Protein membrane-bound, purification

The procedure for purification of Na,K-ATPase in membrane-bound form from the outer renal medulla of mammalian kidney offers the opportunity of exploring the structure of the Na,K-pump proteins in their native membrane environment. The protein remains embedded in the membrane bilayer throughout the purification procedure thus maintaining the asymmetric orientation of the protein in the baso-lateral membrane of the kidney cell in the purified preparation. This preparation has been particularly useful in studies of ultrastructure, protein conformation and for... 

Screening the molecular heterogeneity of receptor expression in endothelial cell surfaces is required for the development of vascular-targeted therapies. First, as opposed to targeting purified proteins as discussed above, membrane-bound receptors are more likely to preserve their functional conformation, which can be lost upon purification and immobilization outside the context of intact cells. Moreover, many cell surface receptors require the cell membrane microenvironment to function so that protein-protein interaction may occur. Finally, combinatorial approaches may allow the selection of cell membrane ligands in a functional assay and without any bias about the cellular surface receptor. Therefore, even as yet unidentified receptors may be targeted. 

In those cases where the intracellular products are required, the cells must first be disrupted. Some products may be present in the solution within the cytoplasm, while others may be insoluble and exist as membrane-bound proteins or small insoluble particles called inclusion bodies. In the latter case, these must be solubilized before further purification. 

I The second purification procedure we examine illus- Vri trates an unusual approach to the purification of a membrane-bound protein. The lactose carrier protein of E. coli is normally tightly bound to the plasma membrane. This protein is involved in the active transport of the dissaccharide lactose across the cytoplasmic membrane. When lactose carrier protein is present, the intracellular concentration of lactose can achieve levels 1,000-fold higher than those found in the external medium. Ron Kaback devised a simple yet elegant procedure for the purification of this protein. 

Purification of the membrane-bound lactose carrier protein is a very different problem from the purification of the soluble OMP synthase. Both the approach to purification and the assays for the protein during purification are quite novel. The assay involves reconstituting a transport system with membranes that are free of lactose carrier protein, then adding the partially purified carrier protein and radioactively labeled lactose. The activity in this assay system is proportional to the transport of radioactive lactose across the membrane in the cell-free reconstituted system. 

The Mg2+-activated ATPase (or ATP synthase) is made up of two parts. The Fj component is the catalytic, Mg2+-binding, extrinsic membrane protein composed of five different subunits, a, (3, y, S and e. The F0 component is an intrinsic membrane complex that contains three subunits, a, b and c, and mediates proton translocation. The F, protein is bound to the membrane through interaction with F0. The complexity of the F,F0 enzyme has presented many difficulties. Hie greatest advances have been made for the bacterial enzymes, notably for thermophiles, Escherichia coli and Rhodospirillum rubrum, where progress has been made in the purification of subunits and their reconstitution into membranes, and the identification of binding sites for Mg2+ and nucleotides on the Fi subunits.300 FiF0 preparations can be incorporated into liposomes and display H+ translocation, ATP-P, exchange and ATP synthesis.301... 

The electron-transfer centres in electron-transfer chains are usually but not always membrane bound, and so present considerable difficulties in purification, particularly from excess phospholipid. These proteins may span the membrane or be embedded from one side only. Other proteins may bind to surface components of the membrane and be separated more readily. 

PBPs are membrane-bound enzymes. In general, PBPs, especially high-molecular-mass PBPs, consist of a short cytoplasmic domain in their N-terminus, followed by a hydrophobic transmembrane domain, then a transglycosylase domain (for class A PBPs) or a non-penicillin-binding domain (also called an unknown functional domain for class B PBPs), and finally a transpeptidase domain located in their C-terminus (Fig. 1 [1,2,15,56]). Thus, purification of PBPs traditionally involved the isolation of membranes and solubilization of PBPs from the membrane preparation by detergents. Since PBPs are present at a very low level (approximately 100-300 molecules per cell) [57-59] and also in multiple forms in the cell, this classical approach was not particularly efficient for producing a significant amount of a purified PBP protein of particular interest. 

The protein has to be obtained in solution prior to its purification. Thus tissues and cells must be disrupted by homogenization or osmotic lysis and then subjected to differential centrifugation to isolate the subcellular fraction in which the protein is located. For membrane-bound proteins, the membrane structure has to be solubilized with a detergent to liberate the protein. 

Chen, Y.L., Ma, Y.T., and Rando, R.R. (1996). Solubilization, partial purification, and affinity labeling of the membrane-bound isoprenylated protein endoprotease. Biochemistry 35 3227-3237. 

Work on the isolation and characterization of the membrane-bound FeS-A/B protein was renewed more than twenty years later by Oh-oka, Takahashi, Kuriyama, Saeki and Matsubara using n-butanol in the isolation procedure and by Wynn and Malkin using acetone. Both groups found partially degraded FeS-clusters, as indicated by the FeS composition, Oh-oka et al finding 4.1Fe -i- 3.2S and Wynn and Malkin 7.9 Fe -i- 6.4 S. Eater, Oh-oka et al used acetone for protein isolation and carried out extended spectroscopic measurements. It was found that only when strictly anaerobic conditions were maintained during the isolation and purification process could the native protein be isolated with intact [4Fe 4S] clusters, conforming strictly to the (8Fe-i-8S) stoichiometry ... 

The sialyltransferases are membrane-bound proteins located in the endoplasmic reticulum (ER) and in the Golgi apparatus. Information about their sequence homology is limited, but they do appear to share a common topography [35]. A catalytic domain resides at the C-terminus followed by an N-terminal segment that anchors the enzyme into the ER or Golgi membrane. Soluble, catalytically active sialyltransferases that lack the anchor segment have been isolated from milk, serum, and other body fluids, suggesting that this N-terminal anchor is not necessary for the enzyme to retain catalytic activity. However, the ability to obtain from natural sources quantities of most sialyltransferases that would be needed for synthesis applications is hampered by low tissue concentrations and difficult purifications. 

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