Abbas Rashidbaigi

Photoaffinity labeling of β-adrenergic receptors: Identification of the β-receptor binding site(s) from turkey, pigeon, and frog erythrocyte

Abstract The β-adrenergic receptors in the erythrocyte membranes from turkey, pigeon, and frog have been identified in situ utilizing the photoaffinity label ±[ 125 I]-iodoazidobenzylpindolol, ±[ 125 I]IABP. The molecular weights determined by SDS-polyacrylamide gel electrophoresis are the following: turkey, 43,500; pigeon, 53,500, 46,000, and 45,000 [labeled in a ratio of 5 (53,500):2 (46,000 plus 45,000)]; and frog, a broad 60,000 to 67,000 dalton band. The data identify the binding site subunit(s) of these β-adrenergic receptors and suggest that the receptor structure from different β-receptor subtypes and different sources may be different. These biochemical differences may contribute to the pharmacologically observed distinction of β-receptor subtypes.

[77] A sandwich radioimmunoassay for human IFN-γ

Publisher Summary This chapter discusses a sandwich radioimmunoassay for human interferon (Hu-IFN)-γ. Two monoclonal antibodies are selected, which recognize a different epitope (antigenic determinant) of human IFN-γ and which bind to IFN-γ simultaneously without mutual interference. One antibody (Ab-1) is immobilized on a solid surface. An interferon solution is incubated with this immobilized Ab-1, then the second antibody (Ab-2), labeled with 125 I or with a covalently conjugated enzyme, is added. Binding of Ab-2 is dependent upon and proportional to the interferon bound by the immobilized Ab-1. After incubation and washing, the interferon concentration is determined by measuring the amount of Ab-2 bound. A standard curve is established with known concentrations of IFN-γ. The interferon assay shown in chapter can detect 20 pg (0.3 units) of Hu-IFN-γ in the 0.10 ml reaction mixture. The assay can be made more sensitive by using M-Ab-γ-127A with a higher specific activity or by derivatizing M-Ab-γ-127A with biotin, peroxidase, or fl-galactosidase. Because pair of monoclonal antibodies is used in this assay, only those IFN-γ molecules that are recognized by both monoclonal antibodies are detected.

[46] Binding of 32P-labeled human recombinant immune interferon to U937 cells

Publisher Summary This chapter describes the measurement of the binding of 32 P-labeled human interferon gamma (Hu-IFN-γ) to human U937 histiocytic lymphoma cells. For studying the interferon receptors, it is necessary to have radioactively labeled interferons with high affinity and specific radioactivity. Several groups have reported the labeling of Hu-IFN-γ with 125 I-labeled Bolton–Hunter reagent. The specific activity of this 125 I-labeled Hu-IFN-γ is barely sufficient for studying the immune interferon receptors in cells with a limited number of binding sites. However, recently it was found that Hu-IFN-γ can be labeled with 32 P from [γ- 32 P]ATP. When recombinant human immune interferon was phosphorylated with [γ- 32 P]ATP and bovine heart muscle protein kinase, a phosphorylated Hu-IFN-γ was obtained with a specific activity of 11,000 Ci/mmol. This is a specific activity at least 10- to 100-fold higher than reported for other 125 I-labeled interferons. This specific activity corresponds to the incorporation of an average of about two molecules of phosphate per molecule of Hu-IFN-γ. The high specific activity of 32 P-labeled Hu-IFN-γ, and its convenient preparation make it a useful reagent for studying the receptor for human immune interferon.

Photoaffinity labeling in the study of lymphoid cell β-adrenergic receptors

Publisher Summary This chapter describes photo-affinity labeling in the study of lymphoid cell β-adrenergic receptors. The β -adrenergic receptor is an integral membrane protein, which modulates adenylate cyclase activity in the plasma membrane of catecholamine- responsive tissues in concert with the guanyl nucleotide-binding protein, N 3 . The S49 lymphoma cell line and the N s -deficient variants, in particular cyc - , have proved very useful in the study of adrenergic regulation of adenylate cyclase. The human lymphocyte β 2 -receptor can be used to monitor β-receptor properties in vivo as a method to study alterations in fl-receptors and fl-adrenergic responsiveness in less accessible target tissues, such as the heart, lungs, vascular smooth muscle. The chapter describes photo-affinity labeling of the β -adrenergic receptor in human lymphocytes using [ 25 I]iodocyanopindolol diazirine ([ 25 I]ICYPD). For preparation, Lymphocytes are separated from whole blood samples. Membranes are prepared according to a modification of described methods.

[41] Labeling of recombinant interferon with [35S]methionine in vivo

Publisher Summary This chapter illustrates a method for uniformally labeling interferon alpha A (IFN-αA) with [ 35 S]methionine by the in vivo maxicell labeling technique. Study of interferon binding to the plasma membrane receptor is the first step in the establishment of interferon function. The number of interferon receptors is relatively low on the cell surface membrane (about 500–5000 receptors per cell). Therefore, to characterize the interferon receptor, it is necessary to obtain radiolabeled interferon with high specific radioactivity. IFN-αA has been iodinated to a reasonably high specific radioactivity with enzymobeads, Bolton–Hunter reagent, and chloramine T. The iodination of interferon does not consistently produce active proteins. This might be due to the oxidation of polypeptide chains or the modification of amino acids, which are required for binding to the receptor and for the functional activity of interferons. Other interferon species (e.g., fibroblast and immune) are highly sensitive to modification by iodination. Any chemical modification procedure produces a heterogeneous population of interferon molecules. The maxicell procedure described in the chapter is a simple method for obtaining [ 35 S]Met-labeled IFN-αA. In this method, chromosomal DNA of E.coli is damaged extensively upon ultraviolet (UV) irradiation. This results in diminished synthesis of E. coli proteins. The plasmid DNA, which survived UV treatment, continues to replicate at a high level.