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

170 Citations (Scopus)

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 monogalactosyldiglyceride, 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, dioleoylmonomethylphosphatidylethanolamine, dioleoyldimethylphosphatidylethanolamine and dioleoylphosphatidylcholine) or increasing degrees of glycosylation (monogalactosyldiglyceride 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/dioleoylphosphatidylcholine or monogalactosyldiglyceride/dioleoylphosphatidylcholine molar ratios in the reconstituted vesicles were reduced. Vesicles reconstituted with high molar ratios of dioleoylphosphatidylethanolamine/dioleoylphosphatidylcholine 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 dioleoylphosphatidylcholine, dioleoylphosphatidylglycerol, cardiolipin, bovine brain phosphatidylserine, phosphatidylinositol, and mixtures of dioleoylphosphatidylcholine 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 monogalactosyldiglyceride).

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

Fingerprint

Calcium-Transporting ATPases
Sarcoplasmic Reticulum
Adenosine
Adenosine Triphosphate
Calcium
Lipids
Chemical analysis
Proteins
triphosphoric acid
1,2-oleoylphosphatidylcholine
Cholates
Glycosylation
Proteolipids
Cardiolipins
Methylation
Deoxycholic Acid
Dialysis
Phosphatidylserines
Distillation columns
Octoxynol

ASJC Scopus subject areas

  • Biochemistry

Cite this

Effect of lipid composition on the calcium/adenosine 5′-triphosphate coupling ratio of the Ca2+-ATPase of sarcoplasmic reticulum. / Navarro, Javier; Toivio-Kinnucan, Maria; Racker, Efraim.

In: Biochemistry, Vol. 23, No. 1, 1984, p. 130-135.

Research output: Contribution to journalArticle

@article{60da07ae72a0465ab95935649a9121de,
title = "Effect of lipid composition on the calcium/adenosine 5′-triphosphate coupling ratio of the Ca2+-ATPase of sarcoplasmic reticulum",
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 monogalactosyldiglyceride, 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, dioleoylmonomethylphosphatidylethanolamine, dioleoyldimethylphosphatidylethanolamine and dioleoylphosphatidylcholine) or increasing degrees of glycosylation (monogalactosyldiglyceride 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/dioleoylphosphatidylcholine or monogalactosyldiglyceride/dioleoylphosphatidylcholine molar ratios in the reconstituted vesicles were reduced. Vesicles reconstituted with high molar ratios of dioleoylphosphatidylethanolamine/dioleoylphosphatidylcholine 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 dioleoylphosphatidylcholine, dioleoylphosphatidylglycerol, cardiolipin, bovine brain phosphatidylserine, phosphatidylinositol, and mixtures of dioleoylphosphatidylcholine 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 monogalactosyldiglyceride).",
author = "Javier Navarro and Maria Toivio-Kinnucan and Efraim Racker",
year = "1984",
language = "English (US)",
volume = "23",
pages = "130--135",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "1",

}

TY - JOUR

T1 - Effect of lipid composition on the calcium/adenosine 5′-triphosphate coupling ratio of the Ca2+-ATPase of sarcoplasmic reticulum

AU - Navarro, Javier

AU - Toivio-Kinnucan, Maria

AU - Racker, Efraim

PY - 1984

Y1 - 1984

N2 - 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 monogalactosyldiglyceride, 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, dioleoylmonomethylphosphatidylethanolamine, dioleoyldimethylphosphatidylethanolamine and dioleoylphosphatidylcholine) or increasing degrees of glycosylation (monogalactosyldiglyceride 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/dioleoylphosphatidylcholine or monogalactosyldiglyceride/dioleoylphosphatidylcholine molar ratios in the reconstituted vesicles were reduced. Vesicles reconstituted with high molar ratios of dioleoylphosphatidylethanolamine/dioleoylphosphatidylcholine 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 dioleoylphosphatidylcholine, dioleoylphosphatidylglycerol, cardiolipin, bovine brain phosphatidylserine, phosphatidylinositol, and mixtures of dioleoylphosphatidylcholine 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 monogalactosyldiglyceride).

AB - 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 monogalactosyldiglyceride, 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, dioleoylmonomethylphosphatidylethanolamine, dioleoyldimethylphosphatidylethanolamine and dioleoylphosphatidylcholine) or increasing degrees of glycosylation (monogalactosyldiglyceride 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/dioleoylphosphatidylcholine or monogalactosyldiglyceride/dioleoylphosphatidylcholine molar ratios in the reconstituted vesicles were reduced. Vesicles reconstituted with high molar ratios of dioleoylphosphatidylethanolamine/dioleoylphosphatidylcholine 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 dioleoylphosphatidylcholine, dioleoylphosphatidylglycerol, cardiolipin, bovine brain phosphatidylserine, phosphatidylinositol, and mixtures of dioleoylphosphatidylcholine 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 monogalactosyldiglyceride).

UR - http://www.scopus.com/inward/record.url?scp=0021321562&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0021321562&partnerID=8YFLogxK

M3 - Article

C2 - 6229280

AN - SCOPUS:0021321562

VL - 23

SP - 130

EP - 135

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 1

ER -