Affinity labeling of a mouse IgB2a myeloma protein with binding affinity for nitrophenyl ligands

Martin, Nancy; Warner, Noel L.; Roeder, Phoebe E.; Singer, S. J.

doi:10.1021/bi00776a020

Cited by 8 publications

(4 citation statements)

References 17 publications

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“…When two polypeptides contribute to an active site, the ratio of affinity label attached to residues in the two chains can vary over a wide range as a result of relatively small differences in the free energy of activation for reaction with groups on the two chains (43). For example, although both the heavy and light chains of immunoglobulin G together form the binding site, it has been observed that the ratio of covalent incorporation of an affinity label into the two chains of different anti-dinitrophenol immunoglobulins varied from 0.1 to 10 (heavy/light) (44). 3 receptors from other sources may also exhibit varied A/ B labeling profiles.…”

Section: Discussionmentioning

confidence: 99%

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

Rashidbaigi¹,

Ruoho²

1981

Proc. Natl. Acad. Sci. U.S.A.

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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 f-adrenergic receptor. When [125I]IABP (specific activity 1300 Ci/ mmol) was photolyzed with crude duck erythrocyte membrane preparations, which contain 3-adrenergic receptor binding sites, highly specific labeling of two polypeptides was observed upon electrophoresis on sodium dodecyl s ate/polyacrylamide gels. The -adrenergic receptor is a protein (1) located in the plasma membrane of various cultured cell lines (2-6), nucleated erythrocytes (7-9), and several mammalian tissues (cardiac muscle, bronchial smooth muscle) that respond to epinephrine. This receptor is functionally coupled to the enzyme adenylate cyclase via the nucleotide regulatory protein (10). Many of the ligand binding properties of this receptor have been studied by utilizing radioactive (3-receptor antagonists (7, 11, 12) and agonists (13,14). One of the competitive antagonists that has demonstrated very high affinity for the ( receptor and has been prepared at the highest specific radioactivity is iodohydroxybenzylpindolol (11, 15, 16). In order to identify the polypeptide or polypeptides that make up this catecholamine receptor, suitable derivatives of ,B receptor antagonists must be available for forming covalent bonds in the binding site. This method for specifically "tagging" a binding site in heterogeneous mixture of binding sites by utilizing chemically reactive site-directed ligands has been described as "affinity labeling" (17)(18)(19); more recently the method of "photoaffinity labeling" has been used (20-23). The use of light-activated compounds possesses several inherent advantages over regular affinity labeling for increasing the specificity of the derivatization reaction (24). Some affinity and photoaffinity labels for the (3receptor have been reported. A bromoacetyl derivative of propranolol (25) and a bromoacetyl derivative of alprenolol (26) were reported as specific irreversible inhibitors of the (3adrenergic receptor.

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Section: Discussionmentioning

confidence: 99%

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

Rashidbaigi¹,

Ruoho²

1981

Proc. Natl. Acad. Sci. U.S.A.

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“…Both compounds described in this paper are a posteriori photolabels. CM cymarin, however, lacks the diazo group, and cannot generate a carbene by the anticipated N2 dissociation mechat Although both the heavy and light chains of IgG immunoglobulin together form the binding site, it has been observed that the ratio of covalent incorporation of an affinity label into the two chains of different anti-Dnp immunoglobulins varies from 0.1 to 10 (heavy/light) (26). Where two polypeptides contribute to an active site, the ratio of affinity label attached to residues in the two chains can vary over a wide range as a result of relatively small differences in the free energy of activation for reaction with groups on the two chains (27).…”

Section: Discussionmentioning

confidence: 99%

Photoaffinity Labeling of the Ouabain-Binding Site on (Na ⁺ + K ⁺ ) Adenosinetriphosphatase

Ruoho

Kyte

1974

Proc. Natl. Acad. Sci. U.S.A.

