Abstract
The key regulatory enzyme of cholesterol, dolichol, and isopentenyl adenosine biosynthesis, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase) is a 97-kilodalton transmembrane glycoprotein which was believed until recently to reside exclusively in the endoplasmic reticulum of mammalian cells. However, several recent publications have shown that the enzyme in liver cells is present not only in the endoplasmic reticulum but also within peroxisomes. In an effort to clarify the role of peroxisomal HMG-CoA reductase, highly purified (95%) rat liver peroxisomes from cholestyramine-treated rats were incubated with RS-[2-14C]mevalonic acid plus cytosolic proteins and then tested for the presence of newly synthesized cholesterol. For comparison, highly purified microsomes from the same liver preparation were incubated at several protein concentrations under the same conditions. A three-step procedure was employed to resolve the newly synthesized cholesterol from the complex mixture of sterol intermediates in cholesterol biosynthesis. After termination of the reaction and addition of a [3H]cholesterol standard, the incubation products were extracted and separated by thin layer chromatography into a number of fractions. The fraction containing C-27 sterols was further resolved by reverse-phase high pressure liquid chromatography. After acetylation, the products were then separated by silicic acid high pressure liquid chromatography. Confirmation of the identity of newly synthesized cholesterol was obtained by recrystallization with added non-radioactive cholestenyl acetate standard. The results indicate that highly purified rat liver peroxisomes are able to convert mevalonic acid to cholesterol in the presence of cytosolic fraction in vitro. An abstract of these results has been published (Krisans, S. K., Thompson, S. L., Burrows, R., and Laub, R. J. (1986) J. Cell Biol. 103, 525 (abstr.).
Highlights
From the $Department of Biology and Molecular BiologyInstitute, San Diego State University, SanDiego, California92182 and the §Department of Chemistry, San Diego State University, SanDiego California92182
The key regulatory enzyme of cholesterol, dolichol, zyme, previouslyconsidered to be exclusively microsomal,has and isopentenyl adenosine biosynthesis, 3-hydroxy-3- recently been found to be localized in rat liver peroxisomes methylglutaryl-coenzyme A reductase (HMG-CoA reductase) is a 97-kilodalton transmembrane glycoprotein which was believed until recentlyto reside esclusively in the endoplasmic reticulum of mammalian cells
It is well known that rat liver peroxisomes contain enzymes for the P-oxidation of long-chain fatty acids, a major pathway of lipid metabolism previously believed to be exclusively mitochondrial [4, 5]
Summary
From the $Department of Biology and Molecular BiologyInstitute, San Diego State University, SanDiego, California92182 and the §Department of Chemistry, San Diego State University, SanDiego California92182. Recent studies indicate that liver peroxisomes are involved in the degradation of cholesterol to bile acids [7,8,9,10]. The results indicate thatfirst fractionated by differential centrifugation [11] to obtain a perhighly purified r a t liver peroxisomes a r e able to con- oxisome-enriched fraction, a microsomal fraction, and a cytosolic vert mevalonic acid to cholesterol in the presence of fraction. Rat liver homogeubiquinone proceed through a common regulatory step, the nates were fractionated by differential centrifugation accordingto the reduction of 3-hydroxy-3-methylglutarylcoenzyme A
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