Nucleotide pyrophosphatase and

It has been reported that preparations of starch grains display very high synthetase activity if they are isolated in 0.5 M sucrose, presumably because they are well preserved after such treatment. However, the possibility that the high reaction rate may be due to the combined action of nucleotide pyrophosphatase and starch phosphorylase has not been adequately ruled out in that study. 

Dephospho-CoA pyrophosphorylase. 4-phosphopanto-thenate was prepared from dephospho-CoA by incubation with commercial nucleotide pyrophosphatase, and isolated by affinity chromatography with Thiopropy1-Sepharose followed by preparative paper chromatography. Dephospho-CoA pyrophosphorylase was estimated at pH 8.5, in a system similar to that used for dephospho-CoA kinase. 

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. 

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). 

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-... 

Structural and functional comparisons of nucleotide pyrophosphatase/phosphodies-terase and alkaline phosphatase implications for mechanism and evolution. Biochemistry 45 9788-9803... 

Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPPl), also known as plasma cell membrane glycoprotein 1 (PC-1), is a member of the NPP family of transmembrane enzymes that catalyzes the hydrolysis of extracellular nucleotides. Different family members demonstrate preference for distinct substrates, but share ability to hydrolyze phosphodiester and pyrophosphatase bonds [1]. ENPPl appears to have preference for ATP, but also demonstrates pyrophosphatase and phosphodiesterase activities toward pyrophosphate and ADP [1, 2],... 

Simao AM, Yadav MC, Narisawa S et al (2010) Proteoliposomes harboring alkaline phosphatase and nucleotide pyrophosphatase as matrix vesicle biomimetics. J Biol Chem 285 7598-7609... 

Cimpean A, Stefan C, Gijsbers R et al (2004) Substrate-specifying determinants of the nucleotide pyrophosphatases/phosphodiesterases NPPl and NPP2. Biochem J 381 71-77... 

Fukui, S., Yoshida, H., and Yamashina, I. Sulfohydrolytic degradation of 3 -phosphoadenosine 5 -phosphosulfate and adenosine 5 -phosphosulfate by enzymes of a nucleotide pyrophosphatase nature. J. Biochem. (Tokyo) 90, 1537-1540 (1981). 

Skidmore, J. R., and Trams, E. G., Nucleotide pyrophosphatase activity of rat liver plasma membranes. Biochim. Biophys. Acta 219, 93-103 (1970). 

Yoshida, H., Fukui, S., Yamashina, 1., Tanaka, T., Sakano, T., Usui, T., Shimotsuhi, T., Yabuuchi, H., Owada, M., and Kitagawa, T., Elevation of nucleotide pyrophosphatase activity in skin fibroblasts from patient with Lowe s syndrome. Biochem. Biophys. Res. Commun. 107, 1144-1150 (1982). 

Clayton, R.A. and L.M. Hanslman Tobacco nucleotide pyrophosphatases Arch. Biochem. Biophys. 87 (1960)... 

Transcription, the synthesis of RNA from a DNA template, is carried out by RNA polymerases (Fig. 14.2). Like DNA polyma-ases, RNA polymerases catalyze the formation of ester bonds between nucleotides that base-pair with the complementary nucleotides on the DNA template. Unlike DNA polymerases, RNA polymerases can initiate the synthesis of new chains in the absence of primers. They also lack the 3 to 5 exonuclease activity found in DNA polymerases. A strand of DNA serves as the template for RNA synthesis and is copied in the 3 to 5 direction. Synthesis of the new RNA molecule occurs in the 5 to 3 direction. The ribonucleoside triphosphates ATP, GTP, CTP, and UTP serve as the precursors. Each nucleotide base sequentially pairs with the complementary deoxyribonucleotide base on the DNA template (A, G, C, and U pair with T, C, G and A, respectively). The polymerase forms an ester bond between the a-phos-phate on the ribose 5 -hydroxyl of the nucleotide precursor and the ribose 3 -hydroxyl at the end of the growing RNA chain. The cleavage of a high-energy phosphate bond in the nucleotide triphosphate and release of pyrophosphate (from the (3 and y phosphates) provides the energy for this polymerization reaction. Subsequent cleavage of the pyrophosphate by a pyrophosphatase also helps to drive the polymerization reaction forward by removing a product. 

Soluble inorganic pyrophosphatase (PPase) is essential for growth of all known organisms. As shown in Equation (8), it catalyzes the simple hydrolytic cleavage of pyrophosphate to two phosphates.This reaction is essential because many key biosynthetic reactions release pyrophosphate rather than phosphate from nucleotide triphosphates, and the subsequent hydrolysis of pyrophosphate makes such reactions functionally irreversible, as well as providing a substantial additional thermodynamic driving force ... 

New DNA strands are synthesized in the 5 —>3 direction. This means that new deoxyribonucleotide triphosphates (dNTPs) are added onto the 3 -OH of the growing chain in a template-directed maimer by DNA polymerase. The polymerization involves the formation of a phosphodiester bond between the a-phosphate of the incoming nucleotide and the 3 -OH of the nascent chain. Only the deoxyribonucleotide monophosphate (dNMP) is incorporated into the growing chain the pyrophosphate (PPi) is cleaved off during polymerization. The cleavage of the phosphodiester bond provides the energy for the polymerization reaction. The resulting pyrophosphate ion is rapidly hydrolyzed to two phosphate ions by the ubiquitous and abundant enzyme pyrophosphatase, and this reaction drives the polymerization in the direction of polymer formation (Fig. 8-1). 

Sugar nucleotides are generally extracted with ethanol or with dilute perchloric or trichloroacetic acids, so that the extracts are essentially free of protein. Ethanol may extract a considerable amount of lipid, while the acids tend to cause appreciable losses of sugar nucleotides by hydrolysis. Trichloroacetic acid can be extracted with ether, while perchloric acid is best removed as its potassium salt. All extractions must be performed in the cold and extraction with ethanol is possible well below 0°C, though it fails to denature all pyrophosphatases and this can lead to losses. Saukkonen (1964) has reviewed a number of procedures for extraction and there is undoubtedly still some room for improvements. 

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