Phospholipase D activity is regulated by product segregation and the structure formation of phosphatidic acid within model membranes.
Phospholipase D from Streptomyces chromofuscus (scPLD) hydrolyzes phosphatidylcholines (PC) to produce choline and phosphatidic acid (PA), a lipid messenger molecule within biological membranes. To scrutinize the influence of membrane structure on scPLD activity, three different substrate-containing monolayers are used as model systems: pure dipalmitoylphosphatidylcholine (DPPC) as well as equimolar mixtures of DPPC/n-hexadecanol (C(16)OH) and DPPC/dipalmitoylglycerol (DPG). The activity of scPLD toward these monolayers is tested by infrared reflection-absorption spectroscopy and exhibits different dependencies on surface pressure. For pure DPPC, the catalytic turnover drastically drops above 20 mN/m. On addition of C(16)OH, this strong decrease starts at 5 mN/m. For the DPPC/DPG system, the reaction yield linearly decreases between 5 and 25 mN/m. The difference in scPLD activity is correlated to the phase state of the monolayers as examined by x-ray diffraction, Brewster angle microscopy, and atomic force microscopy. Because the additives C(16)OH and DPG mediate the miscibility of PC and PA, only a basal activity of scPLD is observed toward the mixed systems at higher surface pressures. At pure DPPC monolayers, scPLD is activated after the segregation of initially formed PA. Furthermore, scPLD is inhibited when the lipids in the PA-rich domains adopt an upright orientation. This phenomenon offers a self-regulating mechanism for the concentration of the second messenger PA within biological membranes.
Wagner
Kerstin
K
Max Planck Institute of Colloids and Interfaces, 14424 Potsdam, Germany. wagner@mpikg.mpg.de
Brezesinski
Gerald
G
D001703:Biophysics / Q000379:methods*; D002118:Calcium / Q000737:chemistry; D002462:Cell Membrane / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D006868:Hydrolysis; D008055:Lipids / Q000737:chemistry; D018625:Microscopy, Atomic Force; D008954:Models, Biological; D010168:Palmitates / Q000737:chemistry; D010712:Phosphatidic Acids / Q000737:chemistry; D010739:Phospholipase D / Q000096:biosynthesis* / Q000737:chemistry*; D013302:Streptomyces / Q000378:metabolism; D013379:Substrate Specificity; D014961:X-Ray Diffraction
D004791:Enzyme Inhibitors; D008055:Lipids; D010168:Palmitates; D010712:Phosphatidic Acids; C031599:dipalmitoylphosphatidic acid; D010739:Phospholipase D; D002118:Calcium