Thiamine pyrophosphatase (nucleoside diphosphatase) in the Golgi apparatus is distinct from microsomal nucleoside diphosphatase

The properties of thiamine pyrophosphatase in the Golgi apparatus of rat liver were studied. Thiamine pyrophosphatase in an extract of the Golgi apparatus was separated into 6 bands of between pH 5.4 and 6.3 by isoelectric focusing on polyacrylamide gel. On the gels all these subforms catalyzed the hydrolyses of GDP, IDP, UDP, and CDP as well as that of thiamine pyrophosphate. The characteristics resembled those of Type B nucleoside diphosphatase of rat brain, though the enzyme did not have 3 subforms of Type B nucleoside diphosphatase in the higher pH region on isoelectric focusing. Thiamine pyrophosphatase of the Golgi apparatus was separated from microsomal nucleoside diphosphatase by DEAE-cellulose column chromatography. The properties of the enzyme were quite similar to those of Type B nucleoside diphosphatase with respect to its substrate specificity, optimum pH for activity, and inhibition by ATP. These findings suggest that thiamine pyrophosphatase in the Golgi apparatus is different from microsomal nucleoside diphosphatase and that it might be basically the same enzyme as Type B nucleoside diphosphatase except for different extents of modification.     

EXISTENCE OF ASCORBIC ACID AS AN ACTIVATOR OF THIAMINE DlPHOSPHATASE IN RAT BRAIN

The effect of the supernatant fraction (105,000 g for 60 min) of rat brain on the microsomal thiamine diphosphatase activity was examined. The thiamine diphosphatase activity was increased by addition of the supernatant fraction. The factor activating the enzyme was a heat-stable and dialyzable substance. It caused lipid peroxidation in the microsomes and the increase of the enzyme activity was mediated through lipid peroxidation of the preparation. When the supernatant fraction was chromatographed on columns of Sephadex G-25 and Dowex 1 × 2, the activator was eluted in fractions containing ascorbic acid. The inhibitory factor of ATPase present in the supernatant fraction was also eluted with the activator. The u.v.-spectrum of the active fraction obtained by these chromatographies was the same as that of ascorbic acid. These findings indicate the existence of ascorbic acid as an activator of thiamine diphosphatase in rat brain and confirm the previous finding that the soluble factor inhibiting ATPase activity is ascorbic acid.     

Thiamine pyrophosphatase and nucleoside diphosphatase in rat brain

Two types of nucleoside diphosphatase were found in rat brain. One (Type L) had similar properties to those of the liver microsomal enzyme with respect to its isoelectric point, substrate specificity, Km values, optimum pH, activation by ATP and molecular weight. The other (Type B), which separated into multiple forms on isoelectric focusing, had lower Km values and a smaller molecular weight than the Type L enzyme, and was inhibited by ATP. The Type B enzyme catalyzed the hydrolysis of thiamine pyrophosphate as well as those of various nucleoside diphosphates at physiological pH, while Type L showed only nucleoside diphosphatase activity at neutral pH. These findings suggest that the two enzymes play different physiological roles in the brain.     

Nucleoside diphosphatase from pig liver: purification and some properties

A procedure is described for purification of nucleoside diphosphatase from pig liver microsomes which avoids exposure of the enzyme to potentially denaturing conditions. The purest fractions obtained have specific activities of approximately 100 units/mg and appear to contain approximately 35% NDPase on a protein basis. Pig liver nucleoside diphosphatase resembles the enzyme obtained from other mammalian tissues in its substrate specificity and in its interaction with MgATP^2? as an allosteric modifier. However the molecular weight of the pig liver enzyme appears higher than that reported for other nucleoside diphosphatases, and activation by MgATP^2? is attributable to an increase in the maximal rate of nucleoside diphosphate hydrolysis rather than to a decrease in K_m. These differences in properties seem to be due to a species difference since similar properties were found with pig liver enzyme prepared by a different extraction procedure. The kinetic parameters which describe the reaction catalyzed by pig liver nucleoside diphosphatase are insensitive to changes in [H⁺]over the range pH 6.5–8.6. The intracellular location of nucleoside diphosphatase is microsomal in both pig and chicken liver.     

