Importance of thiol group(s) in the binding of 125I-labeled neurotensin to membranes from porcine brain.
In a radioreceptor assay employing 125I-labeled neurotensin (125I-NT) and membranes from porcine brain, the reducing agent, dithiothreitol (DTT), was found to enhance binding (ED50, approximately 10 microM), whereas alkylating agents such as N-ethyl-maleimide (NEM) suppressed binding (ED50, approximately 0.7 mM). The enhanced binding appeared to be due to an effect on disulfide group(s) within the NT-receptor or associated protein(s), since the stability of 125I-NT in the presence of membranes was not altered by 2 mM DTT. Scatchard analysis indicated that treatment with 2 mM DTT increased the total number of binding sites approximately 1.6-fold without much effect on the apparent KdS for the high- and low-affinity states. In a similar manner, the effect of 1 mM NEM was shown to result primarily from a decrease (approximately 60%) in the number of binding sites with little change in the KdS. The additional receptors gained by exposure to DTT appeared not to be sensitive to NEM unless pretreated with DTT, suggesting that reduction of disulfide bond(s) converted latent receptors into active receptors. Interestingly, nine Cys residues have been found to be present in the recently cloned rat NT receptor. In other studies, preincubation of membranes with NT prior to treatment with NEM diminished the inhibitory effect of NEM on agonist binding, suggesting that the critical sulfhydryl group(s) were located at the NT binding site or were protected by an allosteric effect.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Physiology, University of Massachusetts Medical Center, Worcester 01655.
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D000478:Alkylation; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D066298:In Vitro Techniques; D007700:Kinetics; D008566:Membranes / Q000378:metabolism; D009496:Neurotensin / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D011869:Radioligand Assay; D018028:Receptors, Neurotensin; D017981:Receptors, Neurotransmitter / Q000187:drug effects / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism; D013552:Swine
D018028:Receptors, Neurotensin; D017981:Receptors, Neurotransmitter; D013438:Sulfhydryl Compounds; D009496:Neurotensin; D005033:Ethylmaleimide; D004229:Dithiothreitol