Purification of a protein binding quinacrine and histrionicotoxin from membrane fragments rich in cholinergic receptors in Torpedo marmorata]

A protein is purified by differential centrifugation from membrane fragments rich in acetylcholine receptor prepared from Torpedo marmorata electric organ after dissolution by a mixture of non denaturing detergents. After polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate and Coomassie blue staining the purified protein yields a single band of apparent molecular weight 43,000. Spectroscopic experiments carried out in the absence of Ca++ and detergents reveal that the 43 K protein interacts with the fluorescent local anesthetic quinacrine and with the frog toxin histrionicotoxin (apparent KD : 7 X 10(-7) M) but not with carbamylcholine and the alpha toxin from N. nigricollis.     

Proteomic analysis of low-abundant integral plasma membrane proteins based on gels

To characterize low-copy integral membrane proteins and offer some methods for human liver proteome projects, we fractionated highly purified rat liver plasma membrane (PM). PM was purified through two sucrose density gradient centrifugations, and treated with 0.1 M Na₂CO₃, chloroform/methanol and Triton X-100. Proteins were separated by electrophoresis and submitted to mass spectrometry analysis. Four hundred and fiftyseven non-redundant membrane proteins were identified, of which 23% (105) were integral membrane proteins with one or more transmembrane domains. One hundred and fifty-three (33.5%) had no location annotation and 68 were unknown-function proteins. The proteins from different fractions were complementory. A database search for all identified proteins revealed that 53 proteins were involved in the cell communication pathway. More interestingly, more than 50% of the proteins had a protein abundance index concentration of less than 0.1 mol/l, and 12% proteins a concentration 100 times less than that of arginase 1 and actin. Received 15 March 2006; received after revision 17 May 2006; accepted 10 June 2006 L.-J. Zhang and X.-e Wang are contributed equally to this work.     

Purification from a total cellular lysate of a protein modifying the sensitivity of Na+/K+ ATPase to ouabain]

A protein (32,000 dalton) has been purified from M2FS cells derived from the murine plasmocytoma MOPC 173. Like tropomyosin, this protein when added with Ca++ to EDTA-treated plasma membranes prepared from the same cell, induced a drastic increase in the Na+/K+ atpase resistance to ouabain.     

Helix insertion into bilayers and the evolution of membrane proteins

Polytopic α-helical membrane proteins cannot spontaneously insert into lipid bilayers without assistance from polytopic α-helical membrane proteins that already reside in the membrane. This raises the question of how these proteins evolved. Our current knowledge of the insertion of α-helices into natural and model membranes is reviewed with the goal of gaining insight into the evolution of membrane proteins. Topics include: translocon-dependent membrane protein insertion, antibiotic peptides and proteins, in vitro insertion of membrane proteins, chaperone-mediated insertion of transmembrane helices, and C-terminal tail-anchored (TA) proteins. Analysis of the E. coli genome reveals several predicted C-terminal TA proteins that may be descendents of proteins involved in pre-cellular membrane protein insertion. Mechanisms of pre-translocon polytopic α-helical membrane protein insertion are discussed.     

Permeability of the isolated human amniotic membrane to divalent cations]

Membrane potential and resistance recordings, in vitro, show that Mg++ does not pass through the amnion from the inside of the amniotic compartment to the outside of the amniotic membrane. Mg++ may become fixed on the surface or in the midst of the amniotic membrane. However, Mg++ diffuses in the opposite direction. Ca++, Ba++, Sr++ diffuse in both directions across the amniotic membrane.     

Lysosomal membrane fragility and catabolism of cytosolic proteins: evidence for a direct relationship

A direct relationship was established between the stability of the lysosomal membrane and an estimate of cytosolic protein catabolism, based on loss of radiolabel from prelabeled protein. Lysosomes in the lysosomally-rich digestive cells of the midgut gland of the marine mussel (Mytilus edulis) were destabilized by experimental treatment with phenanthrene.     

Lysosomal membrane fragility and catabolism of cytosolic proteins: evidence for a direct relationship

Summary A direct relationship was established between the stability of the lysosomal membrane and an estimate of cytosolic protein catabolism, based on loss of radiolabel from prelabeled protein. Lysosomes in the lysosomally-rich digestive cells of the midgut gland of the marine mussel (Mytilus edulis) were destabilized by experimental treatment with phenanthrene.     

