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

Two sarco/endoplasmic reticulum Ca2+-ATPases (SERCAs) have been previously identified in platelets: the 100-kDa SERCA(2b), and the 97-kDa SERCA(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 revealed the presence of an additional 97-kDa Ca2+-ATPase that comigrates with SERCA(3) on SDS-polyacrylamide gels. At a trypsin/membrane protein ratio 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 the PL/IM 430 anti-Ca2+-ATPase antibody that had been shown to recognize a 97-kDa Ca2+-ATPase. The 73- and 68-kDa fragments were precursors of the 40-kDa one. Ca2+-dependent phospholabeling of the 73-kDa fragment and immunostaining of all these proteolytic products by another antibody raised against SERCA(1) established the SERCA nature of the 97-kDa parent enzyme. The SERCA(3)-related E similar to P-forming 80-kDa tryptic fragment appeared during trypsinolysis with a different time course 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 at 5 min of digestion, while the 73-, 68-, and 40-kDa fragments were fully degraded at 2 min of trypsinization. This 80-kDa species was immunostained neither with the PL/IM 430, nor with the anti-SERCA(1) antibodies. Similar results were found in some megakaryoblastoid and lymphoblastoid cell lines. All these data indicate the presence of two distinct tryptic fragmentation patterns attributed to two 97-kDa SERCA isoforms and point to the existence of a multi-SERCA system in different human non-muscle cells.