Both ankyrin and band 4.1 are required to restrict the rotational mobility of band 3 in the human erythrocyte membrane.

A population of band 3 proteins in the human erythrocyte membrane is known to have restricted rotational mobility due to interaction with cytoskeletal proteins. We have further investigated the cause of this restriction by measuring the effects on band 3 rotational mobility of rebinding ankyrin and band 4.1 to ghosts stripped of these proteins as well as spectrin and actin. Rebinding either ankyrin or 4.1 alone has no detectable effect on band 3 mobility. Rebinding both these proteins together does, however, reimpose a restriction on band 3 rotation. The effect on band 3 rotational mobility of rebinding ankyrin and 4.1 are similar irrespective of whether or not band 4.2 is removed from the membrane. We suggest that ankyrin and 4.1 together promote the formation of slowly rotating clusters of band 3.     

Janus kinases and focal adhesion kinases play in the 4.1 band: a superfamily of band 4.1 domains important for cell structure and signal transduction.

The band 4.1 domain was first identified in the red blood cell protein band 4.1, and subsequently in ezrin, radixin, and moesin (ERM proteins) and other proteins, including tumor suppressor merlin/schwannomin, talin, unconventional myosins VIIa and X, and protein tyrosine phosphatases. Recently, the presence of a structurally related domain has been demonstrated in the N-terminal region of two groups of tyrosine kinases: the focal adhesion kinases (FAK) and the Janus kinases (JAK). Additional proteins containing the 4.1/JEF (JAK, ERM, FAK) domain include plant kinesin-like calmodulin-binding proteins (KCBP) and a number of uncharacterized open reading frames identified by systematic DNA sequencing. Phylogenetic analysis of amino acid sequences suggests that band 4.1/JEF domains can be grouped in several families that have probably diverged early during evolution. Hydrophobic cluster analysis indicates that the band 4.1/JEF domains might consist of a duplicated module of approximately 140 residues and a central hinge region. A conserved property of the domain is its capacity to bind to the membrane-proximal region of the C-terminal cytoplasmic tail of proteins with a single transmembrane segment. Many proteins with band 4.1/JEF domains undergo regulated intra- or intermolecular homotypic interactions. Additional properties common to band 4.1/JEF domains of several proteins are binding of phosphoinositides and regulation by GTPases of the Rho family. Many proteins with band 4. 1/JEF domains are associated with the actin-based cytoskeleton and are enriched at points of contact with other cells or the extracellular matrix, from which they can exert control over cell growth. Thus, proteins with band 4.1/JEF domain are at the crossroads between cytoskeletal organization and signal transduction in multicellular organisms. Their importance is underlined by the variety of diseases that can result from their mutations.     

Molecular weight of major component proteins in crude saline extract of adult Paragonimus westermani

When the component proteins in crude saline extract of 13-week old adult Paragonimus westermani were observed by non-denaturing discontinuous-polyacrylamide gel electrophoresis (Disc-PAGE), 8 distinct bands were clearly recognized. Molecular weight (MW) of each band protein, numbered in sequence from cathodal side which appeared in 10% separating gel, was measured first by Ferguson plot utilizing different gel concentrations from 10% to 4.5%. MW of band 1 protein (known as egg protein) was 440 kDa. And MW of other band proteins were: 386 kDa in band 2, 17.4 kDa in band 3, 17 kDa in band 4, 14.3 kDa in band 5, 46 kDa in band 6, 38 kDa in band 7 and 23 kDa in band 8. When the proteins in the crude extract were separated into fractions by molecular sieve chromatography through 1.6(phi) X 70 cm sized Sephacryl S-300 Superfine column and revisualized by Disc-PAGE in 8% gel, the sequence of eluted proteins was band 1, band 2, band 6, band 7 and bands 3, 4, 5 and 8. This elution profile confirmed MW of each band protein in the crude extract as measured by Ferguson plot.     

