Alkaline inorganic pyrophosphatase activity of mammalian-cell alkaline phosphatase

Alkaline phosphatase prepared from mammalian cell cultures was found to have alkaline inorganic pyrophosphatase activity. Both of these activities appear to be associated with a single protein, as demonstrated by: (1) concomitant purification of alkaline phosphatase and alkaline inorganic pyrophosphatase; (2) proportional precipitation of alkaline phosphatase and inorganic pyrophosphatase activities by titrating constant amounts of an enzyme preparation with increasing concentration of antibody; (3) immune electrophoresis, which showed that precipitin bands that have alkaline phosphatase activity also have pyrophosphatase activity; (4) inhibition of pyrophosphatase activity by cysteine, an inhibitor of alkaline phosphatase activity; (5) similar subcellular localization of the two enzyme activities as demonstrated by histochemical methods; (6) hormonal and substrate induction of alkaline phosphatase activity in mammalian cell cultures, which produced a nearly parallel rise in inorganic pyrophosphatase activity.     

Studies on matrix vesicles isolated from chick epiphyseal cartilage. Association of pyrophosphatase and ATPase activities with alkaline phosphatase.

Fractions composed primarily of cells (Fraction I), membrane fragments (Fraction II) and matrix vesicles (Fraction III) were isolated from chick epiphyseal cartilage. The characteristics of the alkaline phosphatase (EC 3.1.3.1), pyrophosphatase (EC 3.6.1.1) and ATPase (EC 3.6.1.3) activities in the matrix vesicle fraction were studied in detail. Mg-2-+ was not absolutely essential to any of the activities, but at low levels was stimulatory in all cases. Higher concentrations inhibited both pyrophosphatase and ATPase activities. Both the stimulatory and inhibitory effects were pH-dependent. Ca-2-+ stimulated all activities weakly in the absence of Mg-2-+. However, when Mg-2-+ was present, Ca-2-+ was slightly inhibitory. Thus, none of the activities appear to have a requirement for Ca-2-+, and hence would not seem to be involved with active Ca-2-+ transport in the typical manner. The distribution of alkaline phosphatase, pyrophosphatase, and Mg-2-+ ATPase activities among the various cartilage fractions was identical, and concentrated primarily in the matrix vesicles. Conversely, the highest level of (Na-+ + K-+)-ATPase activity was found in the cell fraction. All activites showed nearly identical sensitivities to levamisole (4 - 10-3 M) which caused nearly complete inhibition of alkaline phosphatase and pyrophosphatase. About 10-15% of the ATPase activity was levamisole-insensitive. The data are consistent with the concept that the Mg-2-+-ATPase and pyrophosphatase activities of matrix vesicles stem from one enzyme, namely, alkaline phosphatase.     

Methylated and blocked 5' termini in vesicular stomatitis virus in vivo mRNAs.

Methyl groups derived from 3H-methyl methionine were incorporated into vesicular stomatitis virus (VSV) MRNAs isolated from infected cells. Sequential degradation of the 12-18S viral mRNA species with ribonuclease T2, penicillium nuclease, and alkaline phosphatase yielded a single 3H-labeled dinucleotide. A similar resistant 32P-labeled fragment was obtained by digesting VSV mRNA uniformly labeled with 32P. This methylated and blocked oligomer was further cleaved with nucleotide pyrophosphatase, yielding two methylated 5' nucleotides. We postulate that the 5' terminal structure of the vivo 12-18S VSV mRNA contains 7-methylguanosine linked by a 5'-5' pyrophosphate bond to a methylated derivative of adenosine. In contrast to the mRNAs (+ strand), the VSV genome RNA ( MINUS STRAND) IS NOT BLOCKED.     

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.     

