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PubMed1998

Affinity labeling of two nucleotide sites on Na,K-ATPase using 2'(3')-O-(2,4,6-trinitrophenyl)8-azidoadenosine 5'-[alpha-32P]diphosphate (TNP-8N3-[alpha-32P]ADP) as a photoactivatable probe. Label incorporation before and after blocking the high affinity ATP site with fluorescein isothiocyanate.

ATP and its analogues act on the minimal functional unit of Na, K-ATPase, the alpha beta protomer, with high and low affinity effects. Fluorescein isothiocyanate (FITC) irreversibly blocks the high affinity, or catalytic, ATP site, and yet the surviving K+-phosphatase activity of soluble FITC-modified alphabeta protomers can be photoinactivated by 2'(3')-O-trinitrophenyl (TNP)-8N3-ADP (Ward, D. G., and Cavieres, J. D. (1998) J. Biol. Chem. 273, 14277-14284). We have now used TNP-8N3-[alpha-32P]ADP as a photoaffinity label for Na,K-ATPase. The native enzyme can be photolabeled at 5 microM TNP-8N3-[alpha-32P]ADP, and ATP or FITC treatment prevents labeling of the alpha chain. At 25 microM, however, TNP-8N3-[alpha-32P]ADP can be incorporated in the FITC-modified alpha chain, concurrently with the inactivation of the K+-phosphatase activity, to an extrapolated level of 0.5-1.2 mol of 32P-probe per mol of alpha chain. Photoinactivation and labeling are prevented by TNP-ADP, vanadate, or strophanthidin and are promoted by Na+ or Mg2+, but not K+. The cation effects suggest that the fluorescein-modified enzyme incorporates the TNP-8N3-[alpha-32P]ADP. Mg complex preferentially, and the free probe when in the E1 enzyme form and after occupation of a low-affinity Na+ site. Partial trypsinolysis reveals that the point of TNP-8N3-[alpha-32P]ADP attachment is on the C-terminal 58-kDa fragment of the FITC-modified alpha chain. The affinity labeling of the fluorescein enzyme by TNP-8N3-[alpha-32P]ADP endorses the view that two nucleotide sites can be occupied simultaneously in each alpha subunit of Na,K-ATPase.

Adenosine Triphosphate↗
PubMed1978

Photoinduced affinity labeling of the Escherichia coli ribosome puromycin site.

The photoincorporation of puromycin into Escherichia coli ribosomes has been studied in detail. Incorporation into protein L23 as a function of puromycin concentration follows a simple saturation curve and is specifically blocked by structural and functional analogues of puromycin, thus demonstrating that such incorporation proceeds via an affinity labeling process. Incorporation into L23 becomes more specific as the light fluence is reduced, indicating that such incorporation takes place from a native rather than light-denatured puromycin site. L23 remains the major labeled protein using ribosomes prepared by several procedures, suggesting the conservative nature of the site. In addition evidence is presented for affinity labeling of S14 and of a site in the RNA fraction of the 50S particle. Specific incorporation appears to proceed with an anomalously high quantum yield. The detailed photochemical mechanism is not understood, although 8-alkylation of purine moiety has been excluded. Incorporation is largely inhibited in the presence of thiol reagents.

Affinity Labels↗
PubMed1987

[Highly selective affinity labeling of a promoter in a complex with E. coli RNA-polymerase by alkylating derivatives of initiating substrates].

The complex [promoter A2 X E. coli RNA polymerase] was treated with phosphoamides, derivatives of 4-[N-methyl, N-(2-chloroethyl)]-aminobenzylamine and guanosine-5'-mono-, di-, and triphosphates with the alkylating group attached to the terminal phosphates. After this, [alpha-32P]CTP was added. Residues of the affinity reagents bound covalently at the first stage were elongated by radioactive -pC residues due to the catalytic action of the active centre of RNA polymerase. Affinity labelled were beta-and sigma-subunits of the enzyme, and the promoter. The affinity label was localized on -pGpC residues. A guanine residue was alkylated in the promoter as suggested by radioactivity elimination kinetics. As the data obtained and the previously known length of the reagent (maximum distance between the alpha-phosphorus atom of the reagent and the point of alkylation is less than 0.6 nm) indicate, there is a direct rather than protein-mediated contact between the template and the substrate within the complex [promoter X RNA polymerase].