Associations of human erythrocyte band 4.2. Binding to ankyrin and to the cytoplasmic domain of band 3

We have examined the associations of purified red cell band 4.2 with red cell membrane and membrane skeletal proteins using in vitro binding assays. Band 4.2 bound to the purified cytoplasmic domain of band 3 with a Kd between 2 and 8 X 10(-7) M. Binding was saturable and slow, requiring 2-4 h to reach equilibrium. This finding confirms previous work suggesting that the principal membrane-binding site for band 4.2 lies within the 43-kDa cytoplasmic domain of band 3 (Korsgren, C., and Cohen, C. M. (1986) J. Biol. Chem. 261, 5536-5543). Band 4.2 also bound to purified ankyrin in solution with a Kd between 1 and 3.5 X 10(-7) M. As with the cytoplasmic domain of band 3, binding was saturable and required 4-5 h to reach equilibrium. Reconstitution with ankyrin of inside-out vesicles stripped of all peripheral proteins had no effect upon band 4.2 binding to membranes; similarly, reconstitution with band 4.2 had no effect upon ankyrin binding. This shows that ankyrin and band 4.2 bind to distinct loci within the 43-kDa band 3 cytoplasmic domain. Coincubation of ankyrin and band 4.2 in solution partially blocked the binding of both proteins to the membrane. Similarly, coincubation of bands 4.1 and 4.2 in solution partially blocked binding of both to membranes. In all cases, the data suggest the possibility that domains on each of these proteins responsible for low affinity membrane binding are principally affected. The data also provide evidence for an association of band 4.2 with band 4.1. Our results show that band 4.2 can form multiple associations with red cell membrane proteins and may therefore play an as yet unrecognized structural role on the membrane.     

Evidence of monomeric photosystem I complexes and phosphorylation of chlorophyll a/c-binding polypeptides in Chroomonas sp. strain LT (Cryptophyceae)

Thylakoid membranes of the cryptophyte Chroomonas sp. strain LT were solubilized with dodecyl-beta-maltoside and subjected to sucrose density gradient centrifugation. The four pigment protein complexes obtained were subsequently characterized by absorption and fluorescence spectroscopy, SDS-PAGE, and Western immunoblotting using antisera against the chlorophyll a/c-binding proteins of the marine cryptophyte Cryptomonas maculata and the reaction-center protein D2 of photosystem II of maize. Band 1 consisted mainly of free pigments, phycobiliproteins, and chlorophyll-a/c-binding proteins. Band 2 represented a major chlorophyll a/c-binding protein fraction. A mixture of photosystem II and photosystem I proteins comprised band 3, whereas band 4 was enriched in proteins of photosystem I. Western immunoblotting demonstrated the presence of chlorophyll a/c-binding proteins and their association with photosystem I in band 4. Phosphorylation experiments showed that chlorophyll a/c-binding proteins became phosphorylated. Negative staining electron microscopy of band B4 revealed photosystem I particles with dimensions of 22 nm. Our work showed that PSI-LHCI complexes of cryptophytes are similar to those of Chlamydomonas rheinhardtii, the diatom Phaeodactylum tricornutum, and higher plants.     

Transblot identification of biotin-containing proteins in rat liver

Peroxidase-conjugated avidin was used to detect biotin-containing carboxylases in rat liver. By a transblot method, avidin-peroxidase interacted with liver proteins with estimated molecular masses of 120 and 74 kDa. The proteins were identified as pyruvate carboxylase (120 kDa, 6.4 pI) and methylcrotonyl-CoA carboxylase (74 kDa, 7.2 pI) by two-dimensional gel electrophoresis and transblot method. An additional band with estimated molecular mass of 220 kDa was detected in the cytosol fraction of rat liver, compatible with acetyl-CoA carboxylase. Rat liver proteins were prepared and treated with avidin and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transblot with avidin-peroxidase. A 190-kDa band was found with a parallel decrease in the 120-kDa band determined by Coomassie blue staining; however, these proteins did not stain by the transblot avidin-peroxidase method. When the transblot of parallel proteins was incubated with biotin and subsequently with avidin-peroxidase, two additional bands, namely 190 and 145 kDa, were detected while the 74-kDa band disappeared correlated with decreased staining of the 120-kDa band. The present procedure is a simple, rapid, and inexpensive method for detecting biotin-containing proteins in various tissues and organs and in determining the occurrence of nonspecific staining with the avidin-biotin complex method of immunoblot.     