Enzyme cytochemistry of the alveolar sacs and golgian-like cisternae in the ciliate Ichthyophthirius multifiliis

In the cell cortex of the parasitic ciliate Ichthyophthirius multifiliis different kinds of cisternae were observed: the alveolar sacs, thick membrane cisternae and the endoplasmic reticulum. The thick membrane cisternae possess coated dilated rims and sometimes could be observed close to the endoplasmic reticulum. Using cytochemical techniques acid phosphatase, thiamine pyrophosphatase and nucleoside diphosphatase activities were detected in the thick membrane cisternae and in the alveolar sacs of trophozoites. In the endoplasmic reticulum acid phosphatase activity was not detected and only very small amounts of thiamine pyrophosphatase and nucleoside diphosphatase reaction product were observed. After exit from the host, a reduction in acid phosphatase activity was evident in the alveolar sacs. At theront stage acid phosphatase activity is absent from these structures. However, high thiamine pyrophosphatase and nucleoside diphosphatase activities remain in the alveolar sacs during the whole life cycle. On the other hand, acid phosphatase, thiamine pyrophosphatase and nucleoside diphosphatase activities were detected in thick membrane cisternae of theronts. Based on the morphological aspects and enzymatic content the thick membrane cisternae of the cell cortex are designated as golgian-like cisternae. The cytochemical results point out a relationship between the alveolar sacs and the Golgi complex.     

Possible regulation of thiamine diphosphatase activity in rat brain microsomes by lipids.

The effects of various treatments, which affect membrane structure, on microsomal thiamine diphosphatase and thiamine triphosphatase activities of rat brain, were examined. The treatment of micorosomes at alkaline pH caused a 2-fold activation of the thiamine diphosphatase, this being related to a change in membrane structure which was evidenced by a decrease of the turbidity of the microsomal suspension. Repeated freezing and thawing after hypo-osmotic treatment also increased the activity of microsomal thiamine diphosphatase. In addition, the thiamine diphosphatase activity was enhanced by treatment of the microsomes with phospholipase C or acetone. This lipid depletion resulted in a marked reduction in the apparent Km value of the thiamine diphosphatase with a corresponding loss in heat stability of the enzyme. We found further that brain thiamine diphosphatase was solubilized by Triton X-100. This decreased the phospholipid content in the preparation, but did not affect the apparent Km value and heat stability of the enzyme. In contrast with thiamine diphosphatase, thiamine triphosphatase was inactivated by treatment at alkaline pH or with acetone. However, treatment with phospholipase C did not affect the activity of thiamine triphosphatase.     

Enzyme Cytochemistry of the Alveolar Sacs and Golgian-Like Cisternae in the Ciliate Ichthyophthirius multifiliis

In the cell cortex of the parasitic ciliate Ichthyophthirius multifiliis different kinds of cisternae were observed: the alveolar sacs, thick membrane cisternae and the endoplasmic reticulum. The thick membrane cisternae possess coated dilated rims and sometimes could be observed close to the endoplasmic reticulum. Using cytochemical techniques acid phosphatase, thiamine pyrophosphatase and nucleoside diphosphatase activities were detected in the thick membrane cisternae and in the alveolar sacs of trosphozoites. In the endoplasmic reticulum acid phosphatase activity was not detected and only very small amounts of thiamine pyrophosphatase and nucleoside diphosphatase reaction product were observed. After exit from the host, a reduction in acid phosphatase activity was evident in the alveolar sacs. At theront stage acid phosphatase activity is absent from these structures. However, high thiamine pyrophosphatase and nucleoside diphosphatase activities remain in the alveolar sacs during the whole life cycle. On the other hand, acid phosphatase, thiamine pyrophosphatase and nucleoside diphosphatase activities were detected in thick membrane cisternae of theronts. Based on the morphological aspects and enzymatic content the thick membrane cisternae of the cell cortex are designated as golgian-like cisternae. The cytochemical results point out a relationship between the alveolar sacs and the Golgi complex.     

