Partial purification and characterization of the mitochondrial and peroxisomal isozymes of enoyl-coenzyme a hydratase from germinating pea seedlings.

Distinct organellar forms of the beta-oxidation enzyme enoyl-coenzyme A (CoA) hydratase were partially purified and characterized from 2-day germinated pea (Pisum sativum L.) seedlings. The purification was accomplished by disruption of purified mitochondria or peroxisomes, (NH(4))(2)SO(4) fractionation, and gel permeation chromatography using a column of Sephacryl S-300. The organellar isozymes had distinct kinetic constants for the substrates 2-butenoyl-CoA and 2-octenoyl-CoA, and could be easily distinguished by differences in thermostability and salt activation. The peroxisomal isozyme had a native M(r) of 75,000 and appeared to be a typical bifunctional enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase, while the mitochondrial isozyme had a native M(r) of 57,000 and did not have associated dehydrogenase activity. Western blots of total pea mitochondrial proteins gave a positive signal when probed with anti-rat liver mitochondrial enoyl-CoA hydratase antibodies but there was no signal when blots of total peroxisomal proteins were probed.