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Experimental Biology and Medicine 232:281-292 (2007)
© 2007 Society for Experimental Biology and Medicine

ORIGINAL RESEARCH ARTICLE

Insulin-Degrading Enzyme Hydrolyzes ATP

María del Carmen Camberos* and Juan C. Cresto*,{dagger},1

* Centro de Investigaciones Endocrinológicas (CEDIE), Hospital de Niños "Ricardo Gutierrez," Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina; and {dagger} Consejo de Investigaciones del Gobierno de la Ciudad de Buenos Aires, Argentina

To whom requests for reprints should be addressed at 1 Centro de Investigaciones Endocrinológicas (CEDIE), Endocrinología Hospital de Niños "Ricardo Gutierrez" Gallo 1330 (1425), Buenos Aires, Argentina. E-mail: jcresto{at}cedie.org.ar

We reported in a previous work that insulin degradation by insulin-degrading enzyme (IDE) was inhibited by ATP (Exp Biol Med 226:334–341, 2001). Then we studied ATP hydrolysis as a possible mechanism for reversion of this inhibition. ATP hydrolysis was determined by 32P release after hydrolysis of {gamma}[32P]ATP. ATP hydrolysis was studied by Sephadex G200 chromatography, immunoprecipitation, and nondissociating gel electrophoresis. Purified recombinant rat IDE and extractive homogenous IDE showed similar ATP hydrolysis. All results showed concordance between insulin degradation and ATP hydrolysis, suggesting that IDE has both functions. In order to define the type of hydrolysis, we studied inhibitors of IDE, phosphohydrolases, and ATPases. Each substance studied had no effect on ATP hydrolysis, except 1 mM orthovanadate, a known inhibitor of ATPases, phosphatases, and insulin degradation. ATP hydrolysis followed a Michaelis-Menten kinetic with Vmax: 570.45 ± 113.08 pmol Pi/hr and apparent Michaelis constant (Km): 63.13 ± 3.48 µM. ATP binding studies strongly suggested an ATP binding site and enzyme kinetics established only one active hydrolytic ATP binding site per IDE molecule. ATP-induced enzyme aggregation changes as observed by electrophoresis mobility in nondissociating conditions and conformational changes on insulin binding as shown by IDE-insulin cross-linking. We conclude that IDEs have ATPase activity and that insulin-binding and degradation are dependent on ATP concentration; however, insulin does not modify the ATPase activity of IDE.

Key Words: insulin-degrading enzyme • hydrolyzes • ATP




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Structure of Substrate-free Human Insulin-degrading Enzyme (IDE) and Biophysical Analysis of ATP-induced Conformational Switch of IDE
J. Biol. Chem., August 31, 2007; 282(35): 25453 - 25463.
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