Stereochemistry of the alpha-oxidation of 3-methyl-branched fatty acids in rat liver.

The stereochemistry of the alpha-oxidation of 3-methyl-branched fatty acids was studied in rat liver. R- and S-3-methylhexadecanoic acid were equally well alpha-oxidized in intact hepatocytes and homogenates. Subcellular fractionation studies showed that alpha-oxidation of both isomers is confined to peroxisomes. Dehydrogenation of 2-methylpentadecanal, the end-product of the peroxisomal alpha-oxidation of 3-methylhexadecanoic acid, to 2-methylpentadecanoic acid, followed by derivatization with R-1-phenylethylamine and subsequent separation of the stereoisomers by gas chromatography, revealed that the configuration of the methyl-branch is preserved throughout the whole alpha-oxidation process. Metabolism and formation of the 2-hydroxy-3-methylhexadecanoyl-CoA intermediate were also investigated. Separation of the methyl esters of the four isomers of 2-hydroxy-3-methylhexadecanoic acid was achieved by gas chromatography after derivatization of the hydroxy group with R-2-methoxy-2-trifluoromethylphenylacetic acid chloride and the absolute configuration of the four isomers was determined. Although purified peroxisomes are capable of metabolizing all four isomers of 2-hydroxy-3-methylhexadecanoyl-CoA, they can only form the (2S,3R) and the (2R,3S) isomers. Our experiments exclude the racemization of the 3-methyl branch during the alpha-oxidation process. The configuration of the 3-methyl branch does not influence the rate of alpha-oxidation, but determines the side of the 2-hydroxylation, hence the configuration of the 2-hydroxy-3-methylacyl-CoA intermediates formed during the process.

Differential regulation by a peroxisome proliferator of the different multifunctional proteins in guinea pig: cDNA cloning of the guinea pig D-specific multifunctional protein 2.

After our previous report on the cloning of two cDNA species in guinea pig, both encoding the same hepatic 79 kDa multifunctional protein 1 (MFP-1) [Caira, Cherkaoui-Malki, Hoefler and Latruffe (1996) FEBS Lett. 378, 57-60], here we report the cloning of a cDNA encoding a second multifunctional peroxisomal protein (MFP-2) in guinea-pig liver. This 2356 nt cDNA encodes a protein of 735 residues (79.7 kDa) whose sequence shows 83% identity with rat MFP-2 [Dieuaide-Noubhani, Novikov, Baumgart, Vanhooren, Fransen, Goethals, Vandekerckhove, Van Veldhoven and Mannaerts (1996) Eur. J. Biochem. 240, 660-666]. In parallel, we studied the effect of ciprofibrate, a hypolipaemic agent also known as peroxisome proliferator in rodent, on the expression of MFP-1 and MFP-2 (2.6 kb) in rats and guinea pigs. By Northern blotting analysis we demonstrated that three MFP-1-related mRNA species are expressed in the guinea-pig liver. The expression of two of them (3.5 and 2.6 kb) is slightly increased by ciprofibrate, whereas the 3.0 kb MFP-1 mRNA is, unlike the rat one, strongly down-regulated in guinea pigs treated with ciprofibrate. In a similar way, the hepatic expression of the guinea-pig 2.6 kb MFP-2 mRNA is also down-regulated in guinea pigs treated with ciprofibrate. These results demonstrate (1) that in contrast with the unique 3.0 kb MFP-1 rat mRNA, at least three hepatic MFP-1-related mRNA species are co-expressed in guinea pig; and (2) that, opposed to the accepted idea of non-responsiveness of the guinea pig to ciprofibrate, this drug affects MFP-1 and MFP-2 gene expression in this species. Also, the mRNA species for acyl-CoA oxidase and thiolase, two other enzymes of the peroxisomal beta-oxidation pathway that are induced severalfold in responsive species are down-regulated in guinea pig. This paper is the first, to our knowledge, reporting the down-regulation of the expression of genes encoding enzymes involved in the peroxisomal beta-oxidation of fatty acids (MFP-1) and bile acid synthesis (MFP-2) in mammals.

Evidence that multifunctional protein 2, and not multifunctional protein 1, is involved in the peroxisomal beta-oxidation of pristanic acid.

