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At least 649 records · Page 36Linked to original sources
PubMed1984

Enzyme studies on TPPase-reactive cytoplasmic structures observed in early meiotic prophase I of the hamster oocyte.

Ovaries of immature and adult hamsters were incubated in medium containing thiamine pyrophosphate (TPP) to determine the age at which TPPase-reactive cytoplasmic structures first appear in the germ cells, and at what age the structures cease to be present. The structures were found only in oocytes from animals 8-15 days of age. They occur in predictyate germ cells in polyovular follicles and in very early dictyate oocytes in unilaminar follicles. The TPPase-reactive structures were never observed in atretic oocytes, in unilaminar follicles of adult animals, nor in multilaminar follicles of animals at any age. Ovaries of 8-12-day-old animals and adults were then incubated in media in which one of the following substrates was substituted for TPP: uridine diphosphate (UDP), inosine diphosphate (IDP), and adenosine monophosphate (AMP). Half of the samples in each experiment were incubated in medium containing the inhibitor L-p-bromotetramisole. beta-Glycerophosphate was used in control incubations, or the substrate was omitted entirely. The cytoplasmic structures were found to be reactive after incubation in UDP-containing media, but not after incubation in media containing AMP. With IDP as substrate, reactions were atypical and confined to peripheral regions of the cytoplasm. Other sites of enzyme activity after incubation with the various substrates (cell membranes, zona pellucida, endoplasmic reticulum and Golgi apparatus) are also described and discussed.

Aging↗
PubMed1995

Pyruvate: ferredoxin oxidoreductase from the sulfate-reducing Archaeoglobus fulgidus: molecular composition, catalytic properties, and sequence alignments.

Archaeoglobus fulgidus is a hyperthermophilic sulfate-reducing archaeon. In this communication we describe the purification and properties of pyruvate: ferredoxin oxidoreductase from this organism. The catabolic enzyme was purified 250-fold to apparent homogeneity with a yield of 16%. The native enzyme had an apparent molecular mass of 120 kDa and was composed of four different subunits of apparent molecular masses of 45, 33, 25, and 13 kDa, indicating an alpha beta gamma delta structure. Per mol, the enzyme contained 0.8 mol thiamine pyrophosphate, 9 mol non-heme iron, and 8 mol acid-labile sulfur. FAD, FMN, lipoic acid, and copper were not found. The purified enzyme showed an apparent Km for coenzyme A of 0.02 mM, for pyruvate of 0.3 mM, and for clostridial ferredoxin of 0.01 mM, an apparent Vmax of 64 U/mg (at 65 degrees C) with a pH optimum near 7.5 and an Arrhenius activation energy of 75 kJ/mol (between 30 and 70 degrees C). The temperature optimum was above 90 degrees C. At 90 degrees C, the enzyme lost 50% activity within 60 min in the presence of 2 M KCl. The enzyme did not catalyze the oxidation of 2-oxoglutarate, indolepyruvate, phenylpyruvate, glyoxylate, and hydroxypyruvate. The N-terminal amino acid sequences of the four subunits were determined. The sequence of the alpha-subunit had similarities to the N-terminal amino acid sequence of the alpha-subunit of the heterotetrameric pyruvate: ferredoxin oxidoreductase from Pyrococcus furiosus and from Thermotoga maritima, and unexpectedly, to the N-terminal amino acid sequence of the homodimeric pyruvate:ferredoxin oxidoreductase from proteobacteria and from cyanobacteria. No sequence similarities were found, however, between the alpha-subunits of the enzyme from A. fulgidus and the heterodimeric pyruvate:ferredoxin oxidoreductase from Halobacterium halobium.

Amino Acid Sequence↗
PubMed1992

Immunohistochemical demonstration of a new thiamine diphosphate-binding protein in the rat digestive tract.

We purified a new thiamine diphosphate-binding protein (ThDP-BP), produced an antiserum against it, and examined its immunohistochemical distribution in the rat gastrointestinal tract using the avidin-biotin complex method. Positive staining for ThDP-BP was found in the epithelial glands of the stomach, small intestine and large intestine, and in the nuclei of hepatocytes. Measurement of the content of thiamine and its phosphate esters in extracts from the gastric and intestinal mucosa also indicated the presence of ThDP-BP in the stomach and intestinal mucosa. ThDP-BP may be useful for investigating the absorption and metabolism of the thiamine in metabolic disorders.

