Cross-linking of the sarcoplasmic reticulum ATPase protein

Abstract

Treatment of rabbit sarcoplasmic reticulum vesicles with the cross-linking agent, cupric phenanthroline, causes production of high-molecular weight bands on SDS-gel electrophoresis. A plot of log mol wt vs mobility indicates that the main band produced from the ATPase (mol wt = 10 5) has a mol wt of 4 × 10 5 and thus suggests formation of a tetramer. Notably, bands corresponding to dimers, trimers, pentamers, etc., are absent. The bands attributable to calsequestrin and calcium binding protein are unchanged by cupric phenanthroline. With extended treatment, the tetramer itself is polymerized (mol wt>10 6). Partial disruption of the membranes with deoxycholate or Triton X-100 before cross-linking favors tetramer formation; the presence of sodium dodecyl sulfate, on the other hand, prevents intermolecular cross-linking. Our results suggest that the ATPase is at least partially associated within the membrane as a tetramer.