Functional domains of the in situ red cell membrane calcium pump revealed by proteolysis and monoclonal antibodies. Possible sites for regulation by calpain and acidic lipids

Abstract

The functional domains of the in situ red cell membrane calcium pump were mapped by a double labeling technique. In inside-out vesicles (IOVs) the calcium pump was phosphorylated by [gamma-32P]ATP, the proteins blotted onto nitrocellulose and tagged by monoclonal antibodies raised against the purified pump protein. After proteolytic treatment of the IOVs by trypsin, chymotrypsin, or calpain-I, the fragmentation pattern of the enzyme was followed on the double-labeled immunoblots. The changes in the kinetics of the pump were examined by parallel measurements of the active calcium uptake in IOVs. By analysis of the results of tryptic digestion, it was possible to show that the antibodies recognized three different domains of the pump: 1) a Mr = 10,000-15,000 fragment (not seen directly) which includes the calmodulin-binding domain, 2) a nonphosphorylated Mr = 35,000 tryptic fragment, and 3) a phosphorylated fragment of Mr = 76,000-81,000. Chymotrypsin or calpain-I digestion of the membranes produced one major, Mr = 125,000 fragment, which had lost antibody-binding region 1. Production of this fragment coincided with the loss of calmodulin dependence and with a calmodulin-like activation of IOV calcium uptake (high Vmax, cooperativity in calcium activation). The Mr = 125,000 fragment was further activated by acidic lipids producing high Vmax and low K 1/2 (Ca2+) with no cooperativity. Based on these data a kinetic model and a functional map of the plasma membrane calcium pump is suggested.