The production of fructan from sucrose by the action of the fructosyltransferase(s) (FTase) of oral streptococci appears to be dependent upon metal ions, but the exact nature of this dependence has not yet been elucidated'**. In the case of the purified extracellular FTase of Streptococcus mutans, the addition of metal ions to an active preparation had no effect on enzyme activity, although Ca*+ was observed' to prevent inactivation of the enzyme between 40 and 55". EDTA was shown to inhibit the FTase, but this effect was reversed by a number of divdlent ions, including Ca* + , Sr2 ' , Mn' ' , Cd2 ' , Ni2+ , Cu2 ' , or Pb2 ' , thus only implying, amongst other possibilities, a role for a metal ion in enzyme activity'. In contrast, the extracted cell-associated FTase of S. salivarius SS2 was reported to be inhibited by both Ca2+ and NiZf ions.
Recently, it was shown that the cell-associated FTase of S. salivabus ATCC25975 was inactivated by a process mediated by Cu'+. Of interest was the observation that Ca*' could prevent this loss of enzyme activity3. The present study into the role of ions affecting the cell-associated FTase activity of S. safivarius has shown that measurable enzyme activity declines as cells are subcultured in medium essentially devoid of added metal ions except for Na+, K+, Mg*+, Fe*+, and Mn*+. This observation has afforded proof that Ca2+ is a necessary cofactor for the cell-associated FTase activity of S. salivarius and permitted the kinetics of the enzyme to be studied in some detail. Furthermore, the Cu*+-mediated inactivation of the cell-associated enzyme has been confirmed as being due to a process other than simple competition with the activating metal-ion species.
EXPERIMENTAL Organism and medium. -Streptococcus salivarius ATCC25975 was used throughout these studies. Stock cultures were stored at 4" in semi-defined medium containing an excess of calcium carbonate (20 mg/mL). Transfers were made to fresh medium every month. The semi-defined medium was a modification of the strictly defined medium described by Wittenberger et aL4 in which all amino acids