STRUCTURAL BASIS OF INSULIN MIMETIC EFFECT OF VANADYL

通过电子顺磁共振(EPR)和电子核双共振(Endor)光谱的应用，将发展一种新的方法来鉴定血流中与钒(V02+)离子的有机络合物的胰岛素模拟活性有关的分子物种。由于双(乙酰丙酮)氧钒(IV)、双(麦芽)氧钒(IV)和双(3-甲基乙酰丙酮)氧钒(IV)表现出比钒硫酸盐更强的类胰岛素活性，这些化合物将通过EPR和Endor光谱进行表征，以确定这些化合物是否与主要的血清转运蛋白白蛋白、转铁蛋白或转甲状腺素形成特定的分子加合物。将使用分化的3T3-L1脂肪细胞进行胰岛素模拟活性的代谢测试。将比较络合物和它们的蛋白质加合物的胰岛素模拟活性水平。将进行实验，以确定与每种转运蛋白结合的V02+-螯合物的化学计量比是否与在细胞检测中引起最大活性的V02+复合体和转运蛋白的相对浓度相关。初步结果已经表明，二(乙酰丙酮)氧钒(IV)与白蛋白以1：1的加合物结合，细胞检测中V02+络合物与白蛋白的这一比例与最大的胰岛素模拟活性有关。初步研究将确定V02+-螯合物对结合到糖原和脂肪中的放射性葡萄糖的相对量的影响，以及这种影响对胰岛素和其他胰岛素模拟剂的影响有何不同。用双(乙酰丙酮)氧钒(IV)与~(13)C或H~(2+)合成富集物，用Endor光谱测定V02+-螯合物在溶液中的自由结构和与血清转运蛋白的结合。将分析钒(IV)中心与附近磁核的超精细(HF)耦合，以确定这些核相对于V02+离子磁轴的相对位置。每个络合物的中心V02+离子的配位结构和配体几何形状将根据Endor距离约束进行建模。我们将由Endor指定与蛋白质结合时与金属离子配位的蛋白质残基的类型，并确定蛋白质残基在形成加合物时是否取代了螯合原子或改变了它们的几何形状。特别令人感兴趣的是，最近在这个实验室中发现了两种可逆的电离，这两种电离在水溶液中控制着这些V02+螯合物的pH分布，形成四种光谱上可区分的物种。由于这些质子化过程似乎与溶剂分子与赤道氧或轴向氧的氢键有关，因此它们可能在通过扰乱acac配体的互变异构平衡而使蛋白质残基取代螯合物方面起重要作用。因此，我们将努力通过Endor光谱学来指定质子化基团的位置，并使用过氚的V02+-螯合物来消除附近有机氢的重叠共振。由于V02+-螯合物及其蛋白质加合物是顺磁性的，这些研究可能会导致开发一种新的光谱探针来表征亚细胞水平上的大分子相互作用，从而促进细胞中葡萄糖的吸收和代谢。

A new approach to identify the molecular species in the blood stream responsible for insulin-mimetic activity of organic chelates of the vanadyl (V02+) ion will be developed through application of electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopy. Since bis(acetylacetonato)oxovanadium(IV), bis(maltolato)oxovanadium(IV), and bis(3-methyl- acetylacetonato)oxovanadium(IV) exhibit markedly enhanced insulin- mimetic activity over that of vanadyl sulfate, these compounds will be characterized by EPR and ENDOR spectroscopy to determine whether specific molecular adducts are formed with the major serum transport proteins albumin, transferrin, or transthyretin. Metabolic assays of insulin mimetic activity will be made with use of differentiated 3T3-L1 adipocytes. The level of insulin-mimetic activity of the chelated complexes and their protein adducts will be compared. Experiments will be carried out to determine whether the stoichiometry of V02+- chelates bound to each of the transport proteins can be correlated with the relative concentrations of the V02+-complex and the transport protein that elicit maximum activity in cell assays. Preliminary results already indicate that bis(acetylacetonate)oxovanadium(IV) binds to albumin as a 1:1 adduct and that this ratio of V02+-complex : albumin in cell assays is associated with maximal insulin-mimetic activity. Initial studies will be directed to determine the influence of V02+- chelates on the relative amounts of radioactive glucose incorporated into glycogen and lipid and how this may differ for insulin and other insulin mimetic agents. The three-dimensional structure of V02+-Chelates both free in solution and bound to serum transport proteins will be determined by ENDOR spectroscopy with use of bis(acetylacetonato)oxovanadium(IV) synthetically enriched with carbon-13 or deuterium. The hyperfine (hf) couplings of the vanadium(IV) center with nearby magnetic nuclei will be analyzed to assign the relative positions of the nuclei with respect to the magnetic axes of the V02+ ion. The coordination structure and ligand geometry of the central V02+ ion for each complex will be modeled according to ENDOR distance constraints. We shall assign by ENDOR the types of protein residues that coordinate the metal ion in binding to proteins and determine whether protein residues have displaced chelate atoms or altered their geometry upon adduct formation. Of particular interest is the recent identification in this laboratory of two reversible ionizations that govern in aqueous solutions a pH dependent_distnbution of these V02+-chelates into four spectrally distinguishable species. Since these protonation processes appear to be associated with-hydrogen bonding of solvent molecules to either equatorial or axial oxygens, they may be important in displacement of chelate by protein residues by perturbation of the tautomeric equilibrium of the acac ligand. We shall therefore endeavor to assign the location of the protonatable group by ENDOR spectroscopy with use of perdeuterated V02+-chelate to remove overlapping resonances of nearby organic hydrogens. Since the V02+- chelates and their protein adducts are paramagnetic, these studies may lead to development of a new spectroscopic probe to characterize macromolecular interactions at the subcellular level that oven glucose uptake and metabolism in cells.