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

An ethyl diazomalonyl derivative of cymarin was synthesized in order to photoaffinity label the cardiac glycoside-binding site on (Na(+) + K(+)) adenosinetriphosphate (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 occured 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.     

Photoaffinity labeling of corticotropin receptors.

A photoaffinity label for corticotropin (ACTH) receptors was prepared by selective chemical modification of the single tryptophan residue in the hormone by reaction with 2-nitro-5-azidophenylsulfenyl chloride. The photoreactive derivative, [(2-nitro-5-azidophenylsulfenyl)-Trp9]ACTH (2,5-NAPS-ACTH), stimulated corticosterone synthesis to 60% of the maximal rate induced by ACTH in isolated rat adrenocortical cells. 2.5-NAPS-ACTH caused only a marginal stimulation of cyclic AMP production compared to the unmodified hormone. Stimulation of corticosterone production and cyclic AMP accumulation induced by ACTH were both inhibited in a competitive manner by 2,5-NAPS-ACTH. Photolysis of adrenocortical cells in the presence of 2,5-NAPS-ACTH resulted in a 40% inactivation of ACTH receptors mediating steroidogenesis, as shown by the decrease in response to subsequent stimulation with ACTH. No loss of function was observed when photolysis was conducted in the presence of the photoresistant analog [(2,4-dinitrophenylsulfenyl)-Trp9]ACTH. Covalent attachment of the hormone to the receptors was also demonstrated by photolyzing adrenocortical cells in the presence of tritiated 2,5-NAPS-ACTH of high specific radioactivity (90 Ci/mmol) and analyzing the cell proteins by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. A protein with an approximate molecular weight of 100,000 was specifically labeled by this procedure. The unique labeling of an adrenocortical cell protein and the concomitant loss of ACTH responsiveness suggest that physiologically relevant receptors are photolabeled by this method.     

Photoaffinity labeling of retinoic acid-binding proteins.

Retinoid-binding proteins are essential mediators of vitamin A function in vertebrate organisms. They solubilize and stabilize retinoids, and they direct the intercellular and intracellular trafficking, transport, and metabolic function of vitamin A compounds in vision and in growth and development. Although many soluble retinoid-binding proteins and receptors have been purified and extensively characterized, relatively few membrane-associated enzymes and other proteins that interact with retinoids have been isolated and studied, due primarily to their inherent instabilities during purification. In an effort to identify and purify previously uncharacterized retinoid-binding proteins, it is shown that radioactively labeled all-trans-retinoic acid can be used as a photoaffinity labeling reagent to specifically tag two known retinoic acid-binding proteins, cellular retinoic acid-binding protein and albumin, in complex mixtures of cytosolic proteins. Additionally, a number of other soluble and membrane-associated proteins that bind all-trans-[11,12-3H]retinoic acid with high specificity are labeled utilizing the same photoaffinity techniques. Most of these labeled proteins have molecular weights that do not correspond to any known retinoid-binding proteins. Thus, photoaffinity labeling with all-trans-retinoic acid and related photoactivatable retinoids is a method that should prove extremely useful in the identification and purification of novel soluble and membrane-associated retinoid-binding proteins from ocular and nonocular tissues.     

Photoaffinity labeling of rat type I iodothyronine deiodinase

The photoreactive compound p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PAL) was coupled to [125I]rT3, T4, or T3 and incubated with liver and kidney microsomes of hypo-, hyper-, or euthyroid rats to identify the type I iodothyronine deiodinase. Various substrates or inhibitors of the enzyme, including rT3, T4, T3, 6-n-propylthiouracil (PTU), and iopanoic acid, were used as competitors to establish the specificity of protein labeling. The PAL derivatization enhanced the behavior of T4 and T3 as substrates for the type I enzyme. No specific labeling of microsomal proteins was observed with either rT3 or T4-PAL, presumably due to deiodination of the labeled compound. In contrast, T3-PAL labeled a 27-kDa band, the presence of which paralleled thyroid status. The labeling of only this protein was blocked by either substrates or enzyme inhibitors in a dose-dependent fashion, with a rank order of potency predicted by the activity of such compounds in type I enzyme assays. The specific nature of these competitions provides further evidence that this 27-kDa protein, identified in previous studies using N-bromoacetyl [125I]T3 or -T4, contains the active site of the rat type I deiodinase. This is in agreement with the mol wt of the rat type I deiodinase deduced from the recently identified cDNA coding for this protein.     

Photoaffinity labeling of rat androgen binding protein.

