Flip-flop rate

Lipophilicity is intuitively felt to be a key parameter in predicting and interpreting permeability and thus the number of types of lipophilicity systems under study has grown enormously over the years to increase the chances of finding good mimics of biomembrane models. However, the relationship between lipophilicity descriptors and the membrane permeation process is not clear. Membrane permeation is due to two main components the partition rate constant between the lipid leaflet and the aqueous environment and the flip-flop rate constant between the two lipid leaflets in the bilayer [13]. Since the flip-flop is supposed to be rate limiting in the permeation process, permeation is determined by the partition coefficient between the lipid and the aqueous phase (which can easily be determined by log D) and the flip-flop rate constant, which may or may not depend on lipophilicity and if it does so depend, on which lipophilicity scale should it be based ... 

Vesicle size, bilayer fluidity, membrane permeability, microviscosity, ability to bind small molecules, suseeptibility to pore formation, flip-flop rates, extent of water penetration, lateral amphiphile diffusion, vesiele fusion, and kinetic medium effeets (some of which will be discussed briefly below) all depend on the paeking of... 

Within the same context, it is worth mentioning that Menger et al. [65], Ladika et al. [66] and Moss et al. [67] have recently demonstrated that macrocyclic lipids decrease the mobility of the membrane by raising the phase transition temperature and decreasing the permeation of ionic species and the flip-flop rate. 

Eq. 9 leads to spin diffusion with the flip-flop rate limiting the observed T. Below 10 s, the relaxation for the A spin is limited by the relaxation sink, which corresponds to the bonded methylene carbon. The effect of the siqall deviation In the coefficients from 1.0 and 0.0 near t s 10 s is non-exponential recovery, with the negative coefficient leading to a curve which is concave downward. For the M spin, the decay Jumps from theX curve for correlation times <10 " s to the Xg curve for correlation times >10 s. 

Other ESR studies of membrane structure include the determination of phospholipid flip-flop rates (Kornberg and McConnell, 1971), studies of membrane permeability, measurements of vesicle internal volumes and transmembrane potentials (Marsh et aL, 1976 McNamee and McConnell, 1973). 

The use of analytical models for spin dephasing in the solid state [39, 40] is not appropriate for the present case because they do not account for the specific distribution of nuclear spins around the electron spin. In order to consider the real matrix structure close to the chromophore we simulated the HE decay curves numerically by using the known crystal structure [9] and the nuclear flip-flop rate IE as a parameter. The echo decay is calculated as... 

The cylinder stretching protocol appears to work very well for simple solvent-free membrane models [111, 113, 114], but with more refined models this method suffers from two drawbacks, both related to the equifibration of a chemical potential. First, the cylinder separates the simulation volume into an inside and an outside. If solvent is present, its chemical potential must be the same in these two regions, but for more highly resolved models the solvent permeability through the bilayer is usually too low to ensure automatic relaxation. Second, the chemical potential of lipids also has to be the same in the two bilayer leaflets, and again for more refined models the lipid flip-flop rate tends to be too low for this to happen spontaneously. 

Danger and Sekwen (20) investigated the effect of free fatty acids on the permeability of the 1,2-dimyristoyl-5 >72-glycero-3-phosphotidylcholine (DMPC) bilayer at the main phase transition. When vesicles were formed between DMPC and oleic acid, the phase transition temperature was reduced drastically, but at the phase transition from the gel to liquid crystalline phase, membrane permeability reaches a maximum. Saturated fatty acids did not have the same effect on the bilayer as unsaturated fatty acids, in that there was a reduced effect on the phase transition temperature. They also concluded that -bilayer flip-flop is at a maximum at the phase transition temperature of DMPC being in the millisecond range for oleic acid. DMPC was found to have a flip-flop rate of 4 h at the phase transition and even longer rates at other temperatures. 

A cell membrane is illustrated in Fig. 6.1. It is built from a bilayer of lipids, usually phospholipids, associated with which are membrane proteins and polysaccharides. The antiparallel orientation of lipid layers in the bilayer is maintained due to the extremely slow flip-flop rate, i.e. the rate of diffusion transverse to the bilayer. The lipid bilayer is the structural foundation and the proteins and polysaccharides provide chemical functionality. The protein to lipid ratio shows a large variation depending on the cell, but proteins make up at least half of most cell membranes. A prominent exception is mammalian nerve cells which contain only 18 % protein (here also the lipids are sphingomyelins rather than phospholipids). Here, the primary requirement is that the cell membrane should be effective as an electrical... 

