Abstract
Proteolysis of Nereis cuticle collagen by two bacterial collagenases was investigated using viscosimetry, enzyme kinetics, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and ion exchange chromatography of collagenolytic peptides. Collagenase of the marine Vibrio B-30 completely degrades native cuticle collagen at 7 degress C with a turnover number 50 times greater than that of the clostridial collagenase. Although turnover numbers for the two enzymes are comparable when using denatured cuticle collagen as substrate, the vibrial collagenase appears to cleave twice as many peptide bonds per mg of cuticle collagen as does the clostridial enzyme. Sodium dodecyl sulfate gel electrophoresis of collagenase-digested native cuticle collagen reflects the resistance of the collagen to clostridial collagenase; however, the vibrial enzyme completely degrades the cuticle collagen with the formation of one transient intermediate (Mr 400,000). Peptide analysis of fully digested denatured cuticle collagen reveals that the two enzymes have a number of qualitative and quantitative similarities. Despite these, however, only the vibrial collagenase seems capable of extensively degrading native cuticle collagen.
Highlights
From the $ Department of Biochemistry, University of Connecticut Health Center, Farmington, Connecticut 06032, and the Proteolysis of Nereis cuticle collagen by two bacterial collagenases was investigated using viscosimetry, enzyme kinetics, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and ion exchange chromatography of collagenolytic peptides
Sodium dodecyl sulfate gel electrophoresis of collagenase-digested native cuticle collagen reflects the resistance of the collagen to clostridial collagenase; the vibrial enzyme completely degrades the cuticle collagen with the formation of one transient intermediate (Mr400,000)
Only the vibrial collagenase seems capable of extensively degrading native cuticle collagen
Summary
Using three different methods of detection, we can show degradation by two bacterial collagenases. The viscosity of cuticle cuticle collagen by clostridial collagenase was barelydetectacollagen decreases nearly to zero when exposed to each of ble a t enzyme:substrate ratios as high as 1:22. The clostri- trates that a7t"C, the collagenase from Vibrio has a turnover dial collagenaseon the other hand reduces vthisecosity of the number more than 50 times that of the Clostridial enzyme native cuticle collagen by only 20% aft,er a6-h incubation. The using native cuticle collagen assubstrate.Withdenatured slightly sigmoidal initial decrease in viscosity of collagen di- cuticle collagen at 7"C, both enzymes have comparable turngested by the Clostridial enzyme is not yet understood. Clostridial collagenase seems to be slightly better than the vibrial enzyme when denatured calfskin collagen is used as the substrate.
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