Activation of 5-[125I]iodonaphthyl-1-azide via excitation of fluorescent (N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)) lipid analogs in living cells. A potential tool for identification of compartment-specific proteins and proteins involved in intracellular transport and metabolism of lipids

We describe a new technique for analysis of proteins located near fluorescent lipid analogs in intact living cells using the membrane-permeant, photoactivatable probe, 5-[125I]iodonaphthyl-1-azide ([125I]INA). [125I] INA can be activated directly with UV light or indirectly through excitation of adjacent fluorophores (photosensitizers) with visible light to modify nearby proteins covalently with 125I. In this report we demonstrate that fluorescent phospholipids and sphingolipids containing N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-6-aminocaproic acid serve as appropriate photosensitizers for [125I]INA. Using Chinese hamster ovary fibroblasts, we optimized the labeling conditions with respect to lipid concentration and time of irradiation and then examined the profiles of cellular proteins that were labeled when fluorescent analogs of ceramide, sphingomyelin, and phosphatidic acid were used as photosensitizers in living cells. The use of different fluorescent lipids, which label different subcellular compartments of cells as determined by fluorescence microscopy, derivatized different sets of cellular proteins with 125I. The labeled proteins were subsets of the total set of proteins available for derivatization as determined by direct activation of [125I]INA. Most proteins labeled by this procedure were pelleted by centrifugation of cell lysates at high speed (260,000 x g), but several soluble proteins were also labeled under these conditions. The implications of using this technique for identification of compartment-specific proteins and proteins involved in lipid metabolism and transport are discussed.     

Photosensitized labeling of a functional multidrug transporter in living drug-resistant tumor cells

A 170,000-Da glycoprotein (P170 multidrug transporter) becomes specifically labeled in multidrug-resistant human KB carcinoma cells by the photolabile lipophilic membrane probe 5-[125I]iodonaphthalene-1-azide ([125I]INA) when photoactivation of the probe is triggered by energy transfer from intracellular doxorubicin or rhodamine 123. In contrast, in drug-sensitive cells, drug-induced specific labeling of membrane proteins with [125I]INA was not observed. Instead, multiple membrane proteins became labeled in a nonspecific manner. This phenomenon of drug-induced specific labeling of P170 by [125I]INA is observed only in living cells, but not in purified membrane vesicles or lysed cells. It is generated by doxorubicin and rhodamine 123, drugs that are chromophores and to which the cells exhibit resistance; but it is not observed with other drugs or dyes. Verapamil, a calcium channel blocker which reverses resistance to doxorubicin, also abolishes doxorubicin-induced specific [125I]INA labeling of P170. These results reveal that a specific interaction between P170 and doxorubicin takes place in living cells and demonstrate that P170 is directly involved in the mechanism of drug resistance in vivo. They also provide a possible means to label functional domains in the multidrug transporter. The results demonstrate that photosensitized [125I]INA labeling is a technique which provides sufficient spatial and time resolution to detect specific intracellular interactions between chromophores and proteins in vivo.     

Selective radiolabeling of cell surface proteins to a high specific activity

A procedure was developed for selective radiolabeling of membrane proteins on cells to higher specific activities than possible with available techniques. Cell surface amino groups were derivatized with 125I-(hydroxyphenyl)propionyl groups via 125I-sulfosuccinimidyl (hydroxyphenyl)propionate (125I-sulfo-SHPP). This reagent preferentially labeled membrane proteins exposed at the cell surface of erythrocytes as assessed by the degree of radiolabel incorporation into erythrocyte ghost proteins and hemoglobin. Comparison with the lactoperoxidase-[125I]iodide labeling technique revealed that 125I-sulfo-SHPP labeled cell surface proteins to a much higher specific activity and hemoglobin to a much lower specific activity. Additionally, this reagent was used for selective radiolabeling of membrane proteins on the cytoplasmic face of the plasma membrane by blocking exofacial amino groups with uniodinated sulfo-SHPP, lysing the cells, and then incubating them with 125I-sulfo-SHPP. Exclusive labeling of either side of the plasma membrane was demonstrated by the labeling of some marker proteins with well-defined spatial orientations on erythrocytes. Transmembrane proteins such as the epidermal growth factor receptor on cultured cells could also be labeled differentially from either side of the plasma membrane.     

