Clifford Wood

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

Palmitoylcarnitine was oxidised by pea mitochondria.l-carnitine was an essential addition for the oxidation of palmitate or palmitoylCoA. When palmitate was sole substrate, ATP and Mg2+ were also essential additives for maximum oxidation. Additions of CoA inhibited the oxidation of palmitate. It was shown that CoA was acting as a competitive inhibitor of the carnitine-stimulated O2 uptake. It is suggested that palmitoylacarnitine and carnitine passed through the mitochondrial barrier with ease but palmitoylCoA and CoA did not. The presence of carnitine long-chain acyl (palmitoyl)transferase (EC 2.3.1.21) in pea-cotyledon mitochondria was shown. This enzyme may play a role in the transport of long-chain acyl groups through membrane barriers.

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

Abstractβ-Oxidation enzymes were detected both in the mitochondria and microbodies of pea cotyledons. Intact mitochondria did not show β-oxidation enzyme activity but in ruptured mitochondria this activity was high. It is apparent that the mitochondrial membrane barrier prevents rapid access of acyl-CoA substrates to matrix β-oxidation sites. Removal of the membrane barrier permits rapid access of acyl-CoAs and these enzyme activities may then be measured.

The two β-oxidation sites in pea cotyledons : Carnitine palmitoyltransferase: location and function in pea mitochondria.

Two sites for β-oxidation of fatty acids in pea (Pisum sativum L.) cotyledons exist. One site is the microbody, the other the mitochondrion. Mitochondrial β-oxidation of fatty acids is carnitine-dependent. The fatty acid permeates the membrane as palmitoylcarnitine which is formed from cytosolic-side palmitoyl-CoA by a carnitine palmitoyltransferase located on the exterior face of the inner mitochondrial membrane as a peripheral protein. A single-gated pore integral membrane translocator is proposed to exchange the palmitoylcarnitine for carnitine or acetylcarnitine across the membrane. An internal (matrix side) carnitine palmitoyltransferase then reforms palmitoyl-CoA which enters β-oxidation and subsequently the tricarboxylic-acid cycle.

The synthesis of palmitoylcarnitine by etio-chloroplasts of greening barley leaves.

CoASH, Mg2+, ATP and (-)-carnitine were found to be essential for the production of palmitoylcarnitine from palmitate by purified barley etio-chloroplasts. It was concluded that long-chain acyl CoA synthetase (palmitoyl CoA synthetase, EC 6.2.1.3) and carnitine long-chain acyl-transferase (carnitine palmitoyltransferase, EC 2.3.1.21) activity were present in the etio-chloroplasts. It is suggested that the long-chain acylcarnitine formed may move more easily through membrane barriers than the long-chain acyl CoA compound. Also or alternatively this enzyme may spare CoA by transferring long-chain acyl groups from long-chain acyl CoA to carnitine.

Carnitine acyltransferases in chloroplasts of Pisum sativum L.

Carnitine-acetyltransferase (EC 2.3.1.7) and carnitine-palmitoyltransferase (EC 2.3.1.21) activities were shown to be present in chloroplasts of green pea leaves and possibly to occur in leaf mitochondrial and peroxisomal fractions. A role for the enzymes in the transfer of acyl groups across membranes is suggested.

AtJ1, a mitochondrial homologue of the escherichia coli DnaJ protein

The nucleotide sequence of a cDNA clone fromArabidopsis 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 ofEscherichia coli DnaJ, but the glycine/phenylalanine-rich region is not present. Based upon Southern analysis,Arabidopsis appears to have a singleatJ1 structural gene. A single species of mRNA, of 1.5 kb, was detected whenArabidopsis poly(A)+ RNA was hybridized with theatJ1 cDNA. The function ofatJ1 was tested by complementation of adnaJ deletion mutant ofE. coli, allowing growth in minimal medium at 44°C. The AtJ1 protein was expressed inE. 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 bothE. 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 inE. 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.

Carnitine short-chain acyltransferase in pea mitochondria.

Carnitine acetyltransferase was shown to be present in pea-cotyledon mitochondria. Acetyl-carnitine may well be exported, without excessive energy loss, from mitochondrial matrix sites to extra-mitochondrial sites.

The synthesis of short- and long-chain acylarnitine by etio-chloroplasts of greening barley leaves

Etio-chloroplasts of barley, purified on sucrose density gradients were shown to possess carnitine long-chain acyltransferase (carnitine palmitoyltransferase, EC 2.3.1.21) activity and carnitine short-chain acyltransferase (carnitine acetyltransferase EC 2.3.1.7) activity. These enzymes may play a role in the transport of acyl groups as acylcarnitines through the membrane barrier of barley etio-chloroplasts and also ‘or alternatively’ may spare CoA by transferring short- and long-chain acyl groups from short-and long-chain acyl CoA to carnitine.

Mitochondrial and peroxisomal beta-oxidation capacities of organs from a non-oilseed plant.

Until recently, β–oxidation was believed to be exclusively located in the peroxisomes of all higher plants. Whilst this is true for germinating oilseeds undergoing gluconeogenesis, evidence demonstrating mitochondrial β–oxidation in other plant systems has refuted this central dogma of plant lipid metabolism. This report describes a comparative study of the dual mitochondrial and peroxisomal β–oxidation capacities of plant organs. Oxidation of [1–14C] palmitate was measured in the cotyledons, plumules and radicles of Pisum sativum L., which is a starchy seed, over a 14 day period from the commencement of imbibition. Respiratory chain inhibitors were used for differentiating between mitochondrial and peroxisomal β–oxidation. Peroxisomal β–oxidation gave a steady, baseline rate and, in the early stages of seedling development, accounted for 70–100% of the β–oxidation observed. Mitochondrial β–oxidation gave peaks of activity at days 7 and 10–11, accounting for up to 82% of the total β–oxidation activity at these times. These peaks coincide with key stages of seedling development and were not observed when normal development was disrupted by growth in the dark. Peroxisomal β–oxidation was unaffected by etiolation. Since mitochondrial β–oxidation was overt only during times of intense biosynthetic activity it might be switched on or off during seedling development. In contrast, peroxisomes maintained a continuous, low β-oxidation activity that could be essential in removing harmful free fatty acids, e.g. those produced by protein and lipid turnover.

Long-chain acyl CoA synthetase, carnitine and β-oxidation in the pea-seed mitochondrion.

Mitochondria from pea (Pisum sativum L.) seeds were separated into two fractions, mitoplasts (intact inner membrane) and the outer-membrane fraction. The mitoplasts only oxidised palmitate in the presence of carnitine and added outermembrane fraction. Mitoplasts were able to oxidise palmitoylCoA in the presence of carnitine and added outer-membrane fraction had no effect on this oxidation. It was concluded that a long-chain acylCoA synthetase (EC 6.2.1.3) was located on the outer membrane and that the activity of this enzyme in assays was more than sufficient to account for any observed rate of O2 uptake during palmitate oxidation by pea mitochondria. The location of carnitine long-chain acyltransferase (carnitine palmitoyl transferase EC 2.3.1.21) would appear to be the mitoplast i.e. the inner mitochondrial membrane, and confirms the previous work at Newcastle.