Effect of Lipid Composition on the Calcium/Adenosine 5′-Triphosphate Coupling Ratio of the Ca²⁺-ATPase of Sarcoplasmic Reticulum

The Ca²⁺-ATPase of sarcoplasmic reticulum was purified and depleted of proteolipids by solubilization in Triton X-100 and by fractionation on a DE-52 column. The protein reconstituted by deoxycholate-cholate dialysis at low lipid to protein ratios (2-5 mg of lipid/mg of protein), with either dioleoylphosphatidylethanolamine or monogalactosyldi-glycende, exhibited high initial rates of ATP-dependent Ca²⁺ uptake [300-900 nmol min^-1 (mg of protein)^-1] and coupling ratios (Ca²⁺ transported/ATP hydrolyzed) up to 1.2. Ca²⁺-ATPase reconstituted with lipids of increasing degrees of methylation (dioleoylphosphatidylethanolamine, dioleoyl-monomethylphosphatidylethanolamine, dioleoyldimethyl-phosphatidylethanolamine and dioleoylphosphatidylchohne) or increasing degrees of glycosylation (monogalactosyldi-glyceride and digalactosyldiglyceride) revealed a progressive decrease in both ATP-dependent Ca²⁺-uptake and coupling ratios. The rate and extent of Ca²⁺ uptake decreased as the dioleoylphosphatidylethanolamine/dioleoylphosphatidylchoune or monogalactosyldiglyceride/dioleoylphosphatidylcholine molar ratios in the reconstituted vesicles were reduced. Vesicles reconstituted with high molar ratios of dioleoyl-phosphatidylethanolamine/dioleoylphosphatidylchohne or monogalactosyldiglyceride/dioleoylphosphatidylcholine and at a high lipid to protein ratio became leaky and released the Ca²⁺ accumulated inside the vesicles when the temperature of the incubation mixture was increased (e.g., from 20 to 37 °C). Freeze-fracture electron microscopy of reconstituted vesicles incubated at 37 °C demonstrated fusion of vesicles and formation of hexagonal II structures. Reconstitution of the Ca²⁺-ATPase with other phospholipids such as dioleoylphosphatidylchohne, dioleoylphosphatidylglycerol, cardiohpin, bovine brain phosphatidylsenne, phosphatidyhnositol, and mixtures of dioleoylphosphatidylchohne and cholesterol catalyzed Ca²⁺-dependent ATP hydrolysis [0.5-2 μmol of P_i min^-1(mg of protein)^-1] but low rates of Ca²⁺ uptake [5-10 nmol min^-1 (mg of protein)^-1]. Our results suggest that the coupling state of the Ca²⁺ -ATPase as numerically expressed as the Ca²⁺/ATP ratio is stabilized by cone-shaped lipid molecules (e.g., dioleoylphosphatidylethanolamine and mon-ogalactosyldiglycende).