Three diadenosine 5',5'-P1,P4-tetraphosphate hydrolytic enzymes from Physarum polycephalum with differential effects by calcium: a specific dinucleoside polyphosphate pyrophosphohydrolase, a nucleotide pyrophosphatase, and a phosphodiesterase

Two new enzymes that hydrolyze diadenosine tetraphosphate (Ap4A) have been isolated from the acellular slime mold Physarum polycephalum. Both enzymes are different from the Physarum Ap4A symmetrical pyrophosphohydrolase previously described on the basis of their substrate specificities, reaction products, molecular weights, and divalent cation requirements. One enzyme is a nucleotide pyrophosphatase that asymmetrically hydrolyzes Ap4A to AMP and ATP. This enzyme hydrolyzes several mono- and dinucleotides with the corresponding nucleotide monophosphate as one of the products. The percentage hydrolysis of NAD+, Ap4A, and Ap4G, each at 10 microM, was 100, 56, and 51, respectively. A divalent cation is required for activity, with Ca2+ yielding 20-30 times greater activity than Mg2+ or Mn2+. Values of Km for Ap4A and Vmax are similar to the corresponding values for Ap4A symmetrical pyrophosphohydrolase. The second enzyme is a phosphodiesterase I with broad substrate reactivity. This enzyme also asymmetrically hydrolyzes Ap4A, but it does not hydrolyze NAD+. Activity of the phosphodiesterase I is stimulated by divalent cations, with Ca2+ being 50-60 times more stimulatory than Mg2+ or Mn2+. The apparent molecular weights of the nucleotide pyrophosphatase and phosphodiesterase are 184,000 and 45,000, respectively. In contrast, the Ap4A pyrophosphohydrolase hydrolyzes Ap4A to ADP, is inhibited by Ca2+ and other divalent cations, and has an apparent molecular weight of 26,000 as previously reported.     

Location of nucleotide pyrophosphatase and alkaline phosphodiesterase activities on the lymphocyte surface membrane.

1. Isolated mouse spleen lymphocytes hydrolysed UDP-galactose added to the medium. Nucleotide pyrophosphatase activity that accounted for this hydrolysis was enriched to a similar extent as alkaline phosphodiesterase and 5'-nucleotidase in a lymphocyte plasma-membrane fraction. 2. The cell surfaces of mouse spleen and thymus lymphocytes were iodinated with 125I by using the lactoperoxidase-catalysis method. Detergent extracts of the cells were mixed with a purified anti-(mouse liver plasma-membrane nucleotide pyrophosphatase) antiserum and the immunoprecipitates analysed by polyacrylamide-gel electrophoresis. Only one major radioactive component, similar in size (apparent mol.wt 110000-130000) to the liver enzyme, was observed. 3. Electrophoresis of an iodinated spleen plasma-membrane fraction indicated peaks of radioactivity, including one of apparent mol.wt 110000-130000. 4. When detergent extracts of spleen lymphocytes were passed through a Sepharose-bead column containing covalently attached anti-(nucleotide pyrophosphatase) antiserum, the nucleotide pyrophosphatase activity was retained by the beads, whereas protein and leucine naphthylamidase activity were eluted. 5. The results indicate that nucleotide pyrophosphatase and alkaline phosphodiesterase activities are due to the location of the same or similar enzymes at the outer aspect of the lymphocyte plasma membrane. Some possible functions of enzymes at this location are discussed.     

A novel phosphodiesterase I from the soluble fraction of erythrocytes

A previously unrecognized erythrocyte phosphodiesterase I with activity against thymidine-5'-monophospho-p-nitrophenyl ester is described. The enzyme is present in the soluble fraction of the erythrocyte, and was purified about 500-fold by chromatography using DEAE-cellulose, followed by gel chromatography with Sephadex G-200. Erythrocyte phosphodiesterase I has a molecular weight of about 70 000, when fully active as a monomer. Its pI is 5.4 and the pH optimum is 8.5. The Km value for thymidine-5'-monophospho-p-nitrophenyl ester is rather high, about 4 mmol/l. The enzyme has a barely detectable nucleotide pyrophosphatase activity. It is extremely sensitive to SH-inhibitors such as N-ethyl-maleimide, p-chloromercuribenzoate and disulphides (a reversible 50% inhibition was obtained by cystamine, 0.01 mmol/l). It is a metalloenzyme with loosely bound metal, and is stimulated by Mg2+. This activation by Mg2+ is counteracted by Zn2+. Gel chromatography revealed that the enzyme is a monomer in the presence of Mg2+. When inhibited by Zn2+, it forms polymers that can be reconverted to the monomer by thiols. All of the above properties of the erythrocyte enzyme support the conclusion that it is different from plasma membrane phosphodiesterase I (oligonucleate 5'-nucleotidohydrolase, EC 3.1.4.1).     

