Nucleotide Pyrophosphatase Activity Research Articles

Membrane-Associated Nucleotide Pyrophosphatase Activity on Leucocytes from Different Compartments

The distribution of membrane-associated nucleotide pyrophosphatase activity has been investigated on mouse leucocytes of different origin. It is shown that enzyme-specific activity is higher on bone-marrow cells than on cells derived from peripheral organs. Since thymus lymphocytes show the lowest activity, it is obvious that the thymus environment can influence the enzyme activity on bone-marrow stem cells which have entered this organ. Spleen cells from thymus-deficient nude mice displayed more enzyme activity than spleen cells from normal mice, although the percentage of lymphocytes was similar in both donor-cell populations.

Ganglioside biosynthesis in rat liver. Characterization of UDP-N-acetylgalactosamine -- GM3 acetylgalactosaminyltransferase.

UDP-N-acetylgalactosamine--GM3 acetylgalactosaminyltransferase (GM2-synthase) was studied in a Golgi-rich fraction from rat liver. Activity in a cell-free system required the presence of detergents; octyl glucoside was found to be the most effective in stimulating the enzyme. Optimal activity of GM2-synthase was obtained at pH 7.2, in the presence of 0.8% octyl glucoside, 10 mM Mn2+ and 5 mM CDP-choline. The latter was used to counteract the rapid sugar nucleotide hydrolysis caused by a nucleotide pyrophosphatase activity in the Golgi fraction. The apparent Km values for UDP-N-acetylgalactosamine and added GM3 were 0.035 mM and 0.1 mM, respectively. Different results were obtained if endogenous GM3 only was used as the glycolipid acceptor. In this case, the apparent Km value for UDP-N-acetylgalactosamine was 0.18 mM and Co2+ and Fe2+ exceeded Mn2+ in activating GM2-synthase. Under optimal assay conditions and in the presence of added GM3 and 5 mM CDP-choline, the specific activity of the enriched Golgi fraction was measured to be 25-30 nmol X mg protein-1 X h-1; with endogenous GM3 as the sole glycolipid acceptor, V was calculated to be 9 nmol X mg protein-1 X h-1.

Sulfohydrolytic degradation of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) and adenosine 5'-phosphosulfate (APS) by enzymes of a nucleotide pyrophosphatase nature.

The sulfohydrolytic activity to degrade active sulfate (3'-phosphoadenosine 5'-phosphosulfate, PAPS) and its precursor, APS (adenosine 5'-phosphosulfate), with a pH optimum at 9.5 was found to be widely distributed in various tissues of rats. In the liver, the activity was located in plasma membranes and endoplasmic reticula. Triton X-100 solubilized rough and smooth endoplasmic reticula gave two peaks of the activity on gel filtration, both of which had nucleotide pyrophosphatase activities, hydrolyzing the pyrophosphate linkages of ATP, NAD, and UDP-Glc, and the phosphodiester linkage of PNTP (p-nitrophenyl-thymidine 5'-monophosphate) besides PAPS and APS.

Glycosaminoglycan synthesis by cultured skin fibroblasts from a patient with Lowe's syndrome.

Glycosaminoglycan metabolism in cultured skin fibroblasts from a patient with Lowe's syndrome (oculocerebrorenal syndrome) was compared with that in normal fibroblasts. Cells were incubated with [3H]glucosamine and 35SO42-, and then the labeled glycosaminoglycans in the medium, trypsinate, and cell membrane fractions were analyzed by means of enzymatic and chemical degradations and by electrophoresis. Glycosaminoglycans, mostly chondroitin sulfates and dermatan sulfate, were markedly undersulfated in Lowe's syndrome. Undersulfation was most pronounced in the cell membrane fraction of cells which had been incubated at 0.03 mM sulfate concentration. Even at 0.52 mM or a higher sulfate concentration, undersulfation of chondroitin sulfates in the cell membrane fractions was significant. The undersulfation was the result of depressed sulfation rather than that of increased desulfation, as demonstrated by pulse-chase experiments using 35SO42-. Hyaluronate synthesis was not significantly impaired in the Lowe's syndrome fibroblasts. It was inferred, based on the results of the present study and those previously reported that the undersulfation of glycosaminoglycans is a consequence of a lower level of active sulfate (adenosine 3'-phosphate 5'-phosphosulfate) in the Lowe's syndrome fibroblasts, caused by an elevation of nucleotide pyrophosphatase activity degrading adenosine 3'-phosphate 5'-phosphosulfate.