Biosynthesis of a novel bile acid nucleotide and mechanism of 7 alpha-dehydroxylation by an intestinal Eubacterium species

Eubacterium species V.P.I. 12708 has inducible bile acid 7-dehydroxylase activity that can use either 7 alpha or 7 beta bile acids as substrates. Cell extracts prepared from bacteria grown in the presence of cholic acid catalyzed the rapid conversion of free bile acids into a highly polar bile acid metabolite (HPBA). This conjugation activity co-eluted with bile acid 7-dehydroxylase activity on high performance gel filtration chromatography (GFC). The HPBA was purified by a combination of high performance GFC and reverse-phase high performance liquid chromatography (HPLC). The intact HPBA eluted earlier from reverse-phase HPLC than deoxycholyl-CoA and had a Mr of 1102 by Bio-Gel P-2 (GFC). The HPBA had an absorption peak at 255 nm and was sensitive to treatment with phosphodiesterase I or nucleotide pyrophosphatase. The HPBA has a free phosphate as shown by an increase in elution volume on reverse-phase HPLC following treatment with alkaline phosphatase. Treatment of the purified HPBA with nucleotide pyrophosphate plus alkaline phosphatase yielded adenosine, whereas, treatment with nucleotide pyrophosphatase alone generated 5',3'-ADP. A bile acid metabolite was also generated by nucleotide pyrophosphatase treatment. The bile acid metabolite had different chromatographic properties (HPLC and TLC) than the corresponding free bile acid. Gas liquid chromatography-mass spectrometry showed the bile acid metabolite to be 12 alpha-hydroxy-3-oxo-4-cholenoic acid. We hypothesize that the HPBA is an intermediate in 7-dehydroxylation and consists of this compound linked at the C-24 with an anhydride bond to the beta phosphate (5') of ADP-3'-phosphate. These results suggest a novel mechanism of bile acid 7 alpha/7 beta-dehydroxylation in Eubacterium sp. V.P.I. 12708.     

Fluoride inhibition of inorganic pyrophosphatase. I. Kinetic studies in a Mg2+-PPi system using a new continuous enzyme assay.

Reversible inhibition of bakers' yeast inorganic pyrophosphatase (EC 3.6.1.1) by fluoride has been studied as a function of substrate, metal-ion activator and inhibitor concentrations and pH using a new continuous enzyme assay with an automatic phosphate analyzer. The inhibition was shown to be the result of tight binding of fluoride by two catalytically active enzyme-substrate complexes. The reaction between pyrophosphatase and fluoride is relatively slow, so that the rate constants for the binding and release of the inhibitor were derived from phosphate formation curves measured on the time scale of enzyme assays. The pH-dependence of the inhibition reaction in the alkaline medium indicates that both the fluoride-enzyme interaction and the catalytic step of the pyrophosphatase reaction are controlled by the same group on the protein. In the acidic medium, the inhibition is considerably enhanced, presumably because of the protonation of another enzyme group.     

Adenosine triphosphate hydrolysis in rat dental tissues. A histochemical study to differentiate the enzymes involved.

The purpose of this study was to try to differentiate histochemically between the various enzymes which may catalyze the hydrolysis of ATP in developing rat dental tissues. Freeze cut and freeze dried sections of molar and incisor teeth were incubated in lead capture-based media at pH 5.0, 7.2 or 9.4 with one of the following substrates: beta-glycerophosphate, AMP, ADP, ATP, AMP-PNP and tetrasodium pyrophosphate. To establish the enzymatic nature of the hydrolysis parallel sections were incubated after prior fixation in either formaldehyde or glutaraldehyde. By comparing the enzymatic stainings obtained with the various substrates and at the different pH:s, it was concluded that ATP can be visibly hydrolyzed in rat dental tissues by alkaline phosphatase (stratum intermedium, apical part of maturation ameloblasts, basal part of all ameloblasts, odontoblasts and subodontoblastic layer), specific ATPase (apical and basal parts of secretory ameloblasts) and ATP pyrophosphatase and/or adenylate cyclase (stratum intermedium, odontoblasts). Acid phosphatase, specific ADPase, 5'-nucleotidase, inorganic pyrophosphatase, 3':5'-cyclic-AMP-phosphodiesterase and adenylate kinase on the other hand, seem not to be engaged in the ATP hydrolysis to such a degree as to complicate the interpretation of the histochemical staining. The alkaline phosphatase part of the ATP hydrolysis appeared to be rather insensitive to aldehyde fixation, while the hydrolysis effected by specific ATPase and ATP pyrophosphatase and/or adenylate cyclase was extinguished after fixation with formaldehyde for 4 h or glutaraldehyde for 10 min.     

