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PubMed1995

Potent photoaffinity labelled and iodinated antagonists of bradykinin.

Continuing the studies on photoaffinity labelled analogues of the peptide hormone bradykinin (BK), several labelled antagonists were synthesized and characterized regarding their biological activities on rat uterus (RUT) and guinea pig ileum (GPI). The photoreactive amino acid p-benzoyl-phenylalanine (Bpa) was incorporated in potent, iodinated BK analogues at positions -2, -1, 0 and 7. The newly synthesized BK antagonists were derived from HOE 140 ([DArg0, Hyp3, Thi5, D-Tic7, Oic8]-BK) or [D-Phe7]-BK. Because the application of Bpa requires an additional group for the introduction of 125I, iodinated tyrosine was inserted at different positions as a model for radioiodination. Suitable positions for incorporation of tyrosine residues are -1, 0, 3 and 7, whereas the compound with 3-I-Tyr at position 4 had only a low biological activity. The antagonists obtained by modification of HOE 140 generally retained a high antagonistic potency. In this group [D-Bpa-2, 3-I-D-Tyr-1, D-Arg0, Hyp3, Thi5, D-Tic7, Oic8]-BK (pA2 values 8.06 on RUT and 8.15 on GPI) and [Bpa-1, D-Arg0, 3-I-Tyr3, Thi5, D-Tic7, Oic8]-BK (pA2 values 7.55 on RUT and 8.07 on GPI) belong to the most active compounds. The incorporation of D-Bpa at position 7 also resulted in potent analogues. The antagonists [3-I-Tyr-1, D-Arg0, D-Bpa7]-BK (pA2 on RUT 7.69) and [3-I-Tyr-1, D-Arg0, D-Bpa7, Oic8]-BK (pA2 on GPI 7.53) are an alternative to the N-terminal modified HOE 140 analogues. Compounds with D-Bpa7 act as pure competitive antagonists, whereas the HOE 140 derivatives show a mixed antagonism. The comparison of the results between photoaffinity labelled agonists and antagonists suggests that modifications in the series of BK antagonists were better tolerated.

Affinity Labels↗
PubMed1984

Photoaffinity labeling and characterization of the cloned purine-cytosine transport system in Saccharomyces cerevisiae.

8-Azido[2-3H]adenine was used as a photoaffinity label for the purine-cytosine transport system. After irradiation in the presence of the photoaffinity label, the cells were converted into protoplasts, their plasma membranes were purified, and the membrane proteins were extracted and separated by NaDodSO4/PAGE. The radioactivity was specifically incorporated into a protein with a molecular weight of 120,000. Photoaffinity labeling of this protein could be blocked by irradiation in the presence of natural substrates for the transport system. The molecular weight as determined by NaDod-SO4/PAGE was found to be twice the value calculated from mRNA analysis of the cloned gene. Incubation of exponentially growing cells with tunicamycin, an antibiotic that inhibits glycosylation of proteins, resulted in a 40% decrease in the overall initial uptake rate, which correlates with the reduction of the labeled Mr 120,000 protein. Treatment of the extracted labeled plasma membrane proteins with glycosidic enzymes resulted in disappearance of the Mr 120,000 peak and the appearance of new peaks at Mr 60,000 and Mr 73,000. These findings indicate that the purine-cytosine transport protein is a glycoprotein.

Adenine↗
PubMed1989

Photoaffinity labeling with dihydropyridine derivatives of crude membranes from rat skeletal, cardiac, ileal, and uterine muscles and whole brain.