Phenolic glyscosides, a new class of human recombinant nucleotide pyrophosphatase phosphodiesterase-1 inhibitors

Cytotoxicity and kinetic studies of phenolic glycosides, benzoyl salireposide (1) and salireposide (2), isolated from Symplocos racemosa, were performed against phosphodiesterase I enzyme from snake venom and human nucleotide pyrophosphatase phosphodiesterase-1. Lineweaver-Burk and Dixon plots and their secondary replots showed that these compounds are pure non-competitive inhibitors of both enzymes. K(i) Values of compounds 1 and 2 were found to be 360 and 1000 microM, respectively, against human nucleotide pyrophosphatase phosphodiesterase, and 525 and 1100 microM, respectively, against snake venom phosphodiesterase. IC(50) values of compounds 1 and 2 are 90 microM +/- 0.04 and 383 microM +/- 0.03, respectively, against human nucleotide pyrophosphatase phosphodiesterase and 171 microM +/- 0.02 and 544 microM +/- 0.021, respectively, against snake venom phosphodiesterase. Both compounds were found to be nontoxic up to concentration of 500 microM/mL as >90% cells were viable after 3 h of incubation. These compounds are potential candidates for the therapy of arthritis.     

Rat intestinal nucleotide-sugar pyrophosphatase. Localization, partial purification, and substrate specificity

The nucleotide-sugar pyrophosphatase activity of rat small intestine was studied using GDP-[14C]Man as substrate. The highest specific activities in the gastrointestinal tract were in the proximal small intestine, with a preferential localization in villus tip cells. Purified brush-border membranes were highly enriched in nucleotide-sugar pyrophosphatase. After the enzyme was solubilized with detergent and purified 180-fold, it hydrolyzed FAD and p-nitrophenyl-5'-thymidylate, as well as nucleotide sugars. That the same enzyme, a 5'-nucleotide phosphodiesterase, is responsible for nucleotide-sugar pyrophosphatase, phosphodiesterase I, and FAD pyrophosphatase activities is indicated by: co-migration in electrophoresis, parallel thermal inactivation, competitive inhibition studies, and similar regional, cellular, and subcellular localizations.     

Immunoaffinity purification and characterization of nucleotide pyrophosphatase from human placenta

Nucleotide pyrophosphatase [EC 3.6.1.9] was purified to homogeneity from human placenta using a monoclonal antibody affinity column. By sodium dodecylsulfate--polyacrylamide gel electrophoresis, the purified enzyme showed a major band at a molecular size of 130 K. The enzyme was a glycoprotein with N-linked oligosaccharides consisting of both complex- and oligomannoside-types. Substrate specificity to hydrolyze phosphodiester and phosphosulfate linkages as well as other properties were similar to those of nucleotide pyrophosphatase and phosphodiesterase from other sources.     

Nucleotide pyrophosphatase/phosphodiesterase from Euphorbia characias latex: Purification and characterization

An authentic soluble metallo-protein nucleotide pyrophosphatase/phosphodiesterase (ELNPP) was purified to homogeneity from Euphorbia characias latex. The native protein had a molecular mass of 80 ± 5 kDa and was shown to be formed by two apparently identical subunits, each containing 1 Ca²⁺ and 1 Mg²⁺ ion. Whereas Mg²⁺ was shown to be strongly bound to the enzyme, Ca²⁺ was easily removed by treatment with EDTA. Ca²⁺-demetalated enzyme was shown to be almost totally inactive and the activity was fully restored incubating the demetalated ELNPP with Ca²⁺ ions. ELNPP exhibited hydrolytic activities toward pyrophosphate/phosphodiester bonds of a broad range of substrates and very efficiently hydrolyzed the artificial substrate thymidine 5′-monophosphate 4-nitrophenyl ester generating 4-nitrophenolate as a final product, and it has been used for enzyme kinetic experiments. ELNPP represents the first example of a nucleotide pyrophosphatase/phosphodiesterase enzyme purified from the latex of a plant belonging to the large genus Euphorbia.     

