Effects of hyaluronan lyase, hyaluronidase, and chondroitin ABC lyase on mammalian vitreous gel.
OBJECTIVE
To determine the effects of enzymes on mammalian vitreous gel and to thus infer the structural roles of hyaluronan and chondroitin sulfate in the gel.


METHODS
The wet weights of bovine vitreous gels were compared before and after incubation with Streptomyces hyaluronan lyase, chondroitin ABC lyase, testicular hyaluronidase, or buffer alone. The extent of hyaluronan depolymerization was determined by chromatography and that of chondroitin sulfate depolymerization by western blot analysis.


RESULTS
After digestion with Streptomyces hyaluronan lyase (30 U/gel), the gel wet weight was the same as that of controls (incubated with buffer alone) despite 94% of the hyaluronan having been depolymerized; when digested with 100 U/gel, the gel wet weight decreased (to 57% of original wet weight versus 86% for controls, P = < 0.001) and hyaluronan was completely depolymerized. Chondroitin ABC lyase digestion (0.2 U/gel) resulted in a slight reduction in gel wet weight (90% versus 96%, P = < 0.001) and depolymerization of 88% of the hyaluronan; the presence of fully digested chondroitin sulfate chains was established. Digestions with 100 and 500 U/gel of testicular hyaluronidase resulted in a decrease (P = < 0.001, both cases) in gel wet weight (53% versus 82%, 100 U/gel; 57%, versus 86%, 500 U/gel) with 75% and 97% hyaluronan depolymerization, respectively.


CONCLUSIONS
Depolymerization of all vitreous hyaluronan and of chondroitin sulfate resulted in gel wet weight reduction but not gel destruction. Digestion with 30 U/gel of Streptomyces hyaluronan lyase revealed a small pool (6%) of relatively enzyme-resistant hyaluronan that specifically contributed toward maintaining gel wet weight.
Bishop
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Department of Ophthalmology and Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences, University of Manchester, England, UK. paul.bishop@man.ac.uk
McLeod
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D
Reardon
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A
D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D019765:Chondroitin ABC Lyase / Q000494:pharmacology*; D002809:Chondroitin Sulfates / Q000378:metabolism; D002850:Chromatography, Gel; D006820:Hyaluronic Acid / Q000378:metabolism; D006821:Hyaluronoglucosaminidase / Q000494:pharmacology*; D008297:Male; D009929:Organ Size; D011133:Polysaccharide-Lyases / Q000494:pharmacology*; D013302:Streptomyces / Q000201:enzymology; D013737:Testis / Q000201:enzymology; D014822:Vitreous Body / Q000187:drug effects* / Q000378:metabolism
D006820:Hyaluronic Acid; D002809:Chondroitin Sulfates; D006821:Hyaluronoglucosaminidase; D011133:Polysaccharide-Lyases; C100002:hyaluronate lyase; D019765:Chondroitin ABC Lyase