Purification and characterization of the 3'-nucleotidase/nuclease from promastigotes of Leishmania donovani.
The surface membrane-associated 3'-nucleotidase/nuclease (3'-N'ase) of Leishmania donovani has been purified from detergent extracted promastigotes by anion and cation exchange, lectin affinity and gel filtration chromatography. SDS-PAGE analysis of the purified enzyme preparation revealed a 43-kDa polypeptide as well as faster migrating bands. These bands co-migrated, following both one- and two-dimensional electrophoretic analyses, with enzyme activity as determined by an in situ 3'-nucleotidase gel activity assay. It is suggested that the lower molecular weight species arise during purification as a result of proteolytic cleavage of the intact 43-kDa enzyme. The 3'-N'ase exhibited a pI of 5.4, as revealed by 2-dimensional gel electrophoresis. The glycoprotein nature of the 3'-N'ase was suggested by its binding to concanavalin A and by its electrophoretic shift following incubation with N-glycanaseR. In nucleotidase and nuclease assays, the 3'-N'ase was most active with 3'-AMP and poly(A), respectively. Both nucleotidase and nuclease activities exhibited broad pH optima with peaks at 8.5 and 7.5, respectively. At pH 8.5 nucleotidase activity was inhibited by EDTA, Zn2+ and thiols, but was insensitive to tartrate, molybdate and fluoride ions, commonly used inhibitors of phosphatases. The properties of the leishmanial 3'-N'ase was similar to the 3'-N'ase purified from purine-starved Crithidia luciliae, a related trypanosomatid protozoan, and to group of nucleases from fungi and germinating plant seedlings.
Campbell
T A
TA
Department of Immunology and Infectious Diseases, Johns Hopkins University School of Hygiene and Public Health, Baltimore, MD 21205.
Zlotnick
G W
GW
Neubert
T A
TA
Sacci
J B
JB
Gottlieb
M
M
D015720:5'-Nucleotidase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D003421:Crithidia / Q000201:enzymology; D015180:Electrophoresis, Gel, Two-Dimensional; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007893:Leishmania donovani / Q000201:enzymology* / Q000254:growth & development; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015800:Protozoan Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity
D008562:Membrane Glycoproteins; D015800:Protozoan Proteins; D015720:5'-Nucleotidase