Effect of phospholipid, detergent and protein-protein interaction on stability and phosphoenzyme isomerization of soluble sarcoplasmic reticulum Ca-ATPase.
The purpose of the present study was to elucidate the separate roles of lipid, detergent and protein-protein interaction for stability and catalytic properties of sarcoplasmic reticulum Ca-ATPase solubilized in the non-ionic detergent octa(ethylene glycol) monododecyl ether (C12E8). The use of large-zone high-performance liquid chromatography permitted us to define the self-association state of Ca-ATPase peptide at various detergent, phospholipid and protein concentrations, and also during enzymatic turnover with ATP. Conditions were established for monomerization of Ca-ATPase in the presence of a high concentration of phospholipid relative to detergent. The lipid-saturated monomeric preparation was relatively resistant to inactivation in the absence of Ca2+, whereas delipidated enzyme in monomeric or in oligomeric form was prone to inactivation. Kinetics of phosphoenzyme turnover were examined in the presence and absence of Mg2+. Dephosphorylation rates were sensitive to Mg2+, irrespective of whether the peptide was present in soluble monomeric form or was membrane-bound. C12E8-solubilized monomer without added phospholipid was, however, characterized by a fast initial phase of dephosphorylation in the absence of Mg2+. This was not observed with monomer saturated with phospholipid or with monomer solubilized in myristoylglycerophosphocholine or deoxycholate. The mechanism underlying this difference was shown to be a C12E8-induced acceleration of conversion of ADP-sensitive phosphoenzyme (E1P) to ADP-insensitive phosphoenzyme (E2P). The phosphoenzyme isomerization rate was also found to be enhanced by low-affinity binding of ATP. This was demonstrated both in membrane-bound and in soluble monomeric Ca-ATPase. Our results indicate that a single peptide chain constitutes the target for modulation of phosphoenzyme turnover by Mg2+ and ATP, and that detergent effects, distinct from those arising from disruption of protein-protein contacts, are the major determinants of kinetic differences between C12E8-solubilized and membrane-bound enzyme preparations.
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D000252:Calcium-Transporting ATPases / Q000302:isolation & purification / Q000378:metabolism*; D003840:Deoxycholic Acid; D003902:Detergents / Q000494:pharmacology*; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D009132:Muscles / Q000201:enzymology; D010713:Phosphatidylcholines / Q000494:pharmacology*; D010766:Phosphorylation; D011092:Polyethylene Glycols / Q000494:pharmacology*; D011485:Protein Binding; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013501:Surface-Active Agents / Q000494:pharmacology*
D003902:Detergents; D046911:Macromolecular Substances; D010713:Phosphatidylcholines; D013501:Surface-Active Agents; D003840:Deoxycholic Acid; C017272:dodecyloctaethyleneglycol monoether; D011092:Polyethylene Glycols; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D008274:Magnesium