Phospholipase D-Mediated Aggregation, Fusion, and Precipitation of

In the presence of 1 mM Ca2+, however, PLD addition resulted in vesicle aggregation, fusion, and precipitation, originating from the interaction of Ca...
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Langmuir 2004, 20, 941-949

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Phospholipase D-Mediated Aggregation, Fusion, and Precipitation of Phospholipid Vesicles† Sosaku Ichikawa‡ and Peter Walde* Departement Materialwissenschaft, Eidgeno¨ ssische Technische Hochschule (ETH) Zu¨ rich, Universita¨ tstrasse 6, CH-8092 Zu¨ rich, Switzerland Received September 15, 2003. In Final Form: November 6, 2003 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 °C, the terminal phosphate ester bond of POPC was hydrolyzed, yielding the negatively charged POPA (1-palmitoyl-2-oleoyl-sn-glycero-3phosphatidic acid) and the positively charged choline. While the reaction yield in the presence of 1 mM Ca2+ reached 100%, the yield was only ∼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 R-chymotrypsin, catalyzed by trypsin inside the fused vesicles. The R-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 °C.

Introduction In the presence of excess water and around neutral pH, the widely distributed phospholipase D (PLD)1 catalyzes the hydrolysis of the zwitterionic membrane lipid phosphatidylcholine (PC) to yield the negatively charged phosphatidic acid (PA, pKa1 ≈ 3-4, pKa2 ≈ 8-9) and the positively charged choline. Since some of the PLDs are regulated in vivo by a variety of molecules involved in intercellular communication, it is believed that PLD plays a role in cell signal transduction.1-5 From a biophysical and colloidal point of view, the transformation of PC into PA is interesting since this transformation dramatically changes the membrane surface properties. In the case of lipid vesicles (liposomes) used as membrane model systems, it has been shown for example that PA-containing vesicles can undergo vesicle aggregation and/or fusion in the presence of Ca2+, while pure PC vesicles do not fuse.6-9 * To whom correspondence should be addressed (peter.walde@ mat.ethz.ch). † This work was supported by a Japanese Overseas Research Fellowship given to S.I. by Monbukagakusho (the Ministry of Education, Culture, Sports, Science, and Technology of Japan). ‡ Permanent address: Institute of Applied Biochemistry, University of Tsukuba, Tsukuba 305-8572, Japan (sosakui@ sakura.cc.tsukuba.ac.jp). (1) Exton, J. H. Physiol. Rev. 1997, 77, 303-320. (2) Gomez-Cambronero, J.; Keire, P. Cell. Signalling 1998, 10, 387397. (3) Liscovitch, M.; Czarny, M.; Fiucci, G.; Lavie, Y.; Tang, X. Biochim. Biophys. Acta 1999, 1439, 245-263. (4) Shen, Y.; Xu, L.; Foster, D. A. Mol. Cell. Biol. 2001, 21, 595-602. (5) Munnik, T. Trends Plant Sci. 2001, 6, 227-233. (6) Papahadjopoulos, D.; Vail, W. J.; Pangborn, W. A.; Poste, G. Biochim. Biophys. Acta 1976, 448, 265-283. (7) Papahadjopoulos, D.; Vail, W. J.; Newton, C.; Nir, S.; Jacobson, K.; Poste, G.; Lazo, R. Biochim. Biophys. Acta 1977, 465, 579-598. (8) Koter, M.; de Kruijff, B.; van Deenen, L. L. M. Biochim. Biophys. Acta 1978, 514, 255-263. (9) Liao, M.-J.; Prestegard, J. H. Biochim. Biophys. Acta 1979, 550, 157-173.

This Ca2+-mediated fusion of PA-containing vesicles is not yet completely understood.10-12 It seems to be rather complex in its details, as illustrated with a brief summary of some of the earlier findings: (i) the vesicle fusion depends on the PA and Ca2+concentrations13 and only occurs if the lipids are in the liquid-analogue state, i.e., above the main phase transition temperature, Tm;6 (ii) studies on mixed PC-PA vesicles have shown that vesicle fusion only occurs if the molar ratio of Ca2+ to PA is g0.2; otherwise the vesicles only reversibly aggregate, particularly if the PA content in the vesicles is high; (iii) with increasing PC content in mixed PC-PA vesicles, the threshold Ca2+ concentration for fusion increases, while the extent of fusion decreases;13 (iv) it seems that a Ca2+induced lateral phase separation14,15 is important for making the vesicle membranes susceptible to fusion;8 (v) although in some studies extensive leakage of internal contents during the fusion process has not been observed,9 other investigations have shown that Ca2+-induced vesicle leakage is significant and pH dependent, the leakage being more pronounced at pH 8.5 than at pH 6.0;16 (vi) with respect to the mechanism of Ca2+-induced fusion of PAcontaining vesicles, it has been proposed on one hand that the appearance of a hexagonal phase of PA is important, particularly at pH e6.0;17,18 on the other hand, the (10) Papahadjopoulos, D.; Nir, S.; Du¨zgu¨nes, N. J. Bioenerg. Biomembr. 1990, 22, 157-179. (11) Cevc, G.; Richardsen, H. Adv. Drug. Delivery Rev. 1999, 38, 207-232. (12) Ohki, S.; Arnold, K. Colloids Surf., B 2000, 18, 83-97. (13) Sundler, R.; Du¨zgu¨nes, N.; Paphadjopoulos, D. Biochim. Biophys. Acta 1981, 649, 751-758. (14) Ito, T.; Ohnishi, S.-I. Biochim. Biophys. Acta 1974, 352, 29-37. (15) Jacobson, K.; Papahadjopoulos, D. Biochemistry 1975, 14, 152161. (16) Sundler, R.; Paphadjopoulos, D. Biochim. Biophys. Acta 1981, 649, 743-750. (17) Verkleij, A. J.; De Maagd, R.; Leunissen-Bijvelt, J.; De Kruijff, B. Biochim. Biophys. Acta 1982, 684, 255-262.

10.1021/la030357r CCC: $27.50 © 2004 American Chemical Society Published on Web 01/06/2004

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relevance of such a phase transition has been seriously questioned.11,19 In all these basic studies on the aggregation and fusion of vesicles containing PA, phospholipid vesicles with a defined PA content were used. In contrast, we report here on the behavior of phospholipid vesicles in which the PA content changes with time as a result of the action of PLD on PC, starting from a PA-free system. The aim of the present work was to investigate the chemical and physical effects of adding Streptomyces sp. AA586 PLD to large unilamellar vesicles (LUVs) prepared from POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine). To investigate a possible vesicle-vesicle fusion upon PA formation, a novel assay was developed which involved the use of mixtures of vesicles containing entrapped trypsin and vesicles containing entrapped chymotrypsinogen A. Upon fusion, the internal aqueous contents should mix and as a consequence trypsin should catalyze the transformation of the inactive chymotrypsinogen A into the enzymatically active R-chymotrypsin. Leaked enzyme molecules can be inhibited by aprotinin, and it should be possible to monitor the formation of R-chymotrypsin inside the fused vesicles by using the specific, externally added chromogenic substrate benzoylL-Tyr-p-nitroanilide (Bz-Tyr-pNA) for which the vesicle bilayers are permeable.20,21 The principle of this vesicle fusion assay is summarized in Scheme 1.

