Molecular cloning of cDNA for BRab from the brain of Bombyx mori and biochemical properties of BRab expressed in Escherichia coli.
From a brain cDNA library of Bombyx mori, we cloned cDNA for BRab, which encoded a 202-amino-acid polypeptide sharing 60-80% similarity with rab1 family members. To characterize its biochemical properties, cDNA for BRab was inserted into an expression vector (pGEX2T) and expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. The recombinant protein was purified to homogeneity with glutathione S-Sepharose. The purified GST-BRab bound [35S]-GTP gamma S and [3H]-GDP with association constants of 1.5 x 10(6) M-1 and 0.58 x 10(6) M-1, respectively. The binding of [35S]-GTP gamma S was inhibited with GTP and GDP, but with no other nucleotides. The GTP-hydrolysis activity was evaluated to be 5 m mole/min/mole of BRab. In the presence of 6 mM MgCl2, bound [35S]-GTP gamma S and [3H]-GDP were exchanged with GTP gamma S most efficiently. These results suggest that BRab, having a higher affinity for GTP than GDP, converts from the GTP-bound state into the GDP-bound state by intrinsic GTP hydrolysis activity and returns to the GTP-bound state with the exchange of GDP with GTP.
Uno
T
T
Department of Biofunctional Chemistry, Faculty of Agriculture, Kobe University, Hyogo, Japan.
Ueno
M
M
Nakajima
A
A
Shirai
Y
Y
Aizono
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D012831:Bombyx / Q000235:genetics*; D001923:Brain Chemistry / Q000235:genetics*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000032:analysis; D004926:Escherichia coli / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D019476:Insect Proteins; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins; D017422:Sequence Analysis, DNA; D020691:rab GTP-Binding Proteins
C116220:BRab protein, Bombyx mori; D018076:DNA, Complementary; D019476:Insect Proteins; D011993:Recombinant Fusion Proteins; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D005982:Glutathione Transferase; D019204:GTP-Binding Proteins; D020691:rab GTP-Binding Proteins