Studies on ras proteins. Catalytic properties of normal and activated ras proteins purified in the absence of protein denaturants.
Normal (Gly12) and activated (Val12) Ha-ras proteins were produced in Escherichia coli, and purified to an apparent homogeneity without using any protein denaturants. The purified proteins contained an equimolar amount of GDP. They were stable in the presence of 5 mM Mg2+ and 25% (v/v) glycerol when incubated at 60 degrees C for 5 min. The binding of GDP to the protein was greatly stabilized by Mg2+. In the presence of 10 mM Mg2+, the bound GDP hardly exchanged with external guanine nucleotides, even at 30 degrees C. The exchange reaction was markedly enhanced in the presence of 10 mM EDTA or 120 mM ammonium sulfate. The rate-limiting step of the exchange reaction was the dissociation of the bound GDP from the ras protein, and this step was facilitated 40- to 100-fold by the addition of EDTA or ammonium sulfate. The dissociation rate of the normal (Gly12) ras protein was 2- to 3-fold faster than that of the activated (Val12) protein. The dissociation constants (Kd) for GDP of the normal and activated ras proteins were 1.2 X 10(-8) and 3.1 X 10(-9) M, respectively. The overall turnover rate of GTPase activity of the normal ras protein (10.8 mmol.mol-1.min-1) was about 10-fold higher than that of the activated protein (1.1 mmol.mol-1.min-1) in the absence of Mg2+ (less than 10(-8) M).
Satoh
T
T
Institute of Medical Science, University of Tokyo, Japan.
Nakamura
S
S
Nakafuku
M
M
Kaziro
Y
Y
D001483:Base Sequence; D004355:Drug Stability; D004926:Escherichia coli / Q000235:genetics; D020558:GTP Phosphohydrolases / Q000378:metabolism; D011905:Genes, ras; D005822:Genetic Vectors; D005998:Glycine; D006801:Humans; D007700:Kinetics; D010957:Plasmids; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras); D011519:Proto-Oncogenes; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism*; D013816:Thermodynamics; D014633:Valine
D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D020558:GTP Phosphohydrolases; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras); D014633:Valine; D005998:Glycine