Intrinsic membrane proteins separation

Two are hydrophobic intrinsic membrane domains, each with six membrane-spanning helices and two are peripheral membrane ATP binding domains. All four domains may be in a single peptide chain, as in CFTR, or they may be separate smaller proteins as in bacterial periplasmic permeases.431 446... 

A pre-requisite of reconstitution is the isolation of the protein (or glycoprotein) in relatively pure form a process which for intrinsic membrane components requires the use of mild detergents (JJ.) which will separate and solubilize the protein without initiating denaturation. In contrast to solubilization, reconstitution requires the protein to penetrate an aqueous... 

Fig.l SDS-urea PAGE of intrinsic PSII membrane protein fractions. Separation was achieved by ACA 54 gel filtration chromatography. Staining was carried out with Coomassie R 250. 

In filtration processes for the extracorporeal treatment of renal failure, partially rejeeted proteins accumulate at the membrane separation layer [i.e., concentration polarization (CP) phenomena occur] to an extent that depends on protein eoncentralion in the bulk blood, and the fluid dynamics of the blood compartment. Higher protein concentrations at the membrane surface cause the membrane sieving coefficient to be higher than that expeeted, based on the intrinsic membrane separation properties. However, once the latter are known, the actual sieving coefficient can be estimated from the operating conditions and module geometry as follows (Klein et al., 1978) ... 

Three key elements determine the potential and applications of a hollow-fiber membrane (1) pore size and pore size distribution, (2) selective layer thickness, and (3) inherent properties (chemistry and physics) of the membrane material. Pore size and its distribution usually determine membrane applications, separation factor, or selectivity. The selective layer thickness determines the membrane flux or productivity. Material chemistry and physics govern the intrinsic permselectivity for gas separation and pervaporation, fouling characteristics for RO (reverse osmosis), UF (ultrafiltration), and MF (microfiltration) membranes, chemical resistance for membranes used in harsh environments, protein and drug separation, as well as biocompatibUity for biomedical membranes used in dialysis and biomedical and tissue engineering. 

Chlorophyll fluorescence has been extensively utilized as an intrinsic membrane probe to study the photochemistry of photosynthesis. The assignment of the various fluorescence emission bands to specific functional complexes in the membrane has been attempted in many laboratories (Reviewed in Breton, 1982, Butler, 1979 Satoh, this symposium). Two chlorophyll fluorescence emission bands at 685 and 695 nm (at 77 K) have been identified to arise from photosystem II (PS II). In this paper we provide evidence for the fact that these two emission maxima arise from separate pigment-proteins within the PS II reaction center complex. 

An optical immunosensor for continuous T4 measurement has been described, in which the fluorescent indicator protein is separated from the sample flow chamber by a dialysis membrane.024) The indicator is T4-binding globulin (TBG), the intrinsic fluorescence (ex. 290 nm) of which is quenched by T4binding. Due to the high affinity of the TBG for thyroxine, the immunosensor is not reversible, but multiple measurements can be made until the TBG is saturated. Sensitivity is inadequate for clinically useful concentrations of T4, but suggestions for improvement of the method are made. 

Thus, the fat globules are surrounded, at least initially, by a membrane typical of eukaryotic cells. Membranes are a conspicuous feature of all cells and may represent 80% of the dry weight of some cells. They serve as barriers separating aqueous compartments with different solute composition and as the structural base on which many enzymes and transport systems are located. Although there is considerable variation, the typical composition of membranes is about 40% lipid and 60% protein. The lipids are mostly polar (nearly all the polar lipids in cells are located in the membranes), principally phospholipids and cholesterol in varying proportions. Membranes contain several proteins, perhaps up to 100 in complex membranes. Some of the proteins, referred to as extrinsic or peripheral, are loosely attached to the membrane surface and are easily removed by mild extraction procedures. The intrinsic or integral proteins, about 70% of the total protein, are tightly bound to the lipid portion and are removed only by severe treatment, e.g. by SDS or urea. 

Fig. 6-22 Cycle of activation and deactivation of heterotrimeric G-proteins. The ligand-bound receptor acts as a guanine nucleotide-exchange factor that replaces GDP with GTP on the a subunit. The a-GTP and /3-y-GTP subunits then separate to activate downstream target enzymes. The cycle is completed by the action of intrinsic GTPase activity in the a subunit. Free a-GDP subunits readily reassociate with free fiy subunits to form inactive apy heterotrimers. G-protein activation represents a site of amplification in signaling pathways for every ligand bound receptor molecule in the membrane, it is estimated that hundreds or thousands of G-protein molecules are activated.

The extrinsic pathway is activated by tissue injury and is not of major concern in the clinical use of membrane devices. The intrinsic pathway, however, is initiated by a multitude of factors. Including interactions between serum proteins and exogenous materials. Hydrodynamic forces acting on platelets may also lead to the release of platelet factors that trigger the intrinsic pathway. Thus, the selection of membrane materials to minimize thrombogenesls cannot be fully separated from the design of devices to contain them because of this potential for shear forces to activate the clotting cascade. 

Growth factor receptors are stmcturally similar to cytokine receptors, being composed of (1) an N-terminal extracellular domain that provides binding sites for their cytokine ligands (2) a hydrophobic a-helix which spans the cell membrane and (3) a C-terminal cytoplasmic domain. The cytoplasmic domains of growth factor receptors contain an intrinsic protein tyrosine kinase activity, in contrast to cytokine receptors, in which the associated protein tyrosine kinases are separate proteins. 

Two-phase gas-liquid flow clearly reduces concentration polarization, and this can improve membrane separation. For example, Ghosh et al. [91] assessed the effect of gas sparging on protein fraction with BSA (MW 67,000) and lysozyme (MW 14,100) as model solutes. They reported that a nearly complete separation of these two model proteins was achieved with two-phase flow UF (MWCO 100 kDa), indicating an 18-fold increase in selectivity compared to that without air injection. The enhancement in selectivity was believed to be cansed by the disruption of the concentration polarization so that solnte retentions were closer to the intrinsic values. Although the depolarization decreased transmission for both BSA and lysozyme, the theoretical analysis suggested that air injection affected more the transmission of the more rejected component (BSA) so that high separation efficiency was achieved. 

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