Galactosyltransferase activities in rat intestinal mucosa. Inhibition of nucleotide pyrophosphatase.

Nucleotide pyrophosphatase, a Zn2+-dependent enzyme catalyzing the hydrolysis of sugar nucleotides, generally interferes with glycosyltransferase assays. Certain tissues such as intestinal mucosa are particularly rich sources of nucleotide pyrophosphatase activity. The inhibition of nucleotide pyrophosphatase without affecting glycosyltransferase activities by the removal of Zn2+ allows for the assay of galactosyltransferases in rat intestinal particulate fractions. Inactivation of nucleotide pyrophosphatase was achieved by preincubation of particulate enzyme preparations in the presence of EDTA. Addition of Mn2+, required for the galactosyltransferase assays, did not reactivate the nucleotide pyrophosphatase. The presence of 0.05% (w/v) soybean trypsin inhibitor was required during the preparation of intestinal mucosa homogenates and enzyme assays to protect the UDP-galactose: N-acetylglucosaminyl galactosyltransferase from inactivation.

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 K m 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-ethylmaleimide, 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 Mg 2+. This activation by Mg 2+ is counteracted by Zn 2+. Gel chromatography revealed that the enzyme is a monomer in the presence of Mg 2+. When inhibited by Zn 2+, it forms polymers that can be reconverted to the monomer by thiols. All 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).

Stimulatory and inhibitory effects of ATP-regenerating systems on liver adenylate cyclase.

Activity of hepatic adenylate cyclase is significantly influenced by membrane-bound nucleotide pyrophosphatase and by components of ATP-regenerating systems. Nucleotide pyrophosphatase, which hydrolyzes ATP, GTP, and guanyl-5’-yl imidodiphosphate (GPP(NH)P) between the a- and P-phosphates, can affect measured activity by hydrolyzing substrate or modulator, and by facilitating formation of adenosine. This influence was reduced by the preparation of plasma membranes in or their pretreatment with EDTA and dithiothreitol. CAMP formation was linear for a longer time, and ATP regeneration improved while pyrophosphatase activity was reduced. In addition, the measurement of optimal cyclase activity required the inclusion in the reaction mixture of myokinase, to permit rephosphorylation of 5’-AMP, and adenosine deaminase to remove inhibitory levels of adenosine. The nature and magnitude of the effects of myokinase and adenosine deminase could account for much of the effect of a liver cytosol preparation to stimulate the hepatic adenylate cyclase. This suggests that reported effects of cytosol on cyclase activity may be due in part to the influence of these two enzymes. Phosphoenolpyruvate inhibited basal and fluoridestimulated adenylate cyclase activities in a manner that appeared competitive with metal-ATP, but enhanced guanine nucleotide-dependent activities in a free metaldependent manner. However, this enhancement was not seen when membranes were pretreated with EDTA and when cyclase activity was determined in the presence of myokinase and adenosine deaminase. In addition, phosphoenolpyruvate reduced nucleotide pyrophosphatase activity, and the data suggested that the enhancing effect might be due to

Chemical and enzymic degradations of nucleoside mono- and diphosphate sugars: I. Determination of the degradation rate during the glycosyltransferase assays

