TOPOGRAPHICAL DISTRIBUTION OF LIPID BIOSYNTHETIC ENZYMES ON PEROXISOMES (MICROBODIES)

Recent work' has shown that enzymes of the acyl-DHAP (dihydroxyacetone phosphate) pathway for the biosynthesis of both diacyl-glycerolipids and ether lipids are localized to catalase-containing particles (peroxisomes) in liver. Though the data from the fractionation of brains are less conclusive, it appears that in this organ these enzymes are also localized in the catalase-containing particles (microperoxisomes).1.2 In an effort to elucidate the role of acyl-DHAP pathway enzymes in brain microperoxisomal lipid metabolism and in the generation of membrane phospholipid asymmetry, we have determined the distribution of these enzymes in the transverse plane of the microperoxisomal membrane. Specifically, these enzymes are: DHAP acyltransferase (acyl-CoADHAP acyltransferase), acyl-CoA reductase (hexadecano1:NADP' oxidoreductase, CoAacylating), alkyl-DHAP synthase, and alkyl-DHAP reductase (alkyl-G-3-P: NADP' oxidoreductase). The susceptibility of catalase, an enzyme of the microperoxisomal matrix, to proteolysis was used to determine the integrity of the microperoxisomal membrane. A crude microperoxisomal preparation (18,000 x g, 30 min to 40,000 x g, 30 min) from 12-day-old rat brain was preincubated at a concentration of 1 mg protein per ml320 mOs potassium phosphate/sucrose for 30 min at 37'C which contained various amounts of the protease, pronase. When pronase was included in amounts from 0.1-5 mg pronase/mg brain protein, the assay of aliquots for catalase showed that maximally 10% of the activity was destroyed. These data, plus the observation that 5% of the homogenate catalase activity is found in the soluble fraction upon subcellular fraction show that 15% of the microperoxisomes had ruptured membranes. In control experiments to show that catalase was not resistant to pronase digestion, a minimum of 2 mg pronase/mg brain protein was found to completely destroy catalase activity when permeabilizing amounts of the detergent Triton X-100 were included in the preincubation. The minimum amount of Triton required to fully permeabilize the membrane was 0.05%. To determine the transverse distribution of acyl-DHAP pathway enzymes, the crude microperoxisomal preparation was similarly preincubated with trypsin in the presence and absence of permeabilizing amounts of Triton. Trypsin, rather than pronase. was chosen owing to the ease with which it can be inactivated with the addition of trypsin inhibition at the termination of the 30 min preincubation. Assay of aliquots of the preincubation for the four acyl-DHAP pathway