Abstract
The functional significance of the molecular interaction of Ca2+-ATPase in the sarcoplasmic reticulum (SR) membrane was examined using intermolecular cross-linking of Ca2+-ATPase with N, N'-(1, 4-phenylene) bismaleimide (PBM). When SR vesicles were allowed to react with 1mM PBM at pH 7 and 23°C for various intervals and subjected to SDS-PAGE, the amount of the major band of monomeric ATPase decreased with a half life of about 20min. Higher orders of oligomers were concurrently formed without accumulation of any particular species of oligomer. When SR vesicles were allowed to react with 1 mM PBM in the presence of 1 mM adenyl-5'-imidodiphosphate (AMP-PNP), the rate of oligomerization was markedly reduced and the amount of dimeric Ca2+-ATPase increased with time. After 1h, more than 40% of the Ca2+-ATPase had accumulated in the dimeric form. When 1 mol of fluorescein isothiocyanate (FITC) was bound per mol of ATPase, the effects of AMP-PNP on the cross-linking with PBM were completely abolished. When SR vesicles were treated with PBM in the presence of 0.1 mM vanadate in Ca2+ free medium, the oligomerization of the Ca2+-ATPase by PBM was strongly inhibited. The vanadate effect on the cross-link formation was completely removed by the presence of Ca2+ and AMP-PNP in the reaction medium. When SR vesicles were pretreated with PBM in the presence of AMP-PNP and digested with trypsin for a short time, the dimeric ATPase was degraded to a peptide with an apparent molecular mass of about 170kDa. Further digestion resulted in degradation to a 130 kDa peptide. As they were phosphorylated by [γ-32P] ATP in the presence of Ca2+, these peptides contained the subfragment A. In addition, the SDS-PAGE pattern of the dimeric ATPase after the prolonged digestion lacked subfragment A1, suggesting that ATPase molecules were cross-linked by PBM through an SH group in the A1 region of this enzyme.
