Easily reversible desthiobiotin binding to streptavidin, avidin, and other biotin-binding proteins: uses for protein labeling, detection, and isolation.
AuthorsHirsch JD, Eslamizar L, Filanoski BJ, Malekzadeh N, Haugland RP, Beechem JM, Haugland RP
JournalAnal Biochem
PubMed ID12419349
'The high-affinity binding of biotin to avidin, streptavidin, and related proteins has been exploited for decades. However, a disadvantage of the biotin/biotin-binding protein interaction is that it is essentially irreversible under physiological conditions. Desthiobiotin is a biotin analogue that binds less tightly to biotin-binding proteins and is easily displaced by ... More
Identification of proteins containing cysteine residues that are sensitive to oxidation by hydrogen peroxide at neutral pH.
AuthorsKim JR, Yoon HW, Kwon KS, Lee SR, Rhee SG
JournalAnal Biochem
PubMed ID10906242
'A procedure for detecting proteins that contain H(2)O(2)-sensitive cysteine (or selenocysteine) residues was developed as a means with which to study protein oxidation by H(2)O(2) in cells. The procedure is based on the facts that H(2)O(2) and biotin-conjugated iodoacetamide (BIAM) selectively and competitively react with cysteine residues that exhibit a ... More
Presenilin-1 affects trafficking and processing of betaAPP and is targeted in a complex with nicastrin to the plasma membrane.
AuthorsKaether C, Lammich S, Edbauer D, Ertl M, Rietdorf J, Capell A, Steiner H, Haass C
JournalJ Cell Biol
PubMed ID12147673
'Amyloid beta-peptide (Abeta) is generated by the consecutive cleavages of beta- and gamma-secretase. The intramembraneous gamma-secretase cleavage critically depends on the activity of presenilins (PS1 and PS2). Although there is evidence that PSs are aspartyl proteases with gamma-secretase activity, it remains controversial whether their subcellular localization overlaps with the cellular ... More
Cytoplasmic dynein regulation by subunit heterogeneity and its role in apical transport.
AuthorsTai AW, Chuang JZ, Sung CH
JournalJ Cell Biol
PubMed ID11425878
'Despite the existence of multiple subunit isoforms for the microtubule motor cytoplasmic dynein, it has not yet been directly shown that dynein complexes with different compositions exhibit different properties. The 14-kD dynein light chain Tctex-1, but not its homologue RP3, binds directly to rhodopsin''s cytoplasmic COOH-terminal tail, which encodes an ... More
Evidence for allosteric linkage between exosites 1 and 2 of thrombin.
AuthorsFredenburgh JC, Stafford AR, Weitz JI
JournalJ Biol Chem
PubMed ID9325262
'Investigations to date have demonstrated that ligand binding to exosites 1 or 2 on thrombin produces conformational changes at the active site. In this study, we directly compared the effect of ligand binding to exosites 1 and 2 on the structure and function of the active site of thrombin and ... More
The Na+/Ca2+ exchanger NCX1 has oppositely oriented reentrant loop domains that contain conserved aspartic acids whose mutation alters its apparent Ca2+ affinity.
AuthorsIwamoto T, Uehara A, Imanaga I, Shigekawa M
JournalJ Biol Chem
PubMed ID10967097
'We examined the membrane topology and functional importance of residues in regions of the Na(+)/Ca(2+) exchanger NCX1 encompassing the conserved internal alpha repeats by substituted cysteine scanning analysis and kinetic analysis of site-directed mutants. The results suggest that both the alpha-1 repeat and a region encompassing the alpha-2 repeat and ... More
Chicken erythroid AE1 anion exchangers associate with the cytoskeleton during recycling to the Golgi.
AuthorsGhosh S, Cox KH, Cox JV
JournalMol Biol Cell
PubMed ID9950688
'Chicken erythroid AE1 anion exchangers receive endoglycosidase F (endo F)-sensitive sugar modifications in their initial transit through the secretory pathway. After delivery to the plasma membrane, anion exchangers are internalized and recycled to the Golgi where they acquire additional N-linked modifications that are resistant to endo F. During recycling, some ... More
Importin 13: a novel mediator of nuclear import and export.
