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.     

Increase in nucleoside diphosphatase in rat brain striatum lesioned with kainic acid

The activity of ammoniagenesis from guanine nucleotides was found to increase significantly in rat brain after infusion of kainic acid into the striatum. Among the enzymes involved in degrading guanine nucleotides, nucleoside diphosphatase was markedly increased in the lesioned striatum. The enzyme activity began to increase 2 days after the infusion, and reached the maximum on the 13th day, the level being 4 times as high as that of the intact contralateral region. The increased activity was due to Type L enzyme, judging from its substrate specificity. Puromycin and cycloheximide inhibited this increase, indicating that the increased activity resulted from an increase in the net synthesis of the enzyme. These findings suggest that Type L NDPase might play some important roles in gliosis after neuronal lesion.     

Inorganic pyrophosphatase and nucleoside diphosphatase in the parasitic protozoon, Entamoeba histolytica.

Entamoeba histolytica contains two acid pyrophosphatases. One is an inorganic pyrophosphatase with a relatively high K_m ( ? 1 mM) and no cation requirement. The other is a nucleoside diphosphatase with a relatively low K_m ( ? 50 μM) and Ca²⁺ requirement. No Mg²⁺ dependent neutral or alkaline inorganic pyrophosphatase is present. The pyrophosphatases are localized in subcellular particles, display structure-linked latency and are tightly bound to membranes.     

Thiamine pyrophosphatase cytochemistry in rat endometrium during the oestrous cycle

The functional morphology of the Golgi apparatus was studied in various types of cells in the rat endometrium during the oestrous cycle. A cerium-based enzyme-cytochemical method was used for the ultrastructural visualization of the activity of thiamine pyrophosphatase (TPPase). The cerium-based method was evidently superior to the classical lead technique, which was used for comparison. TPPase activity in luminal and glandular epithelial cells displayed cyclical modulation and redistribution. It was restricted to only one or two narrow trans lamellae during dioestrus but extended during proestrus and oestrus into nearly all trans-to-cis lamellae of the well-compartmentated Golgi apparatus. A homogeneous staining reaction, which was particularly intense during the latter two phases and only partly due to unspecific alkaline phosphatase, was confined to the apical and basolateral plasma membranes of luminal epithelial cells. In the stromal fibroblasts, only one short Golgi saccule was positive at dioestrus, whereas three or more trans Golgi lamellae were filled with reaction product during oestradiol-dominated oestrus. TPPase activity was furthermore observed in the lysosomes in epithelial cells, stromal fibroblasts, capillary endothelial cells and pericytes. The present findings of cyclic changes in TPPase activity in epithelial cells and stromal fibroblasts provide the first evidence of cyclic modulation and redistribution of this enzyme in the endometrium.     

Triton-stimulated nucleoside diphosphatase: Characterization

Summary Our previous report indicated a Triton-stimulated NDPase was specifically associated with Golgi membranes isolated from corn roots. Characterization of the enzyme indicates that UDP is the slightly preferred substrate with Mn²⁺ the preferred divalent cation. Monovalent cations do not further activate the Triton-stimulated UDPase activity. The enzyme has a pH optimum at 6.5 and a temperature optimum between 38–40°C. Kinetic analyses indicate that UDP-Mn²⁺ is the substrate for the enzyme.Properties of the Triton-stimulated NDPase are compared to other membrane associated NDPases isolated from plants, animals and fungi. Characteristics and subcellular location of NDPase activity are discussed in relation to the possible biochemical role of the enzyme.This research was supported in part by National Science Foundation grant CDP 79-7927121 and funds provided by Bronfman Science Center, Williams College.     

Affinity purification and some properties of nucleoside diphosphatase from rat liver cytosol.

