Effects of gel phase phospholipid on the Ca(2+)-ATPase.
ATPase activities for the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum reconstituted in dimyristoylphosphatidylcholine [di(C14:0)PC] or dipalmitoylphosphatidylcholine [di(C16:0)PC] are very low at temperatures 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(C14:0)PC in both gel and liquid-crystalline phases; addition of cholesterol at a 1:1 molar ratio with di(C14:0)PC increases Ca2+ binding to two Ca2+ ions bound per ATPase molecule. The affinity of the ATPase for Ca2+ is slightly higher in di(C16:0)PC in the gel phase than in the liquid-crystalline phase, consistent with a shift in the E1/E2 equilibrium toward E1 in gel phase lipid. The rates of dissociation of Ca2+ 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 ATPase in liquid-crystalline lipid increases the rate of dissociation in liquid-crystalline phase lipid, Mg2+ has no effect in gel phase lipid. The fluorescence intensity of the Ca(2+)-ATPase labeled with 4-(bromomethyl)-6,7-dimethoxycoumarin decreases on addition of Mg2+ in liquid-crystalline 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 fluorescence of the ATPase labeled with IAEDANS in gel and liquid-crystalline phase lipids, but changes in tryptophan fluorescence intensity are different.(ABSTRACT TRUNCATED AT 250 WORDS)
Starling
A P
AP
Department of Biochemistry, University of Southampton, U.K.
East
J M
JM
Lee
A G
AG
D015060:1,2-Dipalmitoylphosphatidylcholine; D000818:Animals; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism; D004134:Dimyristoylphosphatidylcholine; D005782:Gels; D066298:In Vitro Techniques; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry; D018482:Muscle, Skeletal / Q000201:enzymology; D010743:Phospholipids / Q000737:chemistry*; D010766:Phosphorylation; D011487:Protein Conformation; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013696:Temperature
D005782:Gels; D008051:Lipid Bilayers; D010743:Phospholipids; D015060:1,2-Dipalmitoylphosphatidylcholine; D000252:Calcium-Transporting ATPases; D002118:Calcium; D004134:Dimyristoylphosphatidylcholine