Molecular cloning and characterization of a soluble inorganic pyrophosphatase in potato.

A cDNA clone encoding a soluble inorganic pyrophosphatase (EC 3.6.1.1) of potato (Solanum tuberosum L.) was isolated by screening a developing tuber library with a heterologous probe. The central domain of the encoded polypeptide is nearly identical at the sequence level with its Arabidopsis homolog (J.J. Kieber and E.R. Signer [1991] Plant Mol Biol 16: 345-348). Computer-assisted analysis of the potato, Arabidopsis, and Escherichia coli soluble pyrophosphatases indicated a remarkably conserved organization of the hydrophobic protein domains. The enzymatic function of the potato protein could be deduced from the presence of amino acid residues highly conserved in soluble pyrophosphatases and was confirmed by its capacity to complement a thermosensitive pyrophosphatase mutation in E. coli. The potato polypeptide was purified from complemented bacterial cells and its pyrophosphatase activity was shown to be strictly dependent on Mg2+ and strongly inhibited by Ca2+. The subcellular location of the potato pyrophosphatase is unknown. Structure analysis of the N-terminal protein domain failed to recognize typical transit peptides and the calculated molecular mass of the polypeptide (24 kD) is significantly inferior to the values reported for the plastidic (alkaline) or mitochondrial pyrophosphatases in plants (28-42 kD). Two unlinked loci could be mapped by restriction fragment length polymorphism analysis in the potato genome using the full-length cDNA as probe.     

Studies on alkaline phosphatase. Phosphorylation of calf intestinal alkaline phosphatase by 32P-labelled pyrophosphate

1. A purified preparation of alkaline phosphatase from calf-intestinal mucosa was phosphorylated by (32)P-labelled PP(i) at a serine residue on the enzyme. Under the conditions employed, up to 0.15mum-labelled sites were obtained from 1mum-[(32)P]PP(i). 2. The phosphorylated enzyme was labile, the rate of dephosphorylation being similar to the overall rate of substrate hydrolysis. 3. A stopped-flow technique was used to determine the number of phosphomonoesterase active sites, which agreed with the number of (32)P-labelled sites. 4. It is concluded that calf-intestinal alkaline phosphatase is both a phosphomonoesterase and a pyrophosphatase.     

Zn2+-Dependent Acid Inorganic Pyrophosphatase Activity as Related to Other Pyrophosphatases in Oryza sativa

Acid inorganic pyrophosphatase on the one hand, and Mg²⁺-dependent alkaline inorganic pyrophosphatase and Zn²⁺-dependent acid inorganic pyrophosphatase on the other hand showed opposite trends in their activities in rice (Oryza sativa L. cv. Ratna) seedlings grown in dark and sun. The opposite trends in their activities were also noted in rice seedlings grown from gamma-irradiated seeds and in detached rice leaves floated on water in dark. The ratios of Mg²⁺ dependent alkaline inorganic pyrophosphatase/acid inorganic pyrophosphatase and Zn²⁺-dependent acid inorganic pyrophosphatase/acid inorganic pyrophosphatase changed significantly in response to the above physical treatments, but the ratio of Mg²⁺ dependent alkaline inorganic pyrophosphatase/Zn²⁺ dependent acid inorganic pyrophosphatase remained relatively stable. The conclusion is that Zn²⁺-dependent acid inorganic pyrophosphatase activity is the same as that of Mg²⁺-dependent alkaline inorganic pyrophosphatase and is different from that of acid inorganic pyrophosphatase, which requires no metal ion for activity. The acid and alkaline inorganic pyrophosphatase activities are due to separate enzyme proteins.     

