Nucleoside Diphosphatase Activity Research Articles

Cytochemical localization of nucleoside diphosphatase and thiamine pyrophosphatase activities in the rabbit corneal endothelium

Localization of NDPase and TPPase activities in the rabbit corneal endothelium was investigated by cytochemical methods. After incubation at both pH 7.0 and 8.0, the NDPase activity was detected in cisternae of ER, nuclear envelope, and Golgi saccules, while the TPPase activity was demonstrated only in Golgi saccules. The reaction products of both enzymes were decreased when incubated at pH 8.0. The NDPase activity was inhibited by uranyl nitrate but not by sodium fluoride, while the TPPase was inhibited by fluoride but not by uranium.

A Kinetic Study of a Solubilized Chitin Synthetase Preparation from Coprinus cinereus

SUMMARY: The solubilized chitin synthetase preparation from the stipes of Coprinus cinereus is activated by its substrate, UDP-N-acetylglucosamine, and by carbohydrates, in particular N-acetylglucosamine, diacetylchitobiose and glucose. Analysis of the results of variation in enzyme activity with UDP-GlcNAc concentration by several methods suggested that this substrate is an allosteric activator, with Hill coefficients close to 2 and 4 at concentrations above and below 0.1 mM respectively, and an [S]0.5 value of 0.9 mM. N-acetylglucosamine lessened but did not abolish the sigmoidal shape of the velocity-substrate concentration plot. The chitin synthetase was inhibited by adenine and uridine nucleotides, and uridine diphosphate was a powerful competitive inhibitor. The enzyme preparation also contained a nucleoside diphosphatase activity and the implications of the removal of the inhibitory UDP formed by the chitin synthetase are considered.

Phosphatase active antigens in sea urchin eggs and embryos. I. Substrate specificity, pH‐optima and inhibitors

Phosphatase activity in sea urchin embryonic antigens was investigated by histochemical staining of immunoprecipitates separated by two-dimensional (crossed) immunoelectrophoresis. Unfertilized eggs were homogenized in a hypotonic medium which solubilized cytoplasmic antigens. Antigens integrated in membranes or enclosed in particles were solubilized by detergent treatment of the residual pellet. Two different phosphatase activities were discerned in the unfertilized eggs, nucleoside diphosphatase (EC 3.6.1.6.) and acid phosphatase (EC 3.1.3.2.). Nucleoside diphosphatase activity was obtained in both the water soluble and detergent extracted protein fractions. This activity was confined to one antigen. Acid phosphatase acitivity on the other hand was almost exclusively obtained in the detergent extracted fraction and about ten distinct antigens displayed this activity. The nucleoside diphosphatase active antigen preferentially hydrolyzed purine nucleoside diphosphates and to a lesser degree triphosphates of these nucleosides. The acid phosphatase active antigens had a broader substrate specificity and hydrolyzed equally well beta-glycerophosphate and nucleotides. Both activities were essentially inactive at neutral or alkaline pH values. The activities were inhibited by p-choloromercuribenzoate and accordingly stimulated by cysteine. Tartrate and sodium fluoride, however, inhibited the acid phosphatase activity while nucleoside diphosphatase activity was either stimulated or not affected at all by these agents.

Phosphatase active antigens in sea urchin eggs and embryos. II. A comparison between the activities in unfertilized eggs and plutei

Phosphatase activities in sea urchin eggs and plutei were investigated by means of histochemical staining of immunoprecipitates. Two protein fractions were obtained by extraction in a hypotonic medium and by detergent treatment of the residual pellet. Three distinctly different phosphatase activities were discerned, nucleoside diphosphatase (EC 3.6.1.6.), acid phosphatase (EC 3.1.3.2.) and alkaline phosphatase (EC 3.1.3.1.). The nucleoside diphosphatase activity, which was confined to one antigen, was present in both water soluble and detergent extracts and at roughly the same concentration in eggs and plutei. By means of a monospecific antiserum the immunological identify of this antigen was established in all instances. The acid phosphatase activity, which was displayed by ten detergent extracted antigens in eggs, was only found in five detergent extracted antigens in plutei. This decrease in number of enzyme active antigens was also reflected by a general decrease in number of enzyme active antigens was also reflected by a general decrease in activity as assessed by quantitative determinations. Furthermore, by means of absorbed antisera it was established that two or three of the acid phosphatase active antigens were "egg specific". Another acid phosphatase active antigen, which was common to both developmental stages, was investigated by a monospecific antiserum. While this antigen was found in both soluble fractions, it was only enzymatically active when extracted with detergent. Alkaline phosphatase active antigens were only found in the detergent extract of plutei. However, immunoprecipitates with this activity appeared both with antiserum against unfertilized eggs and with antiserum against plutei. This suggests that the egg contained the antigens in an enzymatically inactive form.

