Transport of substrates

The presence of a transporter can be assessed by comparing basolateral-to-apical with apical-to-basolateral transport of substrates in polarized cell monolayers. If P-gp is present, then basolateral-to-apical transport is enhanced and apical-to baso-lateral transport is reduced. Transport experiments are in general performed with radioactively labeled compounds. Several studies have been performed with Caco-2 cell lines (e.g. Ref. [85]). Since Caco-2 cells express a number of different transporters, the effects measured are most probably specific for the ensemble of transporters rather than for P-gp alone. P-gp-specific transport has been assayed across confluent cell layers formed by polarized kidney epithelial cells transfected with the MDR1 gene [86], Figure 20.11 shows experimental data obtained with these cell lines. A rank order for transport called substrate quality was determined for a number of compounds [86]. The substrate quality is a qualitative estimate, but nevertheless allows an investigation of the role of the air/water (or lipid/water) partition coefficient, log Kaw, for transport as seen in Fig. 20.11(A). For most of the compounds, a linear correlation is observed between substrate quality and log Kaw- However, four compounds are not transported at all despite their distinct lipophilicity. A plot of the substrate quality as a function of the potential of a... 

Fig. 20.11. Substrate quality obtained by comparing basolateral-to-apical with apical-to-basolateral transport of substrates in polarized cell monolayers of MDR1-transfected cell lines [86] plotted versus (A) the log of the air/water partition coefficient, or (B) H-bond energy (arbitrary units, EUh cf. text). Units of the air/ water partition coefficient were [M ]. Compound (concentrations in Ref. [86] in brackets) were clozapine (50 nM) (1) cyclosporin A (2 tM) (2) daunorubicin (3) dexamethasone (2 tM) (4) digoxin (2 pM) (5) domperidone (2 pM) (6) etoposide (7) flunitrazepam (500 nM) (8) haloperidol (50 nM) (9) ivermectin (50 nM) (10) loperamide (2 pM) (11) morphine (2 pM) (12) ondansetron...

As depicted in Figure 2.8, mass transport of substrate from the bulk water phase takes place through a fluid boundary layer (liquid film) and into a biofilm followed by a combined diffusion and utilization of the substrate in the biofilm. 

The potential production of sulfide depends on the biofilm thickness. If the flow velocity in a pressure main is over 0.8-1 ms-1, the corresponding biofilm is rather thin, typically 100-300 pm. However, high velocities also reduce the thickness of the diffusional boundary layer and the resistance against transport of substrates and products across the biofilm/water interphase. Totally, a high flow velocity will normally reduce the potential for sulfide formation. Furthermore, the flow conditions affect the air-water exchange processes, e.g., the emission of hydrogen sulfide (cf. Chapter 4). 

In many cases, the transport of substrates to the cells and that of metabolites from the surface of the cells to the culture medium are carried out at rates characterised by time constants of the same order of magnitude as those of the biological reactions. Transport or transfer of matter must thus be included in an analysis of the behaviour of a bioreactor as well as the kinetic rates [59, 60]. 

In nature many enzymes are embedded in membranes, which not only serve as a scaffold but also regulate the transport of substrates and products, and control the concentrations of protons and other ions. Instead of embedding molecular catalysts into artificial membranes, as was done for the cytochrome P450 mimic, it is also possible to make amphiphiles that constitute the membranes catalytic themselves. 

Yet another way to alter the effective activity of an enzyme is to change the accessibility of its substrate. The hexokinase of muscle cannot act on glucose until the sugar enters the myocyte from the blood, and the rate at which it enters depends on the activity of glucose transporters in the plasma membrane. Within cells, membrane-bounded compartments segregate certain enzymes and enzyme systems, and the transport of substrate into these compartments may be the limiting factor in enzyme action. 

Because of its cyclic nature, this process presents analogies with molecular catalysis it may be considered as physical catalysis operating a change in location, a translocation, on the substrate, like chemical catalysis operates a transformation into products. The carrier is the transport catalyst which strongly increases the rate of passage of the substrate with respect to free diffusion and shows enzyme-like features (saturation kinetics, competition and inhibition phenomena, etc.). The active species is the carrier-substrate supermolecule. The transport of substrate Sj may be coupled to the flow of a second species S2 in the same (symport) or opposite antiport) direction. 

The principle of the mass transport of substrates/nutrients into the immobilized enzyme/cells, through a solid, porous layer (membrane, biofilm) or through a gel layer of enzyme/cells is the same. The structure, the thickness of this mass-transport layer can be very different, thus, the mass-transport parameters, namely diffusion... 

