Structure and function of bacterial phospholipase C

α毒素和蜡样芽孢杆菌的n结构域（1 -250个残基；CP_<1-250个>）的磷脂酶C（bccp）活性约为野生型α毒素（WT）活性的2%，但不具有溶血活性。bc和bc的溶血活性分别约为WT的10%和1%。由CP_<1 ~ 250>和CbPLC的c结构域（CP_<1 ~ 250>-CB_<251 ~ 372>）组成的杂交蛋白的活性低于WT，由CbPLC的n结构域和α毒素的c结构域（CB_<1 ~ 250>-CP_<251 ~ 370>）组成的杂交蛋白的活性与WT相似。由BcPLC与α -毒素c结构域组成的杂交蛋白（BC- cp_ <251 ~ 370>）或CbPLC（BC- CB_<251 ~ 372>）的PLC活性与BcPLC无显著差异。BC-CP_<251 ~ 370>对兔红细胞有明显溶血作用，而BC-CB_<251 ~ 372>对兔红细胞无明显溶血作用。WT中，CB_<1 ~ 250>-CP_<251 ~ 370>和BC-CP_<251 ~ 370>与红细胞结合较强，CbPLC与BC-CB_<251 ~ 372>结合较弱，而CP_<1 ~ 250>和BcPLC完全不结合。CP_<1-250>与CP_<251-370>孵育完全互补溶血和PLC活性。CP_<251 ~ 370>显著提高BcPLC的溶血活性，但抑制BcPLC的活性。CP_<251 ~ 370>显著刺激CB_<1 ~ 250>的PLC活性，但对溶血活性无影响。CP_<251 ~ 370>显著抑制BcPLC活性。丙烯酰丹附着在263位和365位残基上的c结构域突变体表现出明显的蓝移，表明荧光团向疏水环境移动。这些观察结果表明，毒素的c结构域与磷脂酰胆碱的脂肪酰基残基的相互作用在毒素的n结构域的生物活性中起重要作用。

N-domain(1 -250residues ; CP_<1-250>) of alpha-toxin and Bacillus cereus phospholipase C (BcPLC) possessed phospholipase C(PLC) activity of about 2% of wild-type alpha-toxin (WT) activity, but not hemolytic activity. PLC and hemolytic activities of C.bifermentans PLC(CbPLC) were about 10% and 1% of these activities of WT, respectively. These activities of the hybrid protein consisting of CP_<1-250> and the C-domain of CbPLC(CP_<1-250>-CB_<251-372>) were lower than those of WT and the activities of the hybrid protein consisting of the N-domain of CbPLC and the C-domain of alpha-toxin(CB_<1-250>-CP_<251-370>) were similar to those of WT.On the other hand, PLC activity of the hybrid protein consisting of BcPLC and the C-domain of alpha-toxin(BC-CP_<251-370>) or CbPLC(BC- CB_<251-372>) was not significantly different from that of BcPLC.However, BC-CP_<251-370> significantly hemolyzed rabbit erythrocytes, but BC-CB_<251-372> did not. WT, CB_<1-250>-CP_<251-370> and BC-CP_<251-370> strongly bound to the red cells, CbPLC and BC-CB_<251-372> bound faintly, and CP_<1-250> and BcPLC not at all. Incubation of CP_<1-250> with CP_<251-370> completely complemented hemolytic and PLC activities. Furthermore, CP_<251-370> significantly conferred hemolytic activity on BcPLC, but inhibited PLC activity of BcPLC.CP_<251-370> significantly stimulated PLC activity of CB_<1-250>, but induced no effect on hemolytic activity. CP_<251-370> significantly inhibited PLC activity of BcPLC.The C-domain mutants with acrylodan attached to residues at position 263 and 365 exhibited a marked blue shift, indicative of movement of the fluorophore to a hydrophobic environment. These observations suggest that interaction of the C-domain of alpha-toxin with fatty acyl residues of phosphatidylcholine plays an important role in biological activities of N-domain of alpha-toxin.