Induction of an extracellular cyclic nucleotide phosphodiesterase as an accessory ribonucleolytic activity during phosphate starvation of cultured tomato cells

During growth under conditions of phosphate limitation, suspension-cultured cells of tomato (Lycopersicon esculentum Mill.) secrete phosphodiesterase activity in a similar fashion to phosphate starvation-inducible ribonuclease (RNase LE), a cyclizing endoribonuclease that generates 2':3'-cyclic nucleoside monophosphates (NMP) as its major monomeric products (T. Nürnberger, S. Abel, W. Jost, K. Glund [1990] Plant Physiol 92: 970-976). Tomato extracellular phosphodiesterase was purified to homogeneity from the spent culture medium of phosphate-starved cells and was characterized as a cyclic nucleotide phosphodiesterase. The purified enzyme has a molecular mass of 70 kD, a pH optimum of 6.2, and an isoelectric point of 8.1. The phosphodiesterase preparation is free of any detectable deoxyribonuclease, ribonuclease, and nucleotidase activity. Tomato extracellular phosphodiesterase is insensitive to EDTA and hydrolyzes with no apparent base specificity 2':3'-cyclic NMP to 3'-NMP and the 3':5'-cyclic isomers to a mixture of 3'-NMP and 5'-NMP. Specific activities of the enzyme are 2-fold higher for 2':3'-cyclic NMP than for 3':5'-cyclic isomers. Analysis of monomeric products of sequential RNA hydrolysis with purified RNase LE, purified extracellular phosphodiesterase, and cleared -Pi culture medium as a source of 3'-nucleotidase activity indicates that cyclic nucleotide phosphodiesterase functions as an accessory ribonucleolytic activity that effectively hydrolyzes primary products of RNase LE to substrates for phosphate-starvation-inducible phosphomonoesterases. Biosynthetical labeling of cyclic nucleotide phopshodiesterase upon phosphate starvation suggests de novo synthesis and secretion of a set of nucleolytic enzymes for scavenging phosphate from extracellular RNA substrates.     

Purification of an enzyme aggregate containing 3',5'-cyclic-nucleotide phosphodiesterase and nucleotidase

Several steps of purification (octyl-Sepharose chromatography, Blue Sepharose 6B chromatography and sucrose density gradient centrifugation) led to a highly purified aggregate of the enzymes, 3',5'-cyclic-nucleotide phosphodiesterase (PDE) and nucleotidase. The purified enzyme aggregate showed an S value of 7.3 (SE +/- 0.3, n = 10). Further analysis by SDS-polyacrylamide gel electrophoresis (PAGE) revealed two proteins near 67 and 60 kDa. Dissociation of the 7.3 S enzyme aggregate showed a 3.6 S PDE form and a nucleotidase form at 4.2 S. Additionally, higher S value forms of the nucleotidase up to 17 S have been observed. Apparently, they had formed by self-association. SDS-PAGE of the 17 S nucleotidase form showed only one band at 67 kDa. This was taken as evidence for the homogeneity of the 17 S nucleotidase form and the self-association of the nucleotidase after dissociation from the 7.3 S enzyme aggregate. Furthermore, from this it could be concluded that the 67 kDa protein of the 7.3 S enzyme aggregate should be identified with the nucleotidase, and thus the 60 kDa band represents the PDE.     

A new histo- and cytochemical method for demonstration of cyclic 3',5'-nucleotide phosphodiesterase activity in retinal rod photoreceptor cells of the rat

Cyclic 3',5'-mononucleotide phosphodiesterase (cyclic nucleotide PDEase) activity was studied histo- and cytochemically in the retinal rod photoreceptor cells of the rat by means of a newly developed technique utilizing the intrinsic 5' nucleotidase activity instead of an exogenous 5' nucleotidase source (snake venom). Cyclic GMP and was used as a substrate, the intense activity of phosphodiesterase (PDEase) was distributed over the entire rod outer segments; reaction product was observed on the plasmalemma and on the disk membranes of the outer segments. A slight reaction was also observed on the plasmalemma of the inner segments. However, no precipitate was found in the perinuclear and synaptic regions of the rod photoreceptors. In contrast, when cyclic AMP was utilized as a substrate, a moderate reaction was seen in the synaptic region of the plexiform layer. The intensity of the reaction in the outer segments was much reduced in comparison to the results with cyclic GMP. The enzyme activity was almost completely inhibited by 2 mM 3-isobutyl-1-methylxanthine (IBMX) or 2 mM theophylline, which were potent inhibitors of PDEase. To confirm the propriety of our new cytochemical method, the localization of 5' nucleotidase was also studied utilizing 5' AMP or 5' GMP as substrates. In contrast to the activity of cyclic nucleotide PDEase, the activity of 5' nucleotidase was distributed on all membranes of the photoreceptors from the synaptic outer plexiform layer to the tip of outer segments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Membrane-bound nucleotidase of Bacillus cereus.

