A novel tumor-associated molecular species of 5'-nucleotide phosphodiesterase

5'-Nucleotide phosphodiesterase (5'-NPDase) was partially purified from plasma membranes of murine lymphoma L5178Y. The enzyme was inactivated by N-ethylmaleimide and Zn2+, but stabilized by dithiothreitol, suggesting that it is an SH enzyme. The enzyme, Km 1.54 mM, pI 5.8 and MW 23k, differs from liver 5'-NPDase in MW, Km and sensitivity to some inhibitors. On the contrary, 5'-NPDase, derived from normal mouse organs, is similar to the liver enzyme. The results suggest that tumor cells possess a novel molecular species of 5'-NPDase.     

Organ-specific distribution of isozymes of 5'-nucleotide phosphodiesterase in mouse

1. The distribution of isozymes of 5'-nucleotide phosphodiesterase (E.C.3.1.4.1) was examined in various organs of mouse, including liver, spleen, pancreas, heart, lung, kidney, brain and blood. 2. Five isozymes were identified and designated as isozymes I through V. 3. These isozymes are distributed unevenly with respect to the various organs and clear differences were observed in the patterns of distribution among the organs examined. 4. The level of these isozymes was compared in serum of neonate and adult mice, and a higher level of isozyme I and a lower level of isozyme IV were found in neonates compared to adults. 5. These results suggest that each isozyme has different functional roles in individual organs and that these isozymes may be involved in proliferation and development of cells.     

Hydrolysis of phosphonate esters catalyzed by 5'-nucleotide phosphodiesterase.

4-Nitrophenyl and 2-napthyl monoesters of phenylphosphonic acid have been synthesized, and an enzyme catalyzing their hydrolysis was resolved from alkaline phosphatase of a commerical calf intestinal alkaline phosphatase preparation by extensive ion-exchange chromatography, chromatography on L-phenylalanyl-Sepharose with a decreasing gradient of (NH4) 2SO4, and gel filtration. Detergent-solubilized enzyme from fresh bovine intestine was purified after (NH4)2SO4 fractionation by the same technique. The purified enzyme is homogeneous by polyacrylamide gel electrophoresis and sedimentation equilibrium centrifugation. It has a molecular weight of 108,000, contains approximately 21% carbohydrate, and has an amino acid composition considerably different from that reported from alkaline phosphatase from the same tissue. The homogeneous intestinal enzyme, an efficient catalyst of phosphonate ester hydoolysis but not of phosphate monoester hydrolysis, was identified as a 5'-nucleotide phosphodiesterase by its ability to hydrolyze 4-nitrophenyl esters of 5'-TMP but not of 3'-TMP. Also consistent with this identification was the ability of the enzyme to hydrolyze 5'-ATP to 5'-AMP and PPi, NAD+ to 5'-AMP and NMN, TpT to 5'-TMP and thymidine, pApApApA to 5'-AMP, and only the single-stranded portion of tRNA from the 3'-OH end. Snake venom 5'-nucleotide phosphodiesterase also hydrolyzes phosphonate esters, but 3'-nucleotide phosphodiesterase of spleen and cyclic 3',5'-AMP phosphodiesterase do not. Thus, types of phosphodiesterases can be conveniently distinguished by their ability to hydrolyze phosphonate esters. As substrates for 5'-nucleotide phosphodiesterases, phosphonate esters are preferable to the more conventional esters of nucleotides and bis(4-nitrophenyl) phosphate because of their superior stability and ease of synthesis. Furthermore, the rate of hydrolysis of phosphonate esters under saturating conditions is greater than that of the conventional substrates. At substrate concentrations of 1 mM the rates of hydrolysis of phosphonate esters and of nucleotide esters are comparable and both superior to that of bis(4-nitrophenyl) phosphate.     

On the mechanism of action of bovine intestinal mucosa 5'-nucleotide phosphodiesterase. Stereochemical evidence for a nucleotidyl-enzyme intermediate

The diastereoisomers of adenosine 5'-O-phosphorothioate O-methyl ester have been synthesised. Only the Sp diastereoisomer is a substrate for the 5'-nucleotide phosphodiesterase from bovine intestinal mucosa. The previously unidentified enantiomer of 4-nitrophenyl phenyl phosphonothioate hydrolysed by the enzyme is shown to have the Sp configuration. Digestion of the Sp diastereoisomer of adenosine 5'-O-phosphorothioate O-methyl ester by the enzyme in 18O-labelled water gave 18O-labelled adenosine 5'-O-phosphorothioate which was stereochemically analysed by methylation and subsequent 31P-NMR spectroscopy and shown to possess the Sp configuration. Thus the enzyme-catalysed cleavage proceeded with retention of configuration at phosphorus, presumably via a double-displacement mechanism. This provides strong evidence for the existence of a nucleotidyl-enzyme intermediate on the reaction pathway.     

Phosphorylated threonine as the covalent intermediate in snake venom phosphodiesterase

The covalent intermediate of snake venom phosphodiesterase has been isolated using thymidine 5'-[alpha-32P]triphosphate as substrate. Phosphoamino acid analysis of the labeled enzyme demonstrates that threonine is the active site residue forming the covalent intermediate. 5'-Nucleotide phosphodiesterase is the first enzyme reported to have an active site threonine forming a covalent intermediate.     

