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1.
Nat Commun ; 10(1): 1261, 2019 03 19.
Article in English | MEDLINE | ID: mdl-30890705

ABSTRACT

Cellular functions of arrestins are determined in part by the pattern of phosphorylation on the G protein-coupled receptors (GPCRs) to which arrestins bind. Despite high-resolution structural data of arrestins bound to phosphorylated receptor C-termini, the functional role of each phosphorylation site remains obscure. Here, we employ a library of synthetic phosphopeptide analogues of the GPCR rhodopsin C-terminus and determine the ability of these peptides to bind and activate arrestins using a variety of biochemical and biophysical methods. We further characterize how these peptides modulate the conformation of arrestin-1 by nuclear magnetic resonance (NMR). Our results indicate different functional classes of phosphorylation sites: 'key sites' required for arrestin binding and activation, an 'inhibitory site' that abrogates arrestin binding, and 'modulator sites' that influence the global conformation of arrestin. These functional motifs allow a better understanding of how different GPCR phosphorylation patterns might control how arrestin functions in the cell.


Subject(s)
Arrestin/metabolism , Phosphorylation/physiology , Rhodopsin/metabolism , beta-Arrestin 1/metabolism , beta-Arrestin 2/metabolism , Amino Acid Motifs/physiology , Animals , Arrestin/chemistry , Arrestin/genetics , Arrestin/isolation & purification , Biological Assay , Cattle , Cell Membrane/metabolism , Mutation , Nuclear Magnetic Resonance, Biomolecular , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Rhodopsin/chemistry , Rod Cell Outer Segment/metabolism , beta-Arrestin 1/chemistry , beta-Arrestin 1/isolation & purification , beta-Arrestin 2/chemistry , beta-Arrestin 2/isolation & purification
2.
Methods Cell Biol ; 142: 159-172, 2017.
Article in English | MEDLINE | ID: mdl-28964334

ABSTRACT

Protein purity and yield are two critical parameters for successful protein characterization using structural techniques such as X-ray crystallography, NMR, and several other biophysical methods. The yeast Saccharomyces cerevisiae is one of the popular eukaryotic model systems for overexpression and subsequent purification of recombinant proteins. Here, we describe a protocol for cloning, overexpression, purification, and crystallization of arrestin-1 and its splice variant p44 from yeast. The purification protocol involves a single-affinity chromatography step on a Strep-Tactin column. Highly purified arrestins can be concentrated up to 15mg/mL using ultrafiltration and can be stored in the frozen state for several months without any loss of functionality.


Subject(s)
Arrestin/chemistry , Arrestin/isolation & purification , Chromatography, Affinity/methods , Saccharomyces cerevisiae/metabolism , Arrestin/genetics , Chromatography, Affinity/instrumentation , Crystallization/methods , Protein Splicing , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Ultrafiltration
3.
Protein Expr Purif ; 82(1): 55-60, 2012 Mar.
Article in English | MEDLINE | ID: mdl-22133714

ABSTRACT

Desensitization of agonist-activated G protein-coupled receptors (GPCRs) requires phosphorylation followed by the binding of arrestin, a ~48 kDa soluble protein. While crystal structures for the inactive, 'basal' state of various arrestins are available, the conformation of 'activated' arrestin adopted upon interaction with activated GPCRs remains unknown. As a first step towards applying high-resolution structural methods to study arrestin conformation and dynamics, we have utilized the subtilisin prodomain/Profinity eXact™ fusion-tag system for the high-level bacterial expression and one-step purification of wild-type visual arrestin (arrestin 1) as well as a mutant form (R175E) of the protein that binds to non-phosphorylated, light-activated rhodopsin (Rho∗). The results show that both prodomain/Profinity eXact™ fusion-tagged wild-type and R175E arrestins can be expressed to levels approaching 2-3 mg/l in Luria-Bertani media, and that the processed, tag-free mature forms can be purified to near homogeneity using a Bio-Scale™ Mini Profinity eXact™ cartridge on the Profinia™ purification system. Functional analysis of R175E arrestin generated using this approach shows that it binds to non-phosphorylated rhodopsin in a light-dependent manner. These findings should facilitate the structure determination of this 'constitutively activated' state of arrestin 1 as well as the monitoring of conformational changes upon interaction with Rho∗.