A membrane-bound nucleotidase of Bacillus cereus T was solubilized by digestion with trypsin and subsequently purified more than 300-fold. The purified nucleotidase was most active on ribonucleoside 5'-monophosphates and was slightly less active (40 to 60%) on deoxyribonucleoside 5'-monophosphates and ribonucleoside 3'-monophosphates. In addition to hydrolytic activity, the nucleotidase preparation possessed phosphotransferase activity by which phosphate is transferred from a phosphate donor to the 5' position of nucleosides.     

The class C acid phosphatase of Helicobacter pylori is a 5' nucleotidase

The results from purification and characterization studies of the hppA gene product of Helicobacter pylori confirm its identification as a class C acid phosphatase. The hppA gene of H. pylori ATCC strain 49503 was amplified and modified by PCR, cloned into pET21b, and overexpressed in Escherichia coli. The recombinant protein was liberated from membranes and purified (16x) to apparent homogeneity with cation exchange and Ni-chelate chromatography resulting in a recovery of 39% of total starting activity. The recombinant acid phosphatase exhibited a denatured molecular mass of 24 kDa by SDS-PAGE. Phosphatase activity in both crude and purified samples could be renatured and detected after SDS-PAGE. The native molecular mass of recombinant enzyme was approximately 72 kDa by gel filtration chromatography on Superdex 75. While phosphate and tartrate had little effect on phosphatase activity, molybdate, vanadate, and EDTA had significant inhibitory effects on enzymatic activity. Phosphomonoesterase activity for hydrolysis of p-nitrophenylphosphate (pNPP) as well as other substrates was enhanced in the presence of divalent cations including Cu(2+), Ni(2+), Co(2+), and Mg(2+). Recombinant HppA had narrow substrate specificity with highest activity for arylphosphates and significant activity for 5' nucleoside monophosphates. The pH optimum for enzyme activity was 4.6 and 5.2 for purine and pyrimidine 5' monophosphates, respectively. The affinity constants for the 5' nucleoside monophosphates were found to be 0.5-1 mM. Results from this study confirm HppA inclusion in the class C acid phosphatases and led to its identification as a 5' nucleotidase.     

2',3'-cyclic nucleotide 2'-phosphodiesterase from Fusarium culmorum

We describe the properties of a 2',3'-cyclic nucleotide 2'-phosphodiesterase (EC 3.1.4.16), found in Fusarium culmorum, which hydrolyzes nucleoside 2',3'-cyclic monophosphates to nucleoside 3'-phosphates. In contrast with a similar enzyme found in bacteria, the Fusarium enzyme does not exhibit nucleotidase activity and does not show a requirement for metal ions, but is inhibited by micromolar concentrations of Cu++ and Zn++, and is very stable to heat. This cyclic phosphodiesterase hydrolyzes the four major nucleoside 2',3'-cyclic monophosphates and has greater affinity for purine (Kms for Ado-2',3'-P = 0.3 mM and for Guo-2',3'-P = 0.1 mM) than for pyrimidine nucleotides (Kms for Cyd-2',3'-P = 0.6 mM and for Urd-2',3'-P = 2 mM). The respective Vmax for Urd-2',3'-P; Cyd-2',3'-P; Ado-2',3'-P; and Guo-2',3' are 100:45:16:5. The efficacy of the phosphodiesterase to hydrolyze the four major 2',3' cyclic nucleotides (based on the relative values of Vmax/Km) is not significantly different. The Fusarium enzyme differs from a previously described 2',3' cyclic phosphodiesterase from Neurospora, in that it is inactive on 3',5'-nucleoside monophosphates and nucleoside 2' or 3' phosphates.     

