Purification and characterization of purine nucleoside phosphorylase from developing embryos of Hyalomma dromedarii.
Purine nucleoside phosphorylase from Hyalomma dromedarii, the camel tick, was purified to apparent homogeneity. A molecular weight of 56,000 - 58,000 was estimated for both the native and denatured enzyme, suggesting that the enzyme is monomeric. Unlike purine nucleoside phosphorylase preparations from other tissues, the H. dromedarii enzyme was unstable in the presence of beta-mercaptoethanol. The enzyme had a sharp pH optimum at pH 6.5. It catalyzed the phosphorolysis and arsenolysis of ribo- and deoxyribo-nucleosides of hypoxanthine and guanine, but not of adenine or pyrimidine nucleosides. The Km values of the enzyme at the optimal pH for inosine, deoxyinosine, guanosine, and deoxyguanosine were 0.31, 0.67, 0.55, and 0.33 mM, respectively. Inactivation and kinetic studies suggested that histidine and cysteine residues were essential for activity. The pKa values determined for catalytic ionizable groups were 6-7 and 8-9. The enzyme was completely inactivated by thiol reagents and reactivated by excess beta-mercaptoethanol. The enzyme was also susceptible to pH-dependent photooxidation in the presence of methylene blue, implicating histidine. Initial velocity studies showed an intersecting pattern of double-reciprocal plots of the data, consistent with a sequential mechanism.
Kamel
M Y
MY
Molecular Biology Laboratory, National Research Centre, Dokki, Cairo, Egypt.
Fahmy
A S
AS
Ghazy
A H
AH
Mohamed
M A
MA
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002848:Chromatography, DEAE-Cellulose; D003545:Cysteine / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006639:Histidine / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D009705:Nucleosides / Q000378:metabolism*; D010084:Oxidation-Reduction; D011683:Purine-Nucleoside Phosphorylase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013987:Ticks / Q000196:embryology / Q000201:enzymology*
D000596:Amino Acids; D007527:Isoenzymes; D009705:Nucleosides; D013439:Sulfhydryl Reagents; D006639:Histidine; D011683:Purine-Nucleoside Phosphorylase; D003545:Cysteine