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PubMed1989

Release of the sigma subunit from Escherichia coli RNA polymerase transcription complexes is dependent on the promoter sequence.

The sigma subunit of bacterial RNA polymerase is required for the specific initiation of transcription at promoter sites. However, sigma is released from the transcription complex shortly after transcription is initiated, and elongation proceeds in the absence of sigma. In order to study the position of sigma release, we have developed a method to quantify the photoaffinity labeling produced by an aryl azide positioned at the leading (5'-) end of nascent RNA, as a function of the transcript length [Stackhouse, T.M., & Meares, C.F. (1988) Biochemistry 27, 3038-3045]. Here we compare photoaffinity labeling of transcription complexes containing three natural bacteriophage promoters (lambda PR, lambda PL, and T7 A1) and two recombinant constructs, A1/PR (T7 A1 promoter with the lambda PR transcribed region) and PR/A1 (lambda PR promoter with the T7 A1 transcribed region). Significant photoaffinity labeling of the sigma subunit was observed only on the templates containing the lambda PR promoter region, regardless of the sequence of the transcribed region. These results indicate the molecular interactions responsible for the position of sigma release from the transcription complex mainly involve the nucleotide sequence of the promoter region--rather than the transcribed region--of the DNA template. Further studies on transcription complexes containing the A1/PR and the PR/A1 templates were performed, using polyclonal antibodies against the holoenzyme or against the sigma subunit. These experiments corroborate the promoter dependence of sigma release. They also show a correlation between the release of sigma and stable binding of the transcript by the transcription complex.

Affinity Labels↗
PubMed1988

In situ photolabelling of the human androgen receptor.

In situ photoaffinity labelling of the human androgen receptor has been performed in the LNCaP (Lymph Node Carcinoma of the Prostate) cell line. The covalently labelled receptors were identified by SDS-PAGE. Intact LNCaP cells, incubated with [3H]-R1881 and subsequently irradiated with u.v. light and directly solubilized in SDS-buffer, revealed two photolabelled protein bands at 110 and 50 kDa. Irradiation of intact cells and subsequent isolation of nuclei followed by extraction with 0.5 M NaCl resulted in one major photolabelled protein band at 110 kDa. The labelling of this band could be completely suppressed by a 100-fold molar excess of non-radioactive R1881. Photolabelling of androgen receptors in a cytosolic preparation of LNCaP cells after anion exchange chromatography resulted in a much lower labelling efficiency compared with the in situ labelling procedure, although the androgen receptor was purified 100-fold. The steroid binding domain of the human androgen receptor has been partially mapped with chymotrypsin and S. aureus V8 protease digestion. Proteolytic digestion with chymotrypsin of purified photoaffinity-labelled 110 kDa human androgen receptor resulted in the generation of a 15 kDa peptide which still contains the covalently linked hormone. It is concluded that the in situ photoaffinity labelling technique can be applied successfully for characterization of the steroid binding domain of androgen receptors in prostate cancer cells and in other androgen target cells. Furthermore, it was demonstrated that the human androgen receptor is a monomer with a molecular mass of 110 kDa, of which the steroid binding site is confined to a 15 kDa domain.

Affinity Labels↗
PubMed1996

Determinants of substrate recognition in the protein-tyrosine phosphatase, PTP1.