14C-methylamine-glutaraldehyde conjugation as an alternative to iodination for protein labeling

Radiolabeling of native proteins conventionally has required iodination using 125Iodine (125I). Although radioiodination can result in high specific activity, there are several drawbacks in the use of 125I (e.g., radiological hazards and short half-life). 14C-Methylamine-glutaraldehyde conjugation to proteins offers an alternative for radiolabeling of proteins that is safer and longer-lived alpha-2-Macroglobulin was radiolabeled by conjugation to a 14C-methylamine-glutaraldehyde conjugate. Analysis of the labeling procedure was performed using scintillation counting, gel filtration chromatography, and protein assays. The radiolabeled alpha-2-macroglobulin was activated using established protocols and tested for functional integrity using competitive binding assays in the presence of recombinant receptor associated protein, an alternative ligand for the alpha-2-macroglobulin cellular receptor. The function of alpha-2-macroglobulin was unaffected by the labeling procedure. Comparison of 14C-methylamine-labeling and iodination by Scatchard analysis yielded nonlinear plots that suggested the presence of two sets of receptors with different binding affinities but that do not show cooperativity. This technique offers an alternative to radioiodination for the sensitive labeling of proteins.     

Diazontized (125I) diiodosulafanilic acid as a label for cell surface membranes. Studies on erythrocytes.

1. Diazotized 2,6-diiodosulfanilic acid (DDISA) appears to have properties suitable to serve as an artificial, non-penetrating label of cell surface membranes. Therefore, the conditions for selective labeling of cell surface membranes as compared to intracellular proteins as well as a method for its chemical determination were explored in the present study. 2. DDISA reacts with alpha-naphthol at neutral pH to produce a compound (1-hydroxy-4-(2,6-diiodo-4-sulfo-1-phenylazo-(naphthylene)), DSPN) with a characteristic spectrum in the visible range (Amax 430 nm). The absorbance of the reaction product, DSPN, is linearly proportional to the concentration of DDISA and can be used as a method for the colorimetric determination of DDISA. Reaction of DDISA with a molar excess of alpha-naphthol was also used as a method for inactivating unreacted DDISA to terminate labeling prior to cell fractionation. 3. [125I]DDISA reacts avidly with a variety of basic, neutral and acidic proteins as well as with cell membranes to form an acid-stable covalent azo linkage. 4. Effectiveness of labeling of the surface membrane of intact erythrocytes after incubation with [125I]DDISA was assessed by th ratio of 125I incorporated into membrane proteins compared to intracellular proteins. When intact erythrocytes were exposed to [125I]DDISA, the optimal labeling of membranes occurred at 37 degrees C after 20 min of incubation time and at a concentration of 10(-4) M [125I]DDISA in the incubation media. Under these conditions the ratio of the specific activity (cpm 125I/mg protein) of the membrane fraction to the specific activity of the soluble protein fraction (membrane/supernatant ratio) was greater than 500. When incubations were conducted at 4 degrees C this ratio was less than 50. However, when osmotically lysed erythrocytes were incubated with [125I]DDISA the majority of the label reacted with the soluble protein fraction resulting in a membrane/supernatant ratio of 0.14. 5. The results thus suggest that [125I]DDISA used under the appropriate incubation conditions, including the inactivation and removal of [125I]DDISA by washing with alpha-naphthol, can serve as a highly selective membrane label with minimal incorporation into intracellular soluble proteins. The general applicability of this method for other cell types remains to be explored.     

Two classes of phosphotyrosine-containing proteins detected by labeling with 32P and 125I in HeLa cells

There are two classes of proteins that can be phosphorylated on tyrosine in HeLa cells. One class can be detected by metabolic labeling with [32P]Pi and affinity chromatography using anti-phosphotyrosine antibodies. The other cannot be detected by this technique but can be detected among the proteins which bind to the antibodies by in vitro iodination with 125I. Presumably proteins of the second class contain phosphotyrosine at which the phosphate undergoes very slow turnover. The incubation of cells in phosphate-minus medium caused a marked reduction in the levels of phosphotyrosine-containing proteins, this explaining the failure of detection of the second class proteins even after prolonged labeling with [32P]Pi.     

