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PubMed1975

Purification and properties of a fructose-1,6-diphosphate activated L-lactate dehydrogenase from Staphylococcus epidermidis.

L-(+)-lactate dehydrogenase (LDH) from Staphylococcus epidermidis ATCC 14990 was purified by affinity chromatography. The purified enzyme was specifically activated by fructose-1,6-diphosphate (FDP). The concentration of FDP required for 50% maximal activity was about 0.15 mM. The enzyme activity was inhibited by adenosine diphosphate (ADP) and oxamate. The inhibition by ADP appeared to be competitive with respect to reduced nicotinamide adenine dinucleotide (NADH). The catalytic activity of the LDH for pyruvate reduction exhibited an optimum at pH 5.6. The enzyme is composed of four, probably identical, subunits. Sephadex gel filtration and sedimentation velocity at pH 5.6 Yielded molecular weights of about 130 000 and 126 000, respectively. The molecular weight at pH 6.5 and 7.0 was found to be only about 68 000. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate and sedimentation velocity at pH 2.0 or 8.5 revealed monomeric subunits with an approximate molecular weight of 36000. The thermostability of the heat labile enzyme was increased in the presence of FDP, NADH and pyruvate. The purified LDH exhibited an anomalous type of kinetic behavior. Plots of initial velocity vs. different concentrations of pyruvate, NADH or FDP led to saturation curves with intermediary plateau regions. As a consequence of these plateau regions the Hill coefficient alternated between lower and higher n-values. Some distinguishing properties of the S. epidermidis LDH and other LDHs activated by FDP are discussed.

Adenine Nucleotides↗
PubMed1978

Studies on ATP: thiamine diphosphate phosphotransferase activity in rat brain.

The experiments described in this paper serve as a contribution to the solution of the discrepancies which exist in the assay of ATP:thiamine diphosphate phosphotransferase activity (EC 2.7.4.15), presently in use as a tool for the diagnosis of Leigh's disease (SNE, subacute necrotizing encephalomyelopathy). The results obtained with this phosphotransferase assay can, in part, be explained by the presence of thiamine triphosphate (ThTP) in the preparation of thiamine diphosphate (ThDP) used as a substrate, by the inhibition by ATP of the ThTP phosphohydrolase activity, present in fractions of rat brain homogenates, and by the stimulation by ThDP of the ATPase activity. When [2(-14)C-thiazole]thiamine was used for the synthesis of [14C]ThTP in fractions of rat brain, it was found that after chromatographic separation of thiamine and its phosphates, 14C radioactivity could be demonstrated in the ThTP fractions, even in the absence of an enzyme source. Probably a complex is formed between [14C]thiamine and a phosphate ester which behaves chromatographically as ThTP. It is concluded that the assay system for the measurement of ThTP synthesis in its present form is, in our hands, not suitable for diagnostic purposes.

Adenosine Triphosphate↗
PubMed1985

The histochemical demonstration of fructose diphosphate aldolase activity using a semipermeable membrane technique.

In this communication an enzyme histochemical multistep technique for the demonstration of class 1 fructose-1,6-diphosphate aldolase in heart and skeletal muscle sections is described. With this technique a semipermeable membrane is interposed between the incubating solution and the tissue sections preventing diffusion of the enzyme into the medium during incubation. In the histochemical system the enzyme cleaves the substrate D-fructose-1,6-diphosphate to dihydroxyacetone phosphate and D-glyceraldehyde-3-phosphate. The dihydroxyacetone phosphate is reversibly converted into D-glyceraldehyde-3-phosphate by exogenous and endogenous triose phosphate isomerase. Next the D-glyceraldehyde-3-phosphate is oxidized by exogenous and endogenous glyceraldehyde-3-phosphate dehydrogenase and the electrons are transported concomitantly via NAD+, phenazine methosulphate and menadione to nitro-BT. Sodium azide and amytal are incorporated to block electron transfer to the cytochromes.

