tailieunhanh - Báo cáo khóa học: Quantitative analysis, using MALDI-TOF mass spectrometry, of the N-terminal hydrolysis and cyclization reactions of the activation 2 process of onconase

Onconase, a member of the ribonuclease superfamily, is a potent cytotoxicagent that isundergoingphase II/IIIhuman clinical trials as an antitumor drug. Native onconase from Rana pipiensand its amphibian homologs have an N-ter-minal pyroglutamyl residue that is essential for obtaining fully active enzymes with their full potential as cytotoxins. When expressed cytosolically in bacteria, Onconase is isolated with an additional methionyl (Met1) residue and glutaminyl insteadof a pyroglutamyl residue at position 1 of the N-terminus and is consequently inactivated | Eur. J. Biochem. 271 1163-1171 2004 FEBS 2004 doi Quantitative analysis using MALDI-TOF mass spectrometry of the N-terminal hydrolysis and cyclization reactions of the activation process of onconase Marc Ribo1 Montserrat Bosch1 Gerard Torrent1 Antoni Benito1 Bruno Beaumelle2 and Maria Vilanova1 1Laboratori d Enginyeria de Proteines Departament de Biologia Facultat de Ciencies Universitat de Girona Girona Spain 2UMR 5539 CNRS Department Biologie-Sante Universite Montpellier II Montpellier France Onconase a member of the ribonuclease superfamily is a potent cytotoxic agent that is undergoing phase II III human clinical trials as an antitumor drug. Native onconase from Rana pipiens and its amphibian homologs have an N-ter-minal pyroglutamyl residue that is essential for obtaining fully active enzymes with their full potential as cytotoxins. When expressed cytosolically in bacteria Onconase is isolated with an additional methionyl Met1 residue and glutaminyl instead of a pyroglutamyl residue at position 1 of the N-terminus and is consequently inactivated. The two reactions necessary for generating the pyroglutamyl residue have been monitored by MALDI-TOF MS. Results show that hydrolysis of Me -1 catalyzed by Aeromonas aminopeptidase is optimal at a concentration of 3 m guanidinium-chloride and at pH . The intramolecular cyclization of glutaminyl that renders the pyroglutamyl residue is not accelerated by increasing the concentration of denaturing agent or by strong acid or basic conditions. However temperature clearly accelerates the formation of pyroglutamyl. Taken together these results have allowed the characterization and optimization of the onconase activation process. This procedure may have more general applicability in optimizing the removal of undesirable N-terminal methionyl residues from recombinant proteins overexpressed in bacteria and providing them with biological and catalytic properties identical to those of the .