An inducible 3'-nucleotidase/nuclease from the trypanosomatid Crithidia luciliae. Purification and characterization.
Several species of protozoan parasites of the family Trypanosomatidae have a surface membrane-associated enzyme which is capable of hydrolyzing extracellular 3'-nucleotides and nucleic acids, thereby aiding in the acquisition of nutritionally required purines and Pi from their hosts. In Crithidia luciliae, this 3'-nucleotidase/nuclease previously has been shown to be highly regulated as purine and/or Pi starvation of this trypanosomatid leads to as much as a 1000-fold increase in enzyme activity. We have purified the enzyme to apparent homogeneity from detergent extracts of purine-starved C. luciliae by heparin-agarose chromatography followed by Mono Q and Mono S fast protein liquid chromatography. The enzyme had an apparent molecular weight of 43,000 and a pI of approximately 5.8. The enzyme displayed broad pH optima, with peaks at 8.0, for both nucleotidase and nuclease activities. The pH optima shifted to lower values when the activity was assayed in the presence of sulfhydryl reagents. The enzyme was most active with 3'-AMP and poly(A) in nucleotidase and nuclease assays, respectively. As a nuclease the enzyme hydrolyzed RNA at a faster rate than single-stranded DNA with no detectable hydrolysis of double-stranded DNA. The loss of enzyme activity which occurred upon storage at acid pH was prevented by the inclusion of Zn2+ in storage buffers. The physicochemical and kinetic properties of this trypanosomatid enzyme suggest that it is similar to the class I nucleases found in fungi and in germinating seedlings of higher plants.
Neubert
T A
TA
Department of Immunology and Infectious Diseases, Johns Hopkins University School of Hygiene and Public Health, Baltimore, Maryland 21205.
Gottlieb
M
M
D000818:Animals; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D002853:Chromatography, Liquid; D003421:Crithidia / Q000201:enzymology*; D004229:Dithiothreitol / Q000494:pharmacology; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D009708:Nucleotidases / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism; D009711:Nucleotides / Q000378:metabolism; D013379:Substrate Specificity
D009711:Nucleotides; D009708:Nucleotidases; C040470:3'-nucleotidase; D004229:Dithiothreitol