Animals↗
PubMed2002

Effect of a conserved mutation in uridine diphosphate glucuronosyltransferase 1A1 and 1A6 on glucuronidation of a metabolite of flutamide.

OBJECTIVE: We investigated how the conserved mutation (Y486D) changed the kinetic parameters of uridine diphosphate glucuronosyltransferase 1A1 and 1A6 (UGT1A1 and 1A6) for 2-amino-5-nitro-4-trifluoromethylphenol, which is a major metabolite of flutamide, a nonsteroidal antiandrogenic agent. METHODS: The wild-type or mutant cDNA-expressed UGT was co-incubated with 2-amino-5-nitro-4-trifluoromethylphenol (aglycone) and uridine diphosphate-glucuronic acid (UDP-GA, donor substrate). The glucuronidation of the aglycone was determined. RESULTS: The maximum velocities of the mutant UGT1A1 and UGT1A6 were about 12% and less than 1% of the corresponding wild-type, respectively. The Michaelis constant (K(M)) for the aglycone of the wild-type UGT1A1 was double that of the mutant, but the K(M) for UDP-GA of the wild-type UGT1A1 was not significantly different from that of the mutant. CONCLUSION: Patients with Y486D may accumulate excessive 2-amino-5-nitro-4-trifluoromethylphenol, which might lead to unexpected toxicity.

Androgen Antagonists↗
PubMed2004

Batch and fed-batch production of coenzyme Q10 in recombinant Escherichia coli containing the decaprenyl diphosphate synthase gene from Gluconobacter suboxydans.

Coenzyme Q(10) (CoQ(10)) is a quinine consisting of ten units of the isoprenoid side-chain. Because it limits the oxidative attack of free radicals to DNA and lipids, CoQ(10) has been used as an antioxidant for foods, cosmetics and pharmaceuticals. Decaprenyl diphosphate synthase (DPS) is the key enzyme for synthesis of the decaprenyl tail in CoQ(10) with isopentenyl diphosphate. The ddsA gene coding for DPS from Gluconobacter suboxydans was expressed under the control of an Escherichia coli constitutive promoter. Analysis of the cell extract in recombinant E. coli BL21/pACDdsA by high performance liquid chromatography and mass spectrometry showed that CoQ(10) rather than endogenous CoQ(8) was biologically synthesized as the major coenzyme Q. Expression of the ddsA gene with low copy number led to the accumulation of CoQ(10) to 0.97 mg l(-1) in batch fermentation. A high cell density (103 g l(-1)) in fed-batch fermentation of E. coli BL21/pACDdsA increased the CoQ(10) concentration to 25.5 mg l (-1) and its productivity to 0.67 mg l(-1) h(-1), which were 26.0 and 6.9 times higher than the corresponding values for batch fermentation.

Alkyl and Aryl Transferases↗
PubMed2005

Isopentenyl-diphosphate isomerase is essential for viability of Caenorhabditis elegans.

Homozygosity for a mutation in the idi-1 gene of Caenorhabditis elegans results in paralysis during the first larval stage, followed by an arrest of growth and development late in the first larval stage. Apoptotic corpses, which are apparently the result of normal programmed cell death, persist in the arrested larvae. In genetic mosaics, an additional defect becomes evident upon examination with Nomarski optics: cells that are genotypically mutant enlarge, and their cytoplasm becomes dimpled. Electron microscopy indicates that the dimpling reflects an accumulation of many enlarged lysosomes and autophagosomes. The mosaics demonstrate that the lethal mutation acts cell autonomously with respect to this vesicular abnormality and that there is a maternal effect with respect to the time of developmental arrest of mutant progeny. Cloning of the gene reveals that it is the only gene in C. elegans for isopentenyl-diphosphate isomerase, an enzyme that is important for the synthesis of lipophilic molecules, including farnesyl and geranyl diphosphates.

Amino Acid Sequence↗
PubMed2004

The AtPPT1 gene encoding 4-hydroxybenzoate polyprenyl diphosphate transferase in ubiquinone biosynthesis is required for embryo development in Arabidopsis thaliana.