Effect of a catatoxic steroid pregnenolone-16α-carbonitrile, on rat liver microsomal subfractions

Pregnenolone-16α-carbonitrile (PCN), a potent catatoic steroid without known classical hormonal effects, was administered per os to female rats. Its effects were studied on mixed function oxygenases and on various phosphatases in liver microsomal subfractions: rough microsomes, smooth I, and smooth II microsomes. For comparison, phenobarbital (PB) and 3-methylcholanthrene (MC) were also administered. The inducers increased the protein content in total, rough and smooth I microsomes in the following order: PB, PCN, and MC, whereas the protein amount in smooth II microsomal fraction remained unchanged. The content of cytochrome P-450 was about doubled in all subfractions except the smooth II membranes, following treatment with any of the inducers. PCN differed in inducing specificity from PB in increasing benzo(α)pyrene hydroxylase and from MC in stimulating aminopyrine demethylase in total microsomes. PCN also differed from PB in enhancing the capacity not only for rough and smooth I microsomes, but also for smooth II microsomes to demethylase aminopyrine. No major difference in magnitude of effects among the inducers was noted between rough and smooth I microsomes. In contrast to the altered substrate specificities produced in the monooxygenase system by different inducers, a more uniform pattern of specificities was seen in microsomal phosphatases. PCN, MC and PB all increased the activities of nucleoside diphosphatase (IDPase), whereas G6Pase and ATPase activities were little affected. Cycloheximide was partially effective in depressing the increase of both the monooxygenase system (cytochrome P-450 and benzo(α)pyrene hydroxylase) and nucleoside diphosphatase activity. We conclude that (1) treatment with PCN results in different substrate specificity when compared to PB and MC; that (2) PCN is the most potent inducer of the three in stimulating drug hydroxylation in smooth II microsomes, that (3) various smooth microsomal membranes of liver cells are affected differently by inducers of drug metabolism, and finally (4) that drugs also alter some microsomal phosphatase activities but not all.

Intense phosphatase activity in the developing rete testis of the newborn rat.

The growing rete testis of the newborn rat was shown to exhibit intense 5′ nucleotidase and nucleoside diphosphatase activity. This high activity was confined to the growing rete cords, both intra- and extratesticularly, but not to any other tubular system in that area. ATP-ase and alkaline or acid phosphatase did not show such a marked difference between other duct structures and the rete cords. The striking histochemical differences between rete testis and seminiferous tubules disappear early in puberty.

Localization of nucleoside diphosphatase in the onion root tip.

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.

Nucleotide phosphohydrolase activities of the plasma membranes of embryonic chick liver cells

Plasma membrane fractions of embryonic chick liver cells have been isolated by the techniques of homogenization, differential centrifugation in isotonic, low ionic strength medium, and zonal centrifugation. The purity of the fractions has been determined by electron microscopy and the absence of enzymatic markers for contaminating subcellular fractions. CTPase (EC 3.6.1.4) specific activities are high for plasma membranes isolated from embryonic chick livers. Other nucleoside triphosphatase and nucleoside diphosphatase activities are associated with the plasma membranes. It has not been determined if all nucleotide phosphohydrolase activities are due to a single enzyme or group of enzymes.

Thiamine in neural membranes. enzymic hydrolysis of thiamine diphosphate.

Abstract— The membrane‐associated diphosphatase from rat brain which catalyses the hydrolysis of thiamine diphosphate and nucleoside diphosphate is described. The parallel sub‐cellular distribution of thiamine diphosphatase and nucleoside diphosphatase activity and the equal inhibition of both activities by adenosine methylenediphosphonate, a non‐hydrolysable structural analogue of ADP, suggests that a single enzyme is involved. The divalent cation requirement and basic kinetic properties of this enzyme have been determined. This nucleoside diphosphatase is not activated by ATP.

Localization of phosphatase activities in the rat anterior pituitary gland.

