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PubMed1984

A specific disulfide bond associated with the activity of human urokinase. Its topological identification and reductive cleavage followed by kinetic changes in enzymatic reaction and affinity labeling.

A single SH group in the B chain (33 kDa), generated by the specific reduction of the single interchain SS bond of human urinary urokinase, was alkylated (UK X B) with iodoacetamide to prevent a spontaneous SH-SS interchange. An SS bond in UK X B was exclusively alkylated with iodoacetamide (R X CAM-UK X B) after reduction with dithiothreitol in 0.3 M guanidine X HCl in the presence of the competitive inhibitor N alpha-benzoyl-L- argininamide with concomitant loss of 65-68% of the esterolytic activity towards N-acetyl-glycyl-L-lysine methyl ester. This specific SS bond was located at Cys194 - Cys222 whose SS loop contained the active-site Ser198 , as determined by amino acid analyses and identification of the N and C termini of the tryptic digest. Transformation of UK X B into R X CAM-UK X B induced no shift of the optimal pH in the bell-shaped pH/activity profile; pH values for 50% activity were similar (pH 9.7) for 10-min alkalization of the enzyme but different between UK X B (pH 9.4) and R X CAM-UK X B (pH 8.8) for 18-h alkalization. An unaltered Km value and a decline by 64% in kcat in the esterolytic activity indicate that the pretransition Michaelis complex is formed without degeneration of the primary substrate-binding site, but the catalytic pathway thereafter has deteriorated. In affinity labeling with dansyl chloride or N alpha-tosyl-L-lysine chloromethylketone, which interrupted the catalysis at the latest at a stage involving the abortive acyl intermediate, the second-order rate constant for UK X B was lowered to 28% or 35% for R X CAM-UK X B, respectively, but the labeling yields were similar. The results indicate that indispensable structural elements, such as the catalytic triad and oxyanion hole, are maintained but a local conformation, which is necessary for efficient transition to the acyl intermediate and/or for resistance against alkaline inactivation, is destabilized with Cys194 - Cys222 scission.

Affinity Labels↗
PubMed1990

Affinity labeling of eukaryotic initiation factor 2 and elongation factor 1 alpha beta gamma with GTP analogs.

As part of an attempt to understand the specific function and role of each subunit in multisubunit protein synthesis factors, we have attempted to identify the nucleotide binding peptides of eukaryotic initiation factor 2 (eIF-2). To ensure that the interactions were of a specific nature, two general controls were used: first, other protein factors with characterized GTP binding activity were tested; second, all affinity labeling was checked for nucleotide specificity by protection with the authentic nucleotide at a 10-fold molar excess over the affinity reagent. Results with a number of GTP modifying reagents ([alpha-32P]GTP, [alpha-32P]GDP, oxidized [alpha-32P]GTP, 3'-p-azidobenzoyl-[alpha-32P]GTP, 3'-p-azidobenzoyl-[alpha-32P]GDP, and 5'-p-[8-3H]fluorosulfonylbenzoyl guanosine) indicate that appropriate conditions for both nucleotide and subunit specific labeling have been achieved. Under these conditions all reagents modified the beta subunit of eIF-2. Complementary studies with subunit-deficient forms of eIF-2 also suggest that the beta subunit of eIF-2 is involved with GTP binding. Coupled with other data suggesting that the gamma subunit of eIF-2 might be involved in GTP binding and amino acid sequence data of eIF-2 gamma from which a part of a GTP binding consensus sequence can be localized, support is provided for the concept of alternate GTP binding domains or a GTP binding domain shared between different subunits of eIF-2.

Affinity Labels↗
PubMed1999

Implication of arginine-131 and arginine-303 in the substrate site of adenylosuccinate synthetase of Escherichia coli by affinity labeling with 6-(4-bromo-2,3-dioxobutyl)thioadenosine 5'-monophosphate.

