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D K Novikov

Publications and source records attributed to D K Novikov.

At least 19 recordsLinked to original sources
PubMed2001

Characterization of the promoter region of the human peroxisomal multifunctional enzyme type 2 gene.

Peroxisomal multifunctional enzyme type 2 (perMFE-2) catalyzes conversion of (24E)-3alpha,7alpha, 12alpha-trihydroxy-5beta-cholest-24-enoyl-CoA to (24-keto)-3alpha,7alpha,12alpha-trihydroxy-5beta-cholestanoyl-CoA, which are physiological intermediates in cholic acid synthesis. In contrast to long chain fatty acid oxidizing enzymes clofibrate does not induce peroxisomal enzymes metabolizing bile acid intermediates. We proposed the existence of PPAR-independent regulation of cholesterol side chain oxidation in the process of bile acid synthesis. In the present study, we characterized the promoter region of the human perMFE-2 gene. The promoter contains the Sp1/AP2 binding site (-151/-142) within 197 base pairs upstream of the translation start site. Mutation of the Sp1/AP2 binding site decreases the promoter activity. Analysis by the luciferase assay revealed that the activity of the promoter region is strong in HepG2 and HeLa cell lines, although the activity in HepG2 cells was five- to sixfold higher than that in HeLa cells. Transient transfection assays have confirmed that AP2alpha and AP2gamma were able to transactivate the perMFE-2 promoter/luciferase chimeric gene. Cotransfections with Sp1 expression plasmid decreased the promoter activity. We suggest that perMFE-2 promoter activity is the result of both the abundance of AP2 and Sp1 family members and their relative ratios.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed2000

In mouse alpha -methylacyl-CoA racemase, the same gene product is simultaneously located in mitochondria and peroxisomes.

alpha-Methylacyl-CoA racemase, an enzyme of the bile acid biosynthesis and branched chain fatty acid degradation pathway, was studied at the protein, cDNA, and genomic levels in mouse liver. Immunoelectron microscopy and subcellular fractionation located racemase to mitochondria and peroxisomes. The enzymes were purified from both organelles with immunoaffinity chromatography. The isolated proteins were of the same size, with identical N-terminal amino acid sequences, and the existence of additional proteins with alpha-methylacyl-CoA racemase activity was excluded. A racemase gene of about 15 kilobases was isolated. Southern blot analysis and chromosomal localization showed that only one racemase gene is present, on chromosome 15, region 15B1. The putative initial ATG in the racemase gene was preceded by a functional promotor as shown with the luciferase reporter gene assay. The corresponding cDNAs were isolated from rat and mouse liver. The recombinant rat protein was overexpressed in active form in Pichia pastoris. The presented data suggest that the polypeptide encoded by the racemase gene can alternatively be targeted to peroxisomes or mitochondria without modifications. It is concluded that the noncleavable N-terminal sequence of the polypeptide acts as a weak mitochondrial and that the C-terminal sequence acts as a peroxisomal targeting signal.

Amino Acid Sequence↗
PubMed2000

Human peroxisomal multifunctional enzyme type 2. Site-directed mutagenesis studies show the importance of two protic residues for 2-enoyl-CoA hydratase 2 activity.

Beta-oxidation of acyl-CoAs in mammalian peroxisomes can occur via either multifunctional enzyme type 1 (MFE-1) or type 2 (MFE-2), both of which catalyze the hydration of trans-2-enoyl-CoA and the dehydrogenation of 3-hydroxyacyl-CoA, but with opposite chiral specificity. Amino acid sequence alignment of the 2-enoyl-CoA hydratase 2 domain in human MFE-2 with other MFE-2s reveals conserved protic residues: Tyr-347, Glu-366, Asp-370, His-406, Glu-408, Tyr-410, Asp-490, Tyr-505, Asp-510, His-515, Asp-517, and His-532. To investigate their potential roles in catalysis, each residue was replaced by alanine in site-directed mutagenesis, and the resulting constructs were tested for complementation in a yeast. After additional screening, the wild type and noncomplementing E366A and D510A variants were expressed and characterized. The purified proteins have similar secondary structural elements, with the same subunit composition. The E366A variant had a k(cat)/K(m) value 100 times lower than that of the wild type MFE-2 at pH 5, whereas the D510A variant was inactive. Asp-510 was imbedded in a novel hydratase 2 motif found in the hydratase 2 proteins. The data show that the hydratase 2 reaction catalyzed by MFE-2 requires two protic residues, Glu-366 and Asp-510, suggesting that their catalytic role may be equivalent to that of the two catalytic residues of hydratase 1.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed2000

Substrate specificities of peroxisomal members of short-chain alcohol dehydrogenase superfamily: expression and characterization of dehydrogenase part of Candida tropicalis multifunctional enzyme.

