Proteins translocation

While many biological molecules may be targets for oxidant stress and free radicals, it is clear that the cell membrane and its associated proteins may be particularly vulnerable. The ability of the cell to control its intracellular ionic environment as well as its ability to maintain a polarized membrane potential and electrical excitability depends on the activity of ion-translocating proteins such as channels, pumps and exchangers. Either direct or indirect disturbances of the activity of these ion translocators must ultimately underlie reperfiision and oxidant stress-induced arrhythmias in the heart. A number of studies have therefore investigated the effects of free radicals and oxidant stress on cellular electrophysiology and the activity of key membrane-bound ion translocating proteins. 

The Na/K ATPase has been extensively purified and characterized, and consists of a catalytic a subunit of around 95 kDa and a glycoprotein 0 subunit of approximately 45 kDa (Skou, 1992). The functional transporter exists as a dimer with each monomer consisting of an a and /3 subunit. Hiatt aal. (1984) have su ested that the non-catalytic jS subunit may be involved in the cottect insertion of the a subunit into the lipid bilayer and, therefore, it is conceivable that a modification of the 0 subunit structure may be reflected by changes in the catalytic activity of the a subunit. Therefore, in studies involving the manipulation of tissue glutathione levels, alterations of intracellular redox state may have an effect on substrate binding at an extracellular site on this ion-translocating protein. 

Figure 4.14 Diagrammatic representation of (a) oxy-radical>mediated S-thioiation and (b) thiol/disulphide-initiated S-thiolation of protein suiphydryl groups. Under both circumstances mixed disuiphides are formed between glutathione and protein thiois iocated on the ion-translocator protein resulting in an alteration of protein structure and function. Both of these mechanisms are completely reversible by the addition of a suitabie reducing agent, such as reduced glutathione, returning the protein to its native form.

An assay that produces multiple biological readouts. Most commonly used in relation to the mathematical analysis of an image acquired using an automated microscope whereby analysis algorithms quantify cellular parameters (e.g., number, motility, neurite outgrowth, size, shape) and subcellular events (e.g., receptor internalization, protein translocation, protein expression nuclei shape). 

Staphylococcus carnosus is genetically highly stable, a good secretor (Gram positive) able to translocate proteins containing several hydrophobic transmembrane regions [58], and it has no extracellular proteases, which makes it suitable for production of the secreted enzymes [58],... 

Lavin, A., Hahn, D., and Gasiewicz, T. A., Expression of functional aromatic hydrocarbon receptor and aromatic hydrocarbon nuclear translocator proteins in murine bone marrow stromal cells, Arch. Biochem. Biophys., 352, 9, 1998. 

Stockton, ]. D. Merkert, M. C. Kellaris, K. V. A Complex of Chaperones and Disulfide Isomerases Occludes the Cytosolic Face of the Translocation Protein Sec61p and Affects Translocation of the Prion Protein. Biochemistry 2003, 42, 12821-12834. 

The three subunits of the F0 component have been much studied. Their properties have been reviewed briefly.308 Subunit c is thought to be the proton-translocating protein, as it binds the inhibitor DCCD, which blocks proton translocation, at a single carboxyl group. Indeed H+... 

Pollenz RS, Sattler CA, Poland A. 1994. The aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator protein show distinct subcellular localizations in Hepa lclc7 cells by immunofluorescence microscopy. Mol Pharmacol 45 428-438. 

Martoglio, B. and Dobberstein, B. (1996) Snapshots of membrane-translocating proteins. Trends Cell Biol. 6, 142-147. 

Next let us show how one can compute the proteasome output if the transport rates are given. In our model we assume that the proteasome has a single channel for the entry of the substrate with two cleavage centers present at the same distance from the ends, yielding in a symmetric structure as confirmed by experimental studies of its structure. In reality a proteasome has six cleavage sites spatially distributed around its central channel. However, due to the geometry of its locations, we believe that a translocated protein meets only two of them. Whether the strand is indeed transported or cleaved at a particular position is a stochastic process with certain probabilities (see Fig. 14.5). 

The interesting issue of proton collection at the protein surface is not addressed here, but it does appear that proton antennae are designed surface features of several proton translocating proteins, including the bacterial RC (Adelroth et al., 2001), cytochrome oxidase (Marantz el at, 1998), and bR (Checover et al., 1997). 

The formation of disulfide bonds in proteins synthesized in vitro can be followed by measuring enzymatic activity or by an increased mobility compared to the reduced protein during SDS-PAGF. This increased mobility arises from the fact that, as disulfide-bonded proteins are intra-molecularly cross-linked, they form a more compact structure and occupy a smaller hydrodynamic volume compared to the reduced protein (Gold-enberg and Creighton, 1984). An illustration of this increase in mobility is shown in Fig. 2. Here the mRNA for preprolactin was translated in a cell-free system optimized for the formation of disulfide bonds, and then analyzed by SDS-PAGF. The translocated protein forms disulHde bonds under these conditions whereas the protein synthesized under the same conditions but in the absence of microsomal membranes does not form disulfide bonds. Thus the nascent protein must be translocated into microsomal vesicles for disulfide bond formation to occur. 

The total synthesis of a high affinity TSPO (translocator protein) ligands, DPA-714 (ix), starting from 3-(4-methoxyphenyl)-3-oxopropanenitrile (vii) and 2-chloro-N,N-diethylacetamide (viii) utilizing the microwave assisted organic synthesis (MAOS) has been described by Tang et al. [16]. 

In a number of experimental situations it is desirable to translocate proteins from the exterior into the cytosol, and this may also be desirable for vaccination purposes. Since many proteins retain their function in fusion proteins, cDNA for the protein in question may be linked to the cDNA of a toxin A-moiety and, after expression and reconstitu-... 

ReyesH, Reisz-Porszasz S,HankinsonO. 1992. Identification of the Ah receptor nuclear translocator protein (Arnt) as a component of the DNA binding form of the Ah receptor. Science 256 1193-95... 

Probst MR, Reisz-Porszasz S, Agbunag RV, Ong MS, Hankinson O. 1993. Role of the aryl hydrocarbon receptor nuclear translocator protein in aryl hydrocarbon (dioxin) receptor action. Molec. Pharmacol. 44 511-18... 

Holmes JL, Pollenz RS. 1997. Determination of aryl hydrocarbon receptor nuclear translocator protein concentration and subcellular localization in hepatic and nonhepatic cell culture lines development of quantitative Western blotting protocols for calculation of aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator protein in total cell lysates. Molec. Pharmacol. 52 202-11... 

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