CONTROLLED PROTEOLYSIS OF CA2-ATPASES IN HUMAN PLATELET AND NONMUSCLECELL-MEMBRANE VESICLES - EVIDENCE FOR A MULTI-SARCO()ENDOPLASMIC RETICULUM CA2+-ATPASE SYSTEM/

Two sarco/endoplasmic reticulum Ca2+-ATPases (SERCAs) have been previo usly identified in platelets: the 100-kDa SERCA(2b), and the 97-kDa SE RCA(3) isoforms. Analysis of the acylphosphate intermediate (E similar to P) formation and the immunoreactivity of the platelet Ca2+-ATPases and their proteolytic fragments upon controlled trypsinolysis reveale d the presence of an additional 97-kDa Ca2+-ATPase that comigrates wit h SERCA(3) on SDS-polyacrylamide gels. At a trypsin/membrane protein r atio of 0.025 at 4 degrees C, tryptic fragments of 73-, 68- and 40-kDa , previously unknown in the SERCA family, could be detected by using t he PL/IM 430 anti-Ca2+-ATPase antibody that had been shown to recogniz e a 97-kDa Ca2+-ATPase. The 73- and 68-kDa fragments were precursors o f the 40-kDa one. Ca2+-dependent phospholabeling of the 73-kDa fragmen t and immunostaining of all these proteolytic products by another anti body raised against SERCA(1) established the SERCA nature of the 97-kD a parent enzyme. The SERCA(3)-related E similar to P-forming 80-kDa tr yptic fragment appeared during trypsinolysis with a different time cou rse from that of the 73-, 68-, and 40-kDa ones. At a trypsin/membrane protein ratio of 0.125 at 37 degrees C, it reached its maximum level a t 5 min of digestion, while the 73-, 68-, and 40-kDa fragments were fu lly degraded at 2 min of trypsinization. This 80-kDa species was immun ostained neither with the PL/IM 430, nor with the anti-SERCA(1) antibo dies. Similar results were found in some megakaryoblastoid and lymphob lastoid cell lines. All these data indicate the presence of two distin ct tryptic fragmentation patterns attributed to two 97-kDa SERCA isofo rms and point to the existence of a multi-SERCA system in different hu man non-muscle cells.