Collagenase and Collagenase Inhibitors Secreted by Cultured Human Periodontal Ligament Cells

• Published in: Journal of Nihon University School of Dentistry Vol. 35; no. 2; pp. 109 - 117
• Main Authors: OHSHIMA, Mitsuhiro, KUWATA, Fumiyuki, SASAI, Yoshinori, OTSUKA, Kichibee, SUZUKI, Kantaro
• Format: Journal Article
• Language: English
• Published: Japan Nihon University School of Dentistry 1993
• Subjects: Cells, Cultured; Chromatography, Gel; collagenase; collagenase inhibitor; Collagenases - metabolism; Collagenases - secretion; Dentistry; Dithiothreitol; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Glycoproteins - metabolism; Glycoproteins - secretion; Humans; Matrix Metalloproteinase Inhibitors; Metalloendopeptidases - secretion; Periodontal Ligament - enzymology; periodontal ligament cells; Phenylmercuric Acetate; Tissue Inhibitor of Metalloproteinases
• ISSN: 0029-0432; 1884-2984
• DOI: 10.2334/josnusd1959.35.109

We have developed a new assay method for collagenase activity, in the presence of two different types of collagenase inhibitors, in culture medium of periodontal ligament (PDL) cells. Human PDL cells were cultured with serum-free α-MEM for 5 days after reaching confluency, and the culture medium was then harvested. Collagenase and collagenase inhibitors in the medium were concentrated by ammonium sulfate precipitation. The precipitate was dissolved, and the suspension was applied to an Ultrogel AcA 54 column for partial purification. Collagenase inhibitor activity was detected as two peaks; a small inhibitor molecule of about 32 kDa sensitive only to dithiothreitol (DTT), and a large one of about 70 kDa considerably more sensitive to 4-aminophenylmercuric acetate (APMA) than to DTT. Therefore, pretreatment of the sample preparation with both DTT and APMA was necessary in order to inactivate the inhibitors prior to assay of latent collagenase. This observation indicates that PDL cells secrete collagenase and two collagenase inhibitors without any stimulation in vitro.