The primary structure of superoxide dismutase purified from anaerobically maintained Bacteroides gingivalis.

The superoxide dismutase (SOD) of Bacteroides gingivalis can use either iron or manganese as a cofactor in its catalytic activity. In this study, the complete amino acid sequence of this SOD purified from anaerobically maintained B. gingivalis cells was determined. The proteins consisted of 191 amino acid residues and had a molecular mass of 21,500. The sequence of B. gingivalis SOD showed 44-51% homology with those for iron-specific SODs (Fe-SODs) and 40-45% homology with manganese-specific SODs (Mn-SODs) from several bacteria. However, this sequence homology was considerably less than that seen among the Fe-SOD (65-74%) or Mn-SOD family (42-60%). This indicates that B. gingivalis SOD, which accepts either iron or manganese as metal cofactor, is a structural intermediate between the Fe-SOD and Mn-SOD families.

Biological role of an arginine residue present in a histidine-rich peptide which inhibits hemagglutination of Porphyromonas gingivalis.

Inhibitory effects of synthetic fragments in histatin 8, having the sequence Lys-Phe-His-Glu-Lys-His-His-Ser-His-Arg-Gly-Tyr, on hemagglutination by Porphyromonas gingivalis 381 were examined. The hemagglutinating activity was reduced much more by the peptide Lys-His-His-Ser-His-Arg-Gly-Tyr than by the peptides Lys-His-His-Ser-His and/or Lys-Phe-His-Glu-Lys. These results suggest that the arginine residue may have an important role in the inhibition of hemagglutination by P. gingivalis.

Binding of a histidine-rich peptide to Porphyromonas gingivalis.

Porphyromonas gingivalis 381 cells were incubated with 125I-histidine-rich polypeptide (histatin) 5 in the presence or absence of unlabeled histatin 5, to evaluate the histatin-binding capacity of the cells. The binding of histatin 5 was rapid, reversible, saturable and specific. The number of histatin 5-binding sites per cell was 3,600, and the dissociation constant (Kd) was in the order of 10(-6) M. These findings suggest that histatin interacts with certain bacterial cells through specific binding sites on their surface, and will allow the development of a histatin radioreceptor assay.

Purification and characterization from human parotid secretion of a peptide which inhibits hemagglutination of Bacteroides gingivalis 381.

A peptide from human parotid secretion which inhibited hemagglutination of Bacteroides gingivalis 381 was purified by ultrafiltration followed by DEAE-Sephadex A-25 column chromatography and by gel filtration on Sephadex G-25, and then by reversed-phase HPLC. The complete amino acid sequence of the peptide, determined by automated Edman degradation was as follows; Lys-Phe-His-Glu-Lys-His-His-Ser-His-Arg-Gly-Tyr. The peptide contained 12 residues and the charged amino acids predominated with 4 histidine, 2 lysine, 1 arginine and 1 glutamic acid residues, thus being a histidine-rich peptide. The peptide was an active inhibitor of the hemagglutinating activity of B. gingivalis. Specific binding of tritium-labeled peptide to B. gingivalis cells was demonstrated. These results suggest that the histidine-rich peptide may function as a binding domain for the hemagglutinins of B. gingivalis during agglutination.

Some properties of uridine 5'-diphosphogalactose: N-acetylglucosamine galactosyltransferase in human parotid saliva.

Uridine 5'-diphospho (UDP)-galactose: N-acetylglucosamine galactosyltransferase was separated from human parotid saliva and partially purified. The optimum pH of the enzyme was 7.5. The enzyme required a specific acceptor, and its activity was activated by manganese and magnesium ions.

Substrate specificity of fucosyltransferase purified from human parotid saliva.

The purified fucosyltransferase from human parotid saliva was shown to transfer fucose from GDP-fucose onto the oligosaccharide chains containing the Gal beta 1----3GlcNAc or Gal beta 1----4GlcNAc/Glc sequences. Competition studies between asialotransferrin and either lacto-N-fucopentaose 1 or 2'-fucosyllactose provided evidence that both the substrates competed for a common enzyme active site. These results suggest that the fucosyltransferase activities for the three acceptors may be catalyzed by the same enzyme.

The presence of fucosyltransferases with different substrate specificity in human parotid saliva.

Using glycoproteins and milk oligosaccharides as substrate acceptors, we demonstrated at least two fucosyltransferases in human parotid saliva. One enzyme transferred L-fucose from GDP-fucose to the C-3 position of N-acetylglucosamine or glucose residue of oligosaccharide chains, and the other transferred to the C-4 position of N-acetylglucosamine residue of oligosaccharide chains.

Purification and characterization of galactosyltransferase in human parotid saliva.

