STRUCTURAL BASIS OF INSULIN MIMETIC EFFECT OF VANADYL

通过应用电子顺磁共振（EPR）和电子核双共振（ENDOR）光谱，将开发一种新的方法来鉴定血流中负责钒（VO 2+）离子的有机螯合物的胰岛素模拟活性的分子种类。由于双（乙酰丙酮基）氧代钒（IV）、双（麦芽酚基）氧代钒（IV）和双（3-甲基-乙酰丙酮基）氧代钒（IV）表现出比硫酸氧钒显着增强的模拟胰岛素活性，因此这些化合物将通过EPR和ENDOR光谱进行表征，以确定是否与主要血清转运蛋白白蛋白、转铁蛋白或转甲状腺素蛋白形成特定的分子加合物。将使用分化的3 T3-L1脂肪细胞进行胰岛素模拟活性的代谢测定。将比较螯合复合物及其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VO 2 +-螯合物的化学计量是否可以与在细胞测定中引起最大活性的VO 2 +-复合物和转运蛋白的相对浓度相关。初步结果已经表明，双（乙酰丙酮化物）氧钒（IV）以1：1加合物的形式与白蛋白结合，并且在细胞测定中VO 2 +-复合物：白蛋白的这种比率与最大的胰岛素模拟活性相关。初始研究将针对确定VO 2+螯合物对掺入糖原和脂质中的放射性葡萄糖的相对量的影响，以及这对于胰岛素和其他胰岛素模拟剂可能如何不同。在溶液中游离的和与血清转运蛋白结合的VO 2 +-螯合物的三维结构将通过ENDOR光谱法使用合成富集有碳-13或氘的双（乙酰丙酮）氧钒（IV）来测定。将分析钒（IV）中心与附近磁性核的超精细（hf）耦合，以分配核相对于VO 2+离子的磁轴的相对位置。将根据ENDOR距离约束对每个络合物的中心VO 2+离子的配位结构和配体几何形状进行建模。我们将通过ENDOR指定与金属离子结合的蛋白质残基的类型，并确定蛋白质残基是否在加合物形成时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该实验室中鉴定出两种可逆电离，其在水溶液中控制这些VO 2+螯合物的pH依赖性分布成四种光谱上可区分的物质。由于这些质子化过程似乎与溶剂分子与赤道或轴向氧的氢键结合有关，因此它们可能在螯合物由蛋白质残基通过干扰acac配体的互变异构平衡而置换中是重要的。因此，我们将奋进通过ENDOR光谱法使用全氘代VO 2+螯合物来指定可质子化基团的位置，以去除附近有机氢的重叠共振。由于VO 2+螯合物和它们的蛋白质加合物是顺磁性的，因此这些研究可能导致开发新的光谱探针以表征在亚细胞水平上的大分子相互作用，所述大分子相互作用促进细胞中的葡萄糖摄取和代谢。

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.