The characteristics of photoaffinity labeling with the calcium agonist [3H]Bay K 8644 (Bay) and the calcium antagonists [3H]nitrendipine (Nit) and (+)PN200-110 (PN) of crude membranes from rat skeletal, cardiac, ileal, and uterine muscles and whole brain were investigated. In all these crude membranes, [3H](+)PN (20 nM) was mainly photoincorporated into one protein band with a molecular weight of 30,000 - 41,000 Da. It was also incorporated into some other bands of all these crude membranes. The photoincorporation of [3H](+)PN into these crude membranes was inhibited by the presence of 20 microM unlabeled (+)PN. The photoincorporation of [3H](+)PN into these crude membranes depended on its dose and on the time of UV irradiation. No incorporation of [3H](+)PN was observed in the absence of UV irradiation. The incorporation was not affected by the presence of 1 mM CaCl2 and/or 0.15 M NaCl, but was significantly decreased by 20 microM (+)PN and slightly decreased by 20 microM (-)PN, 20 microM Bay, 1 mM diltiazem, or 1 mM verapamil. Namely, enantiomers of PN caused various extents of stereoselective inhibition of photoaffinity labeling by [3H](+)PN of specific protein bands in these crude membranes. [3H]Nit was photoincorporated into these crude membranes in the same way as [3H](+)PN, but [3H]Bay was not photoincorporated. However, 20 microM unlabeled Nit did not consistently inhibit photoaffinity labeling with [3H]Nit. These findings suggested that measurement of photoaffinity of crude membranes from rat skeletal, cardiac, and uterine muscles and whole brain with [3H](+)PN by UV irradiation is a useful method for investigating the characteristics of the voltage-dependent calcium channels that are affected by 1,4-dihydropyridine derivatives.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1987

Limited proteolysis alters the photoaffinity labeling of adenosine 3',5'-monophosphate dependent protein kinase II with 8-azidoadenosine 3',5'-monophosphate.

Photoaffinity labeling of the regulatory subunits of cAMP-dependent protein kinase with 8-azidoadenosine 3',5'-monophosphate (8-N3cAMP) has proved to be a very specific method for identifying amino acid residues that are in close proximity to the cAMP-binding sites. Each regulatory subunit contains two tandem cAMP-binding sites. The type II regulatory subunit (RII) from porcine heart was modified at a single site, Tyr-381 [Kerlavage, A., & Taylor, S.S. (1980) J. Biol. Chem. 255, 8483-8488]. When a proteolytic fragment of this RII subunit was photolabeled with 8-N3cAMP, two sites were covalently modified. One site corresponded to Tyr-381 and, thus, was analogous to the native RII. The other site of modification was identified as Tyr-196, which is not labeled in the native protein. Photoaffinity labeling was carried out in the presence of various analogues of cAMP that show a preference for one of the two tandem cAMP-binding sites. These studies established that the covalent modification of Tyr-381 was derived from 8-N3cAMP that was bound to the second cAMP-binding site (domain B) and that covalent modification to Tyr-196 was due to 8-N3cAMP that was bound to the first cAMP-binding site (domain A). These sites of covalent modification have been correlated with a model of each cAMP-binding site on the basis of the crystal structure of the catabolite gene activator protein (CAP), which is the major cAMP-binding protein in Escherichia coli.

Adenosine Triphosphate↗
PubMed1984

Photoaffinity labeling of thyroid hormone nuclear receptors. Influence of n-butyrate and analysis of the half-lives of the 57,000 and 47,000 molecular weight receptor forms.

The thyroid hormone receptor is a nuclear-associated protein which appears to mediate the actions of 3,5,3'-triiodo-L-thyronine (L-T3) and 3,5,3',5'-tetraiodo-L-thyronine (L-T4) in mammalian cells. In a previous study we reported that N-2-diazo-3,3,3-trifluoropropionyl-3,5,3'-triiodo-L-thyronine (L-T3-PAL) serves as an effective photoaffinity label probe of the receptor in GH1 cells, a growth hormone producting rat pituitary cell line. Irradiation of cells at 254 nm covalently cross-links L-[125I]T3-PAL to two molecular weight (Mr) nuclear receptor forms, an abundant 47,000 Mr component and a less abundant 57,000 Mr species (Pascual, A., Casanova, J., and Samuels, H. H. (1982) J. Biol. Chem. 257, 9640-9647). In this study we have explored a number of possible interrelationships of the different Mr receptor forms. Denaturing gel electrophoresis and autoradiography indicates that the 57,000 Mr form is a doublet species which differ in Mr by 1,000 to 2,000. The various receptor forms are not an artifact of the L-[125I]T3-PAL probe, and identical forms can be labeled at 310 nm using underivatized L-[125I]T4 with a 15-fold lower coupling efficiency. The 57,000 and 47,000 Mr receptor forms are not generated by indiscriminate proteolysis, UV peptide cleavage, or zero length protein-protein cross-linking by irradiation at 254 nm. Micrococcal nuclease excises both the 57,000 and 47,000 Mr forms, and receptor is not identified in the residual nuclear matrix fraction. Receptor is also not detected in the cytoplasmic fraction. By coupling dense amino acid labeling and photoaffinity labeling of receptor we determined a half-life of 2.4 h for the 57,000 Mr species and 5.6 h for the 47,000 Mr form while both species have similar relative synthetic rates. n-Butyrate has been previously shown to decrease receptor levels in GH1 cells. We demonstrate that n-butyrate decreases receptor levels primarily by shortening the half-life of the 47,000 Mr form.

