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Affinity Labeling

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Narasimha Swamy – One of the best experts on this subject based on the ideXlab platform.

  • 1α 25 dihydroxyvitamin d3 3β 2 bromoacetate an Affinity Labeling derivative of 1α 25 dihydroxyvitamin d3 displays strong antiproliferative and cytotoxic behavior in prostate cancer cells
    Journal of Cellular Biochemistry, 2003
    Co-Authors: Narasimha Swamy, Kelly S Persons, Tai C Chen

    Abstract:

    In this report we describe that 1,25(OH)2D3-3-BE, a VDR-Affinity Labeling analog of 1,25(OH)2D3, showed strong and dose-dependent growth-inhibitory effect in several epithelial cells, i.e., keratinocytes (primary cells), MCF-7 breast cancer, PC-3, and LNCaP prostate cancer and PZ-HPV-7 immortalized normal prostate cell-lines. Furthermore, 10−6 M of 1,25(OH)2D3-3-BE induced apoptosis specifically in LNCaP and PC-3 cells; and the effect was much less pronounced at lower doses. We also showed that the effect (of 1,25(OH)2D3-3-BE) was not due to probable degradation (hydrolysis) of 1,25(OH)2D3-3-BE or random interaction of this molecule with cellular proteins. Tissue- or cell-specific action of 1,25(OH)2D3 and its mimics is not common due to the ubiquitous nature of VDR. Furthermore, variable effects of 1,25(OH)2D3 and its analogs in various cell-lines potentially limits their application as anticancer agents. We showed that 1,25(OH)2D3-3-BE displayed similar growth-inhibitory and cytotoxic activities towards androgen sensitive LNCaP and androgen-independent PC-3 cell-lines. Therefore, these results raise the possibility that 1,25(OH)2D3-3-BE or similar VDR-cross linking analogs of 1,25(OH)2D3 might be considered for further development as potential candidates for prostate cancer. J. Cell. Biochem. 89: 909–916, 2003. © 2003 Wiley-Liss, Inc.

  • Affinity Labeling of the nuclear vitamin D receptor with nonsteroidal alkylating agents.
    Bioorganic & Medicinal Chemistry Letters, 2003
    Co-Authors: Ana Fernandez‐gacio, Carlos Fernandez-marcos, Narasimha Swamy

    Abstract:

    Abstract Synthesis of an Affinity alkylating non steroidal mimic of 1α,25-dihydroxyvitamin D 3 and its radiolabeled counterpart is presented. We also report the Affinity Labeling of the VDR-ligand binding domain (VDR-LBD) with this analogue.

  • molecular modeling Affinity Labeling and site directed mutagenesis define the key points of interaction between the ligand binding domain of the vitamin d nuclear receptor and 1α 25 dihydroxyvitamin d3
    Biochemistry, 2000
    Co-Authors: Narasimha Swamy, Wenrong Xu, Juicheng Hsieh, Mark R Haussler, George J Maalouf, Scott C Mohr

    Abstract:

    We have combined molecular modeling and classical structure−function techniques to define the interactions between the ligand-binding domain (LBD) of the vitamin D nuclear receptor (VDR) and its natural ligand, 1α,25-dihydroxyvitamin D3 [1α,25-(OH)2D3]. The Affinity analogue 1α,25-(OH)2D3-3-bromoacetate exclusively labeled Cys-288 in the VDR-LBD. Mutation of C288 to glycine abolished this Affinity Labeling, whereas the VDR-LBD mutants C337G and C369G (other conserved cysteines in the VDR-LBD) were labeled similarly to the wild-type protein. These results revealed that the A-ring 3-OH group docks next to C288 in the binding pocket. We further mutated M284 and W286 (separately creating M284A, M284S, W286A, and W286F) and caused severe loss of ligand binding, indicating the crucial role played by the contiguous segment between M284 and C288. Alignment of the VDR-LBD sequence with the sequences of nuclear receptor LBDs of known 3-D structure positioned M284 and W286 in the presumed β-hairpin of the molecule, …

R D Hesch – One of the best experts on this subject based on the ideXlab platform.

