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The mechanism of adsorption of the Streptococcus mutans enzymes responsible for the synthesis of insoluble dextran-levan to the S. mutans cell-wall binding sites has been studied. Certain characteristics of these binding sites are presented. The adsorption of these enzymes to the cell surface occurred rapidly without the addition of a source of energy and over a pH range of 3 to 11. The adsorption was inhibited by soluble dextran, probably due to the strong affinity of the polymer to the enzyme. All other polymers and sugars studied showed little or no inhibition. The adsorption was also inhibited by antibody globulin to the a-d immunologically specific group antigen surface polysaccharide of S. mutans and by anti-dextran globulin. The inhibition by anti- a-d globulin is considered to be due to a restriction of access of enzyme to the binding site of the enzyme which may be located in close proximity to the group antigen. On the other hand, anti-dextran globulin appeared to directly inhibit the adsorption by covering the binding site. Dextranase destroyed the binding site and released glucose from the S. mutans cells. These data indicate that S. mutans grown in media containing glucose possesses a small amount of dextran on the cell surface, and that this dextran is, or is a part of, the binding site for enzymes which synthesize the insoluble dextran-levan polymer. Trypsin inhibited the synthesis of insoluble polysaccharide and the adherence of cells. It is not clear in this case that destruction of the binding sites occurred. These data present a partial explanation of the processes which may be concerned in the formation of dental plaque on the smooth surfaces of teeth.

The mechanism of adherence of Streptococcus mutans to smooth glass surfaces has been studied. The results with both viable and heat-killed cells showed that the process required (i) the synthesis of a water-insoluble dextran-levan polymer by cell-bound enzymes and (ii) the participation of a binding site on the surface of the S. mutans cell. Synthesis of the polymer from sucrose in the presence of the cells was required for adherence, and indicates that an "active" form of the polymer was required. Polymer synthesized by cell-free S. mutans enzymes when added to S. mutans cells did not produce adherence. Purified antibody globulin, specific for the ad site in the polysaccharide S. mutans group a antigen, completely inhibited adherence. Antibody to the second antigen present in the polysaccharide molecule, the a antigen, did not inhibit adherence. The evidence indicates that adherence did not require an antigenic binding site which might be common to all S. mutans strains. The orientation of the synthetase enzyme(s), antigenic binding site, and dextran-levan polymer on the cell surface is under study.

An improved affinity-chromatographic method for the preparation of folate-binding protein from cow's milk is described. Under dissociating conditions the protein appeared homogeneous in the ultracentrifuge, with a molecular weight of 35 000 +/- 1500, but it was heterogeneous on electrophoresis and ion-exchange chromatography and evidently consisted of several glycoproteins with similar molecular weights that all bound folic acid. Overall, the protein contained a high proportion of half-cystine (18 residues/molecule) and 10.3% of carbohydrate. At saturation it bound approx. 1 mol of folate/mol of protein at pH 7.2. Equilibrium-dialysis measurements of the binding of folic acid and 5-methyltetrahydrofolate to the purified protein gave non-linear Scatchard plots, the shapes of which depended on pH. The results were interpreted in terms of ligand binding to a polymerizing system in which the affinity of ligand for monomer was greater than its affinity for polymer. When the protein concentration was similar to that in cow's milk, dissociation constants (Kd) for folate and 5-methyltetrahydrofolate were 3 nM and 5 nM respectively at pH 7.2 and 37 degrees C, whereas Kd for the binding of folate to monomer was about 50 pM. The properties of the binding protein are discussed in relation to its possible role in folate absorption in the gut.

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