Fatty Acid Beta-oxidation Enzymes Research Articles

Novel fatty acid beta-oxidation enzymes in rat liver mitochondria. I. Purification and properties of very-long-chain acyl-coenzyme A dehydrogenase.

Freeze-thawed rat liver mitochondria were extensively washed with potassium phosphate, pH 7.5, and the residue was extracted with 10 mM potassium phosphate, pH 7.5, 1% (w/v) sodium cholate, 0.5 M KCl. The four beta-oxidation enzyme activities of the washes and the last extract were assayed with substrates of various carbon chain lengths. Our data suggest that the last extract contains a novel acyl-CoA dehydrogenase and long-chain 3-hydroxyacyl-CoA dehydrogenase. A novel acyl-CoA dehydrogenase was purified. The molecular masses of the native enzyme and the subunit were estimated to be 150 and 71 kDa, respectively. One mole of enzyme contained 2 mole of FAD. These properties and immunochemical properties of the enzyme differed from those of three other acyl-CoA dehydrogenases: short-, medium-, and long-chain acyl-CoA dehydrogenases. Carbon chain length specificity of the enzyme differed from that of other acyl-CoA dehydrogenases. The enzyme was active toward CoA esters of long- and very-long-chain fatty acids, but not toward those of medium- and short-chain fatty acids. The specific enzyme activity was greater than 10 times that of long-chain acyl-CoA dehydrogenase when palmitoyl-CoA was used as substrate. We propose the name "very-long-chain acyl-CoA dehydrogenase" for this enzyme.

Peroxisomal disorders: Complementation analysis using beta-oxidation of very long chain fatty acids

Complementation studies, using fused cell lines from patients with peroxisomal disorders, have shown correction of defective plasmalogen synthesis and phytanic acid oxidation as well as an increase in the number of peroxisomes. At least six complementation groups have been reported. We demonstrate here that complementing cell lines also acquire the ability to oxidize very long chain fatty acids (VLCFA), and that complementation groups defined with this technique are identical to those reported previously when plasmalogen synthesis was used as the criterion for complementation. This VLCFA complementation technique is of particular value in the study of patients in whom defective VLCFA is the only or major enzymatic defect, and we show complementation between cell lines from two patients each with an isolated defect in one of the peroxisomal fatty acid beta-oxidation enzymes.

Peroxisomes induced in Candida Boidinii by methanol, oleic acid and d-alanine vary in metabolic function but share common integral membrane proteins

Peroxisomes massively proliferate in the methylotrophic yeast Candida boidinii when cultured on methanol as the only carbon and energy source. These organelles contain enzymes that catalyze the initial reactions of methanol utilization. The membranes contain abundant proteins of unknown function; their apparent molecular masses are 20, 31, 32 and 47 x 10(3) Mr and are termed PMP20, PMPs31-32 and PMP47. Recently, we reported that peroxisomes in this yeast are also induced by oleic acid and D-alanine as carbon sources, and that these peroxisomes contain increased concentrations of the enzymes of fatty acid beta-oxidation or D-amino acid oxidase, respectively. This report extends these findings and further compares the enzyme composition from peroxisomes induced by methanol, oleic acid and D-alanine. the patterns of matrix proteins represented on SDS-polyacrylamide gels from peroxisomes induced by oleic acid or D-alanine were found to be very different from those of peroxisomes induced by methanol. In order to differentiate between membrane proteins that have specific functions in pathways of substrate utilization from those with more generalized functions, peroxisomal membranes from cultures grown on methanol, oleic acid or D-alanine were purified. Analysis of these fractions demonstrated that while PMP20 is found only in peroxisomes induced by methanol, the PMPs31-32 and PMP47 were the abundant peroxisomal membrane proteins (PMP) regardless of inducing substrate. The data strongly suggest that the function of PMP20 is related to methanol metabolism. In contrast, the functions of PMPs31-32 and PMP47 are 'substrate-nonspecific'. We speculate that they may relate to the structure, assembly or general function of the organelle.

Purification and characterization of fatty acid beta-oxidation enzymes from Caulobacter crescentus

Acetoacetyl coenzyme A (acetoacetyl-CoA) thiolase, an enzyme required for short-chain fatty acid degradation, has been purified to near homogeneity from Caulobacter crescentus. The relative heat stability of this enzyme allowed it to be separated from beta-ketoacyl-CoA thiolase. The purification scheme minus the heating step also permitted the copurification of crotonase and 3-hydroxyacyl-CoA dehydrogenase. These activities are in a multienzyme complex in Escherichia coli, but a similar complex was not observed in C. crescentus. Instead, separate proteins differing in enzymatic activity were detected, analogous to the beta-oxidation enzymes that have been isolated from Clostridium acetobutylicum and from mitochondria of higher eucaryotes. In these cells, as appears to be the case with C. crescentus, the individual enzymes form multimers of identical subunits.