Photoaffinity labeling has been used to identify amino acids involved in recognition of protein substrates by the protein-tyrosine phosphatase PTP1. The photoactive amino acid p-benzoylphenylalanine (Bpa) was incorporated into a phosphotyrosine-containing peptide derived from epidermal growth factor autophosphorylation site Tyr992 (EGFR988 998). This peptide photoinactivated PTP1 in a time- and concentration-dependent manner. Three lines of evidence indicate that the interaction between PTP1 and the photoaffinity label was specific: 1) photoinactivation was inhibited in the presence of a non-Bpa-containing peptide from EGFR Tyr992 in molar excess. 2) The photoaffinity label-containing phosphopeptide was rapidly dephosphorylated by PTP1 with kinetic constants similar to those of the non-Bpa-containing peptide under identical conditions. 3) After complete photoinactivation, the level of incorporation of radioactive photoaffinity label into PTP1 was approximately 0.9 mol of label/mol of enzyme, consistent with a 1:1 stoichiometry of photolabeling. Radiolabeled peptide was used to identify sites of cross-linking to PTP1. Bpa peptide-PTP1 was digested with trypsin, and radioactive fragments were purified by high performance liquid chromatography (HPLC) and analyzed by Edman sequencing. In two parallel experiments which were analyzed using different HPLC columns, a site in the alpha2 region of PTP1, most likely Ile23, was labeled by the Tyr992-derived peptide. The results are discussed in light of the crystal structure of human PTP1B and suggest that an additional mode of substrate recognition must exist for PTP1 catalysis.

Affinity Labels↗
PubMed1983

Topology of the lac carrier protein in the membrane of Escherichia coli.

Proteolysis of topologically sealed right-side-out and inside-out membrane vesicles from Escherichia coli with chymotrypsin, trypsin, or papain inactivates lac carrier function in a symmetrical manner. Concomitantly, the electrophoretic mobility of lac carrier protein photoaffinity labeled in situ with p-nitro[2-3H]phenyl-alpha-D-galactopyranoside is altered from a relative Mr of 33,000 to 20,000, and the time course of proteolysis is almost identical in vesicles of opposite polarities. In contrast, solubilization of the vesicles in NaDodSO4 followed by proteolysis causes fragmentation of the Mr 33,000 band into material that electrophoreses at the solvent front. Notably, proteolysis has no effect whatsoever on the ability of the lac carrier protein to bind substrate, as judged by photoaffinity-labeling experiments. Furthermore, the electrophoretic patterns of samples proteolyzed prior to photoaffinity labeling are the same as those observed when the procedures are reversed. These results show that the lac carrier protein spans the membrane and indicate that the binding site resides within a segment that is embedded in the bilayer.

Affinity Labels↗
PubMed1989

Regulation of chemoattractant receptor interaction with transducing proteins by organizational control in the plasma membrane of human neutrophils.

Isolated purified plasma membrane domains from unstimulated human neutrophils were photoaffinity labeled with F-Met-Leu-Phe-N epsilon-(2-(p-azido-[125I]salicylamido)ethyl- 1,3'-dithiopropionyl)-Lys also referred to as FMLPL-SASD[125I]. Most of the photoaffinity-labeled N-formyl peptide receptors were found in light plasma membrane fraction (PM-L) which has been previously shown to be enriched in guanyl nucleotide binding proteins and the plasma membrane marker alkaline phosphatase (Jesaitis, A. J., G. M. Bokoch, J. O. Tolley, and R. A. Allen. 1988. J. Cell Biol. 107:921-928). Furthermore, the heavy plasma membrane fraction (PM-H), which is enriched in actin and fodrin, was depleted in receptors. Solubilization of PM-L and PM-H in divalent cation-free buffer containing octylglucoside and subsequent sedimentation at 180,000 g in detergent-containing sucrose gradients revealed two receptor forms. The major population, found in PM-L sedimented as a globular protein with an apparent sedimentation coefficient of 6-7S, while a minor fraction found in the PM-H fraction sedimented as a 4S particle. In addition, the 6-7S form could be converted to the 4S form by inclusion of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) in the extraction buffer (ED50 = 10-30 nM). ATP was not effective at doses of up to 10 microM. In contrast, isolation and solubilization of receptors from desensitized cells (photoaffinity labeled after a 15 degrees C incubation with FMLPL-SASD[125I]) revealed that the majority of receptors (greater than 60-90%), which are found in PM-H, sedimented as 4S particles. A minor fraction of receptors found in the PM-L sedimented as 6-7S species. The receptors in the PM-H fraction, however, were still capable of interacting with G-proteins, since addition of unlabeled PM-L membrane fraction as a G-protein source reconstituted a more rapidly sedimenting form showing sensitivity to GTP gamma S. These results suggest that receptors in unstimulated human neutrophils have a higher probability of interacting with G-proteins because they are in the light plasma membrane domain. The results also suggest that receptors that have been translocated to the heavy plasma membrane domain during the process of desensitization or response termination have a lower probability of interacting with G-protein. Since the latter receptors are still capable of forming G protein associations, then their lateral segregation would represent a mechanism of controlling of receptor G-protein interactions. This reorganization of the plasma membrane, therefore, may form the molecular basis for response termination or homologous desensitization in human neutrophils.