The photoinactivation and photoaffinity labeling of androgen binding sites present in cytosol prepared from intact sexually mature rat epididymides have been demonstrated by using unlabeled and [3H]-labeled 17 beta-hydroxy-4,6-androstadien-3-one. Both photoinactivation and photolabeling are dependent upon exposure to light. These processes are inhibited when photolysis is conducted in the presence of the photoinert compound 17 beta-hydroxy-5 alpha-androstan-3-one, suggesting that steroid-specific sites are involved in the reactions. The labeled steroid-specific product of photolysis is macromolecular with a molecular weight of 47,000 as determined by electrophoresis on polyacrylamide gels containing NaDodSO4, and proteinaceous because digestion of the cytosol with pronase before photolysis eliminates steroid-specific binding. After photolysis, the protein-steroid complex has the ability to withstand dissociation during electrophoresis under denaturing conditions, and unlabeled 17 beta-hydroxy-5 alpha-androstan-3-one fails to displace the label from the complex. Thus, the binding of [3H]17 beta-hydroxy-4,6-androstadien-3-one to the cytosolic protein is covalent. This steroid-specific product is identified as an androgen binding protein of testicular origin by its comigration with native androgen binding protein on nondenaturing polyacrylamide gels and by its molecular weight which is within the range reported for androgen binding protein subunits.     

Photoaffinity site-specific covalent labeling of human corticosteroid-binding globulin.

A method was developed for the synthesis of high-specific-activity 21-diazo-21-[6,7-(3)H]deoxycorticosterone, an analog of corticosterone. This analog was used as a photoaffinity label of a high affinity steroid-binding protein, human corticosteroid-binding globulin. Based on direct binding studies and crosscompetition experiments, this diazo derivative exhibited the requisite affinity (within a factor of 1.5 times that of corticosterone) and site specificity to qualify as an affinity labeling legand. Irradiation of corticosteroid-binding globulin with the 21-diazo derivative resulted in irreversible binding to corticosteroid-binding globulin, identified by polyacrylamide gel electrophoresis. Specificity of covalent binding to corticosteroid-binding globulin was established by competition analysis with various steroids. Irreversibility of photodependent binding was shown by persistence of the complex on electrophoresis (in contrast to the noncovalently linked complex), and resistance to exchange with corticosterone or pregnanediol and to solvent extraction. Site specificity of covalent binding was inferred from the effects of a scavenger, Tris-HC1, and fluorescence quenching of a neighboring tryptophan.     

Photoaffinity labeling of pituitary and gonadal receptors for gonadotropin-releasing hormone

A radioactive photoaffinity label for the GnRH receptor was prepared by derivatization of radiodinated [D-Lys6] des-Gly10-GnRH N-ethylamide with the heterobifunctional photolabile reagent N-hydroxysuccinimidyl-4-azido-benzoate. This high affinity photoreactive analog was employed for radiolabeling and characterization of pituitary GnRH receptors in rat, rabbit, mouse, sheep, and cow adenohypophyses and gonadal GnRH receptors in the rat ovary and testis. In rat, rabbit, and mouse pituitary glands, analysis of the GnRH receptor-ligand complex by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography showed two labeled components, both of which were displaced by unlabeled GnRH agonist and antagonist analogs. The larger receptor component was a relatively broad band, with mol wt in rat, rabbit, and mouse of 59,000 +/- 1,900, 62,000 +/- 700, and 60,000 +/- 800, respectively. In the rat pituitary gland, the larger component was composed of 63,000 and 52,000 mol wt components, of which the latter was more heavily labeled and was predominant in purified pituitary gonadotrophs. The mol wts of the smaller components were 40,000 +/- 1,600, 43,000 +/- 1,200, and 41,000 +/- 1,000, respectively. In bovine and ovine pituitary glands, the photolabeled GnRH receptor was a single band with mol wt of 42,000 +/- 1,200 and 39,000 +/- 500, respectively. In the rat ovary and testis, photolabeled GnRH receptors were similar to those in the rat pituitary gland, with two distinct components of 53,000 +/- 1,000 and 42,000 +/- 1,000 mol wt. These findings demonstrate that the pituitary receptors that mediate similar physiological actions of GnRH in different species possess broadly similar structural properties, with minor variations between species. It is also evident that the divergent actions of GnRH in different tissues of the same species, as in the rat pituitary and gonads, are expressed through receptors of similar structure.     

Photoaffinity labeling of Torpedo acetylcholine receptor at multiple sites.

