PSEUDOMONAS-PROTEASE - PURIFICATION, PARTIAL CHARACTERIZATION, AND ITS EFFECT ON COLLAGEN, PROTEOGLYCAN, AND RABBIT CORNEAS

Abstract

The extracellular protease of a virulent strain of P. aeruginosa was purified by DEAE-cellulose chromatography in 2 steps. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the purified enzyme revealed a single band and the enzyme was shown to be the major component of the bacterial filtrate. The protease was fully inhibited by Na2 EDTA, 1,10-o-phenanthroline, L-cysteine and Zn2+ but was insensitive to diisopropylphosphofluoridate. The elastase substrates orcein-elastin and acetyl-L-alanyl-L-alanyl-L-alanine-methyl ester were degraded by the enzyme. The protease activity toward soluble and insoluble collagen was limited to the telopeptide region of the collagen molecule. With soluble collagen, conversion of the .beta. and .gamma. chains into monomeric .alpha. chains was observed. About 60% of the total proteoglycans and 1.5% of the total collagen were solubilized from rabbit corneas following incubation with the enzyme and the solubilized products were nondialyzable. The purified protease has little or no collagenolytic activity and dissolution of the cornea by Pseudomonas protease infection results essentially from the degradation of the protein backbone of the corneal proteoglycans.