AuthorsMingot JM, Kostka S, Kraft R, Hartmann E, Görlich D
JournalEMBO J
PubMed ID11447110
'Importin beta-related receptors mediate translocation through nuclear pore complexes. Co-operation with the RanGTPase system allows them to bind and subsequently release their substrates on opposite sides of the nuclear envelope, which in turn ensures a directed nucleocytoplasmic transport. Here we identify a novel family member from higher eukaryotes that functions ... More
Selective fluorescent labeling of S-nitrosothiols (S-FLOS): a novel method for studying S-nitrosation.
AuthorsSanthanam L, Gucek M, Brown TR, Mansharamani M, Ryoo S, Lemmon CA, Romer L, Shoukas AA, Berkowitz DE, Cole RN,
JournalNitric Oxide
PubMed ID18706513
'Protein S-nitrosation is a reversible post-translation modification critical for redox-sensitive cell signaling that is typically studied using the Biotin Switch method. This method and subsequent modifications usually require avidin binding or Western blot analysis to detect biotin labeled proteins. We describe here a modification of the Biotin Switch assay that ... More
Agrin-induced phosphorylation of the acetylcholine receptor regulates cytoskeletal anchoring and clustering.
AuthorsBorges LS, Ferns M
JournalJ Cell Biol
PubMed ID11285269
'At the developing neuromuscular junction, a motoneuron-derived factor called agrin signals through the muscle-specific kinase receptor to induce postsynaptic aggregation of the acetylcholine receptor (AChR). The agrin signaling pathway involves tyrosine phosphorylation of the AChR beta subunit, and we have tested its role in receptor localization by expressing tagged, tyrosine-minus ... More
Analysis of phosphoinositide-binding proteins using liposomes as an affinity matrix.
AuthorsKnödler A, Mayinger P,
JournalBiotechniques
PubMed ID16018544
'Here we describe a novel affinity binding protocol that uses phosphoinositide-containing liposomes as a matrix for protein binding. Different from other liposome-based assays, this method allows affinity purification of lipid-binding proteins from crude subcellular fractions.' ... More
Purification and characterization of membrane proteins.
AuthorsKraehenbuhl JP, Bonnard C
JournalMethods Enzymol
PubMed ID2167430
Physical proximity and functional association of glycoprotein 1balpha and protein-disulfide isomerase on the platelet plasma membrane.
'Platelet function is influenced by the platelet thiol-disulfide balance. Platelet activation resulted in 440% increase in surface protein thiol groups. Two proteins that presented free thiol(s) on the activated platelet surface were protein-disulfide isomerase (PDI) and glycoprotein 1balpha (GP1balpha). PDI contains two active site dithiols/disulfides. The active sites of 26% ... More
EliCell: a gel-phase dual antibody capture and detection assay to measure cytokine release from eosinophils.
AuthorsBandeira-Melo C, Gillard G, Ghiran I, Weller PF
JournalJ Immunol Methods
PubMed ID11033023
'Eosinophils contain many preformed cytokines and chemokines, which are stored in specific granules along with cationic granule proteins. Mobilization and release of these granule contents can be selective and mediated by vesicular transport. We have developed a sensitive method to detect and quantitate eosinophil vesicular transport-mediated release of specific eosinophil ... More
A solid-phase technique for preparation of no-carrier-added technetium-99m radiopharmaceuticals: application to the streptavidin/biotin system.
AuthorsDunn-Dufault R, Pollak A, Fitzgerald J, Thornback JR, Ballinger JR
JournalNucl Med Biol
PubMed ID11150714
'A high effective specific activity (HESA) formulation of a biotin-containing (99m)Tc ligand [RP488: dimethyl-Gly-Ser-Cys(Acm)-Lys(Biotin)-Gly] conveniently prepared from solid phase was compared to a typical low effective specific activity (LESA) solution formulation to demonstrate improved targeting to streptavidin in an in vitro assay and in an in vivo rat model. RP488 ... More
Iminobiotin affinity columns and their application to retrieval of streptavidin.
