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P M Palosaari

Publications and source records attributed to P M Palosaari.

8 recordsLinked to original sources
PubMed1992

Peroxisomal diseases.

The peroxisomal diseases, which are rare inborn metabolic errors, often have serious effects on the well being of the individual and many of them are fatal at an early age. The Zellweger cerebro-hepato-renal syndrome represents a group consisting of diseases with a generalized loss of peroxisomal functions and is considered as a prototype for peroxisomal dysfunction. The largest group includes those diseases where only a single peroxisomal function is impaired. The most common peroxisomal disease, x-linked adrenoleukodystrophy (ADL), belongs to this group, and neurological symptoms dominate among the patients. The primary diagnosis is usually based on clinical findings and measurement of accumulated or depleted metabolites in the body (e.g. very long chain fatty acids, bile acid intermediates or plasmalogens). Some progress has been made in treating of the peroxisomal diseases. Many patients with x-linked ALD have benefitted from the supplementation of the diet with long chain monounsaturated fatty acids like erucic acid or oleic acid with the simultaneous restriction of very long chain fatty acids. Docosahexenoate (C22:1) has also shown promising results in some studies.

Humans↗
PubMed1991

Amino acid sequence similarities of the mitochondrial short chain delta 3, delta 2-enoyl-CoA isomerase and peroxisomal multifunctional delta 3, delta 2-enoyl-CoA isomerase, 2-enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase enzyme in rat liver. The proposed occurrence of isomerization and hydration in the same catalytic domain of the multifunctional enzyme.

We report the isolation and characterization of a cDNA encoding the mitochondrial short chain delta 3, delta 2-enoyl-CoA isomerase from rat liver. Tryptic fragments of the purified protein were generated, purified, and sequenced. A rat liver cDNA library, constructed in the plasmid vector pUEX1 was screened with oligonucleotides synthesized on the basis of peptide sequences. The obtained clone contained 783 bases predicting to code the entire mature protein of 261 amino acids. The molecular weight of 29,300 predicted from cDNA-derived sequences was consistent with the subunit size determined earlier. A high degree of similarity was noted between the amino acid sequence of isomerase and that of the amino-terminal half of peroxisomal multifunctional isomerase-hydratase-dehydrogenase enzyme and mitochondrial 2-enoyl-CoA hydratase in rat liver. These similarities also appeared at the level of predicted secondary structural elements, suggesting that hte rat multifunctional enzyme has both the isomerization and hydration activities in the amino-terminal domain. This idea is further supported by the proposed existence of only one CoA-binding site in the amino-terminal half of the multifunctional enzyme and by previous studies suggesting that the transfer of the substrate from the isomerization site to the hydration site occurs without aqueous bulk phase (Palosaari P.M., and Hiltunen, J. K. (1990) J. Biol. Chem. 265, 2446-2449).

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1990

Occurrence of a long-chain delta 3,delta 2-enoyl-CoA isomerase in rat liver.

Isoproteins of delta 3,delta 2-enoyl-CoA isomerase (EC 5.3.3.8), an auxiliary enzyme in the beta-oxidation of unsaturated fatty acids having double bonds at odd-numbered positions, were studied in livers of control and clofibrate-treated rats. When liver extracts were applied to a hydroxyapatite column at pH 7.0, the previously characterized peroxisomal trifunctional hydratase-dehydrogenase-isomerase enzyme and the mitochondrial isomerase, which shows a preference for short-chain substrates, were eluted almost in parallel. In addition to these activities, a separate isomerase was observed to elute at a lower potassium phosphate concentration in the gradient. Experiments with extracts of purified mitochondria and peroxisomes demonstrated the mitochondrial origin of this third activity. Studies on the kinetic properties of the third isomerase showed that it has a preference for C10-C12 substrates. An Mr of 200,000 was obtained for the native protein by gel-filtration chromatography. Antibodies to mitochondrial short-chain isomerase and peroxisomal trifunctional enzyme did not recognize this novel mitochondrial isoenzyme. The immunological non-cross-reactivity can be interpreted as suggesting that the different isomerases are not closely related at the level of the primary structure of the polypeptide chain. The present data demonstrate that, similar to many other enzymes of beta-oxidation, delta 3,delta 2-enoyl-CoA isomerase has at least three isoenzymes in rat liver: mitochondrial short- and long-chain isomerases and an additional peroxisomal isoenzyme, which in this case is a part of a multifunctional protein.

Animals↗
PubMed1990

Delta 3,delta 2-enoyl-CoA isomerases. Characterization of the mitochondrial isoenzyme in the rat.

