Purification and Characterization of Mast Cell Tryptase and Chymase from Human Tissues

Abstract

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.

Mast cell chymase impairs bronchial epithelium integrity by degrading cell junction molecules of epithelial cells

“…Prior written consent was obtained from patients by Changzhou general hospital, and the study was approved by the Changzhou University Bio‐Medicine Ethics Committee. The procedure was described by McEuen and Walls . The total protein concentration of purified chymase was measured by bicinchoninic acid protein assay (Sigma, Pool, UK); its purity was assessed by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE), and with silver staining to show the single clear band (30 kDa); its activity was determined using the substrate N‐methoxysuccinyl‐Ala‐Ala‐Pro‐Val 4‐nitroanilide (AAPFpNA) (Sigma) in U/mg/mL and characterized by Western blotting using human chymase‐specific antibody CC1 (Immunopharmacology Group, Southampton).…”

Human lung mast cells modulate the functions of airway smooth muscle cells in asthma

“…Mast cells are a potent source of preformed mediators, as well as many cytokines, chemokines and other newly synthesized lipid mediators. They differ from other inflammatory cells in the lung by releasing large amounts of the proteases tryptase and, depending on location, chymase (16, 19). It is well known that numerous MC‐derived mediators, when used individually, directly affect ASM function [reviewed in (20, 21)].…”

Inhibitory Effects of Cortex Dictamni Aqueous Extract on Dipeptidyl Peptidase I and Chymase Activities and the Screening of Active Ingredients in Cortex Dictamni Based on Molecular Docking Technique

“…where v is the initial velocity of the enzyme reaction, Vmax is the maximal velocity, [S] is the substrate concentration, and Km is the Michaelis-Menten constant (concentration of substrate at 0.5 of Vmax) (A.R. Mceuen et al, 2008 ).…”