AuthorsHofmann K, Wood SW, Brinton CC, Montibeller JA, Finn FM
JournalProc Natl Acad Sci U S A
PubMed ID6933515
'A method is described for the retrieval of streptavidin from the culture broth of Streptomyces avidinii. The key step in this procedure is the adsorption of streptavidin from culture concentrates to an affinity column in which iminobiotin is attached to AH-Sepharose 4B. This column binds streptavbidin at pH 11 and ... More
The quantitation of biotinylated compounds by a solid-phase assay using a 125I-labelled biotin derivative.
AuthorsSmith PJ, Warren RM, von Holt C
JournalFEBS Lett
PubMed ID3582656
The biotin analogue biotinylglycyltyrosine has been synthesized and labelled to a specific activity of 2000 Ci/mmol with 125I. This analogue has been used in conjunction with immobilized streptavidin in an assay which detects as little as 1 fmol biotin or biotinylated molecules in solution. The determination of biotinylated insulin in ... More
High-efficiency labeling of sialylated glycoproteins on living cells.
AuthorsZeng Y, Ramya TN, Dirksen A, Dawson PE, Paulson JC,
JournalNat Methods
PubMed ID19234450
We describe a simple method for efficiently labeling cell-surface sialic acid-containing glycans on living animal cells. The method uses mild periodate oxidation to generate an aldehyde on sialic acids, followed by aniline-catalyzed oxime ligation with a suitable tag. Aniline catalysis dramatically accelerates oxime ligation, allowing use of low concentrations of ... More
Preparation of oligonucleotide-biotin conjugates with cleavable linkers.
AuthorsSoukup GA, Cerny RL, Maher LJ
JournalBioconjug Chem
PubMed ID7711100
A procedure is presented for preparing an oligonucleotide-biotin conjugate that is chemically cleavable through the reduction of a disulfide bond within the linker. Conjugation involves reaction of a primary amine with an N-hydroxysulfosuccinimide ester linked to biotin. The oligonucleotide can be liberated from streptavidin agarose containing immobilized conjugate under mild ... More
alpha-Actinin interacts with rapsyn in agrin-stimulated AChR clustering.
AuthorsDobbins GC, Luo S, Yang Z, Xiong WC, Mei L,
JournalMol Brain
PubMed ID19055765
: AChR is concentrated at the postjunctional membrane at the neuromuscular junction. However, the underlying mechanism is unclear. We show that alpha-actinin, a protein known to cross-link F-actin, interacts with rapsyn, a scaffold protein essential for neuromuscular junction formation. alpha-Actinin, rapsyn, and surface AChR form a ternary complex. Moreover, the ... More
An ent-kaurene that inhibits mitotic chromosome movement and binds the kinetochore protein ran-binding protein 2.
AuthorsRundle NT, Nelson J, Flory MR, Joseph J, Th'ng J, Aebersold R, Dasso M, Andersen RJ, Roberge M
JournalACS Chem Biol
PubMed ID17168522
Using a chemical genetics screen, we have identified ent-15-oxokaurenoic acid (EKA) as a chemical that causes prolonged mitotic arrest at a stage resembling prometaphase. EKA inhibits the association of the mitotic motor protein centromeric protein E with kinetochores and inhibits chromosome movement. Unlike most antimitotic agents, EKA does not inhibit ... More
Detection of protein S-nitrosylation with the biotin-switch technique.
