Molecular Cloning of cDNA for BRab from the Brain of Bombyx mori and Biochemical Properties of BRab Expressed in Escherichia coli

Article abstract of DOI:10.1271/bbb.62.1885

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 rabl 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]-GTPγS and [³H]-GDP with association constants of 1.5 × 10⁶M^-1 and 0.58 × 10⁶ M^-1, respectively. The binding of [³⁵S]-GTPγS 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 MgC1₂, bound [³⁵S]-GTPγS and [³H]-GDP were exchanged with GTPyS 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.