Equilibrative carrier proteins

Dissolve 4 mg of the carrier protein in 400 pi Soln. A. Dissolve about 0.5 mg SMCC (succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carhoxylate, Mr 334.3) in 50 pi DMF and add this solution to the carrier protein. Shake at RT for 1 h, centrifuge and desalt on a Sephadex G-25 column, equilibrated with Soln. B (the activated carrier appears in the void volume). Cool the receiving tube in an ice bath. 

Dissolve 5 mg of ovalbumin, BSA, or another suited carrier protein in 100 pi ddH20. The pH has to be 6, because aggregation occurs above pH 7. Add Soln. B to a final concentration of 2.5% giutaraidehyde and stir at RT for 30 min. Filtrate the reaction mixture on a PD-10 or Sephadex G-25 column, equilibrated with ddH20, and collect the activated protein in the void volume. ... 

The phenomenon of thyroid hormone transport encompasses a number of sequential steps beginning with their secretion by the thyroid follicles and ending with their distribution to multiple sites of metabolism and action within virtually all the body s organs. These steps include equilibration with multiple carrier proteins in the plasma, transcapillary passage of both the hormones and the carrier proteins into extravas-cular spaces, entry into cells through their external membranes, and translocation to subcellular compartments and organelles. Some that are the subject of current inquiry or controversy, include the interaction of the hormones with "minor" transport protein in plasma, the kinetics of transcapillary movement, the presence of specific receptors for the carrier proteins and the hormones on endothelial and epithelial cell membranes, and specific transport mechanisms within cell organelles. 

Transporters comprise carriers and receptors that are located in the plasma membrane of the endothelial cells of the BBB. Carriers are membrane-restricted systems suited to generally transport compounds with a rather fixed size and a molecular mass smaller than 500-600 Da (33). Most of these systems are ATP-driven and therefore possess at least one intracellular ATP-binding domain. Others are equilibrative systems and do not require ATP. The activity of these transporters is temperature sensitive and they can be saturated at higher concentrations of ligands. Their activity can be influenced by competitive and noncompetitive inhibitors and by interfering with their phosphorylation by protein kinases. 

Fig. 3. Chromatography of subtilisin DY on bacitracin-cellulose (prepared with 2,4,6-trichlorotriazine). 200 mg of subtilisin DY dissolved in 3 ml of 50 mM Tris-HCl buffer, pH 8.3, containing 1 mM CaCl2> was applied on a column (30 x 2 cm) of bacitracin-cellulose (inhibitor content 14.9 jjmol/g of dry carrier) equilibrated with 50 mM Tris-HCl buffer, pH 8.3, containing 1 mM CaCl2- After washing the column with the equilibrium buffer, 20% isopropyl alcohol in 1 M NaCl, buffered at pH 8.3 was applied at the position marked with arrow. Fractions (6 ml) were taken at 5-min intervals. Full line, protein dashed line, activity determined with benzyloxycarbonyl-L-alanyl-l-alanyl-L-leucine p-nitro-anilide (Lyublinskaya et al., 1974 1977). 

In the case of a hydrophilic compound that enters the cell by carrier-mediated diffusion, a net increase in concentration inside the cell can sometimes be achieved by binding it to an intracellular protein. Only material in free solution can equilibrate across the membrane, not that which is protein bound. Such binding proteins are important, for example, in the intestinal absorption of calcium (section 4.6.1) and iron (section 4.6.2). 

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