Isolation of a domain of the plasma membrane in Chinese hamster ovary cells which contains iodinatable, acidic glycoproteins of high molecular weight

A light vesicle fraction, apparently derived from the plasma membrane, was obtained following breakage of Chinese hamster ovary (CHO) cells by means of a fluid pump disrupting device. The final preparation was enriched approx. 40-fold over the homogenate in K+,Na+-stimulated ATPase and phosphodiesterase I, but only approx. 10-fold in 125I specific radioactivity after lactoperoxidase-catalyzed iodination. This preparation was compared with another plasma membrane fraction purified as large sheets via a two-phase centrifugation procedure. Two-dimensional polyacrylamide gel electrophoresis followed by Coomassie blue staining indicated that both fractions were fairly similar in polypeptide composition, although a few consistent differences were evident. However, staining of glycoproteins by the periodic acid-Schiff technique or by overlaying with 125I-labeled concanavalin A showed that the vesicle fraction was highly enriched in groups of high molecular weight, acidic glycoproteins which stain only weakly with Coomassie blue. These glycoproteins also bound 125I-labeled ricin I agglutinin and wheat germ agglutinin. They appear to be the major receptors for wheat germ agglutinin on the CHO cell surface. After surface labeling of cells by the 125I-lactoperoxidase technique, the membrane sheet fraction contained a large number of iodinated polypeptides, whereas labeling in the vesicle fraction was restricted almost entirely to the high molecular weight, acidic glycoproteins. It is proposed that the vesicle fraction constitutes a specific domain of the cell surface which is coated on its exterior by this group of glycoproteins. These components probably mask underlying proteins of the plasma membrane from external labeling.     

Rat liver type I iodothyronine deiodinase is not identical to protein disulfide isomerase

This study was done to test the recent hypothesis (Boado et al. (1988) Biochem. Biophys. Res. Commun. 155, 1297-1304) that type I iodothyronine deiodinase (ID-I) is identical to protein disulfide isomerase (PDI). Autoradiograms of rat liver microsomal proteins, labeled with N-bromoacetyl-[125I]triiodothyronine (BrAc[125I]T3) and separated by SDS-PAGE, show predominantly 2 radioactive bands of Mr 27 and 56 kDa. Substrates and inhibitors of ID-I inhibited labeling of the 27 kDa band but not that of the 56 kDa band. Treatment of microsomes with trypsin abolished labeling of the 27 kDa protein and destroyed the activity of ID-I but did not prevent labeling of the 56 kDa protein. Following treatment of microsomes at pH 8.0-9.5 or with 0.05% deoxycholate (DOC) PDI content and labeling of the 56 kDa protein were strongly diminished but ID-I activity and labeling of the 27 kDa protein were not affected. The latter decreased in parallel after treatment at pH greater than or equal to 10. Rat pancreas microsomes contain high amounts of PDI but show no ID-I activity. Reaction of these microsomes with BrAc[125I]T3 results in extensive labeling of a 56 kDa protein but no labeling of a 27 kDa protein. Pure PDI (Mr 56 kDa) was readily labeled by BrAc[125I]T3 but showed no deiodinase activity. These results strongly suggest that the 27 kDa band represents (a subunit of) ID-I while the 56 kDa band represents PDI. From these and other data it is concluded that PDI and ID-I are not identical proteins.     