Ichikawa and Walde Scheme 1. Principle of the System Used To Investigate the Possible Aggregation and Fusion of Phospholipid Vesicles upon External Addition of PLD and Ca2+ a

Experimental Procedures Abbreviations. Bz-Arg-pNA, NR-benzoyl-L-Arg-p-nitroanilide; Bz-Tyr-pNA, benzoyl-L-Tyr-p-nitroanilide; DMPA, 1,2dimyristoyl-sn-glycero-3-phosphate; DPPC, 1,2-dipalmitoyl-snglycero-3-phosphocholine; 410, molar extinction coefficient at 410 nm; HPLC-MS, high-performance liquid chromatography connected to a mass spectrometry detector; LUV, large unilamellar vesicle; 100 nm-LUVET, large unilamellar vesicle prepared by the extrusion technique, using for the final extrusions polycarbonate membranes with a mean pore diameter of 100 nm; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine, PG, phosphatidylglycerol; PI, phosphatidylinositol; PS, phosphatidylserine; PLD, phospholipase D, phosphatidylcholine phosphatidohydolase (EC 3.1.4.4); POPA, 1-palmitoyl2-oleoyl-sn-glycero-3-phosphate; POPC, 1-palmitoyl-2-oleoyl-snglycero-3-phosphocholine; Tm, main solid analogue-liquid analogue lamellar phase transition temperature, also called main solid ordered-liquid disordered phase transition temperature, or main gel-liquid crystalline phase transition temperature; Tris, tris(hydroxymethyl)aminomethane. Materials. Bovine pancreas chymotrypsinogen A (type II), porcine pancreas trypsin (type IX), horseradish peroxidase (type I, 148 purpurogallin U/mg), Arthrobacter globiformis choline oxidase (12.8 U/mg), p-nitrophenyl-p′-guanidinobenzoate HCl, p-nitroaniline, phenol (>99.5%), 4-aminoantipyrine, Triton X-100, Tris ()Trizma Base, >99.9%), CaCl2‚2H2O (>99%), and N-trans-cinnamoylimidazole were obtained from Sigma (Buchs, Switzerland). Bovine pancreas R-chymotrypsin, aprotinin () bovine pancreas trypsin inhibitor), and p-nitroaniline (g99%) were from Fluka (Buchs, Switzerland). Streptomyces sp. AA586 phospholipase D (PLD) (product T-39) was from Asahi Kasei Co. (Tokyo, Japan). The PLD batch used contained 235 U/mg (according to Asahi Kasei, 1 U is defined as the amount of enzyme that catalyzes the hydrolysis of 1 µmol of DOPC (3 mM) to DOPA and choline per minute at 37 °C in the presence of 1.8% (wt/v) Triton X-100 at pH 5.5). POPC (>99%) was from Avanti Polar

Lipids Inc. (Alabaster, AL). Bz-Arg-pNA (>99%) and Bz-Tyr-pNA (>99%) were from Bachem (Bubendorf, Switzerland). Sepharose 4 B was from Pharmacia Biotech (Du¨bendorf, Switzerland). Stock solutions of PLD, aprotinin, CaCl2, sodium cholate, and Triton X-100 were prepared in 50 mM Tris/HCl, pH 8.0. Methods. Trypsin Active Site Titration. The amount of active trypsin in the trypsin sample was determined with p-nitrophenylp′-guanidinobenzoate, following the procedure of Chase and Shaw.22 One gram of trypsin powder contained 27 µmol of active trypsin, corresponding to 64 wt %, as calculated based on a trypsin relative molar mass of 23800.23 R-Chymotrypsin Active Site Titration. The amount of active R-chymotrypsin in the R-chymotrypsin sample was determined with N-trans-cinnamoylimidazole as described before.24 One gram of R-chymotrypsin powder contained 30 µmol of active R-chymotrypsin, corresponding to 75 wt %, as calculated based on a R-chymotrypsin relative molar mass of 25000.23 Chymotrypsinogen A Quantification. Chymotrypsinogen A was quantified after complete conversion to R-chymotrypsin by trypsin.25,26 Chymotrypsinogen A (0.25-7.71 µg/mL) was first incubated for 140 min at 25 °C with trypsin (10 µg/mL) at pH

(18) Farren, S. B.; Hope, M. J.; Cullis, P. R. Biochem. Biophys. Res. Commun. 1983, 111, 675-682. (19) Du¨zgu¨nes, N.; Hong, K.; Baldwin, P. A.; Bentz, J.; Nir, S.; Papahadjopoulos, D. In Cell Fusion; Sowers, A. E., Ed.; Plenum Press: New York, 1987; pp 241-267. (20) Walde, P.; Marzetta, B. Biotechnol. Bioeng. 1998, 57, 216-219. (21) Blocher, M.; Walde, P.; Dunn, I. J. Biotechnol. Bioeng. 1999, 62, 36-43.

(22) Chase, T.; Shaw, E. Biochem. Biophys. Res. Commun. 1967, 29, 508-514. (23) Worthington Enzyme Manual; Worthington, V., Ed.; Worthington Biochemical Corporation: Lakewood, NJ, 1993. (24) Zerner, B.; Bender, M. L. J. Biol. Chem. 1961, 236, 2930-2935. (25) Wright, H. T. J. Mol. Biol. 1973, 79, 13-23. (26) Fadnavis, N. W.; Chandraprakash, Y.; Deshpande, A. Biochimie 1993, 75, 995-999.

a Key: I, inhibitor of trypsin and R-chymotrypsin (aprotinin); S, substrate of R-chymotrypsin (Bz-Tyr-pNA); P1 and P2, products of the hydrolysis of S (p-nitroaniline and Bz-Tyr-OH).

