摘要：

A nuclease of Streptococcus pneumoniae, previously implicated in DNA transport during genetic transformation, was analysed in crude extracts and cell fractions by a novel technique for detecting enzyme activity after gel electrophoresis in the presence of sodium dodecyl sulfate. The nuclease activity corresponded to a polypeptide of 25,000 molecular weight; its absence in mutants defective in DNA transport and its presence in the membrane of normal cells were confirmed. In crude extracts the nuclease was subject to proteolysis; however, the larger proteolytic fragments retained nuclease activity. When protected from proteolysis by addition of α-toluene-sulfonyl fluoride, the membrane nuclease was found by gel filtration to be in a complex with an apparent molecular weight of approximately 250,000. Proteolysis of the complex released both intact and degraded nuclease monomers. The discrete nature of the complex was demonstrated by two-dimensional gel electrophoresis: the first dimension, under native conditions, showed the complex as a single band; the second dimension, in the presence of sodium dodecyl sulfate, allowed identification of the nuclease subunit. A hypothetical model envisages the membrane nuclease complex to be a doughnut-like structure with an aqueous pore through which one strand of DNA passes into the cell as the complementary strand is degraded by the nuclease action.

展开