In incorporation experiments used for the determination of glycosyltransferase activities, we demonstrated that the nucleoside diphosphate sugars are decomposed in three different ways: 1, transfer of the monosaccharide to acceptor molecule, catalyzed by glycosyltransferases; 2, degradation of the glycosyl nucleotides by nucleotide pyrophosphatase into monosaccharide 1-phosphates which are further hydrolyzed into free monosaccharides by phophatases; 3, chemical decomposition of UDP- D-[ 14C]Gal; UDP- D-[ 14C]Glc and UDP- D-[ 14C]GlcUA into 1,2-cyclic phosphate derivatives of the corresponding monosaccharide. All the breakdown products of the nucleoside mono- and diphosphate sugars which are obtained during the incorporation experiments may be separated by paper chromatography and their amounts may be determined. Galactosyltransferase assays on human and rat serum have shown that the three different ways of decomposition of the nucleoside diphosphate sugars are dependent mostly on the concentration of divalent cations (Mn 2+, Mg 2+). Inhibition of the nucleotide pyrophosphatase activity is obtained with low concentrations of UMP, but increasing concentrations of UMP inhibit also the galactosyltransferase activity and consequently enhance the formation of galactose 1,2-monophosphate. A partial elimination of the nucleotide pyrophosphatase activity was achieved by the addition of increasing concentrations of UDP- D-Gal. The results demonstrate that the determination of glycosyltransferase activities in tissues and in biological fluids is not possible without a concomitant determination of the nucleotide pyrophosphatase activity present in the assay.

Nucleotide pyrophosphatase activity in dry and germinated seeds of Triticum, and its relationship to general acid phosphatase activity

A cytochemical investigation has been made of nucleotide pyrophosphatase activity in dry and germinated seeds of Triticum, and its distribution compared to that of general acid phosphatase reactions seen with naphthol AS-BI phosphate and p-nitrophenylphosphate as substrates. Acid phosphatase activity was present in the cytoplasm and in channels through the walls of the aleurone cells in both dry and germinated seeds. The cytoplasmic activity was even more marked with nucleotide pyrophosphatase which was almost entirely absent from the cell walls. Nucleotide pyrophosphatase was active in all endosperm cells but particularly in some cells adjacent to the aleurone layer. In addition, all cells of the scutellum and embryo were positive for nucleotide pyrophosphatase activity, especially the developing fibres and xylem elements of leaves and coleoptiles, mature leaf xylem and phloem elements, scutellar provascular and vascular tissues and the epidermis of dark grown coleoptiles.

Evidence for the biosynthesis of mannosylphosphoryldolichol and N-acetylglucosaminylpyrophosphoryldolichol by an axolemma-enriched membrane preparation from bovine white matter.

AbstractAn axolemma‐enriched membrane fraction prepared by an improved procedure from bovine white matter catalyzes the enzymatic transfer of [14C]mannose and N‐acetyl[14C]glucosamine from their nucleotide derivatives into a mannolipid and an N‐acetylglucosaminyl lipid in the presence of exogenous dolichyl monophosphate. The labeled glycolipid products have the chemical and chromatographic characteristics of mannosylphosphoryldolichol and N‐acetylglucosaminylpyrophosphoryldolichol. The initial rates of synthesis of the glycolipids by the axolemma‐enriched membrane fraction have been compared with the initial rates of glycolipid formation catalyzed by a microsomal preparation and myelin in the presence or absence of dolichyl monophosphate. Essentially no glycolipid synthesis was observed when either GDP‐[14C]mannose or UDP‐N‐acetyl[14C]glucosamine were incubated with myelin in the presence or absence of exogenous dolichyl monophosphate. A comparison of the initial rates of synthesis of the glycolipids using endogenous acceptor lipid revealed that the rate of formation of mannolipid was 7 times faster for the microsomal membranes than the axolemma‐enriched membranes. In the presence of an amount of dolichyl monophosphate approaching saturation the initial rate of glycolipid synthesis was markedly enhanced for both membrane preparations. However, due to a more dramatic enhancement in the axolemma‐enriched membranes the initial rate of mannolipid synthesis was only approx. 2.5 times greater in the microsomal membranes. A similar observation was made when the initial rates of N‐acetylglucosaminyl lipid synthesis were compared for axolemma‐enriched and microsomal preparations in the presence and absence of exogenous dolichyl monophosphate. These studies indicate that the axolemma‐enriched membranes have a relatively lower content of dolichyl monophosphate than the microsomal membranes although the difference in the amount of mannosyltransferase is only two to three‐fold lower.The presence of a sugar nucleotide pyrophosphatase activity capable of degrading GDP‐mannose and UDP‐N‐acetylglucosamine has also been demonstrated in the axolemma‐enriched membrane fraction.

