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Polioencephalomalacia induced with amprolium in buffalo calves--clinicopathologic findings.

Polioencephalomalacia was induced in eight buffalo calves, 6-12 months old, by drenching amprolium (300 mg/kg body weight per day) for 29-55 days. Four buffalo calves of the same age group were drenched with tap water only and served as control. Blood samples were collected at different intervals during amprolium administration until the onset of clinical signs. Cerebrospinal fluid was also collected prior to amprolium administration and at the onset of clinical signs. A significant progressive decrease in erythrocyte transketolase (TK) activity and an increase in the percent of thiamine pyrophosphate (TPP) effect were observed in amprolium-fed calves during amprolium administration until the onset of clinical signs. There was a significant increase in blood lactate and blood pyruvate concentrations and a significant decrease in lactate/pyruvate ratio at the onset of clinical signs. Serum electrolyte (Na, Ca, P, Mg) concentrations showed no significant changes. However, the serum potassium concentration had decreased significantly at the onset of signs. The cerebrospinal fluid analyses revealed a significant increase in lactate and pyruvate concentrations and lactate/pyruvate ratio in amprolium-fed calves. The electrolytes (Na, K, Ca, P and Mg) of cerebrospinal fluid did not show any change. It is concluded that oral administration of amprolium (300 mg/kg body weight daily) for 4-6 weeks produces biochemical changes characteristic of polioencephalomalacia in buffalo calves.

Amprolium↗

Intake of phosphatase-active plasma membrane into the cytoplasm of oocytes from sexually immature hamsters.

When thiamine pyrophosphate is used as substrate, heavily stained complexly branching cytoplasmic structures are seen in developing oocytes of the hamster between 8 and 12 days after birth. This coincides with the onset of the dictyate stage of first meiotic prophase. The structures appear to arise from the oocyte plasma membrane in two ways: (1) by proliferation and infolding of the membrane, and (2) by membrane being pushed into the cytoplasm by invaginating follicle cell processes. This appears to be followed by proliferation of the invaginated plasma membrane within the oocyte cytoplasm. Incubations with ATP or beta-glycerophosphate, with and without the inhibitor L-p-bromotetramisole, indicate that the staining is not due to the action of either ATPase or alkaline phosphatases. The origin and possible developmental significance of the reactive structures are discussed.

Adenosine Triphosphatases↗

Thiamine-dependent enzyme changes in the brains of alcoholics: relationship to the Wernicke-Korsakoff syndrome.

Chronic alcoholism results in thiamine deficiency as a consequence of poor nutrition, impaired absorption, and decreased phosphorylation to the enzyme cofactor form of the vitamin, thiamine pyrophosphate (TPP). Results of this study demonstrate significant reductions of TPP-dependent enzymes [pyruvate dehydrogenase complex, alpha-ketoglutarate dehydrogenase (alpha KGDH), and transketolase] in autopsied cerebellar vermis samples from alcoholic patients with the clinical and neuropathologically confirmed diagnosis of Wernicke-Korsakoff Syndrome (WKS). Enzyme activities in brain samples from alcoholics without WKS were within normal limits and activities of a nonthiamine-dependent enzyme, glutamate dehydrogenase, were not significantly different from control values in brain samples from alcoholics with or without WKS. These findings provide evidence, for the first time, of a direct implication of TPP-related metabolic processes in the pathogenesis of WKS. Decreased activities of alpha KGDH could be the trigger for a sequence of metabolic events resulting in energy compromise, and ultimately neuronal death in this syndrome.

Adult↗

Ultrastructural localization of phosphatase activity in the guinea pig pineal gland by the cerium technique.

Thiamine pyrophosphatase (TPPase), nucleoside diphosphatase (NDPase), and glucose-6-phosphatase (G-6-Pase) were localized by the cerium technique in guinea pig pinealocytes and compared with the corresponding lead technique. NDPase and TPPase were also compared at different pH values using the cerium technique. Vibratome sections of perfusion-fixed tissue were incubated with cerium chloride or lead nitrate. Substrates used were thiamine pyrophosphate (for TPPase), sodium inosine diphosphate (NDPase), and disodium glucose-6-phosphate (G-6-Pase). The 1-2 trans saccules of the Golgi apparatus showed TPPase and NDPase activity but none for G-6-Pase. The endoplasmic reticulum (ER) cisternae and perinuclear space had NDPase and G-6-Pase activity but not TPPase. The abluminal plasmalemma of endothelial cells and the plasmalemma of Schwann cells demonstrated TPPase and NDPase activity but the luminal plasmalemma of the endothelial cells and the plasmalemma of pinealocyte processes showed only NDPase activity. TPPase was active at all pH values tested, but NDPase was most active at pH values of 6.5 and 7.0. Lead phosphate precipitate was frequently seen in nuclei, perinuclear space, ER cisternae, and "synaptic" vesicles when lead was used as the capturing agent. These sites were usually not labeled when cerium was used.

