Pyrophosphatase, nucleotide

Nucleotide Pyrophosphatase. Nucleotide pyrophosphatase was first detected in particles from animal cells, and was subsequently purified from potato tubers. This enzyme, which has been found widely distributed, splits DPN, TPN, and other dinucleotides with a pyrophosphate bridge. As would be expected from the broad specificity, reduction... 

AMP deaminase neuraminidase leucy1-3-naphthylamidase p-nitrophenyIphosphatase inorganic pyrophosphatase nucleotide pyrophosphatase sialyltransferase NADH oxidase... 

Figure 11.4 Analysis of in vitro synthesized RNAs. 32P-Radiolabeled RNAs (48 nucleotides) capped with m7Gp3G (A and C) or m27,3 °Gp3G (B and D) were digested with either RNase T2 (A and C) or RNase T2 plus tobacco acid pyrophosphatase (TAP) (B and D) followed by anion-exchange HPLC on a Partisil 10SAX/25 column as described in the text. Fractions of 1 ml were collected, and the Cerenkov radiation was determined. The elution times of the following standard compounds, detected by ultraviolet (UV) absorption, are indicated with arrows 3,-CMP (Cp), S UMP (Up), 37-AMP (Ap), 3 -GMP (Gp), 3, 5 -m7GDP (pm7Gp), 3, 5 -GDP (pGp), 5 -GDP (p2G), 5 -GTP (p3G), and guanosine-SCtetraphosphate (P4G).

Inosine triphosphate pyrophosphatase (TTPA) deficiency may lead to accumulation of 6-thioinosine triphosphate (ITP) in those treated with AZA or 6-MP. The gene encoding ITPA is located on chromosome 20, and two of five single-nucleotide... 

The reaction is catalyzed by specific enzymes, usually termed204 sugar nucleotide pyrophosphorylases. The equilibrium constant is not far from unity, and addition of inorganic pyrophosphatase, an enzyme that converts inorganic pyrophosphate irreversibly into... 

Hydrolysis of sugar nucleotides with unspecific pyrophosphatases has already been mentioned (Section 11,1, p. 310). A similar reaction is catalyzed by a bacterial enzyme specific for adenosine 5 -(a-D-glucopyranosyl pyrophosphate).459 The specific conversion of uridine 5 -(a-D-glucopyranosyl pyrophosphate) into a-D-glucopyranosyl phosphate, uridine, and inorganic phosphate was observed with an enzyme from Escherichia colt 459,460 a preparation from Bacillus subtilis can act in a similar manner461 on different sugar nucleotides. ... 

To HEPES buffer (100 mL, 200 mM, pH 7.5) were added ManNAc 15 (1.44 g, 6 mmol), PEP sodium salt (1.88 g, 8 mmol), pyruvic acid sodium salt (1.32 g, 12 mmol), CMP (0.64 g, 2 mmol), ATP (11 mg, 0.02 mmol), pyruvate kinase (300 U), myokinase (750 U), inorganic pyrophosphatase (3 U), /V-acctylneuraminic acid aldolase (100 U), and CMP-sialic acid synthetase (1.6 U). The reaction mixture was stirred at room temperature for 2 days under argon, until CMP was consumed. The reaction mixture was concentrated by lyophilization and directly applied to a Bio-Gel P-2 column (200-400 mesh, 3 x 90 cm), and eluted with water at a flow rate of 9 mL/h at 4°C. The CMP-NeuAc fractions were pooled, applied to Dowex-1 (formate form), and eluted with an ammonium bicarbonate gradient (0.1-0.5 M). The CMP-NeuAc fractions free of the nucleotides were pooled and lyophilized. Excess ammonium bicarbonate was removed by addition of Dowex 50W-X8 (H+ form) to the stirred solution of the residual powder until pH 7.5. The resin was filtered off and the filtrate was lyophilized to yield the ammonium salt of CMP-NeuAc 17 (1.28 g, 88%). 

