EFFECTS OF GEL PHASE PHOSPHOLIPID ON THE CA2-ATPASE()

ATPase activities for the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum reconstituted in dimyristoylphosphatidylcholine [di(C14:O)PC ] or dipalmitoylphosphatidylcholine [di(C16:O)PC] are very low at temp eratures below 25 and 30 degrees C, respectively. The stoichiometry of Ca2+ binding to the ATPase is 1 Ca2+ ion bound per ATPase molecule in di(C14:O)PC in both gel and liquid-crystalline phases; addition of ch olesterol at a 1:1 molar ratio with di(C14:O)PC increases Ca2+ binding to two Ca2+ ions bound per ATPase molecule. The affinity of the ATPas e for Ca2+ is slightly higher in di(C16:O)PC in the gel phase than in the liquid-crystalline phase, consistent with a shift in the E1/E2 equ ilibrium toward E1 in gel phase lipid. The rates of dissociation of Ca 2+ from the ATPase in gel and liquid-crystalline phase lipids are the same in the absence of Mg2+, but whereas addition of Mg2+ to the ATPas e in liquid-crystalline lipid increases the rate of dissociation in li quid-crystalline phase lipid, Mg2+ has no effect in gel phase lipid. T he fluorescence intensity of the Ca2+-ATPase labeled with 4-(bromometh yl)-6,7-dimethoxycoumarin decreases on addition of Mg2+ in liquid-crys talline phase lipid, but is unaffected by Mg2+ in gel phase lipid. The rate of phosphorylation of the ATPase in gel phase lipid is very slow , and rates of dephosphorylation of the phosphorylated ATPase are also very slow, p-Nitrophenolphosphatase activity is also very low in gel phase lipid. Binding of ATP results in the same changes in the fluores cence of the ATPase labeled with IAEDANS in gel and liquid-crystalline phase lipids, but changes in tryptophan fluorescence intensity are di fferent. It is concluded that the conformational change following bind ing of ATP to the ATPase (E1Ca(2)ATP reversible arrow E1'Ca(2)ATP) is unaffected by the phase of the lipid, but the rate of phosphate transf er (E1'Ca(2)ATP reversible arrow E1PCa(2)ADP) is decreased.