Alkaline phosphatase and 5'-nucleotide phosphodiesterase from bovine intestine are cross-reactive

Polyclonal antibodies to native alkaline phosphatase and to native 5'-nucleotide phosphodiesterase were found to strongly cross-react with both enzymes. The antibodies also cross-react with both denatured enzymes, with glycopeptides from 5'-nucleotide phosphodiesterase, and with the oligosaccharides remaining after Pronase E digestion of the phosphodiesterase. They do not cross-react with either enzyme after their oligosaccharides have been modified or removed by periodate or trifluoromethanesulfonic acid treatment. Antibodies to denatured 5'-nucleotide phosphodiesterase do not bind to the native phosphodiesterase or alkaline phosphatase but do cross-react with denatured alkaline phosphatase even after removal or modification of the carbohydrate moieties. These results suggest that antibodies to denatured 5'-nucleotide phosphodiesterase may recognize amino acid sequence homology between alkaline phosphatase and 5'-nucleotide phosphodiesterase. However, antibodies to native enzymes apparently recognize cross-reactive determinants of the native enzymes which are carbohydrate in nature. This is the first report of antimammalian alkaline phosphatase antibodies which recognize the carbohydrate moieties of the enzyme.     

Properties of a cyclic 3'5'-nucleotide phosphodiesterase from Vigna mungo

Cyclic AMP phosphodiesterase (PDE) partially purified from roots of Vigna mungo exhibited optimum activity at pH 5.5 to 6.0 and maximum enzyme activity at 50 degrees C. Levels of PDE activity in roots remained relatively constant from the first to the eleventh day after germination; on the twelfth day there was a 400% increase in PDE activity. The enzyme was stable for at least 48 hours at 28 degrees C, retaining 92% of its original activity. Plant growth hormones including gibberellic acid, indoleacetic acid and kinetin at 1.0 and 10.0 microM concentrations did not have any significant effect on enzyme activity. Nucleotides tested including cyclic 2'3' AMP, cyclic 2'3' GMP completely abolished enzyme activity at 1.0mM while cyclic 3'5' GMP, cyclic 3'5' GMP, 2'deoxy 5' ATP, 2'deoxy 5'GTP and 5'ADP were also inhibitory to the enzyme. The enzyme was stimulated by Mg2+, Fe2+ and NH4+ while Cu2+ and Fe3+ were inhibitory. Theophylline, caffeine, phosphate, pyrophosphate and EDTA were inhibitory to the enzyme.     

Particulate cyclic 3',5'-nucleotide phosphodiesterase and calmodulin of cardiac muscle

Cyclic AMP and cyclic GMP phosphodiesterase and calmodulin were measured in purified subcellular fractions of cardiac muscle. Phosphodiesterase activity solubilized by sonication of the nuclear fraction yielded a major 6.6 S form which was calcium-sensitive and cyclic GMP-specific. Phosphodiesterase activity occurring in the nuclear fraction could be further enriched by subfractionation on sucrose density gradients in the presence of MgCl2.     

Hydrolysis of phosphoric acid diesters by snake venom phosphodiesterase and 5'-nucleotide diesters of sinapis alba germs via and covalently enzyme-bound intermediate

Snake venom phosphodiesterase from $\text{Crotalus}\text{durissus}$$\text{terrificus}$ and 5′-nucleotide phosphodiesterase from $\text{Sinapis}\text{alba}$ were incubated with the 1-naphthyl ester of 5′-[methyl-³H]thymidylic acid. After short-time incubation the enzymes were denatured by extraction into phenol and chromatographed on Sephadex G-25. Protein fractions containing radioactivity were collected, dialysed and subjected to ultra-thin-layer isoelectric focussing and autoradiography. The results obtained indicate a hydrolytic course via a covalently-bound intermediate.     

Fluorometric assay of serum 5'-nucleotide phosphodiesterase in normal humans and cancer patients

A sensitive method of assaying serum 5′-nucleotide phosphodiesterase (PDase) is described. A preliminary study did not find significant differences between the levels of PDase in cancer patients and those of normal human sera.     

The reaction of alkylating agents with cyclic 3',5'-nucleotide phosphodiesterase

The reaction of anti-tumour alkylating agents with beef heart cyclic nucleotide phosphodiesterase (adenosine 3',5'-monophosphate phosphohydrolase, EC 3.1.4.c) has been investigated. This enzyme exists in two forms differing in their Michaelis-Menten K_m values. Chlorambucil [p-(di-2-chloroethylamino)-phenyl-butyric acid] inhibits the form of the enzyme with a low K_m value with a velocity constant for inactivation three times that for inhibition of the high K_m form. While the monofunctional N-ethyl analogue of chlorambucil is ineffective as an inhibitor of either form of the enzyme, iodoacetate inhibits both forms, though the velocity constant for inactivation of each form is much less than that for chlorambucil. Also the rate of inactivation of each form does not significantly differ. A cross-linking mechanism for the inactivation of regulatory enzymes is proposed.     

A heat-stable protein inhibitor of cyclic 3',5'-nucleotide phosphodiesterase.

A heat-stable, non-dialyzable inhibitory factor of cyclic nucleotide phosphodieterase was detected in and partially purified from bovine retina. The factor appears to be a protein, since the inhibitory activity was abolished by trypsin digestion but not by DNAase or RNAase treatment. The protein inhibitor from bovine retina effectively inhibits the Ca2+-independent phosphodiesterase from several sources, including bovine retina, bovine rod outer segment, and a human lymphoblastic leukemia cell line, indicating lack of tissue and species specificity.