Immobilization of nucleoside diphosphatase at its allosteric site using immobilized derivatives of pyridoxal 5'-phosphate

3-O-Immobilized and 6-immobilized pyridoxal 5′-phosphate analogs of Sepharose were bound to the allosteric site of nucleoside diphosphatase with very high affinity. Active immobilized nucleoside diphosphatase was prepared by reduction of the Schiff base linkage between the enzyme and pyridoxal 5′-phosphate bound to Sepharose with NaBH₄. 3-O-Immobilized pyridoxal 5′-phosphate analog gave more active immobilized enzyme than the 6-analog; the immobilized enzyme on the 3-O-immobilized pyridoxal 5′-phosphate analog showed about 90% of activity of free enzyme. The immobilized enzyme thus prepared was less sensitive to ATP, an allosteric effector, and showed a higher heat stability than the free enzyme. When an assay mixture containing inosine diphosphate and MgCl₂ was passed through a column of the immobilized enzyme at 37 °C, inosine diphosphate liberated inorganic phosphate almost quantitatively. Properties of the immobilized enzyme on the pyridoxal 5′-phosphate analog were compared with those of the immobilized enzyme on CNBr-activated Sepharose.     

A possible new nucleoside diphosphatase from the cytosol of soybean and alfalfa nodules

Nucleoside diphosphatase activity has been found in the cytosol of soybean (Glycine max) and alfalfa (Medicago satavia) nodules. The enzyme differs from other diphosphatases in that it does not exhibit a strong preference for one nucleoside diphosphate over another. Very little, if any, diphosphatase activity was detected in root extracts of alfalfa and soybean seedlings.     

ENHANCEMENT OF BRAIN THIAMINE DIPHOSPHATASE ACTIVITY OF RATS BY INJECTION OF CHOLINERGIC DRUGS

Abstract— The effects of cholinergic drugs on thiamine diphosphatase (TDPase) in rat brain, liver and kidney were studied to clarify the role of the enzyme in the central nervous system.
Brain TDPase activity was markedly increased by intraperitoneal injection of a sub-lethal dose of physostigmine, ambenonium or pentetrazol. These drugs also increased the activity in the kidney, but not liver. Strychnine, atropine, and scopolamine did not affect the activity of brain TDPase, but decreased the enzyme activity of liver and kidney. Physostigmine also increased the activity of brain thiamine monophosphatase.
Brain TDPase activity reacheda maximum 30 minafterphysostigmine injection (1.0mg/kg). However, inhibition of brain acetylcholinesterase activity was greatest 45 min after physostigmine injection. The TDPase and AChE activities had both returned to normal values 60 min after the injection. The durations of these changes of TDPase and AChE activity corresponded to the duration of the tremor induced by physostigmine. The contents of total and phosphorylated thiamines in the brain but not in the liver or kidney were significantly reduced by physostigmine.
The relationship between ACh and activation of TDPase activity by cholinesterase inhibitors is discussed.     

Beitrag zur Klärung der Spezifität Thiaminpyrophosphat und Nukleosiddiphosphate spaltender Enzyme

Zusammenfassung In kultivierten Hühnerherzzellen besitzt die Thiaminpyrophosphatase des Golgi-Apparates bei pH 5,5 und die Nukleosiddiphosphatase des endoplasmatischen Retikulums bei pH 7,5 ihr Wirkungsoptimum. Im Golgi-Apparat erfolgt die Hydrolyse von Nukleosiddiphosphat bei pH 5,5 und 7,5 durch zwei verschiedene Enzyme.

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Cytochemical characteristics of specificity of enzymes hydrolyzing thiamine pyrophosphate and nucleoside diphosphates

Summary In cultured chicken heart cells the thiamine pyrophosphatase activity localized in the golgi apparatus has a pH optimum of pH 5,5. Nucleoside diphosphatase activity has been demonstrated in the endoplasmic reticulum at a pH optimum of pH 7,5. At pH 5,5 and 7,5 the hydrolysis of nucleoside diphosphate in the golgi apparatus is catalyzed by two enzymes.

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Herrn Prof. Dr. N. Weissenfels danke ich für beratende Hilfe, Frau G. Scheben, Frau S. Rost, Frau H. Gudelius und Frl. I. Rosocha für technische Assistenz.     

