The calcium-transporting ATPase and the calcium- or magnesium-dependent nucleotide phosphatase activities of human placental trophoblast basal plasma membrane are separate enzyme activities.
The properties of calcium-stimulated ATP hydrolysis often differ from those of ATP-dependent calcium transport. We have characterized two components of calcium-stimulated ATP hydrolysis in human placental basal plasma membrane. In the absence of magnesium, component 1 apparently has saturable sites for free calcium in both the nanomolar and low micromolar range. It was stimulated by either calcium or magnesium, was unselective for nucleotide substrate, and its activity was very much greater than that of ATP-dependent calcium transport. Component 1 was inhibited by GTP, permitting measurement of component 2 with activity and magnesium stimulation comparable to ATP-dependent calcium transport. Component 2 was inhibited partially by an antibody against purified erythrocyte calcium transporter and completely by sulfhydryl reagents, whereas component 1 was unaffected. A phosphorylated intermediate of the calcium transporter co-migrated with the erythrocyte transporter on acidic sodium dodecyl sulfate-polyacrylamide electrophoresis gels. Immunostaining after transfer to nitrocellulose revealed a doublet. The band of lower molecular weight co-migrated with that of the human erythrocyte membrane transporter. The addition of GTP permits separate measurement of ATP hydrolysis by the calcium transporter of the placental basal plasma membrane and may be useful in defining its properties in other cell membranes under a variety of conditions.
Kelley
L K
LK
Department of Pediatrics, Washington University School of Medicine, St. Louis Children's Hospital, Missouri 63110.
Borke
J L
JL
Verma
A K
AK
Kumar
R
R
Penniston
J T
JT
Smith
C H
CH
D000251:Adenosine Triphosphatases / Q000276:immunology; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology*; D004912:Erythrocytes / Q000201:enzymology; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006801:Humans; D006868:Hydrolysis; D015151:Immunoblotting; D007074:Immunoglobulin G / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology*; D009708:Nucleotidases / Q000378:metabolism*; D009711:Nucleotides / Q000378:metabolism; D010766:Phosphorylation; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014327:Trophoblasts / Q000201:enzymology*
D007074:Immunoglobulin G; D009711:Nucleotides; D013439:Sulfhydryl Reagents; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D009708:Nucleotidases; D000251:Adenosine Triphosphatases; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D002118:Calcium