Production and characterization of monoclonal antibodies to rat liver microsomal . 33 - hydroxy - 3 - methylglutaryl - coenzyme A reductase ( cholesterol / immunoprecipitation / radioimmunoassay / enzyme purification )

Rat liver microsomal 3-hydroxy-3-methylglutaryl-CoA reductase [HMG-CoA reductase; mevalonate:NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34], the key regulatory enzyme in cholesterol biosynthesis, has been purified to apparent homogeneity. Purified HMG-CoA reductase yields a single diffuse band when NaDodSO4/polyacrylamide gels are stained with Coomassie blue and yields two adjacent bands when gels are stained with silver. Purified reductase was used to elicit the production ofmonoclonal antibodies. Spleen cells from BALB/c mice immunized with purified HMG-CoA reductase were fused with Sp-2/0 myeloma cells. Clones producing monoclonal antibodies to HMG-CoA reductase were identified by using a solid-phase radioimmunoassay and were subcloned in soft agar. The three relatively stable hybridoma lines isolated secrete different Igs as judged by their antibody subclasses and differing abilities to inhibit HMG-CoA reductase in solution. Efficient precipitation of solubilized HMG-CoA reductase was achieved with the two IgG antibodies but not with the IgM. A mixture of all three monoclonal antibodies immunoprecipitates more than 90% of the HMG-CoA reductase activity in solubilized rat liver extracts. These monoclonal antibodies should be useful probes for investigation of the regulation of HMG-CoA reductase and cholesterol synthesis. The level and activity of 3-hydroxy-3-methylglutaryl-CoA reductase [HMG-CoA reductase; mevalonate:NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34], a key regulatory enzyme in cholesterol, dolichol, and isopentenyladenosine biosynthesis, are controlled by cholesterol, low density lipoproteins, oxygenated sterols, hormones, and a variety of effector molecules (1-7). Because cardiovascular disease is the leading cause of death in the United States and hypercholesterolemia represents a major risk factor for the disease, a great deal ofinterest has centered on the regulation ofHMG-CoA reductase and cholesterol synthesis. Regulatory studies based primarily on measurements of HMG-CoA reductase activity have resulted in the proposal of three basic control mechanisms for HMG-CoA reductase: (i) regulation of HMG-CoA reductase synthesis or degradation, or both (1, 8, 9); (ii) interconversion of active and inactive forms of the enzyme by phosphorylation and dephosphorylation (10-12); and (iii) modulation ofHMG-CoA reductase activity by changes in the fluidity of the microsomal membrane (13, 14). Although there have been several reports (15-18) describing the titration of HMG-CoA reductase with antibodies, these studies are in substantial disagreement. In a recent study using immunoprecipitation of HMG-CoA reductase, the antibody was not monospecific, as it precipitated a number of different polypeptides (19). The dearth ofdefinitive regulatory information in this system is largely due to the low levels ofHMGCoA reductase in the endoplasmic reticulum and the resulting difficulty in preparing homogeneous enzyme and monospecific antibodies to reductase. In this report, we describe the purification of HMG-CoA reductase, demonstrate that electrophoretically homogeneous reductase may be composed of subunits, and, most important, describe the preparation and characterization of monoclonal antibodies to HMG-CoA reductase. These monoclonal antibodies, used in the quantitative immunoprecipitation assay we describe, should prove invaluable for studies aimed at distinguishing between the different mechanisms proposed for the regulation ofHMG-CoA reductase and cholesterol synthesis.