Acid Anhydride Hydrolases↗

Phosphatase localization in the endomembrane system of the dinoflagellate Crypthecodinium cohnii.

The distribution of four enzymes within the endomembrane system of the protist Crypthecodinium cohnii has been determined using cytochemical localizations with lead as a capture agent. Nucleoside diphosphatase (NDPase) activity, using inosine diphosphate (IDP) and thiamine pyrophosphate (TPP) as substrates, was observed in the Golgi apparatus, with a gradient of increasing reaction product noted in some cells from the cis to trans cisternae. Tubules and vesicles associated with the trans cisternae also contained reaction product. The endoplasmic reticulum exhibited a high activity of glucose-6-phosphatase [with glucose-6-phosphate (G-6-P) as substrate]. Traces of reaction product were also observed in the cis-most and trans-most cisternae of the dictyosomes. Activity of acid phosphatase (AcPase) was observed in Golgi cisternae as well as in associated cytoplasmic vesicles. Heaviest deposition was localized in medial and trans dictyosome cisternae. The cytoplasmic system of flattened vesicles subtending the surface membranes in these cells did not exhibit reactivity with any of the substrates used. The distribution of these enzymes in this algal cell appears similar to that observed in animal cells and suggests that these enzymes may represent markers for algal cell endomembrane compartments.

Acid Anhydride Hydrolases↗

Structural analysis of Escherichia coli ThiF.

Escherichia coli ThiF is an enzyme in the biosynthetic cascade for generating the essential cofactor thiamin pyrophosphate. In this cascade, ThiF catalyzes adenylation of the C terminus of ThiS. We report here the crystal structures of ThiF, alone and in complex with ATP. The structures provide insight into a preference for ATP during adenylation of the protein ThiS. Additionally, the structures reveal an ordered crossover loop predicted to clamp the flexible tail of ThiS into the ThiF active site during the adenylation reaction. The importance of the crossover loop for ThiF activity is highlighted by mutational analysis. Comparison of ThiF with the structural homologues MoeB, APPBP1-UBA3, and SAE1-SAE2 reveals that the ATP-binding site, including an arginine-finger, is maintained throughout evolution, and shows divergence occurring in protein substrate-binding sites and regions devoted to unique steps in the specific function of each enzyme.

Adenosine Triphosphate↗

Thiamin biosynthesis in Bacillus subtilis: structure of the thiazole synthase/sulfur carrier protein complex.

Thiazole synthase is the key enzyme involved in the formation of the thiazole moiety of thiamin pyrophosphate. We have determined the structure of this enzyme in complex with ThiS, the sulfur carrier protein, at 3.15 A resolution. Thiazole synthase is a tetramer with 222 symmetry. The monomer is a (betaalpha)(8) barrel with similarities to the aldolase class 1 and flavin mononucleotide dependent oxidoreductase and phosphate binding superfamilies. The sulfur carrier protein (ThiS) is a compact protein with a fold similar to that of ubiquitin. The structure allowed us to model the substrate, deoxy-D-xylulose 5-phosphate (DXP), in the active site. This model identified Glu98 and Asp182 as new active site residues likely to be involved in the catalysis of thiazole formation. The function of these residues was probed by mutagenesis experiments, which confirmed that both residues are essential for thiazole formation and identified Asp182 as the base involved in the deprotonation at C3 of the thiazole synthase DXP imine. Comparison of the ThiS binding surface to the surface of ubiquitin identified a conserved hydrophobic patch of unknown function on ubiquitin that may be involved in complex formation between ubiquitin and one of its binding partners.

Bacillus subtilis↗

Ultrastructural localization of tartrate-resistant acid phosphatase (purple acid phosphatase) activity in chicken cartilage and bone.