The second (enoyl-CoA hydratase) and third (3-hydroxyacyl-CoA dehydrogenase) steps of peroxisomal beta-oxidation are catalysed by two separate multifunctional proteins (MFPs), MFP-1 being involved in the degradation of straight-chain fatty acids and MFP-2 in the beta-oxidation of the side chain of cholesterol (bile acid synthesis). In the present study we determined which of the two MFPs is involved in the peroxisomal degradation of pristanic acid by using the synthetic analogue 2-methylpalmitic acid. The four stereoisomers of 3-hydroxy-2-methylpalmitoyl-CoA were separated by gas chromatography after hydrolysis, methylation and derivatization of the hydroxy group with (S)-2-phenylpropionic acid, and the stereoisomers were designated I-IV according to their order of elution from the column. Purified MFP-1 dehydrated stereoisomer IV but dehydrogenated stereoisomer III, so by itself MFP-1 is not capable of converting a branched enoyl-CoA into a 3-ketoacyl-CoA. In contrast, MFP-2 dehydrated and dehydrogenated the same stereoisomer (II), so it is highly probable that MFP-2 is involved in the peroxisomal degradation of branched fatty acids and that stereoisomer II is the physiological intermediate in branched fatty acid oxidation. By analogy with the results obtained with the four stereoisomers of the bile acid intermediate varanoyl-CoA, stereoisomer II can be assigned the 3R-hydroxy, 2R-methyl configuration.

The human peroxisomal multifunctional protein involved in bile acid synthesis: activity measurement, deficiency in Zellweger syndrome and chromosome mapping.

The dehydrogenation of 24R,25R-varanoyl-CoA, the physiological intermediate formed during the peroxisomal breakdown of the bile acid intermediate trihydroxycoprostanic acid, was studied in human liver. The reaction appeared to be catalyzed by two different enzymes. A first one, present in the cytosol, did not discriminate between the four possible varanoyl-CoA isomers and did not require the CoA moiety. The second enzymic activity was associated with peroxisomes and acted only on the 24R,25R-isomer, in which the 24-hydroxy group possesses the D-configuration. The D-specific dehydrogenase is part of a 79 kDa protein which represents the human counterpart of a recently discovered second multifunctional protein in rat liver peroxisomes, named multifunctional protein 2 (MFP-2). Human MFP-2, like its rat counterpart, is also responsible for the formation (by hydratation) of 24R,25R-varanoyl-CoA. A deficiency of MFP-2 in Zellweger liver could be demonstrated immunologically by using antibodies against the rat enzyme and enzymically -- after removal of the cytosol -- by using 24R,25R-varanoyl-CoA. The gene coding for MFP-2 was mapped to chromosome 5q2.3.

Identification and characterization of the 2-enoyl-CoA hydratases involved in peroxisomal beta-oxidation in rat liver.

In this study we attempted to determine the number of 2-enoyl-CoA hydratases involved in peroxisomal beta-oxidation. We therefore separated peroxisomal proteins from rat liver on several chromatographic columns and measured hydratase activities on the eluates with different substrates. The results indicate that rat liver peroxisomes contain two hydratase activities: (1) a hydratase activity associated with multifunctional protein 1 (MFP-1) (2-enoyl-CoA hydratase/delta 3, delta 2-enoyl-CoA isomerase/L-3-hydroxyacyl-CoA dehydrogenase) and (2) a hydratase activity associated with MFP-2 (17 beta-hydroxysteroid dehydrogenase/D-3-hydroxyacyl-CoA dehydrogenase/2-enoyl-CoA hydratase). MFP-1 forms and dehydrogenates L-3-hydroxyacyl-CoA species, whereas MFP-2 forms and dehydrogenates D-3-hydroxyacyl-CoA species. A portion of MFP-2 is proteolytically cleaved, most probably in the peroxisome, into a 34 kDa 17 beta-hydroxysteroid dehydrogenase/D-3-hydroxyacyl-CoA dehydrogenase and a 45 kDa D-specific 2-enoyl-CoA hydratase. Finally, the results confirm that MFP-1 is involved in the degradation of straight-chain fatty acids, whereas MFP-2 and its cleavage products seem to be involved in the degradation of the side chain of cholesterol (bile acid synthesis).

Sugar-Starvation-Induced Changes of Carbon Metabolism in Excised Maize Root Tips.

Excised maize (Zea mays L.) root tips were used to study the early metabolic effects of glucose (Glc) starvation. Root tips were prelabeled with [1-13C]Glc so that carbohydrates and metabolic intermediates were close to steady-state labeling, but lipids and proteins were scarcely labeled. They were then incubated in a sugar-deprived medium for carbon starvation. Changes in the level of soluble sugars, the respiratory quotient, and the 13C enrichment of intermediates, as measured by 13C and 1H nuclear magnetic resonance, were studied to detect changes in carbon fluxes through glycolysis and the tricarboxylic acid cycle. Labeling of glutamate carbons revealed two major changes in carbon input into the tricarboxylic acid cycle: (a) the phosphoenolpyruvate carboxylase flux stopped early after the start of Glc starvation, and (b) the contribution of glycolysis as the source of acetyl-coenzyme A for respiration decreased progressively, indicating an increasing contribution of the catabolism of protein amino acids, fatty acids, or both. The enrichment of glutamate carbons gave no evidence for proteolysis in the early steps of starvation, indicating that the catabolism of proteins was delayed compared with that of fatty acids. Labeling of carbohydrates showed that sucrose turnover continues during sugar starvation, but gave no indication for any significant flux through gluconeogenesis.