An alkaline pyrophosphatase from rat liver cytoplasm has been partially purified and characterized (24) the corresponding enzyme from mice is inhibited by Mg J+-ADP and free PPj, and free Mg2+ has been implicated as an allosteric activator (23). Partial heat inactivation results in loss of the apparent allosteric effects. Rat liver mitochondrial pyrophosphatase, which is inhibited by adenine nucleotides (36), appears to be bound to the inside of the inner mitochondrial membrane (37). This enzyme, after solubilization, has been separated into two fractions which have somewhat different specificity (24, 38). A pyrophosphatase strongly simulated by sulfhydryl reagents (39) has been partially purified from brain tissue (40). The mono-magnesium PPj complex appears to be the true substrate for this enzyme (41). Pynes and Younathan have purified a pyrophosphatase 1800-fold from human erythrocytes (43). The properties of this enzyme are strikingly similar to those of the yeast enzyme the major difference appears to be the more rigid substrate specificity of the erythrocyte enzyme in the presence of Znz. ... 

The equilibria in these phosphoribosyltransferase reactions favor nucleotide synthesis, and since the inorganic pyrophosphate released is rapidly hydrolyzed by inorganic pyrophosphatase, the coupling of these reactions makes the synthesis of nucleotide irreversible. However, the efficiency of salvage is heavily dependent on the intracellular concentration of PRPP. 

It should be noted that many PolyP-dependent enzymes (polyphosphate kinase, exopolyphosphatases, PolyP glucokinase and NAD kinase) are multifunctional and can catalyse reactions both with PolyPs and nucleotide triphosphates. Some PolyP-dependent enzymes, especially exopolyphosphatases, provide excellent examples of cell-compartment specific enzymes. Cell-compartment specificity is a characteristic feature of eucaryotic ATPases (Nelson, 1992) and pyrophosphatases (Baltscheffsky and Baltscheffsky, 1992 Davies et al, 1997 Baykov et al, 1999). This means that the same reaction may be performed in cell compartments by specific enzymes, which differ in their properties, encoding genes and functions. All of the above properties of PolyP-dependent enzymes suggest their important role in the regulation of living cell functions as a whole. 

Most tissues contain very little free riboflavin and, except in the kidneys, where 30% is as riboflavin phosphate, more than 80% is FAD, almost all bound to enzymes. Isolated hepatocytes (and presumably other tissues) show saturable concentrative uptake of riboflavin. The of the uptake process is the same as that of flavokinase, and uptake is inhibited by inhibitors of flavokinase, suggesting that tissue uptake is the result of carrier-mediated diffusion, fol-lowedbymetabolic trapping as riboflavin phosphate, then onward metabolism to FAD, catalyzed by FAD pyrophosphorylase. FAD is a potent inhibitor of the pyrophosphorylase and acts to limit its own synthesis. FAD, which is not protein bound is rapidly hydrolyzed to riboflavin phosphate by nucleotide pyrophosphatase unbound riboflavin phosphate is similarly rapidly hydrolyzed to riboflavin by nonspecific phosphatases (Aw et al., 1983 Yamada et al., 1990). 

The total NADase activity of tissues from these four enzymes is very high, and the total tissue content of nicotinamide nucleotides can be hydrolyzed within a few minutes. Two factors prevent this in vivo. Apart from NAD pyrophosphatase, the enzymes that catalyze the release of nicotinamide from NAD(P) are biosynthetic rather than catabolic, and their activity is highly regulated under normal conditions. Furthermore, the values of K n of the enzymes are of the same order of magnitude as those of many of the NAD(P)-dependent enzymes in the cell, so that there is considerable competition for the nucleotides. Only that relatively small proportion of the nicotinamide nucleotide pool in the cell that is free at any one time will be immediately available for hydrolysis. 

CoA undergoes dephosphorylation, catalyzed by lysosomal acid phosphatase, to dephospho-CoA, followed by pyrophosphatase action to release 4 -phosphopantetheine and 5 -AMP - the reverse of the final stages of CoA synthesis shown in Figure 12.2. CoA is also a substrate for direct pyrophosphatase action, at about 10% of the rate of action on dephospho-CoA. The pyrophosphatase seems to be a general nucleotide pyrophosphatase of plasma membrane rather than an enzyme specific for the degradation of CoA. 

A major factor complicating the quantitative interpretation of most adenylate cyclase measurements is contamination by other enzymes including ATPase, inorganic pyrophosphatase, cyclic nucleotide phosphodiesterase, and various deaminases. Some degree of inhibition of phosphodiesterase is necessary in most preparations, and a methylxan-... 

Enzymatic giycosyiation using sugar nucieotide recyciing systems. Ei = pyruvate kinase, E2 = sugar nucleotide pyrophosphorylase, E3 = pyrophosphatase, E4 = sugar nucleotide synthase, E5 = myokinase, PEP = phosphoenol pyruvate, Pyr = pyruvate... 

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