title: Biochemical characterization of VQ-VII, a cysteine peptidase with broad specificity, isolated from Vasconcellea quercifolia latex.
abstract: The latex from Vasconcellea quercifolia ("oak leaved papaya"), a member of the Caricaceae family, contains at least seven cysteine endopeptidases with high proteolytic activity, which helps to protect these plants against injury. In this study, we isolated and characterized the most basic of these cysteine endopeptidases, named VQ-VII. This new purified enzyme was homogeneous by bidimensional electrophoresis and MALDI-TOF mass spectrometry, and exhibited a molecular mass of 23,984 Da and an isoelectric point >11. The enzymatic activity of VQ-VII was completely inhibited by E-64 and iodoacetic acid, confirming that it belongs to the catalytic group of cysteine endopeptidases. By investigating the cleavage of the oxidized insulin B-chain to establish the hydrolytic specificity of VQ-VII, we found 13 cleavage sites on the substrate, revealing that it is a broad-specificity peptidase. The pH profiles toward p-Glu-Phe-Leu-p-nitroanilide (PFLNA) and casein showed that the optimum pH is about 6.8 for both substrates, and that in casein, it is active over a wide pH range (activity higher than 80 % between pH 6 and 9.5). Kinetic enzymatic assays were performed with the thiol peptidase substrate PFLNA (K m = 0.454 ± 0.046 mM, k cat = 1.57 ± 0.07 s(-1), k cat/K m = 3.46 × 10(3) ± 14 s(-1) M(-1)). The N-terminal sequence (21 amino acids) of VQ-VII showed an identity >70 % with 11 plant cysteine peptidases and the presence of highly conserved residues and motifs shared with the "papain-like" family of peptidases. VQ-VII proved to be a new latex enzyme of broad specificity, which can degrade extensively proteins of different nature in a wide pH range.
authors.affiliation: Departamento de Ciencias Básicas y Experimentales, Universidad Nacional del Noroeste de Provincia de Buenos Aires, Junín, Argentina.
authors.forename: María José
authors.initials: MJ
authors.lastname: Torres
authors.forename: Sebastián Alejandro
authors.initials: SA
authors.lastname: Trejo
authors.forename: Claudia Luisa
authors.initials: CL
authors.lastname: Natalucci
authors.forename: Laura María Isabel
authors.initials: LM
authors.lastname: López
mesh_terms: D000595:Amino Acid Sequence; D029441:Carica / Q000737:chemistry / Q000201:enzymology*; D020134:Catalytic Domain; D057056:Cysteine Proteases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000378:metabolism; D007700:Kinetics; D007840:Latex / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D059748:Proteolysis; D013379:Substrate Specificity; D017186:Titrimetry
chemical_list: D007328:Insulin; D007840:Latex; D010940:Plant Proteins; D057056:Cysteine Proteases