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.     

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.     

THIAMINE IN NEURAL MEMBRANES ENZYMIC HYDROLYSIS OF THIAMINE DIPHBSPHATE

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.     

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.     

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.     

Membranfluß und Nucleosiddiphosphatase-Reaktion in Wurzelhaaren von Lepidium sativum

Summary Root hairs of Lepidium sativum were incubated with a Wachstein-Meisel medium in experiments designed to localize the activity of nucleoside diphosphatase(s). Electron dense precipitates were found in the ER and in Golgi cisternae of the secretory face of the dictyosomes and their adjacent Golgi vesicles. Such precipitates were absent in the Golgi cisternae of the regeneration face of the dictyosomes and in the detached Golgi vesicles which extrude pectic cell wall substances. These results may be the consequence of the normal cycle of membrane compounds associated with the secretion in which the nucleoside diphosphatase(s) participate (by activation and inactivation) as one of the cycling components. Alternatively the nucleoside diphosphatase(s) may undergo a special cycle in which they are transferred from one cisterna or its vesicles to the next as part of the process of cisternal maturation.     

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.     

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.     

Nucleoside triphosphatase from the hen's egg white and vitelline membrane: purification, properties and relation to similar enzymes from the oviduct.

Hen's egg white and vitelline membrane nucleoside triphosphatases were purified resulting in active soluble subunits with MR 260,000 +/- 10,000. PH optima are divalent cation dependent and situated at pH 6.2 and 8.0 with ATP and at pH 6.15 with ADP as substrate. Ca2+ and Mg2+ are activators. Km and Ki values for Pi and PPi were determined. The enzymes are specific neither for ATP nor for ADP alone. No separation between nucleoside triphosphatase and nucleoside diphosphatase could be achieved. Differences found in their action can be due to differences in organization and properties of the (intermediary) enzyme-substrate complexes. A close relationship exists with homologous enzymes found in oviductal secretory cells and in oviductal secretions.     

Uridine diphosphate glucose-sterol glucosyltransferase and nucleoside diphosphatase activities in etiolated pea seedlings.

1. UDP-glucose-sterol glucosyltransferase and nucleoside diphosphatases were isolated in a particulate fraction from 7-day-old etiolated pea seedlings. The glucosyltransferase and UDPase (uridine diphosphatase) are stimulated by Ca2+ cation, less so by Mg2+ cation, and inhibited by Zn2+. 2. Each activity has a pH optimum near 8. 3. The glucosyltransferase is specific for UDP-glucose as the glucosyl donor and is inhibited by UDP. Partial recovery from UDP inhibition is effected by preincubation of the enzyme. 4. Freeze-thaw treatment and subsequent sucrose-density-gradient centrifugation of the particulate fraction shows the glucosyltransferase to be widely distributed among cell fractions but to be most active in particles with a density of 1.15 g/ml. UDPase is most active in particulate material with a density of over 1.18 g/ml but an activity peak also appears at 1.15 g/ml. Of several nucleoside diphosphatase activities, UDPase activity is most enhanced by the freeze-thaw and sucrose-density-gradient-fractionation procedures. 5. Detergent treatment with 0.1% sodium deoxycholate allows the partial solubilization of the glucosyltransferase and UDPase. The two activities are similarly distributed between pellet and supernatant after high-speed centrifugation for two different time intervals. 6. A role for UDPase in the functioning of glucosylation reactions is discussed.     

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.     

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.     

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.     

Nucleoside diphosphatase and 5'-nucleotidase activities of soybean root nodules and other tissues

Nucleoside diphosphatase and 5′-nucleotidase activities were both found to be very high in extracts of soybean (Glycine max L.) root nodules. Both activities increased early in soybean nodule development, prior to the rise in leghemoglobin, and both were found at equivalent levels in nitrogenfixing and nonfixing nodules. Based on a survey of other tissues, these activities were both highest in soybean nodules (1300 nanomoles per milligram protein per minute, nucleoside diphosphatase and 500 nanomoles per milligram protein per minute, 5′-nucleotidase), but they were not always associated with each other; in some tissues one was high and the other low. Neither activity correlated well with ureide production; both seem, rather, to be primarily involved in some other metabolic function. Both the nucleoside diphosphatase and 5′-nucleotidase of soybean nodules were soluble proteins, and neither appeared to be associated with plastids, mitochondria, or bacteroids.     

Guanosine diphosphatase is required for protein and sphingolipid glycosylation in the Golgi lumen of Saccharomyces cerevisiae

Current models for nucleotide sugar use in the Golgi apparatus predict a critical role for the lumenal nucleoside diphosphatase. After transfer of sugars to endogenous macromolecular acceptors, the enzyme converts nucleoside diphosphates to nucleoside monophosphates which in turn exit the Golgi lumen in a coupled antiporter reaction, allowing entry of additional nucleotide sugar from the cytosol. To test this model, we cloned the gene for the S. cerevisiae guanosine diphosphatase and constructed a null mutation. This mutation should reduce the concentrations of GDP-mannose and GMP and increase the concentration of GDP in the Golgi lumen. The alterations should in turn decrease mannosylation of proteins and lipids in this compartment. In fact, we found a partial block in O- and N-glycosylation of proteins such as chitinase and carboxypeptidase Y and underglycosylation of invertase. In addition, mannosylinositolphosphorylceramide levels were drastically reduced.     

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.     