Mechanism of selective release of membrane proteins from human erythrocytes in the presence of liposomes

Incubation of erythrocytes with liposomes results in the release of shed vesicles rich in glycosyl-phosphatidylinositol (GPI)-anchored proteins but poor in transmembranous proteins. We investigated the mechanisms of membrane protein polarization by examining the effect of the interaction between spectrin and membrane proteins on the release of a transmembranous protein, band 3, and a GPI-anchored protein, acetylcholinesterase (AChE), from erythrocyte ghosts. Polymerization of spectrin resulted in a 30-fold decrease in the released amount of band 3 per constant amount of shed vesicles but did not affect the amount of released AChE per constant amount of shed vesicles. On the other hand, the amount of released band 3 per constant amount of shed vesicles increased by cleaving the cytoplasmic part of band 3. Our results first demonstrated that the diffusibility of membrane proteins determined by steric hindrance between membrane proteins and protein mesh primarily determines the ease of localization of membrane proteins into shed vesicles. Taken together with the recent biophysical studies, we built a "fence selection model" that retrograding spectrin mesh sweeps diffusing band 3 molecules from the tip of the membrane crenated area toward the entry of the crenated area, but not AChE molecules. Our study describes a novel method for isolation of a large number of vesicles containing special and intact membrane proteins from cells not by using detergents or organic solvents, but by utilizing the fence effect between the cytoskeleton and membrane proteins.     

Degradation of exogenous membrane proteins implanted into the plasma membrane of cultured hepatoma cells

A method for implanting exogenous membrane proteins into recipient hepatoma cells is described. Red cell band 3 and Sendai virus envelope proteins HN and F were extracted from their respective sources and purified by centrifugation to equilibrium through sucrose step gradients in the presence of octyl-beta-D-glucopyranoside. 0.05-0.15 micron vesicles were formed by adding lipid to combined detergent solubilized, isolated membrane proteins and removing detergent by dialysis. The vesicles were hybrid band 3-Sendai envelope vesicles and not a mixture of two distinct vesicle types as judged by (1) the ability of Sendai specific antibody to immunoprecipitate greater than 99% of band 3 from vesicle suspensions and (2) comigration of band 3 and Sendai envelope proteins on isopyknic sucrose density gradients. The hybrid vesicles (virosomes) were not fusogenic but did bind to cultured hepatoma cells in the cold. Subsequent treatment of virosomes absorbed onto cultured cells with polyethylene glycol resulted in a stable association of 2-10% of added band 3 and Sendai envelope proteins with the cells. Efficient transfer of virosome-associated band 3 to the cells was dependent on both lipid and Sendai envelope proteins. Fluid phase marker transfer, immunofluorescence, and protease digestion experiments demonstrate that the majority of the virosomes were implanted into recipient hepatoma membranes and not simply adsorbed onto their surface or immediately endocytosed. The hybrid membrane protein-viral envelope vesicles thus offer an efficient means for insertion of foreign proteins into the membranes of recipient cultured cells.     

Identification of tubulin-binding proteins of the chicken erythrocyte plasma membrane skeleton which colocalize with the microtubular marginal band

The plasma membrane of nucleated erythrocytes contains a microtubular marginal band which appears to be associated with the plasma membrane skeleton. In this report, we identify two families of cytoskeletal proteins which may be involved in such an association. These proteins, of molecular mass 78 kDa and 48 kDa on SDS-PAGE, are shown to bind tubulin based on a 125I-labeled tubulin binding assay. Solubilization of isolated chicken erythrocyte plasma membranes in Triton X-100 shows that these proteins centrifuge with the pellet, indicating that they are bound to the membrane skeleton. Finally, immunofluorescence studies using antisera raised against the 78 kDa and 48 kDa proteins show that they colocalize with the marginal band in intact cells. Colocalization of cytoskeletal tubulin-binding proteins with the marginal band favors a hypothesis suggesting that the 78 kDa and 48 kDa proteins are involved in the association of the two molecular superstructures.     

