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T Chojnacki

Publications and source records attributed to T Chojnacki.

At least 37 records · Page 2Linked to original sources
PubMed1993

Distribution of prenyltransferases in rat tissues. Evidence for a cytosolic all-trans-geranylgeranyl diphosphate synthase.

The present study describes the presence of two different geranylgeranyl diphosphate (GGPP) synthase activities, one cytosolic and one membrane-associated, in a number of rat tissues. Both enzymes utilize farnesyl diphosphate (FPP) and isopentenyl diphosphate (IPP) as substrates, but they give rise to different products. The membrane-associated activity produces trans,trans,cis-(E,E,Z)-GGPP, involved in the biosynthesis of long-chain polyprenols. The cytosolic activity produces only the all-trans-(E,E,E) isomer of GGPP, which is utilized as substrate in cytosolic protein prenylation reactions. All-trans-GGPP synthase activity was recovered in the cytosolic fraction from all tissues investigated, but the specific activities varied. The highest specific activities were found in brain, spleen, and testis, followed by kidney and liver. The enzyme activity in rat brain cytosol was further characterized and found to exhibit a narrow pH optimum around 5.0-6.0 and to be highly stimulated by Zn2+. Maximal stimulation was attained with 1 mM Zn2+, whereas Mg2+ had no effect on the enzyme activity. The all-trans-GGPP synthase activity exhibited high affinities for its substrates, i.e. the apparent Km values for FPP and IPP were found to be 0.6 and 3.5 microM, respectively. When rats were fed mevinolin (lovastatin), FPP and all-trans-GGPP synthase activities were affected differently in certain tissues. Mevinolin treatment resulted in an increase in FPP but a decrease in all-trans-GGPP synthase activity in rat liver and kidney. In spleen mevinolin treatment caused a greater than 70% decrease in all-trans-GGPP synthase activity, while FPP synthase was almost unaffected. The presence of two different GGPP synthase activities in the cell, together with the fact that FPP and all-trans-GGPP synthesis in the cytosol are regulated independently, may be of significance in the regulation of isoprenoid biosynthesis, as well as of protein isoprenylation.

Alkyl and Aryl Transferases↗
PubMed1992

Phosphatidylethanolamine:dolichol acyltransferase. Characterization and partial purification of a novel rat liver enzyme.

Incubation of rat or human post-heparin plasma with [3H]dolichol incorporated in liposomes consisting of dioleoyl phosphatidylcholine:dioleoyl phosphatidylethanolamine (3:1) resulted in the formation of radioactive dolichyl oleate. Non-heparinized plasma did not esterify dolichol, and, hence, the enzyme involved is probably associated with the cell surface and released into the blood by heparin. The major location of this activity was the liver, and, therefore, a partial purification of the enzyme from heparinized rat liver perfusates was performed using DEAE-Sephacel and heparin-Sepharose chromatography. The dolichol acyltransferase activity copurified with hepatic lipase activity in a lipid-protein complex of 350 kDa. Optimal acylation is achieved at pH 7.5 in the presence of 5% plasma and 20 mM Ca2+. Esterification can only be obtained when dolichol is present in a phospholipid bilayer, and the reaction is strongly stimulated by unsaturated phosphatidylethanolamine or phosphatidylserine. Radiolabeling experiments demonstrated that the primary acyl donor is phosphatidylethanolamine from which the fatty acid is transferred exclusively from position 1. Neither cholesterol nor retinol are esterified by the enzyme, and the reaction is not stimulated by acyl-CoA. Both the extracellular localization and the mechanism of transacylation clearly distinguish this new enzyme from the acyl-CoA:dolichol acyltransferase described earlier in microsomes.

Acyltransferases↗
PubMed1992

Substrate specificity of cis-prenyltransferase in rat liver microsomes.

