Evidence that the effects of phospholipids on the activity of the Ca(2+)-ATPase do not involve aggregation.
The Ca(2+)-ATPase of skeletal-muscle sarcoplasmic reticulum, solubilized in monomeric from in C12E8, has been reconstituted by dialysis into sealed vesicles of dioleoyl phosphatidylcholine [di(C18:1)PC], dimyristoleoyl phosphatidylcholine [di(C14:1)PC], dinervonyl phosphatidylcholine [di(C24:1)PC] or dipalmitoyl phosphatidylcholine [di(C16:0)PC] in the gel phase, at a phospholipid/ATPase molar ratio of 10,000: 1. Cross-linking experiments show that ATPase molecules are present in these reconstituted vesicles as isolated monomeric species. ATPase activities for the reconstituted vesicles are about half of those for the ATPase reconstituted with the same lipid in unsealed membrane fragments, attributed to a close to random orientation for the ATPase molecules in the reconstituted vesicles. ATPase activities for the ATPase in reconstituted vesicles of di(C14:1)PC or di(C24:1)PC are less than in vesicles of di(C18:1)PC, and no activity could be detected for the ATPase in di(C16:0)PC in the gel phase. It is concluded that effects of lipids on the activity of the ATPase are independent of any changes in the state of aggregation of the ATPase. Inhibition of ATPase activity by spermine and by the hydrophilic domain of phospholamban are observed both for the unreconstituted ATPase and for the ATPase in reconstituted vesicles, so that inhibition is independent of any aggregation caused by these polycationic species. Stimulation of ATPase activity by jasmone is also observed both for the unreconstituted ATPase and for the ATPase in reconstituted vesicles, so that stimulation of the ATPase also does not follow from any change in the state of aggregation of the ATPase.
Starling
A P
AP
Department of Biochemistry, University of Southampton, U.K.
East
J M
JM
Lee
A G
AG
D000818:Animals; D002135:Calcium-Binding Proteins / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D003432:Cross-Linking Reagents; D003517:Cyclopentanes / Q000494:pharmacology; D005227:Fatty Acids / Q000737:chemistry; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000378:metabolism; D054883:Oxylipins; D010713:Phosphatidylcholines / Q000494:pharmacology; D010743:Phospholipids / Q000494:pharmacology*; D011485:Protein Binding; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology; D013096:Spermine / Q000494:pharmacology; D000099211:Phospholamban
D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D003432:Cross-Linking Reagents; D003517:Cyclopentanes; D005227:Fatty Acids; D046911:Macromolecular Substances; D008565:Membrane Proteins; D054883:Oxylipins; D010713:Phosphatidylcholines; D010743:Phospholipids; D013096:Spermine; D000099211:Phospholamban; C086299:jasmone