Effect of non-solubilizing SDS concentrations on high affinity Ca2+ binding and steady state phosphorylation by inorganic phosphate of the sarcoplasmic reticulum ATPase.
In this investigation low, non-solubilizing concentrations of the strong anionic detergent SDS were used to perturbate the interaction of Ca2+ and Pi with their respective binding domains on the sarcoplasmic reticulum Ca-transport ATPase. Rising SDS concentrations produce a two-step decline of Ca2+-dependent ATP hydrolysis. At pH 6.15, SDS differently affects high affinity Ca2+ binding and phosphorylation by inorganic phosphate and releases the "mutual exclusion" of these two ligand binding steps. The degree of uncoupling is considerably more pronounced in the presence of 20% Me2SO. The reduction of Ca2+ binding by SDS is demonstrated to be a result of decreased affinity of one of the two specific high affinity binding sites and of perturbation of their cooperative interaction. Higher SDS partially restores the original high Ca2+ affinity but not the cooperativity of binding. Phosphorylation exhibits a higher SDS sensitivity than Ca2+ binding: Increasing SDS competitively inhibits and then completely abolishes phosphoenzyme formation. Thus, SDS binds to the phosphorylation domain, evidently involving the Lys352 residue of the ATPase molecule; this is accompanied by a more unspecific concentration-dependent SDS effect, probably mediated by hydrophobic force, which, finally, suppresses phosphorylation. Me2SO does neither qualitatively affect the SDS-dependent chemical properties of the vesicular material nor the SDS-dependent perturbation of the investigated reaction steps.
Fassold
E
E
Max-Planck-Institut für Medizinische Forschung, Abteilung für Physiologie, Bundesrepublik Deutschland.
Hasselbach
W
W
Küchler
B
B
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D010710:Phosphates / Q000378:metabolism*; D010766:Phosphorylation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology*
D010710:Phosphates; D012967:Sodium Dodecyl Sulfate; D000252:Calcium-Transporting ATPases; D002118:Calcium