Further characterization of the peroxisomal 3-hydroxyacyl-CoA dehydrogenases from rat liver. Relationship between the different dehydrogenases and evidence that fatty acids and the C27 bile acids di- and tri-hydroxycoprostanic acids are metabolized by separate multifunctional proteins.

Recently, we purified five 3-hydroxyacyl-CoA dehydrogenases from isolated rat liver peroxisomal fractions. The enzymes were designated I-V according to their order of elution from the first column used in the purification procedure. Determination of the substrate (L- or D-hydroxyacyl-CoA) stereo-specificity and (de)hydratase measurements with the different 3-hydroxyacyl-CoA stereoisomers of straight-chain fatty acids and the bile acid intermediate trihydroxycoprostanic acid, immunoblotting analysis with antibodies raised against the different enzymes and peptide sequencing, all performed on enzymes I-V and molecular cloning of enzyme III revealed the following picture. Rat liver peroxisomes contain two multifunctional beta-oxidation proteins: (a) multifunctional protein 1 (the classical multifunctional protein; MFP-1) displaying 2-enoyl-CoA hydratase, L-3-hydroxyacyl-CoA dehydrogenase and delta 3, delta 2-enoyl-CoA isomerase activity (enzyme IV) and (b) multifunctional protein 2 (MFP-2) displaying 2-enoyl-CoA hydratase and D-3-hydroxyacyl-CoA dehydrogenase activity (enzyme III). Because of their substrate stereospecificity and because of the stereochemical configuration of the naturally occurring beta-oxidation intermediates, MFP-1 and MFP-2 appear to be involved in the beta-oxidation of fatty acids and bile acids intermediates, respectively. The deduced amino acid sequence of the cloned MFP-2 cDNA is highly similar to that of the recently described porcine endometrial estradiol 17 beta-dehydrogenase [Leenders, F., Adamski, J., Husen, B., Thole, H. H. & Jungblut, P. W. (1994) Eur. J. Biochem. 222, 221-227]. In agreement, MFP-2 also displayed estradiol 17 beta-dehydrogenase activity, indicating that MFP-2 and the steroid dehydrogenase are identical enzymes. MFP-2 is partially cleaved, most probably in vivo, in a estradiol 17 beta-dehydrogenase/D-3-hydroxyacyl-CoA dehydrogenase that forms a dimeric complex (enzyme I) and a hydratase. The physiological significance of enzyme I in bile acid synthesis (and steroid metabolism) remains to be determined. MFP-1 (enzyme IV) is artefactually cleaved during purification giving rise to 3-hydroxyacyl-CoA dehydrogenase V. 3-Hydroxyacyl-CoA dehydrogenase II is a mitochondrial contaminant similar to porcine and murine mitochondrial 3-hydroxyacyl-CoA dehydrogenase.

Quantification of compartmented metabolic fluxes in maize root tips using isotope distribution from 13C- or 14C-labeled glucose.

Metabolic pathways of the intermediate metabolism of maize root tips were identified and quantified after labeling to isotopic and metabolic steady state using glucose labeled on carbon-1, -2, or -6 with 14C or 13C. The specific radioactivity of amino acids and the 13C-specific enrichment of specific carbons of free glucose, sucrose, alanine and glutamate were measured and used to calculate metabolic fluxes. The non-triose pathways, including synthesis of polysaccharides, accumulation of free hexoses, and to a lesser extent starch synthesis, were found to consume 75% of the glucose entering the root tips. The cycle of synthesis and hydrolysis of sucrose was found to consume about 70% of the ATP produced by respiration. The comparison of the specific radioactivities of amino acids and phospholipid glycerol phosphate after labeling with [1-(14)C] or [6-(14)C]glucose revealed the operation of the pentose phosphate pathway. The transfer of label from [2-(14)C]glucose to carbon-1 of starch glucosyl units confirmed the operation of this pathway and indicated that it is located in plastids. It was found to consume 32% of the hexose phosphates entering the triose pathways. The remaining 68% were consumed by glycolysis. The determination of the specific enrichment of carbohydrate carbons -1 and -6 after labeling with [1-(13)C]glucose indicated that both the conversion of triose phosphates back to hexose phosphates and the transaldolase exchange contributed to this randomization. Of the triose phosphates produced by glycolysis and the pentose phosphate pathway, about 60% were found to be recycled to hexose phosphates, and 28% were directed to the tricarboxylic acid cycle. Of this 28%, two-thirds were found to be directed through the pyruvate kinase branch and one-third through the phosphoenolpyruvate branch. The latter essentially has an anaplerotic function since little malate was found to be converted to pyruvate (malic enzyme reaction).