Affinity Labels↗
PubMed2000

The interaction of the B-ring of colchicine with alpha-tubulin: a novel footprinting approach.

Tubulin, the major structural component of the microtubules, participates actively in mitotic spindle formation and chromosomal organization during cell division. Tubulin is the major target for a variety of anti-mitotic drugs. Some of the drugs, such as Vinca alkaloids and taxol, are routinely used for cancer chemotherapy. It is unfortunate that our knowledge of the binding sites on tubulin of these drugs is limited because of lack of a useful and appropriate tool. The photoaffinity labeling approach is the major technique available at present to detect the binding sites of drugs on tubulin. This method, however, has several limitations. First, only part of the binding site can be identified, namely, the residues which react with the photoaffinity label. Second, there are regions of tubulin which are not at the binding site but are affected by the binding of the drug; these regions can not be detected by the photoaffinity labeling approach. The third, and perhaps most serious, limitation is that the traditional approach can detect areas which have nothing to do with the binding of the ligand but which are within a certain distance of the binding site, that distance being less than the length of the photoreactive moiety attached to the ligand. There has been a great deal of controversy on the localization of the binding site of colchicine on tubulin, with some reports suggesting that the binding site is on alpha and some supporting a binding site on beta. Colchicine also has significant effects on tubulin conformation, but the regions which are affected have not been identified. We have attempted here to address these questions by a novel "footprinting" method by which the drug-binding sites and as well as the domain of tubulin affected by drug-induced conformational changes could be determined. Here, we report for the first time that the interaction of the B-ring of colchicine with the alpha-subunit affects a domain of tubulin which appears to be far from its binding site. This domain includes the cysteine residues at positions 295, 305, 315 and 316 on alpha-tubulin; these residues are located well away from the alpha/beta interface where colchicine appears to bind. This is correlated with the stabilizing effect of colchicine on the tubulin molecule. Furthermore, we also found that the B-ring of colchicine plays a major role in the stability of tubulin while the A and the C-rings have little effect on it. Our results therefore, support a model whereby colchicine binds at the alpha/beta interface of tubulin with the B-ring on the alpha-subunit and the A and the C-rings on the beta-subunit.

Amino Acid Sequence↗
PubMed1992

Isoelectric focusing and 2D electrophoresis of the human androgen receptor.

