Identification and Characterization of Ca^<2+>-transport ATPase from Bovine Aortic Smooth Muscle Microsomes

Abstract

Crude microsomal fraction was prepared from bovine aortic smooth muscle. The aortic microsome (AM) retained a membrane bound Ca^<2+>-pump activity that was two orders of magnitude lower than that of skeletal muscle sarcoplasmic reticulum (SR). When AM mambrane proteins, solubilized with polyoxyethyleneglycol-9-laurylether (C_<12>E_9), were separated by a column chromatography in the presence of the detergent, the protein with apparent molecular mass of about 100 kDa was eluted together with other membrane proteins. Adenosine 5'-triphosphate (ATP)-dependent Ca^<2+>-transport activity was restored when proteoliposomes were reconstituted from this fraction with soybean asolectin. Rabbit antiserum raised against the 100 kDa protein crossreacted with the Ca^<2+>-transport ATPase of rabbit skeletal muscle SR. Furthermore, antibody against Ca^<2+>-transport ATPase purified from scallop adductor muscle SR recognized the 100 kDa protein of AM as well as Ca^<2+>-ATPase of rabbit skeletal SR. The 100 kDa protein was phosphorylated with ATP to form an acylphosphate in the same manner as the Ca^<2+>-transport ATPase of skeletal SR. When AM membrane was solubilized in C_<12>E_9 and passed through a calmodulin linked affinity column, the 100 kDa protein was not retained by the column, and almost all this protein was recovered from the passed through fraction. These results suggest that 100 kDa protein of AM membrane has the same characteristics as the Ca^<2+>-transport ATPase of SR in the skeletal muscle.