HISTOCHEMISTRY OF MYELIN XIV PERIPHERAL NERVE MYELIN PROTEINS: ELECTROPHORETIC AND HISTOCHEMICAL CORRELATIONS

Abstract— Myelin from the peripheral nervous system has been shown to contain two basic protein components and an electrophoretically slower-moving major protein, the 'J' band. The 'J' band protein cannot be selectively removed by aqueous or organic solvents and does not correspond to proteolipid or acidic protein. Histochemical stains applied to peripheral nervous systems myelin proteins separated by polyacrylamide electrophoresis indicate that 'J' band protein is analogous with the neurokeratin of the nerve sheath. Trypanophilia observed histochemically in unfixed myelin is principally due to basic proteins. With prolonged tryptic digestion 'J' band protein is degraded. Thus, previous classifications of myelin proteins based on trypsin sensitivity have been modified. All peripheral nervous system myelin proteins should be regarded as trypsin-sensitive, the basic protein being relatively more and the 'J' band protein relatively less susceptible.     

Infrared absorption of tyrosine side chains in proteins

Poly-L -tyrosine absorbs strongly at 1515 cm.^?1, and a band at this frequency has been found in a number of proteins and has been assigned to the tyrosine residue. The assignment was confirmed by examination of spectra of deuterated proteins, which usually exhibit a residual band at 1513 cm.^?1. In proteins, this band correlates linearly with known tyrosine content, but the point corresponding to poly-L -tyrosine itself does not fall on the correlation line. The possibility and limitations of using the infrared method for tyrosine determinations is discussed.     

Detection of the associated state of membrane proteins by polyacrylamide gradient gel electrophoresis with non-denaturing detergents Application to band 3 protein from erythrocyte membranes

Polyacrylamide gradient gel electrophoresis was carried out in micellar solutions of various detergents which differ in degree of potency to denature proteins. From the application of this method to band 3 protein from erythrocyte membranes, it was suggested that the procedure was useful in studying the molecular state of membrane proteins.The electrophoretic behaviors of human and bovine band 3 protein did not show any species specificity in either a denature state and a state resembling the native state. As well as in nonionic detergent solutions, the dimeric and tetrameric structures of bovine band 3 protein were preserved in sodium deoxycholate solution, in which protein complexes maintained in nonionic detergent solutions are frequently dissociated. Even in dodecyltrimethylammonium bromide solution, which is a denaturant for water-soluble proteins, part of the band 3 protein was still present as the oligomer. The results suggest that the oligomeric form of band 3 protein is the stable structure and that the dimer and tetramer possibly coexist in membranes.     

The cytoskeletal system of nucleated erythrocytes. II. presence of a high molecular weight calmodulin-binding protein

Calmodulin was detected in dogfish erythrocyte lysates by means of phosphodiesterase activation. Anucleate dogfish erythrocyte cytoskeletons bound calmodulin. Binding of calmodulin was calcium- dependent, concentration-dependent, and saturable. Cytoskeletons consisted of a marginal band of microtubules containing primarily tubulin, and trans-marginal band material containing actin and spectrinlike proteins. Dogfish erythrocyte ghosts and cytoskeletons were found to contain a calcium-dependent calmodulin-binding protein, CBP, by two independent techniques: (a) 125I-calmodulin binding to cytoskeletal proteins separated by SDS PAGE, and (b) in situ azidocalmodulin binding in whole anucleate ghosts and cytoskeletons. CBP, with an apparent molecular weight of 245,000, co-migrated with the upper band of human and dogfish erythrocyte spectrin. CBP was present in anucleate ghosts devoid of marginal bands and absent from isolated marginal bands. CBP therefore appears to be localized in the trans- marginal band material and not in the marginal band. Similarities between CBP and high molecular weight calmodulin-binding proteins from mammalian species are discussed.     

Potential of 13C and 15N labeling for studying protein-protein interactions using Fourier transform infrared spectroscopy.