PROPERTIES OF THIAMINE DI- AND TRIPHOSPHATASES IN RAT BRAIN MICROSOMES: EFFECTS OF CHLORPROMAZINE

Abstract— The mechanism of the action of chlorpromazine on rat brain thiamine phosphatases were studied to clarify the properties of these enzymes in the CNS. Chlorpromazine at concentrations of 0.25-1.0 m m caused marked decrease of microsomal and soluble thiamine triphosphatase (TTPase) activities and marked increase of microsomal thiamine diphosphatase (TDPase) activity. Imipramine and desipramine also inhibited TTPase but did not cause any marked change in TDPase activities. Addition of chlorpromazine (0.5 m m ) decreased the V_max of microsomal TTPase by about one-half, increased that of TDPase about 3-fold, and lowered the K _m value for TDP but not for TTP.
Acetone treatment of the microsomal fraction lowered the TTPase activity and markedly enhanced the TDPase activity. In acetone-treated microsomes, chlorpromazine also inhibited TTPase activity but did not activate TDPase. Deoxycholate had similar effects to chlorpromazine on these enzyme activities.     

Specific and non-specific phosphatases in the miracidium of Fasciola hepatica L.

Localization and activities of alkaline phosphatase, ATPase, 5-nucleotidase, glucose-6-phosphatase, thiamine pyrophosphatase and nucleoside diphosphatase were studied in the miracidium of Fasciola hepatica L. Except for nucleoside diphosphatase whose activity in the miracidium was not observed, all the enzymes were most active in the archenteron, protonephridia and nerve ganglion. This localization of the reaction intensity allows the inference that the three organs mentioned are sites of both intense carbohydrate metabolism and lively active transport. The role of phosphatases in carbohydrate metabolism is discussed.     

Distribution of Thiamine, Thiamine Phosphates, and Thiamine Metabolizing Enzymes in Neuronal and Glial Cell Enriched Fractions of Rat Brain

The distribution of thiamine, thiamine phosphoesters, and the thiamine pyrophosphate synthetizing [thiamine-pyrophosphokinase (TPKase)] as well as hydrolyzing [thiamine pyrophosphatase (TPPase) and thiamine monophosphatase (TMPase)] enzymes was determined in neuronal and glial enriched fractions prepared from rat brain. Nucleoside diphosphatases [inosine diphosphatase (IDPase) and uridine diphosphatase (UDPase)] and nucleoside monophosphatases [uridine monophosphatase (UMPase) and inosine monophosphatase (IMPase)] were also determined. Thiamine and thiamine mono- and pyrophosphate were present in neuronal enriched fractions at concentrations 2.8, 3.6, and 4.6 times higher than in glial fractions. TMPase was found only in glial enriched fractions, whereas the levels of TPKase, UMPase, IMPase, IDPase, UDPase, and TPPase were 2.0-, 2.2-, 1.3-, 2.8-, 3.7-, and 20.8-fold higher in neuronal than in glial fractions.     

Histochemical studies on Raillietina (Raillietina) johri (Cestoda: Davaineidae). II. Nucleoside diphosphatase and thiamine pyrophosphatase.

Thiamine pyrophosphatase and nucleoside diphosphatase have been studied histochemically in Raillietina (Raillietina) johri. Thiamine pyrophosphatase activity has been observed in the tegument, subtegumental muscle, subtegumental cells, medullary parenchyma, excretory canal and various reproductive structures like testes, ovary, vas deferens, spermatozoa and vitellaria. Eggs exhibit moderate enzyme activity. Various nucleoside diphosphates have been found to be hydrolyzed by thiamine pyrophosphatase. CaCl2, MgCl2 and MnCl2 each activated the enzyme at a final concentration of 6 mM whereas cysteine, reduced glutathione and PCMB inhibited the enzyme activity at a final concentration of 10 mM, 10 mM and 20 mM, respectively. KCN and NaF had no effect on the enzyme staining at concentration as high as 50 mM and 30 mM, respectively. Possible roles of the enzyme in the parasite have been discussed.     

Immunocytochemical localization of galactosyltransferase in HeLa cells: codistribution with thiamine pyrophosphatase in trans-Golgi cisternae

An affinity-purified, monospecific rabbit antibody against soluble human milk galactosyltransferase was used to localize the enzyme in HeLa cells by immunofluorescence and by the protein A-gold technique at the electron microscope level. Specific immunofluorescence was observed in a juxtanuclear cytoplasmic region which was identified, on immunostained thin sections of low-temperature Lowicryl K4M-embedded HeLa cells, as Golgi apparatus. Label by gold particles was limited to two to three trans cisternae of the Golgi apparatus, indicating a compartmentalization of galactosyltransferase in the cisternal stack. Combination of preembedding thiamine pyrophosphatase cytochemistry, with postembedding immunostaining for galactosyltransferase proved codistribution of the two enzymes. However, the acid phosphatase-positive, trans-most cisterna was negative for galactosyltransferase. The close topological association of both galactosyltransferase and thiamine pyrophosphatase (or nucleoside diphosphatase) suggests a concerted action of both enzymes in glycosylation.     

