Characterization and importance of the dimer interface of human calcium-activated nucleotidase

Human calcium-activated nucleotidase (CAN) exists as both a membrane-bound form in the endoplasmic reticulum and pre-Golgi intermediate membranes and as a secreted, soluble form. Although the wild-type human enzyme hydrolyzes ADP poorly, engineered soluble human proteins (SCANs) hydrolyze ADP much more efficiently, making them potentially useful therapeutic proteins for treatment of human clotting pathologies. According to the crystal structure and the recently identified dimeric nature of the soluble nucleotidase, the dimer interface contains a central core of hydrophobic residues. Previously, we demonstrated that the mutation of glutamic acid 130 (located in the dimer interface) to tyrosine increased both the tendency to form dimers and the ADPase activity. In the present study, we investigated the importance of the dimeric state for enzymatic activity and biological function in this nucleotidase by mutating isoleucine 170, which is located in the center of the hydrophobic core of the dimer interface. The results of analytical ultracentrifugation, chemical cross-linking, and tryptophan fluorescence analyses demonstrated that mutation of isoleucine 170 to either positively or negatively charged amino acids (lys or glu) disrupted the calcium-dependent dimerization in soluble CAN. Furthermore, these mutations decreased maximal ADPase activity for both the soluble and membrane-bound enzymes. Although not as critical as the hydrophobic interactions centered at isoleucine 170, the role of hydrophilic interactions in dimer formation was also demonstrated. Thus, mutation of aspartic acid 228 to threonine (D228T) decreased both the tendency to form dimers and ADPase activity, while double mutation of D228T/K224N largely restored the ability to form dimers and the ADPase activity, further indicating that the nucleotidase activity of CAN is linked to its quaternary structure. Since ADPase activity of the soluble form is crucial for its potential development as a therapeutic protein, these findings have implications for engineering the soluble human calcium-activated nucleotidase for clinical applications. In addition, future comparison of monomeric (I170K and I170E mutants) and dimeric (wild-type) crystal structures of SCAN will advance our understanding of its enzymatic mechanism and aid in engineering efforts.     

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.     

Evolution of pyruvate carboxylase and other biotin containing enzymes in developing rat liver and kidney

During contractions, when the rate of ATP hydrolysis exceeds that of ADP phosphorylation, inosine 5'-monophosphate (IMP) accumulates in skeletal muscle. If the cellular energy balance is not promptly restored, subsequent purine degradation to inosine via 5'-nucleotidase can occur, a process that is most robust in the slow-twitch red, as compared to fast-twitch, skeletal muscle. We measured the distribution of 5'-nucleotidase activity among membrane-bound and soluble fractions of fiber specific skeletal muscle sections and found most (80-90%) of the total 5'-nucleotidase activity to be membrane-bound. The 5' IMP nucleotidase activity present in the soluble fraction of muscle extracts differs among fiber types with slow-twitch red > fast-twitch red > mixed fibered > fast-twitch white. Experiments testing the substrate dependence of IMP and AMP dephosphorylation by the soluble fraction of muscle extracts revealed a lower Km toward IMP (approximately 0.7-1.5 mM) than AMP (1.9-2.8 mM). Among skeletal muscle fiber sections, the soluble 5'-nucleotidase activity present in slow-twitch red muscle extracts had the highest substrate affinity, the highest activity with IMP as substrate, and an estimated catalytic efficiency (Vmax/Km) that was > 3-fold higher than calculated for fast-twitch muscle extracts. This is likely due to the Mg2+ dependent cytosolic 5' IMP nucleotidase isoform, since immunoprecipitation experiments revealed 3-4 times more activity in slow-twitch red than in fast-twitch red or fast-twitch white fibers, respectively. These finding are consistent with the previously recognized in vivo pattern of nucleoside formation by muscle where the soleus demonstrated extensive inosine formation at a much lower IMP content than fast-twitch red or fast-twitch white muscle fiber sections.     

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.     

Site-directed mutagenesis of human nucleoside triphosphate diphosphohydrolase 3: the importance of conserved glycine residues and the identification of additional conserved protein motifs in eNTPDases.

