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

At least three different subcellular compartments, including peroxisomes, a re involved in cholesterol biosynthesis. Because proper CNS development dep ends 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 br ain tissue or on the compartmentalization of isoprene metabolism in the CNS . This has been due mainly to the lack of a well-defined isolation procedur e for brain tissue, and also to the presence of myelin in brain tissue, whi ch results in significant contamination of subcellular fractions. As a firs t 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 wit h antibodies against organelle specific proteins that the isolated peroxiso mes are highly purified and well separated from the ER and mitochondria, an d are free of myelin contamination. The isolated peroxisomal fraction was p urified 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 pe roxisomes in the CNS.