Despite our inability to predict quantitatively the influence P-gp may have on the in vivo transport of substrates in normal tissues with respect to other processes, in vitro experiments remain the best means of demonstrating that a compound is a substrate for polarized efflux. Nearly all experiments designed to study the extent of P-gp efflux of test compounds in vivo require adequate in vitro data to support the hypothesis (48,217,226,454). In vitro studies on P-gp substrates such as vinblastine, paclitaxel, cyclosporin A, talinolol, acebutolol, and digoxin have provided a good indication of the effect of P-gp on the in vivo pharmacokinetic behavior of these compounds. These studies show that results from the in vitro studies provide a qualitative estimate of the influence of P-gp on its in vivo pharmacokinetic behavior. Findings such as these give confidence that results from in vitro experiments can be extrapolated to explain modulation of dmg disposition by P-gp efflux. 

In summary, the steady state and transient performance of the poly(acrylamide) hydrogel with immobilized glucose oxidase and phenol red dye (pAAm/GO/PR) demonstrates phenomena common to all polymer-based sensors and drag delivery systems. The role of the polymer in these systems is to act as a barrier to control the transport of substrates/products and this in turn controls the ultimate signal and the response time. For systems which rely upon the reaction of a substrate for example via an immobilized enzyme, the polymer controls the relative importance of the rate of substrate/analyte delivery and the rate of the reaction. In membrane systems, the thicker the polymer membrane the longer the response time due to substrate diffusion limitations as demonstrated with our pAAm/GO/PR system. However a membrane must not be so thin as to allow convective removal of the substrates before undergoing reaction, or removal of the products before detection. The steady state as well as the transient response of the pAAm/GO/ PR system was used to demonstrate these considerations with the more complicated case in which two substrates are required for the reaction. 

In the next experiment we add a small amount, e.g. an initial concentration of 0-001 M, of the organic substrate and record a second voltammetric curve (B in Fig. 1). Still as a simulated situation, let us assume that the substrate is electroactive at a lower potential than that of the SSE alone. In such a case, the voltammetric curve will have a sigmoid shape, first with an exponential increase off and then a gradual flattening out to a plateau value, ijim, at which the rate of transport of substrate molecules by diffusion to the electrode is rate-limiting. This is the region of diffusion control of the rate under properly controlled conditions i im is linearly related to the concentration of the electroactive compound. The potential at flim /2, Ex j2, is an important parameter in that it can be used as a relative measure of the oxidizability of different electroactive compounds (see Section 11). 

Kinetics of Immobilized Enzymes. Another major factor in the performance of immobilized enzymes is the effect of the matrix on mass transport of substrates and products. Hindered access to the active site of an immobilized enzyme can affect the kinetic parameters in several ways. The effective concentration of substrates and products is also affected by the chemistry of the matrix especially with regard to the respective partition coefficients between the bulk solution and the matrix. In order to understand the effects of immobilization upon the rate of an enzyme-catalyzed reaction one must first consider the relationship between the velocity of an enzyme-catalyzed reaction and the... 

Although there are many advantages to the use of whole cells for biotransformations, there are certain limitations that must be considered. One consideration is the transport of substrates and products across the cell membrane. In life, the cell membrane is a proton-tight barrier to the rest of the world. It is generally impermeable to charged molecules and to water, but may have permeability to hydrophobic molecules. Often cells have... 

There are several mechanisms for explaining how biological membranes can transport charged or uncharged substrates against their thermodynamic forces. It is widely accepted that cross-transports by a protein are discrete events. Biomembranes contain enzymes, pores, charges or membrane potentials, and catalytic activities associated with the transport of substrates. It is well established that the electrostatic interactions between the membrane and a charged... 

Many biochemical signaling processes involve the coupled reaction diffusion of two or more substrates. Metabolic biochemical pathways are mainly multicomponent reaction cycles leading to binding and/or signaling and are coupled to the transport of substrates. A reaction-diffusion model can also describe the diffusion of certain proteins along the bacterium and their transfer between the cytoplasmic membrane and cytoplasm, and the generation of protein oscillation along the bacterium (Wood and Whitaker, 2000). 

For the optimal efficiency to occur at steady state, oxidative phosphorylation progresses with a load. Such a load. /L is an ATP-utilizing process in the cell, such as the transport of substrates. A load, which will make the steady state the optimal efficiency state, can be identified through the total exergy loss T,c... 

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