Long chain cis-prenyltransferase in rat liver microsomes was studied using various allylic isoprenoid substrates. Microsomes could utilize trans-geranyl pyrophosphate, but not cis-geranyl pyrophosphate for polyprenyl pyrophosphate synthesis. Both trans, trans-farnesyl pyrophosphate and trans,cis-farnesyl pyrophosphate were used as substrates with Km values of 24 and 5 microM, respectively. trans,trans,cis-Geranylgeranyl pyrophosphate could be used as substrate with an apparent Km of 36 microM. trans,trans,trans-Geranylgeranyl pyrophosphate was also utilized as substrate, but with a very low affinity. After pulse labeling for 4 min, using [3H]isopentenyl pyrophosphate and trans,trans-farnesyl pyrophosphate, the only product formed was trans,trans,cis-geranylgeranyl pyrophosphate, which, upon chasing, yielded polyprenyl pyrophosphate. Independent of the nature of the substrate used, even in the case of polyprenyl 12-pyrophosphate and all-trans-nonaprenyl pyrophosphate, the chain lengths of the products were identical, i.e. polyprenyl pyrophosphates with 15-18 isoprene residues. Microsomes were able to synthesize trans,trans-farnesyl pyrophosphate using trans-geranyl pyrophosphate as substrate. The results indicate that rat liver microsomes contain a farnesyl pyrophosphate synthase activity and that the reaction catalyzed by cis-prenyltransferase may consist of two individual steps, i.e. synthesis of trans,trans,cis-geranylgeranyl pyrophosphate and elongation of this product to long chain polyprenyl pyrophosphates.

Animals↗
PubMed1992

Isoprenoid biosynthesis in rat liver peroxisomes. Characterization of cis-prenyltransferase and squalene synthetase.

Isolated peroxisomes were able to utilize [3H]isopentenyl diphosphate to synthesize farnesyl diphosphate, which then was utilized as substrate by both the peroxisomal squalene synthetase and cis-prenyltransferase. The specific activity of squalene synthetase in peroxisomes was as high as in microsomes, i.e. 160 pmol/mg of protein/min. If NADPH was omitted from the assay medium, presqualene diphosphate accumulated, which indicates that the reaction occurs in two steps, as in microsomes. In the presence of NADPH, incorporation from [3H]farnesyl diphosphate was stimulated 3-fold, and the major products were squalene and cholesterol. The specific activity of cis-prenyl-transferase in peroxisomes was 4-fold higher than in microsomes, i.e. 456 pmol of isopentenyl diphosphate incorporated/mg of protein/h. There were two major products formed from farnesyl diphosphate and [3H] isopentenyl diphosphate, i.e. trans,trans,cis-geranylgeranyl diphosphate and long chain polyprenyl diphosphates. The polyprenyl diphosphates had the same chain length distribution as that of dolichol derivatives in rat liver, with the dominating polyisoprenes being C90 and C95. In contrast to the microsomal enzyme, peroxisomal cis-prenyltransferase did not require detergents for optimal activity. The enzyme was associated primarily with the peroxisomal membrane after sonication of the peroxisomes.

Animals↗
PubMed1992

Biosynthesis of trans,trans,trans-geranylgeranyl diphosphate by the cytosolic fraction from rat tissues.

The cytosolic fractions from rat liver, brain, kidney, spleen and testis demonstrate the capacity to synthesize two products from [3H]isopentenyl diphosphate, i.e., farnesyl diphosphate and geranylgeranyl diphosphate. The highest rate of geranylgeranyl diphosphate synthesis was found in brain, testis and spleen, accounting for up to 30% of the total incorporation of radioactivity under optimal conditions. In all tissues examined the geranylgeranyl diphosphate formed was identified as the trans,trans,trans-isomer. The ratio of geranylgeranyl diphosphate to farnesyl diphosphate produced was specific for the tissue investigated and could be altered by the addition of divalent cations. The results in this study demonstrate the presence of a specific trans,trans,trans-geranylgeranyl diphosphate synthetase showing high affinity for farnesyl diphosphate.

Animals↗
PubMed1992

Transfer of liposomes containing dolichol into isolated hepatocytes.