AuthorsForrester MT, Foster MW, Benhar M, Stamler JS,
JournalFree Radic Biol Med
PubMed ID18977293
Protein S-nitrosylation, the posttranslational modification of cysteine thiols to form S-nitrosothiols, is a principle mechanism of nitric oxide-based signaling. Studies have demonstrated myriad roles for S-nitrosylation in organisms from bacteria to humans, and recent efforts have greatly advanced our scientific understanding of how this redox-based modification is dynamically regulated during ... More
Application of avidin-biotin technology to affinity-based separations.
AuthorsBayer EA, Wilchek M
JournalJ Chromatogr
PubMed ID2205618
During the last decade, avidin-biotin technology has become a commercially viable tool for research, medical and industrial applications. From the beginning, mediation via the avidin-biotin complex was proposed for affinity-based separations. This particular application, however, has been slow in gaining acceptance. One of the reasons is that the strength of ... More
Superoxide dismutase 1 regulates caspase-1 and endotoxic shock.
AuthorsMeissner F, Molawi K, Zychlinsky A,
JournalNat Immunol
PubMed ID18604212
Caspase-1 serves an essential function in the initiation of inflammation by proteolytically maturing the cytokines interleukin 1 beta and interleukin 18. Several Nod-like receptors activate caspase-1 in response to microbial and 'danger' signals by assembling cytosolic protein complexes called 'inflammasomes'. We show here that superoxide dismutase 1 (SOD1) regulates caspase-1 ... More
A functional RNAi screen links O-GlcNAc modification of ribosomal proteins to stress granule and processing body assembly.
AuthorsOhn T, Kedersha N, Hickman T, Tisdale S, Anderson P,
JournalNat Cell Biol
PubMed ID18794846
Stress granules (SGs) and processing bodies (PBs) are microscopically visible ribonucleoprotein granules that cooperatively regulate the translation and decay of messenger RNA. Using an RNA-mediated interference-based screen, we identify 101 human genes required for SG assembly, 39 genes required for PB assembly, and 31 genes required for coordinate SG and ... More
S-glutathionylation of the Rpn2 regulatory subunit inhibits 26 S proteasomal function.
AuthorsZmijewski JW, Banerjee S, Abraham E,
JournalJ Biol Chem
PubMed ID19549781
Although increased intracellular concentrations of hydrogen peroxide (H2O2) are associated with inhibition of 26 S proteasomal activity, the mechanisms responsible for such effects have not been well delineated. In the present studies, we found that direct exposure of purified 26 S proteasomes to H2O2 had negligible effects on their activity, ... More
Approach to profile proteins that recognize post-translationally modified histone
AuthorsLi X, Kapoor TM,
JournalJ Am Chem Soc
PubMed ID20141135
Post-translational modifications (PTMs) of histones, proteins onto which DNA is packaged, are involved in many biological processes, including transcription, recombination, and chromosome segregation. As these PTMs can be dynamic, combinatorial, and mediators of weak interactions, the comprehensive profiling of all proteins that recognize histone PTMs is a daunting task. Here ... More
Identification of a motif in the acetylcholine receptor beta subunit whose phosphorylation regulates rapsyn association and postsynaptic receptor localization.
At the neuromuscular junction, the acetylcholine receptor (AChR) is specifically clustered in the postsynaptic membrane via interactions with rapsyn and other scaffolding proteins. However, it remains unclear where these proteins bind on the AChR and how the interactions are regulated. Here, we define a phosphorylation-dependent binding site on the receptor ... More
Selective enrichment of thiophosphorylated polypeptides as a tool for the analysis of protein phosphorylation.
AuthorsKwon SW, Kim SC, Jaunbergs J, Falck JR, Zhao Y,
JournalMol Cell Proteomics
PubMed ID12734387
A chemoselective alkylation method is described for the isolation and subsequent identification of thiophosphorylated peptides/proteins. The method involves thiophosphorylation of proteins using adenosine 5'-O-(thiotriphosphate) (ATPgammaS) followed by selective in situ alkylation of the newly thiophosphorylated proteins resulting in a stable covalent bond. The chemoselective alkylation exploits the relatively high nucleophilicity ... More
Regulation of calcium/calmodulin-dependent kinase IV by O-GlcNAc modification.
