Purification of brain peroxisomes and localization of 3-hydroxy-3-methylglutaryl coenzyme A reductase

Abstract

At least three different subcellular compartments, including peroxisomes, are involved in cholesterol biosynthesis. Because proper CNS development depends on de novo cholesterol biosynthesis, peroxisomes must play a critical functional role in this process. Surprisingly, no information is available on the peroxisomal isoprenoid/cholesterol biosynthesis pathway in normal brain tissue or on the compartmentalization of isoprene metabolism in the CNS. This has been due mainly to the lack of a well-defined isolation procedure for brain tissue, and also to the presence of myelin in brain tissue, which results in significant contamination of sub-cellular fractions. As a first step in characterizing the peroxisomal isoprenoid pathway in the CNS, we have established a purification procedure to isolate peroxisomes and other cellular organelles from the brain stem, cerebellum and spinal cord of the mouse brain. We demonstrate by use of marker enzymes and immunoblotting with antibodies against organelle specific proteins that the isolated peroxisomes are highly purified and well separated from the ER and mitochondria, and are free of myelin contamination. The isolated peroxisomal fraction was purified at least 40-fold over the original homogenate. In addition, we show by analytical subcellular fractionation and immunoelectron microscopy that HMG-CoA reductase protein and activity are localized both in the ER and peroxisomes in the CNS.