In addition to several other enzymes, the short-chain alcohol dehydrogenase superfamily includes a group of peroxisomal multifunctional enzymes involved in fatty acid and cholesterol side-chain beta-oxidation. Mammalian peroxisomal multifunctional enzyme type 2 (perMFE-2) is a 2-enoyl-CoA hydratase-2/(R)-3-hydroxyacyl-CoA dehydrogenase. As has been shown previously, perMFE-2 hydrates (24E)-3alpha,7alpha, 12alpha-trihydroxy-5beta-cholest-24-enoyl-CoA to (24R, 25R)-3alpha, 7alpha,12alpha,24xi-tetrahydroxy-5beta-choles tanoyl-CoA, which has been characterized as a physiological intermediate in cholic acid synthesis. Out of four possible stereoisomers of 3alpha,7alpha, 12alpha,24xi-tetrahydroxy-5beta-cholestanoyl-CoA , the mammalian perMFE-2 dehydrogenates only the (24R,25R)-isomer. The yeast peroxisomal multifunctional enzyme (MFE) was first described as 2-enoyl-CoA hydratase-2/(R)-3-hydroxyacyl-CoA dehydrogenase. To investigate the stereospecificity of yeast peroxisomal MFE, the two dehydrogenase domains of C. tropicalis MFE were expressed in E. coli as a 65 kDa recombinant protein. This protein catalyzes the dehydrogenation of straight-chain (R)-3-hydroxyacyl-CoAs, but it is devoid of (S)-3-hydroxyacyl-CoA dehydrogenase and 2-enoyl-CoA hydratase activities. The protein dehydrogenates (24R,25R)- and (24R, 25S)-isomers of 3alpha,7alpha, 12alpha, 24xi-tetrahydroxy-5beta-cholestanoyl-CoA. Interestingly, the protein also shows 17beta-estradiol dehydrogenase activity. As a monofunctional (R)-specific 3-hydroxyacyl-CoA dehydrogenase is currently unavailable, this recombinant enzyme can be used to study the stereochemistry of bile acid synthesis.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1998

The crystal structure of dienoyl-CoA isomerase at 1.5 A resolution reveals the importance of aspartate and glutamate sidechains for catalysis.

BACKGROUND: The degradation of unsaturated fatty acids is vital to all living organisms. Certain unsaturated fatty acids must be catabolized via a pathway auxiliary to the main beta-oxidation pathway. Dienoyl-coenzyme A (dienoyl-CoA) isomerase catalyzes one step of this auxiliary pathway, the isomerization of 3-trans,5-cis-dienoyl-CoA to 2-trans,4-trans-dienoyl-CoA, and is imported into both mitochondria and peroxisomes. Dienoyl-CoA isomerase belongs to a family of CoA-binding proteins that share the enoyl-CoA hydratase/isomerase sequence motif. RESULTS: The crystal structure of rat dienoyl-CoA isomerase has been determined at 1.5 A resolution. The fold closely resembles that of enoyl-CoA hydratase and 4-chlorobenzoyl-CoA dehalogenase. Dienoyl-CoA isomerase forms hexamers made up of two trimers. The structure contains a well ordered peroxisomal targeting signal type-1 which is mostly buried in the inter-trimer space. The active-site pocket is deeply buried and entirely hydrophobic, with the exception of the acidic residues Asp176, Glu196 and Asp204. Site-directed mutagenesis of Asp204 revealed that this residue is essential for catalysis. In a molecular modeling simulation, a molecule of 3-trans,5-cis-octadienoyl-CoA was docked into the active site. CONCLUSIONS: The structural data, supported by the mutagenesis data, suggest a reaction mechanism where Glu196 acts as a proton acceptor and Asp204 acts as a proton donor. Asp176 is paired with Glu196 and is important for optimizing the catalytic proton transfer properties of Glu196. In the predicted mode of substrate binding, an oxyanion hole stabilizes the transition state by binding the thioester oxygen. The presence of a buried peroxisomal targeting signal suggests that dienoyl-CoA isomerase is prevented from reaching its hexameric structure in the cytosol.

