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Biomedical subjects

A Rashidbaigi

Publications and source records attributed to A Rashidbaigi.

31 records · Page 2Linked to original sources
PubMed1986

The gene for the human immune interferon receptor is located on chromosome 6.

When 32P-labeled human recombinant immune interferon gamma (Hu-[32P]IFN-gamma) is crosslinked to human cells with disuccinimidyl suberate, a complex with a molecular size of approximately equal to 117,000 Da was identified by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The formation of this complex is inhibited when the binding is performed in the presence of excess unlabeled Hu-IFN-gamma. The specific formation of the 117,000-Da complex is not observed in mouse L cells or Chinese hamster ovary cells. This complex shows all of the criteria that identify it as the Hu-IFN-gamma receptor or its binding subunit. The same complex can be formed following binding and covalent crosslinking of Hu-[32P]IFN-gamma to some hamster-human or mouse-human somatic cell hybrids. The presence of human chromosome 6 in the hybrids is necessary and sufficient for the formation of this complex. More specifically, the long arm of chromosome 6 seems sufficient. Therefore, we have localized the gene for the Hu-IFN-gamma receptor (or its binding subunit) to the long arm of human chromosome 6. The presence of this chromosome in the somatic cell hybrids is not adequate, however, to confer antiviral resistance to the hybrids in the presence of Hu-IFN-gamma.

Biological Assay↗
PubMed1985

Characterization of receptors for immune interferon in U937 cells with 32P-labeled human recombinant immune interferon.

Recombinant human immune interferon (HuIFN-gamma) was labeled with [gamma-32P]ATP and cyclic-AMP-dependent protein kinase from bovine heart to a specific radioactivity of 11,000 Ci/mmol. At least two molecules of phosphate were incorporated per molecule of interferon. The binding of [32P]HuIFN-gamma to human U937 histiocytic lymphoma cells was time dependent, and displaceable by HuIFN-gamma but not by HuIFN-alpha A or HuIFN-beta. The specific binding was saturable with less than 10% nonspecific binding. The dissociation constant of [32P]HuIFN-gamma for U937 interferon receptors was calculated to be 1.5 X 10(-10) M with a total of 1,800 binding sites/cell. Dissociation of bound [32P]IFN-gamma at 24 degrees C exhibited two distinct rates. A fast dissociation with a specific rate constant of 0.141 min-1, and a slow dissociation with a specific rate constant of 0.0027 min-1. The Kd for [32P]HuIFN-gamma was calculated from kinetic constants to be 5.4 X 10(-10) M.

Binding, Competitive↗
PubMed1983

Rapid vesicle reconstitution of alprenolol-Sepharose-purified beta 1-adrenergic receptors. Interaction of the purified receptor with N.

beta-Adrenergic receptors from turkey erythrocyte membranes have been purified 1000-4000-fold using alprenolol-Sepharose affinity chromatography. Addition of deoxycholate solubilized egg phosphatidylcholine to the beta-adrenergic receptor, that is 5-10% pure and in 0.1% digitonin, followed by Sephadex G-50 gel filtration in buffers containing 30 mM MgCl2 results in 65-70% of the receptor being incorporated into phospholipid vesicles. The beta-adrenergic receptor as detected by photoaffinity labeling using [125I]azidobenzylpindolol in membranes and after alprenolol-Sepharose chromatography is a Mr = 40,000 peptide. Addition of deoxycholate extracts of human erythrocyte membranes, which contain the guanine nucleotide stimulatory regulatory protein of adenylate cyclase (Ns) but not beta-adrenergic receptor, were used to reconstitute a guanine nucleotide-mediated change in agonist affinity for the receptor. These results demonstrate that the alprenolol-Sepharose affinity purified beta-adrenergic receptor is functional in both ligand binding and coupling to Ns. The procedure is rapid, efficient and should be generally applicable to beta-adrenergic receptor and Ns from several different membrane systems.

Adenylyl Cyclases↗
PubMed1983

Photoaffinity labeling of the beta-adrenergic receptor from cultured lymphoma cells with [125I]iodoazidobenzylpindolol: loss of the label with desensitization.

The beta-adrenergic antagonist [125I]iodoazidobenzylpindolol ( [125I]IABP) specifically photolabeled two polypeptides in membrane preparations from wild-type (WT) and coupling protein-deficient cyc- cultured lymphoma cells. The molecular weights of the two polypeptides determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis were 65,000 and 55,000. They were labeled in a ratio of approximately 1:1. Pretreatment of intact WT or cyc- cells with 1.0 microM epinephrine for 15 min (desensitization) resulted in a greater loss of the 55,000 Mr polypeptide (40-60%) relative to the 65,000 Mr peptide (10-30% loss). An 18- to 24-hr pretreatment of WT cells with terbutaline (down-regulation) led to a greater than 90% reduction of the photolabeling of both polypeptides, whereas a similar pretreatment of cyc- cells resulted in no further loss of labeled receptor than that observed after only a 15-min pretreatment with epinephrine. There was no indication of a change in the electrophoretic mobility of the [125I]IABP-labeled receptors after either short- or long-term agonist pretreatment. These data provide direct evidence for heterogeneity of the beta-adrenergic receptor in lymphoma cells. The differential loss of the [125I]IABP labeling in the two polypeptides suggests a functional heterogeneity as well.

