Characterization of the inositol trisphosphate-sensitive and insensitive calcium stores by selective inhibition of the endoplasmic reticulum-type calcium pump isoforms in isolated platelet membrane vesicles.
In mixed platelet membrane vesicles the presence of two distinct endoplasmic reticulum-type calcium pump enzymes of 100 and 97 kD molecular mass has been demonstrated. We have previously shown that both calcium pumps were recognized by polyclonal anti-sarcoplasmic reticulum calcium pump antisera [11]. In the present work we studied the effects of several calcium pump inhibitors on active calcium transport and inositol trisphosphate-induced calcium release in these vesicles in an attempt to assign the two calcium pump isoenzymes to specific calcium pools. The effect of the PL/IM 430 inhibitory anti-calcium pump antibody was compared to that of other calcium pump inhibitors acting predominantly on the 100 and the 97 kD calcium pump isoforms, respectively. The PL/IM 430 antibody, which recognized the 97 kD pump on Western blots and 2,5-di-(tert-butyl)-1,4-benzohydroquinone, which inhibited phosphoenzyme formation of the same pump isoform, inhibited calcium accumulation predominantly into an inositol trisphosphate-releasable calcium pool. On the other hand, low concentration of thapsigargin, which inhibited phosphoenzyme formation mainly of the 100 kD pump isozyme, had a more pronounced effect on calcium uptake into an inositol trisphosphate-resistant pool. These data suggest that in platelets the 97 kD calcium pump isoform is likely to be associated with the inositol trisphosphate-sensitive calcium storage organelle.
Papp
B
B
U-348 INSERM, Hospital Lariboisiere, Paris, France.
Pászty
K
K
Kovács
T
T
Sarkadi
B
B
Gárdos
G
G
Enouf
J
J
Enyedi
A
A
D000911:Antibodies, Monoclonal / Q000494:pharmacology; D016227:Benzoquinones / Q000494:pharmacology; D001692:Biological Transport; D001792:Blood Platelets / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors* / Q000276:immunology; D002451:Cell Compartmentation; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D015544:Inositol 1,4,5-Trisphosphate / Q000502:physiology*; D007425:Intracellular Membranes / Q000378:metabolism*; D007527:Isoenzymes / Q000037:antagonists & inhibitors* / Q000276:immunology; D015398:Signal Transduction; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin
D000911:Antibodies, Monoclonal; D016227:Benzoquinones; D007211:Indoles; D007527:Isoenzymes; D013729:Terpenes; C067344:2,5-di-tert-butylbenzoquinone; D019284:Thapsigargin; D015544:Inositol 1,4,5-Trisphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium; C000543:cyclopiazonic acid