The galactosyltransferase has been purified from human parotid saliva by ammonium sulfate precipitation (25-70% saturation), followed by repeated affinity chromatography on Sepharose-alpha-lactalbumin. The molecular weight of the enzyme was estimated to be approximately 56,000. The enzyme catalyzes the transfer of galactose from UDP-galactose to the exposed N-acetylglucosamine residues derived from glycoproteins, forming a Gal beta (1-4)GlcNAc linkage.

Inhibitory effect of human plasma and saliva on co-aggregation between Bacteroides gingivalis and Streptococcus mitis.

The effect of human plasma and saliva on co-aggregation between Bacteroides gingivalis and Streptococcus mitis was studied by means of a turbidimetric assay. The co-aggregation activity was obtained from the maximum slope of the absorbance vs. time curve. Its dependence on pH, temperature, and ionic strength was examined, and the number of Bacteroides cells in relation to the number of Streptococcus cells resulting in optimal co-aggregation was established. Co-aggregation inhibition experiments showed that the co-aggregation activity was inhibited by l-arginine and l-lysine, although the activity was unaffected by the sugars tested. Human plasma and saliva were able to inhibit the co-aggregation in a dose-dependent reaction. Plasma exhibited the most potent inhibitory activity in these fluids. Fibrinogen was the most potent inhibitor of the plasma-derived proteins tested. These data suggest the possibility that the oral fluids may modulate the attachment of B. gingivalis to Gram-positive bacteria in periodontal pockets.

Relationship between enzyme activities involved in oxygen metabolism and oxygen tolerance in black-pigmented Bacteroides.

In this study, the relationship between enzyme activities involved in oxygen metabolism and the degrees of oxygen tolerance in black-pigmented Bacteroides was investigated. All strains of Bacteroides tested possessed the activities of NADH oxidase and superoxide dismutase, whereas the activities of catalase and peroxidases were not detected in the cell-free extracts. There were relatively high correlations between oxygen tolerance and activity of either NADH oxidase or superoxide dismutase. The activity of superoxide dismutase showed a higher correlation with oxygen tolerance than with that of NADH oxidase. Among the species tested, Bacteroides gingivalis showed the highest activities of both the enzymes and was the most tolerant in the presence of oxygen. Furthermore, the activities of these two enzymes increased during aeration of the oxygen-tolerant strain Bacteroides gingivalis 381, but not in the oxygen-sensitive strain Bacteroides denticola ATCC 33185. These results suggest that superoxide dismutase and NADH oxidase might be important for protection of black-pigmented Bacteroides against the toxic effect of oxygen.

Purification and some properties of fucosyltransferase in human parotid saliva.

Fucosyltransferase was purified from human parotid saliva by affinity chromatography on GDP-hexanolamine Sepharose, followed by chromatofocusing on PBE 94 exchanger gel. The purified enzyme had the N-acetyglucosaminide alpha 1----4, the N-acetylglucosaminide alpha 1----3, and the glucoside alpha 1----3 fucosyltransferase activities. The molecular weight of the purified enzyme was estimated to be approximately 20,000. These enzyme activities showed identical pH and divalent metal ion dependencies and identical rates of inactivation upon being heated. The paper chromatographic analysis of the fucosylated products by the purified enzyme and the susceptibility of these products to linkage-specific fucosidase digestion indicated that the transferase formed the Fuc alpha 1----4GlcNAc, Fuc alpha 1----3GlcNAc, and Fuc alpha 1----3Glc linkages.

Changes of serological activity by alpha-L-fucosidase isolated from Streptococcus sanguis ATCC 10557.

alpha-L-Fucosidase isolated from the growth culture of Streptococcus sanguis ATCC 10557 acted on H- and Leb-blood group substances in porcine gastric lining, human gastric lining, human ovarian cyst fluid and human whole saliva, with consequent loss of H- and Leb -activities and a concomitant increase of Lea activity.

Separation of fucosyltransferase and endogenous acceptors of fucose from human parotid saliva.

Using gel filtration on Sephadex G-100 and column chromatography on CM-cellulose, fractions containing either endogenous acceptor of L-fucose or fucosyltransferase were obtained from human parotid saliva. Hydrolysis with mild acid followed by Smith degradation increased the fucosyltransferase activity of the fractions that contained endogeneous acceptor.

Purification and some properties of alpha-L-fucosidase isolated from Streptococcus sanguis.

alpha-L-Fucosidase acting on naturally occurring substrates was highly purified from the growth culture of Streptococcus sanguis ATCC 10557. The molecular weight of the enzyme was approximately 120,000 and the optimal pH was at 5.5. The purified enzyme showed specificity toward the linkage of alpha-(1 leads to 2) fucosides in oligosaccharides and glycoproteins. The enzyme released L-fucose from glycoprotein in human parotid saliva.

Changes in hemoglobin concentration and oxygen saturation in human gingiva with decreasing inflammation.