Delta 3,delta 2-Enoyl-CoA isomerase (EC 5.3.3.8), an obligatory auxiliary enzyme for the metabolism of double bonds at odd-numbered positions of fatty acids during their beta-oxidation, was studied in hearts and livers of normal and clofibrate-treated rats. Hepatic peroxisomal and mitochondrial isoenzymes were separable by dye-ligand chromatography. The mitochondrial one was further purified to apparent homogeneity. An isomerase was also purified from heart muscle, a peroxisome-poor tissue. These enzymes were dimeric basic proteins (pI 9.5) with a subunit molecular weight of 30,000. Both cis- and trans-enoyl-CoA served as substrates for the hepatic enzyme studied. The velocity ratio for the C6-, C10-, and C12-trans-3-enoyl substrates was 9:2.5:1. By immunoelectron microscopy the enzyme protein selected for purification was found to be mitochondrial both in liver and heart. Chromatographic evidence, immunoelectron microscopy, and immunoblotting indicated that in the liver but not in the heart, the enzyme underwent an induction of 1 order of magnitude during clofibrate treatment. Antibodies towards the rat isomerases detected cross-reactive proteins in bovine and pig liver and heart and human placenta. The estimated subunit sizes varied from species to species, being 31,000 in bovine liver and heart, 29,000 in pig liver and heart, and 30,000 in human placenta. The data are in accord with the notion of a dual location of the delta 3,delta 2-enoyl-CoA isomerase. Mitochondrial origin of one of the isoenzymes and its tissue-specific induction by clofibrate were verified by immunochemistry and the identity of the peroxisomal one revealed by the chromatographic behavior of the proteins.

Animals↗
PubMed1990

Peroxisomal bifunctional protein from rat liver is a trifunctional enzyme possessing 2-enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and delta 3, delta 2-enoyl-CoA isomerase activities.

Peroxisomal delta 3, delta 2-enoyl-CoA isomerase (EC 5.3.3.8) was studied in the liver of rats treated with clofibrate. The mitochondrial and peroxisomal isoenzymes were separated chromatographically and the peroxisomal isomerase purified to apparent homogeneity. In addition to the isomerization of 3-enoyl-CoA esters, the purified protein also catalyzed hydration of trans-2-enoyl-CoA and oxidation of L-3-hydroxyacyl-CoA. Incubation of the purified protein with trans-3-decenoyl-CoA, NAD+, and Mg2+ resulted in an increase in absorbance at 303 nm, indicating the formation of 3-ketoacyl-CoA. The protein purified was monomeric, with an estimated molecular weight of 78,000. In immunoblotting it was recognized by the antibody to peroxisomal bifunctional protein from rat liver. Comparison of the amino acid sequences of cyanogen bromide cleaved isomerase with the known sequence of the peroxisomal bifunctional protein from the rat identified them as the same molecule. In control experiments, the peroxisomal bifunctional protein purified according to published methods also catalyzed delta 3, delta 2-enoyl-CoA isomerization. This means that the bifunctional protein of rat liver is in fact a trifunctional enzyme possessing delta 3, delta 2-enoyl-CoA isomerase, 2-enoyl-CoA hydratase (EC 4.2.1.17), and L-3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) activities in the same polypeptide.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1989

Epimerization of 3-hydroxyacyl-CoA esters in rat liver. Involvement of two 2-enoyl-CoA hydratases.

Interconversion of D- and L-isomers of 3-hydroxy-decanoyl-CoA was catalyzed by rat liver homogenate. Cation exchange chromatography followed by ammonium sulfate precipitation and PBE-94 chromatofocusing column was used to separate the peroxisomal bifunctional protein, the classic 2-enoyl-CoA hydratase (crotonase), and a novel 2-enoyl-CoA hydratase. Epimerization activity was lost during the last purification step. None of the above proteins was capable of catalyzing the epimerization by itself, but reconstitution was achieved by recombining crotonase and the novel 2-enoyl-CoA hydratase. Since hydration by the latter enzyme follows a different stereochemical course from that with crotonase, these two hydratases are distinguished as 2-enoyl-CoA hydratase 1 (crotonase) and 2-enoyl-CoA hydratase 2 (the novel hydratase). The data strongly suggested that epimerization in the rat liver proceeds via dehydration-hydration catalyzed by the two different hydratases. The intermediate of this two step mechanism appears to be trans-2-enoyl-CoA.

Acyl Coenzyme A↗