In this study, we examine the interaction between two bacterial proteins, namely HPr and IIAmtl of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system, using FTIR spectroscopy. In an interaction involving a 1:1 molar ratio of these two proteins, when they are unlabeled, the overlap of absorbance of the amide I band arising from the peptide group vibrations of the two proteins is such that it is not possible to determine the contribution which each protein makes to the absorbance. Uniform 15N labeling has little effect on the frequency of the amide I band although there is a significant shift of the amide II band. However, we show that uniform (90%) 13C labeling produces a large shift of bands associated with the carbonyl moiety, especially the amide I band. This opens up windows in different regions of the infrared spectrum. Thus, when the same mixture of the two bacterial proteins is made where one of the proteins is uniformly 13C-labeled (in our case HPr), the amide I maxima of this protein shifts by approximately 45 cm-1 toward lower frequency and reveals the previously overlapped amide I band of the unlabeled IIAmtl. This application of 13C labeling shows the potential of studying protein-protein interactions using FTIR spectroscopy. With thoughtful selection of systems and labeling strategies, numerous studies with proteins should be possible. These could include, among others, enzyme-substrate and protein-ligand interactions.     

Differential phosphorylation of band 3 and glycophorin in intact and extracted erythrocyte membranes

This report presents an analysis of the phosphorylation of human and rabbit erythrocyte membrane proteins which migrate in NaDodSO₄-polyacrylamide gels in the area of the Coomassie Blue-stained proteins generally known as band 3. The phosphorylation of these proteins is of interest as band 3 has been implicated in transport processes. This study shows that there are at least three distinct phosphoproteins associated with the band 3 region of human erythrocyte membranes. These are band 2.9, the major band 3, and PAS-1. The phosphorylation of these proteins is differentially catalyzed by solubilized membrane and cytoplasmic cyclic AMP-dependent and -independent erythrocyte protein kinases. Band 2.9 is present and phosphorylated in unfractionated human and rabbit erythrocyte ghosts but not in NaI- or dimethylmaleic anhydride (DMMA)-extracted membranes. These latter membrane preparations are enriched in band 3 and in sialoglycoproteins. The NaI-extracted ghosts contain residual protein kinase activity which can catalyze the autophosphorylation of band 3 whereas the DMMA-extracted ghosts are usually devoid of any kinase activity. However, both NaI- and DMMA-extracted ghosts, as well as Triton X-100 extracts of the DMMA-extracted ghosts, can be phosphorylated by various erythrocyte protein kinases. The kinases which preferentially phosphorylate the major band 3 protein are inactive towards PAS-1 while the kinases active towards PAS-1 are less active towards band 3. The band 3 protein in the DMMA-extracted ghosts can be cross-linked with the Cu²⁺ -σ-phenanthroline complex. The cross-linking of band 3 does not affect its capacity to serve as a phosphoryl acceptor nor does phosphorylation affect the capacity of band 3 to form cross-links. In addition to band 2.9, the major band 3 and PAS-1, another minor protein component appears to be present in the band 3 region in human erythrocyte membranes. This protein is specifically phosphorylated by the cyclic AMP-dependent protein kinases isolated from the cytoplasm of rabbit erythrocytes. The rabbit erythrocyte membranes lack PAS-1 and the cyclic AMP-dependent protein kinase substrate.     

Identification of a high-affinity binding protein for a hepta-beta-glucoside phytoalexin elicitor in soybean.

A putative receptor protein for a hepta-beta-glucoside phytoalexin elicitor was identified by photoaffinity labeling of detergent-solubilized proteins from soybean root membranes. Incubation of partially purified beta-glucan-binding proteins with a photolabile 125I-labeled 2-(4-azidophenyl)ethyl-amino conjugate of the heptaglucoside elicitor, followed by irradiation with ultraviolet light (366 nm) resulted in specific labeling of a 70-kDa band in SDS/PAGE. Half-maximal inhibition of the 125I-labeling of the protein band by underivatized hepta-beta-glucoside was achieved by 15 nM heptaglucoside. Analysis of the affinity of radiolabel incorporation into the protein by ligand-saturation experiments, gave an apparent Kd value of 3 nM, in full agreement with the results from radioligand-binding studies. Good correlation was also observed between the amount of radiolabel incorporated into the protein and the binding activity of the fractions obtained at different stages in the purification of heptaglucoside-binding activity. Photoaffinity labeling of proteins purified by glucan-affinity chromatography showed the 70-kDa band as the main component along with weak 125I-labeling of a 100-kDa band. The 70-kDa band was also the major protein visualized by silver staining after SDS/PAGE of this fraction, suggesting that it is the predominant form of the heptaglucoside-binding proteins in detergent-solubilized soybean membranes.     