Detection of nearest neighbors to specific fluorescently tagged ligands in rod outer segment and lymphocyte plasma membranes by photosensitization of 5-iodonaphthyl 1-azide

Lima bean agglutinin-fluorescein 5-isothiocyanate conjugate (FluNCS-lima bean lectin) interacts with specific receptor molecules on membranes both from the rod outer segment (ROS) of the frog retina and from S49 mouse lymphoma cells. When [125I]-5-iodonaphthyl 1-azide (125I-INA), which freely and randomly partitions into the lipid bilayer, is added to membranes and the suspension is irradiated at 480 nm, the FluNCS-conjugated lectin photosensitizes the [125I]INA but only at discrete sites. This results in the selective labeling of specific proteins: an 88-kDa protein on ROS membranes and a 56-kDa protein on S49 plasma membranes. Labeling is dependent upon the interaction of the FluNCS-lectin with glycosylated receptor sites, since N-acetylgalactosamine, but not methyl alpha-mannoside, blocked labeling of the 56-kDa protein on S49 membranes. In contrast, a random labeling pattern of membrane proteins was observed upon irradiation at 480 nm using other fluorescein conjugates, such as FluNCS-bovine serum albumin (FluNCS-BSA) or FluNCS-soybean trypsin inhibitor (FluNCS-STI), which interact with cell membranes in a nonselective manner, or with N-(fluorescein-5-thiocarbamoyl)-n-undecyclamine (FluNCS-NHC11), which is freely miscible in the membrane lipid. Random labeling was also obtained by direct photoexcitation of [125I]INA at 314 nm, with no distinct labeling of the 88- and 56-kDa proteins in the respective membranes. These results suggest that protein ligands can be used to guide sensitizers to discrete receptor sites and lead to their selective labeling by photosensitized activation of [125I]INA [Raviv, Y., Salomon, Y., Gitler, C., & Bercovici, T. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 6103-6107].(ABSTRACT TRUNCATED AT 250 WORDS)     

Photolabeling identifies an interaction between phosphatidylcholine and glycerol-3-phosphate dehydrogenase (Gut2p) in yeast mitochondria

In search of mitochondrial proteins interacting with phosphatidylcholine (PC), a photolabeling approach was applied, in which photoactivatable probes were incorporated into isolated yeast mitochondria. Only a limited number of proteins were labeled upon photoactivation, using either the PC analogue [125I]TID-PC or the small hydrophobic probe [125I]TID-BE. The most prominent difference was the very specific labeling of a 70 kDa protein by [125I]TID-PC. Mass spectrometric analysis of a tryptic digest of the corresponding 2D-gel spot identified the protein as the GUT2 gene product, the FAD-dependent mitochondrial glycerol-3-phosphate dehydrogenase. This was confirmed by the lack of specific labeling in mitochondria from a gut2 deletion strain. Only under conditions where the inner membrane was accessible to the probe, Gut2p was labeled by [125I]TID-PC, in parallel with increased labeling of the phosphate carrier (P(i)C) in the inner membrane. A hemagglutinin-tagged version of Gut2p was shown to be membrane-bound. Carbonate extraction released the protein from the membrane, whereas a high concentration of NaCl did not, demonstrating that Gut2p is a peripheral membrane protein bound to the inner membrane via hydrophobic interactions. The significance of the observed interactions between Gut2p and PC is discussed.     

Melatonin binding proteins identified in the rat brain by affinity labeling

N-Bromoacetyl-2-iodo-5-methoxytryptamine (BIM), a novel derivative of the biologically active melatonin analog, 2-iodomelatonin, was prepared and used to identify melatonin binding proteins in rat brain synaptosomes. Incubation of the synaptosomes with BIM resulted in a time and concentration dependent, irreversible inhibition of 2-[125I]iodomelatonin binding. In parallel, the radioactive form of BIM, N-bromoacetyl-2-[125I]iodo-5-methoxytryptamine ([125I]BIM) became incorporated into the synaptosomes. The incorporation of [125I]BIM was inhibited by BIM, 2-iodomelatonin and melatonin but not by 5-methoxytryptamine or N-acetyl serotonin. [125I]BIM became covalently attached to three polypeptides with apparent molecular weight values of 92, 55 and 45 kDa; the labeling of all three proteins was markedly inhibited by melatonin. These results indicate that the 92, 55 and 45 kDa polypeptides are melatonin binding proteins.     

Synthesis and properties of sulfhydryl-group-specific reagents containing 125I.

