Two forms of L-asparaginase in Tetrahymena thermophila.
In T. thermophila two forms of L-asparaginase (EC 3.5.1.1) were extracted and purified to near homogeneity which are associated with membranes. These two forms of L-asparaginase, I or II, act optimally at pH 8.6 and do not present any glutaminase or kinase activity. Both activities reach maximal values at the stationary phase of growth of T. thermophila. L-Asparaginases are solubilized by treatment of the particulates with 2% w/v Triton X-100 and then by sodium phosphate buffer pH 8.0. Both forms cross reacted with antibodies raised against T. pyriformis L-asparaginase and show isoelectric points 7.4 and 8.2. Among the metals tested, Ca2+ is the most effective in activating L-asparaginase I or II activity. Sorbitol alone up to 30% w/v in the assay mixture activates more than 10 x fold the activity of L-asparaginase II. Incubation of L-asparaginase I or II with increasing concentration of phospholipase C results in gradually loss of their activities. The relative effectiveness of a variety of phospholipids to reconstitute enzyme activity is presented as well.
Laboratory of Biochemistry, Faculty of Chemistry, Aristotelian University of Thessaloniki, Greece.
I K
IK
Tsavdaridis
D C
DC
Triantafillidou
D A
DA
Kyriakidis
D000818:Animals; D000918:Antibody Specificity; D001215:Asparaginase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology; D002846:Chromatography, Affinity; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017830:Octoxynol; D010743:Phospholipids / Q000494:pharmacology; D012995:Solubility; D016808:Tetrahymena thermophila / Q000201:enzymology* / Q000254:growth & development
D007527:Isoenzymes; D010743:Phospholipids; D017830:Octoxynol; D001215:Asparaginase; D002118:Calcium