Modulation of nucleotide pyrophosphatase in plasmacytoma cells.

The effect of glucocorticoid hormones on the protein responsible for both nucleotide pyrophosphatase (EC 3.6.1.9) and alkaline phosphodiesterase I (EC 3.1.4.1) activities was examined in murine MOPC 315 plasmacytoma cells. Incubation of these cells with dexamethasone resulted in parallel increases in pyrophosphatase and phosphodiesterase specific activities. The incorporation of [3H]mannose into N-linked oligosaccharide precursors was also analyzed in cells following hormone modulation. In cells treated for 36 hours or cultured continuously with dexamethasone, the resulting increase in enzyme specific activities was accompanied by a decrease in [3H]mannose incorporation, consistent with the hypothesis that in some cell types, nucleotide pyrophosphatase activity is involved in the regulation of glycoprotein synthesis.     

Distribution of two isoforms of NADP-dependent isocitrate dehydrogenase in soybean (Glycine max)

Two different cDNAs that encode NADP-specific isocitrate dehydrogenase (NADP-IDH) isozymes of soybean (Glycine max) were characterized. The nucleotide sequences of the coding regions of these cDNAs have 74% identity to each other and give predicted amino acid sequences that have 83% identity to each other. Using PCR techniques, their coding regions were subcloned into a protein overexpression vector, pQE32, to yield pIDH4 and pIDH1, respectively. Both IDH4 and IDH1 enzymes were expressed in Escherichia coli as catalytically active His6 tagged proteins, purified to homogeneity by affinity chromatography on nickel chelate resin and rabbit polyclonal antibodies to each were generated. Surprisingly, antiserum to IDH4 did not react with IDH1 protein and IDH1 antiserum reacted only very weakly with IDH4 protein. IDH4 antibody reacts with a protein of expected molecular weight in cotyledon, young leaf, young root, mature root and nodules but the reaction with mature leaf tissue was low compared to other tissues. Western blot results show that IDH1 was not expressed in young roots but a protein that reacts with the IDH1 antibody was highly expressed in leaves, showing that there was tissue-specific accumulation of NADP-IDH isozymes in soybean.     

Purification and Developmental Analysis of an Extracellular Proteinase from Young Leaves of Soybean

A proteinase present in intercellular wash fluids from leaves of Glycine max has been purified 600-fold to electrophoretic homogeneity. The native protein is monomeric with a molecular mass of 60 kD, as estimated by denaturing gel electrophoresis, and has an isoelectric point of 7.7. The enzyme has a pH optimum of 9.5 when assayed with Azocoll as a substrate. The proteolytic activity is inhibited by p-chloromercuribenzoic acid and mercuric chloride and requires the presence of reducing agents. The enzyme activity is refractory to other classical sulfhydryl proteinases. The soybean leaf endoproteinase is present within the extracellular space of young leaves, and a portion is bound to the cell wall. Western blot analysis and activity measurements show that the enzyme is present only during the first 15 d postemergence of the leaf and is therefore under strict developmental control. We suggest that the enzyme may play a critical role in the extracellular milieu during rapid cell growth and leaf expansion.     

Maize leaf proteases and their effect on endogenous inorganic pyrophosphatase

Several different proteolytic enzymes are present in leaf and root tissue of maize seedlings. The activity of these enzymes diminishes to a basal level by the time seedling height reaches 20–30 cm. We have partially characterized an endopeptidase with trypsin-like specificity and two aminopeptidases, all from leaf tissue, and compared them to previously reported proteases from maize. Both the endopeptidase and the aminopeptidases degrade the maize leaf enzyme, inorganic pyrophosphatase. Modification of the pyrophosphatase by the peptidases results in the formation of catalytically active, electrophoretically distinct products. The aminopeptidases have little effect on several other maize leaf enzymes, but also modify yeast inorganic pyrophosphatase.     

Human blood platelet 3': 5'-cyclic nucleotide phosphodiesterase. Isolation of low-Km and high-Km phosphodiesterase.

