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Addition of common dietary carbohydrates to Millipore-treated human whole saliva either enhances or inhibits the formation of salivary precipitates, some carbohydrates showing no effect. The purpose of this study was to investigate the precipitation conditions more thoroughly and to elucidate the chemical nature of the precipitates formed. D-Xylose either enhanced precipitation (in long-term incubations) or had no appreciable effect (in 10 minute incubations). Other aldo- and keto-sugars and disaccharides (maltose, sucrose, lactose) generally enhanced precipitation, whereas all polyols (xylitol, D-sorbitol, mannitol, and maltitol) retarded the formation of turbidity in saliva. Xylitol inhibited formation of precipitates also in the presence of D-xylose, dextrans, and starch. Fast protein liquid chromatography (FPLC) of EDTA-soluble pellets obtained by centrifugation of the precipitates produced two major protein fractions (I and II) with a molecular weight of 112,000 and 46,000, respectively. The carbohydrates exerted a selective effect on the relative size of I and II in that polyol incubations resulted in a I to II ratio of 1:3, whereas control incubations (without added sugars) and incubations with other carbohydrates gave ratios of 1:6 to 1:10. Both peaks contained large amounts of acidic amino acids, proline, and glycine. The saliva precipitates contained a substantial portion of a crystalline phase that had the crystal structure of apatite, the individual crystallites being extremely small (less than 1 micron) with a Ca:P ratio of 1.46. The carbohydrates had a similar effect on the overall inorganic composition of the precipitates, but they had a clearly selective effect on the rate of formation of precipitates and on the relative amount of coprecipitating salivary proteins. This selectivity indicates that these carbohydrates, when consumed habitually, may exert different effects on the precipitation of Ca-salts at mineral-deficient enamel and dentine sites. [ABSTRACT FROM AUTHOR]

The cell extracts of two human oral strains (FN2 and FN3) of Fusobacterium nucleatum displayed exceptionally high γ-glutamylpeptidase activity as determined with N-γ- l-glutamyl-2-naphthylamine as substrate. This activity was so dominant that the hydrolysis of other N-aminoacyl-2-naphthylamines progressed at a rate <10% of the former. Two major enzymes (I and II) were partially purified from FN2. I had a molecular weight of 115,000 and did not hydrolyze γ-glutamylcysteinylglycine (glutathione). II had a molecular weight of 70,000 and rapidly liberated only glutamic acid from glutathione. Strain FN3 contained several enzymes hydrolyzing γ-glu-2NA. Direct anion exchange chromatography of FN3 cell extracts separated one enzyme that liberated both glutamic acid and glycine from glutathione, one that was inactive against glutathione (but hydrolyzed γ-glu-2NA), and one that liberated only glutamic acid. Although γ-glu-2NA was a good synthetic substrate, glutathione was hydrolyzed at least 500 times faster by an enzyme present in both strains. These results indicate that the presence of γ-glutamylpeptidase activity is very characteristic of these F. nucleatum strains. [ABSTRACT FROM AUTHOR]

Cell extracts prepared from several oral treponemes isolated from the subgingival plaque of periodontitis patients showed high enzyme activity toward phenylazobenzyl-oxycarbonyl- l-prolyl- l-leucylglycyl- l-prolyl- d-arginine (a compound used as a substrate for microbial collagenases). One major enzyme hydrolyzing this substrate at the Leu-Gly bond only was partially purified from an unspeciated treponeme (strain US), Treponema denticola ATCC 35405, and 29 different clinical isolates of T. denticola. The Treponema US enzyme also hydrolyzed furylacryloyl- l-leucylglycyl- l-prolyl- l-alanine (another substrate of bacterial collagenases) at the Leu-Gly bond. This enzyme also hydrolyzed various collagens and collagen-derived peptides. These treponemal proteases were sensitive to metal chelators and p-chloromercury compounds. The results indicate that human oral treponemes contain enzymes that readily hydrolyze in chromogenic protease substrates the Leu-Gly bond only that is the cleavage site of these substrates also by 'true' microbial collagenases. [ABSTRACT FROM AUTHOR]

Treponema denticola ATCC 35405, a human oral spirochete associated with periodontal disease, was shown to contain three enzymes (I, II, and III) with proline iminopeptidase activity. II and III were considered to be true iminopeptidases, whereas enzyme I was found to be a benzoylarginine peptidase with iminopeptidase activity. Enzyme III, the dominant proline iminopeptidase of T. denticola in terms of its activity toward N- l-prolyl-2-naphthylamine, was considered to be a sulfhydryl peptidase: 0.167 μ M p-chloromercuribenzoic acid totally inactivated the enzyme, and 1.0 m M dithiothreitol restored 92% of activity. The activity of this enzyme was not affected by metal chelators. Chemical modification of enzyme III suggests that tyrosyl (or histidyl) and carboxyl groups may be necessary for its activity. The hydrolysis of N- l-prolyl-2-naphthylamine was found to be very characteristic of T. denticola ATCC 35405; out of 24 different N- l-aminoacyl-2-naphthylamines tested, only the proline derivative was hydrolyzed at a high rate. The substrate specificity of the enzymes discovered indicates that they may be important for the nutrition of T. denticola. The iminopeptidase activity may be related to the pathogenicity of this organism in periodontal disease. [ABSTRACT FROM AUTHOR]

Seven clinical isolates and the ATCC strain 35405 of Treponema denticola, obtained from human periodontal pockets, were studied for peptidase activity with several chromogenic compounds as substrates. The cell sonicates of all strains hydrolyzed phenylazobenzyloxycarbonyl- l-prolyl- l-leucyl-glycyl- l-prolyl- d-arginine (a collagenase substrate), azocasein, and the 2-naphthylamines of l-proline, l-hydroxyproline, l-pyrrolidine, and benzoyl- l-arginine, but the rates of hydrolysis varied considerably from strain to strain. Fast protein liquid chromatography on gel and anion exchange columns revealed further biochemical differences between the strains. The ATCC strain consistently produced several proline iminopeptidases, whereas four of the clinical isolates yielded high and three yielded low iminopeptidase activity. The ATCC strain and six clinical isolates displayed high benzoylarginine peptidase activity. The use of N- l-prolyl-2-naphthylamine as substrate revealed more differences between the strains than other substrates. The substrate specificity of the enzymes discovered suggests that they may be important for the nutrition of the organism or in the protection of the organism against chemical defense factors present in the gingival pocket. [ABSTRACT FROM AUTHOR]