Isolated rat hepatocytes were preincubated with egg lecithin liposomes containing [3H]dolichol and [3H]dolichyl ester, and the intracellular levels and distributions of these lipids were subsequently determined after incubation in a liposome-free medium. [3H]Dolichol was recovered initially mainly in microsomes, and no increase with time in the low level of this compound in the mitochondrial/lysosomal fraction could be observed. A small portion of the labeled dolichol was esterified in the endoplasmic reticulum and transferred to the lysosome-containing fraction. [3H]Dolichyl linoleate was initially localized in microsomes and supernatant, but later accumulated in the mitochondria/lysosomes. Dolichyl linoleate was found in the membrane of microsomes, in the membrane and lumen of lysosomes, and in the soluble cytoplasm. Exogenous dolichol recovered in microsomes was not phosphorylated to any significant extent. Liposomal phosphatidylcholine also showed preferential accumulation in microsomes after incubation with hepatocytes. These results indicate that exogenous or endogenously formed dolichyl esters are transferred from the endoplasmic reticulum to lysosomes, probably through the cytoplasm. It appears that fatty acids play a role in targeting these lipids to their intracellular locations.

Animals↗
PubMed1992

On the specificity of dolichol kinase and DolPMan synthase towards isoprenoid alcohols of different chain length in rat liver microsomal membrane.

1. A wide range of dolichols differing in the length of hydrocarbon chain (from 11 to 32 isoprene residues) were found to be phosphorylated in the presence of CTP in rat liver microsomes. 2. Fully unsaturated polyprenols of the same chain length as dolichols were poor substrates for dolichol kinase at low detergent (Nonidet P-40) concentration. At higher concentration of detergent, both dolichols and polyprenols were equally effective. 3. In the transfer of mannosyl residues from GDPMan, the dolichyl phosphates generated in rat liver microsomes were all good lipid acceptors, while fully unsaturated polyprenyl phosphates were not.

Animals↗
PubMed1992

The occurrence of long chain polyprenols in leaves of plants of Rosaceae family and their isolation by time-extended liquid chromatography.

The long chain polyprenols composed of 30 and more isoprene units from leaves of plants belonging to the genera Potentilla and Rosa have been described. They occur in the form of fatty acid esters. The composition of polyprenol mixture was species dependent and its content reached ca. 0.5% wet weight. Large scale preparation of individual polyprenols from a natural polyprenol mixture was performed using time-extended liquid chromatography on the hydrophobic gel Lipidex-5000.

Chromatography, Liquid↗
PubMed1991

Characterization and distribution of cis-prenyl transferase participating in liver microsomal polyisoprenoid biosynthesis.

The properties of rat liver cis-prenyl transferase, mediating the synthesis of polyisoprenoid pyrophosphate from trans,trans-farnesyl pyrophosphate and [3H]isopentenyl pyrophosphate were studied. The Km values for farnesyl pyrophosphate and isopentenyl pyrophosphate were found to be 25 microM and 4.4 microM, respectively. Appropriate conditions were established to measure the condensation reaction, which was linear during the first hour using 1 mg microsomal protein. Various detergents could solubilize the enzyme, but the presence of Triton X-100 was required during the incubation to obtain full activity. There was also an absolute requirement for Mg2+ and the pH maximum was 7.0. Inorganic phosphate, especially pyrophosphate, proved to be inhibitory. cis-Prenyl transferase is associated mainly with the cytoplasmic surface of rough microsomes and, to some extent, also with smooth I microsomes, but was almost absent from smooth II microsomes. At all localizations, the product is polyprenyl pyrophosphate and to some extent, also polyprenyl monophosphate. The isoprenoids formed contain 15-18 units in the presence of detergents and 16-20 units in the absence of detergents.

Animals↗
PubMed1991

Involvement of sterol carrier protein-2 in dolichol biosynthesis.