Nuclear androgen receptors from cultured genital skin fibroblasts were analyzed by non-denaturing isoelectric focusing (IEF) in ultrathin polyacrylamide gels before and after photoaffinity labeling with [3H]methyltrienolone. Both reversibly and covalently labeled receptors focused at pH 5.28 +/- 0.20 when extracted from nuclei with high salt. Lowering of the salt concentration yielded, in both cases, a second species which focused at pH 7.16. This species became predominant when nuclei were sonicated in IEF sample buffer containing no salt, even after extensive nucleic acid digestion. Low salt cytosols from both prostate and foreskin focused as a single peak of pI: 4.93 +/- 0.31 which remained unchanged when KCl was added to the cytosol up to a concentration of 0.6 M. SDS-polyacrylamide gel electrophoresis of photoaffinity labeled receptors revealed labeled proteins with Mw 90-95 kDa. Two-dimensional electrophoresis of photoaffinity labeled nuclear receptors, extracted in low or high salt, showed that the two isoforms (pI 5.28 and 7.16) contain the same steroid-binding subunit with Mw 90-95 kDa. Nuclear receptors from 4 patients with the receptor positive form of the Complete Androgen Insensitivity Syndrome (CAIS, Rc+) were analyzed by non-denaturing IEF: a single species was observed, focusing at pH 6.0 whether in high or low salt conditions. These results indicate that the nuclear androgen receptor is an acidic protein with pI 5.28 and Mw 90-95 kDa under maximum protein dissociation conditions. When extracted under low salt conditions, it can be isolated in a neutral form (pI 7.16) suggesting its association with a nuclear protein. Receptors of (CAIS, Rc+) patients have an abnormal charge and show no pI shift upon lowering of the salt concentration suggesting that this shift could be a significant step in the mechanism of action of androgens.

Autoradiography↗
PubMed1993

Nucleotide binding properties of a P-glycoprotein homologue from Plasmodium falciparum.

The Plasmodium falciparum P-glycoprotein homologue 1 (PGH1) is structurally similar to several members of the ATP-binding cassette (ABC) superfamily of membrane transporters. We have examined whether the nucleotide binding domains predicted from the deduced amino sequence are functional by photoaffinity labeling of purified parasite digestive vacuoles with the analogue 8-azido-alpha-[32P]ATP (8-N3-ATP). This reagent labels a 160-kDa protein in vacuoles from both a chloroquine sensitive and a chloroquine-resistant parasite isolate. The 160-kDa protein could be immunoprecipitated with affinity-purified antibodies against the P. falciparum P-glycoprotein homologue (PGH1). Inhibition of photoaffinity labeling of PGH1 could be achieved with ATP, ADP, GTP and GDP but not with AMP or GMP. In order to map the 8-N3-ATP binding sites on PGH1, photoaffinity-labeled PGH1 was digested with trypsin and immunoprecipitated with site-specific antibodies. Taken together, these results indicate that 8-N3-ATP specifically labels PGH1 and that one binding site resides within the amino terminal half of the molecule. This supports the contention that PGH1 is involved in a nucleotide-regulated transport function across the membrane of the digestive vacuole.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1986

GTP-binding proteins associated with serotonin-activated adenylate cyclase in Fasciola hepatica.

The liver fluke Fasciola hepatica has serotonin (5-hydroxytryptamine) receptors that function through a transmembrane signalling system requiring GTP which activates adenylate cyclase (ATP pyrophosphate-lyase (cyclising), EC 4.6.1.1). Non-hydrolysable GTP analogs and NaF activate adenylate cyclase in membrane particles of these organisms. The nature of GTP-binding proteins in these membranes was studied using bacterial toxins and photoaffinity labelling. Treatment of membrane particles from flukes with cholera toxin increased basal adenylate cyclase activity, but markedly decreased activation by serotonin, non-hydrolysable GTP analogs, and NaF. [32P]ADP-ribosylation by cholera toxin or photoaffinity labelling with [32P]-8-N3GTP identified a 53 kDa protein and a 45 kDa protein which appeared to be similar to the forms of the alpha-subunit of the GTP-binding protein associated with adenylate cyclase in mammals. Treatment of membrane particles by pertussis toxin did not significantly change basal adenylate cyclase activity and did not change the stimulation of cyclase by activators. A 43 kDa protein which was [32P]ADP-ribosylated by either cholera or pertussis toxin, depending on the conditions used, and photoaffinity labelled by [32P]-8-N3GTP may be part of the transmembrane signalling system in the liver flukes.

Adenosine Diphosphate Ribose↗
PubMed1986

Disulfides of the lutropin receptor.

