaryotic cells by GTP‐blot analysis is hampered by the fact that these proteins differ strongly in their ability to renature after blotting to nitrocellulose.