Rat liver dihydroxyacetone-phosphate acyltransferase: enzyme characteristics and localization studies.
Peroxisomes were isolated from rat liver by pelleting a light mitochondrial (L) fraction over a 30% (w/v) Metrizamide layer. Peroxisomes were recovered as a loose pellet from the bottom of the tube and the purity of the peroxisomal fraction was calculated to be about 90%. The characteristics of dihydroxyacetone-phosphate acyltransferase (DHAP-AT) in the light mitochondrial fraction and the purified peroxisomal fraction were compared. The behaviour of the enzyme in the two fractions was very similar, except for the effect of sodium fluoride, which stimulated the activity in the L fraction 5-10-fold and in the peroxisomal fraction only 1.6-fold. This difference could be explained by the action of fluoride-sensitive acid phosphatases present in the L fraction that dephosphorylate palmitoyl-coenzyme A, a substrate for DHAP-AT. The localizations of DHAP-AT and alkyldihydroxyacetone-phosphate synthase in the rat liver peroxisomal membrane were studied. It is shown that in intact peroxisomes, DHAP-AT and alkyl-DHAP synthase are resistant to proteolytic inactivation by trypsin, as is fatty acid beta-oxidation activity, which served as a marker for the intactness of the peroxisomal membrane. Catalase was found not to be a suitable marker to assess peroxisome intactness in view of its relative insensitivity to trypsin. In 1-lauroyllysophosphatidylcholine-permeabilized peroxisomes, DHAP-AT, alkyl-DHAP synthase and beta-oxidation activities were rapidly inactivated by trypsin. It is concluded that in rat liver peroxisomes, at least the active sites of the integral membrane proteins DHAP-AT and alkyl-DHAP synthase are localized exclusively at the inner surface of the peroxisomal membrane.
Hardeman
D
D
Laboratory of Biochemistry, University of Utrecht, The Netherlands.
van den Bosch
H
H
D000217:Acyltransferases / Q000378:metabolism*; D019883:Alkyl and Aryl Transferases; D000818:Animals; D001665:Binding Sites; D008099:Liver / Q000201:enzymology* / Q000648:ultrastructure; D008274:Magnesium / Q000494:pharmacology; D015636:Magnesium Chloride; D008297:Male; D008566:Membranes / Q000201:enzymology; D008830:Microbodies / Q000201:enzymology; D008930:Mitochondria, Liver / Q000201:enzymology; D051381:Rats; D011919:Rats, Inbred Strains; D012969:Sodium Fluoride / Q000494:pharmacology; D014166:Transferases / Q000378:metabolism
D015636:Magnesium Chloride; D012969:Sodium Fluoride; D014166:Transferases; D000217:Acyltransferases; C020051:glycerone-phosphate O-acyltransferase; D019883:Alkyl and Aryl Transferases; C019654:alkylglycerone-phosphate synthase; D008274:Magnesium