Affinity Labels↗
PubMed1984

Purification of the photoaffinity-labeled glucagon receptor by gel electrophoretic methods.

Rat liver plasma membrane glucagon receptor has been purified with a yield of 0.01% to an estimated homogeneity of 32-60%, using a 2-stage electrophoretic procedure. SDS-solubilized membrane proteins labeled by the photoaffinity-agent, Ne-4-azidophenylamidinoglucagon (APA-glucagon), were separated by polyacrylamide gel electrophoresis in SDS-containing buffers. Gel slices corresponding to the molecular weight of the receptor were excised, electrophoretically extracted and concentrated. The concentrate was subjected to isoelectric focusing on Sephadex to yield a purified product in which the photoaffinity-labeled receptor, with a molecular weight of 56K and a pI' of 5.9, is the sole major component.

Affinity Labels↗
PubMed1992

Photoaffinity labeling of the organic cation/H+ exchanger in renal brush border membrane vesicles.

The brush border membrane of the proximal tubule contains two efflux pathways for organic cations from the cell to the tubular fluid: a P-glycoprotein and an organic cation/H+ exchanger. There is evidence that they transport many of the same substrates. Their structural relatedness is unknown and is the subject of this report. The experimental approach was to identify the exchanger with photoaffinity labeling reagents. The rationale was that if the P-glycoprotein and the organic cation/H+ exchanger transport many of the same substrates, then they might be photoaffinity labeled by the same reagents. [125I]Iodoarylazidoprazosin and [3H]azidopine are two reagents, which have been used, to photoaffinity label the P-glycoprotein. We found that several polypeptides were photolabeled in a time- and concentration-dependent manner. The photoincorporation into only two of these polypeptides (41 and 28 kDa) was blocked extensively by the presence of known substrates for the exchanger. The photoaffinity labeling of only the 41-kDa polypeptide was affected by treatment with the chemical reagents, N-ethylmaleimide and dithiothreitol, which are known to affect the exchanger reaction. The findings are consistent with the interpretation that a 41-kDa polypeptide is, or is a component of, the exchanger.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1990

The gamma 2 subunit is an integral component of the gamma-aminobutyric acidA receptor but the alpha 1 polypeptide is the principal site of the agonist benzodiazepine photoaffinity labeling reaction.

Polyclonal antibodies were raised to a synthetic peptide whose amino acid sequence was derived from the novel gamma-aminobutyric acidA (GABAA) receptor subunit, gamma 2. These anti-gamma 2 1-15 Cys antibodies reacted specifically with the GABAA receptor purified from adult bovine cerebral cortex in an enzyme-linked immunosorbent assay. Anti-gamma 2 1-15 Cys antibodies specifically immunoprecipitated [3H]flunitrazepam photoaffinity-labeled native receptor in parallel with anti-alpha 1 324-341 antibodies. Immunoprecipitation of sodium dodecyl sulphate (SDS) denatured photoaffinity-labeled receptor by anti-gamma 2 1-15 Cys antibodies, however, resulted in a significant decrease in the maximum percentage of radioactivity immunoprecipitated compared to that by anti-alpha 1 324-341 antibodies. In immunoblots, anti-gamma 2 1-15 Cys antibodies reacted with a broad band in the molecular weight range Mr 43,000-49,000 which was distinct from that recognized by anti-alpha 1 324-341 antibodies. The anti-alpha 1 324-341 immunoreactive band was the main subunit irreversibly photoaffinity labeled by [3H]flunitrazepam, i.e. Mr 53,000. These results demonstrate for the first time that the gamma 2 subunit is an integral component of the GABAA receptor but it is the alpha 1 subunit that is the principal site of the agonist benzodiazepine photoaffinity labeling reaction. It supports a role of both the alpha 1 and gamma 2 polypeptides in the formation of the central benzodiazepine binding site within a GABAA receptor oligomer.

Affinity Labels↗
PubMed1983

Photoaffinity labeling: theory and practice.

