Pyrophosphorylase assay and

The assay was used to determine NAD pyrophosphorylase activity in extracts prepared from human chronic myelogenous leukemia cells, and in extracts prepared from rat tissues. 

Dickinson, D.B., Hyman, D., and Gonzales, J.W., 1977, Isolation of uridine 5(-pyrophosphate glucuronic acid pyrophosphorylase and its assay using 32P-pyrophosphate. Plant Physiol. 59 1082-1084. 

Estimates of PRibPP levels from studies with fresh cell lysates indicate that the concentration is about 8 X 10 M which is well below the Km of the enzyme 2 X 10 M (D3, H5). The assay techniques are indirect. In one, C-labeled adenine is added along with excess purified APRT. The amount of AMP- C formed is a measure of the PRibPP present. In a second assay, carboxy-labeled orotic acid is added together with orotidylate pyrophosphorylase and orotidylate decarboxylase. The liberated C02 is a measure of the orotidylic acid which is a measure, in turn, of the PRibPP concentration. 

Rubisco activity was measured by radiochemical method and enzyme quantity by immunorocket as in (3). Extracts for Sucrose-P-synthase were desalted on G-25 Sephadex column and the reaction was run as in (3), except that UDP glucose and Fructose 6 P were adjusted to 25 and 10 mM respectively.The spectrophotometric assay was used to measure ADPgIucose pyrophosphorylase (3). Sucrose synthase and neutral invertase were assayed as in (4). 

Pyrophosphorylase activity of the DNA has been determined by an indirect and a direct approach. In the indirect one, the PPj exchange with deoxynucleo-side triphosphate is measured. In the direct approach, cleavage of DNA to the respective pyrophosphatase is assayed. 

Rat anti-human IMP pyrophosphorylase globulin (AB) was obtained by adding solid ammonium sulfate to immune serum (50% saturation). The reaction was carried out by adding to DEAE-cellulose treated blood to appropriate amounts of DEAE-cellulose treated Lesch-Nyhan blood and potassium phosphate buffer 0.01 M, pH 7.0, to a final volume of 250 [xl. The mixture was incubated at 4 for 16 hours, centrifuged at 2000 RPM for 30 minutes and 50 Jil of the supernatant was assayed for IMP pyrophosphorylase activity. 

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