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EFFECT OF DIVALENT CATIONS ON PORIN INCORPORATION IN PLANAR BLM
No full textBiological membranes contain as a basic structural unit a phospholipid bilayer.
Phospholipid membranes in the form of bilayer films (BLM) are currently used as experimental models for transport phenomena of the biological membranes. In particular, bilayers made of acidic phospholipids, present the advantage to investigate the bioelectrical phenomena across the biological membranes.
This class of phospholipids by having a fixed charge might bind ions that play an important role on many physiological processes. It is known that Ca++ and other alkaline cations change the electrostatic potential of the negatively charged bilayer membranes either absorbing to the phospholipids or accumulating in the aqueous diffuse double layer, and this potential change may induce conductance variation (1).
Cd++ another divalent cation has been shown to exert influence on the permeability of some anaelectrolytes across BLM made of negatively charged phospholipids, but not on the zwitterionic phospholipids (2).
We have studied the effect of divalent cations as Ca++ and Ba++ that exert a cross linking together with a screening effect respectively, on the kinetics of incorporation of the mithocondrial porin in bimolecular lipid membranes made with phosphatydilinositol.
In order to understand how lipids of the bilayer affect protein incorporation at the molecular level we must consider that protein crossing the bilayer has to surmount different regions characterized by different properties as interfacial region, head groups, lipid backbone and hydrocarbon core.
In this study we focused our attention to the role of the interface and head group regions of the BLM in the porin incorporation.
The artificial membranes were made of phosphatydilinositol cromatografically pure in n-decane (1% w/w). The bathing media were KC1 1M and KC1 1M plus CaC12 or BaCl2 10 mM. Porin was added at membrane "black" on the two aqueous bathing solutions. Bilayers conductivity and capacitance measurements were carried out at temperature of 25 ± 2 °C and studied by means of alternate current (f = 1 Hz) recording the voltage after a current to voltage converter in series with the membrane; simultaneously electrical capacitance was measured at 1 KHz before and during porin incorporation (3) .
We noticed that when the membranes are in the presence of Ca++ or Ba++ there was an exceptional stability.
With the different bathing solutions no significant differences were observed in membrane conductance values whereas small capacitance variations were recorded.
Once the membrane has became black porin, at a fixed concentration, was added to the aqueous solutions, and after a lag time due to the diffusion of the protein in the solutions and of the time to overcome the membrane barriers, an increase of the voltage output due to the channels formation was recoded. This phenomenon shows a "S-shaped" kinetics. In contrast to the kinetics of incorporation of the porin in oxidized cholesterol lipid membranes (4), showing an hyperbolic shape. These different kinetics may be explained by the different interface barrier; in fact when the experiment were conducted with porin already present in the bathing solutions before membrane formation, the different kinetics persisted in the phosphatydilinositol and in oxidized cholesterol BLM.
The S shaped kinetics persists also when porin is incorporated in phosphatydilinositol membranes in the presence of Ca++ or Ba++; but the curve in presence of the Ca++ is shifted to the right.
This shift is much more pronounced in the presence of Ba++.
It is known that Ca++ affects the surface potential of BLM containing negatively charged lipids. The protein incorporation into BLM can be regarded as a two steps process: I) the interaction at the surface barrier, and II) the penetration into hydrocarbon region.
In the first step the protein encounter an higher resistance in phosphatydilinositol membrane because it has to overcome the interface region consisting of a diffuse double layer, the membrane surface and the polar head groups.
Ca++, by exerting a cross-linking and screening effect on negatively charged membranes, retards the porin incorporation.
At the moment we are not able to distinguish which of the two effect is prevalent; but results in presence of Ba++, that exerts a screening effect only seem to indicate that the latter effect is prevalent. In order to establish the pure cross-linking effect experiments are in progress.
The influence of the divalent cations on the protein incorporation into BLM, that are sensitive to changes in environmental conditions, make this study appealing in order to provide a better knowledge in the molecular mechanism of the protein incorporation and channel formation.
