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Regulation of chicken gizzard ecto-ATPase activity by modulators that affect its oligomerization status
C C Caldwell1, S C Hornyak, E Pendleton
1Department of Chemistry, San Diego State University, California 92182-1030, USA.
Archives of Biochemistry and Biophysics
|May 23, 2001
Summary
Chicken gizzard smooth muscle ecto-ATPase activity is regulated by protein oligomerization. Modulators affecting enzyme oligomerization alter ecto-ATPase activity, suggesting physiological regulation of enzyme function.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Chicken gizzard smooth muscle membranes contain a major ectonucleoside triphosphate phosphohydrolase.
- This enzyme is an integral membrane glycoprotein belonging to the ecto-E-ATPase (E-NTPDase) family.
Purpose of the Study:
- To investigate the kinetic properties and modulators of the chicken gizzard ecto-ATPase.
- To understand the mechanism of action of various inhibitors and activators on the enzyme.
Main Methods:
- Characterization of enzyme kinetics and response to modulators.
- Use of concanavalin A, chemical cross-linking agents, eosin iodoacetamide, sulfhydryl reagents, xanthene derivatives, NBD-halides, and suramin.
- Preincubation experiments with substrate (ATP) and concanavalin A to determine inhibitor interaction sites.
Main Results:
- The gizzard ecto-ATPase exhibits unusual kinetic properties, temperature dependence, and modulator responses.
- Compounds promoting oligomerization (concanavalin A, cross-linkers) increase enzyme activity.
- Inhibitors of other ATPases also inhibit gizzard ecto-ATPase, with effects distinct from liver ecto-ATP-diphosphohydrolase.
- Inhibitor interaction occurs at a non-catalytic site and is modulated by concanavalin A, suggesting involvement of oligomerization.
Conclusions:
- Structurally diverse modulators affect gizzard ecto-ATPase activity by interfering with protein oligomerization.
- Physiological conditions influencing membrane events can alter enzyme oligomerization status and thus its activity.
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