Purification of a cysteine proteinase from Carica candamarcensis L. and cloning of a genomic putative fragment coding for this enzyme.
We describe the purification of a cysteine proteinase from latex of Carica candamarcensis, hereby designated CC23. The enzyme has been purified by ion-exchange chromatography and behaves electrophoretically as a monomer of M(r) 23,000 and optimal pH of 8.0. It displays a basic isoelectric point, has one cysteine residue in the active site by titration with E-64, confirmed by DNA sequencing, and responds to proteinase inhibitors as a classic cysteine proteinase. The K(m) and k(cat)/K(m) for CC23 using BAPNA were respectively 14.7 +/- 1.8 x 10(-4) M and 1.3 x 10(3) M(-1) s(-1). Therefore, the catalytic efficiency of CC23 is sixfold higher than that of CC-I, another proteinase from the same plant. DNA primers were designed to amplify by PCR a genomic sequence related to this enzyme. An 895-bp DNA fragment was cloned and sequenced. It shows strong homology with chymopapain isoform IV from C. papaya. The translated sequence is similar to that of chymopapain isoform II (73%) and CC-III (77%) from C. candamarcensis.
Pereira
M T
MT
Departamento de Bioquímica e Imunologia, Institutto de Ciências Biológicas, Universidad Federal de Minas Gerais, Brazil.
Lopes
M T
MT
Meira
W O
WO
Salas
C E
CE
D000595:Amino Acid Sequence; D001483:Base Sequence; D029441:Carica / Q000201:enzymology* / Q000235:genetics*; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000235:genetics* / Q000302:isolation & purification*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018745:Genome, Plant; D006023:Glycoproteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000302:isolation & purification*; D007460:Iodoacetamide / Q000494:pharmacology; D007700:Kinetics; D007840:Latex / Q000737:chemistry; D017669:Mercury Compounds / Q000494:pharmacology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000302:isolation & purification*; D017386:Sequence Homology, Amino Acid
D004791:Enzyme Inhibitors; D006023:Glycoproteins; D007840:Latex; D017669:Mercury Compounds; D010446:Peptide Fragments; C090775:Carica candamarencis glycosylated cysteine proteinase; D003546:Cysteine Endopeptidases; D007460:Iodoacetamide