Adenosine triphosphatase site stoichiometry in sarcoplasmic reticulum vesicles and purified enzyme

Publisher Website
Google Scholar
Abstract
The stoichiometry of phosphorylation (catalytic) sites in [rabbit] sarcoplasmic reticulum vesicles (SRV) and SR ATPase purified by differential solubilization with deoxycholate was 4.77 .+-. 0.4 and 6.05 .+-. 0.18 nmol/mg of protein, respectively, when phosphorylation was carried out under conditions permitting 32P labeling of nearly all sites. Assuming that each site corresponds to a single 115K [kilodalton] ATPase chain, the observed site stoichiometry accounts only for 55 and 70% of the total protein. Failure to obtain higher phosphorylation levels was due to the presence of nonspecific protein contaminants in SRV or to the presence of inactive aggregates in the ATPase purified with deoxycholate. This was demonstrated by dissolving SRV and purified ATPase with lithium dodecyl sulfate, subjecting them to molecular sieve HPLC [high performance liquid chromatography], and collecting the elution fractions for determination of protein, measurement of 32P-labeled sites and electrophoretic analysis. In the specific elution peak containing the 115K ATPase chains, phosphorylation levels were 6.62 .+-. 0.33 and 7.03 .+-. 0.18 in SRV and purified ATPase, corresponding to 68 and 86% of the protein in the specific elution peak. An alternate purification method was then developed, based on solubilization of SRV with dodecyl octaethylene glycol monoether (C12E8), separation of delipidated ATPase by anion-exchange chromatography and enzyme reactivation with phosphatidylcholine. This preparation yields 7.3 .+-. 0.44 nmol of phosphorylation site/mg of protein of the SRV fraction before HPLC. Analysis by HPLC demonstrates that the ATPase purified by this method does not have the tendency to form inactive aggregates. It is possible to obtain conditions in which the stoichiometric ratio of phosphorylation (catalytic) sites and 115K chains is nearly 1. Aggregational states that were detected in the native SR membrane are likely to occur by interaction of nonpolar polypeptide segments within the bilayer, permitting polar segments of individual chains to retain enzyme activity at the membrane-water interface.