Carnitine long-chain acyltransferase and oxidation of palmitate, palmitoyl coenzyme A and palmitoylcarnitine by pea mitochondria preparations

Self Cite

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

Section: Resultssupporting

confidence: 89%

“…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).…”

mentioning

confidence: 99%

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

Miernyk

Thomas²,

Wood³

1991

Self Cite

“…Mammalian mitochondria also carry out the oxidation of fatty acids and are thought to contain separate pools of CoA for Krebs cycle activity and fl-oxidation (22). The ability of plant mitochondria to oxidize fatty acids is controversial (26) and may be tissue specific; however, they do contain carnitine: acetyltransferase activity (5, 23, and this report). The role of carnitine and carnitine:acetyltransferase in modulating acetylCoA inhibition in vivo remains to be elucidated.…”

Section: Resultsmentioning

confidence: 95%

Acetyl-Coenzyme A Can Regulate Activity of the Mitochondrial Pyruvate Dehydrogenase Complex in Situ

Budde

Fang²,

Randall³

et al. 1991

In vitro, the pyruvate dehydrogenase complex is sensitive to product inhibition by NADH and acetyl-coenzyme A (CoA). Based upon Km and K1 relationships, it was suggested that NADH can play a primary role in control of pyruvate dehydrogenase complex activity in vivo (JA Miernyk, DD Randall [19871 Plant Physiol 83: 306-310). We have now extended the in vitro studies of product inhibition by assaying pyruvate dehydrogenase complex activity in situ, using purified intact mitochondria from green pea (Pisum sativum) seedlings. In situ activity of the pyruvate dehydrogenase complex is inhibited when mitochondria are incubated with malonate. In some instances, isolated mitochondria show an apparent lack of coupling during pyruvate oxidation. The inhibition by malonate, and the apparent lack of coupling, can both be explained by an accumulation of acetyl-CoA. Inhibition could be alleviated by addition of oxalacetate, high levels of malate, or Lcamitine. The CoA pool in nonrespiring mitochondria was approximately 150 micromolar, but doubled during pyruvate oxidation, when 60 to 95% of the total was in the form of acetyl-CoA. Our results indicate that in situ activity of the mitochondrial pyruvate dehydrogenase complex can be controlled in part by acetyl-CoA product inhibition.The mPDC' occupies a crucial branch point in intermediary metabolism, and activity is regulated by several mechanisms including compartmentation, product and metabolite inhibition, and reversible phosphorylation (reviewed in refs. 13,17). Initial studies of the regulatory properties of plant mPDC have been conducted in vitro using preparations that had been purified 20-to 300-fold (18-21). Recently, we (2-4, 16) have developed a method for assaying mPDC activity in situ using purified intact pea seedling mitochondria. Using this method, several observations on the regulation by reversible phosphorylation have been clarified and extended (2-4). This report continues our investigation of the role of product inhibition in regulation of PDC activity within intact mitochondria.Much like mammalian and microbial PDCs, plant mPDCs

“…In the simplest case of unsaturated fatty acids this is represented by a carboxyl group; in LPC, PAF, and AC it is a more elaborate structure exhibiting negative and positive charges contributed by phosphate or carboxyl and trimethylamine groups, respectively. The physiological function of AC is the transfer of fatty acids through the inner mitochondrial membranes, which probably involves a translocating protein (24). EB is a cyclic hexapeptide antibiotic with a stearic acid side chain, derived from various species ofAspergillus (1 1).…”

Section: Resultsmentioning

confidence: 99%

Influence of Free Fatty Acids, Lysophosphatidylcholine, Platelet-Activating Factor, Acylcarnitine, and Echinocandin B on 1,3-β-d-Glucan Synthase and Callose Synthesis

Kauss¹,

Jeblick²

1986

ABSTRACIDeposition of callose to the cell wall is characteristic of many developmental processes and also occurs during cell or tissue repair mechanism. For example, pollen maturation, sieve pore formation and sealing, pollen tube growth and rejection, gravitropism, virus localization, and the formation of papillae at the site ofattempted penetration by fungi are some relevant processes (2,5,8,13). The major and most characteristic component of callose is 1,3-fl-glucan, which can easily be detected cytochemically and also determined quantitatively by its fluorescence with aniline blue (13). Our recent attempts to understand the regulation of callose formation have therefore concerned studies of the biosynthesis of 1,3-,B-glucan.