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P-C4-45 Kinetic analysis of pore formation by sticholysin I
c4 Lipid-protein interactions and dynamics P-c445 IUNBTIC YsuioFPoREFoRMATION BY ii%!? HOLYStN I MENESTRMA G’, TEJUCA M’s2, DALLA SERRA M’, PERRERAS M’, LAN10 ME2 ‘CNR-ITCCeFSA.POVO (Trcnt~). Italy,*Dep.Bioqui-
P-C446 THE DISTRIBUTION OF PHOSPHATIDYLGLYCEROL ACROSS THE INNER MEMBRANE OF ESCHERICHIA COU
HUIJBREGTS RPH, DE KROON AIPM, DE KRUIJFF B. Dept. Biochemistry of Membranes, CBLE,Utncht University. (NL)
mica, Univ. de la Habana, Cuba
Purpose: Sticholysin I (St-I) was isolated from the venom of the Caribbean sea anemone Stichoda&a hefianrhus. It belongs to the family of actinoporins: basic peptides able to form channels in lipid membranes. To elucidate the molecular mechanismof formation of these pores we have studied the kinetic of interaction with purely lipidic membranes. Methods: The time course of wrmeabilisation was detected by measuring caicein releasefrom uniform-sized large unilamellar vesicles.with a fluorescencemicroplate reader. Channel’sfeatures were determined using volta e-clamped planar lipid bilayers. Partition toe%*lclentsby centrifugation and electrophoresis. Results: Membrane permeabiiisation is optimal under the followin conditions. resenceof 50% sphingomyelin (Sd ), pH arouI3 8, St-I/vesicle ratio < 200. Most of the toxin is bound to the lioid chase. St-I effect was reduced at hiah DH, 0; lo; and high SM content, mainly due’ioi decreasedbindina. From the dose-deoendenceit was inferred that-St-1forms oligome& pores comprising at least 3 monomers. Conclusions: The kinetics of releasewas fully accountedby a model assuming that trimerisation leadsto a conductive pore. All the relevant
Purpose: Phosphatidylglycerol(PC) has several functions in the E.coli cell, both at the cytoplasmicand the periplasmic side of the inner membrane.Knowledge of the distribution of PC over the two leaflets of the inner membrane will lead to a better understandingof these functions. Methods: To discriminatebetween the PG presentin both leaflets, Ecofi cells or derived inner membranevesicleswere incubated with periodate. The disappearanceof PC, due to its oxidation by periodate, was monitored by TLC. Results: Incubations of both intact cells and inside-out or right side-out membrane vesicles with free periodate resulted in a fast, kinetically monophasicoxidation of the total PC pool, due to the permeability of the membrane for this oxidator. Conclusions: The oxidation of PC by free periodate is not a good tool to determine the transmembranedistribution of PC in E.coli. Experimentsusing periodate on a silica support will also be shown and discussed.
P-W-47
P-C448
FATTY ACID DESORPl’ION FROM MONOLAYERS SLOTI-E. J.P. Department of biochemistry and pharmacy, Abe Akademi University, PIN 20520 Turku, Finland
Interrction of spin-hbeled with lipid mn+mbrans
equilibrium
and rate constants were derived.
la&iWifi
Fumimasa Nomura, Kazuaki Takeuchi, Tomohiro Kawamura, Shun-i&i Ohnishiand StroT&ah&&i Faculity of Science and Technology. Ryukoku Univetsity and ‘Institute for Chemical Research,Kyoto University
Purpose: To study ~cyclo&xuin catalyzed desorption of pentadecanoic acid from monolayers at the air/water interface, and to determine whether membmne lateral order (liquid-expanded vs liquid-cohdensed state) affects desotption rates. Met&s: Monolayers were prepared at the air/water interface. btption experiments were FPniard out while maintaining the monolayer at constant surf&~ pressure. fi-Cyclodextrin was pnsent at 0.5 mhl in the subphase. Results: The rate of pentadecanoic acid desorption to 0.5 mM B-CD was a linear function of time. when the monolayer surface pressure was kept constant during the course of the experiment (zero-order kinetics). Tke desotption rate increased curvi-linearly (and monotonously) with increasing temperature (l&l pmol/min. cm’ at 9 “C, and 64*2 pinoWn. cm* at 29 “c). The desorption rate f&o increased uttvi-linearly 1s the lftternl surface pressure of the monolnyer incfcased, and no discontinuity in the desorption function could be observed within the LE+W transition region. Concluston~Theresultssugest thatthe Worption of fatty acids from monolayers to &cyeWextin in the subphasewas insensitive to changes in the laeftd order parameter, since the LIZ-W UM.Sidon did hot *ppW to affect the desmption function.