Glycine residues are recognized as important structural determinants in nucleotide-binding domains of many enzymes. The functional significance of seven glycine residues invariant in all 22 eNTPDase sequences was therefore examined. Glycine-to-alanine mutants of eNTPDase3 were analyzed for nucleotidase activities and tertiary and quaternary structure changes. Mutations G98A and G183A had modest effects on ATPase and ADPase activities. The G141A mutation resulted in 4- to 5-fold decreased nucleotidase activity, while the G222A mutation decreased ATPase activity 20-fold, and ADPase activity 6-fold. Unlike the other five glycine mutants, the G263A and G462A mutations caused significant loss of nucleotidase activity which was observed concomitant with lower protein expression levels, large-scale changes in tertiary and quaternary protein structure, and decreased trafficking to the plasma membrane. Thus, these data identify glycine residues that are essential for enzymatic activity and the tertiary and quaternary structure of eNTPDase3. Further, two additional conserved regions in the eNTPDases are identified, apyrase conserved regions ACR1a and ACR4a, which may be involved in phosphate binding/hydrolysis and protein folding, respectively.     

Entamoeba histolytica: localization and characterization of ca2+-dependent nucleotidases

An activity of Ca²⁺-dependent nucleotidase was detected in axenically-cultivated trophozoites of Entamoeba histolytica. The enzyme was concentrated by differential and sucrose density gradient centrifugation and catalyzed hydrolysis of nucleoside tri- and diphosphates and also thiamine pyrophosphate. Hydrolysis of nucleoside mono-phosphates was not affected by Ca²⁺. Among substrates tested, ATP was most active. Addition of Zn²⁺ or heat treatment almost abolished the enzyme activity. The enzyme exhibited almost the identical activity at acid and neutral pH. Among 6 bands isolated by polyacrylamide gel electrophoresis, 4 were stained with ATP, UTP, CTP and ADP, whereas the other 2 were stained only with ATP, UTP and CTP. The concentrated enzyme preparation, primarily composed of membrane fragments, also had activities of acid phosphatase, acid inorganic pyrophosphatase, 5'-nucleotidase and Mg²⁺-dependent ATPase. These observations suggest that E. histolytica has 2 Ca²⁺-dependent nucleotidases, i.e. one Ca²⁺-dependent ATPase and the other Ca²⁺-dependent nucleoside diphosphatase or an apyrase-like enzyme, and that these nucleotidases are at least partially associated with the plasma membrane or an organelle of lysosomal nature in this parasite.     

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.     

Metabolism of adenine nucleotides by ectoenzymes of vascular endothelial and smooth-muscle cells in culture.

1. Pig aortic endothelial and smooth-muscle cells in culture rapidly catabolize exogenous ATP, ADP or AMP. 2. In both cell types catabolism is due to Mg2+-stimulated ectoenzymes. 3. Inhibition and substrate-specificity studies suggest that both cell types possess three distinct ectonucleotidases, namely nucleoside triphosphatase (EC 3.6.1.15), nucleoside diphosphatase (EC 3.6.1.6) and 5'-nucleotidase (EC 3.1.3.5), as well as nucleoside diphosphate kinase (EC 2.7.4.6). 4. These ectonucleotidase systems could be of importance in the regulation of neurotransmission, blood platelet function and vasodilation.     

Site-directed mutagenesis of human nucleoside triphosphate diphosphohydrolase 3: the importance of residues in the apyrase conserved regions

Ecto-nucleoside triphosphate diphosphohydrolase 3 (eNTPDase-3, also known as HB6 and CD39L3) is a membrane-associated ecto-apyrase. Only a few functionally significant residues have been elucidated for this enzyme, as well as for the whole family of eNTPDase enzymes. Four highly conserved regions (apyrase conserved regions, ACRs) have been identified in all the members of eNTPDase family, suggesting their importance for biological activity. In an effort to identify those amino acids important for the catalytic activity of the eNTPDase family, as well as those residues mediating substrate specificity, 11 point mutations of 7 amino acid residues in ACR1-4 of eNTPDase-3 were constructed by site-directed mutagenesis. Mutagenesis of asparagine 191 to alanine (N191A), glutamine 226 to alanine (Q226A), and arginine 67 to glycine (R67G) resulted in an increase in the rates of hydrolysis of nucleoside diphosphates relative to triphosphates. Mutagenesis of arginine 146 to proline (R146P) essentially converted the eNTPDase-3 ecto-apyrase to an ecto-ATPase (eNTPDase-2), mainly by decreasing the hydrolysis rates for nucleoside diphosphates. The Q226A mutant exhibited a change in the divalent cation requirement for nucleotidase activity relative to the wild-type and the other mutants. Mutation of glutamate 182 to aspartate (E182D) or glutamine (E182Q), and mutation of serine 224 to alanine (S224A) completely abolished enzymatic activity. We conclude that the residues corresponding to eNTPDase-3 glutamate 182 in ACR3 and serine 224 in ACR4 are essential for the enzymatic activity of eNTPDases in general, and that arginine 67, arginine 146, asparagine 191, and glutamine 226 are important for determining substrate specificity for human ecto-nucleoside triphosphate diphosphohydrolase 3.     

