High pressure gel-permeation assay for the proteolysis of human aggrecan by human stromelysin-1: kinetic constants for aggrecan hydrolysis.
The adaptation of an analytical procedure for aggrecan based upon gel-permeation chromatography to an FPLC-based protocol has significantly sped up the analysis. The faster assay has permitted determination of the kinetic constants for digestion of human aggrecan by human stromelysin-1. Monomeric aggrecan appeared to be hydrolyzed by stromelysin-1 to multiple forms with lower molecular weight. The disappearance of high-molecular-weight aggrecan was first-order, showing Km much larger than 2 microM and kc/Km = 4000 M-1 s-1 at pH 7.5. The disappearance of high-molecular-weight aggrecan upon hydrolysis by stromelysin-1 at pH 5.5 was also first-order, with kc/Km = 10,700 M-1 s-1. The disappearance of high-molecular-weight aggrecan at pH 7.5 was first-order for digestion by human leukocyte elastase with kc/Km = 230,000 M-1 s-1, by human cathepsin G with kc/Km = 4200 M-1 S-1, and by human plasma plasmin with kc/Km = 2800 M-1 s-1, all with Km much larger than 2 microM.
Poe
M
M
Department of Enzymology, Merck Research Laboratories, Merck & Co., Rahway, New Jersey 07065.
Stein
R L
RL
Wu
J K
JK
D053674:Aggrecans; D056649:Cathepsin G; D002403:Cathepsins / Q000378:metabolism; D011508:Chondroitin Sulfate Proteoglycans / Q000378:metabolism*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D016326:Extracellular Matrix Proteins; D005341:Fibrinolysin / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D037181:Lectins, C-Type; D019272:Leukocyte Elastase; D019278:Matrix Metalloproteinase 3; D008666:Metalloendopeptidases / Q000235:genetics / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000378:metabolism; D010196:Pancreatic Elastase / Q000378:metabolism; D010446:Peptide Fragments / Q000302:isolation & purification*; D011509:Proteoglycans / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012697:Serine Endopeptidases
D053674:Aggrecans; D011508:Chondroitin Sulfate Proteoglycans; D016326:Extracellular Matrix Proteins; D037181:Lectins, C-Type; D009363:Neoplasm Proteins; D010446:Peptide Fragments; D011509:Proteoglycans; D011994:Recombinant Proteins; D002403:Cathepsins; D012697:Serine Endopeptidases; C534094:CTSG protein, human; D056649:Cathepsin G; D010196:Pancreatic Elastase; D019272:Leukocyte Elastase; D005341:Fibrinolysin; D008666:Metalloendopeptidases; D019278:Matrix Metalloproteinase 3