The effect of sterol carrier protein-2 (SCP-2) on dolichol biosynthesis by rat liver microsomes was investigated. cis-Prenyltransferase activity was stimulated 7-fold in the presence of 5 micrograms of purified SCP-2/mg of microsomal protein, which was similar to the increase obtained by adding detergent. The polyisoprenoid pattern obtained in the presence of SCP-2 was the same as that present in rat liver, in contrast to the pattern appearing upon incubation of microsomes with detergent, which gave shorter polyisoprenoids. Like SCP-2, the cytosolic fraction from rat liver also stimulated cis-prenyltransferase. Incubation with cytosol pretreated with anti-SCP-2 showed no stimulatory effect and led to the accumulation of shorter polyisoprenoids. SCP-2 had no appreciable effect on polyprenol alpha-saturase, dolichol kinase, dolichyl phosphate phosphatase, or acyl-CoA:dolichol acyltransferase. The results demonstrate that SCP-2 greatly stimulates and may regulate the condensation reactions mediated by cis-prenyltransferase in the process of dolichol biosynthesis and permits polymerization of the polyisoprenoid to its natural chain length.

Albumins↗
PubMed1991

Covalent binding of dolichyl phosphate to proteins in rat liver.

Rats were injected via the portal vein with (RS)-[5-3H]-mevalonolactone and the lipids were extracted. From fractions of liver homogenate, all labeled dolichol, cholesterol and ubiquinone could be extracted, but about 40% of microsomal and lysosomal dolichyl phosphate was only released after alkaline hydrolysis. Only a small amount of the non-extractable radioactivity was found to be associated with alpha-unsaturated polyprenyl phosphate. There was no difference in the polyisoprenoid pattern when the two pools of dolichyl phosphate were compared. On the other hand, the specific activity of the bound lipid was only half that of the extractable form. After phenyl-Sepharose chromatography, a peak of protein was isolated exhibiting a 25-fold enrichment in bound radioactive dolichyl phosphate. Treatment with a non-specific protease, followed by chromatography, gave polypeptide fragments associated with bound lipids. On SDS/PAGE a major protein band at 23 kDa and some minor bands with higher molecular masses were found to be associated with this lipid. The results indicate the presence of covalently bound dolichyl phosphate in rat liver.

Animals↗
PubMed1991

Long chain polyisoprenoid alcohols in leaves of Capparis species.

Leaves of twelve species of the genus Capparis were examined for the presence of long chain polyisoprenoid alcohols. In a number of species the accumulation of polyisoprenoid alcohols was up to about 0.3% of dry weight of tissue. In all studied species polyisoprenoid alcohols composed of 12, 13, 14 or 15 isoprene residues formed the main polyprenol family. In the majority of the plants studied lower quantities of an additional polyprenol family were present, in which prenologues composed of 19, 20 or 21 isoprene units were dominating. In one species--Capparis coriacea also the presence of dolichol-like polyprenols with a hydrogenated OH-terminal isoprene unit was documented.

Alcohols↗
PubMed1990

Nonaprenyl-4-hydroxybenzoate transferase, an enzyme involved in ubiquinone biosynthesis, in the endoplasmic reticulum-Golgi system of rat liver.

The properties and distribution of nonaprenyl-4-hydroxybenzoate transferase in rat liver were investigated with subcellular fractions, liver perfusion, and in vivo labeling with [3H]solanesyl-PP. In addition to some ubiquinone-9, only one labeled intermediate, i.e. nonaprenyl-4-hydroxybenzoate, was obtained. In the total microsomal fraction, the enzyme had a pH optimum of 7.5 and was completely inhibited by Triton X-100 and deoxycholate, but not by taurodeoxycholate and beta-octyl glucoside. Liver, kidney, and spleen demonstrated the highest activities of nonaprenyl-4-hydroxybenzoate transferase. Upon subcellular fractionation, high specific activities were found in smooth II microsomes and Golgi III vesicles. The enzyme was also found in lysosomes and plasma membranes, but only at low levels in rough and smooth I microsomes and mitochondria and not at all in peroxisomes and cytosol. When the product of the transferase reaction was used as a substrate in vitro and in a perfusion system, the only product obtained was end product ubiquinone-9. Although the transferase reaction was associated with the inner, luminal surface of microsomal vesicles, the terminal reaction(s) for ubiquinone-9 synthesis are found at the outer cytoplasmic surface. The results suggest that the major site for ubiquinone synthesis is the endoplasmic reticulum-Golgi system, which also participates in the distribution of ubiquinone-9 to other cellular membranes.