125I-containing compounds that react specifically with sulfhydryl groups were prepared in yields of 30 to 40% on the basis of starting 125I quantity. The synthetic precursors were commercially available heterobifunctional crosslinkers and the peptide L-arginyl-L-tyrosine. Two types of sulfhydryl specific reagents were prepared: 3-(2-pyridyldithio)propionylarginyl-[125I]-monoiodotyrosine, which permits reversible incorporation of 125I at sulfhydryl sites, and 3-maleimidopropionylarginyl- [125I]monoiodotyrosine, an irreversible labeling reagent. These products were isolated in a highly radiochemically pure form by C18 HPLC. The second-order rate constants for the reaction of 3-(2-pyridyldithio)propionylarginylmonoiodotyrosine and 3-maleimidopropionylarginylmonoiodotyrosine with N-acetylcysteine were 28 +/- 3 M-1 s-1 and 154 +/- 4 M-1 s-1, respectively, at pH 7.3. Storage of carrier-free 3-(2-pyridyldithio)propionylarginyl-[125I]monoiodotyrosine and 3-maleimidopropionylarginyl-[125I]monoiodotyrosine at -80 degrees C at a radioactive concentration of 0.4 mCi/ml resulted in conversion of 125I to species that did not react covalently with sulfhydryl groups. This process occurred with first-order kinetics and a t1/2 of 5.7 days for the pyridyldithio compound and 7.5 days for the maleimido compound. No conversion was observed during storage at -80 degrees C at radioactive concentrations of 0.02 mCi/ml or less. The labeling properties of these compounds were examined using red blood cell proteins as a test system. 3-(2-Pyridyldithio)propionylarginyl- [125I]monoiodotyrosine and maleimidopropionylarginyl-[125I]monoiodotyrosine reacted preferentially with membrane - associated sulfhydryl groups when incubated with intact red blood cells.     

Reagents for astatination of biomolecules. 2. Conjugation of anionic boron cage pendant groups to a protein provides a method for direct labeling that is stable to in vivo deastatination

Cancer-targeting biomolecules labeled with 211At must be stable to in vivo deastatination, as control of the 211At distribution is critical due to the highly toxic nature of alpha-particle emission. Unfortunately, no astatinated aryl conjugates have shown in vivo stability toward deastatination when (relatively) rapidly metabolized proteins, such as monoclonal antibody Fab' fragments, are labeled. As a means of increasing the in vivo stability of 211At-labeled proteins, we have been investigating antibody conjugates of boron cage moieties. In this investigation, protein-reactive derivatives containing a nido-carborane (2), a bis-nido-carborane derivative (Venus Flytrap Complex, 3), and four 2-nonahydro-closo-decaborate(2-) derivatives (4-7) were prepared and conjugated with an antibody Fab' fragment such that subsequent astatination and in vivo tissue distributions could be obtained. To aid in determination of stability toward in vivo deastatination, the Fab'-borane conjugates were also labeled with 125I, and that material was coinjected with the 211At-labeled Fab'. For comparison, direct labeling of the Fab' with 125I and 211At was conducted. Direct labeling with Na[125I]I and Chloramine-T gave an 89% radiochemical yield. However, direct labeling of the Fab' with Na[211At]At and Chloramine-T resulted in a yield of <1% after quenching with NaS2O5. As another comparison, the same Fab' was conjugated with p-[211At]astatobenzoate NHS ester, [211At]1c-Fab', and (separately) with p-[125I]iodobenzoate NHS ester, [125I]1b-Fab'. An evaluation in athymic mice demonstrated that [211At]1c-Fab' underwent deastatination. In contrast, the high in vivo stability of [125I]1b-Fab' allowed it to be used as a tracer control for the natural distribution of Fab'. Although found to be much more stable in vivo than [211At]1c-Fab', the biodistributions of nido-carborane conjugated Fab' ([125I]2-Fab'/ [211At]2-Fab') and the bis-nido-carborane (VFC) ([125I]3-Fab'/[211At]3-Fab') had very different in vivo distributions than the control [125I]1b-Fab'. Biodistributions of closo-decaborate(2-) conjugates ([125I]4-Fab'/[211At]4-Fab', [125I]6-Fab'/[211At]6-Fab', and [125I]7-Fab'/[211At]7-Fab') demonstrated that they were stable to in vivo deastatination and had distributions similar to that of the control [125I]1b-Fab'. In contrast, a benzyl-modified closo-decaborate(2-) derivative evaluated in vivo ([125I]5-Fab'/[211At]5-Fab') had a very different tissue distribution from the control. This study has shown that astatinated protein conjugates of closo-decaborate(2-) are quite stable to in vivo deastatination and that some derivatives have little effect on the distribution of Fab'. Additionally, direct 211At labeling of Fab' conjugated with closo-decaborate(2-) derivatives provide very high (e.g., 58-75%) radiochemical yields. However, in vivo data also indicate that the closo-decaborate(2-) may cause some retention of radioactivity in the liver. Studies to optimize the closo-decaborate(2-) conjugates for protein labeling are underway.     