  • selective Affinity Labeling of a 27 kda integral membrane protein in rat liver and kidney with n bromoacetyl derivatives of l thyroxine and 3 5 3 triiodo l thyronine
    Journal of Biological Chemistry, 1990
    Co-Authors: Josef Kohrle, Ulla B Rasmussen, H Rokos, Jack L Leonard, R D Hesch

    Abstract:

    Abstract 125I-Labeled N-bromoacetyl derivatives of L-thyroxine and L-triiodothyronine were used as alkylating Affinity labels to identify rat liver and kidney microsomal membrane proteins which specifically bind thyroid hormones. Affinity label incorporation was analyzed by ethanol precipitation and individual Affinity labeled proteins were identified by autoradiography after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Six to eight membrane proteins ranging in size from 17 to 84 kDa were Affinity labeled by both bromoacetyl-L-thyroxine (BrAcT4) and bromoacetyl-L-triiodothyronine (BrAcT3). Affinity Labeling was time- and temperature-dependent, and both reduced dithiols and detergents increased Affinity Labeling, predominantly in a 27-kDa protein(s). Up to 80% of the Affinity label was associated with a 27-kDa protein (p27) under optimal conditions. Affinity Labeling of p27 by 0.4 nM BrAc[125I]L-T4 was blocked by 0.1 microM of the alkylating ligands BrAcT4, BrAcT3, or 100 microM iodoacetate, by 10 microM concentrations of the non-alkylating, reversible ligands N-acetyl-L-thyroxine, 3,3′,5′-triiodothyronine, 3,5-diiodosalicylate, and EMD 21388, a T4-antagonistic flavonoid. Neither 10 microM L-T4, nor 10 microM N-acetyltriiodothyronine or 10 microM L-triiodothyronine blocked Affinity Labeling of p27 or other Affinity labeled bands. Affinity Labeling of a 17-kDa band was partially inhibited by excess of the alkylating ligands BrAcT4, BrAcT3, and iodoacetate, but Labeling of other minor bands was not blocked by excess of the competitors. BrAc[125I]T4 yielded higher Affinity label incorporation than BrAc[125I]T3, although similar banding patterns were observed, except that BrAcT3 Affinity labeled more intensely a 58,000-Da band in liver and a 53,000-55,000-Da band in kidney. The pattern of other Affinity labeled proteins with p27 as the predominant band was similar in liver and kidney. Peptide mapping of Affinity labeled p27 and p55 bands by chemical cleavage and protease fragmentation revealed no common bands excluding that p27 is a degradation product of p55. These data indicate that N-bromoacetyl derivatives of T4 and T3 Affinity label a limited but similar constellation of membrane proteins with BrAcT4 incorporation greater than that of BrAcT3. One membrane protein (p27) of low abundance (2-5 pmol/mg microsomal protein) with a reactive sulfhydryl group is selectively labeled under conditions identical to those used to measure thyroid hormone 5′-deiodination. Only p27 showed differential Affinity Labeling in the presence of noncovalently bound inhibitors or substrates on 5′-deiodinase suggesting that p27 is likely to be a component of type I 5′-deiodinase in rat liver and kidney.