Nucleosidediphosphatase (nucleosidediphosphate phosphohydrolase, EC 3.6.1.6) of rat liver cytosol was purified up to 336--fold by the procedure including affinity chromatographies of concanavalin A- and alanine-Sepharose. The final purified enzyme showed a single protein band upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Its native form was found to be a tetramer with molecular weight of 120 000 which consists of subunit with molecular weight of 30 000. The enzyme was found to be a glycoprotein on the basis of its chromatographic behaviour with concanavalin A-Sepharose and positive staining with periodate-Schiff reaction in polyacrylamide gels. Furthermore, the two molecular forms with isoelectric points of 4.7 and 5.0 were demonstrated by electrofocusing, though they did not show any significant difference with respect to their catalytic properties.     

Localization of nucleoside diphosphatase in the onion root tip

Summary Nucleoside diphosphatase activity occurs in the Golgi apparatus, on the plasmalemma, between the plasmalemma and the definitive cell wall, and in the endoplasmic reticulum of the onion root tip. The presence and/or relative concentrations of reaction product at each of these cellular sites occur in distinctive developmental patterns within the root. Cells with high NDPase activity at any combination of these sites also produce large amounts of a polysaccharide-rich product. A vacuolar activity also occurs in the pericycle and cortex in a developmentally distinctive pattern, but its location has not been correlated to polysaccharide secretory activity.     

Cytochemical detection of nucleoside diphosphatase activity in the central nervous system of the rat.

The cytochemical localization of nucleoside diphosphatase and thiamine pyrophosphatase occurs within the "mature face" of the Golgi apparatus and over the neurilemma in neurons of the cerebellum, the cerebral cortex and the brain stem. The hydrolytic reaction product of the brain enzyme differs from that of the liver in that it is not found in the endoplasmic reticulum or nuclear envelope. Hydrolysis of IDP, UDP or GDP is not greater than that of ADP or CDP in brain homogenates, in contrast to that found in the liver. The NDPase activity of brain homogenates is optimal at pH 7.2, stimulated by heavy metals and inhibited by uranyl nitrate. Thick section cytochemistry suggests that the reaction product is restricted to a network of polygonally shaped compartments. NDPase activity on the neurilemma may reflect the role of this enzyme in the synthesis of glycoproteins involved in neuronal surface recognition.     

Isolation of a Golgi apparatus-rich fraction from rat liver. IV. Thiamine pyrophosphatase

The thiamine pyrophosphatase (the enzyme [s] catalyzing the release of inorganic phosphate with thiamine pyrophosphate as the substrate) activities of Golgi apparatus-, plasma membrane-, endoplasmic reticulum-, and mitochondria-rich fractions from rat liver were compared at pH 8. Activity was concentrated in the Golgi apparatus fractions, which, on a protein basis, had a specific activity six to eight times that of the total homogenates or purified endoplasmic reticulum fractions. However, only 1–3% of the total activity was recovered in the Golgi apparatus fractions under conditions where 30–50% of the UDPgalactose:N-acetylglucosamine-galactosyl transferase activity was recovered. Considering both recovery of galactosyl transferase and fraction purity, we estimate that approximately 10% of the total thiamine pyrophosphatase activity of the liver was localized within the Golgi apparatus, with a specific activity of about ten times that of the total homogenate. Cytochemically, reaction product was found in the cisternae of the endoplasmic reticulum as well as in the Golgi apparatus. This is in contrast to results obtained in most other tissues, where reaction product was restricted to the Golgi apparatus. Thus, enzymes of rat liver catalyzing the hydrolysis of thiamine pyrophosphate, although concentrated in the Golgi apparatus, are widely distributed among other cell components in this tissue.     

Comparative studies on nucleoside diphosphatase of rat ascites hepatoma and rat liver: activity level, purification and properties

1. The activities of nucleoside diphosphatase in various rat ascites cells of hepatoma, and fetal and neonatal rat liver were much lower than that of normal adult rat liver. 2. The enzyme was purified from ascites hepatoma (AH-66 cell lines) to an apparently homogeneous state and the enzymatic properties were studied in comparison with the enzyme from rat liver microsomes. 3. The hepatoma enzyme had less stability based on the results of heat-inactivation experiments. 4. However, the other properties of hepatoma enzyme; Km value, molecular weight, optimal pH, isoelectric point, substrate specificity and antigenicity, were similar to those of rat liver enzyme.