Inhibition of the purified human red-cell Ca2+ pump by a monoclonal antibody

A. J. Caride, A. Enyedi, J. T. Penniston

Research output: Article

1 Citation (Scopus)

Abstract

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 μM-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, Raemae4kers, VanLeuven, Cassiman and 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 M(r) 90,000, 85,000, 50,000 and 33,000. After chymotryptic digestion, the antibody reacts almost exclusively with a fragment of M(r) 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.

Original languageEnglish
Pages (from-to)87-92
Number of pages6
JournalBiochemical Journal
Volume264
Issue number1
Publication statusPublished - 1989

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Calmodulin
Monoclonal Antibodies
Pumps
Antibodies
Adenosine Triphosphate
Digestion
4-Nitrophenylphosphatase
Western Blotting
Cross Reactions
Epitopes
Enzymes

ASJC Scopus subject areas

  • Biochemistry

Cite this

Inhibition of the purified human red-cell Ca2+ pump by a monoclonal antibody. / Caride, A. J.; Enyedi, A.; Penniston, J. T.

In: Biochemical Journal, Vol. 264, No. 1, 1989, p. 87-92.

Research output: Article

Caride, A. J. ; Enyedi, A. ; Penniston, J. T. / Inhibition of the purified human red-cell Ca2+ pump by a monoclonal antibody. In: Biochemical Journal. 1989 ; Vol. 264, No. 1. pp. 87-92.
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