Solubilization and purification of rat pituitary gonadotropin-releasing hormone receptor.

Hazum, Eli; Schvartz, Iris; Waksman, Y; Keinan, Dana

doi:10.1016/s0021-9258(18)69268-3

Cited by 49 publications

(8 citation statements)

References 28 publications

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“…The rat receptor appears to contain 59-and 57-kDa proteins. This is in agreement with the size of the purified rat pituitary GnRH receptor described recently, i.e., 59 and 57 kDa (Hazum et al, 1986), and substantiates the possibility that the intact receptor is a dimer (Conn & Venter, 1985). This receptor molecule could be composed of independent subunits forming a heterodimer or of identical subunits forming a homodimer, in which case the lower molecular size form would be a degradation or possibly a differently glycosylated product of the higher.…”

Section: Discussionsupporting

confidence: 91%

“…Two proteins of close but different size were found to be labeled by the radioactive photoreactive peptide, which was seen consistently in different experiments. Similar size bands were reported earlier using photoreactive GnRH analogues carrying the photoreactive group in the position 6 side chain (Hazum, 1981; Iwashita & Catt, 1986;Hazum et al, 1986). However, protein bands labeled with D-Lys6-GnRH derivatives were diffuse and could not be clearly identified.…”

Section: Discussionsupporting

confidence: 85%

“…However, protein bands labeled with D-Lys6-GnRH derivatives were diffuse and could not be clearly identified. Using radiolabeled D-Phe6,[Orn(2,4-NAPS)]8-GnRH-(l-9)-EA as ligand for photoaffinity labeling resulted in clear protein bands as compared to photolabeling with D-Lys6-GnRH-(l-9)-EA derivatives [compare Figure 4 with the results of Iwashita andCatt (1986) andHazum et al (1986)]. In addition, the D-Lys6 derivative appeared to label only the smaller of the two bands that were labeled by the Orn8 derivative.…”

Section: Discussionmentioning

confidence: 99%

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Photoaffinity labeling of pituitary GnRH receptors: significance of the position of photolabel on the ligand

Nikolics¹,

Szonyi²,

Ramachandran³

1988

Biochemistry

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Photoreactive derivatives of GnRH and its analogues were prepared by incorporation of the 2-nitro-4(5)-azidophenylsulfenyl [2,4(5)-NAPS] group into amino acid residues at positions 1, 3, 6, or 8 of the decapeptide sequence. The modification of Trp3 by the 2,4-NAPS group led to a complete loss of the luteinizing hormone (LH) releasing as well as LH-release-inhibiting activity of the peptide. The [D-Lys(2,4-NAPS)]6 analogue was a very potent agonist that, after covalent attachment by photoaffinity labeling, caused prolonged LH secretion at a submaximal rate. [Orn(2,4-NAPS)]8-GnRH, a full agonist with a relative potency of 7% of GnRH, after photoaffinity labeling caused prolonged maximal LH release from cultured pituitary cells. In contrast, [Orn(2,5-NAPS)]8-GnRH, although being equipotent with the 2,4-NAPS isomer in terms of LH releasing ability, was unable to cause prolonged LH release after photoaffinity labeling. Thus, [Orn(2,4-NAPS)]8-GnRH is a very effective photolabeling ligand of the functionally significant pituitary GnRH receptor. Based on this compound, a pituitary peptidase resistant derivative, D-Phe6,[Orn(2,4-NAPS)]8-GnRH-(1-9)-ethylamide, was synthesized. This derivative showed high-affinity binding to pituitary membranes with a Kd comparable to those of other GnRH analogues. A radioiodinated form of this peptide was used for pituitary GnRH-receptor labeling. This derivative labeled 59- and 57-kDa proteins in rat and 58- and 56-kDa proteins in bovine pituitary membrane preparations, respectively. This peptide also labeled pituitary GnRH receptors in the solubilized state and therefore appears to be a suitable ligand for the isolation and further characterization of the receptor.