Affinity Labels↗
PubMed1977

Affinity labelling of alcohol dehydrogenases. Chemical modification of the horse liver and the yeast enzymes with alpha-bromo-beta(5-imidazolyl)-propionic acid and 1,3-dibromoacetone.

1. DL-alpha-Bromo-beta(5-imidazolyl)-propionic acid is a potential affinity labelling reagent for metallo-enzymes. It has been used with the alcohol dehydrogenases from liver and yeast. The liver enzyme is chemically modified and inactivated in a Michaelis-Menten-type reaction, where one molecule of the reagent is bound per subunit. The enzyme is protected from the inhibitor in a competitive manner by imidazole, 2,2'-dipyridyl, 1,10-phenanthroline and cyclohexanone, which all combine with the active-site zinc. The protection by chloride, acetate and NADH, which are considered to bind at the general anion binding site, is not strictly competitive. Inactivation has an optimum at pH 8.5. For the liver enzyme, the reagent was found to decrease the initial rate of ethanol oxidation. Prior to the irreversible alkylation of Cys-46, reversible binding is shown to occur at the active-site zinc atom. The yeast enzyme was extremely resistant to the reagent and no specific modification was found. 2. The potential affinity labelling and crosslinking reagent, symmetrical 1,3-dibromoacetone although unstable, has also been used for chemical modification. With the liver enzyme, concentrations below 5 mM gave a reaction of the Michaelis-Menten-type at pH 7.0. Several ligands known to complex with the active-site region protect the enzyme against the reagent. Dibromoacetone gave rapid inactivation of the yeast enzyme. Despite the fact that a pseudo-first-order reaction was observed with respect to enzyme as well as inhibitor, no saturating effect was found. In this work, dibromoacetone reacted like a monofunctional reagent.

Acetone↗
PubMed1989

Structure of the Mr 140,000 growth hormone-dependent insulin-like growth factor binding protein complex: determination by reconstitution and affinity-labeling.

To determine the structure of the high molecular weight, growth hormone-dependent complex between the insulin-like growth factors (IGF-I and IGF-II) and their binding proteins in human serum, we have reconstituted the complex from its purified component proteins and analyzed it by gel electrophoresis and autoradiography after covalent cross-linking. The proteins tested in reconstitution mixtures were an acid-labile Mr 84,000-86,000 glycoprotein doublet (alpha subunit), an acid-stable Mr 47,000-53,000 glycoprotein doublet with IGF-binding activity (BP-53 or beta subunit), and IGF-I or IGF-II (gamma subunit). In incubations containing any one of the three subunits 125I-labeled and the other two unlabeled, identical 125I-labeled alpha-beta-gamma complexes of Mr 140,000 were formed. Minor bands of Mr 120,000 and 90,000 were also seen, thought to represent a partially deglycosylated form of the alpha-beta-gamma complex, and an alpha-gamma complex arising as a cross-linking artifact. When serum samples from subjects of various growth hormone status were affinity-labeled with IGF-II tracer, a growth hormone-dependent Mr 140,000 band was seen, corresponding to the reconstituted alpha-beta-gamma complex. Other growth hormone-dependent labeled bands, of Mr 90,000 (corresponding to alpha-gamma), Mr 55,000-60,000 (corresponding to labeled beta-subunit doublet), and smaller bands of Mr 38,000, 28,000, and 23,000-25,000 (corresponding to labeled beta-subunit degradation products), were also seen in the affinity-labeled serum samples and in the complex reconstituted from pure proteins. All were immunoprecipitable with an anti-BP-53 antiserum. We conclude that the growth hormone-dependent Mr 140,000 IGF-binding protein complex in human serum has three components: the alpha (acid-labile) subunit, the beta (binding) subunit, and the gamma (growth factor) subunit.