Human blood platelet contained at least three kinetically distinct forms of 3': 5'-cyclic nucleotide phosphodiesterase (3': 5'-cyclic-AMP 5'-nucleotidohydrolase, EC 3.1.4.17) (F I, F II, and F III) which were clearly separated by DEAE-cellulose column chromatography. Although a few properties of the platelet phosphodiesterases such as their substrate affinities and DEAE-cellulose profile resembled somewhat those of the three 3': 5'-cyclic nucleotide phosphodiesterase in rat liver reported by Russell et al. [10], there were pronounced differences in some properties between the platelet and the liver enzymes: (1) the platelet enzymes hydrolyzed both cyclic nucleotides and lacked a highly specific cyclic guanosine 3': 5'-monophosphate (cyclic GMP) phosphodiesterase and (2) kinetic data of the platelet enzymes indicated that cyclic adenosine 3': 5'-monophosphate (cyclic AMP) and cyclic GMP interact with a single catalytic site on the enzyme. F I was a cyclic nucleotide phosphodiesterase with a high Km for cyclic AMP and a negatively cooperative low Km for cyclic GMP. F II hydrolyzed cyclic AMP and cyclic GMP about equally with a high Km for both substrates. F III was low Km phosphodiesterase which hydrolyzed cyclic AMP faster than cyclic GMP. Each cyclic nucleotide acted as a competitive inhibitor of the hydrolysis of the other nucleotide by these three fractions with Ki values similar to the Km values for each nucleotide suggesting that the hydrolysis of both cyclic AMP and cyclic GMP was catalyzed by a single catalytic site on the enzyme. However, cyclic GMP at low concentration (below 10 muM) was an activator of cyclic AMP hydrolysis by F I. Papaverine and EG 626 acted as competitive inhibitors of each fraction with virtually the same Ki value in both assays using either cyclic AMP or cyclic GMP as the substrate. The ratio of cyclic AMP hydrolysis to cyclic GMP hydrolysis by each fraction did not vary significantly after freezing/thawing or heat treatment. These facts also suggest that both nucleotides were hydrolyzed by the same catalytic site on the enzyme. The differences in apparent Ki values for inhibitors such as cyclic nucleotides, papaverine and EG 626 would indicate that three enzymes were different from each other. Centrifugation in a continuous sucrose gradient revealed sedimentation coefficients F I and II had 8.9 S and F III 4.6 S. The molecular weight of these forms, determined by gel filtration on a Sepharose 6B column, were approx. 240 000 (F I and II) and 180 000 (F III). F III was purified extensively (70-fold) from homogenate, with a recovery of approximately 7%.     

A protein inhibitor of calmodulin-regulated cyclic nucleotide phosphodiesterase in amphibian ovaries

The cytosol fraction of an extract of Xenopus laevis ovaries contains a protein inhibitor that can specifically block the activation of calmodulin-sensitive cyclic nucleotide phosphodiesterase (PDE I) found in that tissue. This inhibitor was purified by DEAE-cellulose chromatography, gel filtration on Sephacryl S-200, and affinity chromatography on calmodulin-Sepharose. It has a molecular weight of approximately 90,000, and is heat-labile and susceptible to inactivation by chymotrypsin. The inhibitor blocks calmodulin activation of cyclic nucleotide phosphodiesterases from amphibian ovary and bovine brain and of the myosin light chain kinase from rabbit smooth muscle, but does not affect the activity of a calmodulin-insensitive cyclic nucleotide phosphodiesterase. The inhibitor not only affects the activation of Xenopus PDE I and of the bovine brain phosphodiesterase by calmodulin, but also inhibits the stimulation of these enzymes by lysophosphatidylcholine. The inhibitor also acts on PDE I activated by partial tryptic proteolysis, but the enzyme fully activated by trypsin is only slightly susceptible to inhibition by this protein. The inhibition of PDE I activation caused by this ovarian factor can be reversed by adding excess amounts of calmodulin or lysophosphatidylcholine. The presence of this inhibitor provides a possible explanation for the previously observed inactivity of PDE I in vivo.     

