Protein synthesis by human gingival fibroblasts in the presence of thiol compounds

• Main Author: Johnson, Paul William
• Language: English
• Published: 2010
• Online Access: http://hdl.handle.net/2429/23954

Hydrogen sulphide and methyl mercaptan are intermediate volatile metabolites produced in the oral cavity through putrefactive action of microorganisms on sulphur-containing proteinaceous substrates. They are the principal cause of intensified malodour associated with peridontal disease and correlate with the depth of sulcular pockets and severity of disease. Their capacity to bind to type I collagen and their ability to depress DNA synthesis in fibroblast tissue cultures suggests that they may play a significant role in the etiology of periodontitis. The present study examines the effect of these compounds on protein synthesis. Human gingival fibroblasts were grown to confluency, attached to dishes in equal numbers, and incubated in serum-free Dulbecco Modified Eagle's Medium supplemented either with L-[¹⁴C]-proline or L-[³H]-proline and L-[¹⁴C]-glycine. Test cultures were incubated in an atmosphere of 5% CO₂/95% air admixed with either hydrogen sulphide or methyl mercaptan (10 ng/ml) and compared to controls free from thiols. Following labelling, media and cell-associated proteins were harvested and analyzed for total labelled protein, collagenase-digestable protein, and procollagens. The results indicate that although both hydrogen sulphide and methyl mercaptan suppress protein synthesis by fibroblasts, the effect of mercaptan is more pronounced. Hydrogen sulphide decreased total [¹⁴C] incorporation into protein of test cultures by 18 to 20% while mercaptan inhibited the process by approximately 30%. These changes were paralleled by decreases in collagenase-digestable proteins. Comparison of control and mercaptan- exposed cultures by DEAE-cellulose chromatography indicated that although test cultures contained twice the amounts of type I procollagens, levels of all other eluted proteins were markedly decreased. Investigations of similar cultures using SDS-poly-acrylamide slab gel electrophoresis demonstrated that exposure of cultures to mercaptan resulted in marked increases of type I procollagen and pC collagen α 2 chains. The identity of these compounds was confirmed by pepsin digestion and provided additional evidence that mercaptan caused accumulation of type I collagen precursors in culture. === Medicine, Faculty of === Pathology and Laboratory Medicine, Department of === Graduate