Localisation of β‐oxidation enzymes in peroxisomes of rice coleoptiles

Pistelli, Laura; Rascio, Nicoletta; Bellis, Luigi De; Alpi, Amedeo

doi:10.1111/j.1399-3054.1989.tb05623.x

Cited by 13 publications

(4 citation statements)

References 19 publications

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“…In our studies the overall rate of b-oxidation determined by NADH formation in the presence of palmitoyl-CoA was not blocked by inhibitors of the mitochondrial electron transport chain, which con®rms data reported for Ricinus communis L., Gossypium hirsutum L. and Helianthus annuus L. (Gerhardt 1992). Isolated glyoxysomes of rape seedlings exhibit acyl-CoA oxidase activity which has recently been characterized (Kock et al 1995) and, in addition, the amounts of activity of the enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogase and thiolase relative to the acyl-CoA oxidase activity (51:18:0.9:1) are in good agreement with previous data reported for the glyoxysomal/peroxisomal b-oxidation enzyme systems (Pistelli et al 1989, Gerhardt 1992. Our results also demonstrate that in rape seed cotyledons b-oxidation of fatty acids seems to be con®ned to glyxysomes.…”

Section: Discussionsupporting

confidence: 87%

Enzymes for lipolysis and fatty acid metabolism in different organelle fractions from rape seed cotyledons

Hoppe

Theimer

1997

Planta

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This study addresses the still open question of whether or not in oily storage tissues, e.g. cotyledons of germinating rape (Brassica napus L.) seedlings' lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) and the b-oxidation system of fatty acids are located in one or more membrane-bounded organelles. The organelles were isolated carefully and identi®ed by marker-enzyme activities. Activities of neither lipase nor acylester acylhydrolase (EC 3.1.1) could be detected either in glyoxysomes or in mitochondria, even when various substrate emulsions were employed. Only after longterm incubations could the presence of a low lipolytic activity be demonstrated for dierent organellar fractions. This alkaline carboxylic ester hydrolase, whose activity is below the detection limit of various standard tests, cannot play a role in the lipolytic function of glyoxysomes. In addition, a complete set of enzyme activities necessary for the conversion of saturated fatty acids to acetyl CoA was found only in the glyoxysomal cell fraction. The low b-oxidation activity discovered in the mitochondrial cell fraction is evidently due to glyoxysomal contamination. Enzyme activities unique to the mitochondrial b-oxidation system such as carnitine palmitoyltransferase (EC 2.3.1.21), carnitine acetyltransferase (EC 2.3.1.7), and acyl-CoA dehydrogenase (EC 1.3.99.3) were absent, indicating that mitochondria are not involved in fatty acid metabolism. In addition, on Western blots, antibodies raised against malate synthase (EC 4.1.3.2) and acyl-CoA oxidase (EC 1.1.3) recognized three polypeptides with molecular masses of 45, 63, and 70 kDa only in glyoxysomal fractions. Obviously, in the fatty rape seed neither glyoxysomes nor mitochondria are involved in triacylglycerol hydrolysis, and b-oxidation of fatty acids occurs exclusively in glyoxysomes.

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Section: Discussionsupporting

confidence: 87%

Enzymes for lipolysis and fatty acid metabolism in different organelle fractions from rape seed cotyledons

Hoppe

Theimer

1997

Planta

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“…The activities of the ,-oxidation enzymes were approximately equally distributed in both organellar fractions (Table I). This result is in marked contrast to reports from other workers on the localization of the $-oxidation enzymes in higher plant cells (6,12,15,27), but is consistent with previous publications from this laboratory (21,30,32,34,35). It has been noted that the substrates of the ,8-oxidation enzymes do not easily cross the intact mitochondrial inner membrane, thus this membrane must be disrupted before detecting enzyme activity (34).…”

Section: Resultscontrasting

confidence: 57%

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

Miernyk

Thomas²,

Wood³

1991

Plant Physiol.

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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

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“…Unspecialized peroxisomes are present in achlorophyllous and non-oil storage tissue; they contain the peroxisomal enzymes catalase, glycolate oxidase and urate oxidase. The function of unspecialized peroxisomes is unknown, though they have been shown unequivocally to contain the fl-oxidation enzymes (Gerhardt 1986;Pistelli et al 1989). Glyoxysomes, common-* To whom correspondence should be addressed ly found in oil-rich tissue of seeds, contain fatty-acid fl-oxidation and glyoxylate-cycle enzymes in addition to the characteristic peroxisomal enzymes, and play a major role in the mobilization of fat reserve.…”

Section: Introductionmentioning

confidence: 98%

Localization of glyoxylate-cycle marker enzymes in peroxisomes of senescent leaves and green cotyledons

Bellis

Picciarelli

Pistelli

et al. 1990

Planta

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Crude particulate homogenates from leaves of barley (Hordeum vulgare L.), rice (Oryza sativa L.), leaf-beet (Beta vulgaris var. cicla L.) and pumpkin (Cucurbita pepo L.) cotyledons were separated on sucrose density gradients. The peroxisomal fractions appeared at a buoyant density of 1.25 g·cm(-3) and contained most of the activities of catalase (EC 1.11.1.6), and hydroxypyruvate reductase (EC 1.1.1.81) on the gradients. In peroxisomal fractions from detached leaves and green cotyledons incubated in permanent darkness we detected the presence of isocitrate lyase (EC 4.1.3.1) and malate synthase (EC 4.1.3.2), key enzymes of the glyoxylate cycle, and β-oxidation activity (except in pumpkin). As proposed by H. Gut and P. Matile (1988, Planta 176, 548-550) the glyoxylate cycle may be functional during leaf senescence, and the presence of two key enzymes indicates a transition from leaf peroxisome to glyoxysome; for pumpkin cotyledons in particular a double transition occurs (glyoxysome to leaf peroxisome during greening, and leaf peroxisome to glyoxysome during senescence).

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Localisation of β‐oxidation enzymes in peroxisomes of rice coleoptiles

Cited by 13 publications

References 19 publications

Enzymes for lipolysis and fatty acid metabolism in different organelle fractions from rape seed cotyledons

Enzymes for lipolysis and fatty acid metabolism in different organelle fractions from rape seed cotyledons

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

Localization of glyoxylate-cycle marker enzymes in peroxisomes of senescent leaves and green cotyledons

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