Animals↗
PubMed1989

Purification and characterization of the pyruvate-ferredoxin oxidoreductase from Clostridium acetobutylicum.

The pyruvate-ferredoxin oxidoreductase from Clostridium acetobutylicum was purified to homogeneity and partially characterized. A 9.2-fold purification was achieved in a three step purification procedure: ammonium sulfate fractionation, chromatography on Phenyl Sepharose and on Procion Blue H-EGN12. The pure enzyme exhibited a specific activity of 25 U/mg of protein. Homogeneity of the pyruvate-ferredoxin oxidoreductase was confirmed by native polyacrylamide gel electrophoresis and sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis. The molecular weight was determined to be 123,000/monomer. The subunit composition of the native enzyme could not be determined because of the instability of the pure enzyme. The pyruvate-ferredoxin oxidoreductase is sensitive to oxygen and dilution during purification. The dilution inactivation could be partially overcome by the addition of 300 microM coenzyme A or 50% ethyleneglycol. A thiamine pyrophosphate content of 0.39 mol per mol of enzyme monomer was found, the iron and sulfur content was 4.23 and 0.91, respectively. The pH-optimum was at pH 7.5 and the temperature optimum was at 60 degrees C. Kinetic constants were measured in the forward reaction. The apparent Km for pyruvate and coenzyme A were 322 microM and 3.7 microM, respectively. With 2-ketobutyrate the pyruvate-ferredoxin oxidoreductase showed 12.5% of the activity compared to pyruvate. No activity was found with 2-ketoglutarate. Ferredoxin from Clostridium pasteurianum could be used as physiological electron acceptor.

Chromatography, Ion Exchange↗
PubMed1987

Enzymatic and morphological response of the thymus to drugs in normal and zinc-deficient pregnant rats and their fetuses.

In the thymus of normally fed pregnant rats the plasma membrane enzymes dipeptidyl peptidase IV (DPP IV) and alkaline phosphatase (alP) were found in cortical and medullary lymphocytes (thymocytes). Plasma membrane aminopeptidase A (APA) and adenosine monophosphate hydrolysing phosphatase (AMPP) were present in cortical reticular cells. In medullary reticular cells, aminopeptidase M (APM), gamma-glutamyl transferase (GGT), adenosine triphosphate (ATPP) and thiamine pyrophosphate (TPPP) cleaving phosphatases were detected. Medullary reticular cells did not contain APA. Lysosomal DPP I and II, acid phosphatase, acid beta-D-galactosidase, beta-D-N-acetyl-glucosaminidase, beta-D-glucuronidase and non-specific esterases occurred especially in macrophages at the corticomedullary junction. The 21-day-old fetal thymus showed a similar reaction pattern as the maternal organ except for APA which was absent before birth. After treatment of the pregnant rats with valproic acid (VPA), salicylic acid (SA), streptozotocin (ST) and retinoic acid (RA) APA showed an increase in activity in the thymic cortex. In addition, ST and RA induced AMPP, ATPP and TPPP activity in cortical reticular cells up to the same pattern as in medullary reticular cells. After ethanol (ET) administration severe damages occurred. The thymic cortex was free of DPP IV-positive lymphocytes; the medullary reticular cells showed reduced or no GGT and occasionally an increased APM activity. Dexamethasone (DEXA) given to normal or zinc-deficient rats produced the most severe lesions; thymocytes with DPP IV activity were completely absent in the cortex and medulla. In Zn-deficient pregnant rats similar alterations were observed as after ET.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
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↗
PubMed1995

Regional alterations of thiamine phosphate esters and of thiamine diphosphate-dependent enzymes in relation to function in experimental Wernicke's encephalopathy.