Acromegaly↗
PubMed1989

Characterization of a cysteine-containing peptide after affinity labelling of Ca2+-ATPase of sarcoplasmic reticulum with the disulfide of 3'(2')-O-biotinyl-thioinosine triphosphate.

3'(2')-O-Biotinyl-thioinosine triphosphate is a substrate of the Ca2+ pump of sarcoplasmic reticulum. Its disulfide inactivates the Ca2+-ATPase with two different velocities. The rapidly inactivated sulfhydryl group cannot be protected by ATP and is therefore considered to be outside the ATP binding site. The slowly reacting sulfhydryl group interacts with the disulfide of 3'(2')-O-biotinyl-thioinosine triphosphate with a dissociation constant of Kd = 137 microM and an inactivation velocity constant of 1.7 X 10(-3) s-1. It is protected by ATP with two different dissociation constants of the enzyme-ATP complex of Kd = 221 microM and 1130 microM. The slowly reacting sulfhydryl group is therefore considered to be part of the ATP binding site. Since it was impossible to isolate a tryptic peptide by affinity purification on matrix-bound avidin after affinity labelling with the disulfide of 3'(2')-O-biotinyl-thioinosine triphosphate, differential labelling with iodo[2-14C]acetic acid after affinity labelling with the disulfide of 3'(2')-O-biotinyl-thioinosine triphosphate was carried out. Tryptic digestion and FPLC purification led to the isolation of a radioactive carboxymethyl derivative of the cysteine-containing peptide ANACNSVIR. This peptide is equivalent to the cDNA-derived sequence 468-476 of Ca2+-ATPase [Brandl et al. (1986) Cell 44, 597-607] and is located between the phosphorylation site, Asp351, and Lys515, a part of the putative purine binding subsite of ATP. Although the carboxymethylation of Cys471 is hindered by (biotinyl-s6ITP)2, the strong dilution of the specific radioactivity of iodo[2-14C]acetic acid in the isolated peptide 468-476 argues against its direct interaction with the ATP analogue. It is therefore proposed that Cys471 undergoes ATP-dependent conformational changes.

Affinity Labels↗
PubMed1992

Functional topography of human ribosomes as studied by affinity labeling with reactive mRNA analogs.

Derivatives of 5'-32P labeled (pU)3 an (pU)6 bearing 4-(N-2-chloroethyl-N-methylamino)benzylmethylamine residue attached to 5'-phosphate via phosphamide bond and (Up)5U[32P]pC and (Up)11U[32P]pC bearing 4-(N-2-chloroethyl-N-methylamino)benzyl residue attached to 3'-end via benzylidene bond were applied for the affinity labeling of 80S ribosomes from human placenta in the presence of a cognate tRNA. The derivatives of 32P-labeled pAUG and pAUGU3 analogous to the 5'-phosphamides of (pU)n were used for affinity labeling of 40S subunits in the presence of ternary complex eIF-2.GTP.Met-tRNA(f). The sites of the reagents' attachment to 18S ribosomal RNA were identified by blot-hybridization of the modified 18S rRNA with restriction fragments of the corresponding rDNA. They were found to be located within positions 976-1057 for (pU)6 and pAUGU3 derivatives and within 976-1164 for (pU)3 and pAUG ones. The sites of 18S rRNA modification with the derivatives of (Up)5UpC and (Up)11UpC were found within positions 1610-1869 at 3'-end of the molecule. All the sites identified here are located presumably within highly conserved parts of the eukaryotic small subunit rRNA secondary structure.

Affinity Labels↗
PubMed1981

Dexamethasone 21-mesylate: an affinity label of glucocorticoid receptors from rat hepatoma tissue culture cells.

