Functional molecular size and structure of dextransucrase by radiation inactivation and gel electrophoresis.

Robyt et al. have proposed a mechanism for dextransucrase in which dextran is synthesized by the cooperative action of two equivalent nucleophiles (Robyt, J.F., Kimble, B.K. and Walseth, T.F. (1974) Arch. Biochem. Biophys. 165, 634-640). To distinguish between the possibilities that the enzyme is a monomer bearing both nucleophiles, or a dimer with each subunit bearing one nucleophile, the molecular weight of the enzyme was determined by SDS-polyacrylamide gel electrophoresis and by radiation inactivation. Two major forms of dextransucrase from Leuconostoc mesenteroides NRRL B-512F were found on SDS-polyacrylamide gel electrophoresis, with Mr 177 000 and 158 000, and sometimes a minor form with Mr 168 000. No form of dextransucrase smaller than Mr 158 000 was found, either in the presence or absence of dextran T10, although levansucrase was detected at Mr 92 000 and 116 000. On irradiation with 60Co, dextransucrase behaved as a single species with a maximum size of Mr 201 000. Because Mr 201 000 is much smaller than the minimum dimer size of Mr 316 000 (= 2 X 158 000), it is concluded that both nucleophiles are probably located on the same peptide, rather than one on each subunit of a dimer, and that peptide association is probably not required for dextran synthesis.