Molecular cloning of a cDNA for a small GTP binding protein, BRho, from the embryo of Bombyx mori and its characterization after expression and purification.
A cDNA clone encoding a small GTP binding protein (Brho) was isolated from an embryonic cDNA library of Bombyx mori that encoded a polypeptide with 202 amino acids sharing 60-80% similarity with the Rho1 family of GTP binding proteins. The effector site and one of the guanine nucleotide binding sites differed from other members of the Rho family. To characterize the biochemical properties of Brho, the clone was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. The recombinant protein was purified to homogeneity with glutathione S-Sepharose. The fusion protein bound [(35)S] GTPgammaS and [(3)H] GDP with association constants of 11x10(6) M(-1) and 6.2x10(6) M(-1), respectively. The binding of [(35)S] GTPgammaS was inhibited by GTP and GDP, but by no other nucleotides. The calculated GTP-hydrolysis activity was 89.6 m mol/min/mol of Brho. Bound [(35)S] GTPgammaS and [(3)H] GDP were exchanged with GTPgammaS most efficiently in the presence of 6 mM MgCl(2). These results suggest that Brho has a higher affinity for GTP than GDP, converts from the GTP-bound state into the GDP-bound state by intrinsic GTP hydrolytic activity, and returns to the GTP-bound state with the exchange of GDP with GTP. Arch.
Uno
T
T
Laboratory of Biological Chemistry, Department of Biofunctional Chemistry, Faculty of Agriculture, Kobe University, Nada-Ku, Kobe, Hyogo, Japan. unotom@kobe-u.ac.jp
Nakasuji
A
A
Hara
W
W
Aizono
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D012831:Bombyx / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004926:Escherichia coli; D015870:Gene Expression; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006868:Hydrolysis; D019476:Insect Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D020741:rho GTP-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D018076:DNA, Complementary; D019476:Insect Proteins; D011993:Recombinant Fusion Proteins; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D005982:Glutathione Transferase; D020741:rho GTP-Binding Proteins