A new member of α(1)-adrenoceptor-coupled Gα(h) (transglutaminase II) family in pig heart: purification and characterization

초록

We previously reported an identification of a 77-kDa GTP-binding protein that co-purified with the alpha(1)-adrenoceptor following ternary complex formation. In the present paper, we report on the purification and characterization of this GTP-binding protein (termed G alpha(h5)) isolated from pig heart membranes. After solubilization of pig heart membranes with NaCl, G alpha(h5) was purified by sequential chromatographies using DEAE-Cellulose, Q-Sepharose, and GTP-agarose columns. The protein displayed high-affinity GTP gamma S binding which is Mg2+-dependent and saturable. The relative order of affinity of nucleotide binding by Gah5 was GTP > GDP > ITP >> ATP greater than or equal to adenyl-5'-yl imidodiphosphate, which was similar to that observed far other heterotrimeric G-proteins involved in receptor signaling. Moreover, the G alpha(h5) demonstrated transglutaminase (TGase) activity that was blocked either by EGTA or GTP gamma S. In support of these observations, the G alpha(h5) was recognized by a specific antibody to G alpha(h7) or TGase II, indicating a homology with G alpha(h) (TGase II) family. These results demonstrate that 77-kDa G alpha(h5) from pig heart is an alpha(1)-adrenoceptor-coupled G alpha(h) (TGase II) family which has species-specificity in molecular mass.

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