We recently described the biological properties of an alpha-keto mesylate derivative of cortisol, cortisol-Mes. Cortisol-Mes exhibited long-term antiglucocorticoid activity, but there was no firm evidence that this activity was irreversible or receptor-mediated. Here we report that dexamethasone mesylate (Dex-Mes), which is the alpha-keto mesylate derivative of the more active glucocorticoid dexamethasone, is a candidate for a steroid-specific affinity label of glucocorticoid receptors. Dex-Mes is relatively stable, like cortisol-Mes, but possesses greater whole-cell antiglucocorticoid activity. However, Dex-Mes also possesses partial agonist activity, which is expressed at somewhat higher concentrations of Dex-Mes than the antagonist activity. Dex-Mes is more efficient than cortisol-Mes in competing for dexamethasone binding to glucocorticoid receptors. Furthermore, Dex-Mes is effective at lower concentrations than cortisol-Mes in causing long-term apparently irreversible antiglucocorticoid effects in whole and broken cells. The cell-free effect of Dex-Mes is specifically prevented by coincubation with an excess of cortisol. These facts argue that the apparently irreversible effects of Dex-Mes are steroid mediated. [3H]Dex-Mes has been used to identify a glucocorticoid-specific, covalently labeled fraction on sodium dodecyl sulfate/polyacrylamide gels with a molecular weight of approximately 85,000. Thus Dex-Mes appears to have been established as an affinity label for glucocorticoid receptors.

Affinity Labels↗
PubMed1989

Use of an affinity label to probe the function of the NADPH binding component of the respiratory burst oxidase of human neutrophils.

The respiratory burst oxidase of neutrophils can be activated in a cell-free system in which solubilized membranes, cytosol, and Mg2+ are required and in which sodium dodecyl sulfate is used to convert the dormant oxidase to an active form. The 2',3'-dialdehyde analog of NADPH was used as an affinity label for the cytosolic NADPH binding component of the respiratory burst oxidase from human neutrophils. When treated with this affinity label in the presence of sodium cyanoborohydride to reduce Schiff bases, neutrophil cytosol was shown to lose at least 90% of its activity in the cell-free system. In contrast to normal cytosol, treated cytosol had lost its ability to abolish the lag time required for activation of the oxidase, suggesting that the treated cytosol was no longer able to participate in the rate-limiting activation step. Furthermore, the treated cytosol had lost its ability to convert the oxidase from a form with a high Km to a form with a low Km for NADPH. The ability of dialdehyde-treated cytosol to activate the oxidase could be restored by untreated cytosol with a concentration dependence suggesting that only one kinetically active component of the oxidase was inhibited by treatment with the NADPH analog. Like the dialdehyde-treated cytosol, cytosols from patients with chronic granulomatous disease caused by a deficiency in a cytosolic Mr = 47,000 protein (pp47) fail to participate in the rate-limiting activation step (Curnutte, J. T., Scott, P. J., and Babior, B. M. (1989) J. Clin. Invest. 83, 1236-1240). These chronic granulomatous disease cytosols were nevertheless able to restore limited activity to the dialdehyde-inactivated cytosol in a cell-free activation system. These results are consistent with a model in which (a) the NADPH binding subunit of the oxidase exists in a very slowly dissociating complex with one or more additional cytosolic components, including pp47, and (b) the NADPH binding component of the oxidase controls the affinity of the enzyme for NADPH, either directly or through the binding of additional cytosolic factors.

Affinity Labels↗
PubMed1992

Affinity labeling of vertebrate oxidosqualene cyclases with a tritiated suicide substrate.

Pig and rat liver oxidosqualene cyclase (OSC) enzymes were purified to homogeneity and showed single bands on SDS-polyacrylamide gel electrophoresis with molecular masses of 75 kDa (pig) and 78 kDa (rat). Pig liver OSC was purified for the first time (441-fold with a yield of 39%). Chemical affinity labeling of pure or crude preparations of the liver cyclases using the mechanism-based irreversible inhibitor of OSC, [3H]29-methylidene-2,3-oxidosqualene ([3H]29-MOS), showed a single radioactive band at 75 kDa (pig) and 78 kDa (rat). Affinity labeling experiments were also performed with dog and human microsomal preparations and with yeast and plant cyclases. All of the vertebrate OSC enzymes were specifically labeled with [3H]29-MOS and gave a single band with molecular masses ranging from 70 to 80 kDa (rat, 78 kDa; dog, 73 kDa; pig, 75 kDa; and human, 73 kDa). In contrast, yeast lanosterol cyclase and plant cycloartenol cyclase were not labeled, demonstrating subtle differences in the active sites of animal, plant, and fungal enzymes.

