Year-end Sale % OFF 
Abstract
An iodinated photosensitive derivative of norepinephine, N-(p-azido-m-iodophenethylamidoisobutyl)-norepinephrine (NAIN), has been synthesized and characterized. NAIN stimulated adenylate cyclase activity in guinea pig lung membranes in a manner similar to (-)-isoproterenol and was inhibited by (-)-alprenolol. NAIN was shown to compete with [125I]iodocyanobenzylpindolol for the beta-adrenergic receptor in guinea pig lung membranes with an affinity which was dependent on the presence of guanyl nucleotides. Carrier-free radioiodinated NAIN ([125I]NAIN) was used at 2 nM to photoaffinity label the beta-adrenergic receptor in guinea pig lung membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of (-)-alprenolol (1 microM) protectable [125I]NAIN labeling showed the same molecular mass polypeptide (65 kDa) that was specifically derivatized with the antagonist photolabel [125I]iodoazidobenzylpindolol. Specific labeling of the beta-adrenergic receptor with [125I]NAIN was dependent on the presence of MgCl2 and the absence of guanyl nucleotide. Guanosine-5'-O-(3-thiotriphosphate (100 microM) abolished specific labeling by [125I]NAIN. N-Ethylmaleimide (2 mM) in the presence of [125I]NAIN protected against the magnesium and guanyl nucleotide effect. These data show that NAIN is an agonist photolabel for the beta-adrenergic receptor.
Highlights
An iodinated photosensitive derivative of norepi- tor. ['251]NAINhas been prepared carrier free at a specific nephine, N-(p-azido-m-iodophenethylamidoisobuty1)-activity of 2200 Ci/mmol and used to photolabel the @-adrenorepinephrine (NAIN), has been synthesized and characterized
Cyanobenzylpindolol for the &adrenergic receptor in guinea pig lung membraneswith an affinity which was dependent on the presence of guanyl nucleotides
Mass spectrometric determinations were performed by the tolabel ['261]iodoazidobenzylpindolol.Specific labeling of the &adrenergic receptor with ['2sI]NAIN was dependent on the presence of MgClz and the absence of guanyl nucleotide
Summary
Label raphy (RF= 0.5) in chloroform/methanol/acetic acid (40:10:2) and on C-18 reverse-phase plates ( R F= 0.5) in ethanol,0.1 N HCl (50:50). The frozen lungs were minced in the presence of a buffer containing 50 mM Tris-HC1,pH 7.4,5 mM EGTA, 100p M phenylmethylsulfonyl fluoride, 100 p~ benzamidine, 5 pg/ml of soybean trypsininhibitor, and 20 pg/ml of leupeptin. W \N / H + N A N 3 carried out in a final volume of 0.25 ml containing 50 mM Tris-HCI, pH 7.5, 1 mMMgC12, 1 mM isobutyl methylxanthine, 0.1 mM ATP, 10 p~ GTP, 5 units of creatine phosphokinase, 2 mM creatine phosphate, 2.2 X 10' cpm of [cx-~'P]ATPa, nd agonist. Reactions were carried out for 10 min at 30°C and terminated by the addition of 0.5 ml of 10% trichloroacetic acid. Samples were centrifuged for 30 min at 1500 rpm at 4°C in an international portable refrigerated Centrifuge. 32P-CyclicAMP was isolated using consecutive columns containing Dowex AG 50 W-X4 resin and 200-400 mesh neutral alumina, as described by Salomon et al [15]. (-)-Isoproterenol and
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