Structural genomics for membrane proteins

Structure-based drug discovery has proven useful in improving and shortening the drug development process. The approach of structural genomics to study a large number of targets in parallel has been commonly applied to protein families and even whole genomes. Paradoxically, although membrane proteins represent the largest type of drug targets, up to 70% today, determination of their structure has been modest compared to that of soluble proteins. Because membrane proteins are important for drug discovery an emphasis has been placed on developing technologies and methods to determine membrane protein structures. Several structural genomics initiatives have been established, focusing on the structural biology of membrane proteins. Received 31 May 2006; received after revision 5 July 2006; accepted 9 August 2006     

Importance of protein-protein interactions for the structural integrity of membrane framents from Torpedo marmorata electric organ]

Electron spin resonance and electron microscopy were used to show that alkaline extraction of a 43,000 dalton protein from torpedo membrane induces structural destabilization of the membrane and rotational diffusion of the cholinergic receptor protein.     

5-hydroxytryptamine-sensitive adenylate cyclase activity in brain synaptosomal membrane preparations]

A serotonine-sensitive adenylate cyclase system has been observed in horse brain membrane preparations. In crude mitochondrial fraction two types of activation sites were characterized, the Ka being = 2 nM and 1 micrometer. In purified synaptosomal membranes, a single adenylate cyclase activation was observed corresponding to the highest apparent affinity (KD = 2nM). This activation site might be related to the high affinity binding site (KD = 2NM) were previously described in the same membrane preparations.     

Increase in membrane conductance by adrenaline in parotid acinar cells.

It is shown that excitation of the alpha- or beta-adrenoceptors in mouse parotid acinar cells causes a marked reduction of surface cell membrane resistance. The alpha-adrenoceptor induced membrane effect is an increase in K conductance. The beta-adrenoceptor induced membrane effect does not seem to be mediated by cyclic AMP.     

Identification of organ-specific glycosylation of a membrane protein in two tissues using lectins

Since glycosylation of proteins is performed by the host cell, and variable sugar groupings can confer heterogeneity on the same polypeptide, we wished to see whether membrane proteins, in particular the ubiquitous transmembrane Na, K-ATPase, could be glycosylated differently in different organs. Using a highly sensitive enzyme-linked antibody detection system of bound digoxigenin-labelled lectins on nitrocellulose sheets containing electroblotted and subunits of kidney and brain Na,K-ATPase, isolated from various rat strains, in combination with isoform-specific immunoblots, we discovered that brain Na,K-ATPase was highly mannosylated in contrast to renal Na,K-ATPase. Thus, we describe the existence of organ-related glycoforms of an integral ubiquitous membrane protein, i.e. diversification of the same polypeptide by organ-typical sugars. At the same time, the presence of the same glycosylation pattern can make distinct protein isoforms occurring in a same organ more homogeneous. Such organ-related glycoforms may serve for tissue identification and as tissue-specific receptors.     

Age-dependent changes of rat liver plasma membrane composition

The chemical composition of liver plasma membrane was studied in Wistar rats aged between 3 and 24 months. Results obtained indicate a significant age-dependent positive correlation of both the protein: phospholipid and cholesterol: phospholipid ratios, whereas the protein: cholesterol ratio seems to remain unaffected. Phospholipid analysis of liver plasma membrane reveals that only the phosphatidylcholine content has a significant negative correlation with age; all other phospholipid species remain basically unchanged.     

Thixotropic effect and electro-osmosis as possible factors of membrane electrical excitability]

Cation motion under the electrical field might break the membrane structure. In the temporary wake of each cation conductivity would be increased. Thus a zone of negative resistance would occur in the intensity-voltage relation. Membrane dehydration, due to electro-osmosis, might induce repeated flip-flops of current at the limits of the negative resistance zone.     

