ATP-dependent degradation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in permeabilized cells.
A system for the assay of 3-hydroxy-3-methyglutaryl (HMG) coenzyme A (CoA) reductase in digitonin-permeabilized Chinese hamster ovary cells is described. Under these conditions, HMG-CoA reductase remained intact and associated with the endoplasmic reticulum, and values for Km (HMG-CoA), Ki (mevinolin), and active/total activity were similar to those seen in sonicated cell preparations. However, the mechanism by which this rapidly turned over (half-life approximately 2 h) enzyme is degraded was disrupted. Addition of ATP at physiological concentrations to digitonin-permeabilized cells resulted in the rapid, irreversible loss of enzyme activity. Immunoblot analysis showed that this loss of activity was followed by cleavage of the intact 97-kilodalton enzyme to a 68-kilodalton fragment which was distinct from the catalytically active fragments generated by nonspecific proteolysis in sonicated cell homogenates. Assay of a lysosomal marker enzyme confirmed that ATP-mediated inactivation and cleavage of reductase was not due to release of lysosomal proteases. The possible role of ATP in phosphorylation, inactivation, and degradation of reductase is discussed.
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D002463:Cell Membrane Permeability; D006903:Hydroxymethylglutaryl CoA Reductases / Q000378:metabolism*; D007700:Kinetics
D000255:Adenosine Triphosphate; D006903:Hydroxymethylglutaryl CoA Reductases; D000469:Alkaline Phosphatase