Precipitation of Phospholipid Vesicles 8.0 (29 mM Tris/HCl) in the presence of 40 mM sodium cholate. The amount of R-chymotrypsin formed was then determined at pH 8.0 (47 mM Tris/HCl) and 25 °C by using Bz-Tyr-pNA (0.42 mM) as substrate as described below and by using an appropriate standard curve prepared with known amounts of R-chymotrypsin under otherwise identical reaction conditions. One gram of chymotrypsinogen A could be converted to 36 µmol of active R-chymotrypsin. R-Chymotrypsin Activity Measurements. The activity of R-chymotrypsin in vesicle-free systems was determined spectrophotometrically at 25 °C with Bz-Tyr-pNA (0.42 mM) as substrate at pH 8.0 (47 mM Tris/HCl),21,27 using an experimentally determined 410 (p-nitroaniline) of 9400 M-1 cm-1. In the case of R-chymotrypsin-containing vesicle samples, two types of measurements were carried out at 25 °C: (a) in the absence of cholate in intact vesicles, and (b) in the presence of 40 mM cholate that destroyed the vesicles and released the entrapped contents. Typically, the experimental conditions were as follows: To 630 µL of 50 mM Tris/HCl, pH 8.0, 200 µL of buffer (case a) or 200 µL of 200 mM sodium cholate in buffer (case b) and 100 µL of R-chymotrypsin-containing vesicle sample were first added and mixed. The reaction was started by adding 70 µL of 6.0 mM Bz-Tyr-pNA (prepared in dimethyl sulfoxide). The absorbance at 410 nm was followed with time, and the optical density changes were quantified by using a separately determined molar extinction coefficient for p-nitroaniline at 410 nm of 8900 M-1 cm-1 (case a, in the presence of LUVs with 0.5 mM POPC) or 9400 M-1 cm-1 (case b, in the presence of 40 mM cholate), respectively. Trypsin Activity Measurements. The activity of trypsin was determined by using Bz-Arg-pNA as chromogenic substrate28 in analogy to what has been described above for R-chymotrypsin, with the exception that a trypsin-containing sample was used and that the reaction was started by adding 70 µL of 6 mM BzArg-pNA (prepared in dimethyl sulfoxide). POPC Determination. POPC was quantified with the Stewart assay,29 using an appropriate calibration curve made with known amounts of POPC (15-145 nmol). Choline Determination. The choline formed upon the phospholipase D-catalyzed hydrolysis of POPC was quantified enzymatically by using choline oxidase and peroxidase:30 0.35 mL of a POPC containing sample in 50 mM Tris/HCl, pH 8.0 (with or without 1 mM CaCl2), was mixed with 0.15 mL of an aqueous solution containing 3.12 mg/mL choline oxidase, 2.64 mg/mL peroxidase, 7 mM phenol, and 5 mM 4-aminoantipyrine. After this mixture was incubated at 37 °C for 60 min, 1 mL of 2% (v/v) Triton X-100 was added and the optical density resulting from the quinoneimine dye formed was recorded spectrophotometrically at 500 nm using a 1 cm quartz cell. Preparation of “Empty” POPC 100 nm-LUVETs. Large unilamellar vesicles (LUVs) were prepared at room temperature from POPC with the extrusion technique31 as described before.21 The POPC concentration was 20 mM, and 50 mM Tris/HCl, pH 8.0, was used as buffer. For final extrusions, Costar Nucleopore membranes from Sterico (Dietikon, Switzerland) with a mean pore diameter of 100 nm were used. These “empty” LUVs (abbreviated as “empty” POPC 100 nm-LUVETs) were stored at 4 °C before use. PLD and CaCl2 Addition to “Empty” POPC 100 nm-LUVETs. The reaction samples were prepared by adding 0.1 mL of 10 mM CaCl2 and 0.1 mL of 2 mg/mL PLD to 0.8 mL of “empty” POPC 100 nm-LUVETs ([POPC] ) 0.625 mM), resulting in suspensions with the following total initial concentrations: [POPC] ) 0.5 mM, [Ca2+] ) 1 mM, [PLD] ) 0.2 mg/mL ()47 U/mL). For control measurements, instead of CaCl2 or PLD stock solutions, 50 mM Tris/HCl, pH 8.0, was added. Preparation of Trypsin-Containing POPC 100 nm-LUVETs. Trypsin-containing LUVs were prepared similarly as in the case of “empty” 100 nm-LUVETs with the exception that only five freezing-thawing cycles were used and that all the preparation (27) Bundy, H. F. Arch. Biochem. Biophys. 1963, 102, 416-422. (28) Nagel, W.; Willig, F.; Peschke, W.; Schmidt, F. H. Hoppe-Seylers Z. Physiol. Chem. 1965, 340, 1-10. (29) Stewart, J. C. M. Anal. Biochem. 1980, 104, 10-14. (30) Imamura, S.; Horiuti, Y. J. Biochem. 1978, 83, 677-680. (31) Mayer, L. D.; Hope, M. J.; Cullis, P. R. Biochim. Biophys. Acta 1986, 858, 161-168.

Langmuir, Vol. 20, No. 3, 2004 943 was carried out at 4 °C. The Tris/HCl buffer contained 10 mg/mL trypsin (corresponding to 270 µM active trypsin). Nonentrapped trypsin was separated from LUVs by size exclusion chromatography at 4 °C using sepharose 4B (column diameter: 1.6 cm, height: 45 cm). One milliliter of the vesicle suspension were applied, a flow rate of 0.4 mL/min was used, and fractions of 2 mL volume were collected. Preparation of Chymotrypsinogen A-Containing POPC 100 nmLUVETs. Chymotrypsinogen A-containing LUVs were prepared in the same way as trypsin-containing LUVs with the exception that (i) the buffer contained 10 mg/mL chymotrypsinogen A (corresponding to 360 µM chymotrypsinogen A) and (ii) the vesicles were prepared at room temperature. Separation of nonentrapped chymotrypsinogen A from chymotrypsinogen A-containing vesicles was performed at 4 °C, as described above in the case of trypsin. PLD and CaCl2 Addition to Chymotrypsinogen A- and TrypsinContaining POPC 100 nm-LUVETs. The reaction samples were prepared by adding 0.12 mL of chymotrypsinogen A-containing vesicles ([POPC] ) 2.1 mM), 0.11 mL of trypsin-containing vesicles ([POPC] ) 2.3 mM), 0.10 mL of 10 mM CaCl2, 0.10 mL of 2 mg/mL PLD, and 0.07 mL of 1.5 mM Bz-Tyr-pNA (prepared in dimethyl sulfoxide) to 0.5 mL of 50 mM Tris/HCl, pH 8.0. The total initial concentrations in the reaction mixture were [POPC] ) 0.5 mM, [Ca2+] ) 1 mM, [PLD] ) 0.2 mg/mL ()47 U/mL), [chymotrypsinogen]overall ) 130 nM, [trypsin]overall ) 86 nM, [BzTyr-pNA] ) 0.11 mM, and [dimethyl sulfoxide] ) 7% (v/v). For control measurements, the corresponding reagents were replaced by 50 mM Tris/HCl. In the cases of measurements with aprotinin, stock solutions of 40 µM (50 µL) were added to result in a total volume of 1 mL and to yield 2 µM aprotinin. HPLC-MS Measurements. The HPLC-MS measurements were carried with a Thermo Finnigan instrument composed of an HPLC (P4000), a diode array detector (UV6000 LP), and an ion-trap mass spectrometry detector (LCQ-Deca). A C18 reversed phase column EC 250/4 Nucleosil 100-5 (from Macherey-Nagel AG, Oensingen, Switzerland) was used at a flow rate of 1 mL/ min. Eluent A was 0.3 mM sodium acetate in a mixture of 1000 mL of methanol and 1 mL of trifluoroacetic acid. Eluent B was 0.3 mM sodium acetate in a mixture of 816 mL of methanol, 81 mL of water, 103 mL of acetonitrile and 1 mL of trifluoroacetic acid. After sample injection, elution was first set for 3 min at 100% eluent A, followed by a linear gradient for 54 min from 100% eluent A to 100% eluent B. Elution at 100% eluent B continued for 13 min. The mass spectrometry detector was set for monitoring m/z values (positive electrospray ionization, z ) 1) of 697.4-698.4 ([POPA - H2 - Na+] adduct) and 782.6-783.6 ([POPC - Na+] adduct). The capillary temperature was 350 °C, and the other settings were as follows: 80 units sheath nitrogen gas flow rate, 20 units auxiliary nitrogen gas flow rate, 4.50 kV I-spray voltage, 3-39 V capillary voltage, and -60 ( 25 V tube lens offsets. The idea to use sodium acetate in the mobile phase for the detection of POPA-H2 as positively charged sodium adduct stems from work on the analysis of a dog plasma sample.32 Other Instrumental Methods. Freeze fracture electron microscopy and dynamic light scattering measurements were performed as described before.20,33 For UV-vis absorption and turbidity measurements, a Cary 1E spectrophotometer (from Varian International AG, Basel, Switzerland) or a Kontron Uvikon 810 instrument was used.

