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PubMedDate not supplied

Identification of bacterial periplasmic glycine betaine-binding protein after electrophoresis and affinity labeling.

Antibodies were elicited in rabbits against periplasmic proteins obtained by cold osmotic shock from the Gram-negative eubacterium Rhizobium meliloti. When analyzed by crossed immunoelectrophoresis (CIE), the periplasmic proteins gave rise to 20 distinct immunoprecipitates corresponding to the same number of bands in polyacrylamide gel electrophoresis (PAGE) under non-denaturing conditions and in SDS-PAGE. The periplasmic glycine betaine-binding protein (GB-BP) was identified by autoradiography after affinity labeling with [14C]glycine betaine in PAGE and in CIE gels. The binding proved to be quite specific to glycine betaine, since the GB-BP was not labeled by choline (a metabolic precursor of glycine betaine in Escherichia coli and Rhizobium meliloti) and 15 distinct L-amino acids, including L-proline which, like glycine betaine is also an osmoprotectant. Affinity labeling of the GB-BP with [14C]glycine betaine after protein separation by PAGE or CIE is a simple and sensitive technique permitting the GB-BP to the unambiguously detected and identified in samples of complex protein mixtures containing down to 2 micrograms of GB-BP in PAGE and only 0.2 micrograms in CIE.

Affinity Labels↗
PubMed1985

A new affinity labeling reagent for the active site of glycogen synthase. Uridine diphosphopyridoxal.

A new affinity labeling reagent for glycogen synthase a from rabbit muscle, uridine diphosphopyridoxal, has been prepared. Incubation of the enzyme with this reagent resulted in a time-dependent, almost complete loss of activity. The inactivation was pseudo-first order, and the results of the kinetic analysis suggested the formation of a noncovalent enzyme-reagent complex prior to the covalent reaction, with a Kinact of 25 microM and a maximal rate constant of 0.22 min-1. The inactivation was pronouncedly protected by UDP-Glc and UDP, but not by the allosteric activator glucose 6-phosphate. The increase in a spectral peak at 425 nm and the decrease in enzymatic activity were well correlated, suggesting that the reagent causes the inactivation of the enzyme by the formation of a Schiff base. The rate of inactivation increased as the pH was raised, giving a pK of 8.85. Almost all the original activity was recovered by the treatment of the inactivated enzyme with cysteamine or any other aminothiol compound. No recovery of the activity, however, was observed with inactivated enzyme which had been treated with NaBH4. A peptide containing the labeled amino acid was isolated for inactivated enzyme after reduction with NaBH4, carboxymethylation, and chymotryptic digestion by fractionation on a Bio-Gel P-6 column and high performance liquid chromatographies. Manual Edman degradation established the sequence as Glu-Val-Ala-Asn-labeled Lys-Val-Gly-Gly-Ile-(Tyr). The introduction of an active site-directing moiety to pyridoxal 5'-phosphate makes the resultant reagent an effective probe for the active site of glycogen synthase.

Affinity Labels↗
PubMed1985

[Synthesis and effect of affinity-labeled analogs and partial sequences of the bradykinin potentiating nonapeptide BPP9 alpha (teprotide)].

Affinity labeled analogues and partial sequences of the bradykinin potentiating nonapeptide BPP9 alpha inhibit the BPP9 alpha induced potentiation of the bradykinin action on the isolated guinea pig ileum. The labeled nonapeptides are more active than the labeled partial sequences. The inhibition of the potentiating action of BPP9 alpha demonstrates, that the influence on bradykinin action is not only a result of the inhibition of peptidyl dipeptide hydrolase.

Affinity Labels↗
PubMed1988

Regulation of pS2 gene expression by affinity labeling and reversibly binding estrogens and antiestrogens: comparison of effects on the native gene and on pS2-chloramphenicol acetyltransferase fusion genes transfected into MCF-7 human breast cancer cells.

