Phosphatidylethanolamine Modulates Ca-ATPase Function and Dynamics

Phospholipids containing phosphoethanolamine (PE) headgroups within biological membranes have been suggested to be important with respect to the functional regulation of membrane proteins, including the Ca-ATPase in sarcoplasmic reticulum (SR). To investigate the role of PE headgroups in modulating...

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Veröffentlicht in:Biochemistry (Easton) 1999-01, Vol.38 (4), p.1356-1364
Hauptverfasser: Hunter, Gregory W, Negash, Sewite, Squier, Thomas C
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Negash, Sewite
Squier, Thomas C
description Phospholipids containing phosphoethanolamine (PE) headgroups within biological membranes have been suggested to be important with respect to the functional regulation of membrane proteins, including the Ca-ATPase in sarcoplasmic reticulum (SR). To investigate the role of PE headgroups in modulating the catalytic activity of the Ca-ATPase, we have reconstituted the Ca-ATPase into unilamellar liposomes containing defined amounts of dioleoylphosphatidylethanolamine (DOPE) and dioleoylphosphatidylcholine (DOPC). The enzymatic activity of the Ca-ATPase progressively increases upon incorporation of increasing amounts of PE into reconstituted vesicles, and approaches that characteristic of native SR membranes. To identify structural changes that correlate with enzyme activation, we have used frequency-domain phosphorescence spectroscopy to measure the rotational dynamics of erythrosin isothiocyanate covalently bound to Lys464 in the phosphorylation domain of the Ca-ATPase. Progressive increases in the rotational dynamics of the phosphorylation domain result from the incorporation of increasing amounts of DOPE, and correlate with enhanced enzymatic function. These results suggest that PE headgroups induce dynamic structural rearrangements involving the phosphorylation domain that modify the rates of nucleotide utilization. In contrast, no changes in the rotational dynamics of the lipid acyl chains are observed irrespective of the PE content. Therefore, the enhanced ATP hydrolytic activity associated with the incorporation of DOPE into these proteoliposomes is the result of specific noncovalent interactions involving PE phospholipid headgroups and the Ca-ATPase.
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To investigate the role of PE headgroups in modulating the catalytic activity of the Ca-ATPase, we have reconstituted the Ca-ATPase into unilamellar liposomes containing defined amounts of dioleoylphosphatidylethanolamine (DOPE) and dioleoylphosphatidylcholine (DOPC). The enzymatic activity of the Ca-ATPase progressively increases upon incorporation of increasing amounts of PE into reconstituted vesicles, and approaches that characteristic of native SR membranes. To identify structural changes that correlate with enzyme activation, we have used frequency-domain phosphorescence spectroscopy to measure the rotational dynamics of erythrosin isothiocyanate covalently bound to Lys464 in the phosphorylation domain of the Ca-ATPase. Progressive increases in the rotational dynamics of the phosphorylation domain result from the incorporation of increasing amounts of DOPE, and correlate with enhanced enzymatic function. 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subjects Calcium-Transporting ATPases - chemistry
Calcium-Transporting ATPases - drug effects
Calcium-Transporting ATPases - metabolism
Calorimetry
Carbonyl Cyanide m-Chlorophenyl Hydrazone - pharmacology
Enzyme Activation
Fluorescence Polarization
Kinetics
Liposomes
Luminescent Measurements
Lysine
Phosphatidylcholines - pharmacology
Phosphatidylethanolamines - pharmacology
Phosphorylation
Proteolipids
Recombinant Proteins - chemistry
Recombinant Proteins - drug effects
Recombinant Proteins - metabolism
Sarcoplasmic Reticulum - enzymology
Thermodynamics
title Phosphatidylethanolamine Modulates Ca-ATPase Function and Dynamics
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