120- and 160-kDa receptors for endogenous mitogenic peptide, phytosulfokine-alpha, in rice plasma membranes

Plant cells in culture secrete a sulfated peptide named phytosulfokine-alpha (PSK-alpha), and this peptide induces the cell division and/or cell differentiation by means of specific high and low affinity receptors. Putative receptor proteins for this autocrine type growth factor were identified by photoaffinity labeling of plasma membrane fractions derived from rice suspension cells. Incubation of membranes with a photoactivable (125)I-labeled PSK-alpha analog, [N(epsilon)-(4-azidosalicyl)Lys(5)]PSK-alpha (AS-PSK-alpha), followed by UV irradiation resulted in specific labeling of 120- and 160-kDa bands in SDS-polyacrylamide gel electrophoresis. The labeling of both bands was completely inhibited by unlabeled PSK-alpha and partially decreased by PSK-alpha analogs possessing moderate binding activities. In contrast, PSK-alpha analogs that have no biological activity showed no competition for (125)I-AS-PSK-alpha binding, confirming the specificity of binding proteins. Analysis of the affinity of (125)I incorporation into the protein by ligand saturation experiments gave apparent K(d) values of 5.0 nm for the 120-kDa band and 5.4 nm for the 160-kDa band, suggesting that both proteins correspond to the high affinity binding site. Treatment of (125)I-AS-PSK-alpha cross-linked proteins with peptide N-glycosidase F demonstrated that both proteins contained approximately 10 kDa of N-linked oligosaccharides. Specific cross-linking of (125)I-AS-PSK-alpha was also observed by using plasma membranes derived from carrot and tobacco cells, indicating the widespread occurrence of the binding proteins. Together, these data suggest that the 120- and 160-kDa proteins are PSK-alpha receptors that mediate the biological activities of PSK-alpha.     

Photoaffinity labeling of the monoamine transporter of bovine chromaffin granules and other monoamine storage vesicles using 7-azido-8-[125I]iodoketanserin

An iodinated azido derivative of ketanserin, 7-azido-8-[125I]iodoketanserin ( [125I]AZIK), has been used to label the monoamine transporter of bovine chromaffin granule membranes by the technique of photoaffinity labeling. In the dark, this derivative was found to bind reversibly to the membranes, with an equilibrium dissociation constant estimated to be 6 nM at 0 degrees C. As for ketanserin, binding occurred at the tetrabenazine site: (i) [125I]AZIK was displaced efficiently from its binding site by tetrabenazine, ketanserin, and 7-azidoketanserin, whereas serotonin, which is a substrate for the transporter but has a low affinity for tetrabenazine binding site, was a poor displacer; pipamperone and pyrilamine, two antagonists of respectively serotonin S2 and histamine H1 receptors, were inactive. (ii) 7-Azidoketanserin was a competitive inhibitor of [3H]dihydrotetrabenazine binding, and it inhibited the ATP-dependent uptake of serotonin by chromaffin granule ghosts. Irradiation of [125I]AZIK with long-wavelength UV light, followed by electrophoresis on sodium dodecyl sulfate/polyacrylamide gels and autoradiography, revealed irreversible labeling of a membrane component with an apparent molecular weight of 73,000. Tetrabenazine inhibited the labeling of this 73-kDa band in a manner parallel to the binding of [125I]AZIK in the dark. Such a labeling is totally compatible with previous results obtained through photolabeling with a tetrabenazine derivative or by target size analysis. Moreover, preliminary experiments showed that [125I]AZIK can label the tetrabenazine binding sites of various sources including rat striatum, rabbit platelets, human pheochromocytoma, and human adrenal medulla. Therefore, this molecule appears to be an excellent probe to label the monoamine transporter of different amine storage vesicles even without purification.     

