Controlled proteolysis of Ca(2+)-ATPases in human platelet and non-muscle cell membrane vesicles. Evidence for a multi-sarco/endoplasmic reticulum Ca(2+)-ATPase system.
Two sarco/endoplasmic reticulum Ca(2+)-ATPases (SERCAs) have been previously identified in platelets: the 100-kDa SERCA2b and the 97-kDa SERCA3 isoforms. Analysis of the acylphosphate intermediate (E-P) formation and the immunoreactivity of the platelet Ca(2+)-ATPases and their proteolytic fragments upon controlled trypsinolysis revealed the presence of an additional 97-kDa Ca(2+)-ATPase that comigrates with SERCA3 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-Ca(2+)-ATPase antibody that had been shown to recognize a 97-kDa Ca(2+)-ATPase. The 73- and 68-kDa fragments were precursors of the 40-kDa one. Ca(2+)-dependent phospholabeling of the 73-kDa fragment and immunostaining of all these proteolytic products by another antibody raised against SERCA1 established the SERCA nature of the 97-kDa parent enzyme. The SERCA3-related E-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-SERCA1 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.
Kovács
T
T
U-348 Institut National de la Santé et de la Recherche Médicale, Hôpital Lariboisière, Paris, France.
Corvazier
E
E
Papp
B
B
Magnier
C
C
Bredoux
R
R
Enyedi
A
A
Sarkadi
B
B
Enouf
J
J
D001792:Blood Platelets / Q000201:enzymology*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D006801:Humans; D006868:Hydrolysis; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D010449:Peptide Mapping; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D014357:Trypsin; D014407:Tumor Cells, Cultured
D007527:Isoenzymes; D014357:Trypsin; D000252:Calcium-Transporting ATPases