Purification and characterization of mast cell tryptase and chymase from human tissues.
Mast cells are key effector cells of the allergic response. When stimulated by specific allergen through the high-affinity IgE receptors or through other stimuli, these cells release a number of potent mediators of inflammation. Amongst these are the serine proteases tryptase and chymase. In humans, tryptase is the most abundant mediator stored in mast cells. Chymase is present in more moderate amounts in a subpopulation of mast cells (MC(TC)). This subtype of mast cells predominates in connective tissue, whereas the other major subtype, the MC(T), predominates in mucosal tissue. Both proteases have been shown to act on specific extracellular proteins and peptides, as well as to alter the behavior of various cell types. Inhibitors of tryptase have been found to be efficacious in animal and human models of asthma, and both proteases are currently being investigated as potential targets for therapeutic intervention. Such pharmacological, physiological, and biochemical studies require the availability of purified tryptase and chymase. In this chapter, we shall describe procedures for the purification of tryptase and chymase from human tissues and provide protocols for monitoring purification and characterization of the final product. The preparation of recombinant proteases will not be covered, though some of the procedures described may be readily adapted for their purification from recombinant expression systems. The procedures described here have been developed for the purification of the human proteases and will require some modification if applied to purify mast cell proteases from the tissues of other species.
McEuen
Alan R
AR
Immunopharmacology Group, Southampton General Hospital, Southampton, UK.
Walls
Andrew F
AF
D002457:Cell Extracts / Q000737:chemistry / Q000302:isolation & purification; D002853:Chromatography, Liquid; D053818:Chymases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D004992:Ethics, Medical; D006801:Humans; D008168:Lung / Q000737:chemistry / Q000201:enzymology*; D008407:Mast Cells / Q000201:enzymology*; D016272:Occupational Health; D012867:Skin / Q000737:chemistry / Q000201:enzymology*; D053802:Tryptases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D002457:Cell Extracts; D053818:Chymases; D053802:Tryptases