Acyl Coenzyme A↗
PubMed1997

Recombinant 2-enoyl-CoA hydratase derived from rat peroxisomal multifunctional enzyme 2: role of the hydratase reaction in bile acid synthesis.

Rat liver peroxisomes contain two multifunctional enzymes: (1) perMFE-1 [2-enoyl-CoA hydratase 1/Delta3,Delta2-enoyl-CoA isomerase/(S)-3-hydroxyacyl-CoA dehydrogenase] and (2) perMFE-2 [2-enoyl-CoA hydratase 2/(R)-3-hydroxyacyl-CoA dehydrogenase]. To investigate the role of the hydratase activity of perMFE-2 in beta-oxidation, a truncated version of perMFE-2 was expressed in Escherichia coli as a recombinant protein. The protein catalyses the hydration of straight-chain (2E)-enoyl-CoAs to (3R)-hydroxyacyl-CoAs, but it is devoid of hydratase 1 [(2E)-enoyl-CoA to (3S)-hydroxyacyl-CoA] and (3R)-hydroxyacyl-CoA dehydrogenase activities. The purified enzyme (46 kDa hydratase 2) can be stored as an active enzyme for at least half a year. The recombinant enzyme hydrates (24E)-3alpha,7alpha,12alpha-trihydroxy- 5beta-cholest-24-enoyl-CoA to (24R,25R)-3alpha,7alpha,12alpha, 24-tetrahydroxy-5beta-cholestanoyl-CoA, which has previously been characterized as a physiological intermediate in bile acid synthesis. The stereochemistry of the products indicates that the hydration reaction catalysed by the enzyme proceeds via a syn mechanism. A monofunctional 2-enoyl-CoA hydratase 2 has not been observed as a wild-type protein. The recombinant 46 kDa hydratase 2 described here survives in a purified form under storage, thus being the first protein of this type amenable to application as a tool in metabolic studies.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1996

[Inhibition of cholesterol biosynthesis in rabbit hepatocytes by 3-beta-(omega-hydroxyalkoxy)cholest-5-enes].

3 beta-(2-Hydroxyethoxy)-, 3 beta-(4'-hydroxybutoxy)-, 3 beta-(6-hydroxyhexyloxy)-, 3 beta-(9-hydroxynonyloxy)-, and 3 beta-(2-hydroxy-2-[3H]ethoxy)cholest-5-enes were synthesized. By means of a spin probe, the influence of the synthesized compounds on the phase transition of dimyristoylphosphatidylcholine were estimated. Time and dose dependences of the incorporation of 3 beta-(2-hydroxy-2-[3H]ethoxy)cholest-5-ene into rabbit hepatocytes (the primary culture) were studied. 3 beta-(2-Hydroxyethoxy)- and 3 beta-(4-hydroxybutoxy)cholest-5-enes were shown to inhibit cholesterol biosynthesis from [14C]acetate in rabbit hepatocyte cultures upon a 24-hour preincubation.

Animals↗
PubMed1996

[Metabolism of 3beta-(2-hydroxyethoxy)cholest-5-ene after intravenous administration to rats].

The intravenous injection of 3 beta-(2-hydroxy-2-[3H]ethoxy)cholest- 5-ene into rats with the cannulated common bile duct resulted in the primary accumulation of radioactivity in liver (24%) and spleen (12%) tissues, bile (7%), and blood serum (15%) after 3 hours. The distribution of 3 beta-(2-hydroxy-2-[3H]ethoxy)cholest-5-ene throughout various tissues was close to that of [14C]cholesterol being administrated under the same conditions. The analysis of radioactive products from blood serum showed that 40-60% of 3 beta-(2-hydroxy-2-[3H]ethoxy)- cholest-5-ene was converted to the acyl derivative under experimental conditions.

Animals↗
PubMed1994

Peroxisomal beta-oxidation. Purification of four novel 3-hydroxyacyl-CoA dehydrogenases from rat liver peroxisomes.