Loss of latent activity of liver microsomal membrane enzymes evoked by lipid peroxidation. Studies of nucleoside diphosphatase, glucose-6-phosphatase, and UDP glucuronyltransferase

The effects of lipid peroxidation on latent microsomal enzyme activities were examined in NADPH-reduced microsomes from phenobarbital-pretreated male rats. Lipid peroxidation, stimulated by iron or carbon tetrachloride, was assayed as malondialdehyde formation. Independent of the stimulating agent of lipid peroxidation, latency of microsomal nucleoside diphosphatase activity remained unaffected up to microsomal peroxidation equivalent to the formation of about 12 nmol malondialdehyde/mg microsomal protein. However, above this threshold a close correlation was found between lipid peroxidation and loss of latent enzyme activity. The loss of latency evoked by lipid peroxidation was comparable to the loss of latency attainable by disrupting the microsomal membrane by detergent. Loss of latent enzyme activity produced by lipid peroxidation was also observed for microsomal glucose-6-phosphatase and UDPglucuronyltransferase. In contrast to nucleoside diphosphatase, however, both enzymes were inactivated by lipid peroxidation, as indicated by pronounced decreases of their activities in detergent-treated microsomes. According to the respective optimal oxygen partial pressure (po2) for lipid peroxidation, the iron-mediated effects on enzyme activities were maximal at a po2 of 80 mmHg and the one mediated by carbon tetrachloride at a po2 of 5 mmHg. Under anaerobic conditions no alterations of enzyme activities were detected. These results demonstrate that loss of microsomal latency only occurs when peroxidation of the microsomal membrane has reached a certain extent, and that beyond this threshold lipid peroxidation leads to severe disintegration of the microsomal membrane resulting in a loss of its selective permeability, a damage which should be of pathological consequences for the liver cell. Because of its resistance against lipid peroxidation nucleoside diphosphatase is a well-suited intrinsic microsomal parameter to estimate this effect of lipid peroxidation on the microsomal membrane.     

Expression and characterization of soluble and membrane-bound human nucleoside triphosphate diphosphohydrolase 6 (CD39L2)

Ecto-nucleoside-triphosphate diphosphohydrolase-6 (eNTPDase6(1), also known as CD39L2) cDNA was expressed in mammalian COS-1 cells and characterized using nucleotidase assays as well as size exclusion, anion exchange, and cation exchange chromatography. The deduced amino acid sequence of eNTPDase6 is more homologous with the soluble E-type ATPase, eNTPDase5, than other E-type ATPases, suggesting it may also be soluble. To test this possibility, both the cell membranes and the growth media from eNTPDase6-transfected COS-1 cells were assayed for nucleotidase activities. Activity was found in both the membranes and the media. Soluble eNTPDase6 preferentially exhibits nucleoside diphosphatase activity, which is dependent on the presence of divalent cations. Western blot analysis of eNTPDase6 treated with PNGase-F indicated both soluble and membrane-bound forms are glycosylated. However, unlike some membrane-bound ecto-nucleotidases, the eNTPDase6 activity was not specifically inhibited by deglycosylation with peptide N-glycosidase F. Soluble eNTPDase6 hydrolyzed nucleoside triphosphates poorly and nucleoside monophosphates not at all. Analysis of the relative rates of hydrolysis of nucleoside diphosphates (GDP = IDP > UDP > CDP > ADP) suggests that soluble eNTPDase6 is a diphosphatase most likely not involved in regulation of ADP levels important for circulatory hemostasis.     

Nucleoside diphosphatase and glycosyltransferase activities can localize to different subcellular compartments in Schizosaccharomyces pombe

Nucleoside diphosphates generated by glycosyltransferases in the fungal, plant, and mammalian cell secretory pathways are converted into monophosphates to relieve inhibition of the transferring enzymes and provide substrates for antiport transport systems by which the entrance of nucleotide sugars from the cytosol into the secretory pathway lumen is coupled to the exit of nucleoside monophosphates. Analysis of the yeast Schizosaccharomyces pombe genome revealed that it encodes two enzymes with potential nucleoside diphosphatase activity, Spgda1p and Spynd1p. Characterization of the overexpressed enzymes showed that Spgda1p is a GDPase/UDPase, whereas Spynd1p is an apyrase because it hydrolyzed both nucleoside tri and diphosphates. Subcellular fractionation showed that both activities localize to the Golgi. Individual disruption of their encoding genes did not affect cell viability, but disruption of both genes was synthetically lethal. Disruption of Spgda1+ did not affect Golgi N- or O-glycosylation, whereas disruption of Spynd1+ affected Golgi N-mannosylation but not O-mannosylation. Although no nucleoside diphosphatase activity was detected in the endoplasmic reticulum (ER), N-glycosylation mediated by the UDP-Glc:glycoprotein glucosyltransferase (GT) was not severely impaired in mutants because first, no ER accumulation of misfolded glycoproteins occurred as revealed by the absence of induction of BiP mRNA, and second, in vivo GT-dependent glucosylation monitored by incorporation of labeled Glc into folding glycoproteins showed a partial (35-50%) decrease in Spgda1 but was not affected in Spynd1 mutants. Results show that, contrary to what has been assumed to date for eukaryotic cells, in S. pombe nucleoside diphosphatase and glycosyltransferase activities can localize to different subcellular compartments. It is tentatively suggested that ER-Golgi vesicle transport might be involved in nucleoside diphosphate hydrolysis.