Inhibition of the purified human red-cell Ca2+ pump by a monoclonal antibody.
1. A monoclonal antibody (1G4) was raised against the red-cell Ca2+ pump, and it reacted with the pump, as verified by Western blot analysis and by the e.l.i.s.a. method. 2. At 1 mM-ATP and 10 microM-Ca2+, 1G4 inhibited the activity of the purified Ca2+ pump by 40%. 3. Ca2+ pump inhibition by the antibody was non-competitive with regard to Ca2+, calmodulin and the high-affinity portion of the ATP curve. Thus its mechanism was quite different from that of the antibody previously reported [Verbist, Wuytack, Raemaekers, VanLeuven, Cassiman & Casteels (1986) Biochem. J. 240, 633-640], which partially caused inhibition by competition at the ATP site. 4. Antibody 1G4 reduced the steady-state level of phosphorylated intermediate and increased by 50% the calmodulin-activated p-nitrophenyl phosphatase activity of the pump. 5. The experimental results are consistent with the hypothesis that 1G4 inhibits the Ca2+ pump by decreasing the rate of the transition from the E2 form to the E1 form, causing a higher concentration of E2. 6. Analysis by Western blot of the pattern of cross-reaction of 1G4 after tryptic digestion of the pump showed that this antibody reacts with bands of Mr 90,000, 85,000, 50,000 and 33,000. After chymotryptic digestion, the antibody reacts almost exclusively with a fragment of Mr 105,000 that is fully active but is not responsive to calmodulin. Altogether, the results indicate that 1G4 binds to an epitope involved in the functional properties of the enzyme but which is not related to the calmodulin-binding domain.
Caride
A J
AJ
Department of Biochemistry and Molecular Biology, Mayo Clinic/Foundation, Rochester, MN 55905.
Enyedi
A
A
Penniston
J T
JT
D000911:Antibodies, Monoclonal / Q000276:immunology*; D000937:Antigen-Antibody Reactions; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors* / Q000276:immunology / Q000378:metabolism; D004910:Erythrocyte Membrane / Q000201:enzymology; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D010446:Peptide Fragments / Q000276:immunology
D000911:Antibodies, Monoclonal; D010446:Peptide Fragments; D000252:Calcium-Transporting ATPases; D002118:Calcium