The fact that mutually different cross sectional patterns were observed in the SC-2D NMR spectrum where 7 was chosen to be 0 ms indicates that both inter- and intramolecular cross relaxation rates and spin flip-flop rates between interacting pairs of protons are relatively slow. This can be understood if one considers that dipolar interactions are partially averaged out by fast translational and rotational molecular motions in the liquid crystalline phase in contrast to the solid phase. 

There is also inside-outside (transverse) asymmetry of the phospholipids. The choline-containing phospholipids (phosphatidylcholine and sphingomyelin) are located mainly in the outer molecular layer the aminophospholipids (phosphatidylserine and phos-phatidylethanolamine) are preferentially located in the inner leaflet. Obviously, if this asymmetry is to exist at all, there must be limited transverse mobility (flip-flop) of the membrane phospholipids. In fact, phospholipids in synthetic bilayers exhibit an extraordinarily slow rate of flip-flop the half-life of the asymmetry can be measured in several weeks. However, when certain membrane proteins such as the erythrocyte protein gly-cophorin are inserted artificially into synthetic bilayers, the frequency of phospholipid flip-flop may increase as much as 100-fold. 

Another A-to-G editing, at a site designated the R/G site, can occur immediately preceding the flip-flop segment in GluR2-GluR4. The flip-flop segment influences the rate of desensitization, while the rate of recovery from the desensitized state depends on the R/G site where the edited form, G, recovers faster than the unedited form, R. 

Kleinfeld, A.M., Chu, P. and Storch, J. (1997) Flip-flop is slow and rate-limiting for the movement of long chain anthroyloxy fatty acids across lipid vesicles. 

The original proposal of the approach, supported by a Monte Carlo simulation study [36], has been further validated with both pre-clinical [38, 39] and clinical studies [40]. It has been shown to be robust and accurate, and is not highly dependent on the models used to fit the data. The method can give poor estimates of absorption or bioavailability in two sets of circumstances (i) when the compound shows nonlinear pharmacokinetics, which may happen when the plasma protein binding is nonlinear, or when the compound has cardiovascular activity that changes blood flow in a concentration-dependent manner or (ii) when the rate of absorption is slow, and hence flip-flop kinetics are observed, i.e., when the apparent terminal half-life is governed by the rate of drug input. 

Computation of oral absorption (kj and elimination (E) rates is often complicated by the flip-flop of the absorption and elimination phases when they differ by less than a factor of 3. Because of these analysis problems, computation of absorption and elimination rates should not be attempted on the basis of oral dosing results alone. 

Note, however that the concepts about the lipid membrane as the isotropic, structureless medium are oversimplified. It is well known [19, 190] that the rates and character of the molecular motion in the lateral direction and across the membrane are quite different. This is true for both the molecules inserted in the lipid bilayer and the lipid molecules themselves. Thus, for example, while it still seems possible to characterize the lateral movement of the egg lecithin molecule by the diffusion coefficient D its movement across the membrane seems to be better described by the so-called flip-flop mechanism when two lipid molecules from the inner and outer membrane monolayers of the vesicle synchronously change locations with each other [19]. The value of D, = 1.8 x 10 8 cm2 s 1 [191] corresponds to the time of the lateral diffusion jump of lecithin molecule, Le. about 10 7s. The characteristic time of flip-flop under the same conditions is much longer (about 6.5 hours) [19]. The molecules without long hydrocarbon chains migrate much more rapidly. For example for pyrene D, = 1.4x 10 7 cm2 s1 [192]. 

Electron spin-electron spin interaction. The transition betwen a and P spin states takes place by the interaction between the A spins and the surrounding off-resonant spins (called B spins). The most important process in this type of the relaxation is cross relaxation. In the cross relaxation, the excess energy of the A spin system is resonantly transferred to the surrounding B spins through a flip-flop process. The relaxation rate depends on either the distance betwen the A and B spins or the number of the B spins surrounding an A spin. It is this relaxation mechanism which provides us with a means for studying the local spatial distribution of radical species. 

The lipids in a lipid bilayer may translocate across the bilayer from one monolayer to the apposed monolayer. This transmembrane translocation process, which is also known as flip-flop, is slow for lipids with large polar head groups such as glyc-erolipids and sphingophospholipids but can be fast in the case of lipids with very small polar moieties such as cholesterol. Typical first-order rate constants for transmembrane translocation of a phospholipid-like molecule in liquid-disordered phase bilayers prepared from l-palmitoyl-2-oleoylphosphatidylcholine (POPC) are s and may be about 10-fold slower in... 

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