J. Kvannes et al., THE PEROXISOMAL BETA-OXIDATION ENZYME-SYSTEM OF RAT-HEART - BASAL LEVEL AND EFFECT OF THE PEROXISOME PROLIFERATOR CLOFIBRATE, Biochimica et biophysica acta (G). General subjects, 1201(2), 1994, pp. 203-216
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.