Affinity Labels↗
PubMed1976

Interaction of integral and peripheral membrane proteins: affinity labeling of yeast cytochrome oxidase by modified yeast cytochrome c.

To identify possible substrate-binding subunit(s) of yeast cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1-9-3-1), the purified enzyme was reacted with yeast iso-1-cytochrome c whose single free sulfhydryl group at position 107 had been activated with 5,5'-dithiobis(2-nitrobenzoate). The resulting cytochrome c derivative appeared to function as an "affinity-label" of cytochrome oxidase, since it rapidly inactivated the enzyme. Inactivation was competitively prevented by underivatized cytochrome c. When the "affinity-labeled" oxidase was analyzed by two-dimensional polyacrylamide electrophoresis in dodecyl sulfate (separation in the second dimension being carried out in the presence of excess sulfhydryl compound), it was found that the derivatized cytochrome c had specifically formed a mixed disulfide with the mitochondrially made subunit III (apparent molecular weight 24,000) of the oxidase. Similar results were obtained when underivatized iso-I-cytochrome c was crosslinked to the oxidase by oxidative disulfide bridge formation in the presence of ortho-phenanthroline and Cu++. These data indicate that the hydrophobic mitochondrially made subunit III of yeast cytochrome c oxidase is in close proximity to the cytochrome c binding site on the enzyme. Since cytochrome c and the mitochondrially made cytochrome oxidase subunit III are typical peripheral and integral membrane proteins, respectively, the present study suggests a useful approach for analyzing specific interactions between these different classes of membrane proteins.

Affinity Labels↗
PubMed1976

Affinity label for beta-adrenergic receptor in turkey erythrocytes.

The compound N-[2-hydroxy-3-(1-naphthoxy)-propyl]-N'-bromoacetylethylenediamine (NHNP-NBE) was found to label covalently the beta-adrenergic receptor in turkey erythrocytes. The compound inhibits irreversibly 1-epinephrine-dependent adenylate cyclase activity [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] in the whole turkey erythrocyte as well as in the erythrocyte membranes possessing the beta-receptor. The affinity label blocks, also irreversibly, the specific [3H] propranolol binding, whereas other bromoacetyl compounds tested have no effect on binding, even at high concentrations, which cause enzyme inactivation. 1-Epinephrine and propranolol offer protection against the affinity label in whole turkey erythrocytes as well as in membranes prepared from these cells. The potential usefulness of an irreversible beta-antagonist is discussed.

Adenylyl Cyclase Inhibitors↗
PubMed1988

Affinity labelling and identification of the high-affinity choline carrier from synaptic membranes of Torpedo electromotor nerve terminals with [3H]choline mustard.

The physiological mechanisms regulating activity of the sodium-dependent, high-affinity choline transporter and the molecular events in the translocation process remain unclear; the protein has not been purified or characterized biochemically. In the present study, [3H]choline mustard aziridinium ion [( 3H]ChM Az), a nitrogen mustard analogue of choline, bound irreversibly to presynaptic plasma membranes from Torpedo electric organ in a hemicholinium-sensitive, and sodium-, time-, and temperature-dependent manner. Specific binding of this ligand was greatest when it was incubated with membranes in the presence of sodium at 30 degrees C. Separation of the 3H-labelled membrane proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that most of the radiolabel was associated with a polypeptide of apparent molecular mass of approximately 42,000 daltons; labelling of this species was abolished in membranes incubated with ligand in the presence of HC-3. Two other 3H-labelled polypeptides were detected, with apparent molecular masses of approximately 58,000 and 90,000 daltons; radiolabelling of the former was also HC-3 sensitive. [3H]ChM Az may be a useful affinity ligand in the purification of the choline carrier from cholinergic neurons.

