Studies on the elastase-serum protein interaction. 3. the elastase inhibitors of swine serum with emphasis on the elastase- 2 -macroglobulin interaction

Baumstark, J.S.

Biochimica et Biophysica Acta 309(1): 181-195

1973


ISSN/ISBN: 0006-3002
PMID: 4122642
DOI: 10.1016/0005-2744(73)90330-6
Accession: 044437668

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Abstract
Impregnation of agar gel electrophoretograms of swine serum with elastase (pancreatopeptidase E, Ec 3.4.4.7) followed by overlaying with elastin particles in agar revealed that swine serum contains three protein inhibitors of elastase, an α0/ρ1-globulin, and α2-globulin and a β1-globulin. The α0/ρ1- and α2-globulins are analogous to the α1-antitrypsin and α2-macroglobulin of human serum while the β1- globulin, the principal elastase inhibitor of swine serum, has no apparent human serum counter part. The molecular weights and electrophoretic mobilities of the α0/ρ1-globulin, α2-macroglobulin and the β1-globulin were found, respectively, to be: 72 000, −6.1·10−5 cm2·V−1·s−1; 960 000 (ref. 15), −4.1·10−5 cm−5·V−1·s−1; 83 600, −3.2·10−5 cm2·V−1·s−1. 1 ml of swine serum was capable of binding 0.6 mg of swine elastase. Swine α2-macroglobulin was purified from swine whole serum by fractional precipitation with (NH4)2SO4 followed by ultracentrifugation, Sephadex G-200 gel filtration and DEAE-cellulose column chromatography. An inhibition curve (elastase activity vs increasing amounts of α2-macroglobulin) revealed that 1.13 mole of α2-macroglobulin (mol. wt 960 000) was capable of binding 1.0 mole of elastase (mol. wt 25 000). The dissociation constant for the α2-macroglobulin elastase complex was 4.5·10−9. p H-stat experiments with α2-macroglobulin-elastase mixtures (molar ratio α2-macroglobulin:elastase > 1.0) revealed an appreciable cleavage of peptide bonds within 16 h. Peptide bond cleavage was also demonstrated by gel filtration of 48-h digests on Sephadex G-50 and G-200 and by peptide mapping.