Characterization of the interactions between the small GTPase RhoA and its guanine nucleotide exchange factors.
A novel spectrophotometric method to study the kinetics of the guanine nucleotide exchange factors-catalyzed reactions is presented. The method incorporates two coupling enzyme systems: (a). GTPase-activating protein which stimulates the intrinsic GTP hydrolysis reaction of small GTPases and (b). purine nucleotide phosphorylase and its chromophoric substrate, 7-methyl-6-thioguanosine, for quantitation of the resultant inorganic phosphate. The continuous coupled enzyme system was used for characterization of the interactions between the small GTPase RhoA and its guanine nucleotide exchange factors, Lbc and Dbl. Kinetic parameters obtained here show that there is no significant difference in kinetic mechanism of these GEFs in interaction with RhoA. The Michaelis-Menten constants were determined to be around 1micro M, and the rate constants k(cat) were around 0.1s(-1).
Tan
Ying-Cai
YC
National Laboratory of Biomacromolecules, Center for Molecular Biology, Institute of Biophysics, Academia Sinica, Beijing 100101, People's Republic of China.
Wu
Hao
H
Wang
Wei-Ning
WN
Zheng
Yi
Y
Wang
Zhi-Xin
ZX
D054758:A Kinase Anchor Proteins; D048868:Adaptor Proteins, Signal Transducing; D002384:Catalysis; D004926:Escherichia coli / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors; D006151:Guanosine / Q000031:analogs & derivatives* / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D015636:Magnesium Chloride / Q000737:chemistry / Q000378:metabolism; D015778:Minor Histocompatibility Antigens; D010710:Phosphates / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000378:metabolism*; D011683:Purine-Nucleoside Phosphorylase / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013872:Thionucleosides / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000737:chemistry* / Q000378:metabolism*
D054758:A Kinase Anchor Proteins; C085467:AKAP13 protein, human; D048868:Adaptor Proteins, Signal Transducing; D020662:Guanine Nucleotide Exchange Factors; C057811:MCF2 protein, human; D015778:Minor Histocompatibility Antigens; D010710:Phosphates; D011518:Proto-Oncogene Proteins; D013872:Thionucleosides; D015636:Magnesium Chloride; D006151:Guanosine; C074696:6-mercapto-7-methylguanosine; D011683:Purine-Nucleoside Phosphorylase; D020742:rhoA GTP-Binding Protein