We have examined the effects of reversibly and irreversibly binding estrogenic and antiestrogenic ligands for the estrogen receptor on pS2 RNA accumulation in MCF-7 human breast cancer cells and on pS2-chloramphenicol acetyl transferase (CAT) fusion gene expression in transfected MCF-7 cells. In MCF-7 cells grown in the absence of estrogens, the reversibly binding estrogen, estradiol, and the affinity labeling estrogen, ketononestrol aziridine, KNA, evoked a 13-fold increase in pS2 RNA level. The reversibly binding antiestrogen trans-hydroxytamoxifen and the affinity labeling antiestrogens tamoxifen aziridine or desmethylnafoxidine aziridine behaved as partial agonists/antagonists. In thymidine kinase-chloramphenicol acetyltransferase (tk-CAT) fusion genes containing a 1000 base pair fragment of the pS2 5'-flanking region encompassing the estrogen responsive element of the gene [pS2 (-1100/-90) tk-CAT], estradiol and ketononestrol aziridine evoked a marked stimulation of CAT activity and, in transfected cells grown in both the presence or absence of the weak estrogen phenol red, the antiestrogens behaved as partial agonists/antagonists. This pS2 5'-flanking region displayed both estrogen-dependent and estrogen-independent enhancer activity as monitored by stimulation of CAT activity. Hormonal regulation of the transfected pS2 fusion gene was similar to that observed in the native pS2 gene of MCF-7 cells; however, antiestrogens, while still partial agonists-antagonists, were relatively more agonistic on the transfected fusion gene than on the native gene. One antiestrogen (ICI 164,384) that behaved as a pure estrogen antagonist on the native gene was a partial agonist-antagonist of pS2 gene expression in the plasmid. This study illustrates that the hormonal regulation of the pS2 gene, as characterized by the agonist-antagonist balance of estrogens and antiestrogens, is influenced by the DNA context of the pS2 estrogen responsive element. Also, the fact that estrogens and antiestrogens that form covalent bonds with the estrogen receptor modulate activity of the native pS2 gene and the pS2-tk-CAT fusion gene in a manner similar to that of their reversibly binding counterparts suggests that it may be possible to use these irreversibly binding ligands to follow the interaction of hormone-receptor complexes with regions regulating estrogenic stimulation of the pS2 gene.

Affinity Labels↗
PubMed1990

Microsomal dexamethasone binding sites identified by affinity labelling.

Binding studies with [3H]dexamethasone identified a class of binding sites on male rat liver microsomes. The binding sites were glucocorticoid-dependent and specific for glucocorticoids and progestins. Scatchard binding parameters, competition studies with triamcinolone acetonide, a synthetic glucocorticoid which competes well for the glucocorticoid receptor, and immunoblotting with an antiglucocorticoid receptor antibody indicated that these sites are distinct from the cytosolic glucocorticoid receptor. Affinity labelling experiments with [3H]dexamethasone 21-mesylate revealed two specifically labelled peptides, one at approx. 66 kDa and a doublet at 45 kDa. The 66 kDa peptide had been previously identified in serum and may be present as a result of serum contamination of the microsomal preparation. The 45 kDa doublet, on the other hand, had been shown to be absent from rat serum. The characteristics of the 45 kDa peptide(s) were identical to those of the dexamethasone binding site identified in the binding studies. [3H]Dexamethasone binding characteristics and affinity labelling of microsomal subfractions, separated by isopycnic centrifugation, showed that the binding sites are located in the endoplasmic reticulum. The identification and role of the 45 kDa peptide doublet remain to be determined.

Adrenalectomy↗
PubMed1991

Affinity labeling of 3 alpha-hydroxysteroid dehydrogenase with 3 alpha-bromoacetoxyandrosterone and 11 alpha-bromoacetoxyprogesterone. Isolation and sequence of active site peptides containing reactive cysteines; sequence confirmation using nucleotide sequence from a cDNA clone.