Molecular structure and subcellular localization of spinach leaf glycolate oxidase

Glycolate oxidase (E.C. 1.1.3.1) was purified from spinach leaves (Spinacia oleracea). The molecular weight of the native protein was determined by sucrose density gradient centrifugation to be 290,000 daltons (13S), whereas that of the monomeric form was 37,000 daltons. The quaternary structure of the holoenzyme is likely to be octameric, analogous to pumpkin cotyledon glycolate oxidase [Nishimura et al, 1982]. The subcellular localization of the enzyme was studied using linear sucrose density gradient centrifugation, and it was found that glycolate oxidase activity is detectable in both leaf peroxisomal and supernatant fractions, but not in chloroplasts and mitochondria; the activity distribution pattern is essentially similar to that for catalase, a known leaf peroxisomal enzyme. Ouchterlony double diffusion and immunotitration analyses, demontrated that the rabbit antiserum against purified spinach leaf glycolate oxidase cross-reacted, identically, with the enzyme molecules present in two different subcellular fractions, i.e, the leaf peroxisome and supernatant fractions. It is thus concluded that the enzyme present in the supernatant is due to the disruption of leaf peroxisomes during the isolation, and hence glycolate oxidase is exclusively localized in leaf peroxisomes in spinach leaves.     

Purification and properties of a mouse liver plasma-membrane glycoprotein hydrolysing nucleotide pyrophosphate and phosphodiester bonds

1. A mouse liver plasma-membrane preparation was solubilized in an N-dodecylsarcosinate-Tris buffer, pH7.8, and the proteins and glycoproteins were separated by a rate-zonal centrifugation in sucrose-detergent gradients. 2. A peak of alkaline phosphodiesterase activity which sedimented ahead of the 5'-nucleotidase peak was associated with a major glycoprotein component of the plasma membrane. 3. The phosphodiesterase activity was then purified further by gel filtration and gave a single glycoprotein band after electrophoresis on polyacrylamide gels. The apparent molecular weight of the polypeptide at pH7.4 and 8.9 was 128000-130000 and was independent of the polyacrylamide concentration. Electrophoresis in gels containing deoxycholate showed that the protein band was coincident with phosphodiesterase activity. 4. After two-dimensional immunoelectrophoresis, with agarose containing rabbit anti-(mouse plasma-membrane) antiserum as second dimension, the enzyme showed one component which was also coincident with the phosphodiesterase activity. 5. An amino acid composition of the glycoprotein is presented. Carbohydrate analysis indicated the presence of glucosamine, neutral sugars and sialic acid. 6. The enzyme was also a nucleotide pyrophosphatase, as shown by a similar enrichment during purification of activity towards ATP, NAD(+), UDP-galactose and UDP-N-acetylglucosamine. The phosphodiesterase activity, measured by using dTMP p-nitrophenyl ester as substrate, was competitively inhibited by nucleotide pyrophosphate substrates. The enzyme showed little or no activity towards RNA, cyclic AMP, AMP, ADP and glycerylphosphorylcholine. 7. The significance of this enzyme activity in the plasma membrane is discussed.     

Molecular structure and subcellular localization of spinach leaf glycolate oxidase

Glycolate oxidase (E.C. 1.1.3.1) was purified from spinach leaves (Spinacia oleracea). The molecular weight of the native protein was determined by sucrose density gradient centrifugation to be 290,000 daltons (13S), whereas that of the monomeric form was 37,000 daltons. The quaternary structure of the holoenzyme is likely to be octameric, analogous to pumpkin cotyledon glycolate oxidase [Nishimura et al, 1982]. The subcellular localization of the enzyme was studied using linear sucrose density gradient centrifugation, and it was found that glycolate oxidase activity is detectable in both leaf peroxisomal and supernatant fractions, but not in chloroplasts and mitochondria; the activity distribution pattern is essentially similar to that for catalase, a known leaf peroxisomal enzyme. Ouchterlony double diffusion and immunotitration analyses, demonstrated that the rabbit antiserum against purified spinach leaf glycolate oxidase cross-reacted, identically, with the enzyme molecules present in two different subcellular fractions, i.e, the leaf peroxisome and supernatant fractions. It is thus concluded that the enzyme present in the supernatant is due to the disruption of leaf peroxisomes during the isolation, and hence glycolate oxidase is exclusively localized in leaf peroxisomes in spinach leaves.     