Affinity Labels↗
PubMed1987

Affinity labeling of the allosteric site of fructose 1,6-bisphosphatase with an AMP analog.

D-Fructose 1,6-bisphosphatase [EC 3.1.3.11, FBPase] is one of the key enzymes in glyconeogenesis and its activity is controlled by various effectors such as substrate, AMP and ATP. To analyze this complex regulation system, we tried an affinity labeling of FBPase with an AMP derivative, since AMP is a potent allosteric inhibitor of this enzyme. The results obtained are as follows. 1. To determine the functional groups which are essential for AMP as an inhibitor, inhibitory activities of some AMP derivatives were examined. These derivatives modified at the purine ring or phosphate group lost the activity while one modified at the ribose ring retained the ability to inhibit FBPase. This shows that an affinity labeling reagent should be an AMP derivative in which the ribose ring is modified. 2. 2',3'-Dialdehyde AMP (dial-AMP) was prepared by periodate oxidation of AMP and was reacted with FBPase. Under appropriate conditions, 1 mol of the reagent was incorporated per mol of enzyme subunit with a concomitant loss of enzyme activity. The reaction was prevented by the presence of AMP but not of ATP. The heat-stability, the kinetic parameters and the UV-absorption spectrum of the modified enzyme were all the same as those of native FBPase in the presence of AMP. Thus it was concluded that the allosteric AMP site in FBPase was modified specifically.

Adenosine Monophosphate↗
PubMed2003

Crystal structures of beta-amylase from Bacillus cereus var mycoides in complexes with substrate analogs and affinity-labeling reagents.

The crystal structures of beta-amylase from Bacillus cereus var. mycoides in complexes with five inhibitors were solved. The inhibitors used were three substrate analogs, i.e. glucose, maltose (product), and a synthesized compound, O-alpha-D-glucopyranosyl-(1-->4)-O-alpha-D-glucopyranosyl-(1-->4)-D-xylopyranose (GGX), and two affinity-labeling reagents with an epoxy alkyl group at the reducing end of glucose. For all inhibitors, one molecule was bound at the active site cleft and the non-reducing end glucose of the four inhibitors except GGX was located at subsite 1, accompanied by a large conformational change of the flexible loop (residues 93-97), which covered the bound inhibitor. In addition, another molecule of maltose or GGX was bound about 30 A away from the active site. A large movement of residues 330 and 331 around subsite 3 was also observed upon the binding of GGX at subsites 3 to 5. Two affinity-labeling reagents, alpha-EPG and alpha-EBG, were covalently bound to a catalytic residue (Glu-172). A substrate recognition mechanism for the beta-amylase was discussed based on the modes of binding of these inhibitors in the active site cleft.

Affinity Labels↗
PubMed1981

Affinity labeling of human placental 17 beta-estradiol dehydrogenase and 20 alpha-hydroxysteroid dehydrogenase with 5'-[p-(fluorosulfonyl)benzoyl]adenosine.

Two pyridine nucleotide linked oxidoreductase activities, 17 beta-estradiol dehydrogenase and 20 alpha-hydroxysteroid dehydrogenase, which were copurified from human placental cytosol as a homogeneous enzyme preparation, may represent dual activity by one enzyme. The affinity labeling nucleotide analogue 5'-[p-(fluorosulfonyl)benzoyl]adenosine, which binds at the cofactor site as a competitive inhibitor of NADH (ki = 1.7 mM), simultaneously and identically inactivated both the 17 beta and 20 alpha activities in a time-dependent and irreversible manner following pseudo-first-order kinetics. NADH and NAD+ markedly protected both activities from inactivation, and the substrate steroids, estrone, estradiol, progesterone, and 20 alpha-hydroxy-4-pregnen-3-one, conferred similar protection, though less than cofactor, against simultaneous loss of both activities. Stoichiometric studies indicated that 2 mol of affinity labeling nucleotide were bound per mol of completely inactivated enzyme dimer. The coincident and identical loss of both activities under all experimental conditions is further evidence that 17 beta-estradiol dehydrogenase and 20 alpha-hydrosteroid dehydrogenase in human placental cytosol represent bifunctional, stereospecific, oxidoreductase activity at one active site on a single protein.