All five known secretory cell types of the rat anterior pituitary gland display nucleoside diphosphatase (NDPase) activity throughout the endoplasmic reticulum (ER), including the nuclear envelope but not the specialized region of ER at the inner aspect of the Golgi apparatus known as GERL. The functions of the ER diphosphatase are currently unknown. However, speculations concerning its association with glucuronyl transferase may focus on the metabolic roles of the ER in pituitary cells other than those directly related to secretory protein transport. The gonadotrophs have been studied for thiamine pyrophosphatase and acid phosphatase activities as well as NDPase activity. The results suggest that the secretory granules of gonadotrophs arise from GERL and not from the inner element of the Golgi apparatus. The innermost Golgi element of this cell type shows NDPase and thiamine pyrophosphatase activities and appears to be composed, in part at least, of anastomosing tubules. Nucleoside phosphatase activity is also present at the surfaces of all five secretory cell types and between the cells and adjacent blood capillaries.

GOLGI APPARATUS, GERL, AND LYSOSOMES OF NEURONS IN RAT DORSAL ROOT GANGLIA, STUDIED BY THICK SECTION AND THIN SECTION CYTOCHEMISTRY

New insights into the ultrastructure and phosphatase localizations of Golgi apparatus and GERL, and into the probable origin of lysosomes in the neurons of fetal dorsal root ganglia and the small neurons of adult ganglia have come from studying thick (0.5-1.0 micro) as well as thin (up to 500 A) sections by conventional electron microscopy. Tilting the thick specimens, by a goniometer stage, has helped to increase our understanding of the three-dimensional aspects of the Golgi apparatus and GERL. One Golgi element, situated at the inner aspect of the Golgi stack, displays thiamine pyrophosphatase and nucleoside diphosphatase activities. This element exhibits regular geometric arrays (hexagons) of interconnected tubules without evidence of a flattened portion (saccule or cisterna). In contrast, GERL shows acid phosphatase activity and possesses small cisternal portions and anastomosing tubules. Lysosomes appear to bud from GERL. Osmium deposits, following prolonged osmication, are found in the outer Golgi element. Serial 0.5-micro and thin sections of thiamine pyrophosphatase-incubated material demonstrate that, in the neurons studied, the Golgi apparatus is a continuous network coursing through the cytoplasm. Serial thick sections of acid phosphatase-incubated tissue suggest that GERL is also a continuous structure throughout the cytoplasm. Tubules of smooth endoplasmic reticulum, possibly part of GERL, extend into the polygonal compartments of the inner Golgi element. The possible physiological significance of a polygonal arrangement of a phosphatase-rich Golgi element in proximity to smooth ER is considered. A tentative diagram of the Golgi stack and associated endoplasmic reticulum in these neurons has been drawn.

Studies on the allosteric modification of nucleoside diphosphatase activity by magnesium nucleoside triphosphates and inosine diphosphate.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTAllosteric modification of nucleoside diphosphatase activity by magnesium nucleoside triphosphates and inosine diphosphateVern L. Schramm and John Francis MorrisonCite this: Biochemistry 1971, 10, 12, 2272–2277Publication Date (Print):June 8, 1971Publication History Published online1 May 2002Published inissue 8 June 1971https://doi.org/10.1021/bi00788a014RIGHTS & PERMISSIONSArticle Views27Altmetric-Citations5LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (613 KB) Get e-Alerts Get e-Alerts

Morphology and cytochemistry of the Golgi apparatus of rat salivary gland acnar cells.

AbstractThe ultrastructure and cytochemistry of the Golgi apparatus was studied in acinar cells of the parotid and von Ebner's glands of the rat. The Golgi apparatus was composed of four to six stacked cisternae, numerous small vesicles, and condensing vacuoles. The latter appeared to form as dilatations of the innermost Golgi cisterna, and their irregular membrane suggested fusion with or fission of small vesicles. Nucleoside diphosphatase and thiamine pyrophosphatase activities were localized to the inner Golgi cisternae. Acid phosphatase activity was localized to condensing vacuoles, coated vesicles, and an irregular, narrow cisterna found in the internal region of the Golgi apparatus and termed the internal Golgi lamella. The morphology and relationships of the Golgi apparatus indicate that it plays an important role in secretory granule formation. The presence of nucleoside diphosphatase activity may reflect the role of the Golgi apparatus in the addition of carbohydrates to the secretory proteins. It is suggested that the internal Golgi lamella develops from the inner Golgi cisterna after secretory granule formation is completed. Acid phosphatase activity in condensing vacuoles may be a mechanism for lysosomal regulation of the secretory process.

