Purification and characterization of a novel transglutaminase from filarial nematode Brugia malayi.
A transglutaminase (pTGase) was purified from filarial nematode, Brugia malayi. The steps used for purification were thermoprecipitation, ammonium sulfate precipitation, gel filtration on Superose 12 HR 10/30, ion-exchange chromatography on a Mono-Q column and further gel filtration on Superose 12 HR 10/30. The last step yielded an electrophoretically homogenous enzyme protein with 2200-fold purification and a reproducible yield of approximately 20%. The purified enzyme had a molecular mass of 56 kDa, specific activity of 2.25 U/mg protein and an isoelectric point of 7.2. The enzyme was active in the basic pH range with an optimum activity at pH 8.5. The pTGase activity was Ca(2+)-dependent and was inhibited by ammonia, primary amines, EDTA, and -SH group blocking reagents. The enzyme activity was also inhibited by high salt (NaCl and KCl) concentrations, detergents, metal ions, and organic solvents. Ampholine (pH 6-8) at 1% (by vol.) caused about 20% inhibition of pTGase activity but at 3% (by vol.) the inhibition increased up to 80%. Similarly, the micromolar concentrations of GTP inhibited the enzyme activity only moderately but at millimolar concentration a significant inhibition was observed. The stability of the pTGase was not affected by 0.1% SDS or other physical parameters such as freezing and thawing. Further, the pTGase was found to be highly thermostable (stable at 60 degrees C for several hours) with optimum activity observed at 55 degrees C. The distinct substrate specificity, unique N-terminal sequence along with the other physico-chemical properties studied, suggested that pTGase is a novel member of transglutaminase family.
Department of Clinical Investigation, University of Texas, M.D. Anderson Cancer Center, Houston.
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Singh
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Mehta
D000818:Animals; D017178:Brugia malayi / Q000201:enzymology*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors; D004795:Enzyme Stability; D005260:Female; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008970:Molecular Weight; D013379:Substrate Specificity; D011503:Transglutaminases / Q000737:chemistry / Q000302:isolation & purification*
D004791:Enzyme Inhibitors; D011503:Transglutaminases