PubMed · 8901398
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E M De Boer, D P Bruynzeel. Patch tests: evaluation by instrumental methods.. https://doi.org/10.1016/0738-081x(95)00107-q
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Heregulin was shown to promote the proteolytic cleavage of its receptor, ErbB-4, in several cell lines. The growth factor also rapidly promoted the transient translocation of ErbB-4 to a detergent-insoluble fraction, in which the receptor was hyper-tyrosine-phosphorylated compared with the receptor present in the detergent-soluble pool. However, an 80-kDa proteolytic fragment of ErbB-4 was found in the detergent-soluble fraction, but not in the detergent-insoluble fraction. Although the heregulin-induced cleavage of ErbB-4 produced a fragment of ErbB-4 very similar to that induced by 12-O-tetradecanoylphorbol-13-acetate or pervanadate (each of which is blocked by metalloprotease inhibitors), the growth factor-induced cleavage was not sensitive to these inhibitors under the same conditions. The heregulin-induced cleavage of ErbB-4 could be blocked by conditions that prevent clathrin-coated pit formation, suggesting that heregulin-mediated ErbB-4 cleavage occurs subsequent to internalization. When reagents that prevent acidification of endosomes were employed, heregulin-induced ErbB-4 cleavage was sensitive to metalloprotease inhibitors. The results imply that during ligand-dependent receptor trafficking, activated ErbB-4 receptors are subject to proteolytic cleavage involving an intracellular metalloprotease.
The quaternary structure of LacS, the lactose transporter of Streptococcus thermophilus, has been determined for the detergent-solubilized and the membrane-reconstituted state of the protein. The quaternary structure of the n-dodecyl-beta-d-maltoside-solubilized state was studied using a combination of sedimentation velocity and equilibrium centrifugation analysis. From these measurements it followed that the detergent-solubilized LacS undergoes reversible self-association with a monomer to dimer mode of association. The association constants were 5.4 +/- 3.6 and 4.4 +/- 1.0 ml mg(-1) as determined from the velocity and equilibrium sedimentation measurements, respectively. The experiments did not indicate significant changes in the shape of the protein-detergent complex or the amount of detergent bound in going from the monomeric to dimeric state of LacS. Importantly, a single Cys mutant of LacS is labeled by 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid in a substrate-dependent manner, indicating that the detergent-solubilized protein exhibits ligand binding activity. The quaternary structure of membrane-reconstituted LacS was determined by freeze-fracture electron microscopy analysis. Recent developments in the analysis of freeze-fracture images (Eskandari, S. P., Wright, E. M., Freman, M., Starace, D. M., and Zampighi, G. A. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 11235-11240) allowed us to directly correlate the cross-sectional area of the transmembrane segment to a dimeric state of the functionally membrane-reconstituted LacS protein. The cross-sectional area of the LacS protein was calibrated using the membrane-reconstituted transmembrane domain of the mannitol transporter enzyme II, an intramembrane particle for which the cross-sectional area was obtained from maps of two-dimensional crystals. The consequences of the determined quaternary structure for the transport function and regulation of LacS are discussed.
Detergent/polymer aqueous two-phase systems are studied as a fast, mild and efficient general separation method for isolation of labile integral membrane proteins. Mechanisms for phase behaviour and protein partitioning of both membrane-bound and hydrophilic proteins have been examined in a large number of detergent/polymer aqueous two-phase systems. Non-ionic detergents such as the Triton series (polyoxyethylene alkyl phenols), alkyl polyoxyethylene ethers (C(m)EO(n)), Tween series (polyoxyethylene sorbitol esters) and alkylglucosides form aqueous two-phase systems in mixtures with hydrophilic polymers, such as PEG or dextran, at low and moderate temperatures. Phase diagrams for these mixtures are shown and phase behaviour is discussed from a thermodynamic model. Membrane proteins, such as bacteriorhodopsin and cholesterol oxidase, were partitioned strongly to the micelle phase, while hydrophilic proteins, BSA and lysozyme, were partitioned to the polymer phase. The partitioning of membrane protein is mainly determined by non-specific hydrophobic interactions between detergent and membrane protein. An increased partitioning of membrane proteins to the micelle phase was found with an increased detergent concentration difference between the phases, lower polymer molecular weight and increased micelle size. Partitioning of hydrophilic proteins is mainly related to excluded volume effects, i.e. increased phase component size made the hydrophilic proteins partition more to the opposite phase. Addition of ionic detergent to the system changed the partitioning of membrane proteins slightly, but had a strong effect on hydrophilic proteins, and can be used for enhanced separation between hydrophilic proteins and membrane protein.