Partial purification and characterization of guanine aminohydrolase fromtrypanosoma cruzi |
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Authors: | Linda L. Nolan |
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Affiliation: | (1) Department of Microbiology, University of Massachusetts, 01003 Amherst, Massachusetts, USA |
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Abstract: | Guanine deaminase (guanine aminohydrolase, EC 3.5.4.3) catalyzes the hydrolytic deamination of guanine to xanthine. A rapid procedure for the partial purification of guanine deaminase fromTrypanosoma cruzi using granulated bed electrofocusing was developed. Supernatants of cell sonicates (40,000 g) were subjected to electrofocusing with a broad range ampholyte (pH 4–9). Sections of the gel were eluted and assayed for xanthine production. Active fractions were pooled, concentrated, and again subjected to electrofocusing with a pH 5–7 range ampholyte. This procedure resulted in over 240-fold purification. The compounds 4-amino-5-imidazolecarboxamide andN6-methyladenine were found to be potent competitive inhibitors of the enzyme. Their respective Ki values were 3.5×10–6M and 9.5×10–6M. Irreversible inactivation of the enzyme was observed upon incubation withp-chloromercurophenylsulfonic acid andN-ethyl-maleamide at 5.0×10–4M. The enzyme was labile to heat; a substantial loss of activity occurred upon incubation at 55°C for 5 min. A broad pH range of activity (pH 7.5–8.5) was observed in Tris, citrate, and phosphate buffers. |
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