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.