  • Affinity Labeling of rat liver and kidney type i 5 deiodinase identification of the 27 kda substrate binding subunit
    Journal of Biological Chemistry, 1990
    Co-Authors: Josef Kohrle, R D Hesch, Ulla B Rasmussen, H Rokos, D M Ekenbarger, Sharon Alex, Jack L Leonard

    Abstract:

    Abstract Extrathyroidal production of 3,3′,5-triiodothyronine from the thyroid secretory product, thyroxine, is catalyzed by tissue-specific iodothyronine 5′-deiodinases. Type I 5′-deiodinase (5’D-I) produces greater than 75% of the T3 found in the circulation and in thyroid hormone-responsive tissues and is most abundant in rat liver and kidney. In this study, we used the bromoacetyl derivatives of T4 (N-bromoacetyl-[125I]L-thyroxine, BrAcT4) and T3 (N-bromoacetyl-[125I]3,3′,5-triiodothyronine, BrAcT3) as alkylating Affinity labels to identify 5’D-I-related protein(s). BrAcT4 and BrAcT3 rapidly and irreversibly inactivated 5’D-I activity in liver and kidney microsomes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of Affinity labeled 5’D-I preparations showed that approximately 80% of the Affinity label was incorporated into a protein with a Mr of 27,000 (p27). 5’D-I substrates and inhibitors specifically blocked Affinity Labeling of p27 with a rank order of potency (BrAcT4 greater than BrAcT3 greater than 3,5,3′-triiodothyronine (rT3) approximately flavone EMD 21388 greater than iodoacetate greater than N-acetyl-T4 (NAcT4) greater than N-acetyl-T3 (NAcT3] identical to that determined for inhibition of 5′-deiodination. Hyper- and hypothyroidism-induced increases and decreases in 5’D-I activity, respectively, were matched by comparable changes in the quantity of Affinity labeled p27. BrAcT3 was a less effective Affinity label for p27 and minor Labeling of a new band with 53 kDa was observed. Molecular sieve chromatography of detergent-solubilized 5’D-I showed coincident peaks of p27 and 5′-deiodinating activity with an apparent Mr approximately 51,000. Two-dimensional gel electrophoresis showed that p27 was a single polypeptide with a pI of 6.1. Approximately 2-5 pmol of p27 were present per mg of liver microsomal protein, equal to previous estimates for 5’D-I content. Our results suggest that p27 represents the substrate binding subunit of type I 5′-deiodinase, the enzyme catalyzing the key reaction in the activation of T4 to the thyromimetically active T3.

Josef Kohrle – One of the best experts on this subject based on the ideXlab platform.

  • selective Affinity Labeling of a 27 kda integral membrane protein in rat liver and kidney with n bromoacetyl derivatives of l thyroxine and 3 5 3 triiodo l thyronine
    Journal of Biological Chemistry, 1990
    Co-Authors: Josef Kohrle, Ulla B Rasmussen, H Rokos, Jack L Leonard, R D Hesch

    Abstract:

    Abstract 125I-Labeled N-bromoacetyl derivatives of L-thyroxine and L-triiodothyronine were used as alkylating Affinity labels to identify rat liver and kidney microsomal membrane proteins which specifically bind thyroid hormones. Affinity label incorporation was analyzed by ethanol precipitation and individual Affinity labeled proteins were identified by autoradiography after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Six to eight membrane proteins ranging in size from 17 to 84 kDa were Affinity labeled by both bromoacetyl-L-thyroxine (BrAcT4) and bromoacetyl-L-triiodothyronine (BrAcT3). Affinity Labeling was time- and temperature-dependent, and both reduced dithiols and detergents increased Affinity Labeling, predominantly in a 27-kDa protein(s). Up to 80% of the Affinity label was associated with a 27-kDa protein (p27) under optimal conditions. Affinity Labeling of p27 by 0.4 nM BrAc[125I]L-T4 was blocked by 0.1 microM of the alkylating ligands BrAcT4, BrAcT3, or 100 microM iodoacetate, by 10 microM concentrations of the non-alkylating, reversible ligands N-acetyl-L-thyroxine, 3,3′,5′-triiodothyronine, 3,5-diiodosalicylate, and EMD 21388, a T4-antagonistic flavonoid. Neither 10 microM L-T4, nor 10 microM N-acetyltriiodothyronine or 10 microM L-triiodothyronine blocked Affinity Labeling of p27 or other Affinity labeled bands. Affinity Labeling of a 17-kDa band was partially inhibited by excess of the alkylating ligands BrAcT4, BrAcT3, and iodoacetate, but Labeling of other minor bands was not blocked by excess of the competitors. BrAc[125I]T4 yielded higher Affinity label incorporation than BrAc[125I]T3, although similar banding patterns were observed, except that BrAcT3 Affinity labeled more intensely a 58,000-Da band in liver and a 53,000-55,000-Da band in kidney. The pattern of other Affinity labeled proteins with p27 as the predominant band was similar in liver and kidney. Peptide mapping of Affinity labeled p27 and p55 bands by chemical cleavage and protease fragmentation revealed no common bands excluding that p27 is a degradation product of p55. These data indicate that N-bromoacetyl derivatives of T4 and T3 Affinity label a limited but similar constellation of membrane proteins with BrAcT4 incorporation greater than that of BrAcT3. One membrane protein (p27) of low abundance (2-5 pmol/mg microsomal protein) with a reactive sulfhydryl group is selectively labeled under conditions identical to those used to measure thyroid hormone 5′-deiodination. Only p27 showed differential Affinity Labeling in the presence of noncovalently bound inhibitors or substrates on 5′-deiodinase suggesting that p27 is likely to be a component of type I 5′-deiodinase in rat liver and kidney.

