Simultaneous presence of two distinct endoplasmic-reticulum-type calcium-pump isoforms in human cells. Characterization by radio-immunoblotting and inhibition by 2,5-di-(t-butyl)-1,4-benzohydroquinone.
Phosphorylation, immunoblotting, limited proteolysis and drug-sensitivity analysis were used to characterize the sarcoendoplasmic-reticulum Ca2+ ATPases in a variety of human cell types. In platelets, several megakaryoblastoid and lymphoblastoid cell lines two distinct autophosphorylated forms of these ATPases with molecular mass of 100 and 97 kDa could be observed, whereas in several other cell types the 97 kDa form was absent. On immunoblots the 97 kDa species was specifically recognized by an inhibitory monoclonal antibody raised against the Ca2+ pump of platelet internal membranes, yielded on trypsinolysis a major fragment of 80 kDa, exhibited a distinct electrophoretic migration pattern as compared with the skeletal-, cardiac- and smooth-muscle Ca2+ pumps, and its autophosphorylation was strongly inhibited by the Ca(2+)-mobilizing agent 2,5-di-(t-butyl)-1,4-benzohydroquinone (tBHQ). The 100 kDa species reacted with an antibody specific for the cardiac- and smooth-muscle Ca2+ pumps, yielded on trypsinolysis fragments of 55 and 35 kDa, and its autophosphorylation was much less sensitive to tBHQ inhibition. These findings indicate the simultaneous presence of two different endoplasmic-reticulum Ca2+ pumps in a variety of human cell types, and may explain the previously observed differences in the Ca(2+)-handling characteristics of different intracellular Ca2+ pools and cell types.
Papp
B
B
U-348 INSERM, Hôpital Lariboisière, Paris, France.
Enyedi
A
A
Pászty
K
K
Kovács
T
T
Sarkadi
B
B
Gárdos
G
G
Magnier
C
C
Wuytack
F
F
Enouf
J
J
D000911:Antibodies, Monoclonal; D001792:Blood Platelets / Q000201:enzymology; D000252:Calcium-Transporting ATPases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000502:physiology*; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D006801:Humans; D006873:Hydroquinones / Q000494:pharmacology*; D015151:Immunoblotting; D008214:Lymphocytes / Q000201:enzymology; D008533:Megakaryocytes / Q000201:enzymology; D008970:Molecular Weight; D009130:Muscle, Smooth / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D014357:Trypsin / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D006873:Hydroquinones; D010446:Peptide Fragments; C019359:2,5-di-tert-butylhydroquinone; D014357:Trypsin; D000252:Calcium-Transporting ATPases