Adenylosuccinate synthetase from Escherichia coli is inactivated in a biphasic reaction by 6-(4-bromo-2,3-dioxobutyl)thioadenosine 5'-monophosphate (6-BDB-TAMP) at pH 7.0 and 25 degrees C. The initial fast-phase inactivation is not affected by the presence of active-site ligands and can be completely eliminated by blocking Cys291 of the enzyme with N-ethylmaleimide (NEM). Reaction of the NEM-treated enzyme with 6-BDB-[32P]TAMP results in 2 mol of reagent incorporated/mol of enzyme subunit. The inactivation kinetics of the slow-phase exhibit an apparent KI of 40.6 microM and kmax of 0.0228 min-1. Active-site ligands, either adenylosuccinate or IMP and GTP, completely prevent inactivation of the enzyme by 6-BDB-TAMP, whereas IMP or IMP and aspartate is much less effective in protection. 6-BDB-TAMP-inactivated enzyme has a 3-fold increase in Km for aspartate with no change in Km for IMP or GTP. Protease digestion of 6-BDB-[32P]TAMP inactivated enzyme reveals that both Arg131 and Arg303 are modified by the affinity-labeling reagent. The crystal structure [Poland, B. W., Fromm, H. J., and Honzatko, R. B. (1996) J. Mol. Biol. 264, 1013-1027] and site-directed mutagenesis [Kang, C., Sun, N., Poland, B. W., Gorrell, A., and Fromm, H. J. (1997) J. Biol. Chem. 272, 11881-11885] of E. coli adenylosuccinate synthetase show that Arg303 interacts with the carboxyl group of aspartate and the 2'-OH of the ribose of IMP and Arg131 is involved in stabilizing aspartate in the active site of the enzyme. We conclude that 6-BDB-TAMP functions as a reactive adenylosuccinate analogue in modifying both Arg131 and Arg303 in the active site of adenylosuccinate synthetase.

Adenosine Monophosphate↗
PubMed1977

Affinity labeling of rabbit muscle pyruvate kinase by 5'-p-fluorosulfonylbenzoyladenosine.

Rabbit muscle pyruvate kinase is irreversibly inactivated upon incubation with the adenine nucleotide analogue, 5'-p-fluorosulfonylbenzoyladenosine. A plot of the time dependence of the logarithm of the enzymatic activity at a given time divided by the initial enzymatic activity(logE/Eo) reveals a biphasic rate of inactivation, which is consistent with a rapid reaction to form partially active enzyme having 54% of the original activity, followed by a slower reaction to yield totally inert enzyme. In addition to the pyruvate kinase activity of the enzyme, modification with 5'-p-fluorosulfonylbenzoyladenosine also disrupts its ability to catalyze the decarboxylation of oxaloacetate and the ATP-dependent enolization of pyruvate. In correspondence with the time dependence of inactivation, the rate of incorporation of 5'-p-[14C]fluorosulfonylbenzoyladenosine is also biphasic. Two moles of reagent per mole of enzyme subunit are bound when the enzyme is completely inactive. The pseudo-first-order rate constant for the rapid rate is linearly dependent on reagent concentration, whereas the constant for the slow rate exhibits saturation kinetics, suggesting that the reagent binds reversibly to the second site prior to modification. The adenosine moiety is essential for the effectiveness of 5'-p-fluorosulfonylbenzoyladenosine, since p-fluorosulfonylbenzoic acid does not inactivate pyruvate kinase at a significant rate. Thus, the reaction of 5'-p-fluorosulfonylbenzoyladenosine with pyruvate kinase exhibits several of the characteristics of affinity labeling of the enzyme. Protection against inactivation by 5'-p-fluorosulfonylbenzoyladenosine is provided by the addition to the incubation mixture of phosphoenolpyruvate. Mg-ADP or Mg2+. In contrast, the addition of pyruvate, Mg-ATP, or ADP and ATP alone has no effect on the rate of inactivation. These observations are consistent with the postulate that the 5'-p-fluorosulfonylbenzoyladenosine specifically labels amino acid residues in the binding region of Mg2+ and the phosphoryl group of phosphoenolpyruvate which is transferred during the catalytic reaction. The rate of inactivation increases with increasing pH, and k1 depends on the unprotonated form of an amino acid residue with pK = 8.5. On the basis of the pH dependence of the reaction of pyruvate kinase with 5'-p-fluorosulfonylbenzoyladenosine and the elimination of cysteine residues as possible sites of reaction, it is postulated that lysyl or tyrosyl residues are the most probably candidates for the critical amino acids.

Adenosine↗
PubMed1984

Affinity labeling of the allosteric ADP activation site of NAD-dependent isocitrate dehydrogenase by 6-(4-bromo-2,3-dioxobutyl)thioadenosine 5'-diphosphate.

