Abstract
Fibroblasts obtained from healthy and diseased human gingiva were labeled with radioactive amino acids and the collagenous proteins synthesized were studied. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of untreated, reduced, and pepsin-treated proteins of the medium and cell extract showed that the collagenous proteins synthesized by these cells exist in the precursor form. Type I collagen was the chief constituent. In addition, cells from normal tissue synthesized type III collagen in amounts varying from 5 to 30%. Type III collagen was not detected in the cultures of fibroblasts from diseased tissue; however, an additional collagen fractionated between 2.5 to 5.0 M NaCl and accounted for 22 to 29% of the total. This collagen had an alpha1/alpha2 ratio of 8.6 and hydroxylysine/lysine ratio and cyanogen bromide peptide pattern were similar to that of alpha1[I]. It is concluded that the fibroblasts derived from disease gingiva synthesize a collagen of composition (alpha1)3, probably of type I.
Highlights
Electrophoresis of untreated, reduced, and pepsin-treated proteins of the medium and cell extract showed that the collagenous proteins synthesized by these cells exist in the precursor form
On CM-cellulose chromatography, this fraction revealed a peak eluting in the region of type III chains and in the experiment presented this peak accounted for 5% of the total collagen (Table II, Fig. 30)
NaCl fractions, illustrated in Figs. 7, C and B, were subjected to cyanogen bromide digestion and the peptide pattern was compared by CM-cellulose chromatography and 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis with those of al [r] and oil [II] chains
Summary
Type I collagen of rat and human fetal skin was prepared by the method of Piez et al [12] and type II collagen from articular cartilage of aborted human fetuses by the method of Miller [13]. J. Miller; additional material was prepared from human fetal skin according to the method of Chung and Miller [14]. All radioactive amino acids and Aquasol were obtained from New. mg) was the product of Worthington Biochemical Corp., Freehold, N.J. P-Aminopropionitrile, iodoacetic acid, and mercaptoethanol were obtained from Calbiochem, La Jolla, Ca., and ion exchange celluloses from Whatman Biochemicals Ltd., Maidstone, Kent, United Kingdom. Chemicals for electrophoresis were the products of Bio-Rad, Richmond, Ca. All other chemicals not listed above were of analytical grade and purchased from Mallinckrodt, Baker, or Fisher Chemical
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