Experiments of the membrane

Validation of membrane characteristics normally requires specialized techniques that lie within the expertise and experience of the membrane suppliers. 

The membrane is the heart of the fuel-cell sandwich and hence the entire fuel cell. It is this electrolyte that makes polymer-electrolyte fuel cells (PEFCs) unique and, correspondingly, the electrolyte must have very specific properties. Thus, it needs to conduct protons but not electrons as well as inhibit gas transport in the separator but allow it in the catalyst layers. Furthermore, the membrane is one of the most important items in dealing with water management. It is for these reasons as well as for others that modeling and experiments of the membrane have been pursued more than any other layer [1],... 

Perfluorosulfonated membranes have a microscopic phase-separated structure with hydrophobic regions and hydrophilic domain. Hydrophobic regions provide the mechanical support and hydrophilic ionic domains provide proton transport channel. Many morphological models for PFSA have been developed based on SAXS and wide-angle x-ray scattering (WAXS) experiments of the membranes. However, because of the random chemical structure of the PFSA copolymer, morphological variation with water content and complexity of coorganized crystalline and ionic domains, limited characteristic detail proved by the SAXS and WAXS experiments, the structure of the PFSl has been still subject of debate. Here, a brief description of seven membrane structure models is provided. 

FIG. 17 Schematic illustration of the setup for a tip-dip experiment. First glycerol dialkyl nonitol tetraether lipid (GDNT) monolayers are compressed to the desired surface pressure (measured by a Wilhehny plate system). Subsequently a small patch of the monolayer is clamped by a glass micropipette and the S-layer protein is recrystallized. The lower picture shows the S-layer/GDNT membrane on the tip of the glass micropipette in more detail. The basic circuit for measurement of the electric features of the membrane and the current mediated by a hypothetical ion carrier is shown in the upper part of the schematic drawing. 

The first column indicates the possible orientations. Dynamic indicates that the site is not fixed at one side of the membrane. Whether or not binding would be measured is indicates by the + and — signs, respectively. The last line in the table gives the results from the actual experiment [30]. RSO, right-side-out ISO, inside-out. 

In conclusion, the steady-state kinetics of mannitol phosphorylation catalyzed by II can be explained within the model shown in Fig. 8 which was based upon different types of experiments. Does this mean that the mechanisms of the R. sphaeroides II " and the E. coli II are different Probably not. First of all, kinetically the two models are only different in that the 11 " model is an extreme case of the II model. The reorientation of the binding site upon phosphorylation of the enzyme is infinitely fast and complete in the former model, whereas competition between the rate of reorientation of the site and the rate of substrate binding to the site gives rise to the two pathways in the latter model. The experimental set-up may not have been adequate to detect the second pathway in case of II " . The important differences between the two models are at the level of the molecular mechanisms. In the II " model, the orientation of the binding site is directly linked to the state of phosphorylation of the enzyme, whereas in the II" model, the state of phosphorylation of the enzyme modulates the activation energy of the isomerization of the binding site between the two sides of the membrane. Steady-state kinetics by itself can never exclusively discriminate between these different models at the molecular level since a condition may be proposed where these different models show similar kinetics. The II model is based upon many different types of data discussed in this chapter and the steady-state kinetics is shown to be merely consistent with the model. Therefore, the II model is more likely to be representative for the mechanisms of E-IIs. 

Kinetic experiments have shown that phloretin binds asymmetrically to the glucose transporter [37]. However, unlike cytochalasin B it binds exclusively to the extracellular surface of the membrane. Similar asymmetries of binding have been reported for a number of steroids that inhibit glucose transport [38]. For example, androsten-4-ene-3,17-dione (Fig. 1) which inhibits with a K of about 20/rM, binds almost exclusively at the inner surface of the membrane. 

Zero-trans experiments. In these experiments the rate of glucose flux from the cis side of the membrane is measured as a function of its concentration, while the concentration on the trans side is kept at zero. Two types of experiment can thus be performed, zero-trans (zt) entry and exit, yielding I max values VqI and and K , values Kol and K o, respectively, where o signifies outside and i inside the erythrocyte. 

Equilibrium exchange experiments. In this situation, the unidirectional flux of isotopically labelled glucose across the membrane is measured as a function of the glucose concentration, which is kept equal on both sides of the membrane. Because the unidirectional influx and efflux at any particular concentration are necessarily equal, the equilibrium-exchange (ee) flux is characterized by a single V ax value, and a single Km value. A . 

Infinite-cis experiments. In this type of experiment, the net flux of glucose from a limitingly high concentration on the cis face of the membrane is measured as a function of the concentration on the trans face. Both entry and exit infinite-m (ic) experiments can be performed, yielding two Am values, A i and K, respectively. Maximal fluxes are obtained when the concentration on the trans side of the membrane is zero. Since this is the zero-trans situation, = Fq and F o =... 

The information obtained from the experiments described above can be used to construct models for the three-dimensional arrangement of the membrane-spanning helices within the transport proteins. One such model, which takes the diameter of an a-helix as 1.1 nm and seems to fit the measured dimensions of lac permease quite nicely, is illustrated in Fig. 5, although it must be emphasized that this is only one of... 

The centrifugal field in the sedimentation equilibrium experiment is the analog of the membrane in an osmometer. 

Another situation is found for the Na+ ions. When the membrane is permeable to these ions, even if only to a minor extent, they will be driven from the external to the internal solution, not only by diffusion but when the membrane potential is negative, also under the effect of the potential gradient. In the end, the unidirectional flux of these ions should lead to a concentration inside that is substantially higher than that outside. The theoretical value calculated from Eq. (5.15) for the membrane potential of the Na ions is -1-66 mV. Therefore, permeabihty for Na ions should lead to a less negative value of the membrane potential, and this in turn should lead to a larger flux of potassium ions out of the cytoplasm and to a lower concentration difference of these ions. All these conclusions are at variance with experience. 

Further progress in understanding membrane instability and nonlocality requires development of microscopic theory and modeling. Analysis of membrane thickness fluctuations derived from molecular dynamics simulations can serve such a purpose. A possible difficulty with such analysis must be mentioned. In a natural environment isolated membranes assume a stressless state. However, MD modeling requires imposition of special boundary conditions corresponding to a stressed state of the membrane (see Refs. 84,87,112). This stress can interfere with the fluctuations of membrane shape and thickness, an effect that must be accounted for in analyzing data extracted from computer experiments. 

If a small amount of gramicidin A is dissolved in a BLM (this substance is completely insoluble in water) and the conductivity of the membrane is measured by a sensitive, fast instrument, the dependence depicted in Fig. 6.15 is obtained. The conductivity exhibits step-like fluctuations, with a roughly identical height of individual steps. Each step apparently corresponds to one channel in the BLM, open for only a short time interval (the opening and closing mechanism is not known) and permits transport of many ions across the membrane under the influence of the electric field in the case of the experiment shown in Fig. 6.15 it is about 107 Na+ per second at 0.1 V imposed on the BLM. Analysis of the power spectrum of these... 

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