Bauer K, van der Ley P, Benz R, Tommassen J
Lehrstuhl für Biotechnologie, Universität Würzburg, Federal Republic of Germany.
J Biol Chem. 1988 Sep 15;263(26):13046-53.
Purified PhoE-porins were reconstituted into black lipid bilayer membranes, and the selectivity and size of the reconstituted pores were determined. Addition of polyphosphates influenced the internal charge situation of the pore resulting in a shift from anion to cation selectivity. However, the pore size as judged from single channel conductances was not influenced by the addition of polyphosphates. A strong inhibition of the pore conductance only occurred when Mg2+ was also present in the aqueous phase. The inhibition of the pore function is presumably caused by the formation of a chelate between the divalent cation and the polyphosphate. Nevertheless, neither this inhibition nor the selectivity shift are specific to phosphate, because both effects can be mimicked by other polyvalent anions such as citrate. Inhibition of the PhoE pore function by polyphosphate in in vivo experiments confirmed the results of in vitro experiments that polyphosphate is only able to affect the permeability of the outer membrane toward beta-lactam antibiotics if Mg2+ is present. The outcome of the in vivo and the in vitro experiments are consistent with the assumption that the PhoE-porins do not contain a specific binding site for phosphate or polyphosphates but are anion selective because of an excess of positively charged amino acids inside or at the surface of the pore.
将纯化的PhoE孔蛋白重构到黑色脂质双分子层膜中，并测定重构孔的选择性和大小。添加多磷酸盐会影响孔的内部电荷状况，导致选择性从阴离子向阳离子转变。然而，从单通道电导判断的孔径不受多磷酸盐添加的影响。仅当水相中也存在Mg2+时，才会强烈抑制孔电导。孔功能的抑制可能是由二价阳离子与多磷酸盐形成螯合物所致。然而，这种抑制和选择性转变都不是磷酸盐特有的，因为其他多价阴离子如柠檬酸盐也能模拟这两种效应。体内实验中多磷酸盐对PhoE孔功能的抑制证实了体外实验的结果，即只有当存在Mg2+时，多磷酸盐才能影响外膜对β-内酰胺抗生素的通透性。体内和体外实验的结果与以下假设一致：PhoE孔蛋白不含有磷酸盐或多磷酸盐的特异性结合位点，而是由于孔内部或表面带正电荷的氨基酸过量而具有阴离子选择性。