Peroxisomes are capable of beta-oxidizing a variety of substrates including the CoA esters of straight chain fatty acids, 2-methyl-branched fatty acids and the bile acid intermediates di- and trihydroxycoprostanic acids. The first reaction of peroxisomal beta-oxidation is catalyzed by an acyl-CoA oxidase. Rat liver peroxisomes contain three acyl-CoA oxidases: 1) palmitoyl-CoA oxidase, oxidizing straight chain acyl-CoAs; 2) pristanoyl-CoA oxidase, oxidizing 2-methyl-branched acyl-CoAs; and 3) trihydroxycoprostanoyl-CoA oxidase, oxidizing the CoA esters of the bile acid intermediates (Van Veldhoven, P.P., Vanhove, G., Asselberghs, S., Eyssen, H. J., and Mannaerts, G. P. (1992) J. Biol. Chem. 267, 20065-20074). We have now investigated whether the third step of peroxisomal beta-oxidation, catalyzed by a 3-hydroxyacyl-CoA dehydrogenase, is also catalyzed by multiple enzymes, using the 3-hydroxyacyl-CoA derivatives of palmitic acid, 2-methylpalmitic acid, and trihydroxycoprostanic acid as the substrates to monitor the dehydrogenase activities. In order to avoid contamination with mitochondrial 3-hydroxyacyl-CoA dehydrogenases, highly purified peroxisomes from untreated rats were employed as the enzyme source. Subfractionation of the peroxisomes revealed that the major portion of the dehydrogenase activities with all three substrates was present in the peripheral membrane protein fraction. Separation of this fraction on various chromatographic columns resulted in the purification of the well known multifunctional protein, a 78-kDa monomeric protein that displays 3-hydroxyacyl-CoA dehydrogenase plus hydratase activity, as well as of four additional novel dehydrogenases with different substrate specificities. Three of the enzymes are monomeric proteins of 35 kDa, 56 kDa, and 79 kDa, respectively. The latter enzyme also displays hydratase activity. The fourth enzyme is a dimer of 89 kDa, the subunits of which form a doublet at 40 kDa. The exact physiological role of each of the 3-hydroxyacyl-CoA dehydrogenases requires further investigation.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1992

[Effect of mevinolin and glycyrrhizinic acid on cholesterol and bile acid metabolism in cultured rabbit hepatocytes].

A comparison of effects of two hypocholesterolemic drugs--mevinolin and glycyrrhizinic acid, on cholesterol and bile acid metabolism in cultured rabbit hepatocytes has been carried out. The following parameters have been determined: i) cholesterol synthesis from [2-14C]acetate; ii) bile acid production from newly synthesized and [4-14C]-labeled HDL2 cholesterol, and, iii) total cholesterol efflux into the incubation medium Mevinolin (0.5 microgram/ml) inhibited [2-14C] acetate incorporation into cholesterol by more than 90%. Conversely, glycyrrhizinic acid did not influence cholesterol synthesis even when used at high (100 micrograms/ml) concentrations but stimulated the conversion of endogenous (by 37%) and exogenous (by 18%) cholesterol into bile acids and increased, in addition, the proportion of bile acids in the total sterol pool released from hepatocytes into the incubation medium. At the same time, mevinolin used at 0.5 microgram/ml decreased the bile acid production by endogenous (by 27%) and exogenous (by 40%) cholesterol. The data obtained suggest that glycyrrhizinic acid exerts hypocholesterolemic action by stimulation of cholesterol conversion into bile acids without any effect on cholesterol synthesis. As for mevinolin, it has a cholesterol-suppressing effect via a mechanism of cholesterol synthesis inhibition only.

Animals↗
PubMed1991

Effect of chylomicron remnants on cholesterol metabolism in cultured rabbit hepatocytes: very low density lipoprotein and bile acid production.

The interrelationship between very low density lipoprotein (VLDL) secretion and bile acid production was studied in primary culture of rabbit hepatocytes. Chylomicron remnants (CR) were added to the cultures to study their effect on VLDL secretion and bile acid production. After 24 hr preincubation of cells with CR (10-50 micrograms protein/mL), intercellular neutral lipid content was increased 1.5-4-fold in a dose-dependent manner. Neutral lipid accumulation was accompanied by a 70-90% reduction of [14C]acetate incorporation into cholesterol, while no stimulation of [14C]oleate incorporation into cholesteryl esters was observed. Incubation of cells with CR increased secretion of free cholesterol, triacylglycerol and apoproteins B and E in VLDL. Stimulation of VLDL cholesterol secretion was accompanied by a reduction of taurocholic acid synthesis. These data demonstrate the existence of an inverse relationship between secretion of VLDL cholesterol and bile acid production under conditions of effective uptake of triacylglycerol-rich CR by hepatocytes.

