PubMed1984
Human gingival fibroblast procollagenase has been purified to apparent homogeneity from serum-free and serum-supplemented fibroblast culture medium by a combination of ammonium sulfate precipitation, CM-cellulose chromatography, and gel-filtration on Bio-Gel P-150. Sodium dodecylsulfate polyacrylamide gel electrophoretic studies suggests that the purified fibroblast proenzyme is comprised of two closely related zymogens with the estimated Mr of 57,000 and 52,000. Upon densitometric scanning of the gels, the ratio of the two proenzyme forms was about 1 : 4 (57 : 52 kdal). Limited proteolysis of the fibroblast procollagenase with trypsin resulted in the conversion of both proenzyme forms into active enzyme forms of Mr 48,000 and 44,000, respectively. Amino acid analysis of the active enzymes and proenzyme forms revealed that the active enzymes contained fewer basic amino acids than do the proenzyme forms. The purified trypsin-activated fibroblast collagenase hydrolyzed type I collagen fibrils, cleaved tropocollagen in solution at 24 degrees C into TCA and TCB fragments, and cleaved the synthetic peptide substrate, DNP-peptide III, at the Gly-Ile bond. The gingival fibroblast collagenase exhibited a pH optimum of 7.5, was completely inhibited by EDTA or dithioerythritol but was not inhibited by N-ethylmaleimide or phenylmethylsulfonyl fluoride, and appeared to cleave human type III collagen approximately 10-fold faster than homologous type I collagen. In addition, comparison of the biochemical properties of the precursor and active forms of human gingival fibroblast collagenase with the precursor and active forms of human skin fibroblast collagenase, previously characterized by Stricklin and co-workers (Biochemistry 17: 2331-2337, 1978), revealed that they were similar in Mr, amino acid composition, and substrate specificity. Furthermore, the human gingival and skin fibroblast procollagenases were immunologically identical.