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), contain 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 interfering side reactions. The presence of polypeptides with electrophoretic and immunological properties similar to the beta-oxidation enzymes of liver peroxisomes, was demonstrated by immunoblotting using polyclonal antibodies. The level of 72 and 52 kDa subunits of fatty acyl-CoA oxidase (FAO), quantitated by an anti-FAO1-16 peptide antibody, was only 1% of the level in liver (expressed per g of tissue). Immunoblots of one-dimensional (1-D) SDS-PAGE of rat heart and liver peroxisomal 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-dimensional (2-D) IEF/SDS-PAGE revealed that all subunits are strongly basic polypeptides, with a microheterogeneity, which probably represents deamidations of the polypeptides. The 2-D immunoblot also revealed another group of polypeptides with M(r) 72 kDa of less basic isoelectric point, possibly representing an isoform of fatty acyl-CoA oxidase. Substrate specificity studies revealed the highest Vmax values with C10-C12. For the very long-chain fatty acids C20-C24, the monoenes revealed much higher Vmax values than the saturated fatty acids. Administration of the classical peroxisome proliferator clofibrate resulted in 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 ratio of the increase in FAO mRNA to the increase in FAO activity was about 4, this ratio in heart was about 0.5.
Kvannes
J
J
Department of Biochemistry, University of Bergen, Norway.
Eikhom
T S
TS
Flatmark
T
T
D015094:3-Hydroxyacyl CoA Dehydrogenases / Q000378:metabolism*; D042962:Acyl-CoA Oxidase; D000375:Aging; D000595:Amino Acid Sequence; D000818:Animals; D002994:Clofibrate / Q000494:pharmacology*; D004746:Enoyl-CoA Hydratase / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D006321:Heart / Q000187:drug effects*; D007535:Isomerases / Q000378:metabolism*; D008099:Liver / Q000201:enzymology; D008297:Male; D008830:Microbodies / Q000201:enzymology; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism*; D009206:Myocardium / Q000201:enzymology*; D010088:Oxidoreductases / Q000032:analysis; D063994:Peroxisomal Bifunctional Enzyme; D051381:Rats; D017208:Rats, Wistar; D013379:Substrate Specificity
D009097:Multienzyme Complexes; D010088:Oxidoreductases; D015094:3-Hydroxyacyl CoA Dehydrogenases; D042962:Acyl-CoA Oxidase; D004746:Enoyl-CoA Hydratase; D063994:Peroxisomal Bifunctional Enzyme; D007535:Isomerases; D002994:Clofibrate