Subject(s)
Arrestin/isolation & purification , Arrestin/metabolism , Animals , Arrestin/chemistry , Arrestin/genetics , Cattle , Cloning, Molecular , Escherichia coli/genetics , Gene Expression , Genetic Vectors , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Rhodopsin/metabolism , Subtilisin/chemistry , Subtilisin/genetics , Subtilisin/isolation & purification
4.
Arch Biochem Biophys ; 507(2): 219-31, 2011 Mar 15.
Article in English | MEDLINE | ID: mdl-21176771

ABSTRACT

A 50-kDa-polypeptide band peripherally bound to retinal rod outer segment (ROS) membranes was purified by anion-exchange chromatography. When the 50-kDa protein was compared with purified arrestin-1, it was observed that: (1) both proteins comigrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and were recognized by either anti-50-kDa protein polyclonal antibodies or anti-arrestin-1 monoclonal antibodies; (2) protein fragments and peptide fingerprint maps obtained following limited and complete proteolysis with specific proteases were very similar for both molecules; and (3) several chromatographically-purified tryptic peptides from the 50-kDa protein possessed the same amino acid composition as tryptic peptides deduced from the reported arrestin-1 primary structure. Consequently, arrestin-1 and the purified 50-kDa protein must correspond to variants of the same molecule. However, in contrast to arrestin-1 that associated to the ROS membranes only in the presence of light and ATP, the 50-kDa protein interacted with the ROS membranes in a light-independent manner, either in the presence or absence of ATP. These results clearly established that phosphorylated and illuminated rhodopsin is not the membrane anchor for this variant of arrestin-1.


Subject(s)
Arrestin/metabolism , Cell Membrane/metabolism , Light , Rod Cell Outer Segment/metabolism , Animals , Arrestin/chemistry , Arrestin/isolation & purification , Cattle , Cell Membrane/radiation effects , Molecular Weight , Phosphorylation/radiation effects , Protein Binding/radiation effects , Rod Cell Outer Segment/radiation effects , Solubility , Substrate Specificity
5.
PLoS One ; 5(12): e15249, 2010 Dec 13.
Article in English | MEDLINE | ID: mdl-21179207

ABSTRACT

BACKGROUND: Visual and ß-arrestins are scaffolding proteins involved in the regulation of receptor-dependent intracellular signaling and their trafficking. The arrestin superfamilly includes several arrestin domain-containing proteins and the structurally related protein Vps26. In Dictyostelium discoideum, the arrestin-domain containing proteins form a family of six members, namely AdcA to -F. In contrast to canonical arrestins, Dictyostelium Adc proteins show a more complex architecture, as they possess, in addition to the arrestin core, other domains, such as C2, FYVE, LIM, MIT and SAM, which potentially mediate selective interactions with either lipids or proteins. METHODOLOGY AND PRINCIPAL FINDINGS: A detailed analysis of AdcA has been performed. AdcA extends on both sides of the arrestin core, in particular by a FYVE domain which mediates selective interactions with PI(3)P, as disclosed by intrinsic fluorescence measurements and lipid overlay assays. Localization studies showed an enrichment of tagged- and endogenous AdcA on the rim of early macropinosomes and phagosomes. This vesicular distribution relies on a functional FYVE domain. Our data also show that the arrestin core binds the ADP-ribosylation factor ArfA, the unique amoebal Arf member, in its GDP-bound conformation. SIGNIFICANCE: This work describes one of the 6 arrestin domain-containing proteins of Dictyostelium, a novel and atypical member of the arrestin clan. It provides the basis for a better understanding of arrestin-related protein involvement in trafficking processes and for further studies on the expanding roles of arrestins in eukaryotes.


