Kinetics of interaction of nucleotides with nucleotide-free H-ras p21

✍ Scribed by John, Jacob; Sohmen, Roland; Feuerstein, Juergen; Linke, Rosita; Wittinghofer, Alfred; Goody, Roger S.

• Book ID: 121348467
• Publisher: American Chemical Society
• Year: 1990
• Tongue: English
• Weight: 1014 KB
• Volume: 29
• Category: Article
• ISSN: 0006-2960
• DOI: 10.1021/bi00477a025

✦ Synopsis

A method is described for the convenient preparation of substantial quantities of nucleotide-free p21 or of 1:l complexes with nucleotides other than GDP. The nucleotide-free protein has been used for kinetic studies of the binding of G D P and GTP, making use of the fluorescent analogues 3'-(methylanthraniloyl)-2'-deoxy-GDP and -GTP. Stopped-flow studies have led to the formulation of a two-step binding ' Abbreviations: p21, protein product of the human c-Ha-ras protooncogene; GTP(B,y-CH,), guanosine S-(&y-methylenetriphosphate); GPPNP, guanosine S-(P,y-imidotriphosphate); mantdGDP/GTP or mdGDP/GTP, 3'-O-(N-methylanthraniloyl)-2'-deoxyguanosine di/triphosphate; EDTA, ethylenediaminetetraacetic acid; DTE, dithioerythritol.