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Expression and Characterization of Chicken Muscle Ecto‐ATPase in Mammalian COS Cells
Author(s) -
Kirley Terence L.,
Gerber Lamar K.,
Smith Thomas M.
Publication year - 1999
Publication title -
iubmb life
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.132
H-Index - 113
eISSN - 1521-6551
pISSN - 1521-6543
DOI - 10.1080/713803482
Subject(s) - microbiology and biotechnology , atpase , chemistry , biology , biochemistry , enzyme
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 (IC 10 mu M). The active, expressed enzyme = 50 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.