Rat Mast Cell Proteinase II (RMCPII) from mucosal mast cell was titrated into rat serum and the resulting serpin:enzyme complex (SEC) was purified by affinity chromatography on anti-RMCPII Sepharose 4B and by MonoQ anion exchange. The purified complex was used to raise polyclonal antibodies which, after cross-absorption against RMCPII Sepharose 4B, were specific for serpin and were used to affinity purify two rat serpin molecules (RSI and RSII) which inhibit RMCPII in rat serum. The kinetic and thermodynamic constants characterising the interaction between RMCPII and RSI and RSII are: k<SUB>ass</SUB> 2.2.x10<SUP>5</SUP> M<SUP>-1</SUP>s<SUP>-1</SUP> and 1.65x10<SUP>5</SUP>M<SUP>-1</SUP>s<SUP>-1</SUP> respectively; K<SUB>i</SUB>, 3.6x10<SUP>-10</SUP>M and 1.0x10<SUP>-9</SUP>M; k<SUB>diss</SUB>, 7.9x10<SUP>-5</SUP>s<SUP>-1</SUP> and 1.65x10<SUP>-4</SUP>s<SUP>-1</SUP>. Amino-terminal sequence analysis indicated that RSI and II were distinct, differing at the N-terminal residues and were products of the rat SPI-1 locus. Rat Mast Cell Proteinase I (RMCPI) from connective tissue mast cells cleaved both RSI and RSII and was not inhibited. Further antibodies were generated against RSI and II, and partially successful attempts were made to raise a monoclonal antibody against rat serpin in complex with Mouse Mast Cell Proteinase Ie. Two polyclonal antibody preparations, raised in rabbit and sheep, were used to develop an ELISA that was specific for rat serpins, although no assay developed in this work would differentiate RSI from RSII. The change in serpin concentrations in serum and perfused tissues during helminth infection was monitored with the ELISA. Serpins concentrations in control plasma were determined to be 3mg/mL. Helminth infection caused a significant (p< 0.001) increase in this serpin concentration by day 7 of infection.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:660638 |
| Date | January 1993 |
| Creators | Pirie-Shepherd, Steven Robert |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/20112 |
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