Increased resistance of the collagen in avian dystrophic muscle to collagenolytic attack: evidence for increased crosslinking.
Fibrous collagen from normal (line 412) and dystrophic (line 413) chicken pectoral muscles was examined for susceptibility to bacterial collagenase by a pH-stat titration method to assess the kinetics of peptide bond hydrolysis. These experiments showed that there was a profound difference in the kinetics of hydrolysis using fibrous collagen purified from normal and dystrophic muscle. Although normal collagen was readily hydrolyzed by the collagenase, dystrophic collagen was not hydrolyzed in the presence of 200 microM Ca and was hydrolyzed slowly in the presence of 5 mM Ca. At this high Ca concentration, the Km for collagenase was six times higher in dystrophic collagen than in normal, whereas the maximum reaction rate (Vmax) remained the same. After CNBr digestion, peptides from both sources were readily hydrolyzed by collagenase in the presence of 200 microM Ca, and there was no noticeable difference in the reaction kinetics. The above results were not modified whether the chicken was sacrificed on day 4 or day 98 post-hatching, and the insusceptibility to collagenase preceded the clinical manifestations of dystrophy. The structure of both fibrous and CNBr-treated collagen from normal and dystrophic muscle was compared. One and two-dimensional gel electrophoreses showed identical peptide maps. Gel filtration of CNBr peptides showed in the excluded volume the presence of twice as much high-molecular-weight (presumably crosslinked) peptides from dystrophic collagen compared with normal. We conclude that the intermolecular crosslinks are more extensively formed in dystrophic collagen and that this increased crosslinking results in the increased resistance of the dystrophic collagen to collagenase.
Feit
H
H
Department of Neurology, Henry Ford Hospital, Detroit, MI 48202.
Kawai
M
M
Mostafapour
A S
AS
D000818:Animals; D002645:Chickens; D003094:Collagen / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D003012:Microbial Collagenase / Q000494:pharmacology*; D009132:Muscles / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D009137:Muscular Dystrophy, Animal / Q000201:enzymology / Q000378:metabolism*; D010449:Peptide Mapping
D003094:Collagen; D003012:Microbial Collagenase