Selective affinity labeling of a 27-kDa integral membrane protein in rat liver and kidney with N-bromoacetyl derivatives of L-thyroxine and 3,5,3'-triiodo-L-thyronine

125I-Labeled N-bromoacetyl derivatives of L-thyroxine and L-triiodothyronine were used as alkylating affinity labels to identify rat liver and kidney microsomal membrane proteins which specifically bind thyroid hormones. Affinity label incorporation was analyzed by ethanol precipitation and individual affinity labeled proteins were identified by autoradiography after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Six to eight membrane proteins ranging in size from 17 to 84 kDa were affinity labeled by both bromoacetyl-L-thyroxine (BrAcT4) and bromoacetyl-L-triiodothyronine (BrAcT3). Affinity labeling was time- and temperature-dependent, and both reduced dithiols and detergents increased affinity labeling, predominantly in a 27-kDa protein(s). Up to 80% of the affinity label was associated with a 27-kDa protein (p27) under optimal conditions. Affinity labeling of p27 by 0.4 nM BrAc[125I]L-T4 was blocked by 0.1 microM of the alkylating ligands BrAcT4, BrAcT3, or 100 microM iodoacetate, by 10 microM concentrations of the non-alkylating, reversible ligands N-acetyl-L-thyroxine, 3,3',5'-triiodothyronine, 3,5-diiodosalicylate, and EMD 21388, a T4-antagonistic flavonoid. Neither 10 microM L-T4, nor 10 microM N-acetyltriiodothyronine or 10 microM L-triiodothyronine blocked affinity labeling of p27 or other affinity labeled bands. Affinity labeling of a 17-kDa band was partially inhibited by excess of the alkylating ligands BrAcT4, BrAcT3, and iodoacetate, but labeling of other minor bands was not blocked by excess of the competitors. BrAc[125I]T4 yielded higher affinity label incorporation than BrAc[125I]T3, although similar banding patterns were observed, except that BrAcT3 affinity labeled more intensely a 58,000-Da band in liver and a 53,000-55,000-Da band in kidney. The pattern of other affinity labeled proteins with p27 as the predominant band was similar in liver and kidney. Peptide mapping of affinity labeled p27 and p55 bands by chemical cleavage and protease fragmentation revealed no common bands excluding that p27 is a degradation product of p55. These data indicate that N-bromoacetyl derivatives of T4 and T3 affinity label a limited but similar constellation of membrane proteins with BrAcT4 incorporation greater than that of BrAcT3. One membrane protein (p27) of low abundance (2-5 pmol/mg microsomal protein) with a reactive sulfhydryl group is selectively labeled under conditions identical to those used to measure thyroid hormone 5'-deiodination. Only p27 showed differential affinity labeling in the presence of noncovalently bound inhibitors or substrates on 5'-deiodinase suggesting that p27 is likely to be a component of type I 5'-deiodinase in rat liver and kidney.     

Thyroid hormone effect on rat heart mitochondrial proteins and affinity labeling with N-bromoacetyl-3,3',5-triiodo-L-thyronine. Lack of direct effect on the adenine nucleotide translocase

N-bromoacetyl-3,3',5-tri[3'-125I]iodo-L-thyronine was used to label intact heart mitochondria from eu, hypo- and hyperthyroid rats in order to identify proteins involved in T3-regulated mitochondrial processes. The results show strong labeling, competed for by T3 and other analogues, of two proteins with a molecular mass of 48,000 and 49,200 Da. No labeling is seen of the adenine nucleotide translocase, a likely target, neither at 0 degree C, at room temperature, nor after preincubation with the substrates or specific inhibitors. No difference in labeling intensity or distribution is seen in mitochondria from eu-, hypo- or hyperthyroid rats, and the abundance of the adenine nucleotide translocase is unchanged, but five other proteins show differential abundance.     

