Human mast cell proteases: activity assays using thiobenzyl ester substrates.
Human mast cells contain proteases that are important functional components and serve as markers of mast cell activation or degranulation. Although tryptase is the best recognized mast cell protease, chymase and Cathepsin G also are found in some human mast cells. Methods for measuring the activities of these enzymes using sensitive synthetic peptide thiobenzyl ester substrates are described in this chapter. Using a visible plate reader with kinetic software femtomole quantities of these proteases can be measured. These methods are demonstrated in assays of tryptase and chymase in cell-free extracts of the HMC-1 and 5C6 human mast cell lines, as well as in extracts of cord blood derived mast cells.
Johnson
David A
DA
Department of Biochemistry and Molecular Biology, Quillen College of Medicine, East Tennessee State University, Johnson City, TN, USA.
D002460:Cell Line; D053818:Chymases; D004228:Dithionitrobenzoic Acid / Q000737:chemistry; D004952:Esters / Q000737:chemistry; D006801:Humans; D008407:Mast Cells / Q000737:chemistry / Q000201:enzymology*; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D013439:Sulfhydryl Reagents / Q000737:chemistry; D053802:Tryptases
D004952:Esters; D013439:Sulfhydryl Reagents; D004228:Dithionitrobenzoic Acid; D012697:Serine Endopeptidases; D053818:Chymases; D053802:Tryptases