Pig heart NAD-dependent isocitrate dehydrogenase is allosterically activated by ADP which reduces the Km of isocitrate. The new ADP analogue 6-(4-bromo-2,3-dioxobutyl)thioadenosine 5'-diphosphate (BDB-TADP) reacts irreversibly with the enzyme at pH 6.1 and 25 degrees C, causing a rapid loss of the ability of ADP to increase the initial velocity of assays conducted at low isocitrate concentrations and a slower inactivation measured using saturating isocitrate concentrations. The rate constant for loss of ADP activation exhibits a nonlinear dependence on BDB-TADP concentration; in the presence of 0.2 mM MnSO4, KI for the reversible enzyme-reagent complex is 0.069 mM with kmax at saturating reagent concentrations equal to 0.031 min-1. For reaction at the site causing overall inactivation, KI for the initial reversible enzyme-reagent complex is estimated to be 0.018 mM with kmax = 0.0083 min-1 in the presence of 0.2 mM MnSO4. Total protection against both reactions is provided by 1 mM ADP plus 0.2 mM MnSO4 or by 0.1 mM ADP plus 0.2 mM MnSO4 plus 0.2 mM isocitrate, but not by NAD, ATP, or ADP plus EDTA. The BDB-TADP thus appears to modify two distinct metal-dependent ADP-binding sites. Incubation of isocitrate dehydrogenase with 0.14 mM BDB-[beta-32P]TADP at pH 6.1 in the presence of 0.2 mM MnSO4 results in incorporation of 0.81 mol of reagent/mol of average subunit when the ADP activation is completely lost and the enzyme is 68% inactivated. The time-dependent incorporation is consistent with the postulate that covalent reaction of 0.5 mol of BDB-TADP/mol of average enzyme subunit causes complete loss of ADP activation, while reaction with another 0.5 mol of BDB-TADP would lead to total inactivation. The enzyme is composed of three distinct subunits in the approximate ratio 2 alpha:1 beta:1 gamma. The distribution of BDB-[beta-32P]TADP incorporated into modified enzyme is 63:30:7% for alpha:beta:gamma throughout the course of the reaction. These results indicate the 6-(4-bromo-2,3-dioxobutyl)thioadenosine 5'-diphosphate functions as an affinity label of two types of potential metal-dependent ADP sites of NAD-dependent isocitrate dehydrogenase and that these allosteric sites are present on two (alpha and beta) of the enzyme's three types of subunits.

Adenosine Diphosphate↗
PubMed1984

Structures of the somatotropin receptor and prolactin receptor on rat hepatocytes characterized by affinity labelling.

Human somatotropin competed for 125I-human somatotropin binding to hepatocytes from female or male rats. Bovine somatotropin and prolactin each inhibited part, but not all, of the uptake of 125I-human somatotropin. The binding of 125I-prolactin was inhibited by human somatotropin and prolactin, but not by bovine somatotropin. Bovine somatotropin and human somatotropin, but not prolactin, competed for 125I-bovine somatotropin binding sites. 125I-labelled hormones were covalently coupled to membrane receptors with higher efficiency on hepatocytes from female than from male rats, allowing structural descriptions of lactogenic and somatogenic binding sites that had not been possible previously. Disuccinimidyl suberate covalently coupled 125I-human somatotropin into saturable complexes of Mr 300 000, 220 000, 130 000, 65 000 and 50 000. Bovine somatotropin inhibited the incorporation of 125I-human somatotropin into complexes of Mr 300 000, 220 000 and 130 000, whereas low concentrations of prolactin competed for incorporation into the 65 000- and 50 000-Mr species. 125I-bovine somatotropin was incorporated into complexes of Mr 300 000, 220 000 and 130 000. Human somatotropin and bovine somatotropin, but not prolactin, inhibited the production of these complexes. 125I-prolactin binding produced complexes of Mr 65 000 and 50 000. Native prolactin and human somatotropin, but not bovine somatotropin, inhibited uptake of 125I-prolactin into these species. Thus direct affinity labelling, as well as competition for covalent coupling, suggests that the 300 000-, 220 000- and 130 000-Mr species are components of the somatotropin receptor and that the 65 000- and 50 000-Mr complexes result from hormone binding to the prolactin receptor. By subtracting the Mr of prolactin, it was calculated that the hormone was bound to species of Mr 43 000 and 28 000. These Mr values were not affected by reduction of solubilized membranes, suggesting that the structure of the prolactin receptor is not stabilized by interchain disulphide bonds between subunits. Subtracting the Mr of somatotropin from somatogenic complexes indicated that the hormone had bound to species of Mr 280 000, 200 000 and 100 000. The 300 000- and 220 000-Mr complexes were not isolated from reduced membranes, whereas the amount of the 130 000-Mr species was augmented. These observations could suggest that a major component of the somatotropin receptor is a trimeric aggregate in which some subunits are retained in a larger complex by interchain disulphide bonds.