AuthorsDias WB, Cheung WD, Wang Z, Hart GW,
JournalJ Biol Chem
PubMed ID19506079
Similar to phosphorylation, GlcNAcylation (the addition of O-GlcNAc to Ser(Thr) residues on polypeptides) is an abundant, dynamic, and inducible post-translational modification. GlcNAcylated proteins are crucial in regulating virtually all cellular processes, including signaling, cell cycle, and transcription. Here we show that calcium/calmodulin-dependent kinase IV (CaMKIV) is highly GlcNAcylated in vivo. ... More
Acute regulation of Na+/H+ exchanger NHE3 by parathyroid hormone via NHE3 phosphorylation and dynamin-dependent endocytosis.
AuthorsCollazo R, Fan L, Hu MC, Zhao H, Wiederkehr MR, Moe OW
JournalJ Biol Chem
PubMed ID10866993
Parathyroid hormone (PTH) is a potent inhibitor of mammalian renal proximal tubule Na(+) transport via its action on the apical membrane Na(+)/H(+) exchanger NHE3. In the opossum kidney cell line, inhibition of NHE3 activity was detected from 5 to 45 min after PTH addition. Increase in NHE3 phosphorylation on multiple ... More
Concentration of MHC class II molecules in lipid rafts facilitates antigen presentation.
AuthorsAnderson HA, Hiltbold EM, Roche PA
JournalNat Immunol
PubMed ID11248809
The plasma membranes of eukaryotic cells are not uniform and possess distinct cholesterol- and sphingolipid-rich raft microdomains that are enriched in proteins known to be essential for cellular function. Lipid raft microdomains are important for T cell receptor (TCR)-mediated activation of T cells. However, the importance of lipid rafts on ... More
Identification of a 40-kDa cell surface sialoglycoprotein with the characteristics of a major influenza C virus receptor in a Madin-Darby canine kidney cell line.
AuthorsZimmer G, Klenk HD, Herrler G
JournalJ Biol Chem
PubMed ID7629082
Infection of cells by influenza C virus is known to be initiated by virus attachment to cell surface glycoconjugates containing N-acetyl-9-O-acetylneuraminic acid. Using an in vitro virus binding assay, we have detected this carbohydrate on several glycoproteins of Madin-Darby canine kidney cells (type I), a polarized epithelial cell line permissive ... More
Regulation of membrane type-1 matrix metalloproteinase activation by proprotein convertases.
AuthorsYana I, Weiss SJ
JournalMol Biol Cell
PubMed ID10888676
Membrane type-1 matrix metalloproteinase (MT1-MMP) is the prototypical member of a subgroup of membrane-anchored proteinases that belong to the matrix metalloproteinase family. Although synthesized as a zymogen, MT1-MMP plays an essential role in extracellular matrix remodeling after an undefined process that unmasks its catalytic domain. We now report the existence ... More
Contact-dependent acquisition of transferrin-bound iron by two strains of Haemophilus parasuis.
AuthorsCharland N, D'Silva CG, Dumont RA, Niven DF
JournalCan J Microbiol
PubMed ID7728656
Two strains of Haemophilus parasuis, namely, the type strain (ATCC 19417) and strain E751, were investigated with respect to iron acquisition. Both strains produced iron-repressible outer membrane proteins and could acquire iron from porcine transferrin but not from porcine lactoferrin. Neither strain used bovine transferrin, and human transferrin was used ... More
Use of biotin-labeled nucleic acids for protein purification and agarose-based chemiluminescent electromobility shift assays.
AuthorsRodgers JT, Patel P, Hennes JL, Bolognia SL, Mascotti DP
JournalAnal Biochem
PubMed ID10625515
We have employed biotin-labeled RNA to serve two functions. In one, the biotin tethers the RNA to streptavidin-agarose beads, creating an affinity resin for protein purification. In the other, the biotin functions as a label for use in a modified chemiluminescent electromobility shift assay (EMSA), a technique used to detect ... More
Erbin is a protein concentrated at postsynaptic membranes that interacts with PSD-95.
