Diacylglycerol partitioning and mixing in detergent micelles: relevance to enzyme kinetics.
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.
Zhou
C
C
Mekert Chemistry Center, Boston College, Chestnut Hill, MA 02167, USA.
Roberts
M F
MF
D001409:Bacillus cereus / Q000201:enzymology*; D003902:Detergents; D019852:Diacylglycerol Kinase / Q000378:metabolism*; D004075:Diglycerides / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D005960:Glucosides; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008823:Micelles; D008968:Molecular Conformation; D017830:Octoxynol; D012995:Solubility; D010738:Type C Phospholipases / Q000378:metabolism*
D003902:Detergents; D004075:Diglycerides; D005960:Glucosides; D008823:Micelles; C018619:octyl-beta-D-glucoside; D017830:Octoxynol; D019852:Diacylglycerol Kinase; D010738:Type C Phospholipases