Inhibition studies of alkaline phosphatase in hard tissue-forming cells.

The effects of the alkaline phosphatase inhibitors levamisole and R 8231 on p-nitro-phenylphosphatase, inorganic pyrophosphatase and adenosine triphosphatase (ATPase) activities in dentingenically active odontoblasts were studied. The p-nitrophenylphosphatase and inorganic pyrophosphatase activities were inhibited, while 40% of the ATP-splitting enzyme activity remained under the assay condition used. This finding, togeather with earlier studies, indicates that at least two different phosphatase are active at alkaline pH in hard tissue-forming cells; on nonspecific alkaline phosphatase and one specific ATPase. The ATPase activity is uninfluenced by ouabain and ruthenium red and is activated by Ca-2+ ions.     

Association of inorganic-pyrophosphatase activity with human alkaline-phosphatase preparations

1. The inorganic-pyrophosphatase activity of alkaline phosphatases prepared from human liver and small intestine was investigated at different stages of purification. 2. Both liver and intestinal preparations possessed pyrophosphatase activity at all stages of purification, and the two types of activity were not separated by gel filtration or by anion-exchange or cation-exchange chromatography. 3. After starch-gel electrophoresis of the tissue extracts, the zones of pyrophosphatase activity coincided exactly with alkaline-phosphatase zones. 4. Hydrolysis of each type of substrate was inhibited by the presence of the other, and a constant ratio of alkaline-phosphatase activity to pyrophosphatase activity was maintained during inactivation of the enzymes by incubation at 55°. 5. These results are consistent with the view that alkaline phosphatases are also inorganic pyrophosphatases.     

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.     

Use of biotinylated inorganic pyrophosphatase for detection of biotin bound to solid support

A colorimetric procedure to detect biotin bound to microtiter plates with a sensitivity down to 10(-16) mol was developed using biotinylated inorganic pyrophosphatase of Escherichia coli. Reaction of pyrophosphatase with 1 mM N-biotinyl-6-aminocaproic acid N-hydroxy-sulfonosuccinimide ester yielded a stable 87% active enzyme containing 5.6 mol biotin/mol. In the measurements of human immunoglobulin G, a biotinylated pyrophosphatase.streptavidin complex provided a sensitivity superior to that of conventional enzyme immunoassay due to low nonspecific binding. The new procedure was also more sensitive compared with that using biotinylated alkaline phosphatase. Together with high thermostability of pyrophosphatase and its substrate, low background staining allowed measurement of enzymatic activity to be performed at 60 degrees C for 4 h resulting in a marked increase in assay sensitivity.     

End labeling of enzymatically decapped mRNA.

A method is presented for rapid and efficient 5' end labeling with 32P of capped mRNAs, by a series of three enzymatic reactions: the blocking nucleotide of the cap structure is removed by tobacco acid pyrophosphatase, and after dephosphorylation with alkaline phosphatase the 5' end is labeled with gamma-32-P-ATP and T4 polynucleotide kinase.     

The influence of metal ions on the orthophosphatase and inorganic pyrophosphatase activities of human alkaline phosphatase

1. The differential effects of adding Zn(2+) and Mg(2+) on the orthophosphatase and inorganic pyrophosphatase activities of human intestinal alkaline phosphatase were studied. 2. In the presence of excess of Zn(2+), inorganic pyrophosphatase activity is inhibited. At higher concentrations of pyrophosphate, hydrolysis of this substrate takes place, but is inhibited competitively by the Zn(2+)-pyrophosphate complex. This complex also acts as a competitive inhibitor of orthophosphate hydrolysis. 3. Excess of Mg(2+) also inhibits pyrophosphatase action by removal of substrate; at low concentrations, this ion activates pyrophosphatase, as is the case with orthophosphatase. 4. It is concluded that, when interactions between metal ions and pyrophosphate are taken into account, the effects of these ions are consistent with the view that alkaline phosphatases possess both orthophosphatase and inorganic pyrophosphatase activities.     