156

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An ethyl diazomalonyl derivative of cymarin was synthesized in order to photoaffinity label the cardiac glycoside-binding site on (Na+ + K+) adenosinetriphosphatase (EC 3.6.1.3). When a noncovalent complex of the enzyme and this cardiac glycoside derivative was photolyzed, a covalent bond was formed between the ligand and the larger of the two polypeptide subunits of the enzyme. Several control experiments demonstrate that this photochemical reaction occurred while the ligand was bound to the site at which it inhibits the enzyme activity. Another specific inhibitor, tentatively identified as the ethyl chloromalonyl derivative of cymarin, produced similar photoaffinity labeling of the larger subunit, demonstrating that the photolytic dissociation of the diazo group may not be responsible for the photochemical reaction. Since the cardiac glycoside-binding site, which is accessible from the outside surface of the plasma membrane, and the phosphorylation site, which is accessible from the inside surface, are both on the larger polypeptide subunit of (Na+ + K+) adenosinetriphosphatase, this polypeptide has sequences exposed to both sides of the membrane.(Na+ + K+) adenosinetriphosphatase is the membrane-bound enzyme that transports sodium and potassium in opposite directions across the cell membrane to create the ion gradients that are utilized to perform physiological functions. The cardiac glycosides and aglycones, such as digoxin, ouabain, and strophanthidin, are natural products that have been used therapeutically for centuries in the treatment of heart disease. Their only known biochemical effect is to inhibit specifically (Na+ + K+) adenosinetriphosphatase and consequently decrease or completely stop active cation flux.The purified enzyme is a specific complex of two polypeptide chains (1). In order to determine which polypeptide contains the cardiac glycoside-binding site, a radioactive affinity label can be used (2). Haloacetyl derivatives of the cardiac glycosides strophanthidin and hellebrigenin (3, 4) were synthesized for affinity labeling of the cardiac glycoside-binding site of (Na+ + K+) adenosinetriphosphatase. They did not, however, react covalently and specifically with the binding site (5). One disadvantage of such electrophilic reagents is the requirement for a suitably positioned nucleophile in the active site. On the other hand, a photoaffinity label can be converted by photolysis into an exceedingly reactive intermediate, and under appropriate circumstances even insertion into a C-H bond is possible (6). Compounds that generate carbenes or nitrenes upon photolysis have been used for the photoaffinity labeling of enzyme active sites (7), antibody ligand sites (8), and membrane-bound adenosine 3': 5'-cyclic monophosphate binding sites (9). We have synthesized and used an ethyl diazomalonyl derivative of the cardiac glycoside, cymarin (DAMN cymarin, Fig. 1) for photoaffinity labeling of this specific binding site in (Na+ + K+) adenosine triphosphatase. MATERIALS AND METHODS Synthesis of pho...

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“…These affinity labeling reagents have had either a diazonium (Martin et al, 1972) or a bromoacetyl moiety (Haimovich et al, 1972), as the reactive group. Such reagents are restricted in their reactivity to amino acid residues having nucleophilic " Monomeric protein 460 (3.35 X 10-5 m) in 0.2 m Tris-HCl (pH 8.0) buffer at 4°in the presence of Dnp-AD (1.05 X 10-4 m) was irradiated with six 15-W General Electric F15T8-B1 lamps at a distance of 3 cm.…”

Section: Discussionmentioning

confidence: 99%

“…Affinity reagents with two types of reactive groups, diazonium fluoroborate (Metzger and Potter, 1968; Goetzl and Metzger, 1970;Ray and Cebra, 1972;Martin et al, 1972) and bromoacetyl (Haimovich et al, 1970(Haimovich et al, , 1972Givol et al, 1971), have been synthesized for the purpose of investigating anti-Dnp1 *myeloma proteins and anti-Dnp antibodies of limited heterogeneity. These reagents have a rather narrow spectrum of reactivity, forming covalent bonds with Tyr, His, Lys, and Cys residues.…”

mentioning

confidence: 99%

Combining region of protein 460, a mouse γA immunoglobulin which binds several haptens. Binding and reactivity of two types of photoaffinity labeling reagents

Yoshioka¹,

Lifter²,

Hew³

et al. 1973

Biochemistry

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Affinity labeling of a mouse IgB2a myeloma protein with binding affinity for nitrophenyl ligands

Cited by 8 publications

References 17 publications

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

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

Photoaffinity Labeling of the Ouabain-Binding Site on (Na ⁺ + K ⁺ ) Adenosinetriphosphatase

Combining region of protein 460, a mouse γA immunoglobulin which binds several haptens. Binding and reactivity of two types of photoaffinity labeling reagents

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Affinity labeling of a mouse IgB2a myeloma protein with binding affinity for nitrophenyl ligands

Cited by 8 publications

References 17 publications

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

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

Photoaffinity Labeling of the Ouabain-Binding Site on (Na + + K + ) Adenosinetriphosphatase

Combining region of protein 460, a mouse γA immunoglobulin which binds several haptens. Binding and reactivity of two types of photoaffinity labeling reagents

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Photoaffinity Labeling of the Ouabain-Binding Site on (Na ⁺ + K ⁺ ) Adenosinetriphosphatase