Homogeneous 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD, EC 1.1.1.50) of rat liver cytosol is potently inhibited at its active site by nonsteroidal anti-inflammatory drugs (NSAIDs). Using 3 alpha-bromoacetoxy-5 alpha-androstan-17-one (BrAnd, a substrate analog) and 11 alpha-bromoacetoxyprogesterone (Br11P, a glucocorticoid analog) as affinity-labeling agents, kinetic evidence was obtained that these agents alkylate this site. Inactivation of 3 alpha-HSD with either [14C]BrAnd or [14C] Br11P led to the incorporation of 1 mol of affinity-labeling agent per enzyme monomer. Complete acid hydrolysis of 3 alpha-HSD radiolabeled with either agent followed by amino acid analysis led to the identification of [14C]carboxymethylcysteine indicating that [14C]BrAnd and [14C]Br11P covalently tag discrete reactive cysteine(s) at the enzyme active site. Trypsin digestion of [14C]BrAnd-inactivated 3 alpha-HSD followed by peptide mapping led to the purification of a single radiolabeled peptide (3A1) which gave the following sequence: H2N-Ser-Ile-Gly-Val-Ser-Asn-Phe-Asn-X-Arg-CO2H. Identical experiments on [14C] Br11P-inactivated 3 alpha-HSD led to the purification of three radiolabeled peptides (11P1-11P3). The major radiolabeled peptide (11P1) had an identical sequence to 3A1 which was tagged with [14C]BrAnd. The minor radiolabeled peptides had the following sequences: H2N-Ser-Lys-Asp-Ile-Ile-Leu-Val-Ser-Tyr-X-Thr-Leu-Gly-Ser-Ser-Arg-CO2H (11P2) and H2N-Ser-Pro-Val-Leu-Leu-Asp-Asp-Pro-Val-Leu-X-Ala-Ile-Ala-Lys-CO2H (11P3). In each peptide group X was identified as carboxymethylcysteine. Alignment of the peptide sequences with the primary structure of 3 alpha-HSD, deduced from its cDNA clone, assigned peptide 11P1 to residues 162-171, peptide 11P2 to residues 208-223, and peptide 11P3 to residues 232-246 of the amino acid sequence. The reactive cysteines correspond to Cys170, Cys217, and Cys242. We propose that Cys170 labeled by BrAnd may lie within the catalytic pocket of the enzyme. By contrast the 11 alpha-bromoacetoxy group in Br11P labeled several reactive cysteines which may be involved in the binding of glucocorticoids and NSAIDs.

3-Hydroxysteroid Dehydrogenases↗
PubMed1980

Different reactivity of mitochondrial and cytoplasmic aspartate aminotransferases toward an affinity labeling reagent analog of the coenzyme.

The two isoenzymes of aspartate aminotransferase from pig heart have been reacted with a derivative of the coenzyme, 4'-N-(2,4-dinitro-5-fluorophenyl) pyridoxamine-5'-phosphate, which is a potential affinity labeling reagent. The derivative has a great affinity for both isoapoenzymes. In the cytosolic isoenzyme, the reversible binding is followed by a covalent labeling of the epsilon amino group of lysine 258, which usually forms an aldimine bond with pyridoxal-5'-phosphate. In the mitochondrial isoenzyme, no labeling occurs at the active site. The different reactivity indicates that a small but definite difference exists in the geometry of the two active sites. In the cytosolic isoenzyme also a sulfhydryl group outside the active site region, namely cysteine 45, reacts, but not by an affinity labeling mechanism. In both isoenzymes, the reversibly bound reagent slowly undergoes a splitting reaction by which pyridoxal-5'-phosphate is regenerated and activity re-established; the rate of this reaction is not fast enough to impaire the labeling potential of the reagent.

Affinity Labels↗
PubMed1983

Identification of the catalytic subunit of an oligomeric casein kinase (G type). Affinity labeling of the nucleotide site using 5'-[p-(fluorosulfonyl)benzoyl]adenosine.

Identification of the catalytic subunit of a G type [using guanosine 5'-triphosphate (GTP) as well as adenosine 5'-triphosphate (ATP) as phosphate donor], oligomeric, cyclic nucleotide independent casein kinase purified from bovine lung was carried out after reaction with 5'-[p-(fluorosulfonyl)-benzoyl]adenosine (FSBA) and isolation of the subunit components of the enzyme. FSBA exhibited the major characteristics of an affinity label reacting at the nucleotide (ATP, GTP) site of the casein kinase. FSBA acted as a competitive inhibitor of ATP (and GTP), led to complete inactivation of the enzyme in a reaction showing two kinetic steps, and became irreversibly bound to the protein. After being labeled with FSBA, the casein kinase (apparent molecular weight of 140 000) was separated into its two monomeric components of apparent molecular weights 38 000 (alpha) and 27 000 (beta), respectively, after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Use of radioactive FSBA showed that specific affinity labeling was limited to the alpha casein kinase subunit. This result was in agreement with the fact that casein kinase activity was found associated with the alpha monomer after electrophoretic separation of the alpha and beta subunits. It may thus be concluded that the largest (alpha) subunit contains the catalytic site of the casein kinase G. Electrophoretic analysis of purified protein kinase under denaturing conditions suggested an alpha 3 beta 2 combination for an apparent molecular weight of 130 000-140 000. However, a maximum of 2 mol of FSBA could be specifically bound to the alpha subunit per mol of enzyme, with a concomitant complete inactivation. These data would be in agreement with an alpha 2 beta 2 subunit composition for casein kinase G, as proposed by other research groups for a similar type of protein kinase of different sources. These observations suggest that the alpha subunits are functionally similar, each of them containing a nucleotide (ATP, GTP) binding site. The possible role of the beta subunit in the enzyme activity remains to be established.