AuthorsHuang YZ, Wang Q, Xiong WC, Mei L
JournalJ Biol Chem
PubMed ID11279080
Neuregulin is a factor essential for synapse-specific transcription of acetylcholine receptor genes at the neuromuscular junction. Its receptors, ErbB receptor tyrosine kinases, are localized at the postjunctional membrane presumably to ensure localized signaling. However, the molecular mechanisms underlying synaptic localization of ErbBs are unknown. Our recent studies indicate that ErbB4 ... More
Affinity chromatography of DNA labeled with chemically cleavable biotinylated nucleotide analogs.
AuthorsHerman TM, Lefever E, Shimkus M
JournalAnal Biochem
PubMed ID3740417
DNA labeled with the chemically cleavable biotinylated nucleotide Bio-12-SS-dUTP was chromatographed on biotin cellulose affinity columns using either avidin or streptavidin as the affinity reagent. Although both proteins were equally effective in binding the Bio-12-SS-DNA to the affinity resin, two important differences were found. First, nonbiotinylated DNA bound to avidin, ... More
Proteolytic cleavage of the ectodomain of the L1 CAM family member Tractin.
AuthorsXu YZ, Ji Y, Zipser B, Jellies J, Johansen KM, Johansen J
JournalJ Biol Chem
PubMed ID12446717
Tractin is a member of the L1 family of cell adhesion molecules in leech. Immunoblot analysis suggests that Tractin is constitutively cleaved in vivo at a proteolytic site with the sequence RKRRSR. This sequence conforms to the consensus sequence for cleavage by members of the furin family of convertases, and ... More
Histamine treatment induces rearrangements of orthogonal arrays of particles (OAPs) in human AQP4-expressing gastric cells.
AuthorsCarmosino M, Procino G, Nicchia GP, Mannucci R, Verbavatz JM, Gobin R, Svelto M, Valenti G
JournalJ Cell Biol
PubMed ID11564760
To test the involvement of the water channel aquaporin (AQP)-4 in gastric acid physiology, the human gastric cell line (HGT)-1 was stably transfected with rat AQP4. AQP4 was immunolocalized to the basolateral membrane of transfected HGT-1 cells, like in native parietal cells. Expression of AQP4 in transfected cells increased the ... More
Activation and functional characterization of the mosaic receptor SorLA/LR11.
We previously isolated and sequenced the approximately 250-kDa type 1 receptor sorLA/LR11, a mosaic protein with elements characterizing the Vps10p domain receptor family as well as the low density lipoprotein receptor family. The N terminus of the Vps10p domain comprises a consensus sequence for cleavage by furin ((50)RRKR(53)) that precedes ... More
Expression of macrophage MARCO receptor induces formation of dendritic plasma membrane processes.
AuthorsPikkarainen T, Brännström A, Tryggvason K
JournalJ Biol Chem
PubMed ID10196178
MARCO is a novel macrophage-specific receptor structurally related to macrophage class A scavenger receptors. It is constitutively expressed in macrophages of the marginal zone of the spleen and in lymph nodes and is up-regulated in other tissues during systemic bacterial infections. In this study, we show that ectopic expression of ... More
Role for the third intracellular loop in cell surface stabilization of the alpha2A-adrenergic receptor.
AuthorsEdwards SW, Limbird LE
JournalJ Biol Chem
PubMed ID10347190
Previous studies have shown that alpha2A-adrenergic receptor (alpha2A-AR) retention at the basolateral surface of polarized MDCKII cells involves its third intracellular (3i loop). The present studies examining mutant alpha2A-ARs possessing short deletions of the 3i loop indicate that no single region can completely account for the accelerated surface turnover of ... More
A dileucine motif targets E-cadherin to the basolateral cell surface in Madin-Darby canine kidney and LLC-PK1 epithelial cells.