Subject(s)
Arrestin/chemistry , Amino Acid Sequence , Animals , Antibodies, Monoclonal/chemistry , Arrestin/isolation & purification , Dictyostelium , Endosomes/metabolism , Green Fluorescent Proteins/chemistry , Lipids/chemistry , Mice , Microscopy, Fluorescence/methods , Molecular Sequence Data , Protein Structure, Tertiary , Rabbits , Sequence Homology, Amino Acid , Subcellular Fractions
6.
Mol Pharmacol ; 75(1): 19-26, 2009 Jan.
Article in English | MEDLINE | ID: mdl-18820126

ABSTRACT

Dopamine D(2) and D(3) receptors are similar subtypes with distinct interactions with arrestins; the D(3) receptor mediates less agonist-induced translocation of arrestins than the D(2) receptor. The goals of this study were to compare nonphosphorylated arrestin-binding determinants in the second intracellular domain (IC2) of the D(2) and D(3) receptors to identify residues that contribute to the differential binding of arrestin to the subtypes. Arrestin 3 bound to glutathione transferase (GST) fusion proteins of the D(2) receptor IC2 more avidly than to the D(3) receptor IC2. Mutagenesis of the fusion proteins identified a residue at the C terminus of IC2, Lys149, that was important for the preferential binding of arrestin 3 to D(2)-IC2; arrestin binding to D(2)-IC2-K149C was greatly decreased compared with wild-type D(2)-IC2, whereas binding to the reciprocal mutant D(3)-IC2-C147K was enhanced compared with wild-type D(3)-IC2. Mutating this lysine in the full-length D(2) receptor to cysteine decreased the ability of the D(2) receptor to mediate agonist-induced arrestin 3 translocation to the membrane and decreased agonist-induced receptor internalization in human embryonic kidney 293 cells. The reciprocal mutation in the D(3) receptor increased receptor-mediated translocation of arrestin 3 without affecting agonist-induced receptor internalization. G protein-coupled receptor crystal structures suggest that Lys149, at the junction of IC2 and the fourth membrane-spanning helix, has intramolecular interactions that contribute to maintaining an inactive receptor state. It is suggested that the preferential agonist-induced binding of arrestin3 to the D(2) receptor over the D(3) receptor is due in part to Lys149, which could be exposed as a result of receptor activation.


Subject(s)
Arrestin/chemistry , Arrestin/metabolism , Receptors, Dopamine D2/metabolism , Receptors, Dopamine D3/metabolism , Amino Acid Sequence , Amino Acid Substitution , Animals , Arrestin/genetics , Arrestin/isolation & purification , Binding Sites , Biophysical Phenomena , Cell Line , Cysteine/metabolism , Glutathione Transferase/metabolism , Humans , Hydrogen Bonding , Kidney/cytology , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Rats , Receptors, Dopamine D2/chemistry , Receptors, Dopamine D2/genetics , Receptors, Dopamine D3/chemistry , Receptors, Dopamine D3/genetics , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Sequence Homology, Amino Acid
7.
Mol Pharmacol ; 75(1): 113-23, 2009 Jan.
Article in English | MEDLINE | ID: mdl-18809670

ABSTRACT

Arrestins mediate G protein-coupled receptor desensitization, internalization, and signaling. Dopamine D(2) and D(3) receptors have similar structures but distinct characteristics of interaction with arrestins. The goals of this study were to compare arrestin-binding determinants in D(2) and D(3) receptors other than phosphorylation sites and to create a D(2) receptor that is deficient in arrestin binding. We first assessed the ability of purified arrestins to bind to glutathione transferase (GST) fusion proteins containing the receptor third intracellular loops (IC3). Arrestin3 bound to IC3 of both D(2) and D(3) receptors, with the affinity and localization of the binding site indistinguishable between the receptor subtypes. Mutagenesis of the GST-IC3 fusion proteins identified an important determinant of the binding of arrestin3 in the N-terminal region of IC3. Alanine mutations of this determinant (IYIV212-215) in the full-length D(2) receptor generated a signaling-biased receptor with intact ligand binding and G-protein coupling and activation, but deficient in receptor-mediated arrestin3 translocation to the membrane, agonist-induced receptor internalization, and agonist-induced desensitization in human embryonic kidney 293 cells. This mutation also decreased arrestin-dependent activation of extracellular signal-regulated kinases. The finding that nonphosphorylated D(2)-IC3 and D(3)-IC3 have similar affinity for arrestin is consistent with previous suggestions that the differential effects of D(2) and D(3) receptor activation on membrane translocation of arrestin and receptor internalization are due, at least in part, to differential phosphorylation of the receptors. In addition, these results imply that the sequence IYIV212-215 at the N terminus of IC3 of the D(2) receptor is a key element of the arrestin binding site.