Tartrate-resistant acid adenosine triphosphatase activity at pH 6.5, using a lead-salt method, was localized at light and electron microscopic levels in cartilage and bone matrices, osteoclasts, and chondroclasts. Cartilage matrix staining occurred after vascular invasion of the growth plate. In osteoclasts, activity was present in lysosomes, extracellular ruffled border channels, and the underlying cartilage and bone matrices. Staining artifacts occurred at lower pH levels (pH 5.4, 5.0). Adenosine diphosphate, p-nitrophenylphosphate, thiamine pyrophosphate, and alpha-naphthylphosphate also acted as substrates; but no activity was observed when adenosine monophosphate, adenylate-(beta, gamma-methylene) diphosphate, and beta-glycerophosphate were used. The activity was inhibited by NaF, dithionite, and a high concentration of p-chloromercuribenzoic acid, and activated by simultaneous addition of FeCl2 and ascorbic acid, as has been shown in biochemical studies. These histochemical results support the view that the adenosine triphosphate hydrolyzing activity at pH 6.5 is due to tartrate-resistant acid phosphatase (TRAP). There were some differences in ultrastructural localization between TRAP and tartrate-sensitive acid phosphatase (TSAP) activities in osteoclasts: TSAP activity was more intense in lysosomes and Golgi complexes and TRAP was stronger in the cartilage and bone matrices. It is suggested, therefore, that most of TRAP is in an inactive form in cells and is activated when secreted.

Acid Phosphatase↗

Brain thiamine, its phosphate esters, and its metabolizing enzymes in Alzheimer's disease.

Clinical data suggest that high-dose thiamine (vitamin B1) may have a mild beneficial effect in some patients with Alzheimer's disease (AD). Since this action could be related to a brain thiamine deficiency, we measured directly levels of free (nonphosphorylated) thiamine and its phosphate esters, thiamine monophosphate and thiamine diphosphate (TDP), and activities of three TDP-metabolizing enzymes (thiamine pyrophosphokinase, thiamine diphosphatase, and thiamine triphosphatase) in autopsied cerebral cortex of 18 patients with AD and 20 matched controls. In the AD group, mean levels of free thiamine and its monophosphate ester were normal, whereas levels of TDP were significantly reduced by 18 to 21% in all three cortical brain areas examined. Activities of the TDP-metabolizing enzymes were normal in the AD group, suggesting that decreased TDP is not due to altered levels of these enzymes. The TDP decrease could be explained by a cerebral cortical deficiency in AD of ATP, which is needed for TDP synthesis. Although the magnitude of the TDP reduction is slight, a chronic subclinical TDP deficiency could contribute to impaired brain function in AD and might provide the basis for the modest improvement by thiamine in cognitive status of some patients with AD.

Acid Anhydride Hydrolases↗

Profiles in altered metabolism I--the organic acids accumulating in acute non-diabetic ketoacidosis associated with alcoholism.

Several organic acids, among them the acidic catabolites of the branched chain amino acids and tyrosine, have been found to be elevated in the sera of non-diabetic patients presenting acute ketoacidosis associated with alcohol abuse. These findings are interpreted in terms of insufficiency of dietary co-factors required for their further catabolism. Butane-2,3-diol is also found frequently elevated in the urine of these patients and suggests interception of hydroxyethyl thiamine pyrophosphate by circulating high levels of acetaldehyde.

Acidosis↗

Preparation and in vitro cytotoxicity of a methotrexate-anti-MM46 monoclonal antibody conjugate via an oligopeptide spacer.

A method was developed by which conjugates of methotrexate (MTX) with antibody were prepared via an oligopeptide spacer which, after internalization of the conjugates into the target cells, would be cleaved by lysosomal enzymes to liberate MTX or its derivative(s) for entry of the drug into the cytoplasm through the lysosomal membrane. The conjugate of MTX with a monoclonal antibody (MAb) (aMM46) to mouse mammary carcinoma MM46 cells prepared by this method via Leu-Ala-Leu-Ala showed potent, antibody-directed cytotoxicity through lysosomal degradation of the conjugate, most likely at the tetrapeptide spacer. This was supported by the following observations: (a) the cytotoxicity of the aMM46 conjugate was more potent than that of the corresponding normal gamma-globulin conjugate, the antibody alone not being cytotoxic; (b) the conjugate retained its potent cytotoxicity to MM46 cells even after removal, by hydroxylamine treatment, of a less stably bound MTX derivative which might have been released extracellularly and have caused non-specific cytotoxicity by entering the cells via the membrane active transport system for MTX; (c) the cytotoxicity was not inhibited by thiamine pyrophosphate, an inhibitor of the MTX transport system; (d) the cytotoxicity was inhibited significantly with ammonium chloride, which inactivates lysosomal enzymes by raising the pH; and (e) the cleavability of the Leu-Ala-Leu-Ala spacer by the lysosomal enzymes was verified by using bio-undegradable poly(D-lysine) and biodegradable poly(L-lysine) as the lysosomotropic macromolecular carriers: unlike the poly(L-lysine) counterpart, the direct MTX-poly(D-lysine) conjugate showed very weak cytotoxicity while the tetrapeptide-mediated conjugate of MTX with poly(D-lysine) exhibited potent cytotoxicity.