Results and Discussion Characterization of “Empty” POPC 100 nm-LUVETs. On the basis of dynamic light scattering measurements, the “empty” vesicles prepared in this work had a mean hydrodynamic diameter of about 103 nm with a low scattering angle dependency and a relatively low polydispersity index; see the values reported in Table 1. Freeze fracture electron micrographs confirmed the relative (32) Jemal, M.; Almond, R. B.; Teitz, D. S. Rapid Commun. Mass Spectrom. 1997, 11, 1083-1088. (33) Dorovska-Taran, V.; Wick, R.; Walde, P. Anal. Biochem. 1996, 240, 37-47.

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Figure 1. Effect of Ca2+ on the yields in the PLD-catalyzed hydrolysis of “empty” POPC 100 nm-LUVETs (initial [POPC] ) 0.5 mM) at 40 °C (50 mM Tris/HCl, pH 8) as determined either enzymatically from the free choline formation as a function of incubation time (A) or analyzed by HPLC-MS after an incubation of 96 h (B): (1, 9) no PLD, no CaCl2 (control); (2, O) no PLD, 1 mM CaCl2; (3, 0) 0.2 mg/mL PLD, no CaCl2; (4, b) 0.2 mg/mL PLD, 1 mM CaCl2. Table 1. Dynamic Light Scattering Measurements of the “Empty” POPC 100 nm-LUVETs Prepared, As Measured at 40 °C scattering angle (deg)

hydrodynamic radius (nm)

polydispersity index (-)

60 90 120

53.0 ( 0.3 51.6 ( 0.2 50.5 ( 0.2

0.019 ( 0.006 0.027 ( 0.007 0.033 ( 0.003

homogeneity of the vesicle preparation and showed that most of the vesicles were unilamellar (not shown), as expected for this type of 100 nm-LUVETs.21,31,33 PLD Addition to “Empty” POPC 100 nm-LUVETs. The extent of POPC hydrolysis to POPA (quantified as choline release) upon addition of Streptomyces sp. AA586 PLD to POPC vesicles in the absence and in the presence of 1 mM Ca2+ was measured as a function of incubation time at 40 °C, as shown in Figure 1A. The presence of Ca2+ had no influence on the initial reaction rates (data not shown) but had a significant influence on the equilibrium reaction yields, which were ∼68% in the absence of Ca2+ (reaction 3 in Figure 1A) and 100% in the presence

of 1 mM Ca2+ (reaction 4 in Figure 1A). In both cases, these equilibrium values were reached after about 24-48 h. No significant hydrolysis was observed in the absence of PLD and Ca2+ (reaction 1 in Figure 1A) or in the absence of PLD but presence of 1 mM Ca2+ (reaction 2 in Figure 1A). A HPLC-MS analysis of the reaction mixtures carried out after reaching equilibrium confirmed that a complete POPC hydrolysis upon PLD addition only occurred in the presence of Ca2+ (see Figure 1B). The reaction yields determined in the absence of Ca2+ (reaction 3 in Figure 1) agree quite well with a previous investigation in our laboratory, using the same type of PLD, 1H NMR spectroscopy as analytical tool, and slightly different experimental conditions.33 A 100% conversion of POPC into POPA could never be reached in the absence of Ca2+. In the presence of 1 mM Ca2+ (reaction 4 in Figure 1), visible lipid precipitation occurred after about 100 h of incubation at 40 °C. If all the reactions were performed at 25 °C, lipid precipitation was already visible after 5070 h. This precipitation is most likely due to the binding of Ca2+ ions to the negatively charged POPA which leads to a dehydration of the headgroups, and in turn to a reduction of the phospholipid alkyl chain mobility, as discussed in the case of DMPA/Ca2+ systems.14,34 The Ca2+-POPA complex formation results supposedly (i) in a dramatic increase in Tm,35 (ii) in a lateral phase separation, and finally (iii) in the precipitation of the POPA-Ca2+ complexes, as again discussed in the case of DMPA-Ca2+ systems.34,36-39 The Tm value of POPA at pH 7 and in the absence of Ca2+ is around 28 °C,40 in comparison with POPC that has a Tm of about -3 °C.41 On the basis of our experimental observations about the vesicle precipitation, the Tm value of POPA in the presence of Ca2+ must be above 40 °C. Freeze fracture electron micrographs taken after an incubation time of 24 h at 40 °C confirmed the changes in the aggregation state of the phospholipids in all those samples containing both PLD and 1 mM CaCl2 (reaction 4 in Figure 1); see Figure 2. It looks as if small, flattened disklike aggregates first formed (Figure 2B), before further aggregation led to visible lipid precipitation. There is no evidence that so-called cochleate cylinders (large rolledup planar lamellae) formed through vesicle fusion as described in the case of Ca2+-treated sonicated unilamellar vesicles prepared from PS.42 In the absence of Ca2+, but in the presence of PLD (case 3 in Figure 1) and in the two control measurements without PLD (cases 1 and 2 in Figure 1), no obvious changes in vesicle size and morphology were observed, at least during the first 140 h (freeze fracture electron microscopy data not shown). Figure 3 shows how the turbidity of the POPC 100 nmLUVET suspension changed upon adding PLD in the presence of 1 mM CaCl2 at 40 °C (curve 4). The turbidity (34) Laroche, G.; Dufourc, E. J.; Dufourc, J.; Pe´zolet, M. Biochemistry 1991, 30, 3105-3114. (35) Van Dijck, P. W. M.; de Kruijff, B.; Verkleij, A. J.; van Deenen, L. L. M.; de Gier, J. Biochim. Biophys. Acta 1978, 512, 84-96. (36) Garidel, P.; Blume, A. Langmuir 2000, 16, 1662-1667. (37) Graham, I.; Gagne´, J.; Silvius, J. R. Biochemistry 1985, 24, 71237131. (38) Liao, M. J.; Prestegard, J. H. Biochim. Biophys. Acta 1981, 645, 149-156. (39) Konaouci, R.; Silvius, J. R.; Graham, I.; Pe´zolet, M. Biochemistry 1985, 24, 7132-7140. (40) Demel, R. A.; Yin, C. C.; Lin, B. Z.; Hauser, H. Chem. Phys. Lipids 1992, 60, 209-223. (41) Koynova, R.; Caffrey, M. Biochim. Biophys. Acta 1998, 1376, 91-145. (42) Papahadjopoulos, D.; Vail, W. J.; Jacobson, K.; Poste, G. Biochim. Biophys. Acta 1975, 394, 483-491.