Affinity cross-linking of the lutropin receptor with 125I-human choriogonadotropin (hCG) on porcine granulosa cells produced four distinct homone-receptor complexes under reducing conditions. They contain 18-, 24-, 28-, and 34-kDa components (Ji, I., Bock, J. H., and Ji, T. H. (1985) J. Biol. Chem. 260, 12815-12821). Photoaffinity labeling and cross-linking produced 136-, 102-, and 74-kDa hCG-receptor complexes under reducing conditions and the 136-kDa complex under nonreducing conditions. In addition, the unreduced 102-kDa complex was seen in photoaffinity labeling but not in cross-linking. When the unreduced 136-kDa complex was reduced, the 102- and 74-kDa complexes were generated, indicating release of the 34- and the 28-kDa components in two steps. When the unreduced 102-kDa complex was reduced, the 74-kDa complex was produced, indicating the release of a 28-kDa component. The 74-kDa complex could not be reduced but was cleaved by alkaline treatment to produce the hCG alpha beta dimer. The results indicate that the 24-kDa component is released from the 74-kDa complex, since the apparent mass of the hCG alpha beta dimer on gels is 50 kDa. The 24-kDa component appears to be the initial site for photoaffinity labeling or cross-linking and to be disulfide linked to the 28-kDa component which is in turn disulfide linked to the 34-kDa component. These intercomponent disulfides exist in some receptors but not all. Formation of the disulfide-linked 136-kDa band required the presence of a sulfhydryl-blocking agent, N-ethylmaleimide. In particular, the 34-kDa component was vulnerable to reduction. There was no significant evidence of disulfides between the hormone and any of the receptor components.

Affinity Labels↗
PubMed1989

Hormone binding site of the insulin receptor: analysis using photoaffinity-mediated avidin complexing.

A trifunctional reagent was designed which allows derivatization of ligands, particularly peptides and proteins, for subsequent photoaffinity labelling of receptors and specific isolation of the covalent complex or its fragments. B29-(2-nitro-4-azidophenyl)-biocytinyl-insulin (NB-insulin) was synthesized, radioiodinated, and the B26-mono-iodo derivative isolated by HPLC. It was used to photoaffinity label human placental membranes and the purified insulin receptor. Extensive digestion of the covalent insulin-receptor complex with trypsin (EC 3.4.21.4) led to the generation of a fragment of Mr 14,000. Specific complexing with avidin, derivatized avidin or streptavidin could be demonstrated for the photoaffinity labelled alpha-subunit and the 14,000 core fragment. The latter was isolated (approx. 100 pmol from 3-4 placentae) by streptavidin affinity chromatography and HPLC. According to microsequencing based on the known primary structure of the insulin receptor, the N-terminus of the core peptide appears to be Leu20-His21-Glu22-Leu23. We thus conclude: a part of the insulin-binding region of the receptor is located close to the N-terminus of its alpha-subunit in a remarkably stable domain of the sequence 20--(approx.) 120.

Affinity Labels↗
PubMed1996

p-Azidosalicyl-5-amino-6-phenoxybenzimidazole photolabels the N-terminal 63-103 amino acids of Haemonchus contortus beta-tubulin 1.

Benzimidazoles (BZ) are broad spectrum anthelmintics thought to exert their effects by interacting with and disrupting the functions of microtubules. However, direct biochemical evidence for binding between BZ and tubulin has not been shown nor is it known what sequences in tubulin interact with BZ. In this study, a photoactive analogue of 2-acetamido-5-(3-aminophenoxy)benzimidaz ole that has biological activity similar to other benzimidazoles was synthesized and used to photoaffinity label cell lysates from the parasitic nematode of sheep Haemonchus contortus. The photoactive analogue, 2-acetamido-5-[3-(4-azido-3-125I-salicyl amido)phenoxy]benzimida zol e or 125I-ASA-BZ, was shown to photolabel a 54-kDa protein that was specifically immunoprecipitated with anti-tubulin monoclonal antibodies. Tubulin photoaffinity labeling by 125I-ASA-BZ was also inhibited with molar excess of various BZ analogues and colchicine. Interestingly, 125I-ASA-BZ photoaffinity-labeled the beta- and not the alpha-subunits of tubulin. Proteolytic digestion of 125I-ASA-BZ-labeled tubulin with Staphylococcus aureus V8 proteinase revealed one major peptide with an apparent molecular mass of 3.5 kDa. Exhaustive digestion of 125I-ASA-BZ-labeled beta-tubulin with trypsin resulted in two fractions containing radioactive peptides. Protein sequencing of the high performance liquid chromatography-purified tryptic ASA-BZ-photolabeled peptides identified the N-terminal 63-77 and 78-103 sequences as the BZ binding domain.