Subject(s)
Arrestin/metabolism , GTP-Binding Proteins/metabolism , Mutation , Receptors, Dopamine D2/genetics , Signal Transduction , Animals , Arrestin/isolation & purification , Cell Line , Cyclic AMP/biosynthesis , Dopamine/pharmacology , Extracellular Signal-Regulated MAP Kinases/immunology , Fluorescent Antibody Technique, Indirect , Glutathione Transferase/metabolism , Horseradish Peroxidase/immunology , Humans , Kidney/cytology , Protein Binding , Radioligand Assay , Rats , Receptors, Dopamine D2/metabolism , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Sulpiride/metabolism , Time Factors , Transfection
8.
J Neurochem ; 101(1): 223-31, 2007 Apr.
Article in English | MEDLINE | ID: mdl-17394465

ABSTRACT

Invertebrate visual signal transduction involves photoisomerization of rhodopsin, activating a guanine nucleotide binding protein (G protein) of the G(q) class, iG(q), which stimulates a phospholipase C, increasing intracellular Ca2+. Arrestin binding to photoactivated rhodopsin is a key mechanism of desensitization. We have previously reported the cloning of a retina-specific arrestin cDNA from Loligo pealei displaying 56-64% sequence similarity to other reported arrestin sequences. Here, we report the purification of the 55-kDa squid visual arrestin. Purified squid visual arrestin is able to inhibit light-activated GTPase activity dose-dependently in arrestin-depleted rhabdomeric membranes and associate with the membrane in a light-dependent manner. Membrane association can be partially inhibited by inositol 1,2,3,4,5,6-hexakisphosphate (IP6), a soluble analog of the membrane lipid phosphatidylinositol 3,4,5-triphosphate. In reconstitution assays, we demonstrate arrestin phosphorylation by squid rhodopsin kinase, a novel function among the G protein-coupled receptor kinase family. Phosphorylation of purified arrestin requires squid rhodopsin kinase, membranes, light-activation, and the presence of Ca2+. This is the first large-scale purification of an invertebrate arrestin and biochemical demonstration of arrestin function in the invertebrate visual system.


Subject(s)
Arrestin/isolation & purification , Arrestin/metabolism , Decapodiformes/metabolism , G-Protein-Coupled Receptor Kinase 1/metabolism , Photoreceptor Cells, Invertebrate/metabolism , Rhodopsin/metabolism , Animals , Arrestin/pharmacology , Cell Membrane/drug effects , Cell Membrane/metabolism , Decapodiformes/cytology , Dose-Response Relationship, Drug , GTP Phosphohydrolases/metabolism , GTP Phosphohydrolases/radiation effects , Membrane Lipids/metabolism , Phosphatidylinositol Phosphates/metabolism , Phosphorylation/drug effects , Photoreceptor Cells, Invertebrate/cytology , Vision, Ocular/drug effects , Vision, Ocular/physiology
9.
J Mol Biol ; 354(5): 1069-80, 2005 Dec 16.
Article in English | MEDLINE | ID: mdl-16289201

ABSTRACT

Arrestins play a fundamental role in the regulation and signal transduction of G protein-coupled receptors. Here we describe the crystal structure of cone arrestin at 2.3A resolution. The overall structure of cone visual arrestin is similar to the crystal structures of rod visual and the non-visual arrestin-2, consisting of two domains, each containing ten beta-sheets. However, at the tertiary structure level, there are two major differences, in particular on the concave surfaces of the two domains implicated in receptor binding and in the loop between beta-strands I and II. Functional analysis shows that cone arrestin, in sharp contrast to its rod counterpart, bound cone pigments and non-visual receptors. Conversely, non-visual arrestin-2 bound cone pigments, suggesting that it may also regulate phototransduction and/or photopigment trafficking in cone photoreceptors. These findings indicate that cone arrestin displays structural and functional features intermediate between the specialized rod arrestin and the non-visual arrestins, which have broad receptor specificity. A unique functional feature of cone arrestin was the low affinity for its cognate receptor, resulting in an unusually rapid dissociation of the complex. Transient arrestin binding to the photopigment in cones may be responsible for the extremely rapid regeneration and reuse of the photopigment that is essential for cone function at high levels of illumination.