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Figure 2. Freeze fracture electron micrographs of the PLD- and Ca2+-containing reaction mixture 4 described in the legend of Figure 1. The samples were frozen either 24 h (left-hand side of A, B) or 44 h (right-hand side of A) after the start of the reaction. The lengths of the bars correspond to 400 nm (in A) and 200 nm (in B), respectively.

Figure 3. Time-dependent changes in the turbidity (measured at 300 nm) of the reaction mixtures 1 (2), 2 (3), 3 (9, 0, @), and 4(O, b, s (continuous recording)), as described in the legend of Figure 1. For the reaction mixtures 3 and 4, data from three independent measurements are included in the figure.

reached a maximum after 70-80 h and then decreased due to phospholipid-Ca2+ precipitation, as described

above. In the absence of Ca2+ (but with PLD) or in the absence of PLD (with or without Ca2+), the turbidity remained constant for at least 140 h, indicating again that changes in the size and/or morphology of the vesicle suspension only occurred if both PLD and Ca2+ were added. The turbidity behavior is also reflected-now more quantitatively-in the dynamic light scattering measurements; see Figure 4. While the size and size distribution of the two control samples (1 and 2) and the sample containing PLD without added CaCl2 (3) did not change significantly up to about 140 h of incubation at 40 °C (data not shown), the reaction mixture containing PLD and Ca2+ (4)ssee curves b and c in Figure 4sgot more polydisperse with the formation of smaller aggregates as well as larger structures with a radius centered around 500 nm. In summary, all measurements with “empty” vesicles showed that in the presence of PLD significant size changes in the vesicle sample only occurred if CaCl2 (1 mM in our case) was coadded with PLD. The question whether these changes involved true vesicle fusion was approached by a novel vesicle fusion assay (Scheme 1) which is based on the use of vesicles containing entrapped

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Table 2. Characteristic Properties of Trypsin-Containing and Chymotrypsinogen A-Containing POPC 100 nm-LUVETs, As Obtained in the Pooled Chromatography Fractions after Separation from Free Trypsin or Chymotrypsinogen A, Respectively

vesicle type

no.

mean hydrodynamic diametera (nm)

trypsin-containing vesicles

1 2 3 4d 1 2d

99 99 97 109 96 110

chymotrypsinogen A containing vesicles

POPC concn (mM) 2.1 1.5 1.7 2.3 1.7 2.1

vesicle concnb(nM)

overall enzyme concn (µM)

mean intravesicular enzyme concn (µM)

mean no. of enzyme molecules per vesicle

entrapment yield of enzymec (%)

26 19 23 24 23 22

0.53 0.35 0.42 0.78 0.70 1.07

86 75 81 99 141 148

21 18 18 33 31 50

1.6 1.1 1.0 1.8 1.2 1.9

a As determined by dynamic light scattering measurements at a scattering angle of 90°. b For the calculation, monodispersity, unilamellarity, and a spherical vesicle shape were considered. Furthermore, a bilayer thickness of 3.7 nm, a mean POPC headgroup area of 0.72 nm2, and a negligible critical POPC aggregate concentration of 1.0 nM were taken into account. c Entrapment yields are related to the total amount of trypsin (or chymotrypsinogen A) used during the vesicle preparation. d Preparation used for the fusion tests.

Figure 4. Dynamic light scattering measurements (reported as relative scattering intensity vs size measured at a scattering angle of 90°) of the reaction mixture 4 described in the legend of Figure 1. The sample was measured before PLD and CaCl2 addition (a, O) and 30 h (b, 9) and 140 h (c, 3) after PLD and CaCl2 addition.

Figure 5. Stability of free trypsin (O) and trypsin-containing POPC 100 nm-LUVETs (b) prepared in 50 mM Tris/HCl, pH 8.0, and stored at 4 °C. For trypsin-containing vesicles: [POPC] ) 2.1 mM; [trypsin]overall ) 0.53 µM. Mean trypsin concentration inside the vesicles was 86 µM. Determination of active trypsin after 40 mM cholate addition to the vesicles using Bz-Arg-pNA as substrate. For free trypsin in 50 mM Tris/HCl, pH 8.0: [trypsin] ) 78 µM.

trypsin and vesicles containing entrapped chymotrypsinogen A. The results on the preparation of these two protein-containing vesicles and on the fusion tests are reported and discussed in the following. Characterization of Trypsin-Containing POPC 100 nm-LUVETs. The loading of POPC vesicles with trypsin occurred passively during the vesicle preparation, resulting in a 1.0-1.8% entrapment yield under the conditions used (20 mM POPC during the vesicle formation process); see Table 2. The separation of free trypsin from trypsin-containing vesicles was very efficient, which allowed the preparation of a suspension of vesicles containing only trypsin trapped inside the vesicles, completely rid of free enzyme molecules (see Figure 1A, Supporting Information). The hydrodynamic vesicle diameter varied between 97 and 109 nm. There were about 18-33 trypsin molecules present per vesicle, corresponding to a trypsin concentration inside the vesicles of about 75-99 µM (see Table 2). If stored at 4 °C, the trypsincontaining vesicles lost about 8% of their activity within the first 3 days. After 20 days, the enzyme activity decreased by 20% (see Figure 5). This loss in activity is not surprising since it is well-known that trypsin undergoes autolysis (self-degradation), particularly in the absence of Ca2+.23 A similar extent of inactivation was observed with free trypsin at [trypsin] ) 78 µM in 50 mM Tris/HCl, pH 8.0 (Figure 5), indicating that trypsin entrapment in the vesicles led neither to inactivation nor to stabilization of this enzyme. This shows that there is