Deciphering the BAR code of membrane modulators

The BAR domain is the eponymous domain of the “BAR-domain protein superfamily”, a large and diverse set of mostly multi-domain proteins that play eminent roles at the membrane cytoskeleton interface. BAR domain homodimers are the functional units that peripherally associate with lipid membranes and are involved in membrane sculpting activities. Differences in their intrinsic curvatures and lipid-binding properties account for a large variety in membrane modulating properties. Membrane activities of BAR domains are further modified and regulated by intramolecular or inter-subunit domains, by intermolecular protein interactions, and by posttranslational modifications. Rather than providing detailed cell biological information on single members of this superfamily, this review focuses on biochemical, biophysical, and structural aspects and on recent findings that paradigmatically promote our understanding of processes driven and modulated by BAR domains.     

Challenges and approaches to understand cholesterol-binding impact on membrane protein function: an NMR view

Experimental evidence for a direct role of lipids in determining the structure, dynamics, and function of membrane proteins leads to the term ‘functional lipids’. In particular, the sterol molecule cholesterol modulates the activity of many membrane proteins. The precise nature of cholesterol-binding sites and the consequences of modulation of local membrane micro-viscosity by cholesterol, however, is often unknown. Here, we review the current knowledge of the interaction of cholesterol with transmembrane proteins, with a special focus on structural aspects of the interaction derived from nuclear magnetic resonance approaches. We highlight examples of the importance of cholesterol modulation of membrane protein function, discuss the specificity of cholesterol binding, and review the proposed binding motifs from a molecular perspective. We conclude with a short perspective on what could be future trends in research efforts targeted towards a better understanding of cholesterol/membrane protein interactions.     

Evidence of a direct action of triiodothyronine (T3) on the cell membrane of GH3 cells: an electrophysiological approach

Electrophysiological experiments demonstrate that triiodothyronine (T3) exerts a direct effect on the membrane of a strain of cultured rat pituitary tumor cells, GH3/B6. These cells respond to pressure application of T3 (2-5 nl, concentration 1 X 10(-10) M) with an increase in the membrane resistance (Rm) and a hyperpolarization. Spontaneously firing cells become silent.     

Delta opioid receptors recycle to the membrane after sorting to the degradation path

Soon after internalization delta opioid receptors (DOPrs) are committed to the degradation path by G protein-coupled receptor (GPCR)-associated binding protein. Here we provide evidence that this classical post-endocytic itinerary may be rectified by downstream sorting decisions which allow DOPrs to regain to the membrane after having reached late endosomes (LE). The LE sorting mechanism involved ESCRT accessory protein Alix and the TIP47/Rab9 retrieval complex which supported translocation of the receptor to the TGN, from where it subsequently regained the cell membrane. Preventing DOPrs from completing this itinerary precipitated acute analgesic tolerance to the agonist DPDPE, supporting the relevance of this recycling path in maintaining the analgesic response by this receptor. Taken together, these findings reveal a post-endocytic itinerary where GPCRs that have been sorted for degradation can still recycle to the membrane.     

Coronavirus envelope protein: A small membrane protein with multiple functions

Coronavirus envelope protein is a small membrane protein and minor component of the virus particles. It plays important roles in virion assembly and morphogenesis, alteration of the membrane permeability of host cells and virus-host cell interaction. Here we review recent progress in characterization of the biochemical properties, membrane topology and functions of the protein. Received 27 February 2007; received after revision 4 April 2007; accepted 26 April 2007     

Many mechanisms, one entrance: membrane protein translocation into the nucleus

The inner nuclear membrane harbors a unique set of membrane proteins, many of which interact with nuclear intermediate filaments and chromatin components and thus play an important role in nuclear organization and gene expression regulation. These membrane proteins have to be constantly transported into the nucleus from their sites of synthesis in the ER to match the growth of the nuclear membrane during interphase. Many mechanisms have evolved to enable translocation of these proteins to the nucleus. The full range of mechanisms goes from rare autophagy events to regulated translocation using the nuclear pore complexes. Though mechanisms involving nuclear pores are predominant, within this group an enormous mechanistic range is observed from free diffusion through the peripheral channels to many distinct mechanisms involving different nucleoporins and other components of the soluble protein transport machinery in the central channels. This review aims to provide a comprehensive insight into this mechanistic diversity.