Substrate permeation

Nollert, P., Harries, W. E., Fu, D., Miercke, L. J. and Stroud, R. M. (2001). Atomic structure of a glycerol channel and implications for substrate permeation in aqua(glycero)porins, FEBS Lett., 504, 112-117. 

The recent identification of selective protease substrates to simultaneously measure markers of both viable and dead cells led to the development of optional methods for HTS that provide flexibility and added advantages (Niles et al. 2007a). The assay to measure viable cells is based on a cell-permeable protease substrate called glycyl-phenylalanyl-aminofluorocoumarin (GF-AFC). The procedure is a homogeneous add-incubate-measure method that is faster, more sensitive, and less toxic to cells than the tetrazolium and resazurin reduction assays. The substrate can be prepared in an aqueous buffer and is added directly to samples containing cells. The substrate permeates viable cells where constitutive protease activity in the cytoplasm rapidly removes the amino acids, yielding free AFC. The amount of AFC released is directly proportional to viable cell numbers and shows improved sensitivity compared to the resazurin assay (Figure 6.5). The AFC is detected via a microplate fluo-rometer equipped with a (380- to 400-nm excitation/505-nm emission) filter set. 

In several cases, when the electrocatalytic pathway involves adduct formation between immobilized units of the catalyst and the mobile substrate permeating the porous solid, the mechanism can be described in terms of a charge transfer process preceded by a relatively slow chemical reaction. Here, the chronoamperometric current can be approached by (Koutecky and Brdicka, 1947 Bard and Faulkner, 2001) ... 

With regard to electrocatalytic processes, it is pertinent to note that in the case of insulating oxides, there is, in general, no possibility of substrate permeation... 

The determination of nitrophenyl phosphate using alkaline phosphatase (Arnold, 1985) has been used as a model system for analytically important substrates. The enzyme is entrapped in the reagent layer of the optical sensor depicted in Fig. 7. The nitrophenyl phosphate substrate permeates from the sample into the layer and is there converted to the strongly yellow nitrophenol. The change in light absorption is proportional to the substrate concentration. 

Single and parallel dual-membrane structures are successfully prepared by using multilayer flow such as organic/aqueous two-layer flow and organic/aqueous/organic three-layer flow inside a microchannel, and this method can be applied to the preparation of surface-modified polymer membranes (Fig. 42). For example, horseradish peroxidase is immobilized on one side of the membrane surface, and this enzyme-modified membrane realizes substrate permeation and a subsequent reaction. 

The PPIE model fits the kinetic data representing the effects of vesicles on thiolysis and hydrolysis. However, several of the parameters used in the fitting procedure were derived from the effect of micelles on the same reaction or depended on assumptions regarding solubilization sites or rates of substrate permeation. In addition, use of a single rate constant to represent reactivity in the vesicle ignores the fact that a solution containing vesicles has, in principle, several potential reaction sites, and no a priori theory predicts that reactivity at all the sites is equal or comparable. 

Substrate permeation, reaction with substrate water and with substrate conponents... 

Porins. In natural cell walls the rate and selectivity of substrate permeation is controlled by porins. Porins are transmembrane proteins that form trimeric water-filled channels that allow passive diffusion of small solutes, such as ions. 

A bioreactor in which living yeast cells are sandwiched between a cellulose acetate ultrafiltration membrane and a cellulose acetate reverse osmosis membrane (Figure 9.1) was proposed [280], [281]. A solution containing glucose substrate and nutrients is in contact with the ultrahltration (UP) membrane, and pressure is applied on the substrate solution. The substrate permeates the UF membrane freely, together with solvent (water), and arrives at the cell layer where the bioreaction starts to occur, leading to the ethanol product. When the solution is forced by pressure out of the cell layer from the side that is in contact... 

Catalyst honey comb substrate permeated with precious metals... 

Fig. 20.19 Two limiting scenarios representing (a) facile catalysis and (b) slow charge transport (and/or facile substrate permeation) through the polymer coating.

The capability of a diffusion-controlled enzyme membrane to completely convert the substrate permeating the membrane offers an interesting analytical application. In other words, interfering substances are converted into non-disturbing products by "filtering" the analyte flux to the indicator electrode. 

The remaining parameter needed to proceed further with the kinetic analysis is the characteristic substrate permeation current density, is. 

Equation 3 indicates that for redox-polymer mediated reactions that comply with "SR kinetics, fast mediator-substrate cross-reaction rates (large ik) and poor substrate permeation through the polymer phase (low is) will tend to decrease the thickness of the reaction zone. Situations may thus arise in which only the outermost layers of mediator sites attached to the coating participate in the reaction with substrate. Such a surface process is observed for the oxidation of catechol at a carbon electrode modified with a Nation film containing Fe(bipy)3 + as mediator. The homogeneous solution rate constant for the oxidation of catechol by Fe(bipy)3 is estimated a to be 2 x 10 and is roughly 1000 times... 

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