The localization in rat liver of alkaline phosphodiesterase to a discrete organelle implicated in ligand internalization

The subcellular distribution of alkaline phosphodiesterase and NADH pyrophosphatase, two activities thought to be expressed by the same enzyme, was investigated. Although the two activities share a localization to a low-density vesicular membrane (equilibrium density = 1.12 g.cm-3), little NADH pyrophosphatase activity, in contrast to alkaline phosphodiesterase, was found in plasma membrane (equilibrium density = 1.18 g.cm-3), as reflected by the distribution of 5'nucleotidase. The binding and uptake of 125I-labelled insulin in perfused rat liver was also investigated. This ligand was found to bind to sinusoidal plasma membrane at 4 degrees C, but was rapidly internalized at 37 degrees C to the low-density membrane, which is rich in alkaline phosphodiesterase and NADH pyrophosphatase. These vesicular membranes were shown to belong to none of the enzymatically characterized subcellular bodies, and it is proposed that they represent discrete organelles participating in the subcellular processing of receptor-ligand complexes.     

Inhibition of the orthophosphatase and pyrophosphatase activities of human alkaline-phosphatase preparations

1. Purified human liver and small-intestinal alkaline orthophosphatases release inorganic phosphate at appreciable rates from a variety of organic pyrophosphate substrates. 2. The pyrophosphatase action is inhibited by Mg²⁺ ions at concentrations that activate the hydrolysis of orthophosphate substrates by these enzymes. 3. The results of mixed-substrate experiments, denaturation studies with heat or urea and starch-gel electrophoresis suggest that both orthophosphatase and pyrophosphatase activities are, in each preparation, properties of a single enzyme. 4. Intestinal phosphatase shows greater pyrophosphatase activity relative to orthophosphatase than the liver enzyme.     

Enzymatic changes in gourd and bean cotyledons during ageing and the effect of detopping

Cotyledons of gourd (Cucurbita maxima Duchesne) and bean (Phaseolus vulgaris L.) were used to study the changes in the activities of catalase, peroxidase, acid inorganic pyrophosphatase and alkaline inorganic pyrophosphatase during ageing and the diversion in such changes that occur when cotyledon senescence was retarded by detopping the seedlings above the cotyledons. Catalase, acid inorganic pyrophosphatase and alkaline inorganic pyrophosphatase activities declined during the senescence of the cotyledons. When cotyledon senescence was retarded by detopping as marked by the increase in the levels of chlorophyll and protein, there was also an increase in the activities of these enzymes. Peroxidase activity, on the other hand, increased during the senescence of the cotyledons and detopping the seedlings resulted in a further increase in the peroxidase activity. It can be suggested that some root factor(s) probably cytokinin(s) is (are) mobilised into the cotyledons of the detopped seedlings which otherwise would have been mobilised into the shoot apices, and help retard or even reverse the senescence of the cotyledons.     

Changes in pyrophosphatase activity during the de novo mineralization associated with cartilage and bone formation

Pyrophosphatase was extracted from implants undergoing de novo mineralization in an in vivo model of matrix-induced endochondral bone formation. Before the onset of the mineralization of the plaques and after the mineralization process had been completed only one form of pyrophosphatase activity was observed. During the active deposition of calcium phosphate, however, a new, higher molecular weight form of pyrophosphatase activity was produced suggesting that this enzyme activity is associated with biological mineralization. This observation gives support to the earlier suggestion that inhibitors of calcium phosphate precipitation, such as pyrophosphate, must be removed from the site of mineralization before calcification can occur. This high-molecular-weight activity also appears to be associated with alkaline and/or acid phosphatase activity as determined by molecular exclusion chromatography.