Expression and characterization of chicken muscle ecto-ATPase in mammalian COS cells.
Chicken muscle ecto-ATPase has unusual enzyme kinetics and properties not found in many other E-type ATPases. To determine whether the unique properties of the chicken ecto-ATPase are inherent in the protein sequence and not mediated by some unique property of the chicken system, we have spliced together two partial cDNAs encoding the ecto-ATPase. The enzymatic properties of the COS (green monkey kidney) cell-expressed protein are indistinguishable from the purified chicken gizzard ecto-ATPase, including a 2- to 3-fold stimulation of membrane-bound activity by crosslinking and lectins, properties not shared by most other E-type ATPases. The expressed enzyme is specific for nucleotide triphosphates (ATPase:ADPase hydrolysis ratio of 26:1) and is inhibited by Cibacron Blue (IC50 = 10 microM). The active, expressed enzyme can be affinity-purified with Cibacron Blue, is relatively resistant to deglycosylation, and is less stable than other E-type ATPases. Expression in the presence of tunicamycin resulted in an inactive, unfolded enzyme.
Kirley
T L
TL
Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Ohio 45267-0575, USA. kirleytl@email.uc.edu
Gerber
L K
LK
Smith
T M
TM
D000251:Adenosine Triphosphatases / Q000235:genetics* / Q000378:metabolism*; D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000201:enzymology; D002645:Chickens; D003432:Cross-Linking Reagents; D004229:Dithiothreitol / Q000494:pharmacology; D007700:Kinetics; D037102:Lectins; D018482:Muscle, Skeletal / Q000201:enzymology*; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D014162:Transfection; D014227:Triazines / Q000494:pharmacology
D003432:Cross-Linking Reagents; D037102:Lectins; D011994:Recombinant Proteins; D014227:Triazines; C012842:Cibacron Blue F 3GA; D000251:Adenosine Triphosphatases; C027201:ectoATPase; D004229:Dithiothreitol