Affinity labeling of the carbohydrate binding site of the lectin discoidin I using a photoactivatable radioiodinated monosaccharide

N-(4-Azidosalicyl)galactosamine (GalNASA), a photoactivatable, radioiodinatable analogue of N-acetylgalactosamine (GalNAc), has been prepared and characterized. We have used this reagent for labeling of the carbohydrate binding site of discoidin I, an endogenous lectin produced by Dictyostelium discoideum. GalNASA behaved as a ligand for discoidin I, as judged by its ability to compete in an assay measuring the carbohydrate binding activity of discoidin I. In this assay, it exhibited a Ki,app of 800 microM, comparable to that of GalNAc. The Ki,app of GalNASA decreased to 40 microM upon prior photolysis with ultraviolet light. In contrast, N-(4-azidosalicyl)ethanolamine produced no inhibition of carbohydrate binding regardless of photolysis. Covalent labeling of discoidin I with 125I-GalNASA was entirely dependent upon ultraviolet light. A portion of the labeling, representing 40-60% of the total, was sensitive to reagents which were known to inhibit carbohydrate binding by discoidin I, including GalNAc, asialofetuin, and ethylenediaminetetraacetic acid. N-Acetylglucosamine, which is not a ligand of discoidin I, was without effect. As a control, no carbohydrate-sensitive labeling was observed upon incubation of 125I-GalNASA with bovine serum albumin. The carbohydrate-sensitive fraction of discoidin I photolabeling with 125I-GalNASA exhibited a Kd of 15-40 microM, in agreement with the Ki,app of prephotolyzed GalNASA observed in the carbohydrate binding assay. Some labeling occurred if 125I-GalNASA was photolyzed prior to incubation with discoidin I, suggesting the involvement of long-lived species in the labeling reaction. Partial proteolytic digestion of photolabeled discoidin I revealed specific fragments whose labeling was completely blocked by GalNAc.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of thyroid hormone receptors in rat liver nuclei by photoaffinity labeling with L-thyroxine and triiodo-L-thyronine

Photoaffinity labeling of rat liver nuclear extract with underivatized thyroid hormones was performed after incubation with 1 nM [3',5'-125I]thyroxine ([125I]T4) or [3'-125I]triiodothyronine [( 125I]T3) by irradiation with light above 300 nm. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the covalently photolabeled nuclear extract revealed four distinct hormone binding proteins of molecular masses 96, 56, 45, and 35 kilodaltons (kDa), respectively. Distribution of the hormone among these proteins was similar for T4 and T3. The 56- and 45-kDa proteins were the most prominently labeled. The specificity of the photoattachment of thyroid hormones to these nuclear proteins was verified by the irradiation of eight randomly chosen proteins and two proteins known to have thyroid hormone binding sites, human thyroxine binding globulin and bovine serum albumin. Only the latter two were photolabeled with [125I]T4. Competition studies performed by incubating nuclear extracts with [125I]T4 or [125I]T3 in the presence of increasing amounts of the corresponding unlabeled hormone (10-, 100-, and 1000-fold molar excess) demonstrated that (1) photoattachment of labeled T3 or T4 to the 56- and 45-kDa proteins was inhibited by 67-78% and 73-85%, respectively, after incubation with a 1000-fold molar excess of unlabeled hormone, (2) in the presence of lower molar excesses of the corresponding competitor (10- and 100-fold), photoattachment of labeled T3 or T4 to the 56- and 45-kDa receptors was gradually inhibited to a similar extent on both proteins, and (3) the 35- and 96-kDa proteins, although having thyroid hormone binding sites, display lower binding activities since the inhibition of photoattachment of labeled T3 or T4 by a 1000-fold molar excess of unlabeled hormone did not exceed 30-42% and 26-49%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)