Powell J T, Brew K
J Biol Chem. 1976 Jun 25;251(12):3653-63.
Sedimentation velocity ultracentrifugation and other procedures have been used to investigate macromolecular interactions of bovine colostrum galactosyltransferase with a glycoprotein substrate (ovalbumin) and with the lactose synthase regulatory protein, alpha-lactalbumin. The determination of equilibrium binding constants for these interactions and the effects of ligands and combinations of ligands on the equilibria have clarified several aspects of the mechanism of galactosyltransferase and its regulation by alpha-lactalbumin. 1. The attachment of Mn2+ at the tight binding site on galactosyltransferase (site I, Kd 2.3 muM) is an essential prerequisite for interactions with ovalbumin and with alpha-lactalbumin. 2. The attachment of Mn2+ or Ca2+ at the weaker metal binding site (site II, Kd 1 to 2 mM) does not significantly affect the interaction of galactosyltransferase with either protein. This is consistent with the hypothesis derived from kinetic studies that site II is functionally connected with the binding of UDP-derivatives. 3. While the binding of ovalbumin to galactosyltransferase in the presence of Mn2+ alone can be observed by ultracentrifugation, this interaction is too weak to cause binding of galactosyltransferase to ovalbumin-Sepharose. Binding to ovalbumin-Sepharose could only be detected by affinity chromatography in the presence of both Mn2+ (10 mM) and UDP (0.3 MM). Sedimentation studies showed that the association of galactosyltransferase with ovalbumin is pressure-dependent, and that the presence of UDP in the complex increases the equilibrium association constant by a factor of 46. The enzyme Mn2+-UDP-ovalbumin complex has unusual hydrodynamic properties. 4. The presence of saturating concentrations of UDP-galactose potentiates the binding of alpha-lactalbumin at high concentrations of Mn2+, as shown by a 25-fold increase in the association constant. Competitive inhibition by alpha-lactalbumin, with respect to ovalbumin that is observed by steady state kinetics, is attributed to the mutally exclusive binding of the proteins with an enzyme complex containing Mn2+ and UDP-galactose. 5. Monsaccharides (N-acetylglucosamine and glucose) strongly enhance the binding of alpha-lactalbumin to enzyme complexes containing Mn2+, in the presence or absence of UDP-glucose. The binding of alpha-lactalbumin and monosaccharide to form enzyme complexes containing both is random, and evaluation of the four associated equilibrium constants shows that the binding is strongly synergistic. 6. Although the significance of some of the many equilibria studied cannot be ascertained, it appears than an element of randomness may be present in reactions catalyzed by galactosyltransferase...
沉降速度超速离心法及其他方法已被用于研究牛初乳半乳糖基转移酶与糖蛋白底物（卵清蛋白）以及与乳糖合酶调节蛋白α-乳白蛋白之间的大分子相互作用。对这些相互作用的平衡结合常数的测定以及配体和配体组合对平衡的影响，阐明了半乳糖基转移酶的作用机制及其受α-乳白蛋白调节的几个方面。1. Mn2+在半乳糖基转移酶上的紧密结合位点（位点I，解离常数Kd为2.3 μM）的附着是与卵清蛋白和α-乳白蛋白相互作用的必要前提。2. Mn2+或Ca2+在较弱的金属结合位点（位点II，解离常数Kd为1至2 mM）的附着不会显著影响半乳糖基转移酶与任何一种蛋白质的相互作用。这与动力学研究得出的假设一致，即位点II在功能上与UDP衍生物的结合相关。3. 虽然仅在存在Mn2+的情况下通过超速离心可观察到卵清蛋白与半乳糖基转移酶的结合，但这种相互作用太弱，无法使半乳糖基转移酶与卵清蛋白-琼脂糖结合。只有在同时存在Mn2+（10 mM）和UDP（0.3 mM）时，才能通过亲和色谱检测到与卵清蛋白-琼脂糖的结合。沉降研究表明，半乳糖基转移酶与卵清蛋白的缔合是压力依赖性的，并且复合物中UDP的存在使平衡缔合常数增加了46倍。酶-Mn2+-UDP-卵清蛋白复合物具有异常的流体动力学性质。4. 如缔合常数增加25倍所示，饱和浓度的UDP-半乳糖的存在增强了在高浓度Mn2+下α-乳白蛋白的结合。稳态动力学观察到的α-乳白蛋白对卵清蛋白的竞争性抑制，归因于蛋白质与含有Mn2+和UDP-半乳糖的酶复合物的互斥结合。5. 单糖（N-乙酰葡糖胺和葡萄糖）在存在或不存在UDP-葡萄糖的情况下，都能强烈增强α-乳白蛋白与含有Mn2+的酶复合物的结合。α-乳白蛋白和单糖结合形成同时含有两者的酶复合物的过程是随机的，对四个相关平衡常数的评估表明这种结合具有强烈的协同作用。6. 尽管所研究的众多平衡中某些平衡的意义尚无法确定，但在半乳糖基转移酶催化的反应中似乎可能存在一定程度的随机性……