Affinity Labels↗
PubMed1984

Characterization of the rat liver glucocorticoid receptor purified by DNA-cellulose and ligand affinity chromatography.

Two rapid and high yield purification methods for the rat liver glucocorticoid receptor based on differential DNA affinity (method A) and ligand affinity (method B) chromatography are described. In method A, the amount of receptor in rat liver cytosol that can be activated and subsequently eluted from a DNA-cellulose column has been increased to 80% by introducing a second heat activation step. Using this method, 1.5 nmol of 25% pure glucocorticoid receptor can be routinely obtained per day from 15-20 rat livers. Method B yields about 2.2 nmol of 60% pure receptor with an overall yield of congruent to 60%. The quality of these purifications has been controlled by affinity labeling. In each case, more than 95% of purified binding activity represented the intact 92,000 +/- 400-Da glucocorticoid receptor polypeptide as shown by sodium dodecyl sulfate-gel electrophoresis and fluorography. No difference in the labeling pattern was observed using either [3H]triamcinolone acetonide (photoaffinity labeling) or [3H]dexamethasone 21-mesylate (electrophilic labeling). The electrophilic labeling step was performed in the cytosol prior to purification by method A to compare the labeled components thus purified with those obtained when the photoaffinity labeling was performed after the purification. Using this approach, distinct breakdown products of the glucocorticoid receptor were revealed, co-purifying during DNA affinity chromatography. Cross-linked receptor obtained by method A has been further purified to homogeneity by preparative sodium dodecyl sulfate-gel electrophoresis and successfully used as immunogen to raise glucocorticoid receptor antibodies in rabbits. These antibodies raised against glucocorticoid receptor, as well as those previously obtained using affinity chromatography-purified receptor, react with the receptor molecules irrespective of their method of purification. Glucocorticoid receptors purified by methods A and B have been analyzed for specific DNA-binding properties by the nitrocellulose filter binding assay.

Animals↗
PubMed1980

Interactions of cytochrome c with mitochondrial membranes. Binding to succinate-cytochrome c reductase.

Methyl-4-azidobenzoimidate was reacted with horse heart cytochrome c to give a photoaffinity-labeled derivative of this heme protein. The modified cytochrome c bound to cytochrome c-depleted mitochondria with the same Kd as native cytochrome c and restored oxygen uptake to the same extent. Irradiation of cytochrome c-depleted mitochondrial membranes with 3- to 4-fold excess of photoaffinity-labeled cytochrome c over cytochrome c oxidase resulted in covalent binding of the derivative to the membranes. Fractionation of the irradiated mitochondria in the presence of detergents and salts followed by chromatography on an agarose Bio-Gel-A-5m showed that the labeled cytochrome c was bound covalently to succinate-cytochrome c reductase. The covalently bound cytochrome c was active in mediating electron transfer between its reductase and oxidase. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the succinate-cytochrome c reductase containing photoaffinity-labeled 125I-cytochrome c showed that the reductase contained a protein binding site for cytochrome c. It is suggested that cytochrome c1 is the most likely site for the cytochrome c binding in mitochondria in situ.

Affinity Labels↗
PubMed2005

The leucotriene C4 binding sites in multidrug resistance protein 1 (ABCC1) include the first membrane multiple spanning domain.

