Biochemical characteristics of a rice (Oryza sativa L., IR36) G-protein alpha-subunit expressed in Escherichia coli.
A cDNA encoding the alpha-subunit of the heterotrimeric G-protein in rice (RGA1) was overexpressed in Escherichia coli and then isolated by Ni2+-nitrilotriacetic acid affinity chromatography. The molecular mass of RGA1 bearing a His tag was approx. 49 kDa. Immunoblot analysis using anti-RGA1 revealed that the RGA1 protein is most abundant in seedling leaves and least abundant in mature roots. It exists at particularly high levels in the immature embryo after pellicle extrusion. In addition, the RGA1 antiserum exhibited a difference in binding affinity for Galpha proteins from monocots (maize and rice) and dicots (Arabidopsis, pea, soya bean and tomato); whereas it cross-reacted with Galpha proteins of monocots, it did not with those of dicot plants. When bound to guanosine 5'-(gamma-thio)triphosphate (GTP[S]), the RGA1 protein was partially protected from tryptic proteolysis. In the presence of GTP[S], trypsin cleaved the RGA1 protein into four fragments 24, 14, 11 and 5 kDa in size. When RGA1 was bound to GDP, only the 5 kDa polypeptide was seen on SDS/PAGE after trypsin digestion. Photoaffinity labelling with [alpha-32P]GTP and a GTP[S]-binding assay revealed that RGA1 incorporated 32P and showed specific binding to a guanine nucleotide. Guanidine binding of RGA1 was affected by the concentration of MgCl2 (maximum at 2 mM). The rate of guanine nucleotide binding of RGA1 (kon,GTP[S]=0.0141+/-0.0014 min-1) and, at steady state, the kcat value for GTP hydrolysis (0.0075+/-0.0001 min-1) were very low even at 2 mM MgCl2. The binding affinity for the nucleotides examined was in the order GTP-S- >/= GTP > GDP > CTP > ATP >/= dTTP.
Seo
H S
HS
Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Chinju 660-701, Korea.
Choi
C H
CH
Lee
S Y
SY
Cho
M J
MJ
Bahk
J D
JD
D000345:Affinity Labels; D001667:Binding, Competitive; D003001:Cloning, Molecular; D004926:Escherichia coli; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D020690:GTPase-Activating Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D015151:Immunoblotting; D007700:Kinetics; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism; D012275:Oryza / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D016324:Sequence Tagged Sites; D013379:Substrate Specificity
D000345:Affinity Labels; D020690:GTPase-Activating Proteins; D046911:Macromolecular Substances; D008565:Membrane Proteins; D011994:Recombinant Proteins; C085379:rho GTPase-activating protein; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins