Purification and analysis of DNases of Tritrichomonas foetus: evidence that these enzymes are glycoproteins.
Tritrichomonas foetus is the causative agent of trichomoniasis. In cattle, infection results in economic losses to the beef and dairy industries due to abortion and infertility. Soluble DNases of T. foetus that play a role in pathogenesis and are potential therapeutic targets, were extracted and purified utilising lectin affinity chromatography. The DNases were bound to and eluted from Concanavalin A (Con A)-sepharose indicating that they are glycoproteins with alpha-linked mannose or glucose residues. The nature of the glycans carried on the eluted proteins in the fraction containing DNase activity was assessed using an enzyme-linked lectin assay. The lectin binding studies predict the presence of both N- and O-type glycans. Manganese was a potent (33%) activator of the DNase(s) whereas zinc inhibited enzyme activity by approximately 66%. The DNase(s) had a pH optimum of 4 and a molecular weight of 160 kDa. The DNase(s) were able to completely degrade DNA from animal, plant, fungal, yeast and bacterial sources, but did not significantly degrade RNA.
Greenwell
Pamela
P
School of Biosciences, University of Westminster, 115 New Cavendish Street, London, UK. greenwp@wmin.ac.uk
Younes
Maha
M
Rughooputh
Sanjiv
S
D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods; D003851:Deoxyribonucleases / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D004587:Electrophoresis, Agar Gel / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D037102:Lectins / Q000032:analysis / Q000737:chemistry; D008562:Membrane Glycoproteins / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D008970:Molecular Weight; D012685:Sepharose / Q000031:analogs & derivatives; D016835:Tritrichomonas foetus / Q000201:enzymology*
D037102:Lectins; D008562:Membrane Glycoproteins; C028326:concanavalin A-sepharose; D012685:Sepharose; D003851:Deoxyribonucleases