Subject(s)
Arrestin/chemistry , Biological Evolution , Crystallography, X-Ray , Retinal Cone Photoreceptor Cells/chemistry , Alanine/metabolism , Amino Acid Sequence , Amino Acid Substitution , Animals , Anura , Arginine/chemistry , Arginine/metabolism , Arrestin/genetics , Arrestin/isolation & purification , Arrestin/metabolism , Arrestins/chemistry , Asparagine/chemistry , Cattle , Conserved Sequence , Electrophoretic Mobility Shift Assay , Escherichia coli/genetics , GTP-Binding Proteins/metabolism , Humans , Hydrogen Bonding , Hydrophobic and Hydrophilic Interactions , Models, Molecular , Molecular Sequence Data , Mutagenesis , Mutation , Phosphates/metabolism , Proline/chemistry , Protein Folding , Protein Structure, Secondary , Protein Structure, Tertiary , Retinal Cone Photoreceptor Cells/metabolism , Retinal Rod Photoreceptor Cells/chemistry , Sensitivity and Specificity , Sequence Homology, Amino Acid , Signal Transduction , Spectrum Analysis, Raman , Urodela , Valine/chemistry
10.
Invest Ophthalmol Vis Sci ; 44(7): 2858-67, 2003 Jul.
Article in English | MEDLINE | ID: mdl-12824223

ABSTRACT

PURPOSE: To elucidate the antigen recognized by monoclonal antibody (mAb) 7G6, a widely used cone-specific marker. METHODS: 7G6 immunocytochemistry was performed on sections of human, primate, and bovine retina. The antigen was immunoprecipitated from human retinal lysates and purified with protein G. Edman degradation and liquid chromatography of tryptic peptides combined with tandem mass spectrometry (LC-MS/MS) identified the antigen. RESULTS: Sequencing of peptides derived from the immunoprecipitated 7G6 antigen identified it as cone arrestin. The identity was confirmed by Western blot analysis with recombinant human cone arrestin and competition with the antibody in immunocytochemistry. Subcellular localization of cone arrestin in dark-adapted and bleached bovine retinas showed that cone arrestin accumulated in cone outer segments of light-adapted retina but was more concentrated in the inner segments of dark-adapted retina. By expression of truncated human cone arrestin mutants systematically deleting areas divergent from bovine and primate cone arrestins, the epitope of 7G6 was identified as a divergent loop exposed at the surface within the N-domain of cone arrestin. CONCLUSIONS: Several independent methods established that the 7G6 antigen is cone arrestin. The 7G6 epitope is contained in a divergent loop, the sequence of which is conserved in bovine and primates but not other vertebrate species consistent with the specificity of the antibody. The light-dependent translocation of cone arrestin suggests a role in light-dark adaptation of cones. Because of the location of its gene on the X-chromosome, cone arrestin is a candidate gene for X-linked cone dystrophies.


Subject(s)
Antibodies, Monoclonal , Arrestin/metabolism , Autoantigens/metabolism , Light , Retinal Cone Photoreceptor Cells/metabolism , Retinal Cone Photoreceptor Cells/radiation effects , Amino Acid Sequence , Animals , Antibody Specificity , Arrestin/genetics , Arrestin/isolation & purification , Autoantigens/genetics , Autoantigens/isolation & purification , Blotting, Western , Cattle , Dark Adaptation , Epitope Mapping , Fluorescent Antibody Technique, Indirect , Humans , Macaca , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Transport/radiation effects , Retinal Cone Photoreceptor Cells/cytology , Sequence Analysis, Protein , Vision, Ocular
11.
FEBS Lett ; 470(3): 336-40, 2000 Mar 31.
Article in English | MEDLINE | ID: mdl-10745092