Modulation in nucleoside diphosphatase activity of mammotrophic cells of the rat adenohypophysis during secretion.

The modulations in nucleoside diphosphatase (NDPase) activity which occur during protein secretion have been investigated in mammotrophic hormone-producing cells (MT) of the female rat adenohypophysis. Pituitaries were fixed by perfusion or immersion in glutaraldehyde, and nonfrozen sections were incubated for NDPase. Tissue was examined from lactating and estrogen-treated animals in which mammotrophic hormone secretion (MTH) is high and from postlactating rats in which secretion of MTH is suppressed. In all experimental groups, NDPase reaction product was found: ( a) in Golgi cisternae, ( b) around forming and mature secretion granules, ( c) in pericapillary and intercellular spaces and ( d) along the outer surface of endothelial and MT cell membranes. The staining intensity of both the intracellular and extracellular sites paralleled the level of MTH secretion; it was greatest after estrogen treatment, moderate during lactation and minimal in the postlactating animal. In animals given estrogen, the Golgi apparatus was increased in size and up to six successive cisternae along the concave Golgi face were filled with reaction product in virtually every cell. In postlactating animals, the Golgi apparatus was small, and there was only patchy staining of a few cisternae in some cells. From its location and fluctuations with secretion, it seems likely that NDPase activity may be associated with the condensation and/or discharge of secretory granules.

ISOLATION OF β-GLUCAN SYNTHETASE PARTICLES FROM PLANT CELLS AND IDENTIFICATION WITH GOLGI MEMBRANES

A variety of particle-bound synthetases that use sugar nucleotides as glycosyl donors for the formation of polysaccharides similar to those of the cell wall have been demonstrated in mung beans and other plant tissues,(1) but the particles in question have not been previously identified.(2, 3) The polysaccharide synthetase particles from peas that form mainly beta-1,4-glucan from UDPG and GDPG have now been separated from other cell particles by combinations of velocity and isopycnic density gradient centrifugation. The particles have an effective density of about 1.15 gm cm(-3), exhibit latent nucleoside diphosphatase activity upon IDP, UDP, GDP, and to a lesser extent upon ADP, and also possess acid phosphatase and weak ATPase activity. The isolated synthetase particles consist of somewhat condensed Golgi dictyosomes and free dictyosomal membranes bearing vesicles. It is concluded that the synthetase particles are Golgi membranes. The nucleoside diphosphatase activity of these particles may represent inactivated polysaccharide synthetase.

Nucleosidediphosphatase activity in plasma membrane of rat liver

1. 1. The intracellular distribution of nucleosidediphosphatase activity has been investigated in rat liver with ADP and IDP as substrates and compared with those of reference enzymes. 2. 2. After differential centrifugation in 0.25 M sucrose, the distribution of the enzyme acting on IDP is similar to that of glucose-6-phosphatase; ADPase distribution is comparable to that of 5′-AMPase. 3. 3. Deoxycholate considerably increases nucleosidephosphatase activity on IDP, but has little effect when ADP is used as the substrate. The detergent does not affect the distribution of ADPase; on the contrary, significant changes of the IDPase distribution are observed if the test is performed with or without deoxycholate. 4. 4. Enzyme distribution curves have been determined after centrifugation in a sucrose gradient of a particle preparation obtained by centrifuging the homogenate at 3.7·10 6 × g· min . When granules are layered at the top of the gradient no clear distinction is seen between the enzyme distribution curves. When granules are deposited at the bottom of the gradient, a striking separation of the nucleosidediphosphatase distribution curves can be observed. The behaviour of IDPase is similar to that of glucose-6-phosphatase; ADPase behaves like 5′-AMPase. 5. 5. In purified plasma membrane preparations, ADPase and 5′-AMPase show a specific activity approx. 25 times higher than that fund in the homogenate. IDPase is only 5 times purer than in the homogenate. Some properties of the nucleosidediphosphatase associated with the plasma membrane are given. 6. 6. The results are discussed with respect to the presence in rat liver of two nucleosidediphosphatases, one associated with the plasma membrane and the other with endoplasmic-reticulum membranes.