The multiple drug resistance protein 1 (MRP1 or ABCC1) transports anticancer drugs and normal cell metabolites. Leucotriene C(4) (LTC(4)) is one of the highest affinity substrates of MRP1. In this study, we have synthesized and characterized a novel photoreactive azido analogue of LTC(4) (AALTC(4)). The specificity of AALTC(4) binding to MRP1 was confirmed using an LTC(4)-specific monoclonal antibody. Moreover, binding with radioiodinated [(125)I]AALTC(4) (or IAALTC(4)) to MRP1 was dramatically competed with unmodified LTC(4) and to a lesser degree by glutathione (GSH). Oxidized glutathione (GSSG) slightly increased IAALTC(4) binding to MRP1, while MK571, verapamil, and vincristine inhibited IAALTC(4) binding to MRP1. Using AALTC(4) together with a panel of epitope-specific and LTC(4)-specific monoclonal antibodies, we identified LTC(4) binding sites in MRP1. Western blotting of large tryptic fragments of MRP1 with three well-characterized epitope-specific mAbs (MRPr1, QCRL1, and MRPm6) showed LTC(4) binding in both the N- and C-terminal halves of MRP1. Furthermore, a peptide corresponding to the N-terminal membrane-spanning domain of MRP1 (MSD0) was photoaffinity labeled by AALTC(4), indicating that MSD0 contains an LTC(4) binding site. Higher resolution mapping of additional LTC(4) binding sites was obtained using eight MRP1 variants with each containing hemaglutanin A (HA) epitopes at different sites (at amino acid 4, 163, 271, 574, 653, 938, 1001, or 1222). MRP1 variants were photoaffinity labeled with IAALTC(4) and digested with trypsin to isolate specific regions of MRP1 that interact with LTC(4). These results confirmed that sequences in MSD0 interact with IAALTC(4). Other regions that were photoaffinity labeled by IAALTC(4) include TM 10-11, TM 16-17, and TM 12, shown previously to encode MRP1 drug binding site(s). Together, our results show a high-resolution map of LTC(4) binding domains in MRP1 and provide the first direct evidence for LTC(4) binding within MSD0.

Amino Acid Sequence↗
PubMed1987

Purification and partial characterization of a corticosteroid-binding globulin from hamster serum.

Our objective was to characterize and purify the corticosteroid-binding proteins in hamster pregnancy serum. When [3H]cortisol-labeled pregnancy and proestrous serum were subjected to native polyacrylamide gel electrophoresis, a single peak of specific steroid-binding activity was detected in each, with identical electrophoretic mobility. The steroid-binding affinity (Ka = 1.07.10(8) M-1 for cortisol) is typical of corticosteroid-binding globulin from other species, but the steroid-binding specificity (cortisol greater than testosterone greater than progesterone) is not. An ultraviolet photoaffinity-labeling protocol was developed using 17 beta-hydroxy-4,6-[1,2-3H]androstadiene-3-one ([3H]androstadienolone), permitting analysis of ultraviolet photoaffinity-labeled proestrous and pregnancy serum by two-dimensional polyacrylamide gel electrophoresis and fluorography. Both sera contained the same labeled protein species. Corticosteroid-binding globulin was purified from pregnancy serum by DEAE-cellulose chromatography followed by steroid affinity chromatography on androstadienolone-17 beta-hemisuccinate-ethylenediamine-AffiGel 10. The purified protein (Mr = 62,250; pI = 3.95; n = 1; Stokes radius = 3.5; S = 4-5) was determined to be a glycoprotein. When analyzed by gel filtration and two-dimensional polyacrylamide gel electrophoresis, purified corticosteroid-binding globulin behaved the same as in unfractionated serum, and when ultraviolet photoaffinity-labeled with [3H]androstadienolone, purified corticosteroid-binding globulin produced the same fluorogram spot pattern seen in unfractionated serum. A specific corticosteroid-binding globulin antiserum was raised in rabbits, and this antiserum reacted with a single spot in Western blots of unfractionated serum. Thus, hamster pregnancy serum was determined to have one corticosteroid-binding protein. This protein is identical to the corticosteroid-binding globulin found in proestrous serum, with a higher titer in pregnancy serum. No other steroid-binding component is observed in hamster serum.