The acetylcholine receptor from Torpedo californica electroplax was labeled with the photoaffinity reagent bis(3-azidopyridinium)decane perchlorate. All four receptor subunits (alpha, beta, gamma, and delta) were specifically labeled. In the presence of cholinergic agonists the gamma-, beta-, and delta-subunit labeling was decreased significantly, whereas labeling of the alpha subunit was minimally affected. Full occupancy of the two high-affinity sites involving the alpha subunits in the vicinity of alpha-Cys-192-Cys-193 by covalent reaction with bromoacetylcholine also caused a large decrease of gamma-subunit labeling by the photoaffinity reagent and lesser but significant decreases in beta- and delta-subunit labeling. No decrease in labeling of the alpha subunit was seen. Labeling of the alpha subunit could, however, be inhibited by high concentrations of the agonist carbamoylcholine. We conclude that the binding sites of high-affinity reside at interfaces of the alpha subunit and other subunits and that the alpha subunit also contributes to formation of a low-affinity site(s) for cholinergic compounds.     

Photoaffinity labeling of indole-3-acetic acid-binding proteins in maize

The photoaffinity labeling agent 5-azidoindole-3-acetic acid, an analog of the endogenous plant hormone indole-3-acetic acid (an auxin), was used to identify indole-3-acetic acid-binding proteins in maize. Two peptides with subunit molecular masses of 24 and 22 kilodaltons are specifically labeled in a saturable manner. Both peptides are slightly acidic and behave as dimers under nondenaturing conditions. The possibility that one of these peptides is the auxin receptor that mediates cell elongation in maize is discussed.     

Photoaffinity labeling of the androgen receptor from human skin fibroblasts

The reproducible photolabeling of the androgen receptor from human skin fibroblasts, using [3H]methyltrienolone (R-1881) as ligand is described. Crude nuclei were irradiated for 2 min using a UV lamp with an emission line at 352 nm and a CuSO4 filter. After KCl extraction, proteins were precipitated with trichloroacetic acid, washed with ether and assayed for radioactivity. Specific binding was determined as the difference in bound radioactivity between cells incubated with [3H]R-1881 +/- a 200-fold excess of unlabeled dihydrotestosterone (DHT). The photolabeled proteins were analyzed on SDS-polyacrylamide gel electrophoresis yielding one peak of 90 kDa and in several cases, one of 43 kDa. These peaks comprised 60 +/- 20% of the saturable binding recovered on the gels. The overall efficiency of photolabeling was between 1 and 5%. The amount of covalently bound radioactivity was proportional to the number of cells used. The labeling was inhibited by R-1881, DHT, the anti-androgens hydroxyflutamide and cyproterone acetate and to a lesser extent by estradiol and progesterone. No covalent attachment of R-1881 to any protein was observed when nuclei from patients with androgen insensitivity were irradiated, whether or not the cells were receptor positive or negative. In conclusion the androgen receptor from human skin fibroblast can be efficiently photolabeled and could be used as a marker to follow receptor purification. The absence of photolabeling of nuclear extracts from receptor-positive androgen-insensitive patients may reflect some abnormality of the receptor.     

Photoaffinity labeling of an herbicide receptor protein in chloroplast membranes

2-Azido-4-ethylamino-6-isopropylamino-s-triazine (azido-atrazine) inhibits photosynthetic electron transport at a site identical to that affected by atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine). The latter is a well-characterized inhibitor of photosystem II reactions. Azido-atrazine was used as a photoaffinity label to identify the herbicide receptor protein; UV irradiation of chloroplast thylakoids in the presence of azido[(14)C]atrazine resulted in the covalent attachment of radioactive inhibitor to thylakoid membranes isolated from pea seedlings and from a triazine-susceptible biotype of the weed Amaranthus hybridus. No covalent binding of azido-atrazine was observed for thylakoid membranes isolated from a naturally occurring triazine-resistant biotype of A. hybridus. Analysis of thylakoid polypeptides from both the susceptible and resistant A. hybridus biotypes by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, followed by fluorography to locate (14)C label, demonstrated specific association of the azido[(14)C]atrazine with polypeptides of the 34- to 32-kilodalton size class in susceptible but not in resistant membranes.     

Photoaffinity labeling of the somatostatin receptor: identification of molecular subtypes.