3',5'-Cyclic nucleotide phosphodiesterase activity of the sulphatase A of ox liver

The sulphatase A (aryl-sulphate sulphohydrolase, EC 3.1.6.1) of ox liver hydrolyses adenosine 3',5'-monophosphate (cyclic AMP) to adenosine 5'-phosphate at an optimum pH of approx. 4.3, close that for the hydrolysis of cerebroside sulphate, a physiological substrate for sulphatase A. The Km is 11.6 mM for cyclic AMP. On polyacrylamide gel electrophoresis sulphatase A migrates as a single protein band which coincides with both the arylsulphatase and phosphodiesterase activities, suggesting that these are due to a single protein. Cyclic AMP competitively inhibits the arylsulphatase activity of sulphatase A, showing that both activities are associated with a single active site on the enzyme. sulphatase A also hydrolyses guanosine 3',5'-monophosphate, but not uridine 3',5'-monophosphate nor adenosine 2',3'-monophosphate.     

Properties of cyclic nucleotide phosphodiesterase of the rabbit myometrium in various functional states

Chromatography on DEAE-cellulose of a soluble sulfate-precipitated fraction of cyclic nucleotide phosphodiesterase from rabbit myometrium revealed two 3':5'-GMP and 3':5'-AMP-hydrolase activities. 3':5'-GMP phosphodiesterase (fraction I) was eluted with 0.15-0.23 M NaCl, while 3':5'-AMP phosphodiesterase (fraction II) with 0.2-0.35 M NaCl. 3':5'-GMP phosphodiesterase hydrolyzed 3':5'-GMP with Km = 14 microM and V = 5.25 nmol . min . mg of protein, while 3':5'-AMP phosphodiesterase hydrolyzed both cyclic nucleotides with Km for 3':5'-GMP equal to 12 microM and V = 1.33 nmol . min . mg of protein; the Km value for 3':5'-AMP was 3.6 and 30.5 microM, respectively; the corresponding values of V were 0.28 and 0.97 nmol . min . mg of protein. In late pregnancy, the level of the 3':5'-AMP hydrolase activity of rabbit myometrium was significantly elevated in parallel with an increase in V, predominantly for the enzyme with a low affinity for 3':5'-AMP. The 3':5'-GMP hydrolase activity and V were largely decreased for both phosphodiesterase fractions; the Km value for fraction I was also diminished. During labour, the rate of 3':5'-AMP hydrolysis by myometrium phosphodiesterase was decreased down to the level typical of functional rest. The rate of 3':5'-GMP hydrolysis during the same period by fraction I remained at a low level, i. e., as in pregnancy, while that of fraction II was increased up to the level typical of functional rest.(ABSTRACT TRUNCATED AT 250 WORDS)     

(2')3',5'-Bisphosphate nucleotidase

(2')3',5'-Bisphosphate nucleotidase has been prepared in electrophoretically homogeneous form from guinea pig liver. The enzyme catalyzes the hydrolysis of the 2'- or 3'-phosphate from the appropriate nucleoside 2',5'- and 3',5'-bisphosphates and is active with 3'-phosphoadenosine 5'-phosphosulfate and with coenzyme A but not with ATP. The 40,000-dalton protein is a monomer that requires Mg2+ for activity.     

The sequential 2',3'-cyclic phosphodiesterase and 3'-phosphate/5'-OH ligation steps of the RtcB RNA splicing pathway are GTP-dependent

The RNA ligase RtcB splices broken RNAs with 5'-OH and either 2',3'-cyclic phosphate or 3'-phosphate ends. The 3'-phosphate ligase activity requires GTP and entails the formation of covalent RtcB-(histidinyl)-GMP and polynucleotide-(3')pp(5')G intermediates. There are currently two models for how RtcB executes the strand sealing step. Scheme 1 holds that the RNA 5'-OH end attacks the 3'-phosphorus of the N(3')pp(5')G end to form a 3',5'-phosphodiester and release GMP. Scheme 2 posits that the N(3')pp(5')G end is converted to a 2',3'-cyclic phosphodiester, which is then attacked directly by the 5'-OH RNA end to form a 3',5'-phosphodiester. Here we show that the sealing of a 2',3'-cyclic phosphate end by RtcB requires GTP, is contingent on formation of the RtcB-GMP adduct, and involves a kinetically valid RNA(3')pp(5')G intermediate. Moreover, we find that RtcB catalyzes the hydrolysis of a 2',3'-cyclic phosphate to a 3'-phosphate at a rate that is at least as fast as the rate of ligation. These results weigh in favor of scheme 1. The cyclic phosphodiesterase activity of RtcB depends on GTP and the formation of the RtcB-GMP adduct, signifying that RtcB guanylylation precedes the cyclic phosphodiesterase and 3'-phosphate ligase steps of the RNA splicing pathway.     

