IDENTIFICATION OF 2 FURTHER CATIONIC PEROXIDASE ISOENZYMES SECRETED BY PEANUT CELLS IN SUSPENSION-CULTURE

Two cationic peroxidase isoenzymes have been identified, using ion-exc hange and hydrophobic column chromatography, in the spent media of pea nut cells maintained in suspension culture. One isoenzyme, designated CPz-I, has a molecular mass of 34 000 Da determined by SDS-PAGE and 33 610 +/- 12 Da by MALDI-TOF mass spectrometry and a pI > 9.5 determine d by isoelectric focusing. The N-terminal, and some internal amino aci d sequences from trypsin-generated peptides have been obtained, but da ta-base sequence comparisons have identified no other peroxidases. The second isoenzyme, designated CPz-II, is a glycoprotein as determined by recognition by a monoclonal antibody (mAb 2.23) which is specific f or the xylose/fucose containing complex plant N-glycan structure, and has a molecular mass of 33 500 Da determined by SDS-PAGE and 33 164 +/ - 17 Da determined by MALDI-TOF mass spectrometry, and a basic pI of 9 .0 as determined by isoelectric focusing. N-terminal, and some interna l amino acid sequences from trypsin-generated peptides, have been obta ined and data-base searching revealed highest, but not complete, ident ity with the translated sequence of prxPNC-2, a cDNA isolated previous ly from the same peanut cell suspension culture system. This partial i dentity is confirmed by recognition of CPz-II by an antibody raised ag ainst the protein product of prxPNC-2 obtained by the expression of th e cDNA in Escherichia coli. (C) Elsevier, Paris.