Glycoprotein reglucosylation and nucleotide sugar utilization in the secretory pathway: identification of a nucleoside diphosphatase in the endoplasmic reticulum.

UDP is generated in the lumen of the endoplasmic reticulum (ER) as a product of the UDP-glucose-dependent glycoprotein reglucosylation in the calnexin/calreticulin cycle. We describe here the identification, purification and characterization of an ER enzyme that hydrolyzes UDP to UMP. This nucleoside diphosphatase is a ubiquitously expressed, soluble 45 kDa glycoprotein devoid of transmembrane domains and KDEL-related ER localization sequences. It requires divalent cations for activity and hydrolyzes UDP, GDP and IDP but not any other nucleoside di-, mono- or triphosphates, nor thiamine pyrophosphate. By eliminating UDP, which is an inhibitory product of the UDP-Glc:glycoprotein glucosyltransferase, it is likely to promote reglucosylation reactions involved in glycoprotein folding and quality control in the ER.     

THE EFFECT OF THIAMINE DEFICIENCY ON THE ACETYLCOENZYMEA AND ACETYLCHOLINE LEVELS IN THE RAT BRAIN

Abstract— It is shown that transketolase activities in red blood cells and whole brain of normal and thiamine-deficient rats correlate well with heart frequencies.
The effect of thiamine depletion on the levels of acetylcoenzyme A (acetyl-CoA) and acetylcholine (ACh), and on the activities of pyruvate dehydrogenase, choline acetyl-transferase and acetylcholine esterase was studied in whole brains of thiamine-deficient, thiamine-supplemented ad libitum and pair-fed rats. The concentrations of acetyl-CoA and ACh decreased in thiamine-deficient brains by 42 and 35 per cent, respectively.
Total pyruvate dehydrogenase activity did not change during vitamin B₁ deficiency. The 'resolved' enzyme, reconstituted with thiamine diphosphate, had an association constant of 5.4 × 10⁻⁶ m . Choline acetyltransferase and acetylcholine esterase activities remained unchanged in thiamine deficiency.
Possible mechanisms which could explain the reduced Ach levels in vitamin B₁ deficiency are discussed.     

Three new Nudix hydrolases from Escherichia coli

Three members of the Nudix (nucleoside diphosphate X) hydrolase superfamily have been cloned from Escherichia coli MG1655 and expressed. The proteins have been purified and identified as enzymes active on nucleoside diphosphate derivatives with the following specificities. Orf141 (yfaO) is a nucleoside triphosphatase preferring pyrimidine deoxynucleoside triphosphates. Orf153 (ymfB) is a nonspecific nucleoside tri- and diphosphatase and atypically releases inorganic orthophosphate from triphosphates instead of pyrophosphate. Orf191 (yffH) is a highly active GDP-mannose pyrophosphatase. All three enzymes require a divalent cation for activity and are optimally active at alkaline pH, characteristic of the Nudix hydrolase superfamily. The question of whether or not Orf1.9 (wcaH) is a bona fide member of the Nudix hydrolase superfamily is discussed.     

Three-dimensional structure of transketolase, a thiamine diphosphate dependent enzyme, at 2.5 A resolution.

The crystal structure of Saccharomyces cerevisiae transketolase, a thiamine diphosphate dependent enzyme, has been determined to 2.5 A resolution. The enzyme is a dimer with the active sites located at the interface between the two identical subunits. The cofactor, vitamin B1 derived thiamine diphosphate, is bound at the interface between the two subunits. The enzyme subunit is built up of three domains of the alpha/beta type. The diphosphate moiety of thiamine diphosphate is bound to the enzyme at the carboxyl end of the parallel beta-sheet of the N-terminal domain and interacts with the protein through a Ca2+ ion. The thiazolium ring interacts with residues from both subunits, whereas the pyrimidine ring is buried in a hydrophobic pocket of the enzyme, formed by the loops at the carboxyl end of the beta-sheet in the middle domain in the second subunit. The structure analysis identifies amino acids critical for cofactor binding and provides mechanistic insights into thiamine catalysis.