Partial Purification and Characterization of the Mitochondrial and Peroxisomal Isozymes of Enoyl-Coenzyme A Hydratase from Germinating Pea Seedlings 1

Distinct organellar forms of the j#-oxidation enzyme enoylcoenzyme 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, (NH4)2SO4 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 Mr of 75,000 and appeared to be a typical bifunctional enoyl-CoA hydratase/3-hydroxyacylCoA dehydrogenase, while the mitochondrial isozyme had a native Mr 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. characteristic of animal cell mitochondrial p-oxidation that distinguishes it from the peroxisomal pathway (18). It has been suggested that the pea mitochondrial ,8-oxidation activity observed by Wood and Thomas could be attributed to peroxisomal contamination (12, 20). This criticism ignores the fact that pea mitochondrial f-oxidation requires L-carnitine while the peroxisomal pathway is unable to utilize acylcarnitines (23, 34). Nevertheless, the criticism of peroxisomal contamination was directly addressed and, using an improved purification procedure, it was shown that pea mitochondria devoid of peroxisomal contamination (5) were still fully capable of carnitine-dependent fatty acid f-oxidation (31). Having established the validity of the mitochondrial localization of f-oxidation in peas, it was desirable to compare the physicochemical and catalytic properties of the organelle-specific isozymes. EH3 was chosen for the initial studies because it has the highest in vitro catalytic activity among the f-oxidation enzymes. The subcellular localization of fl-oxidation of fatty acids in plant cells has been a point of some controversy. It was initially assumed that the f-oxidation sequence was localized within mitochondria (14), as was thought to be the case at that time in animal tissues. It was then demonstrated that in the endosperm ofgerminating castor oil seeds the f-oxidation enzymes were colocalized with the enzymes of the glyoxylate cycle within specialized peroxisomes (6). Subsequently, it was demonstrated that fl-oxidation has a dual localization in animal cells: mitochondria and peroxisomes (18). However, despite occasional reports to the contrary (e.g. ref. 19), for many years the consensus has been that there is no fl-oxidation in plant mitochondria and that this pathway in plant cells is localized exclusively within peroxisomes (reviewed in ref. 14). Thomas and associates (21, 30, 35) reported that mitochondria from germinating pea seedlings were capable of the foxidation of fatty acids, and that this process was absolutely dependent upon added carnitine. Carnitine dependence is one This research was supported in part by the Newcastle University Small Grants Committee. J. A. M. was a collaborator via a fellowship under Organisation for Economic Co-operation and Development Project on Food Production and Preservation. 2 Permanent address: Seed Biosynthesis Research Unit, U.S. Department of Agriculture, Agricultural Research Service, Northern Regional Research Center, Peoria, IL 61604. MATERIALS AND METHODS