A distinct zinc binding site in the alpha-lactalbumins regulates calcium binding. Is there a physiological role for this control?
A distinct zinc binding site in the alpha-lactalbumins regulates calcium binding. Is there a physiological role for this control?
复制标题
α-乳清蛋白中独特的锌结合位点调节钙结合。
DOI：
10.1021/bi00283a010
复制
发表时间：
1983
期刊：
影响因子：
2.9
通讯作者：
Berliner,LJ
中科院分区：
文献类型：
--
作者：
Murakami,K;Berliner,LJ
摘要：在牛、人、豚鼠和兔等几种乳蛋白物种中发现了一个独特的锌结合位点。锌（II）或铝（III）结合到这些蛋白质的钙形式会导致钙的排除，并使蛋白质返回到荧光发射光谱参数确定的“载脂蛋白构象”。Zn （II）和Al （III）的解离常数在低微摩尔范围内。另外，通过观察游离的未配位的Mn （II）在Zn （II）结合时发生位移，用电子自旋共振法测定了Zn （II）的结合。Co （II）和Cu （II）也被证明与锌位点结合，同时也排出Ca （II）。描述阳离子与a-乳清蛋白结合的最合适的模型是钙和锌的两个物理上不同但相互排斥的位点，蛋白质不能同时结合两个位点的阳离子。乳糖生物合成的动力学参数表明，载脂蛋白或锌（II）和钙（II）形式的乳清蛋白完全没有差异。在锌（~ 50µ）和钙（~ 1 mM）的生理浓度下，观察到钙导致的约40%的速率增强，这完全是由钙激活半乳糖转移酶引起的。虽然乳清蛋白的构象[Ca （II）或Zn (II)]在动力学上是等效的，但在生理条件下Ca （II）形式可能占主导地位。e蛋白-乳白蛋白（a-LA） 1是“乳糖合成酶”复合物的非催化调节亚基。la与半乳糖转移酶的结合使后者的特异性发生了变化，从末端的a -乙酰基-gliicosaminyl受体到葡萄糖。我们之前对几种乳蛋白物种的研究记录了钙和几种镧系元素（Kdiss a 1 (T10-10“12 M)）在特定位点的极强结合，这导致了独特的构象变化，通过内在荧光光谱中明显的蓝移来监测（Murakami et al, 1982）。这个钙位点也结合Mn （II）、Cd （II）和Mg (II)，尽管亲和力较弱。由于牛奶中游离钙的浓度范围至少在1-5毫米水平（Altman & Dittmer, 1971），乳蛋白的钙形式应该是生理上重要的构象，这似乎很简单。本文用Zn （II）、Co （II）和其他金属的结果证实了第二阳离子位点的存在，该位点与Ca （II）位点在蛋白质的载脂蛋白构象上表现出竞争行为。金属Zn （II）和Co （II）也是半乳糖转移酶的主要阳离子活化剂（O 'Keefe etal ., 1980）。由于锌（II）的生理浓度远高于其与a-LA的平衡解离常数和半乳糖转移酶激活的Km值，我们还研究了锌（II）和钙（II）在乳糖生物合成中的作用。
Kentaro Murakami* and Lawrence J. Berliner* abstract: A distinct zinc binding site has been found in several-lactalbumin species: bovine, human, guinea pig, and rabbit. Binding of Zn (II) or Al (III) to the calcium forms of these proteins causes exclusion of calcium and return of the protein to its “apo conformation” as determined by fluorescence emission spectral parameters. Zn (II) and Al (III) dissociation constants are in the low micromolar range. In addition, de-terminations of Zn (II) binding were made by electron spin resonance by observing free unliganded Mn (II), which was displaced upon Zn (II) binding. Co (II) and Cu (II) were also shown to bind to the zinc site while also expelling Ca (II). The most appropriate model that describes cation binding to a-lactalbumins is of two physically distinct but mutually ex-clusive sites for calcium and zinc, respectively, where the protein cannot bind cations at both sites simultaneously. Kinetic parameters for lactose biosynthesis show absolutely no difference between theapo or Zn (II) and Ca (II) forms of-lactalbumin. At physiological concentrations of zinc (~ 50 µ) and calcium (~ 1 mM), a ca. 40% rate enhancement due to calcium was observed, which was totally accounted for by calcium activation of galactosyl transferase. While either conformer of-lactalbumin [Ca (II) or Zn (II)] is kinetically equivalent, the Ca (II) form probably dominates underphys-iological conditions. e protein-lactalbumin (a-LA) 1 is the noncatalytic reg-ulatory subunit of the “lactose synthase” complex. The as-sociation of-LA with galactosyl transferase imparts a change in specificity of the latter enzyme from terminal A-acetyl-gliicosaminyl acceptorsto glucose. Our previous work with several-lactalbumin species documented the extremely strong binding of calcium and several lanthanides (Kdiss a 1 (T10-10” 12 M) at a specific site, which caused a unique conformational change monitored by a distinct blue shift in the intrinsic fluorescence spectra (Murakami et al., 1982). This calcium site also bound Mn (II), Cd (II), and Mg (II), albeit with a weaker affinity. Since the concentration range of free calcium in milk is at least at the 1-5 mM level (Altman & Dittmer, 1971), it might seem straightforward that the calcium form of-lactalbumin should be the physiologically important conformer. The results presented here with Zn (II), Co (II), and other metals confirm thepresence of a second cation site that displays competitive behavior with the Ca (II) site for the “apo conformation” of the protein. The metals Zn (II) and Co (II) are also primary cation activators of the galactosyl transferase (O’Keefe et al., 1980a). Since the physiological concentration of Zn (II) is well above both its equilibrium dissociation constant with a-LA and its Km value for galactosyl transferase activation, we have also examined the role or Zn (II) and Ca (II) in lactose biosynthesis.