Adenylyl Cyclases↗
PubMed1983

Use of photolabels to probe the Na,K-ATPase and the beta-adrenergic receptor.

Radioactive photoaffinity labels have been used to probe the cardiac glycoside-binding site of Na,K-ATPase and the catecholamine-binding site of the beta-adrenergic receptor. For the Na,K-ATPase, a systematic positioning of the photoactive group on the first, second, and third digitoxoses of digitoxin showed that the specific radioactivity in the alpha subunit decreased 5- to 20-fold as the photoactive group was extended further away from the steroid nucleus, whereas the beta subunit is positioned very close to the alpha subunit in the region of the cardiac glycoside-binding site. For the beta-adrenergic receptor, a new class of orthoiodophenylazide derivatives of pindolol was prepared with carrier-free 125I. Photolysis of the beta-adrenergic receptor of duck, turkey, pigeon, and frog erythrocyte membrane with one of these compounds (iodoazidobenzylpindolol) allowed identification of the receptor polypeptides. It was found that the size of the polypeptides and the number of polypeptides varied.

Affinity Labels↗
PubMed1983

Effect of dithiothreitol on the beta-adrenergic receptor of S49 wild type and cyc- lymphoma cells: decreased affinity of the ligand-receptor interaction.

Dithiothreitol (DTT) treatment of WT or cyc- lymphoma membranes resulted in the simultaneous loss of epinephrine-stimulated adenylate cyclase activity and beta-adrenergic antagonist binding. The treatment produced no significant decrease in NaF-stimulated activity and only a partial loss of the PGE1 stimulation. Epinephrine partially protected against the loss of epinephrine-stimulated cyclase activity. The decrease in beta-adrenergic stimulation of adenylate cyclase was characterized by over a 100-fold increase in Kact for epinephrine stimulation of adenylate cyclase (10 mM DTT for 30 min) with no effect on the Vmax. We have previously shown that two polypeptides (Mr = 55,000 and 65,000 daltons) are specifically labeled by [125I]iodoazidobenzylpindolol (IABP) in WT and cyc-. The IABP photolabeling of the 55,000 dalton beta-receptor polypeptide was preferentially reduced by 1.0 mM DTT, whereas 10 mM DTT eliminated the photolabeling of both polypeptides. The effects of DTT were not due to either scavenging of the nitrene or reduction of the azide. A reduction in epinephrine stimulation of adenylate cyclase was also observed after treatment of intact WT, and the effects were readily reversed in the cells by washout of the DTT. The DTT effects on membranes were not reversed by washout. Our results demonstrate that the oxidized state of the lymphoma beta-receptor is necessary for maximum sensitivity to agonist stimulation of adenylate cyclase and that low concentrations of reducing agent selectively decrease specific photo-labeling of the 55,000 dalton beta-receptor polypeptide.

Adenylyl Cyclases↗
PubMed1982

Synthesis and characterization of iodoazidobenzylpindolol.

A high affinity beta-adrenergic ligand, iodoazidobenzylpindolol, was synthesized and characterized. The absorption spectrum of this compound changed markedly upon photolysis, consistent with decomposition of the azide group. This compound has a KD of 5--7 x 10(-10) M for the duck erythrocyte ghost beta-adrenergic receptor when measured in a competitive binding assay.

Affinity Labels↗
PubMed1981

Iodoazidobenzylpindolol, a photoaffinity probe for the beta-adrenergic receptor.

A high-affinity pindolol derivative, (+/-)-1-(indol-4-yloxy)-3-[1-(p-azido-m-iodophenyl)-2-isobutylamine]-2-propanol (IABP), has been prepared; it contains an iodide and an azide functional group and acts as a photoaffinity label for the beta-adrenergic receptor. When [125I]IABP (specific activity 1300 Ci/mmol) was photolyzed with crude duck erythrocyte membrane preparations, which contain beta-adrenergic receptor binding sites highly specific labeling of two polypeptides was observed upon electrophoresis on sodium dodecyl sulfate/polyacrylamide gels. These two polypeptides, A (Mr approximately 45,000) and B (Mr approximately 48,500), and B (Mr approximately 48,500), were photolabeled in a ratio of approximately 4:1 A/B. Binding of [125I]IABP and covalent derivatization of the beta receptor was at least 70% specific. The data indicate that [125I]IABP is a very effective compound for identification of polypeptides containing the beta-receptor binding site even in crude membrane preparations. The beta receptor of the duck erythrocyte plasma membrane may be composed of more than one subunit.

Affinity Labels↗