The potential for generating a reactive reagent in situ by photoexcitation and for labeling sites not containing reactive nucleophilic residues provides photoaffinity labeling with a special advantage in the study of ligand-binding sites. There are, however, problems in the application of the photoaffinity technique. A number of concerns about this approach are examined by using as a background studies with arylazido-beta-alanyl nucleotide probes developed in this laboratory. It is concluded that photoaffinity labeling is likely to emerge as one of the major techniques for elucidating structure-function relationships within biological membranes. In the future the design of photoprobes with shorter activation half-lives may make it possible to investigate time-dependent (i.e., conformational) changes occurring at the active site of enzymes and within complex ligand-binding sites.

Adenosine Triphosphate↗
PubMed1995

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.

Affinity Labels↗
PubMed1983

Photoaffinity-labelling of the glycine receptor of rat spinal cord.

The irreversible incorporation upon ultraviolet illumination of the glycine receptor antagonist, [3H]strychnine, into synaptic membrane fractions of rat spinal cord has been investigated. The specificity of this photoaffinity-labelling reaction for the glycine receptor was demonstrated by the following results: (a) the Kd value (9.7 nM) of the glycine-displaceable irreversible incorporation of [3H]strychnine was similar to the previously reported Kd of [3H]strychnine binding to the glycine receptor; (b) pre-illumination of the membranes with unlabelled strychnine led to a corresponding reduction in the number, but not the affinity, of reversible glycine-displaceable [3H]strychnine binding sites; (c) the ultraviolet light-induced incorporation into the membranes of [3H]strychnine was inhibited by different glycine receptor agonists; other neurotransmitter substances had little or no effect. Also, [3H]strychnine alone was shown to be stable upon illumination with ultraviolet light; this suggests that photocrosslinking of [3H]strychnine may require energy transfer from specific groups of its high-affinity receptor binding site. Upon sodium dodecyl sulphate/polyacrylamide gel electrophoresis a single labelled polypeptide with a relative molecular mass of 48000 was revealed from spinal cord membranes photoaffinity-labelled with [3H]strychnine. Spinal cord membranes photoaffinity-labelled with the gamma-aminobutyric acid receptor ligand [3H]flunitrazepam, however, gave a single polypeptide with a relative molecular mass of 5- 0000. Treatment of membranes, labelled with [3H]strychnine, by endoglycosidase H did not alter the relative molecular mass of the 48000-Mr labelled polypeptide. Trypsin treatment, on the other hand, successively produced major fragments of relative molecular masses of 42000 and 37000. Also, even after extensive treatment with trypsin or chymotrypsin, greater than or equal to 90% of the radioactivity incorporated into the labelled membranes remained membrane-associated. It is concluded that the strychnine binding site of the glycine receptor is located on a protease-inaccessible, i.e. probably hydrophobic domain of the 48000-Mr subunit.

Affinity Labels↗
PubMed1985

Photoaffinity labelling of androgen receptors with 17 beta-hydroxy-17 alpha-[3H]methyl-4,9,11-estratrien-3-one.

The synthetic androgen 17 beta-hydroxy-17 alpha-[3H]methyl-4,9,11-estratrien-3-one (R1881) has been used as photoaffinity label to characterize androgen receptors in calf uterus and rat prostate. Polyacrylamide gel electrophoresis under denaturing conditions showed that the DNA-binding form of the androgen receptor in calf uterus cytosol is a protein with a molecular mass of 98 kD. In rat prostate cytosol an androgen receptor with a molecular mass of 46 kD could be photoaffinity labelled with R1881. The photoaffinity labelling procedure described here provides a method for studying the hormone binding domain of androgen receptors in partial purified preparations.

Affinity Labels↗
PubMed1986

Photoaffinity labeling of the gonadotropin receptor with native, asialo, and deglycosylated choriogonadotropin.

