Potential thyroliberin affinity labels. 1. Chloroacetyl-substituted phenylalanylpyrrolidines

Goebel, R; Currie, Bruce L.; Bowers, Cyril Y.

doi:10.1021/jm00136a003

Cited by 7 publications

(2 citation statements)

References 4 publications

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“…A facile synthetic route for preparation of TRH-ASA was decided on after surveying several published syntheses of TRH and analogues (Goebel et al, 1981;Grim et al, 1981;Labroo et al, 1983;Szirtes et al, 1984Szirtes et al, , 1986. The synthesis was facilitated by the availability of pre-activated proline and pyroglutamyl esters, and no unusual difficulties were encountered during the synthetic procedures.…”

Section: Discussionmentioning

confidence: 99%

Synthesis and characterization of a high-affinity photoactivatable analogue of thyrotropin-releasing hormone

Brady

Tashjian

1992

Biochemical Journal

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An analogue of thyrotropin-releasing hormone (TRH, pGlu-His-ProNH2), i.e. pGlu-His-ProNH-(CH2)6-(4-azidosalicylamide) (TRH-ASA), has been synthesized and, in a radioiodinated form (TRH-IASA), characterized and used as a photoaffinity reagent to label the TRH receptor on rat pituitary GH4C1 cells. TRH-IASA bound to GH4C1 cells with high affinity (Kd = 8 nM), comparable with that of TRH binding. The binding of TRH-IASA was competitive with binding of TRH, two TRH analogues and a TRH receptor antagonist, chlordiazepoxide. TRH-IASA did not bind to or label GH12C1 cells, which lack functional TRH receptors. Labelling of GH4C1 cells with TRH-IASA followed by SDS/PAGE and autoradiography of membrane proteins demonstrated labelling of a single polypeptide which ran as a diffuse band between 71 and 91 kDa, centred at 76 kDa. No change in this labelling pattern was observed as a function of the length of time (between 5 min and 2 h) that GH4C1 cells were incubated with 3 nM-TRH-IASA. Using either a very short (5 s) photolysis interval or low TRH-IASA concentrations, only the 76 kDa band was labelled. Minor bands appeared only after extended photolysis and use of high TRH-IASA concentrations. We conclude that the TRH receptor from rat pituitary GH4C1 cells is a single peptide with an apparent molecular mass of 76 kDa. Details of the chemical synthesis of TRH-ASA are given in Supplementary Publication SUP 50167 (5 pages), which has been deposited at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1992) 281, 5.

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Section: Discussionmentioning

confidence: 99%

Synthesis and characterization of a high-affinity photoactivatable analogue of thyrotropin-releasing hormone

Brady

Tashjian

1992

Biochemical Journal

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“…Synthesis of 2,5,carbonyl]-t-phenylalanine tert-Butyl Ester (SL-Phe). A mixed carboxylic acid anhydride method similar to that reported for other iV-acylphenylalanine derivatives was used to prepare SL-Phe from 3-carboxy-2,2,5,5-tetramethylpyrrolidine-l-oxyl (2.69 mmol) and L-phenylalanine iert-butyl ester (2.69 mmol) (Goebel et al, 1982). Slightly yellow needles of SL-Phe were obtained that showed one spot on TLC with an Rf of 0.67 in chloroform-methanol (9:1) on silica gel.…”

Section: Methodsmentioning

confidence: 99%

Interaction of a spin-labeled phenylalanine analog with normal and sickle hemoglobins: detection of site-specific interactions through spin-label-induced proton NMR relaxation

Lee¹,

Currie²,

Johnson³

1986

Biochemistry

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In a preliminary report, we have previously shown that N-[(2,2,5,5-tetramethyl-1-oxypyrrolidin-3-yl)carbonyl]-L-phenyl ala nine tert-butyl ester (SL-Phe) exhibits specific binding to hemoglobin and an antiaggregation activity more than 2 orders of magnitude greater than that of phenylalanine [Lu, H.-Z., Currie, B. L., & Johnson, M. E. (1984) FEBS Lett. 173, 259-263]. Transverse 1H NMR relaxation measurements have been used to investigate the interaction of SL-Phe with hemoglobin molecules by use of the resonances assigned to the C2 protons of the beta 2 His, the beta 143 His, and the beta 146 or beta 97 His residues as intrinsic probes. Distance calculations using the paramagnetically induced relaxation data suggest that the SL-Phe binding site is approximately 12-16 A away from the C2 protons of the beta 2 His and the beta 146 or beta 97 His residues in the (carbonmonoxy)hemoglobin tetramer; for deoxyhemoglobin, the distances are approximately 14-17 A between the SL-Phe binding site and the C2 protons of the beta 2 His, the beta 143 His, and the beta 146 His residues. Calculations using the (carbonmonoxy)hemoglobin crystal atomic coordinates only restrict the probable SL-Phe binding region to the full F and H helices of the beta-chain and a small section of the alpha-chain. For deoxyhemoglobin, the distance calculations provide greater restrictions on the probable binding region, limiting it to small sections of the beta-chain F, G, and H helices near the EF bend and to a few residues on the alpha-chain.(ABSTRACT TRUNCATED AT 250 WORDS)

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ChemInform Abstract: POTENTIAL THYROLIBERIN AFFINITY LABELS. 1. CHLOROACETYL‐SUBSTITUTED PHENYLALANYLPYRROLIDINES

Goebel¹,

Currie²,

Bowers³

1981

Chemischer Informationsdienst

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Potential thyroliberin affinity labels. 1. Chloroacetyl-substituted phenylalanylpyrrolidines

Cited by 7 publications

References 4 publications

Synthesis and characterization of a high-affinity photoactivatable analogue of thyrotropin-releasing hormone

Synthesis and characterization of a high-affinity photoactivatable analogue of thyrotropin-releasing hormone

Interaction of a spin-labeled phenylalanine analog with normal and sickle hemoglobins: detection of site-specific interactions through spin-label-induced proton NMR relaxation

ChemInform Abstract: POTENTIAL THYROLIBERIN AFFINITY LABELS. 1. CHLOROACETYL‐SUBSTITUTED PHENYLALANYLPYRROLIDINES

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