John H. Highberger

Animal collagenases: Specificity of action, and structures of the substrate cleavage site☆

Abstract Two highly purified animal collagenases, one derived from homogenates of the rabbit V 2 ascites cell carcinoma growing in muscle and the second isolated from the media of tadpole tissue cultures cleaved isolated non helical α chains from chick and rat skin collagen, and the CNBr peptide CB7 from chick skin α1 chains at one, and the same peptide bond. Although two other Gly-Ile bonds exist elsewhere in the α1 chain they were not cleaved.

Isolation and characterization of A peptide containing the site of cleavage of the chick skin collagen α1[I] chain by animal collagenases☆

Abstract A 36-residue peptide containing the bond cleaved by animal collagenases was isolated from a digest of chick skin collagen α1-CB7 by Staphylococcus V8 protease. This cleavage site peptide, in contrast to the 36-residue α1-CB2, showed no tendency to renature to the triple helical form, as monitored by molecular sieve chromatography and the determination of circular dichroism spectra. These results provide a direct demonstration that the conformation of the α1[I] chain immediately around the collagenase cleavage site in the native molecule must be of a lower degree of helicity than other portions of the chain. This is considered to be an important factor in the collagenase specificity, in providing access to the sensitive bonds, but enzyme binding sites, probably located in the adjacent region(s) of maximum helicity, are also considered necessary to produce the maximum reaction rate.

The amino acid sequence of chick skin collagen α1-CB7: The presence of a previously unrecognized triplet

Abstract Because alignment of the amino acid sequences of chick skin collagen α2-CB3 (1) with the relevant portion of chick skin collagen α1-CB7 (2) suggested that a Gly-X-Y triplet may have been missed in the latter, the peptide TM-2, produced by tryptic digestion of maleylated α1-CB7, was reinvestigated. Cleavage by trypsin at the unblocked lysine at position 18, and isolation of the resulting COOH-terminal peptide, showed this to be a 15-residue peptide containing a previously unrecognized Gly-Pro-Hyp triplet. Sequencing of the peptide showed this to occupy positions 4 through 6, or 56 through 58 of α1-CB7. The latter thus has 271 instead of 268 residues, and the α1[I] chain 1055 instead of 1052.