The purine-2-deoxyribonucleosidase from Crithidia luciliae. Purification and trans-N-deoxyribosylase activity.
Crude extracts of Crithidia luciliae catalysed a deoxyribosyl transfer from purine deoxynucleosides to free purine bases. Fractionation of a 0-80% (NH4)2SO4 fraction from C. luciliae on DEAE-cellulose resulted in the separation of three nucleosidase activities. Two of these were ribonucleosidases, one specific for inosine, uridine and xanthosine and the other for inosine and guanosine, whereas the third activity was specific for purine deoxyribonucleosides. This pattern is similar to that found in Leishmania donovani. Significant deoxyribosyltransferase activity was, however, associated with the purine-2'-deoxyribonucleosidase from C. luciliae. The purine-2'-deoxyribonucleosidase was purified to homogeneity by a six-step procedure involving (NH4)2SO4 fractionation and chromatography on DEAE-cellulose, hydroxyapatite, Sephadex G-75, and a chromatofocusing resin. The purified enzyme migrated as a single band of 17 kDa on SDS/polyacrylamide gel electrophoresis. The enzyme catalysed the hydrolysis of deoxyinosine, deoxyguanosine and deoxyadenosine with Km values of 80 +/- 10.5 microM, 20.7 +/- 3.2 microM and 17.3 +/- 5.3 microM, respectively, and V values for these substrates in the ratio 1:0.5:0.39. The pH optimum for deoxyribosyl transfer from deoxyinosine to guanine was at pH 7.7, while deoxyinosine hydrolysis in the presence of guanine was optimal in the range pH 6-7. During the synthesis of deoxyinosine from hypoxanthine and deoxyadenosine two products were formed. One of these coeluted with deoxyinosine on HPLC, while the second was tentatively identified as the positional isomer, 7-(beta-D-2'-deoxyribofuranosyl)hypoxanthine.
Steenkamp
D J
DJ
Department of Chemical Pathology, University of Cape Town Medical School, South Africa.
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003421:Crithidia / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D010430:Pentosyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet
D010430:Pentosyltransferases; C021888:nucleoside deoxyribosyltransferase