Identification and characterization of a novel calcium-activated apyrase from Cryptosporidium parasites and its potential role in pathogenesis

Herein, we report the biochemical and functional characterization of a novel Ca(2+)-activated nucleoside diphosphatase (apyrase), CApy, of the intracellular gut pathogen Cryptosporidium. The purified recombinant CApy protein displayed activity, substrate specificity and calcium dependency strikingly similar to the previously described human apyrase, SCAN-1 (soluble calcium-activated nucleotidase 1). CApy was found to be expressed in both Cryptosporidium parvum oocysts and sporozoites, and displayed a polar localization in the latter, suggesting a possible co-localization with the apical complex of the parasite. In vitro binding experiments revealed that CApy interacts with the host cell in a dose-dependent fashion, implying the presence of an interacting partner on the surface of the host cell. Antibodies directed against CApy block Cryptosporidium parvum sporozoite invasion of HCT-8 cells, suggesting that CApy may play an active role during the early stages of parasite invasion. Sequence analyses revealed that the capy gene shares a high degree of homology with apyrases identified in other organisms, including parasites, insects and humans. Phylogenetic analysis argues that the capy gene is most likely an ancestral feature that has been lost from most apicomplexan genomes except Cryptosporidium, Neospora and Toxoplasma.     

Mammalian plasma membrane ecto-nucleoside triphosphate diphosphohydrolase 1, CD39, is not active intracellularly. The N-glycosylation state of CD39 correlates with surface activity and localization

CD39 is a member of the membrane-bound ecto-nucleoside triphosphate diphosphohydrolase family. The active site for native CD39 is located on the outer surface of the cellular plasma membrane; however, it is not yet known at what stage this enzyme becomes active along the secretory pathway to the plasma membrane. In this study, sucrose density fractionations performed on CD39-transfected COS-7 cell membranes suggest that CD39 activity resides primarily in the plasma membrane. Furthermore, we have created recombinant, soluble versions of CD39, one that is secreted and others that are retained in the endoplasmic reticulum, to demonstrate that CD39 is not active until it reaches the plasma membrane both in yeast and COS-7 cells. Moreover, the secreted active soluble CD39 in COS-7 cells is found to receive a higher degree of N-glycan addition than the inactive form retained intracellularly. When COS-7 cells were treated with tunicamycin to prevent N-glycosylation, soluble CD39 was not detected in the extracellular medium and remained inactive intracellularly. Surface biotinylation analysis also revealed that surface-expressed wild type CD39 receives a higher degree of N-glycosylation than intracellular forms and that inhibition of N-glycosylation prevents its plasma membrane localization. In addition, both intact and digitonin-permeablized COS-7 cells transfected with CD39 possess similar ecto-ATPase activities, further supporting the conclusion that only surface-expressed CD39 is enzymatically active. All of these data suggest that intracellular CD39 is inactive and that only a fully glycosylated CD39 has apyrase activity and is localized at the cell surface.     

Distribution,properties, and functions of midgut carboxypeptidases and dipeptidases from Musca domestica larvae

Dipeptidase and carboxypeptidase A activities were determined in cells and luminal contents of the fore-, mid-, and hind-midgut of Musca domestica larvae. Dipeptidase activity was found mainly in hind-midgut cells, whereas carboxy-peptidase activity was recovered in major amounts in both cells and in luminal contents of hind-midguts. The subcellular distribution of dipeptidase and part of the carboxypeptidase A activities is similar to that of a plasma membrane enzyme marker (aminopeptidase), suggesting that these activities are bound to the microvillar membranes. Soluble carboxypeptidase A seems to occur both bound to secretory vesicles and trapped in the cell glycocalyx. Based on density-gradient ultracentrifugation and thermal inactivation, there seems to be only one molecular species of each of the following enzymes (soluble in water or solubilized in Triton X-100): membrane-bound dipeptidase (pH optimum 8.0; Km 3.7 mM GlyLeu, M_r 111,000), soluble carboxypeptidase (pH optimum 8.0; Km 1.22 mM N-carbobenzoxy-glycyl-L-phenylalanine (ZGlyPhe), M_r45,000) and membrane-bound carboxypeptidase (pH optimum 7.5, Km 2.3 mM ZGlyPhe, M_r58,000). The results suggest that protein digestion is accomplished sequentially by luminal trypsin and luminal carboxypeptidase, by membrane-bound carboxypeptidase and aminopeptidase, and finally by membrane-bound dipeptidase.     