Adenosine↗
PubMed2000

Steroidal affinity labels of the estrogen receptor alpha. 4. Electrophilic 11beta-aryl derivatives of estradiol.

Ten electrophilic estradiol 11beta-aryl derivatives were synthesized, with three different types of 11beta-substituent: (i) pOO(CH(2))(2)X (compounds: 6, X = OSO(2)CH(3); 7, X = I; 13, X = NHCOCH(2)Cl; 15, X = N(CH(3))COCH(2)Br; and 16, X = N(CH(3))COCH(2)Cl); (ii) pOO(CH(2))(5)X (compounds: 17, X = I; 20, X = NHCOCH(2)Br; and 22, X = N(CH(3))COCH(2)Br); and (iii) pOC(triple bond)CCH(2)X (compounds: 27, X = NHCOCH(2)Cl; and 29, X = N(CH(3))COCH(2)Cl). The range of their apparent affinity constants for binding the lamb uterine estrogen receptor alpha (ERalpha) was 3-40% that of estradiol. Six electrophiles, chloroacetamides 13, 16, 27, and 29, iodide 17, and bromoacetamide 20 (whose arm linking the electrophilic carbon to the 11beta-phenyl group includes at least six bonds), were able to irreversibly inhibit the binding of [(3)H]estradiol to ER (25-60% decrease in binding sites), with the following compound effectiveness order: 17 < 13 < 16 approximately 20 approximately 27 approximately 29. Mesylate 6, iodide 7 (whose linking arm includes only three bonds), and bromoacetamides 15 and 22 (which differ from 16 by the Cl to Br change and from 20 by the NH to NCH(3) change, respectively) were much less effective (<10% decrease in binding sites, if any). The fact that the inactivation of estradiol-binding sites by the six electrophiles was totally prevented by estradiol indicated that they were ER affinity labeling agents. When ER was modified by methyl methanethiosulfonate, an SH-specific reagent, the different compounds led to very contrasting results in ER affinity labeling. With modified ER, iodide 17 and chloroacetamides 27 and 29 were practically inactive, chloroacetamides 13 and 16 and bromoacetamide 20 were still active but less effective than on the native ER, whereas tertiary bromoacetamides 15 and 22, found to be practically inactive on native ER, became the most effective electrophiles ( approximately 45% and approximately 65% binding sites inactivated, respectively). The results indicate that in the steroid-filled hormone-binding pocket: (i) nucleophilic residues are localized on the beta-side but relatively remote from the steroid nucleus (distance from C-11 > "seven bonds"); (ii) relatively discrete changes in the electrophilic functionality, such as Cl to Br or NH to NCH(3) of haloacetamido compounds, can markedly modify the positioning of the electrophilic center which could no longer react with the nucleophilic residues; and (iii) cysteine residues (probably homologues of human ERalpha cysteine 381 and/or cysteine 530) are, at least partly, the covalent attachment sites of the electrophiles. Moreover, modification of cysteine residues by methyl methanethiosulfonate changes the structure of the hormone-binding pocket, whose labeling by the various electrophiles is profoundly altered.

Affinity Labels↗
PubMed1982

Stable, soluble, model immune complexes made with a versatile multivalent affinity-labeling antigen.

We describe here the synthesis of a family of multivalent affinity-labeling antigens based on the soluble carbohydrate polymer Ficoll. Ficoll was derivatized successively with chloroacetate, ethylenediamine, and glutaric anhydride and finally esterified with 2,4-dinitrophenol. Prior to esterification, the polymer could also be derivatized with tyramine to allow trace iodination and with the monosaccharides galactose or mannose. The numbers of substituent groups could be controlled at several points in the synthesis. The resulting multiple dinitrophenyl esters on a Ficoll or glycosylated Ficoll polymer specifically cross-linked anti-dinitrophenyl antibodies to form covalently cross-linked antigen-antibody complexes. The glycosylated Ficolls were particularly made for studies of the influence of antigen structure on the behavior of immune complexes. The intermediates in the synthesis are suitable for other derivatizations as well. These model immune complexes are stable and soluble, they can be separated by size, and they overcome some of the limitations on the study of complexes imposed by previous techniques of preparing them.

