Reaction sequence and molecular mass of a Cl(-)-translocating P-type ATPase.
The basolateral membranes of Aplysia californica foregut absorptive cells contain both Cl(-)-stimulated ATPase and ATP-dependent Cl- transport activities, and each was inhibited by orthovanadate. Both of these orthovanadate-sensitive activities were reconstituted into proteoliposomes. The reaction sequence kinetics were determined by [gamma-32P]ATP-induced phosphorylation of the reconstituted Cl- pump. Rapid phosphorylation and dephosphorylation kinetics of acyl phosphate bonding were confirmed by destabilization of the phosphoprotein by either hydroxylamine or high pH. Mg2+ caused phosphorylation of the enzyme; Cl- caused dephosphorylation. Orthovanadate almost completely inhibited the Mg(2+)-driven phosphorylation reaction. The molecular mass of the catalytic unit (subunit) of the enzyme appeared to be 110 kDa, which is in agreement with molecular masses of all other catalytic units (subunits) of P-type ATPases.
Gerencser
G A
GA
Department of Physiology, College of Medicine, University of Florida, Gainesville 32610-0174.
Zelezna
B
B
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D027321:Anion Transport Proteins; D001048:Aplysia; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology*; D002712:Chlorides / Q000378:metabolism* / Q000494:pharmacology; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008051:Lipid Bilayers; D008081:Liposomes; D008970:Molecular Weight; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D011510:Proteolipids / Q000378:metabolism; D014638:Vanadates / Q000494:pharmacology
D027321:Anion Transport Proteins; D002712:Chlorides; D008051:Lipid Bilayers; D008081:Liposomes; D010750:Phosphoproteins; D011510:Proteolipids; C020485:proteoliposomes; D014638:Vanadates; D004024:Dicyclohexylcarbodiimide; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D017301:Ca(2+) Mg(2+)-ATPase; C031652:anion-sensitive ATPases