Histochemical localization of nucleotide pyrophosphatase and cyclic nucleotide phosphodiesterase in seeds and shoots of Triticum

The activities of potato nucleotide pyrophosphatase and cyclic nucleotide phosphodiesterase against a common substrate, p-nitrophenyl thymidine 5-phosphate and its histochemical analogue, AS-BI-naphthyl thymidine 5-phosphate, were determined with the aid of relatively specific inhibitors, NAD and 2,3-cAMP, respectively. These inhibitors were utilized to reexamine wheat (Triticum aestivum L. cv. Mironovska 808) seeds and 3–5-d old shoots for the occurrence and histochemical localization of nucleotide pyrophosphatase, and to establish the localization of cyclic nucleotide phosphodiesterase. Nucleotide pyrophosphatase is a cytoplasmic enzyme found to be particularly active in the coleoptile epidermis and hypodermis, leaf mesophyll, as well as in developing fibres and phloem. Cyclic nucleotide phosphodiesterase is also a cytoplasmic enzyme active in the shoot vascular bundles, particularly the xylem, and in the seed. Within the seed it is highly active in the crushed cell layer adjacent to the scutellum and in endosperm cells adjacent to the aleurone layer. Within the embryo, cyclic nucleotide phosphodiesterase is most active in epithelial cells adjacent to the crushed cell layer, the suspensor, radicle and root-cap, as well as in the pro-vascular tissues of the scutellum.     

d-Glyceraldehyde 3-Phosphate Dehydrogenases of Higher Plants

The d-glyceraldehyde 3-P dehydrogenases of spinach leaf, pea seed, and pea shoot were purified. The NADP and NAD-linked enzymes of either spinach leaves and pea shoots could not be separated. Changes in the ratio of NADP- to NAD-linked activity of the spinach leaf and pea shoot enzymes were observed during both purification and storage of crude extracts. The spinach leaf, pea shoot, and pea seed enzymes differ electrophoretically from each other and from the rabbit muscle enzyme.The pea seed and shoot enzymes contain bound nucleotide cofactor, resist proteolytic attack, have similar Michaelis-Menton kinetic constants and are competitively inhibited by d-sedoheptulose-7-phosphate and d-sedoheptulose 1,7-diphosphate. Charcoal removes the bound nucleotide from the pea seed enzyme but not from the pea shoot enzymes. NADP and NADPH were found to inhibit the reductive but not oxidative reaction catalyzed by the charcoal treated seed enzyme. The function of the pea shoot NADP and NAD-linked enzymes in chloroplast metabolism is discussed in regard to their location and catalytic properties. Although the NADP-linked activity can be assigned a primary, if not exclusive function in photosynthesis, the assignment of a distinct metabolic function to the NAD-linked activity cannot be made at present.     

ISOLATION OF RAT LIVER PLASMA MEMBRANES : Use of Nucleotide Pyrophosphatase and Phosphodiesterase I as Marker Enzymes

Nucleotide pyrophosphatase and phosphodiesterase I of rat liver have been found to be localized primarily in cell particulates highly enriched with respect to the most commonly accepted plasma membrane marker, 5'-nucleotidase, and therefore should themselves be assigned a plasma membrane localization. The observation that plasma membranes sediment in isotonic sucrose with both nuclear and microsomal fractions was exploited to obtain plasma membrane preparations from each fraction. Both preparations are similar in chemical and enzymic composition. Moreover, the preparative method developed in this study appears to give the best combination of yield, purity, and reproducibility available. The question of the possible identity of nucleotide pyrophosphatase and phosphodiesterase I is considered, and evidence is presented suggesting that these activities may be manifestations of the same enzyme.     

A hypothesis concerning the structure of cAMP-and cGMP-dependent protein kinases.

cAMP-and cGMP-dependent protein kinases have been purified. Each enzyme demonstrates high specificity and affinity for the cyclic nucleotide with binding of two moles of nucleotide per holoenzyme and each enzyme is an ATP: phosphotransferase. The holoenzymes have similar molecular weights and demonstrate similar molecular asymmetry. A structural model relating the two enzymes is proposed. cGMP-dependent protein kinase is proposed to be a dimer composed of two identical protomers in isologous association with the chains arranged in anti-parallel fashion. cAMP-dependent protein kinase is proposed to have a similar structure with a dyad axis of symmetry but with a discontinuity in each chain. These structures account for the differing mechanisms of cyclic nucleotide activation of the two enzymes.