Reference "Biochemical characteristics of a rice (Oryza sativa L., IR36) G-protein alpha-subunit expressed in Escherichia coli"

Reference ID

3985

Title

Biochemical characteristics of a rice (Oryza sativa L., IR36) G-protein alpha-subunit expressed in Escherichia coli

Source

Biochemical Journal, 1997, vol. 324, pp. 273-281

Authors (5)

Seo-H-S	Choi-C-H
Lee-S-Y	Cho-M-J
Bahk-J-D

Abstract

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.