The cytochemical localization of nucleotide pyrophosphatase activity in plant tissues using naphthyl esters of thymidine-5'-phosphate.

A cytochemical method for the localization of nucleotide pyrophosphatase activity in plants employing naphthol AS-BI thymidine 5'-monophosphate and alpha-naphthyl thymidine 5'-monophosphate as specific substrates is reported. Biochemical evidence for the validity of this method is presented and the synthesis of the naphthol AS-BI ester is described. The application of this cytochemical technique to shoots of Triticum sp. and roots of Vicia faba has shown nucleotide pyrophosphatase to be ubiquitous in its distribution in these organs and to occur in a structurally-bound form in the cytoplasm. The highest activity was detected in developing fibres adjacent to the leaf vascular bundles, in the coleoptile epidermal and hypodermal cells and in the coleoptile and leaf xylem.

Pyridine nucleotide involvement in rat hepatic microsomal drug metabolism—I. Factors that influence NADPH kinetic estimations during mixed function oxidase reactions

The localisation of significant amounts of nucleotide pyrophosphatase activity in the rat hepatic microsomal fraction results in erroneous values of apparent K m (NADPH) for aminopyrine- Ndemethylase. This was demonstrated by the inclusion of 20 mM pyrophosphate, which inhibits nucleotide pyrophosphatase activity and reduces the apparent K m (NADPH) value from 27.9 μM to 7.92 μM. The apparent K m (NADPH) values determined in the presence of three Type I substrates were not statistically different from each other, but the value in the presence of aniline was lower. The kinetic constants of NADPH for NADPH cytochrome P450-reductase in the presence of either aminopyrine, ethylmorphine or aniline, are also reported.

Evidence for the enzymatic transfer of N-acetylglucosamine from UDP- N-acetylglucosamine into dolichol derivatives and glycoproteins by calf brain membranes

Incubating white matter membranes with UDP- N-acetyl-[ 14C]glucosamine in the presence of Mg 2+ and AMP resulted in the labeling of two major glycolipids, a minor glycolipid and several membrane-associated glycoproteins. The addition of AMP protected the labeled sugar nucleotide from degradation by a membrane-bound sugar nucleotide pyrophosphatase activity. While no labeled oligosaccharide lipid was recovered in a CHCl 3CH 3OHH 2O (10:10:3) extract after incubating with only UDP- N-acetyl-[ 14C] glucosamine, Mg 2+, and AMP, the inclusion of unlabeled GDP-mannose led to the formation of an N-acetyl-[ 14C]glucosamine-labeled oligosaccharide lipid that was soluble in CHCl 3CH 3OHH 2O (10:10:3). The [ GlcNAc- 14C]oligosaccharide unit was released by treatment with 0.1 N HCl in 80% tetrahydrofuran at 50 °C for 30 min and appears to have the same molecular size as the lipid-linked [ mannose- 14C] oligosaccharide, formed enzymatically by white matter membranes as judged by their elution behavior on Bio-Gel P-6. The incorporation of N-acetyl-[ 14C]glucosamine into glycolipid was stimulated by exogenous dolichol monophosphate, but inhibited by UMP or tunicamycin, a glucosamine-containing antibiotic. Although UMP and tunicamycin drastically inhibited the labeling of glycolipid, these compounds had very little effect on the labeling of glycoproteins. The major glycolipids have the chemical and Chromatographic characteristics of N-acetylglucosaminylpyrophosphoryldolichol and N,N′-diacetylchitobiosylpyrophosphoryldolichol. When the labeled glycoproteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, four labeled polypeptides were observed, having apparent molecular weights of 145,000, 105,000, 54,000, and 35,000. Virtually all of the N-acetyl-[ 14C]glucosamine was released when the labeled glycopeptides, produced by pronase digestion, were incubated with an exo-β- N-acetylglucosaminidase, indicating that all of the N-acetyl-[ 14C]glucosamine incorporated under these conditions is attached to white matter membrane glycoproteins at nonreducing termini.