Animals↗

Assay values for thiamine or thiamine phosphate esters in whole blood do not depend on the anticoagulant used.

We compared the whole blood, plasma, and erythrocyte (red blood cell (RBC)) concentrations of thiamine and thiamine phosphate esters in the presence of heparin or EDTA as anticoagulants. Three blood specimens were collected from each of 24 healthy volunteers into evacuated collection tubes containing the following anticoagulants: heparin, Na2EDTA, or K2EDTA. The concentrations of nonphosphorylated free thiamine (T), thiamine monophosphate (TMP), thiamine diphosphate (TDP), and thiamine triphosphate (TTP) were determined by the NH2-column HPLC method. The anticoagulant used had no effect on the concentrations obtained in whole blood and plasma of thiamine or any of the above thiamine compounds (P>0.05). RBCs were isolated by centrifugation and washed with isotonic saline, and the cell counts of the washed cells were adjusted to their whole blood values. In the washed RBCs with any anticoagulant, the concentrations of T, TMP, and TDP expressed either as nmol/L of whole blood or a ratio to hemoglobin were significantly lower (P<0.05) than those in whole blood.

Adult↗

Pyruvate dehydrogenase complex from ribbed mussel gill mitochondria.

The pyruvate dehydrogenase complex has been demonstrated in high speed pellet preparations from sonicated ribbed mussel gill mitochondria. The activity of the complex is inhibited by low chloride (less than 100 mM) concentrations, EDTA (1 mM), succinate, ATP, and NAD/NADH ratios below 4. Inhibition by EDTA is relieved by addition of 10 mM MgCl2-1 mM CaCl2. ATP inhibition was enhanced by NaF and reversed by high Mg++ concentrations in the absence of NaF. Pyruvate and thiamine pyrophosphate inhibited the inactivation by ATP. The nonhydrolyzable ATP analog AMP-PNP caused inhibition of the overall catalytic activity that was identical to ATP. Factors involved in the ATP inhibition and Mg++ reversal are lost with freezing or cold storage. Preliminary results using gamma-32P-ATP indicate that a protein kinase that phosphorylates the alpha subunit of E1 (pyruvate dehydrogenase) from the mammalian PDC is associated with the gill PDC. The activity of the complex may be regulated by a phosphorylation/dephosphorylation mechanism and by the relative levels of substrates, products, and other metabolites in the mitochondria.

Animals↗

Involvement of alpha-cysteine-62 and beta-tryptophan-135 in human pyruvate dehydrogenase catalysis.

Pyruvate dehydrogenase (E1), a heterotetramer (alpha(2)beta(2)), is the first catalytic component of the mammalian pyruvate dehydrogenase complex (PDC). To investigate the roles of cysteine-62 of E1alpha (alphaC62) and tryptophan-135 of E1beta (betaW135) (identified previously as active site residues using chemical modifications) in E1 catalysis, two recombinant human E1 mutants were generated using site-directed mutagenesis: alphaC62A and betaW135L. Compared to wild-type, k(cat) values for alphaC62A and betaW135L measured by PDC assay were markedly reduced to 7.2 and 11. 6%, respectively. Apparent K(m) values for thiamin pyrophosphate (TPP) were increased approximately sixfold for both mutants, resulting in catalytic efficiency for TPP of only 1-2% of the wild-type E1. K(m) values for pyruvate increased only moderately (twofold). The alphaC62A and betaW135L mutants were less thermostable than wild-type E1. The conformations of the mutant apo-E1s determined by spectral analysis were different from that of the wild-type apo-E1. CD spectral analysis indicated that TPP binding was affected for both the alphaC62A and betaW135L mutant E1s. The substrate analogs, fluoropyruvate and bromopyruvate, were shown to be active site-directed inhibitors of human E1; in the absence of TPP, bromopyruvate (but not fluoropyruvate) inhibited human E1 due to SH-group modification. Pyruvate induced inactivation of human E1 could be restored by thiol reagents. Cysteine-62 (and maybe another group) is proposed to be involved in E1 inhibition by the substrate and substrate analogs. Taken together these results indicate that alphaC62 and betaW135 facilitate coenzyme binding, and alphaC62 could be near the substrate-binding site.