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Section: Discussionsupporting

confidence: 91%

Section: Discussionsupporting

confidence: 85%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Photoaffinity labeling of pituitary GnRH receptors: significance of the position of photolabel on the ligand

Nikolics¹,

Szonyi²,

Ramachandran³

1988

Biochemistry

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“…Biotinylation of specific ligands could allow, among many other possible applications, receptor visualization [with possible amplified detection sensitivity (Hsu et al, 1981)], or analysis and sorting of discrete cell subpopulations bearing the corresponding receptors (Wilchek & Bayer, 1984;Berenson et al, 1986). These approaches have been achieved or at least attempted with biotinylated derivatives of hormones, which might also represent valuable tools for the purification of their specific receptors either by direct (Finn et al, 1984;Hazum et al, 1986) or indirect (Redeuilh et al, 1985) affinity chromatography strategies.…”

Section: Introductionmentioning

confidence: 99%

New probes for angiotensin II receptors. Synthesis, radioiodination and biological properties of biotinylated and haptenated angiotensin derivatives

Bonnafous

Tencé

Seyer

et al. 1988

Biochemical Journal

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The present work delineates the basis for chemical modifications which can be introduced on the angiotensin II (AII) molecule to design probes suitable for indirect affinity techniques, especially for receptor purification. Using the solid-phase synthesis strategy, biotin or dinitrophenyl moieties have been added at the N-terminus of AII, with aminohexanoic acid as spacer arm. The resulting probes, (6-biotinylamido)hexanoyl-AII (Bio-Ahx-AII) and dinitrophenylaminohexanoyl-AII (Dnp-Ahx-AII), were prepared in their monoiodinated and highly labelled radioiodinated forms, with possible sulphoxidation of biotin. In addition to their ability to interact with streptavidin and anti-Dnp antibodies respectively, the two ligands displayed almost unchanged affinities for hepatic AII receptors as compared with AII. Bio-Ahx-AII and Dnp-Ahx-AII behaved as agonists on several AII-sensitive systems. The potential applications of these probes, receptor purification, cell labelling and sorting and histochemical receptor visualization, are discussed.

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“…Recently, we have succeeded in solubilizing the GnRH receptor from rat and bovine pituitary membrane preparations in an active form (Hazum et al, 1986(Hazum et al, , 1987 by using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1 -propanesulfonate (CHAPS). In the present study, we report the characterization of the binding properties of the solubilized GnRH receptor, as well as the role of carboxylic groups of the receptor in both GnRH binding and the biological activity.…”

mentioning

confidence: 99%

Binding properties of solubilized gonadotropin-releasing hormone receptor: role of carboxylic groups

Hazum

1987

Biochemistry

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The interaction of 125I-buserelin, a superactive agonist of gonadotropin-releasing hormone (GnRH), with solubilized GnRH receptor was studied. The highest specific binding of 125I-buserelin to solubilized GnRH receptor is evident at 4 degrees C, and equilibrium is reached after 2 h of incubation. The soluble receptor retained 100% of the original binding activity when kept at 4 or 22 degrees C for 60 min. Mono- and divalent cations inhibited, in a concentration-dependent manner, the binding of 125I-buserelin to solubilized GnRH receptor. Monovalent cations require higher concentrations than divalent cations to inhibit the binding. Since the order of potency within the divalent cations was identical with that of their association constants to dicarboxylic compounds, it is suggested that there are at least two carboxylic groups of the receptor that participate in the binding of the hormone. The carboxyl groups of sialic acid residues are not absolutely required for GnRH binding since the binding of 125I-buserelin to solubilized GnRH receptor was only slightly affected by pretreatment with neuraminidase and wheat germ agglutinin. The finding that polylysines stimulate luteinizing hormone (LH) release from pituitary cell cultures with the same efficacy as GnRH suggests that simple charge interactions can induce LH release. According to these results, we propose that the driving force for the formation of the hormone-receptor complex is an ionic interaction between the positively charged amino acid arginine in position 8 and the carboxyl groups in the binding site.

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Solubilization and purification of rat pituitary gonadotropin-releasing hormone receptor.

Cited by 49 publications

References 28 publications

Photoaffinity labeling of pituitary GnRH receptors: significance of the position of photolabel on the ligand

Photoaffinity labeling of pituitary GnRH receptors: significance of the position of photolabel on the ligand

New probes for angiotensin II receptors. Synthesis, radioiodination and biological properties of biotinylated and haptenated angiotensin derivatives

Binding properties of solubilized gonadotropin-releasing hormone receptor: role of carboxylic groups

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