  • Affinity Labeling of rat liver and kidney type i 5 deiodinase identification of the 27 kda substrate binding subunit
    Journal of Biological Chemistry, 1990
    Co-Authors: Josef Kohrle, R D Hesch, Ulla B Rasmussen, H Rokos, D M Ekenbarger, Sharon Alex, Jack L Leonard

    Abstract:

    Abstract Extrathyroidal production of 3,3′,5-triiodothyronine from the thyroid secretory product, thyroxine, is catalyzed by tissue-specific iodothyronine 5′-deiodinases. Type I 5′-deiodinase (5’D-I) produces greater than 75% of the T3 found in the circulation and in thyroid hormone-responsive tissues and is most abundant in rat liver and kidney. In this study, we used the bromoacetyl derivatives of T4 (N-bromoacetyl-[125I]L-thyroxine, BrAcT4) and T3 (N-bromoacetyl-[125I]3,3′,5-triiodothyronine, BrAcT3) as alkylating Affinity labels to identify 5’D-I-related protein(s). BrAcT4 and BrAcT3 rapidly and irreversibly inactivated 5’D-I activity in liver and kidney microsomes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of Affinity labeled 5’D-I preparations showed that approximately 80% of the Affinity label was incorporated into a protein with a Mr of 27,000 (p27). 5’D-I substrates and inhibitors specifically blocked Affinity Labeling of p27 with a rank order of potency (BrAcT4 greater than BrAcT3 greater than 3,5,3′-triiodothyronine (rT3) approximately flavone EMD 21388 greater than iodoacetate greater than N-acetyl-T4 (NAcT4) greater than N-acetyl-T3 (NAcT3] identical to that determined for inhibition of 5′-deiodination. Hyper- and hypothyroidism-induced increases and decreases in 5’D-I activity, respectively, were matched by comparable changes in the quantity of Affinity labeled p27. BrAcT3 was a less effective Affinity label for p27 and minor Labeling of a new band with 53 kDa was observed. Molecular sieve chromatography of detergent-solubilized 5’D-I showed coincident peaks of p27 and 5′-deiodinating activity with an apparent Mr approximately 51,000. Two-dimensional gel electrophoresis showed that p27 was a single polypeptide with a pI of 6.1. Approximately 2-5 pmol of p27 were present per mg of liver microsomal protein, equal to previous estimates for 5’D-I content. Our results suggest that p27 represents the substrate binding subunit of type I 5′-deiodinase, the enzyme catalyzing the key reaction in the activation of T4 to the thyromimetically active T3.