Acetates↗
PubMed1991

[The effect of oxidized cholesterol derivatives on the absorption and degradation of very low density beta-lipoproteins by human and rabbit hepatocytes].

In our study the influence of oxidated cholesterol derivatives (7 alpha, 7 beta-oxicholesterol, 7-ketocholesterol, cholestene-3-one) on binding and degradation of beta-VLDL by human and rabbit hepatocytes was investigated. Cholesterol oxy derivatives inhibit the degradation of beta-VLDL in rabbit hepatocyte culture. There is no such effect with human hepatocytes. However beta-VLDL binding with human hepatocytes is significantly decreased under oxidated cholesterol derivatives influence.

Absorption↗
PubMed1990

[The use of the direct basophil degranulation test for the diagnosis of allergy].

Basophil degranulation test (BDT) was used in examinations of patients suffering from allergies. A modified technique was employed with toluidine blue staining and basophil scintillation in a counter chamber. The examinees were divided into three groups. Group 1 (n = 34) consisted of patients suffering from atopic bronchial asthma with suspected sensitization to home dust. Group 2 included 118 patients with a history of allergy to penicillin. Group 3 consisted of 51 patients with drug allergies (streptomycin, vitamin B1, procaine, analgin intolerance). In all the cases specific diagnosis was confirmed by skin and provocation test. 71.4 percent of the patients with sensitization to home dust allergen showed positive results in BDT, as did 68.6 percent of Group 3 subjects. Of the cases with penicillin sensitization confirmed by application test 91.7 percent were positive in BDT, of those with positive intradermal tests, 66.7 percent were BDT-positive, this being probably explained by individual sensitivity of the patients. These results recommend BDT, an informative and simple technique, for wide practice.

Asthma↗
PubMed1990

[Effect of chylomicron remnants on cholesterol metabolism in cultured rabbit hepatocytes: production of very low density lipoproteins and bile acids].

Primary cultures of rabbit hepatocytes were used to investigate the effect of purified (B-100 free) chylomicron remnants (CR) on lipid and bile acid metabolism. ApoB-100-containing lipoproteins were removed from the CR-enriched plasma fraction by affinity column chromatography on Sepharose 4B conjugated with anti-apoB-100 monoclonal antibodies. CR were shown to stimulate the accumulation of neutral lipids in hepatocytes in a dose-response manner. After 24-hour preincubation of rabbit hepatocytes with 50 micrograms protein/ml CR the cellular neutral lipid content increased: 1.9-4-fold for triglycerides, 1.5-3.7-fold for free cholesterol and 1.5-2.5-fold for esterified cholesterol. This accumulation was accompanied by a decreasing incorporation of [14C] acetate into cholesterol (80-90%) and triglycerides (70-80%). At the same time the incorporation of [14]oleate into triglycerides increased by 50-65%. The inhibited biosynthesis of fatty acids might account for this effect. No effect of CR on cholesterol esterification by [14C]oleate was observed. CR increased the amount of triglycerides and free cholesterol secreted in very low density lipoproteins (VLDL). The secretion of taurocholic acid was decreased. These data confirm our hypothesis that dietary cholesterol is preferentially secreted by hepatocytes within VLDL but is not accumulated as cholesterol esters or oxidized to bile acids.

Animals↗
PubMed1990

[Study of HDL2-dependent synthesis of bile acids in culture of rabbit hepatocytes: effects of oxidized cholesterol derivatives].

The effect of individual oxysterols--products of auto-oxidation of cholesterol on bile acid synthesis by cultivated rabbit hepatocytes was studied. Relative rates of bile acid synthesis were measured as the conversion of 4-14C cholesterol-HDL2 into total 4-14C labeled bile acids. 7 beta-hydroxycholesterol and 3,5-cholestane-7-dione strongly inhibited bile acid synthesis at concentrations 1-10 micrograms/ml. These data support the hypothesis that oxidized cholesterol derivatives accelerate the development of hypercholesterolemia in rabbits fed on cholesterol containing diet.

Animals↗