Thiamine phosphate esters (thiamine monophosphate-TMP; thiamine diphosphate-TDP and thiamine triphosphate-TTP) were measured as their thiochrome derivatives by High Performance Liquid Chromatography in the brains of pyrithiamine-treated rats at various stages during the development of thiamine deficiency encephalopathy. Severe encephalopathy was accompanied by significant reductions of all three thiamine phosphate esters in brain. Neurological symptoms of thiamine deficiency appeared when brain levels of TMP and TDP fell below 15% of normal values. Activities of the TDP-dependent enzyme alpha-ketoglutarate dehydrogenase were more severely reduced in thalamus compared to cerebral cortex, a less vulnerable brain structure. On the other hand, reductions of TTP, the non-cofactor form of thiamine, occurred to a greater extent in cerebral cortex than thalamus. Early reductions of TDP-dependent enzymes and the ensuing metabolic pertubations such as lactic acidosis impaired brain energy metabolism, and NMDA-receptor mediated excitotoxicity offer rational explanations for the selective vulnerability of brain structures such as thalamus to the deleterious effects of thiamine deficiency.

Animals↗
PubMed1988

Anion specificity of the jejunal folate carrier: effects of reduced folate analogues on folate uptake and efflux.

We previously reported that 3H-folate uptake by rabbit jejunal brush-border membrane (BBM) vesicles was markedly stimulated by an outwardly directed OH- gradient (pHin 7.7, pHout 5.5), inhibited by anion exchange inhibitors (DIDS, SITS, furosemide), and saturable (folate Km = 0.19 microM) suggesting carrier-mediated folate/OH- exchange (or H+/folate cotransport). In the present study, the anion specificity of this transport process was examined. Under conditions of an outwardly directed OH- gradient, DIDS-sensitive folate uptake was cis inhibited (greater than 90%) by reduced folate analogues: dihydrofolate (IC50 = 0.40 microM), folinic acid (IC50 = 0.50 microM), 5-methyltetrahydrofolate (IC50 = 0.53 microM), and (+)amethopterin (IC50 = 0.93 microM). In contrast, 10 microM (-)amethopterin had only a modest effect on folate uptake (18% inhibition) suggesting stereospecificity of the folate/OH- exchanger. The nonpteridine compounds which are transported by the folate carrier in L1210 leukemic cells (phthalate, thiamine pyrophosphate, and PO4(-3] did not inhibit jejunal folate uptake. Furthermore, folate uptake was not inhibited by SO4(-2) (4 mM) or oxalate (4 mM) thereby distinguishing this carrier from the previously described intestinal SO4(-2)/OH- and oxalate/Cl- exchangers. After BBM vesicles were loaded with 3H-folate, the initial velocity of 3H-folate efflux was stimulated by unlabeled folate in the efflux medium. The transstimulation of 3H-folate efflux by unlabeled folate was furosemide (or DIDS) inhibitable and temperature sensitive. Half-maximal stimulation of furosemide-sensitive 3H-folate efflux was observed with 0.25 +/- 0.05 microM unlabeled folate, a concentration similar to the Km for folate uptake. These data suggest that folate-stimulated 3H-folate efflux is mediated by the folate/OH- exchanger.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2005

New mitochondrial carriers: an overview.

The transport of metabolites, nucleotides and cofactors across the mitochondrial inner membrane is performed by members of mitochondrial carrier family (MCF). These proteins share marked structural features that have made feasible the functional characterization of numerous MCs in the last years. The MCs responsible for transport activities in mitochondria known for decades such as glutamate uptake or ATP-Mg/Pi exchange have recently been identified as well as novel carriers such as those involved in S-adenosylmethionine or thiamine pyrophosphate uptake. Here, after a brief review of the novel data on structural characteristics and import mechanisms of MCF members, we present an exhaustive compilation of human MC sequences, including previously characterized carriers, together with their respective Saccharomyces cerevisiae orthologues, ordered according to the phylogenetic analysis of el Moualij and co-workers [Yeast (1997) 13: 573-581]. We have detected the existence of at least 49 human MC sequences, including those of yet unknown function. An overview of novel MCF members functionally characterized in recent years in mammals and in yeast genomes is presented.

Biological Transport↗
PubMed2005

Cloning and characterization of the gene encoding phosphoketolase in Leuconostoc mesenteroides isolated from kimchi.