Alkyl and Aryl Transferases↗
PubMed1990

Voltage-dependent behaviour of dolichyl phosphate-phosphatidylcholine bilayer lipid membranes.

The current-voltage steady-state characteristics, cyclic voltammograms and capacitance-voltage steady-state relationships of bilayer lipid membranes made from dioleoylphosphatidylcholine or its mixtures with dolichyl-12 phosphate have been studied. Sustained fluctuations of the capacitance of dolichyl phosphate modified bilayers under applied voltage were observed. The results suggest that the dynamics of dolichyl phosphate molecules in membranes can be regulated by transmembrane electrical potential.

Chemical Phenomena↗
PubMed1989

Uptake and modification of dietary polyprenols and dolichols in rat liver.

Short and long dolichols and polyprenols in free form or esterified with fatty acids were incorporated into liposomes and administered to rats through a gastric tube. The free alcohols were taken up by the liver to different extents. While uptake in other organs was less, it also involved the fatty acid esters. The use of systems other than liposomes did not increase the efficiency of uptake. Most of the administered lipids were recovered in the lysosomes. Exogenous dolichols and polyprenols were both partly esterified in the liver and, to some extent, also phosphorylated; a portion of the polyprenols was also alpha-saturated. These results indicate that various polyisoprenes are taken up, to a small extent, from the diet by tissues under normal conditions and in liver these dietary lipids undergo terminal modifications.

Animals↗
PubMed1989

Hydrolysis of dolichyl esters by rat liver lysosomes.

Dolichyl ester hydrolase activity is broadly distributed among the organs of the rat. The highest activity was found in spleen, brain, lung, and thyroid tissues, whereas this activity is very low in stomach and intestine. The esterase involved is localized to the lumen of lysosomes and, to some extent, in the plasma membranes. Hydrolysis occurs with both alpha-saturated and alpha-unsaturated polyisoprenes esterified with different fatty acids, but the rate of hydrolysis is strongly dependent on the nature of the substrate. The enzyme involved is inhibited by divalent cations, EDTA and EGTA and also by one of the products, dolichol. The esterase is activated by 3-[(3-cholamidopropyl) dimethylammonio]-1-propranesulfonic acid and taurodeoxycholate and inhibited by Triton X-100. Dolichyl esterase activity is completely inhibited by alpha- and beta-naphthyl acetate, phenylmethylsulfonyl fluoride, and beta-chloromethylmercurisulfate. These inhibitors, as well as the pH optimum for dolichyl ester hydrolysis, clearly differentiate the enzyme involved from cholesteryl esterase and triglyceride lipase. Microsomal phospholipase A hydrolyzes dolichyl esters at a slow rate only. In vivo labeling experiments with [3H]mevalonate demonstrated that newly synthesized dolichol is transported in esterified form to the lysosomes, where this lipid is slowly hydrolyzed by the esterase. The possibility is raised that the role of the fatty acyl moiety may be to target dolichol to its final location in the cell.

Animals↗
PubMed1989

The occurrence of unique, long-chain polyprenols in the leaves of Potentilla species.

Polyprenols are accumulated in the leaves of Potentilla anserina at concentration up to 0.3% fresh weight. They constitute a mixture of poly-cis fully unsaturated analogues of up to 29 isoprene units long. In this and other species of Potentilla the polyprenol mixture is composed of two families, one grouping the medium chain-length polyprenols (built up of about 20 isoprene units), and the second one, composed mainly of very long prenolgues from 24 to approx. 28 isoprene units. This is a first report on the occurrence of polyprenyl alcohols of this chain length in plant material and the first one on the presence of multiple polyprenol mixture in angiosperms. A useful modification of polyprenols preparation from plant material, based on solid phase extraction with hydrophobic gel Lipidex-5000 is described.

Biomarkers↗