Phospholipase D-mediated aggregation, fusion, and precipitation of phospholipid vesicles.
Large unilamellar vesicles with a diameter of 100 nm were prepared from the zwitterionic phospholipid POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) at pH 8.0. After addition to these vesicles of the enzyme phospholipase D (PLD) from Streptomyces sp. AA586 at 40 degrees C, the terminal phosphate ester bond of POPC was hydrolyzed, yielding the negatively charged POPA (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidic acid) and the positively charged choline. While the reaction yield in the presence of 1 mM Ca2+ reached 100%, the yield was only approximately 68% in the absence of Ca2+. Furthermore, in the absence of Ca2+, the size of the vesicles did not change significantly with time upon PLD addition, as judged from turbidity, dynamic light scattering, and electron microscopy measurements. In the presence of 1 mM Ca2+, however, PLD addition resulted in vesicle aggregation, fusion, and precipitation, originating from the interaction of Ca2+ ions with the negatively charged phospholipids formed in the membranes. Vesicle fusion was monitored by using a novel fusion assay system involving vesicles containing entrapped trypsin and vesicles containing entrapped chymotrypsinogen A. After vesicle fusion, chymotrypsinogen A transformed into a-chymotrypsin, catalyzed by trypsin inside the fused vesicles. The alpha-chymotrypsin formed could be detected with benzoyl-L-Tyr-p-nitroanilide as a membrane permeable chymotrypsin substrate. The observed vesicle precipitation occurring after vesicle fusion in the presence of 1 mM Ca2+ was correlated with an increase of the main phase transition temperature, Tm, of POPA to values above 40 degrees C.
Ichikawa
Sosaku
S
Departement Materialwissenschaft, Eidgenössische Technische Hochschule (ETH) Zürich, Universitätstrasse 6, CH-8092 Zürich, Switzerland. sosakui@sakura.cc.tsukuba.ac.jp
Walde
Peter
P
D002118:Calcium / Q000737:chemistry; D002413:Cations, Divalent; D011232:Chemical Precipitation; D002919:Chymotrypsinogen / Q000737:chemistry; D008051:Lipid Bilayers / Q000737:chemistry; D008561:Membrane Fusion; D008854:Microscopy, Electron; D010712:Phosphatidic Acids / Q000737:chemistry; D010713:Phosphatidylcholines / Q000737:chemistry; D010739:Phospholipase D / Q000737:chemistry*; D010743:Phospholipids / Q000737:chemistry*; D013696:Temperature; D013997:Time Factors; D014357:Trypsin / Q000737:chemistry
C103238:1-palmitoyl-2-oleoyl-glycero-3-phosphatidic acid; D002413:Cations, Divalent; D008051:Lipid Bilayers; D010712:Phosphatidic Acids; D010713:Phosphatidylcholines; D010743:Phospholipids; D002919:Chymotrypsinogen; D010739:Phospholipase D; D014357:Trypsin; D002118:Calcium; C028694:1-palmitoyl-2-oleoylphosphatidylcholine