Studies of phosphorus metabolism by isolated nuclei: X. Nucleolar nucleosidediphosphatase activity

1. 1. Isolated rat liver nucleoli catalyze formation of P i with both ribo- and 2-deoxyribonucleoside diphosphates as substrates. Nucleolar GDPase activity is maximal at pH 7.2 to 7.8 and 40°; it is stimulated maximally by magnesium or manganese. The principal products of nucleolar GDPase are GMP and P i; no evidence has been obtained for a contribution to this activity by polynucleotide phosphorylase. 2. 2. Nucleoli also contain glucose-6-phosphatase and inorganic pyrophosphatase. Although such a content of phosphatases is analogous to that of microsomes, the absence of microsomes in the isolated nucleoli, and the non-identity of the nucleolar phosphatases with microsomal phosphatases, has been established by several means. 3. 3. The results of this work verify previous histochemical demonstrations of nucleoside diphosphatase activity in liver and hepatoma nucleoli.

Studies of phosphorus metabolism by isolated nuclei: IX. Nucleolar nucleosidediphosphatase activity

Studies of phosphorus metabolism by isolated nuclei: IX. Nucleolar nucleosidediphosphatase activity

Immunochemical characterization of subcellular fractions from isolated parenchymal and reticulo-endothelial rat liver cells

Rat livers were divided into two fractions, one containing parenchymal cells, and the other reticulo-endothelial cells. The two cell types were fractionated separately into microsomal and cell sap fractions. Detergent extracts of these preparations were analysed for soluble antigens in gel diffusion experiments using rabbit antisera against the 4 different fractions. The cell type specificity of the antisera was also assessed by fluorescent antibody staining of liver sections. Parenchymal microsomes and cell sap contained both fraction specific and common antigens. No clear cut results were obtained with the fractions of the reticulo-endothelial cells. However, all fractions also contained cell type specific antigens. As shown by double diffusion experiments and fluorescent antibody staining, this cell type specificity was primarily confined to the microsomes. Most of the liver specific antigens were found in the microsomes of the parenchymal cells. The parenchymal cell fractions contained at least 5 electrophoretically distinct antigens which exhibited non-specific esterase activity with α-naphtyl propionate as substrate. One of these antigens was also present in kidney microsomes and another in testis microsomes. The esterase which was found both in liver and kidney was equally distributed between parenchymal microsomes and cell sap. The other four were mainly associated with the microsomal fraction. The concentration of these esterases in the reticulo-endothelial extracts was variable, and usually much lower than in the parenchymal extracts. The parenchymal extracts contained three additional antigens which showed acid phosphatase activity when assayed with α-naphtyl phosphate or β-glycerophosphate. While one was present in both cell sap and microsomes, two were recovered only from the microsomal extracts. No acid-phosphatase active antigens were found in the reticulo-endothelial extracts. Another parenchymal antigen present in microsomes and cell sap exhibited nucleoside diphosphatase activity with uridine- or inosine diphosphate as substrate. It did not split adenosine diphosphate or the nucleoside triphosphates. The parenchymal cell sap fraction also contained an antigen of slow electrophoretic mobility with lactic acid dehydrogenase activity.

ELECTROPHORETIC STUDIES ON PHOSPHATASES FROM THE PANCREATIC ISLETS OF OBESE-HYPERGLYCAEMIC MICE

ABSTRACT Disc-electrophoresis on polyacrylamide gels was used for the separation of phosphatases from the pancreatic islets of American obese-hyperglycaemic mice. Three sites of enzyme activity involving inosine diphosphate, adenosine diphosphate and thiamine pyrophosphate were observed and their apparent substrate specificity and sensitivity to various ions examined. By comparison with the epididymis, it was concluded that the different enzyme activities most probably represented alkaline phosphatase, thiamine pyrophosphatase and Golgi-associated nucleoside diphosphatase. The activity of the last enzyme was measured in the endocrine and exocrine pancreas of fed and starved animals by densitometric scanning of the stained gels. The Golgi-associated nucleoside-diphosphatase activity was found to be about 4 times higher in the islets than in the acinar tissue, starvation of the animals for 7 days being without effect. The relative activities of two different molecular varieties of non-specific acid phosphatase were assayed after the isoenzymes had been extracted from the polyacrylamide gels. In the liver and endocrine pancreas the most rapidly migrating enzyme had the highest activity. Most of the enzyme activity from the exocrine pancreas, on the other hand, was found in the slow band.