A nucleotide phosphodiesterase with preference for 2',5'-phosphodiester bonds from Ehrlich ascites carcinoma

We have previously reported that many tumor cell lines express a 5'-nucleotide phosphodiesterase (phosphodiesterase I, EC 3.1.4.1) with properties clearly distinguishable from enzymes of normal tissues (Biochim. Biophys. Acta (1988) 966, 99-106). Such an enzyme with 5'-nucleotide phosphodiesterase activity was purified from Ehrlich ascites carcinoma by measuring the cleavage of thymidine 5'-monophosphate p-nitrophenyl ester (TMP-NP). The enzyme is a soluble protein, has a pH optimum of 7.5, and the molecular mass estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 67 kDa. The enzyme does not hydrolyze other chromogenic substrates for phosphodiesterases, nor pyrophosphate bond of various nucleotides which are cleaved by 5'-nucleotide phosphodiesterases of normal tissues. But, it hydrolyzes dinucleotides to form 5'-phosphates, and is more active on 2',5'- than on 3',5'-phosphodiester bonds. These results indicate that the TMP-NP splitting enzyme in Ehrlich ascites carcinoma cells is a 2',5'-phosphodiesterase.     

Purification and characterization of cytoplasmic 5'(3')-nucleotidase from rabbit spleen: characteristic differences of the enzyme from the rat spleen nucleotidase.

1. A 5'(3')-nucleotidase, which preferably hydrolyzed 3'-dTMP and 3'-dUMP, was highly purified from rabbit spleen cytosol. 2. The enzyme also hydrolyzes 3'-UMP, 5'-dUMP and guanine nucleotides, but does not hydrolyze any adenine nucleotides or cytosine nucleotides. 3. The activity is dependent upon Mg2+, Co2+ or Mn2+; the addition of deoxyinosine stimulates the activity, and the pH optimum for the hydrolysis of 3'-dTMP is 7.0. 4. Although the catalytic properties of the enzyme are similar to the 5'(3')-nucleotidase from rat spleen, these nucleotidases differ in their molecular disposition. 5. The charge state of the rabbit enzyme is slightly basic, and the subunit M(r) is about 27 kDa, while the value of the rat enzyme is 26 kDa. 6. Immunochemical experiments with the use of antibodies against the purified nucleotidase indicate that enzymes from rabbit spleen and from rat spleen are composed of different polypeptides.     

Differences and similarities between guanosine 3',5'-cyclic monophosphate phosphodiesterase and adenosine 3',5'-cyclic monophosphate phosphodiesterase activities in neuroblastoma cells in culture.

There are phosphodiesterase activities in both particulate and supernatant fractions which hydrolyze guanosine 3',5'-cyclic monophosphate (cGMP) and adenosine 3',5'-cyclic monophosphate (cAMP) with an apparent Km of 2-8 muM and with an apparent Km of 44-222 muM. 4-(3-Butoxy-4-methoxybenzyl-2-imidazolidinone (RO20-1724) did not inhibit cGMP phosphodiesterase activity in homogenates of mouse neuroblastoma cells, but markedly inhibited cAMP phosphodiesterase activity. Papaverine and theophylline inhibited both cGMP and cAMP phosphodiesterase activities to about the same extent. The former was more potent than the latter. The specific activity of cGMP phosphodiesterase as a function of protein concentrations first increased and then decreased. The specific activity of cAMP phosphodiesterase decreased under a similar experimental condition.     