4-Hydroxybenzoate polyprenyl diphosphate transferase (4HPT) is the key enzyme that transfers the prenyl side chain to the benzoquione frame in ubiquinone (UQ) biosynthesis. The Arabidopsis AtPPT1 cDNA encoding 4HPT was cloned by reverse transcription-polymerase chain reaction (RT-PCR) based on the information of the Arabidopsis genomic sequence, and the function of the gene was determined. Heterologous expression of the AtPPT1 gene enabled restoration of the respiratory ability and UQ synthesis in a yeast mutant that was defective in 4HPT activity. The mitochondrial fraction that was prepared from the yeast mutant, which expressed the AtPPT1 gene, exhibited 4HPT enzymatic activity with geranyl diphosphate (GPP) as the prenyl substrate. This indicated that the AtPPT1 gene encodes active 4HPT with a broad substrate specificity in terms of the prenyl donor. The AtPPT1 mRNA was predominantly expressed in the flower cluster, and the green fluorescent protein (GFP) fused with the signal peptide of AtPPT1 was translocated into the mitochondria. T-DNA insertion mutation that disrupts the AtPPT1 gene in Arabidopsis resulted in the arrest of embryo development at an early stage of zygotic embryogenesis. These results demonstrate that the AtPPT1 gene involved in the biosynthesis of mitochondrial UQ plays an essential role in embryo development in Arabidopsis .

Alkyl and Aryl Transferases↗
PubMed2005

The maize An2 gene is induced by Fusarium attack and encodes an ent-copalyl diphosphate synthase.

Using the technique of differential display, a maize transcript was identified whose silk tissue expression is induced in the presence of the ear rot pathogen Fusarium graminearum. The 3445 nt transcript includes a 727 nt 5' untranslated leader with the potential for extensive secondary structure and represents the maize gene An2. An2 encodes a copalyl diphosphate synthase (CPS)-like protein with 60% amino acid sequence identity with the maize An1 gene product involved in gibberellin (GA) biosynthesis. Recombinant expression and functional analysis demonstrated that both AN1 and AN2 are ent-copalyl diphosphate (ent-CPP) synthases (ent-CPS). Notably, the presence of an additional ent-CPS gene is consistent with previous reports that maize GA biosynthesis can proceed in the absence of An1. In addition, northern blot analysis showed that An2 transcript levels were strongly up-regulated by Fusarium attack, with an increase in silk, husk and ear tip tissues as early as 6 h after inoculation of silk channels with spore suspensions of various Fusarium sp. Gene expression of a third maize CPS-like gene, Cpsl1, is not affected by Fusarium infection. The Fusarium-inducible nature of An2 is also consistent with a previous report that cell-free extracts from maize seedlings produce ent-CPP derived diterpenes in response to Fusarium infection. However, it is not known whether An2 is involved in defense-related secondary metabolism in addition to GA synthesis.

5' Untranslated Regions↗
PubMed1976

A mutant strain of Scenedesmus obliquus deficient in ribulose diphosphate carboxylase, cytochrome f and photosystem II activity.

The partial reactions of photosynthesis shown by strain F208, a non-photosynthetic mutant strain of Scenedesmus obliquus, have been compared with those performed by other mutant strains which lacked; Photosystem II activity (strains 11 and F131), cytochrome f (strain 50), P-700 and cytochrome f (strain F 119), and P-700 (strains F139 and 199). In this respect the properties of strain F208 were those that would be expected if Photosystem II activity and cytochrome f were not present in this strain. Examination of the composition of strain F208 has shown the absence of cytochrome f in both the soluble and the membrane-bound form. The considerably lower level of plastoquinone compared to that found in the wild type is characteristic of the strains which lack Photosystem II activities. Fraction 1 protein could not be detected in extracts of strain F208 by sedimentation velocity experiments in the ultracentrifuge, and only 7% of the wild type ribulose diphosphate carboxylase activity was found after chromatography of these extracts on DEAE-cellulose. The properties of strain F208 are compared with those of the ac-20 and cr-1 strains of Chlamydomanas rheinhardi, both of which have a deficiency of ribulose diphosphate carboxylase which is considered to result from a deficiency of chloroplast ribosomes. Strain F208 resembles these strains in its abnormal chloroplast ultrastructure and its decreased levels of the RNA forms derived from the chloroplast ribosomes when compared with the wild type. Chloroplast fragments isolated from strains of S. obliquus which lacked cytochrome f (strains 50 and F208) were able to use diaminodurene and ascorbate as an electron donor to Photosynstem I. Since this reaction was inhibited by mercuric salts it would appear that plastocyanin, but not cytochrome f, was involved in this electron transfer.

