Isolation and characterization of a cysteine protease from the latex of Araujia hortorum fruits.
A new protease (araujiain h I) was purified to mass spectroscopy homogeneity from the latex of Araujia hortorum Fourn. (Asclepiadaceae) fruits by ultracentrifugation and ion exchange chromatography. The enzyme has a molecular mass of 24,031 (mass spectrometry) and an iso-electric point higher than 9.3. The optimum pH range for casein hydrolysis was 8.0-9.5. The enzyme showed remarkable caseinolytic activity at high temperatures, although its thermal stability decayed rapidly. The proteinase was activated by thiol compounds and inhibited by common thiol-blocking reagents, particularly E-64 and HgCl2, suggesting the enzyme belongs to the cysteine protease family. The concentration of active sites as determined by titration with E-64 was 3.3 microM. When assayed on N-alpha-CBZ-amino acid-p-nitrophenyl esters, the enzyme showed higher preference for the glutamine derivative, followed by those of alanine, asparagine, glycine, and leucine, in decreasing order. Partial homology (36-48%) with other plant cysteine proteinases was observed in an internal fragment obtained by Protease V8 treatment.
Priolo
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N
Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, Argentina. priolo@biol.unlp.edu.ar
Morcelle del Valle
S
S
Arribére
M C
MC
López
L
L
Caffini
N
N
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002364:Caseins / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005638:Fruit / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007700:Kinetics; D007840:Latex / Q000737:chemistry*; D008969:Molecular Sequence Data; D010946:Plants, Medicinal / Q000201:enzymology*; D016415:Sequence Alignment; D014461:Ultracentrifugation
D002364:Caseins; D015853:Cysteine Proteinase Inhibitors; D007840:Latex; D003546:Cysteine Endopeptidases