Zhou C, Roberts M F
Mekert Chemistry Center, Boston College, Chestnut Hill, MA 02167, USA.
Biochim Biophys Acta. 1997 Oct 18;1348(3):273-86. doi: 10.1016/s0005-2760(97)00066-0.
For many of the enzymes that utilize or produce diacylglycerols, detergent mixed micelles are often used in assay systems to solubilize the lipophilic substrates or products. The assumption is often made that the diacylglycerol (DAG) is solubilized and well mixed throughout the population of micelles during the time course of the assay. In the present work the partitioning and exchange dynamics of diacylglycerols (from dihexanoyl-DAG to didecanoyl-DAG) in a variety of detergent micelles have been studied by NMR and fluorescence methods. In all detergents, the longer the DAG chain lengths, the more detergent is required for solubilization. However, efficiency of solubilization varies tremendously with Triton X-100 the most efficient (i.e. the least detergent is required), and deoxycholate the least efficient in solubilizing DAG. The mixing and exchange dynamics of pyrene-labeled DAG molecules in these micelles (measured by stopped-flow fluorescence) were fastest for Triton X-100 and slowest with charged bile salt micelles. Of the detergent systems characterized, Triton X-100 appears to be the optimal detergent for use in assays of enzymes that interact with DAG (beta-octylglucoside and diheptanoylphosphatidylcholine have good exchange dynamics, but higher amounts of these detergents are needed to solubilize DAG). Bile salt micelles provide the least solubilization and the slowest exchange kinetics (so slow that this could be a significant problem in some enzyme assays). This information on DAG behavior in micelles is discussed with respect to assays of an enzyme that generates DAG as product (phospholipase C) and one that uses DAG as substrate (DAG kinase). Although slow exchange of DAG occurs in some micelle systems, this does not appear to be a rate-limiting step in the kinetics for either of these enzymes.
对于许多利用或产生二酰基甘油的酶而言，在分析系统中常使用去污剂混合胶束来溶解亲脂性底物或产物。人们通常假定在分析过程中，二酰基甘油（DAG）在整个胶束群体中被溶解且混合均匀。在本研究中，通过核磁共振（NMR）和荧光方法研究了多种去污剂胶束中二酰基甘油（从二己酰基 - DAG到二十二酰基 - DAG）的分配和交换动力学。在所有去污剂中，DAG链长越长，溶解所需的去污剂就越多。然而，溶解效率差异极大，Triton X - 100效率最高（即所需去污剂最少），而脱氧胆酸盐溶解DAG的效率最低。芘标记的DAG分子在这些胶束中的混合和交换动力学（通过停流荧光测量）对于Triton X - 100最快，而对于带电荷的胆盐胶束最慢。在所表征的去污剂体系中，Triton X - 100似乎是用于与DAG相互作用的酶分析的最佳去污剂（β - 辛基葡萄糖苷和二庚酰磷脂酰胆碱具有良好的交换动力学，但溶解DAG需要更多量的这些去污剂）。胆盐胶束的溶解能力最差且交换动力学最慢（慢到在某些酶分析中可能成为一个重大问题）。关于DAG在胶束中的行为信息，结合以DAG为产物的酶（磷脂酶C）和以DAG为底物的酶（DAG激酶）的分析进行了讨论。尽管在某些胶束系统中DAG发生缓慢交换，但这似乎不是这两种酶动力学中的限速步骤。