Ascorbic Acid↗
PubMed1991

Modification of the radiochemical assay of rat liver mevalonate-5-diphosphate decarboxylase and induction and stabilization of the activity.

Problems encountered in attempts to purify mevalonate-5-diphosphate decarboxylase from rat liver are addressed. These are the quantitative, facile separation of [14C]isopentenol in the radiochemical assay (2) the instability of the enzyme activity and (3) the very low activity in rat liver. The assay was modified by using Sep Pac C18 filters to bind and release [14C]isopentenol. Authentic isopentenol was quantitated by absorbance at 210 nm wavelength and the extinction coefficient estimated to be epsilon m = 3.26 X 10(3). Recovery of authentic isopentenol from aqueous solution after binding and elution into methanol was quantitative from 10-100 nmols. Recovery of [14C]ispentenol from assay mixtures using 2-[14C]mevalonate-5-diphosphate and alkaline phosphatase to hydrolyze phosphate was quantitative using Sep Pac filter but not using petroleum ether extraction. Enzyme activity was stabilized by phenylmethylsulfonyl fluoride, aprotinin and leupeptin and was stable at -73 degrees C for 3 months. Activity of the decarboxylase was increased by 5-fold after feeding young rats 2.5% cholestyramine for ten days to four weeks.

Animals↗
PubMed1987

Synthesis of the tetrasaccharide lipid intermediate P1-dolichyl P2-[O-alpha-D-mannopyranosyl-(1----6)-O-beta-D-mannopyranosyl-(1----4) -O-(2- acetamido-2-deoxy-beta-D-glucopyranosyl)-(1----4)-2-acetamido-2- deoxy-alpha-D-glucopyranosyl] diphosphate.

O-alpha-D-Mannopyranosyl-(1----6)-O-beta-D-mannopyranosyl-(1----4)-O-(2- acetamido-2-deoxy-beta-D-glucopyranosyl)-(1----4)-2-acetamido-2-deoxy- D-glucopyranose was isolated from bovine or ovine mannosidosis urine. After peracetylation, treatment with trimethylsilyl trifluoromethanesulfonate gave a high yield of a peracetyl oxazoline, which was phosphorylated with dibenzyl phosphate to give a dibenzyl glycosyl phosphate that was converted into a peracetyl tetrasaccharide phosphate by catalytic hydrogenolysis. A coupling reaction with P1-dolichyl P2-diphenyl diphosphate, prepared in two stages from pig-liver dolichol, yielded a peracetyl diphosphoric diester, which on O-deacetylation gave P1-dolichyl P2-[O-alpha-D-mannopyranosyl-(1----6)-O-beta-D-mannopyranosyl-(1----4)-O -(2- acetamido-2-deoxy-beta-D-glucopyranosyl)-(1----4)-2-acetamido-2-deoxy- alpha-D- glucopyranosyl] diphosphate. This tetrasaccharide lipid intermediate was active as an acceptor of D-mannose residues from GDP-D-mannose in the presence of calf pancreas microsomes.

Acetylation↗
PubMed1989

Characterization of dolichyl diphosphate phosphatase from rat liver.

