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

Javier Navarro, Maria Toivio-Kinnucan, Efraim Racker

Research output: Contribution to journalArticle

172 Scopus citations

Abstract

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 Pi 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).

Original languageEnglish (US)
Pages (from-to)130-135
Number of pages6
JournalBiochemistry
Volume23
Issue number1
DOIs
StatePublished - Jan 1984
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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