THE PEROXISOMAL BETA-OXIDATION ENZYME-SYSTEM OF RAT-HEART - BASAL LEVEL AND EFFECT OF THE PEROXISOME PROLIFERATOR CLOFIBRATE

Peroxisomes, isolated from homogenates of rat hearts (myocard), contai n a beta-oxidation enzyme system which is indistinguishable from that found in liver, but the total capacity of beta-oxidation is only 0.8% of the liver value (expressed per g of tissue). Fatty acyl-CoA oxidase was assayed by an H2O2 based fluorescent assay avoiding important int erfering side reactions. The presence of polypeptides with electrophor etic and immunological properties similar to the beta-oxidation enzyme s of liver peroxisomes, was demonstrated by immunoblotting using polyc lonal antibodies. The level of 72 and 52 kDa subunits of fatty acyl-Co A oxidase (FAG), quantitated by an anti-FAO(1-16) peptide antibody, wa s only 1% of the level in liver (expressed per g of tissue). Immunoblo ts of one-dimensional (1-D) SDS-PAGE of rat heart and liver peroxisoma l fractions revealed a 60 kDa subunit of the fatty acyl-CoA oxidase in addition to the known 72 and 52 kDa subunits. Immunoblots of two-dime nsional (2-D) IEF/SDS-PAGE revealed that all subunits are strongly bas ic polypeptides, with a microheterogeneity, which probably represents deamidations of the polypeptides. The 2-D immunoblot also revealed ano ther group of polypeptides with M(r) 72 kDa of less basic isoelectric point, possibly representing an isoform of fatty acyl-CoA oxidase. Sub strate specificity studies revealed the highest V-max values with C-10 -C-12. For the very long-chain fatty acids C-20-C-24, the monoenes rev ealed much higher V-max values than the saturated fatty acids. Adminis tration of the classical peroxisome proliferator clofibrate resulted i n a similar increase in the fatty acyl-CoA oxidase activity and the 72 and 52 kDa subunits of FAO in the heart. The response (activity) was found to change from 2.2-fold increase in young (34 days) to 11.1-fold increase in adult (76 days) rats. In contrast to liver, where the rat io of the increase in FAO mRNA to the increase in FAG activity was abo ut 4, this ratio in heart was about 0.5.