ABSTRACT

To elucidate the quenching mechanism of phototransduction in vertebrate cone photoreceptors, a cDNA clone encoding cone specific arrestin (cArr) was isolated from a bovine retinal cDNA library using a human cArr cDNA probe. Affinity-purified anti-peptide antibody specific to cArr was prepared. Immunohistochemical staining displayed specific labeling of cArr in cone photoreceptors and immunoblotting identified a 46 kDa protein band. We purified cArr from bovine retinas by sequential column chromatography using DEAE-cellulose, gel filtration and mono Q columns. Binding studies revealed no binding of cArr to rhodopsin regardless of whether it was bleached and/or phosphorylated. cArr also failed to bind to heparin-Sepharose under conditions which rod arrestin (rArr) bound to the column. The present data suggest that cArr may play a role in the quenching of phototransduction in cone photoreceptors and that its activity therein is different to that of rArr.


Subject(s)
Arrestin/isolation & purification , Arrestin/metabolism , Retinal Cone Photoreceptor Cells/chemistry , Amino Acid Sequence , Animals , Arrestin/chemistry , Arrestin/genetics , Cattle , Chromatography, Affinity , Cloning, Molecular , Heparin/metabolism , Humans , Molecular Sequence Data , Molecular Weight , Phosphorylation , Protein Binding , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Retinal Cone Photoreceptor Cells/cytology , Rhodopsin/metabolism , Sequence Alignment
12.
Methods Enzymol ; 315: 238-51, 2000.
Article in English | MEDLINE | ID: mdl-10736706

ABSTRACT

The data collected with the techniques discussed in this chapter suggest significant differences between the active conformation(s) of the opsin/atr complex, which are reversibly formed in the dark, and the active conformation (R*) of the meta-II photoproduct. First, there is good evidence for noncovalent opsin/atr complexes with considerable activity (although covalent binding of atr is found in mutant opsins. Even more intriguing, all-trans-retinal in an amount that saturates the activity of the opsin/atr complex toward Gt does not measurably inhibit the access of 11-cis-retinal to the light-sensitive binding site during regeneration (Fig. 2C). On the other hand, forced protonation at or near Glu-134 appears to be an integral mechanism for both the meta-II and the opsin-like activities (Fig. 4). Thus, it is not inconceivable that these two activities of the receptor arise from two fundamentally different conformations, one meta-II-like and one opsin-like. They would be similar with respect to the Gt (or RK) protein-protein interaction but different in their mode of retinal-protein interaction.


Subject(s)
Eye Proteins , Retinaldehyde/metabolism , Rhodopsin/metabolism , Rod Opsins/metabolism , Animals , Arrestin/isolation & purification , Arrestin/metabolism , Cattle , Cell Membrane/chemistry , Cell Membrane/metabolism , G-Protein-Coupled Receptor Kinase 1 , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Hydrogen-Ion Concentration , Kinetics , Phosphorylation , Protein Kinases/isolation & purification , Protein Kinases/metabolism , Retina/metabolism , Rhodopsin/chemistry , Rhodopsin/isolation & purification , Rod Cell Outer Segment/chemistry , Rod Cell Outer Segment/metabolism , Rod Opsins/chemistry , Rod Opsins/isolation & purification , Spectrophotometry/methods , Transducin/isolation & purification , Transducin/metabolism
14.
Curr Eye Res ; 18(5): 327-34, 1999 May.
Article in English | MEDLINE | ID: mdl-10372993

ABSTRACT

PURPOSE: A variety of methods have been developed for the purification of S-antigen but a simple and rapid procedure based on hydroxyapatite-agarose (HA) adsorbtion is most widely used. In the present study, we investigated the nature of proteins purified with the aid of HA chromatography. METHODS: After elimination of retinal S-antigen by HA, the soluble extract of retinal tissue was readsorbed on HA. The proteins were thereafter desorbed by 10 to 500 mM phosphate buffer gradient. Two peaks obtained by SDS-PAGE were used for the generation of specific antisera for subsequent analysis by ELISA and Western blotting. RESULTS: Four proteins (two 48 kDa , one 50 kDa and one 46 kDa) were obtained in this manner. Partial amino acid sequencing permitted the identification of these proteins as alpha-enolase (48 kDa), gamma-enolase (48 kDa), Glucose-6-phosphate-Isomerase (50 kDa) and aspartate-amino-transferase (46 kDa). CONCLUSION: The selected glycolytic enzymes co-purified with retinal S-antigen by hydroxyapatite agarose chromatography of bovine retina.