Mercuric and cadmium ions stimulate phosphorylation of band 4.2 protein on human erythrocyte membranes

In this study, we found that Hg2+ and Cd2+ enhanced the phosphorylation of human erythrocyte membranous proteins, especially band 4.2 protein, which was hardly phosphorylated in the absence of the metal ions. p-Chloromercuribenzenesulfonate and p-chloromercuribenzoate had effects similar to those of Hg2+ and Cd2+ on band 4.2 protein phosphorylation, while other metal ions and sulfhydryl agents, such as N-ethylmaleimide, 5,5'-dithiobis-(2-nitrobenzoic acid), or iodoacetate, did not. The Hg2+-stimulated phosphorylation of band 4.2 protein required a millimolar concentration of Mg2+, and it was inhibited by Ca2+ dose-dependently. Phosphoserine was identified from a hydrolysate of the phosphorylated band 4.2 protein by high-voltage electrophoresis. A specific protein inhibitor against cAMP-dependent protein kinase decreased the Hg2+-stimulated phosphorylation of band 4.2 protein. This protein had more binding sites for 203Hg2+ than any other membrane proteins. A spectrin complex from the Hg2+-treated membranes contained the band 4.2 protein, which was not detected in the complex from untreated membranes. Furthermore, protein kinase, which could phosphorylate the band 4.2 protein, was also contained in the cytoskeletal fraction from the Hg2+-treated membranes. These results suggest that Hg2+ may bind certain sulfhydryl groups of band 4.2 and other proteins to make band 4.2 protein susceptible to the endogenous cAMP-dependent protein kinase.     

Alterations in iodinated cell surface proteins during myogenesis

Lactoperoxidase catalysed iodination was used to label surface proteins of chick embryo muscle cells during myogenesis. Both quantitative and qualitative changes were observed between 125I-labelled surface proteins of pre-fusion, mid-fusion, and post-fusion cells. Significantly, two bands at 245,000 molecular weight were present at pre-fusion but were observed as a single band at mid- and post-fusion. Radioactivity in this band increased selectively at post-fusion with a concomitant increase in lower molecular weight labelled proteins.     

Band 3 and ankyrin homologues are present in eye lens: evidence for all major erythrocyte membrane components in same non-erythroid cell

Although immunological homologues of erythrocyte membrane proteins have been individually discovered in a wide variety of tissues and cultured cells, the major structural components of the membrane have not yet been demonstrated simultaneously in the same cell type. Thus, considerable uncertainty continues to exist concerning whether the red cell homologues form elements of a structure which is similar to or unique from the framework which supports the erythrocyte membrane. Because the red cell cytoskeletal proteins, spectrin, actin and band 4.1, have been previously found in the superficial cortex of the lens, we decided to determine whether the corresponding membrane anchoring components of band 3 and ankyrin also occur in this cell type. Using antiserum specific for band 3 and ankyrin, we report the existence of immunologically cross-reactive proteins of similar molecular weight. Because these anchoring proteins appear and disappear coordinately with the aforementioned cytoskeletal proteins during the intermediate stages of lens cell maturation, it is conceivable that an erythrocyte-like membrane structural organization may occur transiently in the eye lens.     

HISTOCHEMISTRY OF MYELIN–XV. CHANGES IN THE MYELIN PROTEINS OF THE PERIPHERAL NERVE UNDERGOING WALLERIAN DEGENERATION–ELECTROPHORETIC AND MICRODENSITOMETRIC OBSERVATIONS

The chronological order of changes in rat peripheral nerve proteins during Wallerian degeneration has been investigated by microdensitometric and electrophoretic techniques. Both methods revealed an early loss of myelin proteins. The histochemical microdensitometric study showed a very substantial early loss of stainable protein basic groups and a somewhat slower progressive loss of the major protein component of peripheral nerve myelin (the J band). The electrophoretic study showed an early loss of both the J band protein and the slower-moving basic protein band. The histochemical study also suggested that some cerebroside may be lost in the early stage of Wallerian degeneration. It is concluded that degradation of myelin proteins is an initial event in the process of myelin breakdown.