GSBS Dissertations and Theses

Approval Date

March 1986

Document Type

Doctoral Dissertation

Department

Graduate School of Biomedical Sciences, Biochemistry

Subjects

Trypsin Inhibitors; Hemolymph; Biochemistry; Trypsin Inhibitors; Academic Dissertations; Dissertations, UMMS

Abstract

Trypsin inhibitory activity from the hemolymph of the tobacco hornworm, Manduca sexta, was purified by affinity chromatography on immobilized trypsin and resolved into two fractions with molecular weights of 13700 (inhibitor A) and 8000 (inhibitor B) by Sephadex G-75 gel filtration. SDS-polyacrylamide gel electrophoresis under non-reducing conditions gave a molecular weight estimate of 15000 for inhibitor A and 8500 for inhibitor B. Electrophoresis of these inhibitors under reducing conditions on polyacrylamide gels gave molecular weight estimates of 8300 and 9100 for inhibitor A and inhibitor B, respectively, suggesting that inhibitor A is a dimer. Isoelectro-focusing on polyacrylamide gels focused inhibitor A as a single band with pI of 5.7, whereas inhibitor B was resolved into two components with pIs of 5.3 and 7.1. Both inhibitors A and B are stable at 100° C and at pH 1.0 for at least 30 minutes, but both are inactivated by dithiothreitol even at room temperature and non-denaturing conditions. Inhibitors A and B inhibit trypsin, chymotrypsin, plasmin, and thrombin but they do not inhibit elastase, papain, pepsin, subtilisin BPN' and thermolysin. In fact, subtilisin BPN' completely inactivated both inhibitors A and B. Inhibitor A and inhibitor B form stable complexes with trypsin. Stoichiometric studies showed that inhibitor A combines with trypsin and chymotrypsin in a 1:1 molar ratio. The inhibition constants (Ki) for trypsin and chymotrypsin inhibition by inhibitor A were estimated to be 1.45 x 10-8 M and 1.7 x 10-8 M, respectively. Inhibitor A in complex with chymotrypsin does not inhibit trypsin (and vice versa) suggesting that inhibitor A has a common binding site for trypsin and chymotrypsin. The amino terminal amino acid sequences of inhibitors A and B revealed that both these inhibitors are homologous to the bovine pancreatic trypsin inhibitor (Kunitz) .

Quantitation of the trypsin inhibitory activity in the hemolymph of the larval and the pupal stages of Manduca sexta showed that the trypsin inhibitory activity decreased from larval to the pupal stage. Further, inhibitor A at the concentration tested caused approximately 50% reduction in the rate of proteolytic activation of prophenoloxidase in a hemocyte lysate preparation from Manduca sexta, suggesting that inhibitor A may be involved in the regulation of prophenoloxidase activation. However, inhibitor B was not effective even at three times the concentration of inhibitor A. Since activation of prophenoloxidase has been suggested to resemble the activation of alternative pathway of complement, the effect of inhibitors A and B and the hemolymph of Manduca sexta on human serum alternative pathway complement activity was evaluated. The results showed that, although inhibitors A and B do not affect human serum alternative complement pathway, other proteinaceous component(s) in Manduca sexta hemolymph interact(s) and cause(s) an inhibition of human serum alternative complement pathway when tested using rabbit erythrocyte hemolytic assay.

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