most likely no interaction between POPC and the positively charged enzyme molecules; the pI of trypsin is about 10.5.23 Characterization of Chymotrypsinogen A-Containing POPC 100 nm-LUVETs. The separation of free chymotrypsinogen A from the vesicles by size exclusion chromatography was again very efficient (Figure 1B, Supporting Information), and the entrapment yield (1.21.9%) and the vesicle diameter (96-110 nm) were comparable to the case of trypsin-containing vesicles; see Table 2. Each vesicle contained about 31-50 chymotrypsinogen A molecules, corresponding to a chymotrypsinogen A concentration inside the vesicles of 141-148 µM. Chymotrypsinogen A in this vesicle preparation was rather stable if stored at 4 °C (Figure 2, Supporting Information). For at least 16 days, there was no loss in the amount of active R-chymotrypsin which could be obtained from chymotrypsinogen A through activation with trypsin. PLD and CaCl2 Addition to a Mixture of TrypsinContaining POPC 100 nm-LUVETs and Chymotrypsinogen-Containing POPC 100 nm-LUVETs. The addition of PLD, CaCl2 (1 mM), and the chymotrypsin substrate Bz-Tyr-pNA (initial concentration 0.11 mM) to a mixture of trypsin-containing and chymotrypsinogen A-containing vesicles led to the hydrolysis of Bz-Tyr-pNA (formation of p-nitroaniline, as monitored spectrophotometrically at 410 nm); see curve 1 in Figure 6. This clearly indicates that R-chymotrypsin was formed in this vesicle system. After about 2-6 h of incubation at 40 °C, all

Precipitation of Phospholipid Vesicles

Figure 6. Hydrolysis of Bz-Tyr-pNA caused by the addition of PLD and CaCl2 to a mixture of trypsin-containing POPC 100 nm-LUVETs and chymotrypsinogen A-containing POPC 100 nm-LUVETs at 40 °C (50 mM Tris/HCl, pH 8.0): 1, complete reaction mixture; 2, no PLD added; 3, no Ca2+ added; 4, no chymotrypsinogen A-containing vesicles present; 5, no trypsincontaining vesicles present. The total initial concentrations were [POPC] ) 0.5 mM, [trypsin] ) 0.086 µM, [chymotrypsinogen A] ) 0.13 µM, [PLD] ) 0.2 mg/mL (47 U/mL), [Ca2+] ) 1 mM, [Bz-Tyr-pNA] ) 0.11 mM, and [dimethyl sulfoxide] ) 7.0% (v/ v).

substrate molecules added were hydrolyzed. In the absence of PLD or in the absence of Ca2+, the rate of hydrolysis of Bz-Tyr-pNA was rather low; see Figure 6, curves 2 and 3, respectively. Furthermore, if PLD, CaCl2, and Bz-TyrpNA were added (i) to chymotrypsinogen A-containing vesicles alone (in the absence of trypsin-containing vesicles) or (ii) to trypsin-containing vesicles alone (in the absence of chymotrypsinogen A-containing vesicles), the rate of p-nitroaniline formation was very low; see Figure 6, curves 4 and 5, respectively. All this indicates that the action of PLD on POPC vesicles in the presence of Ca2+ (and not in the absence of Ca2+) led to a fusion of the vesiclessat least during the first hours of the reaction, before the above-described phospholipid rigidification and precipitation occurred. This fusion involved the mixing of the aqueous vesicle contents, resulting in the transformation of chymotrypsinogen A into R-chymotrypsin, catalyzed by trypsin molecules which originally were present in a different population of vesicles, as indicated in Scheme 1. One general point of concern in vesicle fusion which we considered was the question whether vesicle-vesicle interactionssaggregation and or/fusionsled to a release of trapped molecules; see for example the experiments on the Ca2+-induced fusion of PS vesicles.43,44 To avoid (or minimize) a possible leakage from the vesicles, we used in our study vesicles containing large molecules, proteins with relative molar masses in the range of 25000, which can be considered as being beneficial with respect to the general leakage concern.45 Nevertheless, to inhibit potentially released enzyme molecules from the vesicles’ interior into the external aqueous space, the inhibitor protein aprotinin was used. This protein has a relative molar mass of 6500 and an isoelectric point around 10.5.46 Since aprotinin inhibits (43) Wilschut, J.; Du¨zgu¨nes, N.; Fraley, R.; Papahadjopoulos, D. Biochemistry 1980, 19, 6011-6021. (44) Hoeckstra, D.; Yaron, A.; Carmel, A.; Scherphof, G. FEBS Lett. 1979, 106, 176-180. (45) Du¨zgu¨nes, N.; Allen, T. M.; Fedor, J.; Papahadjopoulos, D. In Molecular Mechanism of Membrane Fusion; Ohki, S., Doyle, D., Flanagan, T. D., Hui, S. W., Mayhew, E., Eds.; Plenum Press: New York, 1988; pp 543-555.

Langmuir, Vol. 20, No. 3, 2004 947

Figure 7. Influence of aprotinin on the hydrolysis of Bz-TyrpNA caused by the addition of PLD and CaCl2 to a mixture of trypsin-containing POPC 100 nm-LUVETs and chymotrypsinogen A-containing POPC 100 nm-LUVETs at 40 °C (50 mM Tris/HCl, pH 8.0). The total concentrations were [POPC] ) 0.5 mM, [trypsin] ) 0.086 µM, [chymotrypsinogen A] ) 0.13 µM, [PLD] ) 0.2 mg/mL (47 U/ml), [Ca2+] ) 1 mM, [Bz-Tyr-pNA] ) 0.11 mM, and [dimethyl sulfoxide] ) 7.0% (v/v). (1) Chymotrypsinogen A-containing vesicles and trypsin-containing vesicles were first incubated with PLD and Ca2+ for 24 h at 40 °C. Aprotinin and Bz-Tyr-pNA were then added, and the formation of p-nitroaniline was monitored. (2) Chymotrypsinogen A-containing vesicles and trypsin-containing vesicles were mixed with PLD, Ca2+, aprotinin, and Bz-Tyr-pNA, and the formation of p-nitroaniline was followed. (3) Chymotrypsinogen A-containing vesicles and trypsin-containing vesicles were first incubated with PLD and Ca2+ for 24 h at 40 °C. Bz-Tyr-pNA was then added (without aprotinin), and the formation of p-nitroaniline was monitored. (4) Chymotrypsinogen A-containing vesicles and trypsin-containing vesicles were first incubated with PLD and Ca2+ in the presence of externally added aprotinin for 24 h at 40 °C. Bz-Tyr-pNA was then added, and the formation of p-nitroaniline was monitored.

