GTP-BLOT ANALYSIS OF SMALL GTP-BINDING PROTEINS - THE C-TERMINUS IS INVOLVED IN RENATURATION OF BLOTTED PROTEINS
GTP-BLOT ANALYSIS OF SMALL GTP-BINDING PROTEINS - THE C-TERMINUS IS INVOLVED IN RENATURATION OF BLOTTED PROTEINS
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DOI：
10.1111/j.1432-1033.1994.00099.x
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发表时间：
1994-10-01
期刊：
影响因子：
--
通讯作者：
KLINZ, FJ
中科院分区：
文献类型：
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作者：
KLINZ, FJ
Recombinant c-Ha-ras, ralA and rap2, but not raplA or raplB proteins retained their ability to bind [alpha-P-32]GTP after SDS/PAGE and transfer to nitrocellulose. Recombinant c-Ha-ras missing the C-terminal 23 amino acid residues failed to bind [alpha-P-32]GTP after the blot, and the ability of recombinant ralA missing the C-terminal 28 amino acid residues to bind [alpha-P-32]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 [alpha-P-32]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 [alpha-P-32]GTP-binding and immunoreactivity for cytosolic and membrane-bound c-Ha-ras revealed that binding of [alpha(-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.