Human choriogonadotropin (hCG) is a heterodimeric hormone composed of an alpha and a beta subunit. hCG and its asialo (ashCG) and deglycosylated (dghCG) forms vary in their ability to stimulate hormone responsive adenylate cyclase. ashCG is a partial agonist, and dghCG is an antagonist. Photoactivatable moieties were coupled to hCG, ashCG, and dghCG, and the derivatives were radioiodinated. Competitive binding studies indicate that all of the derivatives had a similar affinity for the gonadotropin receptor on porcine granulosa cell membranes. Radiolabeled derivatives were used to photoaffinity label the gonadotropin receptor. Radiolabeled complexes were separated by NaDodSO4/PAGE. All of the derivatives produced similar autoradiographic patterns, except that dghCG produced an additional 48-kDa complex. To investigate the structure of the complexes further, peptide mapping of proteolytic digests was used. All, except for the 48-kDa complex, generated similar peptide maps indicating a relationship between those complexes in which the smaller components are part of the larger. The 48-kDa complex contained both subunits of 40-kDa dghCG. Therefore, this complex is expected to contain an additional component of 8 kDa. The complex was generated whether the hormone-receptor complex was photoaffinity labeled on cells, on isolated membranes, or after solubilizing in detergent. Formation was blocked by excess hCG and did not occur in the absence of UV irradiation. We conclude that the hCG derivatives are able to photoaffinity label the hCG receptor but that the dghCG derivative can photoaffinity label an additional component that was not observed when derivatives of hCG or ashCG were used to label the receptor.

Adenylyl Cyclases↗
PubMed1992

Effects of GTP analogues and activation of endogenous protein kinases on photoaffinity labeling with [3H](+)PN200-110 of crude membranes from rat heart and brain.

The effects of GTP analogues and conditions in which various endogenous protein kinases were activated on photoaffinity labeling with [3H](+)PN200-110 (PN) of crude membranes from rat cardiac muscle and whole brain were investigated. Photoaffinity labeling with 20 nM [3H](+)PN of these crude membranes was decreased by 100 microM GTP-gamma-S, but not by 100 microM GTP or 100 microM GDP-beta-S. Similar results were obtained on the effects of GTP and its analogues on the specific binding of 20 nM [3H](+)PN to these crude membranes under the same conditions. Activation of endogenous protein kinases in these crude membranes did not influence the photoaffinity labeling with [3H](+)PN. These results suggested the binding sites, or DPH-sensitive, or L-type, calcium channels in curde membranes from rat cardiac muscle and whole brain are directly or indirectly modulated by endogenous GTP-binding protein, but not by various endogenous protein kinases in these crude membranes.

Affinity Labels↗
PubMed1985

8-Azidoflavins as photoaffinity labels for flavoproteins.

8-Azidoflavins have been synthesized and their potential as photoaffinity labels for flavoproteins has been explored. They are very photolabile, and in aqueous media they react with solvent to yield 8-aminoflavins and 8-hydroxlaminoflavins as the main products. They fulfill the criteria expected of a good photoaffinity label, since they bind stoichiometrically at the flavin-binding site of flavoproteins, thus minimizing problems of nonspecific labeling. Second, they absorb strongly in the visible, so that the reactive nitrene can be generated without short wavelength light, minimizing the possibility of light-induced damage of the protein. Third, in the absence of light, 8-N3-flavins are stable, permitting a study of their binding to apoproteins. 8-Azidoflavins have been bound to hen egg white riboflavin-binding protein, Megasphera elsdenii flavodoxin, yeast Old Yellow Enzyme, Aspergillus niger, glucose oxidase, and pig kidney D-amino acid oxidase, and the effect of exposure to visible light has been determined. Only small extents of covalent attachment of the flavin to the protein were found with flavodoxin, D-amino acid oxidase, and Old Yellow Enzyme; much more extensive labeling was obtained with glucose oxidase and riboflavin-binding protein. In addition to their photoreactivity, 8-azidoflavins have been found to be converted to 8-aminoflavins by reaction with sulfite or upon reduction. Similar reactions occur with 8-hydroxylamino-, 8-(O-methyl)hydroxylamino-, and 8-hydrazinoflavins, which serve as models for possible flavin-protein covalent linkages which could be formed in the photolabeling procedure. Some of the properties of these flavins, which were obtained by reaction of 8-F-flavin with the corresponding nucleophiles, are also described.

Affinity Labels↗
PubMed1988

Photoaffinity labeling of functional states of the nicotinic acetylcholine receptor.

The nicotinic acetylcholine receptor was subjected to photoaffinity labeling in different conformational and functional states. The photolabel used was the ion-channel blocker [3H]-TPMP+. A procedure is described for isolating labeled delta-polypeptide chains from the receptor complex by preparative SDS-polyacrylamide gel electrophoresis. The photolabel was localized in the primary structure of the delta-chain. The site of labeling was found to be identical when photoaffinity labeling was performed in the resting, desensitized, or antagonist state, respectively.

Affinity Labels↗