Concentrative carrier proteins

Using any of the carrier proteins available in highly purified form, eg, TBG or TBPA, a convenient and accurate quantitative determination of and is possible by displacement of radioiodinated or T. This procedure enables their quick determination at low concentrations even in the presence of coundess other substances that occur in body duids (31). In a similar fashion, intact cell nuclei or solubilized proteins from rat fiver cell nuclei, which display high affinities for thyroid hormones, especially T, have been used to establish relative binding affinities of many thyromimetic compounds (7). 

FIGURE 25.7 The pathway of palmhate synthesis from acetyl-CoA and malonyl-CoA. Acetyl and malonyl building blocks are introduced as acyl carrier protein conjugates. Decarboxylation drives the /3-ketoacyl-ACP synthase and results in the addition of two-carbon units to the growing chain. Concentrations of free fatty acids are extremely low in most cells, and newly synthesized fatty acids exist primarily as acyl-CoA esters. 

Hapten density is important for both immunization and assay performance, and hence the extent of conjugation or hapten density should be confirmed by established methods. A characteristic ultraviolet (UV) or visible absorbance spectrum that distinguishes the hapten from the carrier protein or use of a radiolabeled hapten can be used to determine the degree of conjugation. If the hapten has a similar A. iax to the protein, the extent of incorporation can still be estimated when the concentration of the protein and the spectral characteristics of the hapten and protein are known. The difference in absorbance between the conjugate and the starting protein is proportional to... 

Dissolve the carrier protein in 0.1M MES, 0.15 M NaCl, pH 4.7, at a concentration of lOmg/ml. If using native, multi-subunit KLH, increase the NaCl concentration of all buffers to 0.9 M (yes, 0.9 M, not 0.9 percent) to maintain solubility of the protein. If using KLH subunits, the high-salt concentration is not necessary. For neutral pH conjugations, substitute 0.1 M sodium phosphate, 0.15M NaCl, pH 7.2, for the MES buffer. 

Dissolve sulfo-SMCC (Thermo Fisher) at a concentration of lOmg/ml in the activation buffer. Immediately transfer the appropriate amount of this crosslinker solution to the vial containing the dissolved carrier protein. 

Dissolve the carrier protein (or another carrier that contains amine groups) in 0.1M sodium carbonate, 0.15 M NaCl, pH 8.5, at a concentration of 2mg/ml. 

Extracellular ligands (hormones, neurotrophins, carrier protein, adhesion molecules, small molecules, etc.) will bind to specific transmembrane receptors. This binding of specific ligand induces the concentration of the receptor in coated pits and internalization via clathrin-coated vesicles. One of the best studied and characterized examples of RME is the internalization of cholesterol by mammalian cells [69]. In the nervous system, there are a plethora of different membrane receptors that bind extracellular molecules, including neurotrophins, hormones and other cell modulators, being the best studied examples. This type of clathrin-mediated endocytosis is an amazingly efficient process, capable of concentrating... 

These methods of solute transfer usually rely on a relatively low intracellular concentration of the solute of interest, so that it will readily diffuse into the cell down the electrochemical gradient (as in the case of ion channels). Alternatively, the solute may be moved into the cell using chemical energy derived from another solute moved in the same direction (co-transport) or opposite direction (countertransport) on the carrier protein (symporters and antiporters respectively). The transfer of the second solute is in turn dependent on an inward electrochemical gradient. Ultimately, these gradients are established by primary, energy-requiring solute pumps (e.g. ATPases), which, on most epithelia, are located on the basolateral/serosal membrane (see Section 5.2 for discussion of ATPases). 

Active transport of a drug is mediated by a specific carrier. This is of particular interest in neural tissues and choroid plexus. In this case, the actual molecular size and shape are important, because it involves binding to a specific carrier protein that transports it. Active transport is an energy-dependent process, and may work against a concentration gradient. It is also a saturable mechanism and may be competitively inhibited by other ligands. 

Medication Transportation interactions. Recall that carrier proteins in the bloodstream escort medications and that over 80% of the circulating concentration of most psychiatric medicines is bound to serum proteins. When there are not enough protein binding sites to go around, however, the biologically active free fraction of the drug is increased. 

The transport mechanisms that operate in distribution and elimination processes of drugs, drug-carrier conjugates and pro-drugs include convective transport (for example, by blood flow), passive diffusion, facilitated diffusion and active transport by carrier proteins, and, in the case of macromolecules, endocytosis. The kinetics of the particular transport processes depend on the mechanism involved. For example, convective transport is governed by fluid flow and passive diffusion is governed by the concentration gradient, whereas facilitated diffusion, active transport and endocytosis obey saturable MichaeUs-Menten kinetics. 

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