Determination of the Oligomeric Status of the Sarcoplasmic Reticulum Ca2+-ATPase Using Optimized Chemical Crosslinking

In determining protein-protein interactions in native muscle membranes biochemically, bifunctional crosslinking agents of various length and solubility have been established as effective tools. Gel electrophoretic and immunoblotting methodology have proven to be highly suitable for the analysis of crosslinked products. Here, we have analysed the oligomeric status of a well established muscle membrane complex, the sarcoplasmic reticulum Ca-ATPase, employing a rapid optimization procedure of chemical crosslinking. Both, the fast SERCA1 and the slow SERCA2 isoforms of this enzyme appear to exist predominantly as a tetrameric complex under native conditions. Direct proteinprotein interactions are postulated to be important for cooperative kinetics and protection against proteolytic degradation. In order to keep artifacts of random crosslinking and hydrolysis of crosslinkers to a minimum, an efficient and swift experimental scheme is described to improve reaction conditions with respect to concentration ratios between crosslinkers and biomembranes, length of incubation time, pH and temperature. Employing the 11.4-Å probe bis-sulfosuccinimidyl-suberate and a mini-gel system for the analysis of microsomal vesicles, highly reproducible and optimal results could be obtained with relatively small amounts of skeletal muscle tissue. Thus, the reaction scheme of optimization of crosslinking described in this study is generally suitable for the analysis of supramolecular complexes in biomembranes and should improve the initial determination of the quaternary protein structures within muscle membrane microdomains.