Affinity Labels↗
PubMed1982

Affinity labeling of the plasma membrane 3,3',5-triiodo-L-thyronine receptor in GH3 cells.

The binding of 3,3',5-triiodo-L-thyronine (T3) to GH3 rat pituitary tumor cells was studied at 15 degrees C and was shown to be saturable, reversible, and stereospecific. Least-squares analysis of the binding data showed two classes of binding sites with dissociation constants of 1.8 +/- 0.2 nM and 260 +/- 30 nM and binding capacities of (5.2 +/- 0.2) X 10(4) and (1.6 +/- 0.2) X 10(6) sites per cell, respectively. Affinity labeling of intact cells was carried out by incubation of cells with 0.3 nM N-bromoacetyl-[125I]T3 at 15 degrees C for 1 hr. Analysis of the cellular extracts by sodium dodecyl sulfate gel electrophoresis showed three labeled protein bands with apparent molecular masses of 55, 47, and 33 kilodaltons (kDal) in a ratio of 86:7:7. The labeling of only the 55-kDal protein band was selectively reduced to 50% by 20 microM unlabeled T3. Highly purified plasma membranes of GH3 cells were prepared and shown to be free of nuclei. Affinity labeling of the purified plasma membranes gave the same labeling pattern as with intact cells. Peptide mapping by Staphylococcus aureus V8 digestion of the 55-kDal protein from cells or plasma membranes gave the identical peptide fragments. Thus the 55-kDal protein labeled from intact cells is the same protein as that from purified plasma membranes. These results together with our earlier findings [Horiuchi, R., Cheng, S.-y., Willingham, M. & Pastan, I. (1982) J. Biol. Chem. 257, 3139-3144] suggest that the 55-kDal protein may be involved in mediating the uptake of T3 in GH3 cells.

Affinity Labels↗
PubMed1982

Inactivation of interferons: halomethyl ketone derivatives of phenylalanine as affinity labels.

Antiviral activity of rabbit and mouse fibroblast interferons was irreversibly destroyed by treatment with halomethyl ketone derivatives of phenylalanine but not by treatment with a halomethyl ketone derivative of lysine. The inactivation reaction was pH dependent, suggesting the involvement of an amino acid residue ionizing in the region of pH 7. Tryptophan and phenylalanine, known ligands of interferons, protected rabbit interferon substantially against inactivation by the chloromethyl ketone derivative of N-tosylphenylalanine. Mixed bovine brain gangliosides protected rabbit and mouse interferons against inactivation by this reagent. Although halomethyl ketone derivatives of phenylalanine were originally designed and used for affinity labeling of the active site of chymotrypsin and similar enzymes, no evidence was found for a chymotrypsin-like activity of interferons. It is proposed that halomethyl ketone derivatives of phenylalanine inactivate interferon by an affinity labeling mechanism, first binding to a hydrophobic binding site and then reacting irreversibly with a nearby nucleophilic amino acid residue, which appears to be a histidine. This conclusion implies that a hydrophobic site on interferons is necessary for their antiviral activity.

Affinity Labels↗
PubMed2000

Affinity labeling of rat glutathione S-transferase isozyme 1-1 by 17beta -iodoacetoxy-estradiol-3-sulfate.

Rat liver glutathione S-transferase, isozyme 1-1, catalyzes the glutathione-dependent isomerization of Delta(5)-androstene-3,17-dione and also binds steroid sulfates at a nonsubstrate inhibitory steroid site. 17beta-Iodoacetoxy-estradiol-3-sulfate, a reactive steroid analogue, produces a time-dependent inactivation of this glutathione S-transferase to a limit of 60% residual activity. The rate constant for inactivation (k(obs)) exhibits a nonlinear dependence on reagent concentration with K(I) = 71 microm and k(max) = 0.0133 min(-1). Complete protection against inactivation is provided by 17beta-estradiol-3,17-disulfate, whereas Delta5-androstene-3,17-dione and S-methylglutathione have little effect on k(obs). These results indicate that 17beta-iodoacetoxy-estradiol-3-sulfate reacts as an affinity label of the nonsubstrate steroid site rather than of the substrate sites occupied by Delta5-androstene-3,17-dione or glutathione. Loss of activity occurs concomitant with incorporation of about 1 mol 14C-labeled reagent/mol enzyme dimer when the enzyme is maximally inactivated. Isolation of the labeled peptide from the chymotryptic digest shows that Cys(17) is the only enzymic amino acid modified. Covalent modification of Cys(17) by 17beta-iodoacetoxy-estradiol-3-sulfate on subunit A prevents reaction of the steroid analogue with subunit B. These results and examination of the crystal structure of the enzyme suggest that the interaction between the two subunits of glutathione S-transferase 1-1, and the electrostatic attraction between the 3-sulfate of the reagent and Arg(14) of subunit B, are important in binding steroid sulfates at the nonsubstrate steroid binding site and in determining the specificity of this affinity label.