trypsin as well as R-chymotrypsinsthe dissociation constants of the enzyme-aprotinin complex being in the order of 10-11-10-12 M in the case of trypsin and 10-7 M in the case of chymotrypsin, respectively47swe considered this inhibitor to be well suited for the present work. Furthermore, aprotinin was already successfully used in the previous studies on R-chymotrypsin-containing POPC 100 nm-LUVETs.20,21 Figure 7 shows product-time curves of four different sets of experiments. In the first (curve 1) chymotrypsinogen A-containing vesicles and trypsin-containing vesicles were first incubated with externally added PLD and Ca2+ (1 mM) for 24 h. After that, aprotinin and Bz-Tyr-pNA were added, and the formation of p-nitroaniline was followed spectrophotometrically at 410 nm. The reaction clearly proceeded for at least 24 h, with an initial velocity of about 0.5 µM/min. After 24 h, 73% of the substrate molecules were hydrolyzed. To our first surprise, however, no significant substrate hydrolysis occurred if chymotrypsinogen A-containing vesicles and trypsin-containing vesicles were mixed in the presence of PLD, Ca2+ (1 mM), Bz-Tyr-pNA, and aprotinin (curve 2 in Figure 7). Furthermore, only a very slow substrate hydrolysis was found if chymotrypsinogen A-containing vesicles and trypsin-containing vesicles were first incubated in the presence of PLD and Ca2+ (1 mM) for 24 h, and Bz-Tyr-pNA was then added in the absence of aprotinin (curve 3 in Figure 7). The same was observed, if chymotrypsinogen A-containing vesicles and trypsin(46) Kassell, B. Methods Enzymol. 1970, 19, 844-852. (47) Laskowski, M., Jr.; Sealock, R. W. In The Enzymes; Boyer, P. D., Ed.; Academic Press: New York, 1971; Vol. III, pp 375-473.

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Langmuir, Vol. 20, No. 3, 2004

Figure 8. Effect of disrupting a mixture of chymotrypsinogen A- and trypsin-containing POPC 100 nm-LUVETs with cholate on the activation of chymotrypsinogen A by trypsin in the absence (1) or presence (2) of aprotinin at 40 °C (50 mM Tris/ HCl, pH 8.0). The total initial concentrations were [POPC] ) 0.5 mM, [trypsin] ) 0.086 µM, [chymotrypsinogen A] ) 0.13 µM, [PLD] ) 0.2 mg/mL (47 U/mL), [aprotinin] ) 2.0 µM (curve 2 only), [Ca2+] ) 1 mM, [Bz-Tyr-pNA] ) 0.11 mM, [dimethyl sulfoxide] ) 7.0% (v/v), and [cholate] ) 40 mM.

containing vesicles were first incubated in the presence of PLD, Ca2+ (1 mM), and aprotinin for 24 h at 40 °C, and Bz-Tyr-pNA was then added (curve 4 in Figure 7). Before discussing these observations, let us mention that in control experiments shown in Figure 8, chymotrypsinogen A-containing vesicles and trypsin-containing vesicles were incubated in the presence of Ca2+, Bz-TyrpNA, and sodium cholate (40 mM)seither in the absence of aprotinin (curve 1) or in the presence of aprotinin (curve 2). The presence of high concentrations of sodium cholate led to a transformation of the POPC vesicles into mixed cholate-POPC micelles48 and a concomitant release of the aqueous contents. This release of trypsin and chymotrypsinogen A resulted in an activation of chymotrypsinogen A if aprotinin was not present (curve 1 in Figure 8). On the other hand, the presence of aprotinin led to an almost complete inhibition of trypsin under the conditions used: chymotrypsinogen A was not activated to R-chymotrypsin (curve 2 in Figure 8). All the experiments carried out with aprotinin can be explained as follows. It is likely that the positively charged aprotinin molecules interact with the outer monolayer of the phospholipid membrane, particularly if the membrane has a negative charge (presence of POPA). On the other hand, entrapped trypsin, chymotrypsinogen A, and the formed R-chymotrypsin are also positively charged at pH 8, the pI values being between 9 and 10.5;23 these proteins are therefore probably also getting adsorbed to some extent onto the vesicle membrane during the course of the PLDcatalyzed POPC-POPA transformation. The site of adsorption is most likely the inner surface of the vesicle membrane. Due to the partial hydrophobicity of the externally added aprotinin,49 this small protein penetrates into and possibly crosses the phospholipid membrane from the outer layer into the inner layer of POPA-containing vesicles, allowing interaction with trypsin, chymotrypsinogen A, or the formed R-chymotrypsin. This hypothesis is supported by (i) the fact that aprotinin has bactericidal activity,50,51 strongly interacting with nega(48) Treyer, M.; Walde, P.; Oberholzer T. Langmuir 2002, 18, 10431050. (49) Ilyina, E.; Roongta, V.; Pan, H.; Woodward, C.; Mayo, K. H. Biochemistry 1997, 36, 3383-3388. (50) Pellegrini, A.; Thomas, U.; von Fellenberg, R.; Wild P. J. Appl. Bacteriol. 1992, 72, 180-187.

