3-Hydroxy-3-methylglutaryl coenzyme A reductase localization in rat liver peroxisomes and microsomes of control and cholestyramine-treated animals: quantitative biochemical and immunoelectron microscopical analyses.

Abstract

Abstract. 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, a key regulatory enzyme involved in cholesterol biosynthesis, has recently been reported to be present in rat liver peroxisomes (Keller, G. A., M. C. Barton, D. J. Shapiro, and S. J. Singer, 1985, Proc. Natl. Acad. Sci. USA, 82:770-774). Immunoelectron labeling of ultrathin frozen sections of normal liver, using two monoclonal antibodies to purified rat liver microsomal HMG-CoA reductase, indicated that the enzyme is present in the matrix of peroxisomes. This study is a quantitative biochemical and immunoelectron microscopical analysis of HMGCoA reductase in rat liver peroxisomes and microsomes of normal and cholestyramine-treated animals. Cholestyramine treatment produced a six-to sevenfold increase in the specific activity of peroxisomal HMGCoA reductase, whereas the microsomal HMG-CoA reductase specific activity increased by about twofold. Using a computer program that calculates optimal linear combinations of marker enzymes, it was determined that between 20 and 30% of the total reductase activity was located in the peroxisomes of cholestyramine-treated animals. Less than 5 % of the reductase activity was present in peroxisomes under control conditions. Quantitation of the immunoelectron microscopical data was in excellent agreement with the biochemical results. After cholestymmine treatment there was an eightfold increase in the density of gold particles per peroxisome, and we estimate about a threefold increase in the labeling of the ER.T HE key regulatory enzyme of cholesterol, dolichol, and isopentenyl adenosine biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) 1 reductase, is a 97-kD transmembrane glycoprotein that was believed until recently to reside exclusively in the endoplasmic reticulum (ER) of mammalian cells (7,8, 27,28). However, a recent publication showed that the enzyme in liver cells is present not only in the ER but also within peroxisomes (18). Immunoelectron labeling of ultrathin frozen sections of normal liver, using two monoclonal antibodies to purified rat liver microsomal HMG-CoA reductase, indicated that the enzyme is concentrated in the matrix of peroxisomes.This study was designed to determine what percentage of the total liver HMG-CoA reductase activity is attributable to the peroxisomal enzyme and to determine if the peroxisomal and the ER enzymes are dependently or independently regulated. Normal rat livers and livers obtained from animals in which the total HMG-CoA reductase activity was increased 1. Abbreviations used in this paper: ER, endoplasmic reticulum; HMGCoA, 3-hydroxy-3-methylglutaryl coenzyme A.by cholestyramine treatment were fractionated by differential and density gradient centrifugation.Two completely independent analyses of the fractions were performed: (a) immunoelectron microscopy with quantitation of the antigenic sites of HMG-CoA reductase; and (b) quantitative enzyme activity measurements of the subcellular fractions with computer-assisted analyses. Materials and Methods Anim...

Peroxisomes and peroxisomal disorders: The main facts

“…Although HMG-CoA reductase is lacking of peroxisomal targeting information, a number of studies indicate that it is located not only in the ER but also in peroxisomes (Keller et al, 1985;Keller, 1986;Engfelt et al, 1997; reviewed by Olivier et al, 2000 andKovacs et al, 2007).…”

Characterization of an Acyl-CoA Thioesterase That Functions as a Major Regulator of Peroxisomal Lipid Metabolism

“…The initial enzymes involved in cholesterol synthesis have been demonstrated to be present in peroxisomes. These enzymes include acetoacetyl-CoA thiolase (56), HMG-CoA synthase (57), and HMG-CoA reductase (58,59). PTE-2 hydrolyzes acetyl-CoA (substrate for acetoacetyl-CoA thiolase), acetoacetyl-CoA (substrate for HMG-CoA synthase), and HMG-CoA (substrate for the HMGCoA reductase).…”

Biogenesis of peroxisomes in regenerating rat liver. II. Immunoelectron microscopical investigation of catalase and acylCoA oxidase.

“…On the other hand, in all recent studies on immunoelectron microscopic localization of peroxisomal proteins, from our laboratory (6, 30,50), and by others (10,(22)(23)(24) no evidence of ribosomal, or as a matter of fact, cytosolic labeling has been observed. Since in regenerating rat liver the new formation of peroxisomes is associated with the increased rate of synthesis of their enzyme proteins, we have extended our immunocytochemical studies to this model of peroxisomal biogenesis with the objective of identifying the sites of biosynthesis of peroxisomal enzymes in the cytosol of rat liver cells.…”