ATP: Urea Amidolyase (ADP) (Candida utilis)

Robert J. Roon, Bruce Levenberg

Research output: Contribution to journalArticlepeer-review

15 Scopus citations

Abstract

This chapter describes the assay, purification, and properties of urea amidolyase. Urea serves as the sole nitrogen source for the growth of certain yeasts and unicellular green algae that contain no detectable urease activity. Such cells possess urea amidolyase, which catalyzes Mg2+- and K+-dependent cleavage of a mole of urea to two moles of ammonia and one mole of carbon dioxide, concomitant with the breakdown of a mole of adenosine triphosphate (ATP) to adenosine diphosphate (ADP) and inorganic phosphate. The formation of urea amidolyase is repressed by ammonia. Two types of assays are recognized for routine use. The first involves the measurement of the rate of release of 14CO2 from urea-14C and offers the advantage of convenience and high sensitivity. This assay is, particularly useful with crude extracts, because it is largely unaffected by the presence of contaminating enzymes, such as myokinase, ATPase, and 2,4-Dinitrophenylhydrazine (DPNH) oxidase. The second method is based on a coupled spectrophotometric determination of ADP using phosphoenolpyruvate, pyruvate kinase, and lactate dehydrogenase. The decrease in absorbance at 340 mμ, resulting from the oxidation of DPNH, which accompanies the conversion of pyruvate to lactate, allows a continuous determination of the rate of cleavage of ATP to ADP. This method is limited to purified amidolyase preparations, because cruder fractions often contain high levels of ATPase and other interfering activities. In Tris buffers, the optimum pH for amidolyase activity (assay method 1) is 7.8–7.9.

Original languageEnglish (US)
Pages (from-to)317-324
Number of pages8
JournalMethods in enzymology
Volume17
DOIs
StatePublished - Jan 1 1970
Externally publishedYes

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