Degree Name

PhD (Doctor of Philosophy)

Program

Biomedical Sciences

Date of Award

May 1993

Abstract

Tryptases are trypsin-like enzymes found in mast cell granules. Although in vivo substrates have not been positively identified, tryptases cleave a limited number of potential physiological substrates in vitro, including high molecular weight kininogen (HMWK) and vasoactive intestinal peptide (VIP). Purified human lung mast cell tryptase (HLT) apparently exists as a tetramer with an M$\sb{\rm r}$ of 135-144 kDa by gel filtration, whereas SDS-PAGE yielded two bands of M$\sb{\rm r}$ 29 Kda and 33 Kda. Tryptases are resistant to inhibition by most natural trypsin inhibitors and display some affinity for heparin. The existence of tryptase isozymes has been implied from the cloning of two tryptase cDNAs from human lung tissue, but distinct isozymes have not been isolated and characterized. This knowledge gap has been filled by isolating and characterizing two electrophoretically different forms of human lung mast cell tryptase, designated high-HLT (high molecular weight HLT) and low-HLT (low molecular weight HLT). These two forms of HLT have been separated by chromatography on a cellulose phosphate column, with the high M$\sb{\rm r}$ form eluting with 10 $\mu$M heparin and the low M$\sb{\rm r}$ form subsequently eluting with 1 M NaCl. Using HMWK and VIP as substrates, these two forms of HLT were found to differ with regard to specificity and rate of cleavage. High-HLT initially cleaved HMWK at a single Arg residue, whereas low-HLT cleaved HMWK simultaneously at multiple sites. Both isozymes cleaved VIP at multiple sites, but differed with regard to the preferential site of cleavage. Low-HLT was, on an active site basis, 25 and 2 times more active than high-HLT on HMWK and VIP, respectively. In addition, gel filtration of the isozymes yielded M$\sb{\rm r}$s of 125 Kda for high-HLT and 28 kDa for low-HLT, indicating tetrameric and monomeric quaternary structures, respectively. Both isozymes were inhibited by human secretory leukocyte proteinase inhibitor (SLPI), but not by other trypsin inhibitors tested. This work provides the first evidence for the existence of distinct tryptase isozymes, with supposedly different in vivo functions, and identification of an inhibitor that may control tryptase activity in vivo.

Document Type

Dissertation - Open Access

Included in

Biochemistry Commons

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