Subject(s)
Arrestin/isolation & purification , Chromatography, Agarose/methods , Durapatite , Enzymes/isolation & purification , Glycolysis , Retina/chemistry , Amino Acid Sequence/genetics , Animals , Aspartate Aminotransferases/genetics , Aspartate Aminotransferases/isolation & purification , Cattle , Enzymes/genetics , Glucose-6-Phosphate Isomerase/genetics , Glucose-6-Phosphate Isomerase/isolation & purification , Glycolysis/physiology , Molecular Sequence Data , Phosphopyruvate Hydratase/genetics , Phosphopyruvate Hydratase/isolation & purification
15.
Nature ; 391(6670): 918-21, 1998 Feb 26.
Article in English | MEDLINE | ID: mdl-9495348

ABSTRACT

Retinal arrestin is the essential protein for the termination of the light response in vertebrate rod outer segments. It plays an important role in quenching the light-induced enzyme cascade by its ability to bind to phosphorylated light-activated rhodopsin (P-Rh*). Arrestins are found in various G-protein-coupled amplification cascades. Here we report on the three-dimensional structure of bovine arrestin (relative molecular mass, 45,300) at 3.3 A resolution. The crystal structure comprises two domains of antiparallel beta-sheets connected through a hinge region and one short alpha-helix on the back of the amino-terminal fold. The binding region for phosphorylated light-activated rhodopsin is located at the N-terminal domain, as indicated by the docking of the photoreceptor to the three-dimensional structure of arrestin. This agrees with the interpretation of binding studies on partially digested and mutated arrestin.


Subject(s)
Arrestin/chemistry , Rod Cell Outer Segment/chemistry , Amino Acid Sequence , Animals , Arrestin/isolation & purification , Binding Sites , Cattle , Crystallography, X-Ray , Humans , Models, Molecular , Molecular Sequence Data , Phosphorylation , Protein Conformation , Protein Structure, Secondary
16.
Ophthalmologe ; 93(6): 732-8, 1996 Dec.
Article in German | MEDLINE | ID: mdl-9081534

ABSTRACT

UNLABELLED: S-antigen (AgS) and interphotoreceptor retinoid-binding protein (IRBP) are two highly potent uveitopathogenic autoantigens of the retina frequently used to trigger autoimmune uveitis in animal trials. PURPOSE: The aim of this study was the simultaneous isolation of both proteins from bovine retina, additionally avoiding time-consuming dialysis and employing prepacked, commercially available cartridges. METHOD: Retina extract, obtained in the usual manner, was precipitated with ammonium sulfate. Both the desalting and the buffer exchange of dissolved precipitate were carried out using a Bio-Gel P6 column. Subsequent separation with Econo-Pac Q gave prepurified AgS and IRBP fractions. Further purification was realized predominantly with Econo-Pac Q, -HTP and -HIC (5-ml cartridges). RESULTS: AgS and IRBP were isolated in high purity (sodium dodecylsulfate polyacrylamide gel electrophoresis, silver stain) from bovine retina using prepacked cartridges with yields of approximately 0.25 (for AgS) and 0.15 (for IRBP) mg/g retina, respectively. DISCUSSION: The application of ready-to-use cartridges allows simultaneous isolation of AgS and IRBP in milligram amounts under simplified conditions. This approach might be of particular interest for small samples of retina.


Subject(s)
Arrestin/isolation & purification , Eye Proteins/isolation & purification , Retina/chemistry , Retinol-Binding Proteins/isolation & purification , Animals , Cattle , Tissue Extracts/chemistry
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