Catalytic Domain↗

Synthesis of the quinoline-linked triazolopyrimidine analogues and their interactions with the recombinant tobacco acetolactate synthase.

Acetolactate synthase (ALS) is the first common enzyme in the biosynthesis of L-leucine, L-isoleucine, and L-valine. Triazolopyrimidine sulfonamide (TP) is a mixed-type inhibitor of ALS with respect to both pyruvate and thiamine pyrophosphate. In this study, we synthesized new substituted quinoline-linked TP analogues and several TP analogues which contained either unsubstituted aminoquinolines or amino isoquinolines. In addition, we examined the interactions of both the wild-type and the sulfonylurea-resistant recombinant tobacco ALS enzymes in a highly pure and active form with the quinoline-linked TP analogues, respectively. The wild-type tobacco ALS was extremely sensitive to inhibition by the quinoline-linked TP analogues. In contrast, the mutant tobacco ALS was insensitive to both the quinoline-linked triazolopyrimidine and the sulfonylurea herbicides. The results indicate that the ability of the quinoline-linked TP analogues to inhibit ALS is highly sensitive to substitution at the ortho position (C-7) and to the position of the ring nitrogen around the sulfonamide functionality (C-8).

Acetolactate Synthase↗

The genes for anabolic 2-oxoglutarate: ferredoxin oxidoreductase from Hydrogenobacter thermophilus TK-6.

2-Oxoglutarate: ferredoxin oxidoreductase (OGOR) of a thermophilic, chemolithoautotrophic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, is involved in carbon dioxide fixation via a reductive TCA cycle. The enzyme is a heterodimer comprising subunits of 70 and 35 kDa. The structural genes for the subunits (korAB) were cloned with primers designed from N-terminal sequences of the purified enzyme. The korAB genes were followed by two open reading frames of unknown function (orf3 and orf4). KorA carried a binding motif for coenzyme A, and KorB carried binding motifs for Fe-S cluster and thiamine pyrophosphate. Active recombinant enzyme from korAB was produced in E. coli under the control of the lac promoter. orf3 and orf4 were not necessary for the expression of active enzyme in E. coli. The recombinant enzymes had high substrate specificity toward 2-oxoglutarate as in the case of the native enzyme purified from strain TK-6.

Amino Acid Sequence↗

Simultaneous detection of thiamine and its phosphate esters from microalgae by HPLC.

We present an easy and sensitive method for measuring thiamine and its phosphate esters in small biological samples of microalgae (Amphidinium carterae Hulburt and Nitzschia microcephala Grun). The method consists of extraction of thiamine and its derivatives in acid solution, followed by liquid chromatography with fluorescence detection. The detection limit is as low as 15 fmol of thiamine. For comparison to microalgae, the method has been applied to evaluate thiamine levels in the crustacean Artemia salina Leach and is suitable for nutritional studies of the food web of the Baltic salmon, which suffers from thiamine deficiency. This method of HPLC analysis can be readily utilized to follow uptake and interconversion of thiamine and its phosphate esters in many micro- and macroalgae.

Animals↗

Pisum sativum mitochondrial pyruvate dehydrogenase can be assembled as a functional alpha(2)beta(2) heterotetramer in the cytoplasm of Pichia pastoris.

Pea (Pisum sativum) mitochondrial pyruvate dehydrogenase (E1) was produced by coexpression of the mature alpha and beta subunits in the cytoplasm of the yeast Pichia pastoris. Size-exclusion chromatography of recombinant E1, using a Superose 12 column, yielded a peak at M(r) 160,000 that contained both alpha and beta subunits as well as E1 activity. This corresponds to the size of native alpha(2)beta(2) E1. Recombinant E1 alpha (His(6))-E1 beta was purified by affinity chromatography using immobilized Ni(+), with a yield of 2.8 mg L(-1). The pyruvate-decarboxylating activity of recombinant E1 was dependent upon added Mg(2+) and thiamin-pyrophosphate and was enhanced by the oxidant potassium ferricyanide. Native pea mitochondrial E1-kinase catalyzed phosphorylation of Ser residues in the alpha-subunit of recombinant E1, with concomitant loss of enzymatic activity. Thus, mitochondrial pyruvate dehydrogenase can be assembled in the cytoplasm of P. pastoris into an alpha(2)beta(2) heterotetramer that is both catalytically active and competent for regulatory phosphorylation.

Autoradiography↗