Dolichyl diphosphate phosphatase (DolPPase) has been characterized in rat liver. Subcellular distribution studies indicate that the enzyme is localized in the endoplasmic reticulum. The in vitro enzymatic activity is stimulated by EDTA, due to release of inhibition by trivalent cations found in the assay tubes. All di- and trivalent cations tested were inhibitory, with the trivalent ions Al3+ and Fe3+ showing greater than 70% inhibition at a concentration of 10 microM. The assay requires the presence of a detergent for optimal activity, with Triton X-100 giving maximum activity at 0.1%. The substrate specificity of DolPPase toward polyprenyl diphosphates has been determined and indicates that there is little preference of the enzyme for substrates of different chain length, and either stereochemical orientation or degree of saturation of the alpha-isoprene unit. Km values of 11-14 microM were obtained for all substrates tested. Preliminary studies on the transmembrane topology of the DolPPase using latency assays, indicate that the active site of the enzyme may reside on the cytoplasmic face of the endoplasmic reticulum.

Animals↗
PubMed1992

Effects of substitution of aspartate-440 and tryptophan-487 in the thiamin diphosphate binding region of pyruvate decarboxylase from Zymomonas mobilis.

A tryptophan residue at position 487 in Zymomonas mobilis pyruvate decarboxylase was altered to leucine by site-directed mutagenesis. This modified Z. mobilis pyruvate decarboxylase was active when expressed in Escherichia coli and had unchanged kinetics towards pyruvate. The enzyme showed a decreased affinity for the cofactors with the half-saturating concentrations increasing from 0.64 to 9.0 microM for thiamin diphosphate and from 4.21 to 45 microM for Mg2+. Unlike the wild-type enzyme, there was little quenching of tryptophan fluorescence upon adding cofactors to this modified form. The data suggest that tryptophan-487 is close to the cofactor binding site but is not required absolutely for pyruvate decarboxylase activity. Substitution of asparagine, threonine or glycine for aspartate-440, a residue which is conserved between many thiamin diphosphate-dependent enzymes, completely abolishes enzyme activity.

Amino Acid Sequence↗
PubMed1993

Crystal structure of transketolase in complex with thiamine thiazolone diphosphate, an analogue of the reaction intermediate, at 2.3 A resolution.

The crystal structure of the complex of transketolase and thiamine thiazolone diphosphate has been determined at 2.3 A resolution. The complex has a structure which closely resembles that of this enzyme with the cofactor ThDP. This is consistent with the observation that the binding of the analogue to transketolase involves ground state rather than transition state interactions. Since thiamine thiazolone diphosphate resembles an expected intermediate in the catalytic pathway, the structure of the intermediate was modelled from the crystal structure. Based on this model, enzymic groups responsible for binding of the intermediate and proton transfer during catalysis are suggested.

Binding Sites↗
PubMed1987

Characterization and tissue specificity of a monoclonal antibody against human uridine 5'-diphosphate-glucuronosyltransferase.

A monoclonal antibody against human liver uridine 5'-diphosphate-glucuronosyltransferase (UDPGTase) was developed. Enzyme inhibition studies with this monoclonal antibody showed inhibition of human liver UDPGTase activity with bilirubin, 4-methylumbelliferone, and 4-nitrophenol as substrates. Testosterone, estrone, and phenolphthalein UDPGTase activity was not inhibited. The monoclonal antibody probably recognizes the uridine 5'-diphosphate-glucuronic acid binding site at 4-nitrophenol UDPGTase. Proteins with a molecular mass of 54,000 daltons were immunopurified from solubilized human liver using the monoclonal antibody as ligand coupled to Sepharose 4B beads. The distribution of UDPGTase isoforms in various human tissues was studied by immunofluorescence. The enzyme could be detected in liver, kidney, stomach, small intestine, colon, and skin. In liver, only hepatocytes contain UDPGTase. In kidney, the enzyme was localized exclusively in proximal tubuli and in stomach in epithelial mucous cells. In small intestinal epithelium, maximal fluorescence was seen at the villous tip. In colon, all lining epithelial cells were stained. In colon polyps, staining for UDPGTase was clearly decreased, and in colon carcinoma it was undetectable. In skin, the stratum corneum was intensely stained, whereas the stratum basale showed no fluorescence.

