title: Kinetic constants for the hydrolysis of aggrecan by the papaya proteinases and their relevance for chemonucleolysis.
abstract: The four known proteinases from papaya latex, namely papain (EC 3.4.22.2), chymopapain (EC 3.4.22.6), caricain (EC 3.4.22.30), and glycyl endopeptidase (EC 3.4.22.25), were purified to homogeneity and fully characterized by single radial immunodiffusion and active-site titration. A modified HPLC gel permeation assay was used to determine the kinetic constants for aggrecan hydrolysis by the papaya proteinases. The disappearance of intact aggrecan monomer was first-order, indicating that for the four enzymes studied the Km was much larger than 0.5 microM and that kcat/Km = 1.2 +/- 0.1 x 10(6) M-1 s-1 for chymopapain, 1.20 +/- 0.08 x 10(6) M-1 s-1 for caricain, 0.90 +/- 0.02 x 10(6) M-1 s-1 for papain, and 0.120 +/- 0.005 x 10(6) M-1 s-1 for glycyl endopeptidase. Chymodiactin, the chymopapain preparation used for chemonucleolysis, consists of a mixture of chymopapain (70%), caricain (20%), and glycyl endopeptidase (4%). The rate constant for the aggrecan hydrolysis by such a mixture was not significantly different from the rate constant for pure chymopapain. As a result of these observations, we predict that pure chymopapain could replace partially purified chymopapain preparations for chemonucleolysis.
authors.affiliation: Department of Pharmaceutics, University of Gent, Belgium.
authors.forename: P M
authors.initials: PM
authors.lastname: Dekeyser
authors.forename: D J
authors.initials: DJ
authors.lastname: Buttle
authors.forename: B
authors.initials: B
authors.lastname: Devreese
authors.forename: J
authors.initials: J
authors.lastname: Van Beeumen
authors.forename: J
authors.initials: J
authors.lastname: Demeester
authors.forename: A
authors.initials: A
authors.lastname: Lauwers
mesh_terms: D053674:Aggrecans; D010450:Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D016326:Extracellular Matrix Proteins; D006868:Hydrolysis; D007404:Intervertebral Disc Chemolysis; D007700:Kinetics; D037181:Lectins, C-Type; D010944:Plants / Q000201:enzymology*; D011509:Proteoglycans / Q000378:metabolism*
chemical_list: D053674:Aggrecans; D016326:Extracellular Matrix Proteins; D037181:Lectins, C-Type; D011509:Proteoglycans; D010450:Endopeptidases