A new member of α1-adrenoceptor-coupled Gαh (transglutaminase II) family in pig heart: Purification and characterization

Abstract

We previously reported an identification of a 77-kDa GTP-binding protein that co-purified with the α1-adrenoceptor following ternary complex formation. In the present paper, we report on the purification and characterization of this GTP-binding protein (termed Gαh5) isolated from pig heart membranes. After solubilization of pig heart membranes with NaCl, Gαh5 was purified by sequential chromatographies using DEAE-Cellulose, Q-Sepharose, and GTP-agarose columns. The protein displayed high-affinity GTPγS binding which is Mg2+-dependent and saturable. The relative order of affinity of nucleotide binding by Gαh5 was GTP> GDP > ITP ≫ ATP ≥ adenyl-5′-yl imidodiphosphate, which was similar to that observed for other heterotrimeric G-proteins involved in receptor signaling. Moreover, the Gαh5 demonstrated transglutaminase (TGase) activity that was blocked either by EGTA or GTPγS. In support of these observations, the Gαh5 was recognized by a specific antibody to Gαh7 or TGase II, indicating a homology with Gαh (TGase II) family. These results demonstrate that 77-kDa Gαh5 from pig heart is an α1-adrenoceptor-coupled Gαh (TGase II) family which has species-specificity in molecular mass.