Animals↗
PubMed1990

Effects of uridine diphosphate glucuronosyltransferase activity on the maximal secretion rate of bilirubin conjugates in the rat.

It is generally accepted that biliary secretion is rate-limiting for the plasma-to-bile transport of a saturating load of bilirubin. Previous studies from this laboratory have suggested a relationship between the hepatic bilirubin uridine diphosphate glucuronosyltransferase activity and the apparent maximal rate of bilirubin secretion (excretory transport maximum). The present study was undertaken to further investigate this relationship in rats with a wide range of transferase levels and to analyze the effects of transferase activity on the formation of bilirubin monoglucuronides and diglucuronides in vivo and in vitro. Animals with moderately decreased enzyme activity still showed a near-normal excretory transport maximum but decreased fractional amounts of conjugated bilirubins in plasma and liver and a decreased ratio of bilirubins diconjugates to monoconjugates in bile, liver, and plasma. A more pronounced decrease in the level of transferase activity yielded lower excretory transport maximum values as well. Enhanced transferase activities were obtained by pretreatment with a series of enzyme-inducing agents, usually at dosages not influencing bile flow. An enhanced transferase activity produced significant increases in bilirubin excretory transport maximum, decreased concentrations of both unconjugated and conjugated bilirubins in plasma, and an increased ratio of diconjugates to monoconjugates in bile and in liver. In all animals, the ratio of disconjugates to monoconjugates observed in bile was correlated with the ratio found after incubation of liver homogenates in vitro at low concentrations of bilirubin substrate. Overall, the relationship between bilirubin excretory transport maximum and uridine diphosphate glucuronosyltransferase activity can best be described by a hyperbolic curve. At "saturation" of the excretory transport maximum values, no increase of conjugates in liver or in plasma could be observed, suggesting that not the biliary secretion, per se, but rather the conjugation rate in vivo is rate-limiting. The latter seems lower than the in vitro assayed enzyme activity. The present investigations point to an equilibration of bilirubin pigments between plasma, liver, and bile. The in vivo conjugation rate can be estimated from the in vitro assay (but not at high enzyme activities) and determines the maximal biliary secretion rate, the ratio of diconjugates to monoconjugates in the three compartments studied, and the relative amount of conjugates in plasma. Our studies suggest that the conjugation rate in vivo rather than the biliary secretion step is the major determinant of the maximal bilirubin secretion rate.

Animals↗
PubMed1975

Management of adenocarcinoma of prostate with diethylstilbestrol diphosphate and cyclophosphamide.

Eleven patients with terminal metastatic prostatic carcinoma were selected for a pilot study to evaluate how effective diethylstilbestrol diphosphate (Stilphostrol) and cylcophosphamide (Cytoxan) are in this disease. Six of 7 patients had a good response when treated with diethylstilbestrol diphosphate. The mean duration of response was 6.4 months. All 6 patients given cyclophosphamide had marked relief of pain and increased mobility. Of these, 3 died twelve, sixteen, and nineteen weeks, respectively, following initiation of therapy while still benefiting from pain relief. The mean duration of response was 14.8 weeks.

Adenocarcinoma↗
PubMed1976

Regression of prostatic cancer metastasis by high doses of diethylstilbestrol diphosphate.

Diethylstilbestrol diphosphate (DES-P) has shown effective symptomatic relief in patients with metastatic carcinoma of the prostate. Although there is little known about its role in soft tissue metastasis, our experience in 3 patients with advanced carcinoma of the prostate infiltrating the trigone and ureterovesical junction revealed significant improvement of hydronephrosis. All patients failed to respond to conventional doses of stilbestrol. Diethylstilbestrol diphosphate is recommended in the treatment of advanced carcinoma of the prostate with soft tissue metastasis. It is safe and effective, and the tumor responses outweigh the side effects of the drug. The mechanism of action of this compound is discussed.

Aged↗