Pore Binding

The Nayl a-subunit is a large protein, and several receptor sites for natural product neurotoxins have been identified by photoaffinity labeling and site-directed mutagenesis [79]. The small hydrophilic guanidines TTX and STX and the p,-conotoxin peptides bind at the extracellular mouth of the conduction pathway and are thought to physically occlude the pore. Binding of these toxins shows little state- or use-dependence. A number of a scorpion, sea anemone, and spider toxins also bind to an extracellular site, but act as... 

Membrane Membrane OalNac Pore Binding Binding In 1Ac Selectivity... 

Imamoto, N Tachibana, T.. Matsubae, M., and Yoneda, Y. (1995b). A karyophilic protein forms a stable complex with cytoplasmic proteins prior to nuclear pore binding. J. Biol. Chem. 270,8559-8656. 

Other immobilization methods are based on chemical and physical binding to soHd supports, eg, polysaccharides, polymers, glass, and other chemically and physically stable materials, which are usually modified with functional groups such as amine, carboxy, epoxy, phenyl, or alkane to enable covalent coupling to amino acid side chains on the enzyme surface. These supports may be macroporous, with pore diameters in the range 30—300 nm, to facihtate accommodation of enzyme within a support particle. Ionic and nonionic adsorption to macroporous supports is a gentle, simple, and often efficient method. Use of powdered enzyme, or enzyme precipitated on inert supports, may be adequate for use in nonaqueous media. Entrapment in polysaccharide/polymer gels is used for both cells and isolated enzymes. 

The reaction kinetics approximation is mechanistically correct for systems where the reaction step at pore surfaces or other fluid-solid interfaces is controlling. This may occur in the case of chemisorption on porous catalysts and in affinity adsorbents that involve veiy slow binding steps. In these cases, the mass-transfer parameter k is replaced by a second-order reaction rate constant k. The driving force is written for a constant separation fac tor isotherm (column 4 in Table 16-12). When diffusion steps control the process, it is still possible to describe the system hy its apparent second-order kinetic behavior, since it usually provides a good approximation to a more complex exact form for single transition systems (see Fixed Bed Transitions ). 

Figure 12.10 Diagram showing two subunits of the channel, illustrating the way the selectivity filter is formed. Main-chain atoms line the walls of this narrow passage with carbonyl oxygen atoms pointing into the pore, forming binding sites for ions. (Adapted from D.A. Doyle et al., Sdence 280 69-77, 1998.)...

Ultrafiltration of micellar solutions combines the high permeate flows commonly found in ultrafiltration systems with the possibility of removing molecules independent of their size, since micelles can specifically solubilize or bind low molecular weight components. Characteristics of this separation technique, known as micellar-enhanced ultrafiltration (MEUF), are that micelles bind specific compounds and subsequent ultrafiltration separates the surrounding aqueous phase from the micelles [70]. The pore size of the UF membrane must be chosen such, that the micelles are retained but the unbound components can pass the membrane freely. Alternatively, proteins such as BSA have been used in stead of micelles to obtain similar enan-tioselective aggregates [71]. 

The anthrax toxin is a tripartite toxin and consists ofthe binding component protective antigen (PA), the lethal factor (LF), which is a metalloprotease, and the edema factor (EF), which is a calmodulin-dependent adenylyl-cyclase. Both enzyme components are translocated via PA into target cells. PA is activated by furin-induced cleavage and forms heptamers, which are similar to the binding components of C2 toxin and iota toxin. In the low pH compartment of endosomes, the heptamers form pores to allow translocation of LF and EF. LF cleaves six of the seven MEKs (MAPK-kinases) thereby inhibiting these enzymes. The functional consequence is the blockade of the MAPK pathways that control cell proliferation, differentiation, inflammation, stress response, and survival. Whether this is the reason for the LT-induced cell death of macrophages is not clear [1]. 

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