Work supported by 40% grant from MURST 1991
1) S. G. A. McLaughlin, G. Szabo and G. Eisenman, 1971: J. Gen. physiol., 58, 667.
2) S. Micelli, E. Gallucci and C. Lippe, 1978: Arch. Intern. Physiol. Biochim., 86, 755-759.
3) G. Monticelli, E. Gallucci and S. Micelli, 1990: Proc. X School on Biophysics of Membrane Transport, 1: 328-343, Poland.
4) S. Micelli, G. Monticelli and E. Gallucci, 1990: IBST 1: 221
PORIN INCORPORATION KINETICS IN PHOSPHATIDYLINOSITOL AND OXIDIZED CHOLESTEROL MEMBRANES
No full textConductivity of planar lipid bilayer membranes can be modulated by insertion of protein molecules (1).
Mithocondrial porins form water filled pores in the outer membrane and these channels act selectively on the permeation of hydrophilic solutes.
Aims of this research were: 1) the reconstitution of porin channels in planar lipid bilayer membranes, 2) the analysis and the comparison of porin incorporation kinetics in different lipid membranes: phosphatidylinositol (PI) and oxidized cholesterol, 3) the study of protein concentration effect on the incorporation kinetics, 4) the analysis of the channel reconstitution dependence on the applied voltage.
The artificial membranes were formed, with lipid in n-decane (1% w/w), brushing the solution on a small circular hole between two compartments, 4 ml volume, of a teflon experimental chamber. The two compartments were filled with KCl 1M solution and continuously stirred.
Pt electrodes in bathing solutions were connected to a current to voltage converter and to a sinusoidal voltage source (Vs=VS sin2nt; n=1Hz) so that the voltage at the converter (Vl) resulted directly related to the electrical current crossing the membrane (2).
Experiments were performed at room temperature: 24 ± 1 °C.
Membrane electrical conductances and capacitances were higher in oxidized cholesterol than in PI membranes.
Addition of porin to the solutions bathing a completely black membrane induced an increase of membrane current with time indicating protein fusion and channel formation into the lipid matrix.
In PI membranes the time course of current was S-shaped, irrespective of porin addition before or after membrane formation, but in the first case a delay in channel formation and a slower kinetics were observed (3).
In oxidized cholesterol membranes the current time behaviours were S-shaped when porin was added after membrane formation but resulted of exponential type when porin was present before membrane formation. In this second case faster kinetics were observed and the incorporation started with an higher conductance value.
The time lag between porin addition and the first current variation was shorter in oxidized cholesterol than in PI membranes. For a same porin concentration the incorporation kinetics were faster and higher steady levels were reached in oxidized cholesterol than in PI membranes.
In both types of membranes porin incorporation rate decreased lowering porin concentration; five kinetics of porin incorporation (protein added after membrane formation) in oxidized cholesterol membranes are reported in figure.
Besides the porin concentration also the applied voltage affects the incorporation kinetics and the channel formation.
At a given porin concentration faster kinetics are recorded and higher steady levels are reached increasing the voltage to a critical value after which incorporation is less effective, the kinetics are slower and accompanied by lower steady levels.
The voltage dependence of functional pore reconstitution was more evident in PI than in oxidized cholesterol membranes.
In oxidized cholesterol membrane, at the steady state, nonlinear current-voltage characteristics were obtained. The conductance increases with the voltage to reach a critical value after which inactivation occurs.
At a given applied voltage higher membrane conductances were calculated increasing porin concentration.
Significative differences in the current-voltage relations were obtained depending on the absence or the presence of porin at the membrane formation. In this latter case the membranes had higher conductance values but, at the tested voltages, the range of variability was smaller.
The nature of the lipid matrix can be considered responsible of the different protein incorporation; in particular the surface charge of the lipid bilayer membrane and the interface potential, due to the local accumulation of ions at the membrane-solution interface, play a key role in the incorporation of porin in PI planar bilayer lipid membranes.
These observations suggest the possibility to insert protein channels in lipid layers built on a solid substrate finalized to realize organic semiconductors to be used as biosensor or transducers.