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.

Higher-plant cyclic nucleotide phosphodiesterases. Resolution, partial purification and properties of three phosphodiesterases from potato tuber

1. Three phosphodiesterases that are capable of hydrolysing 3':5'-cyclic nucleotides were purified from potato tubers. 2. The phosphodiesterases were fractionated by (NH4)2SO4 precipitation and CM-cellulose chromatography. The phosphodiesterases were resolved from each other and further purified by gel filtration in high- and low-ionic-strength conditions. 3. All three enzymes lacked significant nucleotidase activity. 4. Enzymes I and II had mol. wts. 240,000 and 80,000 respectively, determined by gel filtration, whereas enzyme III showed anomalous behaviour on gel filtration, behaving as a high- or low-molecular-weight protein in high- or low-ionic-strength buffers respectively. 5. All enzymes hydrolysed 2':3'-cyclic nucleotides as well as 3':5'-cyclic nucleotides. The enzymes also had nucleotide pyrophosphatase activity, hydrolysing NAD+ and UDP-glucose to various extents. Enzymes I and II hydrolyse cyclic nucleotides at a greater rate than NAD+, whereas enzyme III hydrolyses NAD+ at a much greater rate than cyclic nucleotides. All three enzymes hydrolysed the artificial substrate bis-(p-nitro-phenyl) phosphate. 6. The enzymes do not require the addition of bivalent cations for activity. 7. Both enzymes I and II have optimum activity at pH6 with 3':5'-cyclic AMP and bis-(p-nitrophenyl) phosphate as substrates. The products of 3':5'-cyclic AMP hydrolysis were 3'-AMP and 5'-AMP, the ratio of the two products being different for each enzyme and varying with pH. 8. Theophylline inhibits enzymes I and II slightly, but other methyl xanthines have little effect. Enzymes I and II were competitively inhibited by many nucleotides containing phosphomonoester and phosphodiester bonds, as well as by Pi. 9. The possible significance of these phosphodiesterases in cyclic nucleotide metabolism in higher plants is discussed.

Nucleotide pyrophosphatase of rat liver. A comparative study on the enzymes solubilized and purified from plasma membrane and endoplasmic reticulum.

Studies on the subcellular distribution of rat liver nucleotide pyrophosphatase activity revealed its presence in the plasma membrane and the endoplasmic reticulum only. The enzymes from either source were solubilized specifically with trypsin without an apparent change of their catalytic properties. A 200-fold and 1600-fold purification, respectively, was achieved by a procedure including DEAE-cellulose and affinity-chromatography with AMP as ligand, gel filtration on Sephadex G-200 and gel electrophoresis. Both nucleotide pyrophosphatases were isolated as electrophoretically homogeneous soluble proteins. They were shown to contain carbohydrate moieties. The electrophoretic mobility of both enzymes in polyacrylamide gels was identical at three pH values. Dodecylsulfate gel electrophoresis indicated a molecular weight of 137 000 for both glycoproteins. The enzymes hydrolyze a variety of purine and pyrimidine nucleotides yielding a 5'-nucleoside monophosphate. Adenosine 3':5'-monophosphate, nucleic acids and phosphate monoesters are not cleaved, but p-nitrophenyl-thymidine5'-monophosphate is readily hydrolyzed. In view of their substrate and inhibitor specificities the enzymes are considered nucleotide pyrophosphatases rather than phosphodiesterases.

