June 1977
Volume 16, Issue 6
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Articles  |   June 1977
Pseudomonas protease. Purification, partial characterization, and its effect on collagen, proteoglycan, and rabbit corneas.
Investigative Ophthalmology & Visual Science June 1977, Vol.16, 488-497. doi:
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      E Kessler, H E Kennah, S I Brown; Pseudomonas protease. Purification, partial characterization, and its effect on collagen, proteoglycan, and rabbit corneas.. Invest. Ophthalmol. Vis. Sci. 1977;16(6):488-497.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

The extracellular protease of a virulent strain of Pseudomonas aeruginosa was purified by DEAE-cellulose chromatography in two steps. 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-orthophenanthroline, L-cysteine and Zn+2 ions but was insensitive to dissopropylphosphofluoridate. The elastase substrates orcein-elastin and acetyl-L-alanyl-L-alanyl-L-alamine-methyl ester were degraded by the enzyme. The protease activity toward soluble and insoluble collagen was found to be 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. It was concluded that the purified protease has little or no collagenolytic activity and that dissolution of the cornea by Pseudomonas protease infection results essentially from the degradation of the protein backbone of the corneal proteoglycans.

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