Pharmacological studies have suggested that the somatostatin (SS) receptor is heterogeneous and may exhibit subtypes selective for SS-14 and SS-28. Whether this heterogeneity can be explained by separate molecular forms of the receptor protein is unclear. In the present study, we have developed a novel photosensitive azido derivative of the octapeptide SS analog Tyr3 SMS (EE 581) and used it as a photoaffinity probe to characterize the molecular components of the SS receptor in five receptor positive tissues (normal rat brain, pituitary, pancreas, and adrenal cortex, and mouse AtT-20 pituitary tumor cells). [125I]EE-581 labeled specific high affinity binding sites in all these tissues (Kd range 1.3-1.67 nM). Photoaffinity labeled membrane SS receptors were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography. Three specifically labeled SS receptor proteins of 80 kilodaltons (kDa), 58 kDa, and 32 kDa were identified and exhibited a tissue-specific distribution. The 58 kDa species was the exclusive form in pancreas, adrenal cortex, and AtT-20 cells and the dominant form in brain. The 32 kDa receptor protein was expressed as a minor form (ratio of 58 kDa:32 kDa 3:1), exclusively in brain. The 80 kDa receptor was found only in the pituitary where it occurred as the sole SS receptor species. Competition experiments showed that the 58 kDa and 32 kDa receptor proteins in brain reacted with SS-14 greater than SS-28; in contrast, the 58 kDa protein in AtT-20 cells bound SS-28 greater than SS-14 suggesting the existence of distinct subtypes of the 58 kDa receptor in these two tissues. These data represent the first systematic evaluation of the molecular forms of SS receptor proteins by photoaffinity labeling in different target tissues and provide direct evidence for molecular heterogeneity and SS-14/SS-28 selectivity; a major 58 kDa protein present in most tissues, an additional 32 kDa protein uniquely expressed in brain, and an 80 kDa protein exclusive to the normal pituitary.     

Pulse Labeling of RNA of Mammalian Cells

When cells from a hypotetraploid strain of Ehrlich ascites tumor are exposed to uridine-³H either in vivo or in vitro, the amount of radioactivity incorporated into RNA reaches a maximum within ten minutes, after which any further incorporation stops. ³H-uridine triphosphate disappears from the acid soluble pool within 30 minutes and the findings indicate that the RNA of these cells can be pulse labeled without the use of any antibiotic or the need of a „chase.” The stability of the pulse labeled RNA in the presence of pentobarbital (an inhibitor of RNA synthesis) indicates the virtual absence of RNA breakdown. However, actinomycin D, at a dosage of 250 μg/mouse in vivo and 10 μg/ml in vitro produces breakdown of labeled RNA, thus confirming earlier observations that the drug is not a suitable tool for RNA kinetics determinations. The pulse-labeled RNA leaves the nucleus slowly and some radioactive RNA is still present in the nuclear fraction after 24 hours. Radioactivity begins to appear in cytoplasmic ribosomal RNA after 20 minutes and continues to increase up to six hours.     

Photoaffinity labeling of the multidrug-resistance-related P-glycoprotein with photoactive analogs of verapamil.

Verapamil, a phenylalkylamine calcium channel blocker, has been shown to reverse multidrug resistance in tumor cells, possibly by increasing drug retention through interaction with an outward drug transporter of the resistant cells. In this study two photoactive radioactive analogs of verapamil, N-(p-azido[3,5-3H]benzoyl)aminomethyl verapamil and N-(p-azido[3-125I]salicyl)aminomethyl verapamil, were synthesized and used to identify the possible biochemical target(s) for verapamil in multidrug-resistant DC-3F/VCRd-5L Chinese hamster lung cells selected for resistance to vincristine. The results show that a specifically labeled 150- to 180-kDa membrane protein in resistant cells was immunoprecipitated with a monoclonal antibody specific for P-glycoprotein. Phenylalkylamine binding specificity was established by competitive blocking of specific photolabeling with the nonradioactive photoactive analogs as well as with verapamil. Photoaffinity labeling was also inhibited by 50 microM concentrations of the calcium channel blockers nimodipine, nifedipine, nicardipine, azidopine, bepridil, and diltiazem and partially by prenylamine. Bay K8644, a calcium channel agonist, also inhibited P-glycoprotein photolabeling. Moreover, P-glycoprotein labeling was inhibited in a dose-dependent manner by vinblastine with half-maximal inhibition at 0.2 microM compared to that by verapamil at 8 microM. Photolabeling was also partially inhibited by two of the drugs to which these cells are cross-resistant, doxorubicin and actinomycin D, at 100 microM, but not by colchicine. These data provide direct evidence that P-glycoprotein has broad drug recognition capacity and that it serves as a molecular target for calcium channel blocker action in reversing multidrug resistance.     

Leukocyte Labeling With 51Chromium. III. The Kinetics of Normal Lymphocytes

The kinetics of radiochromate-labeledautologous blood lymphocytes werestudied in normal subjects. The labeledcells equilibrated within hours with arecirculating lymphocyte pool (RLP)distributed between the blood, thespleen, and the bone marrow. The RLPwas found to be about twice the sizeof the blood lymphocyte pool contained within it and turned overthrough the blood about 12 times daily.The labeled cells had an average halflife in the RLP of 18 days.

Submitted on October 13, 1971 Revised on January 24, 1972 Accepted on January 26, 1972