Distribution of liver plasma membrane 5′ nucleotidase as indicated by its reaction with anti-plasma membrane serum

Antiserum against mouse liver plasma membranes was used to investigate the properties and distribution of the surface membrane enzyme 5′ nucleotidase. The antiserum inhibited 5′ nucleotidase but had no effect on alkaline phosphodiesterase, nucleotide pyrophosphatase, or insulin-binding activity. 5′ Nucleotidase was purified from mouse liver plasma membranes and the purified enzyme was shown to be inhibited by the antiserum. The membrane-bound and the purified enzyme were both inhibited in a noncompetitive manner. The reaction of the antiserum with 5′ nucleotidase activity of mouse liver plasma membrane “light” and “heavy” subfractions, and of rat liver and pig lymphocyte surface-membrane fractions was investigated. In each case the enzyme was inhibited by the antiserum. Since a protein must be partially exposed on the membrane surface in order to react with its antibody, the results are discussed in terms of the disposition of 5′ nucleotidase within the membrane.

Phosphatase activity in epiphyseal cartilage of the chicken ( Gallus gallus)

1. 1. Epiphyseal cartilage was separated into resting, ossifying and new bone regions. 2. 2. An acid phosphatase with sharp peaks at pH 3·0 and 6·0 was found in the soluble cartilage preparation. 3. 3. Activities of alkaline phosphatase, nucleotide pyrophosphatase, 5-nucleotidase and glycerophosphatase activities were higher in ossifying cartilage and new bone than in resting cartilage. 4. 4. It is suggested that phosphatases in the ossifying cartilage and new bone may play a very active role in preparing tissue for calcification.

Enzymatic Hydrolysis of Uridine Diphosphate-N-acetyl-d-galactosamine and Uridine Diphosphate-N-acetyl-d-glucosamine by Normal Cells, and Blocks in This Hydrolysis in Transformed Cells and Their Revertants

Abstract The two sugar nucleotides, UDP-N-acetyl-d-galactosamine and UDP-N-acetyl-d-glucosamine, are enzymatically hydrolyzed by normal hamster cell extracts to yield free N-acetyl-d-galactosamine and N-acetyl-d-glucosamine, respectively. A nucleotide pyrophosphatase activity splits the sugar nucleotides to UMP and the appropriate N-acetyl-d-hexosamine-1-phosphate, and another enzymatic step with orthophosphoric monoester phosphohydrolase activity then hydrolyzes the phosphorylated intermediate sugar to free N-acetyl-d-hexosamine. The two enzymatic activities differ in their divalent ion requirement and optimal pH and show the same hydrolytic activity on both sugar nucleotides. Different lines of hamster transformed cells and their revertants showed a block in either one or the other enzyme. Cell lines transformed by Rous sarcoma virus or Simian virus 40 were blocked in the nucleotide pyrophosphatase activity, whereas cell lines transformed by polyoma virus or after treatment with the chemical carcinogen dimethylnitrosamine were blocked in the second phosphohydrolase step. Variants from the polyoma- and dimethylnitrosamine-transformed cells, which had reverted in the biological properties characteristic of transformation, showed the same enzymatic block as their parental transformed cells. The block in enzyme activities may thus be due to a common genetic change in transformed cells and their revertants that is not found in the normal cells.

Nucleotide pyrophosphatase activity of rat liver plasma membranes

1. 1. Palmityl-CoA is metabolised to S- palmityl pantetheine by plasma membrane preparations. The metabolism of other nucleotide pyrophosphates by the plasma membrane of liver cells was found to be similar. UDPG was converted to glucose i-phosphate and UMP; NAD + and NADH were hydrolysed at the pyrophosphate bond. 2. 2. The subcellular distribution of the nucleotide pyrophosphatase was studied with NADH as substrate. The similarity in distribution to 5′-nucleotidase, a plasma membrane marker enzyme, indicates that nucleotide pyrophosphatase is an authentic plasma membrane enzyme. 3. 3. NADH breakdown was competitively inhibited by UDPG, CoA and NAD +, and the properties of the enzyme were very similar to those of other mammalian nucleotide pyrophosphatases.