A semicontinuous, high-performance liquid chromatography-based assay for stromelysin.
A search for low molecular weight peptide substrates for the metalloendoproteinase, human fibroblast stromelysin, resulted in the discovery that substance P (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2) is a substrate for this enzyme and is cleaved exclusively at the Gln6-Phe7 bond. On the basis of this observation, a semicontinuous HPLC-based assay was developed that monitors the production of the hydrolysis product, fragment 7-11 (SP7-11). Steady-state velocities for the production of SP7-11 have been determined as a function of substrate concentration and obey simple, Michaelis-Menten kinetics. For a 1-ml reaction volume, Vmax = (2.4 nmol SP7-11/min)/micrograms protein and Km = 0.38 mM.
Harrison
R
R
Department of Enzymology, Merck Sharp and Dohme Research Laboratories, Rahway, New Jersey 07065.
Teahan
J
J
Stein
R
R
D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004789:Enzyme Activation / Q000187:drug effects; D006868:Hydrolysis; D007700:Kinetics; D019278:Matrix Metalloproteinase 3; D008666:Metalloendopeptidases / Q000032:analysis* / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D010662:Phenylmercuric Acetate / Q000031:analogs & derivatives / Q000494:pharmacology; D013373:Substance P / Q000378:metabolism; D013379:Substrate Specificity
D010446:Peptide Fragments; D013373:Substance P; C061335:substance P (7-11); C010024:4-aminophenylmercuriacetate; D008666:Metalloendopeptidases; D019278:Matrix Metalloproteinase 3; D010662:Phenylmercuric Acetate