Transformation of Collagen to "Elastin" in Dermal Collagens with Varying Sensitivity towards Collagenase
Authors: M. K. Keech
Journal: Annals of the Rheumatic Diseases (1958)

It has been reported previously (Keech, 1955) that the dermal collagen from eight out of sixty indivi- duals failed to show any evidence of digestion by collagenase under the electron microscope, and that two others reacted excessively for their particular age groups.Indications of variable reactivity towards collagenase were also obtained from nitrogen determinations of the material digested (Keech, 1954a).It was also shown that in one case of dermatomyositis the rash-bearing skin was collagenase-resistant, whereas the abdominal skin from the same case digested normally.All these findings remained unexplained and appeared to be independent of the disease, therapy, and all the experimental procedures.A small quantity of eight out of the ten abnor- mally-reacting substrates still remained, so a more detailed electron microscope investigation was undertaken to find out whether the samples shown to be resistant to the action of collagenase would also prove resistant to the action of alkali and periodate which apparently transforms collagen to elastin-like structures (Burton, Hall, Keech, Reed, Saxl, Tunbridge, and Wood, 1955; Hall, Keech, Reed, Saxl, Tunbridge, and Wood, 1955).Micro- shrinkage temperature experiments were performed as well as analytical studies on the amino acid composition of the various samples.More detailed characterization was accomplished by chromato- graphy and hydroxyproline determinations.Materials and Methods Prepared Collagen.-Six of the eight collagenase- * Empire Rheumatism Council Research Fellow.resistant substrates and the two exhibiting excessive digestion which were used in previous work (Keech, 1955), together with five controls of comparable age showing average collagenase digestion, constituted the starting materials.Each was examined as described below and then treated in one of the following ways:(a) Suspended in sterile distilled water pH 5 6, heated at 370 C., and samples taken after lI, 3, and 24 hrs.(b) Suspended in 02 M potassium hydrogen phthalate buffer, pH 5-0, heated at 370 C., and samples taken after 1J and 3 hrs.(c) Suspended in a mixture of 1 per cent.sodium metaperiodate in phthalate buffer, pH 5 0, incubated at 370 C., and samples taken at Ij and 3 hrs.(d) Suspended in 0-2 M borate buffer, pH 8-8, and incubated at 370 C. for 24 hrs.Antibiotics.-O05 ml. of a penicillin and streptomycin mixture was added to each test tube in the above experiments.This successfully prevented bacterial con- tamination.Samples for Electron Microscopic Examination were ground gently in a glass tissue grinder until a milky sus- pension was obtained; drops were placed on collodioncovered 200-mesh copper grids, drained with filter paper, allowed to dry, washed with distilled water, shadowed with chromium, and examined in a Siemens electron microscope, type UM 60 C.Counts of the different elastin-like structures were made by carefully scanning two grids from each sample.The specimen holder had been adjusted to visualize 25 squares of the grid, and each part of each square was examined in sequence.As each square was scanned at a magnification of 11,000 and as about 25 fields were needed to cover each square, a minimum of 2 x 625 fields were scrutinized from each specimen.23