Work supported in part by CNR grant 92.02202.CT14.
1) G. Monticelli - "Pore formation in planar lipid bilayer membranes" Proc. X School on Biophysics of Membrane Transport, Szczyrk (Poland) 1990, 1: 315-327.
2) G. Monticelli, E. Gallucci and S. Micelli - "Experimental data on incorporation of porin molecules in lipid bilayers" Proc. X School on Biophysics of Membrane Transport, Szczyrk (Poland) 1990, 1: 328-343.
3) G. Monticelli, E. Gallucci and S. Micelli - "Pore formation in lipid membranes" Proc. Int. Cong. Memb. and Memb. Processes, Chicago 1990, 1: 175-177
Tecnologie di rete e distretti industriali: una convergenza possibile o inevitabile?
No full textIl presente scritto si propone di affrontare due questioni di fondo: 1) se l'avvento e la diffusione delle tecnologie di rete, che migliorano significativamente e rendono meno costosa la comunicazione a distanza, non annullino i vantaggi della prossimità territoriale tipici del distretto industriale; 2) quali siano le ragioni all'origine dell'inerzia delle imprese distrettuali nel dotarsi delle tecnologie dell'informazione e della comunicazione (ICT), inerzia che rende tutt'altro che naturale e scontato il processo di convergenza fra reti territoriali e reti virtuali
Evidence for a different assembly of Gramicidin A in oxidized cholesterol black lipid membrane
No full textGramicidin A (GA) is a hydrophobic linear pentadocapeptide that forms cation- selective channels in natural and black lipid membranes (BLM). The channels are believed to consist of single-stranded helices with the NH2 terminals linked together by hydrogen bonds. Linear gramicidins require a lipid bilayer to fold properly. If the phospholipid acyl chains are less than eight carbons long, channel folding is not observed; if the acyl chains are too long, the membrane- spanning channels are destabilized. Besides, embedded proteins might influence the molecular packing lipids (R. E. Koeppe II et al. Biol. Skr. Dan. Selsk, 49-93-98, 1998). In our experiments, we chose oxidized cholesterol black lipid membranes because their thickness (40±10 A) (Ti Tien and A. L. Diana, In Chemistry and Physics of Lipids, 2, 1968) is suited to both gramicidin length (25-30 A) and folding. The time course of gramicidin incorporation into BLM, studied by means of alternating current (E. Gallucci et al., Biophys. J. 71:824- 831, 1986), was sigmoidal independently of the KCI concentration used, indicating a cooperative process of incorporation into BLM. By means of a simple mathematical model (S. Micelli et al, submitted) we estimated the tm value, i.e. the parameter that describes a phase transition process (or similar phenomenon). This phenomenon has been observed by AA as a function of temperature and GA concentration (J. A. Killian and B. De Kruijff, Biophys. 1. 53: 111-117, 1988; Mingtao Ge and J. H. Freed, Biophys. J., 76: 266-280, 1999). On the other hand, the average values of the steady-state conductance against gramicidin concentration, for different KCI concentrations, shows a sigmoidal behavior too. These curves were fitted by means of a four-parameter equation which allowed us to obtain the Hill coefficient, D, or slope factor. The Hill coefficient gives the number of single-stranded helices which form the channel. Our results suggest that the number of channel sub-units depends on the KCI concentration. In our case, at a KCI concentration of 1M, the channel is characterized by two sub-units, in accordance with AA's finding (D. U. Urry, Proc. Natl. Acad. Sci. USA, 86:672, 1971, W. R. Veatch and R. Blond, Biochemisty, 13:5249, 1974), while for KCI concentrations of 0.1 and 0.5 M. the channel is formed by more than two sub-units
Digitale da toccare. Sette designer per sette aziende artigiane: l'autoproduzione al tempo di internet
No full textAuto-prodotti all'italiana. Chi fa da sé fa per tre? Ne parliamo con i designer protagonisti di Autoproduzioni Italiane
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