DOI
https://doi.org/10.25772/7E1Y-N341
Defense Date
1986
Document Type
Dissertation
Degree Name
Doctor of Philosophy
Department
Microbiology & Immunology
First Advisor
Shaun Ruddy
Abstract
Purified human C3a was iodinated (125I-C3a) and used to study the interaction of labeled peptide with rat peritoneal mast cells (RMC). Cellular binding of 125I-C3a occurred within 30 sec, followed by a rapid dissociation from the cell. Both the binding of 125I-C3a and the rate of dissociation from the cell were temperature dependent. At 0°C, the binding of 125I-C3a was increased and the rate of dissociation reduced, as compared to 37°C. Once 125I-C3a was exposed to RMC, it lost the ability to rebind to a second batch of RMC. Analysis of the supernatants by trichloroacetic acid (TCA) precipitation and electrophoresis in sodium dodecyl sulfate polyacrylamide gels (SDS PAGE) revealed a decrease in the fraction of 125I precipitable by TCA and the appearance of 125I-C3a cleavage fragments. Pretreatment of RMC with enzyme inhibitors specific for chymotrypsin, but not trypsin, abrogated the degradation of 125I-C3a. Treatment of RMC bearing 125I-C3a with Bis (sulfosuccinimidyl) suberate (BS3) covalently crosslinked the 125I-C3a to chymase, the predominant enzyme found in the secretory granules. Antiserum directed against chymase precipitated 125I-C3a from extracts of RMC treated with BS3. Indirect immunofluorescence of RMC using the 1gG fraction of goat anti-rat chymase showed that chymase is present on the surface of unstimulated cells. Neither purified chymase nor heparin proteoglycan alone had any appreciable effect on 125I-C3a, but together they resulted in prompt degradation of the 125I-C3a. Immunoabsorption of RMC sonicates with specific antibody for chymase completely abrogated the ability of these sonicates to degrade 125I-C3a. Intact RMC were pretreated with serine esterase inhibitors prior to 125I-C3a and BS3 exposure. The cells to which 125I-C3a had been crosslinked to were solublized and analyzed by SDS PAGE and autoradiography. There were three bands visualized, a 35,000 dalton band which was defined as chymase, and two undefined 45,000 and 55,000 dalton bands. The results indicate that 125I-C3a binds to RMC and is promptly degraded by chymase in the presence of heparin proteoglycan. In addition, this proteolysis of 125I-C3a by chymase must be blocked in order to detect plasma membrane C3a binding components on RMC.
Rights
© The Author
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
12-15-2016
Comments
Scanned, with permission from the author, from the original print version, which resides in University Archives.