Animals↗
PubMed1999

Synthesis of the nanomolar photoaffinity GABA(B) receptor ligand CGP 71872 reveals diversity in the tissue distribution of GABA(B) receptor forms.

A radioiodinated probe, [125I]-CGP 71872, containing an azido group that can be photoactivated, was synthesized and used to characterize GABA(B) receptors. Photoaffinity labeling experiments using crude membranes prepared from rat brain revealed two predominant ligand binding species at approximately 130 and approximately 100 kDa believed to represent the long (GABA(B)R1a) and short (GABA(B)R1b) forms of the receptor. Indeed, these ligand binding proteins were immunoprecipitated using a GABA(B) receptor-specific antibody confirming the receptor specificity of the photoaffinity probe. Most convincingly, [125I]-CGP 71872 binding was competitively inhibited in a dose-dependent manner by cold CGP 71872, GABA, saclofen, (-)-baclofen, (+)-baclofen and (L)-glutamic acid with a rank order and stereospecificity characteristic of the GABA(B) receptor. Photoaffinity labeling experiments revealed that the recombinant GABA(B)R2 receptor does not bind [125I]-CGP 71872, providing surprising and direct evidence that CGP 71872 is a GABA(B)R1 selective antagonist. Photoaffinity labeling experiments using rat tissues showed that both GABA(B)R1a and GABA(B)R1b are co-expressed in the brain, spinal cord, stomach and testis, but only the short GABA(B)R1b receptor form was detected in kidney and liver whereas the long GABA(B)R1a form was selectively expressed in the adrenal gland, pituitary, spleen and prostate. We report herein the synthesis and biochemical characterization of the nanomolar affinity [125I]-CGP 71872 and CGP 71872 GABA(B)R1 ligands, and differential tissue expression of the long GABA(B)R1a and short GABA(B)R1b receptor forms in rat and dog.

Affinity Labels↗
PubMed1993

Nascent transcript-binding protein of the pea chloroplast transcriptionally active chromosome.

This study describes the nascent RNA-binding protein of the pea chloroplast transcriptional complex. The protein has been identified by photoaffinity labelling of the transcriptionally active chromosome (TAC) which utilizes the endogenous plastid DNA as template. UV irradiation of lysed chloroplast or the isolated TAC under conditions optimized for transcription photocross-links nascent radiolabelled transcripts (up to 250 nucleotides in length) to a 48 kDa protein. The photoaffinity labelling of the transcript-binding protein is dependent on UV irradiation, is maximal after about 30 min of irradiation, and is completely dependent on transcriptional activity; no cross-linkage has been observed with pre-synthesized RNA. Cross-linkage is influenced by salts and inhibitors in accordance with their effects on transcription. The photoconjugate is composed of protein and RNA moieties, and can be hydrolysed by several proteases. However, the cross-linked transcript is protected from nucleases until the protein is removed. Manganese enhances photoaffinity labelling of the transcript-binding protein, and this is paralleled by an increase in total transcriptional activity of TAC. This protein was isolated by 2-dimensional polyacrylamide gel electrophoresis and the sequence of 15 amino acid residues at the amino terminus was determined. The nascent transcript-binding protein appears to be involved in the transcription of all three classes of chloroplast genes. We also found a polypeptide of identical molecular weight to get cross-linked to nascent transcripts in chloroplasts isolated from other legumes such as Cicer arietenum, Vigna radiata and Phaseolus vulgaris, and monocots like Zea mays, Oryza sativa and Pennisetum americanum.

Affinity Labels↗