Fatty acid metabolism in renal ischemia

The increase in free fatty acids in the ischemic tissue is a consistent observation and these free fatty acids are considered to play a role in the cellular toxicity. To elucidate the cause of higher levels of free fatty acids in ischemic tissue, we examined the catabolism of fatty acids. The beta-oxidation of lignoceric (24:0), palmitic (16:0) and octanoic (8:0) acids and the peroxidation of fatty acids were measured at different times of renal ischemia in whole kidney homogenate. The enzymatic activities for the oxidation of fatty acids decreased with the increase in ischemia time. However, the lipid peroxide levels increased 2.5-fold of control with ischemic injury. Sixty min of ischemia reduced the rate of oxidation of octanoic, palmitic and lignoceric acids by 57, 59 and 69%, respectively. Almost similar loss of fatty acid oxidation activity was observed in the peroxisomes and mitochondria. These data suggest that loss of mitochondrial and peroxisomal fatty acid beta-oxidation enzyme activities from ischemic injury may be one of the factors responsible for the higher levels of free fatty acids.

Transcription regulation of peroxisomal fatty acyl-CoA oxidase and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase in rat liver by peroxisome proliferators.

The structurally diverse peroxisome proliferators ciprofibrate, clofibrate, and bis(2-ethylhexyl) phthalate [(EtHx)2 greater than Pht] increase the activities of hepatic catalase and peroxisomal fatty acid beta-oxidation enzymes in conjunction with profound proliferation of peroxisomes in hepatocytes. In order to delineate the level at which these enzymes are induced in the liver, the transcriptional activity of specific genes for fatty acyl-CoA oxidase (FAOxase) and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme (PBE), the first two enzymes of the peroxisomal beta-oxidation system, and for catalase were measured in isolated hepatocyte nuclei obtained from male rats following a single intragastric dose of ciprofibrate, clofibrate, or (EtHx)2 greater than Pht. All three peroxisome proliferators rapidly increased the rate of FAOxase and PBE gene transcription in liver, with near maximal rates (9-15 times control) reached by 1 hr and persisting until at least 16 hr after administration of the compound. FAOxase and PBE mRNA levels, measured by blot-hybridization analysis and FAOxase and PBE protein content, analyzed by immunoblotting, increased concurrently up to at least 16 hr following a single dose of peroxisome proliferator. The catalase mRNA level increased about 1.4-fold, but the transcription rate of the catalase gene was not significantly affected. The results show that the peroxisome proliferators clofibrate, ciprofibrate, and (EtHx)2 greater than Pht selectively increase the rate of transcription of peroxisomal fatty acid beta-oxidation enzyme genes. Whether the transcriptional effects are mediated by peroxisome proliferator-receptor complexes remains to be elucidated.

Specific changes in the protein composition of rat liver in response to the peroxisome proliferators ciprofibrate, Wy-14,643 and di-(2-ethylhexyl)phthalate.

The hypolipidaemic agents ciprofibrate and Wy-14,643 ([4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid) and the phthalate-ester plasticizer di-(2-ethylhexyl)-phthalate (DEHP), like other peroxisome proliferators, produce a significant hepatomegaly and induce the peroxisomal fatty acid beta-oxidation enzyme system together with profound proliferation of peroxisomes in hepatic parenchymal cells. Changes in the profile of liver proteins in rats following induction of peroxisome proliferation by ciprofibrate, Wy-14,643 and DEHP have been analysed by high-resolution two-dimensional gel electrophoresis. The proteins of whole liver homogenates from normal and peroxisome-proliferator-treated rats were separated by two-dimensional gel electrophoresis using isoelectric focusing for acidic proteins and nonequilibrium pH gradient electrophoresis for basic proteins. In the whole liver homogenates, the quantities of six proteins in acidic gels and six proteins in the basic gels increased following induction of peroxisome proliferation. Peroxisome proliferator administration caused a repression of three acidic proteins in the liver homogenates. By the immunoblot method using polyspecific antiserum against soluble peroxisomal proteins and monospecific antiserum against peroxisome proliferation associated Mr 80000 polypeptide (polypeptide PPA-80), the majority of basic proteins induced by these peroxisome proliferators appeared to be peroxisomal proteins. Polypeptide PPA-80 becomes the most abundant protein in the total liver homogenates of peroxisome-proliferator-treated rats. These results indicate that ciprofibrate, DEHP and Wy-14,643 induce marked changes in the profile of specific hepatic proteins and that some of these changes should serve as a baseline to identify a set of gene products that may assist in defining the specific 'peroxisome proliferator domain'.

Binding of the enzymes of fatty acid beta-oxidation and some related enzymes to pig heart inner mitochondrial membrane.

The binding of crotonase (enoyl-CoA hydratase), beta-hydroxyacyl-CoA dehydrogenase, beta-ketothiolase, succinyl-CoA transferase, and carnitine acetyltransferase to inner mitochondrial membranes, mitoplasts, intact mitochondria, erythrocyte membranes, and phosphatidylcholine liposomes was studied. Succinyl-CoA transferase does not bind to any of these membranes. Carnitine acetyltransferase, on the other hand, binds to all of these membranes. The enzymes of fatty acid beta-oxidation, thiolase, crotonase, and beta-hydroxyacyl-CoA dehydrogenase bind to inner membrane, but not to liposomes. The binding shows a moderate dependence on ionic strength (2-200 mM) and pH (6.5-8). These data indicate the possibility of an organization of the enzymes of fatty acid beta-oxidation on the inner mitochondrial membrane, but do not support the idea of an organization of the enzymes of ketone body catabolism.