The purpose of this study was to determine if functional changes in the human gingival vasculature were reversible following the resolution of gingival inflammation. Ten patients with 40 inflamed gingival sites were evaluated before and 2 weeks after the completion of treatment. We determined the hemoglobin concentration and the oxygen saturation of hemoglobin at each site by tissue reflectance spectrophotometry. With the use of treatment including motivation, oral hygiene instruction, and scaling, clinical parameters such as the gingival and plaque indices, the Periotron score, and the probing depth were altered toward a healthier state. With the resolution of gingival inflammation, the increased hemoglobin concentration and decreased oxygen saturation in the inflamed gingiva were restored to normal levels. These findings suggest that reversible changes in the local hemoglobin concentration and oxygen saturation are associated with decreasing gingival inflammation in human subjects.

Interaction of parotid saliva basic glycoprotein with Streptococcus sanguis ATCC 10557.

It is postulated that an initial step in dental plaque formation is the adherence of oral bacteria to the salivary pellicle. Recently, we have found that a proline-rich and basic glycoprotein (MGP) from human parotid saliva, which is successfully purified by Concanavalin A-Sepharose affinity chromatography, binds to some oral streptococci such as S. mitis and S. sanguis. This paper deals with some inhibitors which affect the binding of the MGP to S. sanguis ATCC 10557. The assay for the binding ability of the radioactive MGP to the bacterial cells was performed by incubation of the reaction mixture containing 10 microgram of [3H]MGP (6000 dpm) and about 4.5 mg of bacterial cells (dry weight) in 0.5 ml of 0.05 M Tris-HCl buffer containing 0.05 M NaCl, pH 8.0 with a final volume of 0.51 ml. After 1 hour standing at 4 degrees C, the cells were washed five times with the same buffer. The resulting sediment was solubilized in 0.5 ml of NCS tissue solubilizer and the radioactivity was measured. The binding of the radioactive MGP to bacterial cells was specifically inhibited by galactose, lactose and N-acetyllactosamine. Applications of heat and trypsin on the cell surface, strikingly reduced the binding ability. These findings strongly suggested that a lectin-like substance may be present on the bacterial cell surface.

Purification and characterization of galactosephilic component present on the cell surfaces of Streptococcus sanguis ATCC 10557.

Previous studies have indicated that a galactosephilic component present on the bacterial cell surfaces of Streptococcus sanguis ATCC 10557 may be responsible for the salivary glycoprotein-mediated binding of the cells. The purpose of this study was to investigate the purification and characterization of galactosephilic cell surface component from S. sanguis ATCC 10557. A galactosephilic component involving fibrils on the cell surfaces was isolated by the techniques of freezing and thawing, and purified by an affinity chromatography on beta-D-galactose binding-Bio-Gel P-2 followed by gel filtrations on Bio-Gel P-150 and on Bio-Gel P-30. Both disk gel electrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the purified product was homogeneous. The isoelectric point of the purified sample was 8.5 to 9.0. Treatment of the purified sample with pronase E reduced remarkably either the hemagglutinating activity or the precipitation reaction with proline-rich glycoprotein in human parotid saliva, suggesting that the active site may be present on the peptide moieties. When sugar specificity was examined by hemagglutination-inhibition test, D-galactose was the strongest inhibitor. The results of this study suggest that the galactosephilic component may be a bacterial lectin.

Inhibitory effects of synthetic histidine-rich peptides on haemagglutination by Bacteroides gingivalis 381.

The haemagglutinating activity of Bacteroides gingivalis 381 was significantly inhibited by the synthetic peptide, Asp-Ser-His-Ala-Lys-Arg-His-His-Gly-Tyr-Lys-Arg-Lys-Phe-His- Glu-Lys-His-His-Ser-His-Arg-Gly-Tyr. However, bradykinin potentiator C, which does not contain cationic amino acids such as L-histidine, L-arginine and L-lysine, had no inhibitory effect.

Haemoglobin concentration and oxygen saturation in dog gingiva with experimentally induced periodontitis.

The index of haemoglobin concentration (Hb index) and apparent oxygen saturation (apparent SO2) were determined by a new, non-invasive method, tissue reflectance spectrophotometry. The Hb index was positively correlated with the haemoglobin concentration. The relationship between the apparent SO2 and oxygen partial pressure was a sigmoid curve resembling the haemoglobin oxygen dissociation curve. The Hb index and the apparent SO2 were monitored continuously by tissue reflectance spectrophotometry during the induction of experimental periodontitis with silk ligatures. The Hb index increased rapidly during the first 7 days after ligation and then decreased gradually during the remaining period. The apparent SO2 decreased during the first 7 days but gradually rose during the final 9 weeks. The maximum level of the deoxyhaemoglobin concentration after ligation was elevated about two times over that found before ligation, whereas the increase in oxyhaemoglobin concentration was relatively small. These results suggest that the oxygen supply to inflamed gingiva may increase to some extent, but not sufficiently to compensate for the increased oxygen consumption.