ATP inhibits insulin-degrading enzyme activity.
We studied the ability of ATP to inhibit in vitro the degrading activity of insulin-degrading enzyme. The enzyme was purified from rat skeletal muscle by successive chromatographic steps. The last purification step showed two bands at 110 and 60 kDa in polyacrylamide gel. The enzyme was characterized by its insulin degradation activity, the substrate competition of unlabeled to labeled insulin, the profile of enzyme inhibitors, and the recognition by a specific antibody. One to 5 mM ATP induced a dose-dependent inhibition of insulin degradation (determined by trichloroacetic acid precipitation and insulin antibody binding). Inhibition by 3 mM adenosine 5'-diphosphate, adenosine 5'-monophosphate, guanosine 5'-triphosphate, pyrophosphate, beta-gamma-methyleneadenosine 5'-triphosphate, adenosine 5'-O-(3 thiotriphosphate), and dibutiryl cyclic adenosine 5'-monophosphate was 74%, 4%, 38%, 46%, 65%, 36%, and 0%, respectively, of that produced by 3 mM ATP. Kinetic analysis of ATP inhibition suggested an allosteric effect as the plot of 1/v (insulin degradation) versus ATP concentration was not linear and the Hill coefficient was more than 1 (1.51 and 2.44). The binding constant for allosteric inhibition was KiT = 1.5 x 10(-7) M showing a decrease of enzyme affinity induced by ATP. We conclude that ATP has an inhibitory effect on the insulin degradation activity of the enzyme.
Camberos
M C
MC
CEDIE-Endocrinology, Htal. de Niños R. Gutiérrez, Buenos Aires, Argentina.
Pérez
A A
AA
Udrisar
D P
DP
Wanderley
M I
MI
Cresto
J C
JC
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D000906:Antibodies; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D015151:Immunoblotting; D007328:Insulin / Q000276:immunology / Q000378:metabolism*; D007339:Insulysin / Q000037:antagonists & inhibitors* / Q000276:immunology / Q000302:isolation & purification; D008297:Male; D018482:Muscle, Skeletal / Q000201:enzymology; D051381:Rats; D017208:Rats, Wistar; D014238:Trichloroacetic Acid
D000906:Antibodies; D004791:Enzyme Inhibitors; D007328:Insulin; D014238:Trichloroacetic Acid; D000255:Adenosine Triphosphate; D007339:Insulysin