Affinity Labels↗
PubMed1996

Catalytic role of the amino-terminal proline in 4-oxalocrotonate tautomerase: affinity labeling and heteronuclear NMR studies.

4-Oxalocrotonate tautomerase (EC 5.3.2-; 4-OT), a hexamer consisting of 62 residues per subunit, catalyzes the isomerization of unsaturated alpha-keto acids, converting unconjugated ketones to the conjugated isomers via a dienolic intermediate. The recently solved crystal structure of an isozyme of 4-OT suggests that the amino-terminal proline is the catalytic base [Subramanya, H. S., Roper, D. I., Dauter, Z., Dodson, E. J., Davies, G. J., Wilson, K. S., & Wigley, D. B. (1996) Biochemistry 35, 792-802]. In support of this proposed role, we have found that the active-site-directed irreversible inhibitor 3-bromopyruvate (3-BP) blocks the amino terminus of 4-OT to Edman degradation and results in the disappearance of the 15N resonance of Pro-1 (delta = 49.2 ppm at pH 6.40 and 42 degrees C) in the 15N NMR spectrum of uniformly 15N-labeled 4-OT. Furthermore, covalent bonding between a 15N resonance of 4-OT and the methylene carbon of the reduced, 3-(13)C-labeled lactyl adduct derived from [3-(13)C]-bromopyruvate was then directly demonstrated using two heteronuclear NMR methods, an 1H-(13)C HSQC experiment and a novel inverse correlation experiment which we call H(C)N. The chemical shift of the modified 15N resonance (delta = 86.5 ppm) is consistent with that of an alkylated and cationic, amino-terminal proline. Affinity labeling with 2-(14)C-labeled bromopyruvate indicates that the ultimate stoichiometry of modification is I equiv of 3-BP per 4-OT monomer. However, an analysis of the residual enzyme activity after differing extents of fractional modification with 3-BP indicates that modification of three active sites per hexamer abolishes essentially all activity of the hexamer. Thus, 4-OT exhibits half-of-the-sites stoichiometry with 3-BP. Finally, the pH dependence of kinact/KI for affinity labeling by 3-BP yields a pKa value of 6.7 +/- 0.3, in reasonable agreement with the pKa values found for kcat/KM for the non-sticky substrate 2-hydroxy-2,4-pentadienoate and by direct NMR titration of Pro-1 [Stivers, J. T., Abeygunawardana, C., Mildvan, A. S., Hajipour, G., & Whitman, C. P. (1996) Biochemistry 35, 814-823]. These results strongly implicate the amino-terminal proline as the general-base catalyst on 4-OT.

Affinity Labels↗
PubMed1991

Affinity labeling of mu and kappa receptors with naloxone benzoylhydrazone.

Naloxone benzoylhydrazone (NalBzoH) labels both mu and kappa receptors in standard homogenate binding assays. We now report that [3H]NalBzoH can effectively photoaffinity label opioid receptors. By modifying [3H]NalBzoH binding conditions, we can selectively label either mu or kappa 3 receptors in calf striatal membranes or classical U50,488-sensitive kappa 1 receptors in guinea pig cerebellar membranes. After removal of unbound radioligand, the [3H]NalBzoH-labeled membranes were irradiated with UV light to couple the bound radioligand to its binding site. No specific mu, kappa 1, or kappa 3 binding remained after a 20-hr dissociation at 25 degrees without UV irradiation. In contrast, approximately 45% of mu and 40% of kappa 1 and kappa 3 binding remained after 2 min of UV exposure. In time course studies, increasing the UV exposure from 30 sec to 3 min produced a progressive increase in radioligand incorporation, which did not increase further with UV exposure times up to 5 min. A portion of the binding in irradiated membranes appeared to be covalently coupled to proteins. Following solubilization of irradiated membranes with sodium dodecyl sulfate, approximately 30-40% of the specifically bound radioactivity precipitated with trichloroacetic acid (TCA). These levels of TCA-precipitable binding corresponded to the amount of dissociation-resistant binding described above. No specific binding could be TCA precipitated in samples that were not exposed to UV. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [3H]NalBzoH-labeled membranes revealed a number of labeled peaks with levels of radioactivity that did not correspond to the intensity of the protein bands, suggesting that this technique was not randomly labeling proteins. This approach may be a useful method to affinity label, characterize, and purify mu and kappa opiate receptor subtypes.