17-Hydroxysteroid Dehydrogenases↗
PubMed1996

Origin of carbohydrate recognition specificity of human lysozyme revealed by affinity labeling.

In order to reveal the origin of carbohydrate recognition specificity of human lysozyme by clarifying the difference in the binding mode of ligands in the active site, the inactivation of human lysozyme by 2',3'-epoxypropyl beta-glycoside derivatives of the disaccharides, N,N'-diacetylchitobiose [GlcNAc-beta-(1-->4)-GlcNAc] and N-acetyllactosamine [Gal-beta-(1-->4)-GlcNAc], was investigated and the three-dimensional structures of the affinity-labeled enzymes were determined by X-ray crystallography at 1.7 A resolution. Under the conditions comprising 2.0 x 10(-3) M labeling reagent and 1.0 x 10(-5) M human lysozyme at pH 5.4, 37 degrees C, the reaction time required to reduce the lytic activity against Micrococcus luteus cells to 50% of its initial activity was lengthened by 3.7 times through the substitution of the nonreducing end sugar residue, GlcNAc to Gal. The refined structure of human lysozyme labeled by 2',3'-epoxypropyl beta-glycoside derivatives of N,N'-diacetylchitobiose (HL/NAG-NAG-EPO complex) indicated that the interaction mode of the N,N'-diacetylchitobiose moiety in substites B and C in this study was essentially the same as in the case of the complex of human lysozyme with the free ligand. On the other hand, the hydrogen-bonding pattern and the stacking interaction at subsite B were remarkably different between the HL/NAG-NAG-EPO complex and human lysozyme labeled by the 2',3'-epoxypropyl beta-glycoside of N-acetyllactosamine (HL/GAL-NAG-EPO complex). The reduced number of possible hydrogen bonds as well as the less favorable stacking between the side chain of Tyr63 in human lysozyme and the galactose residue in the HL/GAL-NAG-EPO complex reasonably explained the less efficient ability of the 2',3'-epoxypropyl beta-glycoside of N-acetyllactosamine as compared to that of N,N'-diacetylchitobiose as an affinity labeling reagent toward human lysozyme.

Affinity Labels↗
PubMed1990

Affinity labeling of the virginiamycin S binding site on bacterial ribosome.

Virginiamycin S (VS, a type B synergimycin) inhibits peptide bond synthesis in vitro and in vivo. The attachment of virginiamycin S to the large ribosomal subunit (50S) is competitively inhibited by erythromycin (Ery, a macrolide) and enhanced by virginiamycin M (VM, a type A synergimycin). We have previously shown, by fluorescence energy transfer measurements, that virginiamycin S binds at the base of the central protuberance of 50S, the putative location of peptidyltransferase domain [Di Giambattista et al. (1986) Biochemistry 25, 3540-3547]. In the present work, the ribosomal protein components at the virginiamycin S binding site were affinity labeled by the N-hydroxysuccinimide ester derivative (HSE) of this antibiotic. Evidence has been provided for (a) the association constant of HSE-ribosome complex formation being similar to that of native virginiamycin S, (b) HSE binding to ribosomes being antagonized by erythromycin and enhanced by virginiamycin M, and (c) a specific linkage of HSE with a single region of 50S, with virtually no fixation to 30S. After dissociation of covalent ribosome-HSE complexes, the resulting ribosomal proteins have been fractionated by electrophoresis and blotted to nitrocellulose, and the HSE-binding proteins have been detected by an immunoenzymometric procedure. More than 80% of label was present within a double spot corresponding to proteins L18 and L22, whose Rfs were modified by the affinity-labeling reagent. It is concluded that these proteins are components of the peptidyltransferase domain of bacterial ribosomes, for which a topographical model, including the available literature data, is proposed.

Affinity Labels↗