PhD (Doctor of Philosophy)
Date of Award
Human lung tryptase (HLT), a mast cell derived trypsin-like enzyme, was isolated from whole human lung tissue obtained at autopsy. Increased yields from this purification process allowed extensive characterization of the enzyme. One of the critical steps in the purification scheme was the use of a linear heparin gradient to elute active material from cellulose phosphate. Gel filtration studies in 1.0 M NaCl yielded an apparent M(,r) of 135,000, and subsequent electrophoresis on sodium dodecyl sulfate-polyacrylamide gels demonstrated the presence of two active species with apparent M(,r) = 30,900 and 31,600. Enzymatic activity was sensitive to NaCl concentrations above 0.05 M and was only 50% in 0.15 M NaCl, decreasing to 18% in 0.6 M NaCl. The effects of synthetic and natural inhibitors were studied, confirming the enzyme's trypsin-like characteristics and demonstrating that naturally occurring serum inhibitors are incapable of diminishing its activity. A complete amino acid analysis showed a high tryptophan content. Antisera to human lung tryptase was generated, and the immunological identity of active fractions was investigated. The stability of HLT in various buffer systems was extensively studied, and 10 mM MES buffer, pH 6.1 appeared to provide the best conditions during extended storage and purification. The effect of heparin on the enzyme's activity using the synthetic substrates Z-Lys-SBzl was studied, and heparin concentrations of 10 micromolar stabilized HLT and allowed full expression of activity even at low ionic strengths. In the presence of heparin the enzyme retained full activity after 24 hours at 37(DEGREES)C, whereas in the absence of heparin, activity was lost after 30 min at this temperature. Heparin had a similar effect on HLT's ability to cleave natural substrates such as fibronectin. Assays comparing the activity of HLT on the substrates Z-Lys-SBzl and Z-Arg-SBzl were performed. The K(,m) and V(,max) of HLT for the above substrates were determined. The substrate 4-methylumbelliferyl-p-guanidinobenzoate (MUG-B) has been used to perform an active site titration on HLT, and a k(,cat) of 610/sec was calculated for the substrate Z-Arg-SBzl.
Dissertation - unrestricted
Smith, Timothy J., "Human Lung Tryptase Purification and Characterization" (1985). Electronic Theses and Dissertations. Paper 2796. https://dc.etsu.edu/etd/2796