The dual location of ?-oxidation enzymes in germinating pea cotyledons

Wood, C.; Burgess, N.; Thomas, David

doi:10.1007/bf00446368

Cited by 28 publications

(19 citation statements)

References 15 publications

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“…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). When mitochondria isolated by centrifugation in sucrose density gradients are added to cuvettes for spectrophotometric enzyme assays, sufficient sucrose is carried along to maintain Having established the occurrence of both mitochondrial and peroxisomal isozymes in 2 to 3 d germinated pea seedlings, a partial purification of the EH isozymes was accomplished by disruption of the organelle-enriched fractions, removal of membranous material by centrifugation, (NH4)2SO4 fractionation, and gel permeation chromatography (Table II).…”

Section: Resultssupporting

confidence: 89%

“…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: Resultssupporting

confidence: 89%

“…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).…”

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confidence: 99%

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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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Section: Resultssupporting

confidence: 89%

Section: Resultssupporting

confidence: 89%

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confidence: 99%

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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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“…Although we do not exclude the possibility that a (3-oxidation activity is also present in the mitochondria (23), it appears likely that the activities observed in the present work are peroxisomal because the assay conditions used (high CoA concentration and absence of carnitine in the overall (3-oxidation assay, high osmotic pressure of the media in all enzyme assays) would not allow the detection of the mitochondrial (-oxidation activities (32). Moreover, the simultaneous increase of catalase, an ancillary enzyme of fl-oxidation that degrades the H202 formed at the oxidase step, and of malate synthase, a glyoxylate cycle enzyme, suggests that glucose starvation is associated with a general increase of peroxisomal activities.…”

Section: Discussionmentioning

confidence: 77%

Increased Fatty Acid β-Oxidation after Glucose Starvation in Maize Root Tips

Dieuaide¹,

Brouquisse²,

Pradet³

et al. 1992

Plant Physiol.

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The effects of glucose starvation on the oxidation of fatty acids were studied in excised maize (Zea mays L.) root tips. After 24 hours of glucose starvation, the rate of oxidation of palmitic acid to CO2 by the root tips was increased 2.5-fold. Different enzyme activities were tested in a crude particulate fraction from nonstarved root tips and those starved for 24 hours. It is usually thought that carbohydrates are the major respiratory substrates of plant cells, whereas lipids and proteins make a negligible contribution to the respiratory carbon flux (1). However, there is growing evidence that the carbohydrate reserves quickly decrease to low levels in darkness (4,20). This suggests that carbohydrates may become limiting, and the contribution of lipids and proteins to respiration increase, not only in senescing tissues (1) but also during the normal plant life.The metabolic consequences of carbohydrate starvation have been studied in a number of plant species. The depletion of intracellular carbohydrate is associated with a decrease of the respiration rate and a decline in the respiratory quotient from 1 to 0.75. After a long period of starvation, a delay is needed for the recovery of respiration (27) and fermentation (28). These results suggested that cellular components were degraded to sustain respiration. More recently, the decrease of total proteins and fatty acids and increasing levels of phosphorylcholine (9) and asparagine (12) provided more direct evidence for phospholipid and protein breakdown during sugar starvation in sycamore cells in culture. Similar results were obtained with maize root tips, in which proteins and fatty acids were also found to decrease during starvation (5). These results suggested that, during starvation, fatty acids would be degraded by ,8-oxidation, thus producing acetylCoA necessary to the tricarboxylic acid cycle. However the role of,-oxidation is not well established. This pathway has been generally associated with neoglucogenesis, but it has been recently shown that, in lettuce embryos, the ,8-oxidation of fatty acids directly feeds the tricarboxylic acid cycle, providing the substrate for respiration during the early steps of germination (29). It can be hypothesized that ,8-oxidation could play a similar role in sugar-starved tissues.The cellular localization of ,3-oxidation is still a matter of debate. It was considered as exclusively peroxisomal (14, 17), but previous works indicating a mitochondrial localization of 13-oxidation (33) have been recently reinforced (24). In the roots of maize seedlings (13), the activities of the four 13-oxidation enzymes have been previously detected in the peroxisomal, not in the mitochondrial, fraction.The aim of the present work was to determine whether the overall 13-oxidation pathway was functional in maize root tips and to compare the peroxisomal 1-oxidation and glyoxylic cycle activities before and after a starvation treatment. Our results indicate that the 13-oxidation of palmitic acid is active in maize root tips and...

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“…Recent works have reported that the presence of the /3-oxidation system is exclusively localized in peroxisomes from the non-fatty tissues of potato tubers, mung bean hypocotyls, Jerusalem artichoke tubers and algae (Gerhardt 1983, Macey and Stumpf 1983, Stabenau et al 1984, although some authors claim that mitochondria are involved as in mammalian cells (Wood et al 1986).…”

Section: Introductionmentioning

confidence: 99%

Localisation of β‐oxidation enzymes in peroxisomes of rice coleoptiles

Pistelli¹,

Rascio

Bellis³

et al. 1989

Physiologia Plantarum

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1989. Localisation of/S-oxidation enzytnes in peroxisomes of rice coleoptiles. -Physiol. Plant. 76: 177-148. Enzymes of the /3-oxidation pathway in rice {Oryza sativa L., cv. Arborio) coleoptiles were investigated. The coleoptiles contain acyl-CoA oxidase (EC 1.3.99.3), 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35), enoyl-CoA hydratase (EC 4.2.1.17)and thiolase (EC 2.3.1.9). Analysis of coleoptile homogenates by sucrose density fractionation showed a preferential distribution of these enzymes in the unspecialized peroxisomes. The enzymatic activity found in the mitochondrial fraction was due to peroxisomal contamination since electron micrographs show the peroxisomes to be intact and pure whereas the mitochondrial fraction was contaminated by other organelles. It appears that the /3-oxidation pathway is localized in the unspecialized peroxisomes of rice coleoptiles, extending the number of plant species in which such a localization has been observed.

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The dual location of ?-oxidation enzymes in germinating pea cotyledons

Cited by 28 publications

References 15 publications

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

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

Increased Fatty Acid β-Oxidation after Glucose Starvation in Maize Root Tips

Localisation of β‐oxidation enzymes in peroxisomes of rice coleoptiles

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