E-cadherin is a major adherens junction protein of epithelial cells, with a central role in cell-cell adhesion and cell polarity. Newly synthesized E-cadherin is targeted to the basolateral cell surface. We analyzed targeting information in the cytoplasmic tail of E-cadherin by utilizing chimeras of E-cadherin fused to the ectodomain of ... More
Nox1-dependent reactive oxygen generation is regulated by Rac1.
AuthorsCheng G, Diebold BA, Hughes Y, Lambeth JD
JournalJ Biol Chem
PubMed ID16636067
Rac1 has been implicated in the generation of reactive oxygen species (ROS) in several cell types, but the enzymatic origin of the ROS has not been proven. The present studies demonstrate that Nox1, a homolog of the phagocyte NADPH-oxidase component gp91(phox), is activated by Rac1. When Nox1 is co-expressed along ... More
Comparative study of iron acquisition by biotype 1 and biotype 2 strains of Actinobacillus pleuropneumoniae.
AuthorsD'Silva CG, Archibald FS, Niven DF
JournalVet Microbiol
PubMed ID7667902
Four strains of the swine pathogen, Actinobacillus pleuropneumoniae, namely, the type strain (ATCC 27088; biotype 1), the 'reference' strain of biotype 2 (Bertschinger 2008/76), and two additional biotype 1 strains, strain BC181, which is less virulent than the type strain, and strain K17, which was isolated from a lamb, were ... More
A macrophage protein, Ym1, transiently expressed during inflammation is a novel mammalian lectin.
AuthorsChang NC, Hung SI, Hwa KY, Kato I, Chen JE, Liu CH, Chang AC
JournalJ Biol Chem
PubMed ID11297523
Oral infections of mice with Trichinella spiralis induce activation of peritoneal exudate cells to transiently express and secrete a crystallizable protein Ym1. Purification of Ym1 to homogeneity was achieved. It is a single chain polypeptide (45 kDa) with a strong tendency to crystallize at its isoelectric point (pI 5.7). Co-expression ... More
Staurosporine inhibits agrin-induced acetylcholine receptor phosphorylation and aggregation.
AuthorsWallace BG
JournalJ Cell Biol
PubMed ID7513708
Agrin, a protein that mediates nerve-induced acetylcholine receptor (AChR) aggregation at developing neuromuscular junctions, has been shown to cause an increase in phosphorylation of the beta, gamma, and delta subunits of AChRs in cultured myotubes. As a step toward understanding the mechanism of agrin-induced AChR aggregation, we examined the effects ... More
Inhibition of glucose metabolism sensitizes tumor cells to death receptor-triggered apoptosis through enhancement of death-inducing signaling complex formation and apical procaspase-8 processing.
AuthorsMuñoz-Pinedo C, Ruiz-Ruiz C, Ruiz de Almodóvar C, Palacios C, López-Rivas A
JournalJ Biol Chem
PubMed ID12556444
Tumors display a high rate of glucose uptake and glycolysis. We investigated how inhibition of glucose metabolism could affect death receptor-mediated apoptosis in human tumor cells of diverse origin. We show that both substitution of glucose for pyruvate and treatment with 2-deoxyglucose enhanced apoptosis induced by tumor necrosis factor (TNF)-alpha, ... More
A cell-free assay for glycosylphosphatidylinositol anchoring in African trypanosomes. Demonstration of a transamidation reaction mechanism.
AuthorsSharma DK, Vidugiriene J, Bangs JD, Menon AK
JournalJ Biol Chem
PubMed ID10347210
We established an in vitro assay for the addition of glycosyl-phosphatidylinositol (GPI) anchors to proteins using procyclic trypanosomes engineered to express GPI-anchored variant surface glycoprotein (VSG). The assay is based on the premise that small nucleophiles, such as hydrazine, can substitute for the GPI moiety and effect displacement of the ... More
Synaptic-like microvesicles of neuroendocrine cells originate from a novel compartment that is continuous with the plasma membrane and devoid of transferrin receptor.