Ichikawa and Walde

tively charged bacterial membranes, and by (ii) a report on the binding of aprotinin to negatively charged multilamellar vesicles prepared from soybean phospholipid mixtures.52 A passage of aprotinin across the POPA-containing bilayers and the resulting enzyme inhibition seems only to be possible at the early stages of the reaction, within the first hours. After complete POPC hydrolysis to POPAs which occurred after less than 20 hsunder the conditions used (Figure 1), the interaction of aprotinin with the phospholipid membrane resulted in an increase in the membrane permeability for the substrate without inhibition of the enzymes. This effect on the permeability of the bilayer may be a direct consequence of the Ca2+-dependent lipid rigidification during the course of the reaction as discussed above. With POPC vesicles alone (without POPA) in the fluid state (above Tm), there was no detectable effect of aprotinin on the permeability for Bz-Tyr-pNA (Marzetta and Walde, unpublished). On the basis of the above considerations, the measurements reported in Figure 7 show that (i) vesicle fusion indeed occurred as a result of the PLD action on POPC in the presence of Ca2+ and that (ii) the contents of the vesicles did not leak out from the vesicles during the fusion. If leakage would have occurred, the experiments in which chymotrypsinogen A-containing vesicles and trypsincontaining vesicles were incubated for 24 h at 40 °C, followed by Bz-Tyr-pNA addition, significant hydrolysis should have occurred by the leaked proteins (curve 3 in Figure 7). This was, however, not the case. In summary, the present investigation showed that the addition of Streptomyces sp. AA586 PLD and 1 mM CaCl2 to POPC 100 nm-LUVETs led to an initial vesicle aggregation and fusion with an increase in the vesicle sizes from initially about 100 nm to a mixture of lipid aggregates with diameters varying between about 40 and 500 nm. During the PLD reaction (in the presence of 1 mM Ca2+) POPC was quantitatively hydrolyzed to POPA and choline (100% reaction yield). The POPA molecules formed caused a decrease in the fluidity of the phospholipid membranes due to the interaction with Ca2+, until the system collapsed under precipitation. If the experiments were carried out in the absence of Ca2+ but otherwise identical conditions, only about 68% of the POPC was hydrolyzed and there was no detectable vesicle aggregation or fusion. Concluding Remarks The effect of the divalent cations Ca2+ and Mg2+ on the physicochemical and colloidal properties of lipid vesicles containing negatively charged phospholipids (PS, PG, and PA) was studied extensively in the past. With respect to the present investigation, some of the important earlier findings are as follows: (i) the effects of Ca2+ strongly depend on the pH51,53 and on the concentration of the phospholipid9 and Ca2+;7-9,15,35 (ii) the binding of Ca2+ to negatively charged lipid vesicles can lead to vesicle fusions,6-9,42,43 to an increase in Tm,15,34,49 to phase separations,14,15,36,37,39,54 and to phospholipid precipitation, e.g., cochleate formation;36,42 and (iii) the rate of phase separa(51) Pellegrini, A.; Thomas, U.; Bramaz, N.; Klauser, S.; Hunziker, P.; von Fellenberg, R. Biochem. Biophys. Res. Commun. 1996, 222, 559565. (52) Tiourina, O. P.; Sharf, T. V.; Selishcheva, A. A.; Sorokoumova, G. M.; Shevets, V. I.; Larinova, N. I. Biochemistry (Moscow) 2001, 66, 340-344. (53) Tra¨uble, H.; Eibl, H. Proc. Natl. Acad. Sci. U.S.A. 1974, 71, 214-219.

Precipitation of Phospholipid Vesicles

tion37 greatly depends on the content of negatively charged lipids in the membrane.54 In the system that we studied, neutral (zwitterionic) POPC vesicles ([POPC] ) 0.5 mM) were continuously transformed into lipid vesicles containing POPA through the action of added Streptomyces sp. AA586 PLD in the absence and in the presence of Ca2+ (1 mM). We clearly showed that the presence of Ca2+ influences not only the reaction yields but also the aggregation state of the phospholipids. Using a mixture of trypsin- and chymotrypsinogen A-containing vesicles, we particularly demonstrated that vesicle aggregation and fusion only occur if Ca2+ (1 mM) is added together with PLD. The fusion process occurred without leakage of the water soluble probe molecules from the vesicles. This was certainly due to the macromolecular size and the positive overall charge of the proteins used at the pH of the experimentssboth factors contributing to a lowering of the leakage rate. The general advantage of having large probe molecules in vesicle fusion assays was pointed out before45 and fusion assays using mixtures of enzyme- and small substratecontaining vesicles were investigated in the past.44,55 The experiments we describe here, however, are the first vesicle fusion studies in which enzyme (trypsin) and substrate (chymotrypsinogen A) are both macromolecules. The phospholipase D used in our work was a sample isolated from Streptomyces sp AA586, a PLD which is catalytically active against PC in the absence of Ca2+ 56,57 as well as in the presence of 1 mM Ca2+ (Figure 1). From the kinetic measurements reported in Figure 1, and in agreement with earlier observations,33 there is no indication that Streptomyces sp. AA586 PLD shows a so-called “lag-phase” in the hydrolysis-time profile, as observed in the case of monolayer studies with DPPC as substrate and Streptomyces chromofuscus PLD.58 There is also no (54) Ito, T.; Ohnishi, S.-I.; Ishinaga, M.; Kito, M. Biochemistry 1975, 14, 3064-3069. (55) Ingolia, T. D.; Koshland, D. E., Jr. J. Biol. Chem. 1978, 253, 3821-3829. (56) Imamura, S.; Matsumura, E.; Misaki, H.; Mutoh, N. Jpn. Kokai Tokyo Koho JP-58152481, 1982. (57) Bonaccio, S.; Walde, P.; Luisi, P. L. J. Phys. Chem. B 1994, 98, 6661-6663 and 10376. (58) Estrela-Lopis, I.; Brezesinski, G.; Mo¨hwald, H. Biophys. J. 2001, 80, 749-754.

Langmuir, Vol. 20, No. 3, 2004 949

evidence that the POPA formed acted as an activator58,59 or as inhibitor,58,60 as again described before in the case of S. chromofuscus PLD. Therefore, the PLD from S. chromofuscus seems to be structurally different from the PLD isolated from Streptomyces sp. AA586; see refs 61 and 62 for a recent account about Streptomyces PLDs. With respect to PLD-induced fusion of phospholipid vesicles in general, it was reported before that the action of S. chromofuscus PLD in the presence of Ca2+ on PC vesicles,59 on vesicles composed of PC/PE/PA (20:50:30, molar ratio),63 or on lipid vesicles containing 50 mol % PC/PE/PI/PS (44:27:16:13, molar ratio) and 50 mol % cholesterol64 can result in vesicle fusion. Although the systems were quite different, containing the fusogenic phospholipid PE, our investigation is in line with these findings. Acknowledgment. We thank Thomas Hitz (at ETH) for helping in the HPLC-MS analysis, Dr. Ernst Wehrli (at ETH) for the electron micrographs, and Professor Francis C. Szoka (University of California, San Francisco) and Professor Gerrit L. Scherphof (University of Groningen, The Netherlands) for useful discussions about vesicle fusion. S.I. acknowledges the receipt of a Monbukagakusho overseas research fellowship. Supporting Information Available: Size exclusion chromatograms illustrating the efficiency of separating trypsincontaining vesicles from free trypsin and chymotryposinogen A-containing vesicles from free chymotrypsinogen A, as well as stability data of chymotrypsinogen A-containing vesicles. This material is available free of charge via the Internet at http://pubs.acs.org. LA030357R (59) Geng, D.; Chura, J.; Roberts, M. F. J. Biol. Chem. 1998, 273, 12195-12202. (60) Yamamoto, I.; Nishii, M.; Tokuoka, E.; Handa, T.; Miyajima, K. Colloid Polym. Sci. 1997, 275, 627-633. (61) Leiros, I.; Secundo, F.; Zambonelli, C.; Servi, S.; Hough, E. Sturcture 2000, 8, 655-667. (62) Yang, H.; Roberts, M. F. Protein Sci. 2003, 12, 2087-2098. (63) Park, J.-B.; Lee, T.-H.; Kim, H. Biochem. Int. 1992, 27, 417422. (64) Blackwood, R. A.; Smolen, J. E.; Transue, A.; Hessler, R. J.; Harsh. D. M.; Brower, R. C.; French, S. Am. J. Physiol. 1997, 272 (Cell Physiol. 41), C1279-C1285.