Affinity Labels↗
PubMed1995

Affinity labeling of a sulfhydryl group in the cardiacglycoside receptor site of Na+/K(+)-ATPase by N-hydroxysuccinimidyl derivatives of digoxigenin.

Na+/K(+)-ATPase from pig kidney is inactivated by protein-reactive N-hydroxysuccinimidyl derivatives of digoxigenin. Like digoxigenin, its protein-reactive derivatives N-hydroxysuccinimidyl digoxigenin-3-methylcarbonyl-epsilon-aminocaproate (HDMA), 3-amino-3-deoxydigoxigenin hemisuccinimide succinimidyl ester (ADHS), 3-iodoacetylamino-3-deoxydigoxigenin (IAD) and digoxigenin-3-O-succinyl-[2-(N-maleimido)]ethylamide (DSME) inhibited the sodium pump in the presence of Na+, Mg2+ and ATP. At 37 degrees C, half-maximal inhibition of Na+/K(+)-ATPase was seen by HDMA at 0.47 microM, by ADHS at 5.8 microM, by IAD at 8 microM and by DSME at 94 microM. Thus, all compounds bind to the cardiac steroid receptor site of Na+/K(+)-ATPase. Affinity labeling of the alpha subunit by 'front door' or 'back door' phosphorylation was only seen with HDMA or ADHS in the range 0.1 microM. Excess of ouabain protected against affinity labeling. All the other protein-reactive derivatives of digoxigenin labeled the enzyme independent of the formation of a phosphointermediate at much higher concentrations. This labeling was not suppressed by an excess of ouabain. Tryptic hydrolysis of the HDMA-modified Na+/K(+)-ATPase gave peptides of the apparent molecular masses 20, 12.5 and 11.2 kDa. The 11.2-kDa and 12.5-kDa peptides started amino-terminally with Asp68, and the 20-kDa peptide with Asp24. Thus, the HDMA-labeled peptides originate from the cardioactive steroid-binding site formed by the first and second transmembrane helix. N-Hydroxysuccinimidyl esters such as HDMA are normally thought to modify lysine and arginine residues covalently. Since such residues do not exist in the putative cardiac glycoside-binding site, the possibility of a thioester formation of the digoxigenin derivatives HDMA and ADHS with Cys104 in the H1 transmembrane domain was tested. In fact, hydroxylaminolysis led to the release of the covalently bound HDMA, and the formation of a free sulfhydryl group. This could be labeled by [2-14C]ICH2COOH. We therefore propose, consistent with a recent conclusion from a site-directed mutagenesis experiment [Canessa, C. M., Horisberger, J.-D., Louvard, D. & Rossier, B. C. (1992) EMBO J. 11, 1681-1687], that a cysteine residue (probably Cys104) participates in the structure and function of the cardiac glycoside binding.

Affinity Labels↗
PubMed1975

Affinity labeling of the ribosomal decoding site with an AUG-substrate analog.

The trinucleotide AUG was condensed at the 5'-end with N-bromoacetyl-p-aminophenylphosphate. This bromoactylated AUG analog reacted irreversibly with the mRNA binding site of Escherichia coli 70S ribosomes. After reaction of 70S ribosomes with the AUG analog, labeled 30S subunits could be isolated that were programmed for initiation-factor-dependent binding of fMet-tRNAfMet. This shows that this AUG-affinity label reacted in the decoding site for fMet-tRNAfMet. By combination of sodium dodecyl sulfate-, Sarkosyl-, and ureapolyacrylamide gel electrophoresis the AUG-affinity label was found to be irreversibly bound to ribosomal proteins S4, the ram gene product, and S18.

Binding Sites↗