Methods: We synthesized two modified melittins with replacement of Cys at Ser 18 (Mell) or Cys at Leu 9 (MelZ), respectively. These peptides were labeied with 4 maleimide-TEMPO. Results: ESR spectra of spin-labeled melittins (abbreviated as Mell’ and MeW) were three sharp lines, but addition of m phosphatidylcholine(egg PC) vesicles caused decease in the sharp lines. In the case of Me12”, the broad component appeared at higher lipidlpeptide molar ratios. Addition of phosphatidylserine to egg PC increaeed the broad component. Chromium acetate decreased the sharp componenta for M&l’ and Me12’, but no effect for the broad component of Me12’. Conclueions: The hydrophobic aide of the helix containing Leu 9 is loWed on the f&tty acyl chains in membranes, wbmas the hydrophilic &de cot&r&g &r 18 is exposed to aqueos phase. 128
P-C446 THE DISTRIBUTION OF PHOSPHATIDYLGLYCEROL ACROSS THE INNER MEMBRANE OF ESCHERICHIA COU
HUIJBREGTS RPH, DE KROON AIPM, DE KRUIJFF B. Dept. Biochemistry of Membranes, CBLE,Utncht University. (NL)
mica, Univ. de la Habana, Cuba
Purpose: Sticholysin I (St-I) was isolated from the venom of the Caribbean sea anemone Stichoda&a hefianrhus. It belongs to the family of actinoporins: basic peptides able to form channels in lipid membranes. To elucidate the molecular mechanismof formation of these pores we have studied the kinetic of interaction with purely lipidic membranes. Methods: The time course of wrmeabilisation was detected by measuring caicein releasefrom uniform-sized large unilamellar vesicles.with a fluorescencemicroplate reader. Channel’sfeatures were determined using volta e-clamped planar lipid bilayers. Partition toe%*lclentsby centrifugation and electrophoresis. Results: Membrane permeabiiisation is optimal under the followin conditions. resenceof 50% sphingomyelin (Sd ), pH arouI3 8, St-I/vesicle ratio < 200. Most of the toxin is bound to the lioid chase. St-I effect was reduced at hiah DH, 0; lo; and high SM content, mainly due’ioi decreasedbindina. From the dose-deoendenceit was inferred that-St-1forms oligome& pores comprising at least 3 monomers. Conclusions: The kinetics of releasewas fully accountedby a model assuming that trimerisation leadsto a conductive pore. All the relevant
Purpose: Phosphatidylglycerol(PC) has several functions in the E.coli cell, both at the cytoplasmicand the periplasmic side of the inner membrane.Knowledge of the distribution of PC over the two leaflets of the inner membrane will lead to a better understandingof these functions. Methods: To discriminatebetween the PG presentin both leaflets, Ecofi cells or derived inner membranevesicleswere incubated with periodate. The disappearanceof PC, due to its oxidation by periodate, was monitored by TLC. Results: Incubations of both intact cells and inside-out or right side-out membrane vesicles with free periodate resulted in a fast, kinetically monophasicoxidation of the total PC pool, due to the permeability of the membrane for this oxidator. Conclusions: The oxidation of PC by free periodate is not a good tool to determine the transmembranedistribution of PC in E.coli. Experimentsusing periodate on a silica support will also be shown and discussed.
P-W-47
P-C448
FATTY ACID DESORPl’ION FROM MONOLAYERS SLOTI-E. J.P. Department of biochemistry and pharmacy, Abe Akademi University, PIN 20520 Turku, Finland
Interrction of spin-hbeled with lipid mn+mbrans
equilibrium
and rate constants were derived.
la&iWifi
Fumimasa Nomura, Kazuaki Takeuchi, Tomohiro Kawamura, Shun-i&i Ohnishiand StroT&ah&&i Faculity of Science and Technology. Ryukoku Univetsity and ‘Institute for Chemical Research,Kyoto University
Purpose: To study ~cyclo&xuin catalyzed desorption of pentadecanoic acid from monolayers at the air/water interface, and to determine whether membmne lateral order (liquid-expanded vs liquid-cohdensed state) affects desotption rates. Met&s: Monolayers were prepared at the air/water interface. btption experiments were FPniard out while maintaining the monolayer at constant surf&~ pressure. fi-Cyclodextrin was pnsent at 0.5 mhl in the subphase. Results: The rate of pentadecanoic acid desorption to 0.5 mM B-CD was a linear function of time. when the monolayer surface pressure was kept constant during the course of the experiment (zero-order kinetics). Tke desotption rate increased curvi-linearly (and monotonously) with increasing temperature (l&l pmol/min. cm’ at 9 “C, and 64*2 pinoWn. cm* at 29 “c). The desorption rate f&o increased uttvi-linearly 1s the lftternl surface pressure of the monolnyer incfcased, and no discontinuity in the desorption function could be observed within the LE+W transition region. Concluston~Theresultssugest thatthe Worption of fatty acids from monolayers to &cyeWextin in the subphasewas insensitive to changes in the laeftd order parameter, since the LIZ-W UM.Sidon did hot *ppW to affect the desmption function.
Methods: We synthesized two modified melittins with replacement of Cys at Ser 18 (Mell) or Cys at Leu 9 (MelZ), respectively. These peptides were labeied with 4 maleimide-TEMPO. Results: ESR spectra of spin-labeled melittins (abbreviated as Mell’ and MeW) were three sharp lines, but addition of m phosphatidylcholine(egg PC) vesicles caused decease in the sharp lines. In the case of Me12”, the broad component appeared at higher lipidlpeptide molar ratios. Addition of phosphatidylserine to egg PC increaeed the broad component. Chromium acetate decreased the sharp componenta for M&l’ and Me12’, but no effect for the broad component of Me12’. Conclueions: The hydrophobic aide of the helix containing Leu 9 is loWed on the f&tty acyl chains in membranes, wbmas the hydrophilic &de cot&r&g &r 18 is exposed to aqueos phase. 128