Effects of con A and other lectins on pure 5'nucleotidase isolated from lymphocyte plasma membranes.

Inhibition of purified or membrane-bound 5′nucleotidase by various lectins was studied in lymphocytes from pig mesenteric lymph nodes. Con A or $\text{Lens culinaris}$ lectin LcH inhibited (75 %) purified 5′nucleotidase by a non-competitive process without cooperativity. Inhibition by these lectins of 5′ nucleotidase activity in whole lymphocytes, plasma membranes (untreated or solubilized) and LcH-receptor fraction displayed high positive cooperativity, reached higher level (90 %) and was of mixed type. An interaction between lectin receptors and 5′nucleotidase accounted for these differences. Wheat germ agglutinin (WGA) and divalent Con A which are not mitogenic for T lymphocytes had no effect on 5′nucleotidase; pokeweed mitogen (PWM), mitogen of T and B cells, was not inhibitor. When membrane proteins were cross-linked by glutaraldehyde, Con A inhibition of whole lymphocyte 5′nucleotidase presented the same properties as the purified enzyme. Possible correlation between 5′nucleotidase inhibition and lymphocyte stimulation is discussed.     

Mutagenesis of two conserved tryptophan residues of the E-type ATPases: inactivation and conversion of an ecto-apyrase to an ecto-NTPase

A human brain E-type ATPase (HB6 ecto-apyrase) was subjected to site-directed mutagenesis to assess the functional significance of two highly conserved tryptophan residues (Trp 187 and Trp 459), the only two tryptophans conserved in nearly all E-type ATPases. Mutation of tryptophan 187 to alanine yielded a poorly expressed ecto-apyrase completely devoid of nucleotidase activity. Immunolocalization of the W187A mutant in mammalian COS cells showed a cellular distribution clearly different from that of the wild-type enzyme, with the majority of the immunoreactivity concentrated in the interior of the cell. Unlike the wild-type enzyme, this mutant did not bind the nucleotide analogue Cibacron Blue and was sensitive to proteolytic digestion by chymotrypsin. These results suggest alteration of the tertiary structure, causing the enzyme to be improperly folded and retained within the cell. In contrast, mutation of tryptophan 459 to alanine resulted in an ecto-apyrase with enhanced NTPase activity, but diminished NDPase activity. Immunolocalization of this active mutant ecto-apyrase revealed a cellular pattern similar to that of the wild-type enzyme, distributed along the cell periphery and in cell processes. Coupling this active W459A mutation to a previously described mutation (D219E) resulted in an enzyme which preferentially hydrolyzes nucleoside triphosphates over diphosphates. The D219E/W459A double mutant had an ATPase:ADPase ratio of 11:1 and a UTPase:UDPase ratio of 148:1. In addition, the double mutant is substantially less sensitive to inhibition by azide, a more potent inhibitor of ecto-apyrases than ecto-ATPases. Thus, mutation of only two amino acids of an E-type ATPase essentially converts an ecto-apyrase to an ecto-NTPase.     

Enzymes of carbohydrate metabolism in the developing rice grain

The levels of reducing and nonreducing sugars, starch, soluble protein, and selected enzymes involved in the metabolism of sucrose, glucose-1-P, and glucose nucleotides were assayed in dehulled developing rice grains (Oryza sativa L. line IR1541-76-3) during the first 3 weeks after flowering. The level of reducing sugars in the grain was highest 5 to 6 days after flowering. The level of nonreducing sugars and the rate of starch accumulation were maximum 11 to 12 days after flowering, when the level of soluble protein was also the highest. The activities of bound and free invertase, sucrose-UDP and sucrose-ADP glucosyltransferases, hexokinase, phosphoglucomutase, nucleoside diphosphokinase, and UDP-glucose and ADP-glucose pyrophosphorylases were high throughout starch deposition, and were maximum, except for nucleoside diphosphokinase which did not increase in activity, between 8 and 18 days after flowering. Soluble primed phosphorylase and ADP glucose-α-glucosyltransferase (starch synthetase) were both present during starch accumulation. Phosphorylase activity was at least 2-fold that of soluble starch synthetase but the synthetase followed more closely the rate of starch accumulation in the grain. The activity of starch synthetase bound to the starch granule also increased progressively with increased starch content of the grain.     

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.     

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.     

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.