AuthorsSchmidt A, Hannah MJ, Huttner WB
JournalJ Cell Biol
PubMed ID9128254
We have characterized the compartment from which synaptic-like microvesicles (SLMVs), the neuroendocrine counterpart of neuronal synaptic vesicles, originate. For this purpose we have exploited the previous observation that newly synthesized synaptophysin, a membrane marker of synaptic vesicles and SLMVs, is delivered to the latter organelles via the plasma membrane and ... More
Mutations of Arg440 and Gly455/Gly456 oppositely change pH sensing of Na+/H+ exchanger 1.
AuthorsWakabayashi S, Hisamitsu T, Pang T, Shigekawa M
JournalJ Biol Chem
PubMed ID12562776
To identify important amino acid residues involved in intracellular pH (pH(i)) sensing of Na(+)/H(+) exchanger 1, we produced single-residue substitution mutants in the region of the exchanger encompassing the putative 11th transmembrane segment (TM11) and its adjacent intracellular (intracellular loop (IL) 5) and extracellular loops (extracellular loop 6). Substitution of ... More
Effects of a sphingolipid synthesis inhibitor on membrane transport through the secretory pathway.
AuthorsRosenwald AG, Machamer CE, Pagano RE
JournalBiochemistry
PubMed ID1567816
We investigated the effects of an inhibitor of sphingolipid biosynthesis, 1-phenyl-2-(decanoyl-amino)-3-morpholino-1-propanol (PDMP), on cells in culture. Two Golgi-associated enzymes were affected by incubation of cells with PDMP. The synthesis of glucosylceramide was inhibited at low concentrations of PDMP (2.5-10 microM), and in the presence of higher concentrations (greater than or ... More
Metabolic behavior of cell surface biotinylated proteins.
AuthorsHare JF, Lee E
JournalBiochemistry
PubMed ID2713332
The turnover of proteins on the surface of cultured mammalian cells was measured by a new approach. Reactive free amino or sulfhydryl groups on surface-accessible proteins were derivatized with biotinyl reagents and the proteins solubilized from culture dishes with detergent. Solubilized, biotinylated proteins were then adsorbed onto streptavidin-agarose, released with ... More
Strategies and solid-phase formats for the analysis of protein and peptide phosphorylation employing a novel fluorescent phosphorylation sensor dye.
Protein kinases represent one of the largest families of regulatory enzymes, with more than 2,000 of them being encoded for by the human genome. Many cellular processes are regulated by the reversible phosphorylation of proteins and upwards of 30% of the proteins comprising the eukaryotic proteome are likely to be ... More
Immobilization of nicotinic acetylcholine receptors in mouse C2 myotubes by agrin-induced protein tyrosine phosphorylation.
AuthorsMeier T, Perez GM, Wallace BG
JournalJ Cell Biol
PubMed ID7593170
Agrin induces the formation of highly localized specializations on myotubes at which nicotinic acetylcholine receptors (AChRs) and many other components of the postsynaptic apparatus at the vertebrate skeletal neuromuscular junction accumulate. Agrin also induces AChR tyrosine phosphorylation. Treatments that inhibit tyrosine phosphorylation prevent AChR aggregation. To examine further the relationship ... More
The paramyxovirus simian virus 5 hemagglutinin-neuraminidase glycoprotein, but not the fusion glycoprotein, is internalized via coated pits and enters the endocytic pathway.
AuthorsLeser GP, Ector KJ, Lamb RA
JournalMol Biol Cell
PubMed ID8741847
The hemagglutinin-neuraminidase (HN) and fusion (F) glycoproteins of the paramyxovirus simian virus 5 (SV5) are expressed on the surface of virus-infected cells. Although the F protein was found to be expressed stably, the HN protein was internalized from the plasma membrane. HN protein lacks known internalization signals in its cytoplasmic ... More