Isolation of the Central Type IV Collagen Triple-Helical Domain by Chymotrypsin Treatment

概要

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The full length triple-helical domain of type IV collagen could not be isolated with pepsin treatment owing to susceptibility of the interrupted sites. We here report a simple and effective method to obtain the central type IV collagen triple-helical domain. Chymotrypsin treatment of the acid extract from bovine lens capsule yielded essentially a single band with Mr of 400k under nonreducing conditions in SDS-PAGE. Two bands were recovered after reduction with polypeptide sizes with Mr 140k and 115k, corresponding to nearly the full length of collagenous sequences of α1(IV)and α2(IV). The chymotrypsin-treated type IV collagen retained the triple-helical conformation and consisted of two α1(IV)-derived chains and one α2(IV)-derived chain. An extensive treatment inserted a new nick only in the α2(IV)chain, but did not change the triple-helical structure, suggesting that the interruptions in the -Gly-Xaa-Yaa- triplet repeats except those in the α2(IV)chain are not susceptible to chymotrypsin, contrary to the current hypothesis that the interruptions distort the triple-helical conformation and are therefore susceptible to proteolytic treatment in general. Another characteristic is monodispersity of the chymotrypsin-treated type IV collagen in contrast with the pepsin-treated bovine lens capsule extract that contained mostly 7S tetrameric or higher aggregates of type IV collagen.
日本結合組織学会の論文
1998-09-25