Mutation of aspartic acid-351, lysine-352, and lysine-515 alters the Ca2+ transport activity of the Ca2+-ATPase expressed in COS-1 cells.
Full-length cDNAs encoding neonatal and adult isoforms of the Ca2+-ATPase of rabbit fast-twitch skeletal muscle sarcoplasmic reticulum were expressed transiently in COS-1 cells. The microsomal fraction isolated from transfected COS-1 cells contained immunoreactive Ca2+-ATPase and catalyzed Ca2+ transport at rates at least 15-fold above controls. No differences were observed in either the rates or Ca2+ dependency of Ca2+ transport catalyzed by the two isoforms. Aspartic acid-351, the site of formation of the catalytic acyl phosphate in the enzyme, was mutated to asparagine, glutamic acid, serine, threonine, histidine, or alanine. In every case, Ca2+ transport activity and Ca2+-dependent phosphorylation were eliminated. Ca2+ transport was also eliminated by mutation of lysine-352 to arginine, glutamine, or glutamic acid or by mutation of Asp351-Lys352 to Lys351-Asp352. Mutation of lysine-515, the site of fluorescein isothiocyanate modification in the enzyme, resulted in diminished Ca2+ transport activity as follows: arginine, 60%; glutamine, 25%; glutamic acid, 5%. These results demonstrate the absolute requirement of acylphosphate formation for the Ca2+ transport function and define a residue important for ATP binding. They also demonstrate the feasibility of a thorough analysis of active sites in the Ca2+-ATPase by expression and site-specific mutagenesis.
Maruyama
K
K
Banting and Best Department of Medical Research, Charles H. Best Institute, University of Toronto, ON, Canada.
MacLennan
D H
DH
D000818:Animals; D001224:Aspartic Acid; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D005796:Genes; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008239:Lysine; D008861:Microsomes / Q000378:metabolism; D024510:Muscle Development; D009132:Muscles / Q000201:enzymology; D009154:Mutation; D011817:Rabbits; D014162:Transfection
D007527:Isoenzymes; D001224:Aspartic Acid; D000252:Calcium-Transporting ATPases; D008239:Lysine; D002118:Calcium