Homogeneous 3'----5'-exonucleases and their multienzyme complexes from the rat liver

Isolation and general properties of 3'-5' exonucleases I and II (EC 3.1.4.26), which are specific to single-stranded DNA, are described. Such enzymes, being components of replication complexes, could correct replication errors. Homogeneous exonucleases I and II consist of a single subunit with molecular mass of 50 and 40 kDa, respectively. These enzymes are located preferentially in the nuclear membrane and chromatin. They form complexes with nuclear DNA polymerases and some other proteins and are not observed practically in a free state. Molecular masses of the complexes amount from 70 to 1.500 kDa. The complexes dissociate as a result of solution hydrophobization and can be reconstituted after the decrease of hydrophobization. The heavy membrane complex form of 3'----5' exonuclease I manifests enzymatic activities of DNA polymerase alpha (EC 2.7.7.7), non-specific nucleoside triphosphatase (EC 3.1.3.2), nucleotidase (EC 3.1.3.31) and faint activity of endonuclease (EC 3.1.4.5). Complexes under study do not display activity of thymidine kinase (EC 2.7.1.21), marker protein of replitase, neither in G0 nor in S-period.     

Genome-wide transcriptional analysis of the phosphate starvation stimulon of Bacillus subtilis

Bacillus subtilis responds to phosphate starvation stress by inducing the PhoP and SigB regulons. While the PhoP regulon provides a specific response to phosphate starvation stress, maximizing the acquisition of phosphate (P(i)) from the environment and reducing the cellular requirement for this essential nutrient, the SigB regulon provides nonspecific resistance to stress by protecting essential cellular components, such as DNA and membranes. We have characterized the phosphate starvation stress response of B. subtilis at a genome-wide level using DNA macroarrays. A combination of outlier and cluster analyses identified putative new members of the PhoP regulon, namely, yfkN (2',3' cyclic nucleotide 2'-phosphodiesterase), yurI (RNase), yjdB (unknown), and vpr (extracellular serine protease). YurI is thought to be responsible for the nonspecific degradation of RNA, while the activity of YfkN on various nucleotide phosphates suggests that it could act on substrates liberated by YurI, which produces 3' or 5' phosphoribonucleotides. The putative new PhoP regulon members are either known or predicted to be secreted and are likely to be important for the recovery of inorganic phosphate from a variety of organic sources of phosphate in the environment.     

Nucleotidase and DNase activities in Brazilian snake venoms

Among the myriad of enzymes present in animal venoms, nucleotidases and nucleases are poorly investigated. Herein, we studied such enzymes in 28 crude venoms of animals found in Brazil. Higher levels of ATPase, 5'-nucleotidase, ADPase, phosphodiesterase and DNase activities were observed in snake venoms belonging to Bothrops, Crotalus and Lachesis genera than to Micrurus genus. The venom of Bothrops brazili snake showed the highest nucleotidase and DNase activities, whereas that of Micrurus frontalis snake the highest alkaline phosphatase activity. On the other hand, the venoms of the snake Philodryas olfersii and the spider Loxosceles gaucho were devoid of most nucleotidase and DNase activities. Species that exhibited similar nucleotidase activities by colorimetric assays showed different banding pattern by zymography, suggesting the occurrence of structural differences among them. Hydrolysis of nucleotides showed that 1 mol of ATP is cleaved in 1 mol of pyrophosphate and 1 mol of orthophosphate, whereas 1 mol of ADP is cleaved exclusively in 2 mol of orthophosphates. Pyrophosphate is barely hydrolyzed by snake venoms. Phosphodiesterase activity was better correlated with 5'-nucleotidase, ADPase and ATPase activities than with DNase activity, evidencing that phosphodiesterases are not the main agent of DNA hydrolysis in animal venoms. The omnipresence of nucleotidase and DNase activities in viperid venoms implies a role for them within the repertoire of enzymes involved in immobilization and death of preys.     

