The Effect of Carnitine on Palmitate Oxidation by Pea Cotyledon Mitochondria

McNeil, Philip H.; Thomas, David

doi:10.1093/jxb/27.6.1163

Cited by 29 publications

(7 citation statements)

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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: Resultssupporting

confidence: 89%

“…Protein concentrations were estimated by the method of Bradford (4), using purified fraction V BSA (21) as the standard.…”

Section: Other Analytical Methodsmentioning

confidence: 99%

“…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 Octenoyl-CoA was prepared by the mixed anhydride method as described by Miernyk and Trelease (24).…”

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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%

“…Protein concentrations were estimated by the method of Bradford (4), using purified fraction V BSA (21) as the standard.…”

Section: Other Analytical Methodsmentioning

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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“…Pea seeds (Pisum sativum L. cv. Bunting) were a generous gift of Batchelors' Foods, Worksop, Notts, England, and germinated as previously described [23].…”

Section: Biological Materialsmentioning

confidence: 99%

AtJ1, a mitochondrial homologue of theEscherichia coli DnaJ protein

et al. 1996

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The nucleotide sequence of a cDNA clone from Arabidopsis thaliana ecotype Columbia was determined, and the corresponding amino sequence deduced. The open reading frame encodes a protein, AtJ1, of 368 residues with a molecular mass of 41 471 Da and an isoelectric point of 9.2. The predicted sequence contains regions homologous to the J- and cysteine-rich domains of Escherichia coli DnaJ, but the glycine/phenylalanine-rich region is not present. Based upon Southern analysis, Arabidopsis appears to have a single atJ1 structural gene. A single species of mRNA, of 1.5 kb, was detected when Arabidopsis poly(A)+ RNA was hybridized with the atJ1 cDNA. The function of atJ1 was tested by complementation of a dnaJ deletion mutant of E. coli, allowing growth in minimal medium at 44 degrees C. The AtJ1 protein was expressed in E. coli as a fusion with the maltose binding protein. This fusion protein was purified by amylose affinity chromatography, then cleaved by digestion with the activated factor X protease. The recombinant AtJ1 protein was purified to electrophoretic homogeneity. In vitro, recombinant AtJ1 stimulated the ATPase activity of both E. coli DnaK and maize endosperm cytoplasmic Stress70. The deduced amino acid sequence of AtJ1 contains a potential mitochondrial targeting sequence at the N-terminus. Radioactive recombinant AtJ1 was synthesized in E. coli and purified. When the labeled protein was incubated with intact pea cotyledon mitochondria, it was imported and proteolytically processed in a reaction that depended upon an energized mitochondrial membrane.

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“…This occurs through the reversible exchange of carnitine and CoASH on acetate moieties by a carnitine transferase present in both organelles, and by carnitine translocase activities. The discovery of (acetyl-, octanoyl-or palmitoyl-) transferase activities in preparations of mitochondria and chloroplasts from seedlings of legume species (McNeil and Thomas 1976;Wood et al 1983Wood et al , 1984McLaren et al 1985; Thomas and Wood 1986;Burgess and Thomas 1986;Gerbling and Gerhardt 1988;Schwabedissen-Gerbling and Gerhardt 1995;Wood 2000a, 2009) and the quantiWcation of acylcarnitines alongside free carnitine in tissues of Arabidopsis thaliana and other plant species (Bourdin et al 2007) demonstrate the involvement of carnitine in plant lipid metabolism. The link between carnitine and FA appears to be partly inherent to a mitochondrial -oxidation process in plant cells (Panter and Mudd 1973;Thomas and Wood 1986;Masterson et al 2000) connected to some speciWc metabolic requirements, notably for chloroplast maturation during the greening of the emerging plumules of pea seedlings (Thomas et al 1981;Wood et al 1992a, b;Wood 2000b, 2009).…”

Section: Introductionmentioning

confidence: 99%

The effect of carnitine on Arabidopsis development and recovery in salt stress conditions

Charrier

Rippa

et al. 2011

Planta

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Carnitine exists in all living organisms where it plays diverse roles. In animals and yeast, it is implicated in lipid metabolism and is also associated with oxidative stress tolerance. In bacteria, it is a major player in osmotic stress tolerance. We investigate the carnitine function in plants and our present work shows that carnitine enhances the development and recovery of Arabidopsis thaliana seedlings subjected to salt stress. Biological data show that exogenous carnitine supplies improve the germination and survival rates of seedlings grown on salt-enriched medium, in a manner comparable to proline. Both compounds are shown to improve seedling survival under oxidative constraint meaning that they may act on salt stress through antioxidant properties. A transcriptome analysis of seedlings treated with exogenous carnitine reveals that it modulates the expression of genes involved in water stress and abscisic acid responses. Analyses of the abscisic acid mutants, aba1-1 and abi1-1, indicate that carnitine and proline may act through a modulation of the ABA pathway.

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The Effect of Carnitine on Palmitate Oxidation by Pea Cotyledon Mitochondria

Cited by 29 publications

References 1 publication

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

AtJ1, a mitochondrial homologue of theEscherichia coli DnaJ protein

The effect of carnitine on Arabidopsis development and recovery in salt stress conditions

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