The binding of monoclonal and polyclonal antibodies to the Ca2+-ATPase of sarcoplasmic reticulum: effects on interactions between ATPase molecules

Elek Molnar, Norbert W. Seidler, I. Jóna, Anthony N. Martonosi

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

We analyzed the interaction of 14 monoclonal and 5 polyclonal anti-ATPase antibodies with the Ca2+-ATPase of rabbit sarcoplasmic reticulum and correlated the location of their epitopes with their effects on ATP-ase-ATPase interactions and Ca2+ transport activity. All antibodies were found to bind with high affinity to the denatured Ca2+-ATPase, but the binding to the native enzyme showed significant differences, depending on the location of antigenic sites within the ATPase molecule. Of the seven monoclonal antibodies directed against epitopes on the B tryptic fragment of the Ca2+-ATPase, all except one (VIE8) reacted with the enzyme in native sarcoplasmic reticulum vesicles in both the E1 and E2V conformations. Therefore these regions of the Ca2+-ATPase molecule are freely accessible in the native enzyme. The monoclonal antibody VIE8 bound with high affinity to the Ca2+-ATPase only in the E1 conformation stabilized by 0.5 mM Ca2+ but not in the E2V conformation stabilized by 0.5 mM EGTA and 5 mM vanadate. Several antibodies that reacted with the B fragment interfered with the crystallization of Ca2+-ATPase in the presence of EGTA and vanadate and at least two of them destabilized preformed Ca2+-ATPase crystals, suggesting inhibition of interactions between ATPase molecules. Of five monoclonal antibodies with epitopes on the A1 tryptic fragment of the Ca2+-ATPase only one gave strong reaction with the native enzyme, and none interfered with ATPase-ATPase interactions as measured by the polarization of fluorescence of FITC-labeled Ca2+-ATPase. Therefore the regions of the molecule containing these epitopes are relatively inaccessible in the native structure. Partial tryptic cleavage of the Ca2+-ATPase into the A1, A2 and B fragments did not promote the reaction of anti-A1 antibodies with sarcoplasmic reticulum vesicles, but solubilization of the membrane with C12E8 rendered the antigenic site fully accessible to several of them, suggesting that their epitopes are located in areas of contacts between ATPase molecules. Two monoclonal anti-B antibodies that interfered with ATPase-ATPase interactions, produced close to 50% inhibition of the rate of ATP-dependent Ca2+ transport, with significant inhibition of ATPase activity; this may suggest a role for ATPase oligomers in the regulation of Ca2+ transport. The other antibodies that interact with the native Ca2+-ATPase produced no significant inhibition of ATPase activity even at saturating concentrations; therefore their antigenic sites do not undergo major movements during Ca2+ transport.

Original languageEnglish
Pages (from-to)147-167
Number of pages21
JournalBBA - Biomembranes
Volume1023
Issue number2
DOIs
Publication statusPublished - Apr 13 1990

Fingerprint

Calcium-Transporting ATPases
Sarcoplasmic Reticulum
Adenosine Triphosphatases
Monoclonal Antibodies
Molecules
Antibodies
Epitopes
Conformations
Anti-Idiotypic Antibodies
Vanadates
Egtazic Acid
Enzymes
Adenosine Triphosphate
Fluorescence Polarization
Fluorescein-5-isothiocyanate
Crystallization
Oligomers
varespladib methyl
Fluorescence

Keywords

  • Antibody
  • ATPase, Ca-
  • ATPase-antibody interaction
  • ATPase-ATPase interaction
  • Monoclonal antibody
  • Polyclonal antibody
  • Sarcoplasmic reticulum

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Cell Biology

Cite this

The binding of monoclonal and polyclonal antibodies to the Ca2+-ATPase of sarcoplasmic reticulum : effects on interactions between ATPase molecules. / Molnar, Elek; Seidler, Norbert W.; Jóna, I.; Martonosi, Anthony N.

In: BBA - Biomembranes, Vol. 1023, No. 2, 13.04.1990, p. 147-167.

Research output: Contribution to journalArticle

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N2 - We analyzed the interaction of 14 monoclonal and 5 polyclonal anti-ATPase antibodies with the Ca2+-ATPase of rabbit sarcoplasmic reticulum and correlated the location of their epitopes with their effects on ATP-ase-ATPase interactions and Ca2+ transport activity. All antibodies were found to bind with high affinity to the denatured Ca2+-ATPase, but the binding to the native enzyme showed significant differences, depending on the location of antigenic sites within the ATPase molecule. Of the seven monoclonal antibodies directed against epitopes on the B tryptic fragment of the Ca2+-ATPase, all except one (VIE8) reacted with the enzyme in native sarcoplasmic reticulum vesicles in both the E1 and E2V conformations. Therefore these regions of the Ca2+-ATPase molecule are freely accessible in the native enzyme. The monoclonal antibody VIE8 bound with high affinity to the Ca2+-ATPase only in the E1 conformation stabilized by 0.5 mM Ca2+ but not in the E2V conformation stabilized by 0.5 mM EGTA and 5 mM vanadate. Several antibodies that reacted with the B fragment interfered with the crystallization of Ca2+-ATPase in the presence of EGTA and vanadate and at least two of them destabilized preformed Ca2+-ATPase crystals, suggesting inhibition of interactions between ATPase molecules. Of five monoclonal antibodies with epitopes on the A1 tryptic fragment of the Ca2+-ATPase only one gave strong reaction with the native enzyme, and none interfered with ATPase-ATPase interactions as measured by the polarization of fluorescence of FITC-labeled Ca2+-ATPase. Therefore the regions of the molecule containing these epitopes are relatively inaccessible in the native structure. Partial tryptic cleavage of the Ca2+-ATPase into the A1, A2 and B fragments did not promote the reaction of anti-A1 antibodies with sarcoplasmic reticulum vesicles, but solubilization of the membrane with C12E8 rendered the antigenic site fully accessible to several of them, suggesting that their epitopes are located in areas of contacts between ATPase molecules. Two monoclonal anti-B antibodies that interfered with ATPase-ATPase interactions, produced close to 50% inhibition of the rate of ATP-dependent Ca2+ transport, with significant inhibition of ATPase activity; this may suggest a role for ATPase oligomers in the regulation of Ca2+ transport. The other antibodies that interact with the native Ca2+-ATPase produced no significant inhibition of ATPase activity even at saturating concentrations; therefore their antigenic sites do not undergo major movements during Ca2+ transport.

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