The gene encoding phosphoketolase, which is 2749 bp long and contains 814 amino acid polypeptides with a total molecular mass of 91.9 kDa, was cloned from Leuconostoc mesenteroides C7 (LMC7) and expressed in Escherichia coli. It exhibited a homology of >58% with phosphoketolases from other lactic acid bacteria. The phosphoketolase of LMC7 belongs to the xylulose 5-phosphate (X5P)/fructose 6-phosphate (F6P) phosphoketolase (Xfp) family, which is an enzyme with dual specificity for X5P and F6P. The members of this family contain typical thiamin pyrophosphate (TPP) binding sites as reported for other TPP-dependent enzymes, and several highly conserved regions as signature patterns for phosphoketolases. The plasmid pGPK containing the Xfp gene (xfp) exhibits phosphoketolase activity in E. coli. The specific activities of the enzyme from E. coli BL21 and E. coli EC101 harboring xfp were 0.28 and 0.14 units/mg, respectively. They both exhibited a 1.5-fold increase in the production of acetic acid from acetyl phosphate compared with their corresponding original strain.

Aldehyde-Lyases↗
PubMed1990

Measurement of alpha-ketoglutarate dehydrogenase activity in tissue extracts and human platelets using reversed-phase high-performance liquid chromatography.

A new method for the determination of the activity of alpha-ketoglutarate dehydrogenase complex (KGDHC) in mouse brain and liver mitochondria and in human platelets using reversed-phase high-performance liquid chromatography is described. This method is based on the quantification of succinyl-CoA formed in the reaction catalyzed by KGDHC. Succinyl-CoA was separated using a YMC-Pack C8 column employing isocratic elution and detected spectrophotometrically at 254 nm. The detection limit of succinyl-CoA was 0.05 nmol. Succinyl-CoA in the supernatant of the assay mixture was stable for several hours at 4 degrees C and for a week when stored at -20 degrees C. The KGDHC assay showed good linearity with time and added protein, and all tissues demonstrated an absolute requirement for added alpha-ketoglutarate, nicotinamide dinucleotide, and coenzyme A and partial or no requirement for thiamine pyrophosphate, magnesium chloride, and dithiothreitol. The specific activities in liver and brain mitochondria and platelet homogenates determined by the present method were 19.2 +/- 0.9, 18.1 +/- 2.8, and 2.6 +/- 0.3 nmol/min/mg protein, respectively. In human platelets, the present method gives higher specific activity and lower blank values than a prior method using 14CO2 and may be useful in the diagnosis of KGDHC deficiency. This method is simple, rapid, and can be readily employed for the determination of KGDHC activity in various animal tissues and human platelets.

Acyl Coenzyme A↗
PubMed1991

Determination of thiamin and its phosphate esters in cultured neurons and astrocytes using an ion-pair reversed-phase high-performance liquid chromatographic method.

A sensitive method, based on fluorescence detection, for the determination of thiamin derivatives after precolumn derivatization is described. The separation is achieved on a PRP-1 column using ion-pair reversed-phase HPLC. This method is especially well adapted to the detection of thiamin triphosphate in complex mixtures such as tissue extracts. The detection limit for TTP is 50 fmol. The contents of thiamin derivatives were determined in primary cultures of rat cerebellar granule neurons and cerebral astrocytes. The amount of TTP is about five times higher in neurons than in astrocytes. Thus in rat brain TTP seems to be essentially associated with neurons and the intracellular concentration is estimated to be about 0.2 microM. Our results suggest the existence, in nerve cells, of specific regulatory mechanisms not related to the blood-brain barrier and responsible for the maintenance of thiamin homeostasis in brain.

Animals↗
PubMed1983

Purification and properties of transketolase from fresh rat liver.

Transketolase has been purified from rat liver. The final product is judged to be homogeneous by the criteria of gel filtration on Sephadex G-200 and Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The estimated Mr's are 139,000 for the native protein and 69,000 for the dissociated subunits. The purified enzyme does not require either Mg2+ or thiamine pyrophosphate for optimum activity. The best C2-donor substrate is D-xylulose 5-phosphate, with Km = 25 microM. D-Fructose 6-phosphate is an active C2-donor, but beta-hydroxypyruvate was found to be inactive, even at high concentration. The product of the C2-transfer from D-xylulose 5-phosphate or D-fructose 6-phosphate to D-ribose 5-phosphate has been identified as D-sedoheptulose 7-phosphate. Neither D-glucose 6-phosphate nor D-arabinose 5-phosphate showed C2-acceptor activity. This enzyme does not appear to be responsible for the formation of octulose 8-phosphates which have been detected in liver extracts.

Animals↗