Expression of a Brassic napus glutamate 1-semialdehyde aminotransferase in Escherichia coli and characterization of the recombinant protein

Glutamate 1-semialdehyde aminotransferase (GSA-AT) is a key regulatory enzyme, which converts glutamate 1-semialdehyde (GSA) to 5-aminolevulinic acid (ALA) in chlorophyll biosynthesis. ALA is the universal precursor for the synthesis of chlorophyll, heme, and other tetrapyrroles. To study the regulation of chlorophyll biosynthesis in Brassica napus, two cDNA clones of GSA-AT were isolated for genetic manipulation. A SalI-XbaI fragment from one of the two cDNA clones of GSA-AT was used for recombinant protein expression by inserting it at the 3' end of a calmodulin-binding-peptide (CBP) tag of the pCaln vector. The CBP tagged recombinant protein, expressed in Escherichia coli, was purified to apparent homogeneity in a one step purification process using a calmodulin affinity column. The purified CBP tagged GSA-AT is biologically active and has a specific activity of 16.6 nmol/min/mg. Cleavage of the CBP tag from the recombinant protein with thrombin resulted in 9.2% loss of specific activity. However, removal of the cleaved CBP tag from the recombinant protein solution resulted in 60% loss of specific activity, suggesting possible interactions between the recombinant protein and the CBP tag. The enzyme activity of the CBP tagless recombinant protein, referred as TR-GSA-AT hereafter, was not affected by the addition of pyridoxamine 5' phosphate (PMP). Addition of glutamate and pyridoxal 5' phosphate (PLP) to the TR-GSA-AT enhanced the enzyme activity by 3-fold and 3.6-fold, respectively. Addition of both glutamate and PLP increased the enzyme activity by 4.6-fold. Similar to the GSA-AT of B. napus, the active TR-GSA-AT is a dimeric protein of 88 kDa with 45.5 kDa subunits. As the SalI-XbaI fragment encodes a biologically active GSA-AT that has the same molecular mass as the native GSA-AT, it is concluded that the SalI-XbaI fragment is the coding sequence of GSA-AT. The highly active polyclonal antibodies generated from TR-GSA-AT were used for the detection of GSA-AT of B. napus.     

Purification and properties of a ribonuclease in human urine that hydrolyses polycytidylic acid.

Human urine RNase was purified about 2000-fold. The preparation is free from phosphatase, phosphodiesterase and DNase activities. On electrophoresis through polyacrylamide gel at pH 8.3, it migrates toward the anode and stains with periodic acid-Schiff reagent, suggesting that it is acidic and glycoprotein in nature. Its isoelectric point is at pH 4.1. It has a molecular weight of about 21,500. It is thermostable at pH 4.2 and thermolabile at pH 8.5. It has a pH optimum at 6.5. It exhibits highest preference for cytidine 3'-phosphate linkages. Its activity on poly (C) is endonucleolytic. It cleaves poly (C) via intramolecular transphosphorylation. It has no action on cytidine 2': 3'-cyclic phosphate or uridine 2':3'-cyclic phosphate. Its rate of hydrolysis of poly (U) is less than 2% of that of poly C). Poly (A) and poly (G) are totally inert to its action. Its action on poly (C) is inhibited by poly (G), poly (A) and poly (U). It differs from bovine pancreatic Rnase A in its physical, chemical and catalytic properties. It is, however, similar to human serum and pancreatic RNase in all its properties, suggesting that pancreas is its likely source.     

Phosphodiesterase activity of CvfA is required for virulence in Staphylococcus aureus

We previously identified the cvfA gene (SA1129) as a novel virulence regulator in Staphylococcus aureus using the silkworm infection model. The cvfA gene, which is conserved among various pathogenic bacteria, contributes to the expression of the agr locus, a global virulence regulator that controls the expression of genes encoding various exoproteins, such as hemolysin. CvfA protein has a transmembrane domain, an RNA binding domain (KH domain), and a metal-dependent phosphohydrolase domain (HD domain). We report here the purification of recombinant CvfA protein from a membrane fraction of Escherichia coli by measuring its phosphodiesterase activity. Purified CvfA protein hydrolyzed the phosphodiester linkage of 2',3'-cyclic AMP, 2',3'-cyclic GMP, and 2',3'-cyclic phosphate at the 3'-terminal of RNA in the presence of Mn(2+). CvfA mutant proteins with amino acid substitutions in the HD domain had significantly decreased phosphodiesterase activity. Furthermore, mutated cvfA genes encoding proteins with low phosphodiesterase activity did not complement the decreased hemolysin production or the attenuated killing ability against silkworms in the cvfA deletion mutant. These results suggest that the phosphodiesterase activity of CvfA protein is required for virulence in S. aureus.