Affinity Labels↗
PubMed2005

Novel fluorescent labelled affinity probes for diadenosine-5',5'''-P1,P4-tetraphosphate (Ap4A)-binding studies.

Tandem synthetic-biosynthetic procedures were used to prepare two novel fluorescent labelled affinity probes for diadenosine-5',5'''-P1,P4-tetraphosphate (Ap4A)-binding studies. These compounds (dial-mant-Ap4A and azido-mant-Ap4A) are shown to clearly distinguish known Ap4A-binding proteins from Escherichia coli (LysU and GroEL) and a variety of other control proteins. Successful labelling of chaperonin GroEL appears to be allosteric with respect to the well-characterized adenosine 5'-triphosphate (ATP)-binding site, suggesting that GroEL possesses a distinct Ap4A-binding site.

Allosteric Site↗
PubMed1984

Short-wave ultraviolet irradiation increases photo-affinity labeling of benzodiazepine sites.

Photoaffinity labeling of benzodiazepine sites with [3H]-flunitrazepam was examined using either long-wave (366 nm) or short-wave (254 nm) ultraviolet irradiation. A multiple exposure protocol was employed so that the time course of the process could be determined as well as the fraction of total sites labeled. At 366 nm, approximately 20% of the total sites present were labeled and the remainder showed reduced affinity for flunitrazepam, in agreement with published reports. When membranes from either cortex or cerebellum were irradiated at 254 nm, however, the fraction of sites labeled increased above 40%. The change in the ratio of labeled sites to those showing reduced affinity, as well as differences in the time courses of the two phenomena, are taken as evidence that the labeling and affinity change are independent processes rather than two effects of a single event. Inhibition of labeling by clonazepam and Ro15-1788 indicated the "central" nature of the labeled sites.

Affinity Labels↗
PubMed2004

Photo-affinity labeling strategies in identifying the protein ligands of bioactive small molecules: examples of targeted synthesis of drug analog photoprobes.

The discovery of many new targets by chemical genetics has frequently exploited the fact that their biologically active chemical ligands were reactive and thus could covalently bind to their protein target(s). When experimental compounds or therapeutic agents with unidentified mechanisms of action do not contain reactive groups that can covalently label the putative site of molecular action, it may be possible to create a reactive photo-affinity probe if there is sufficient knowledge of the structure-activity relationship of the chemical series. Two specific examples are presented. These include the use of photo-affinity probes in the identification of the mechanism of action of synthetic oxazolidinones, a class of novel acting antibiotics and in the identification of a novel target for the insulin-sensitizing thiazolidinediones. Developments in photo-affinity labeling and combinatorial library design now imply that the parallel incorporation of photo-probes into screening library design could, at least in principle, greatly facilitate reverse pharmacological and chemical genetics approaches to protein target discovery.

Animals↗
PubMed1990

Glucocorticoid receptor identified on nuclear envelopes of male rat livers by affinity labeling and immunochemistry.

To exert their action at the genome, steroids must traverse the nuclear envelope, either alone or complexed to their receptor. Our previous studies identified two classes of dexamethasone-binding sites on male rat liver nuclear envelopes: a low capacity, high affinity site and a high capacity, low affinity site. The affinity reagent, [3H]dexamethasone mesylate, labeled peptides at 35-85 kDa, which may be the low affinity glucocorticoid-binding peptides, as these peptides showed the same response to hormonal manipulation as the low affinity [3H]dexamethasone-binding sites previously characterized. With dexamethasone mesylate and a monoclonal antibody against the glucocorticoid receptor, we have confirmed that the high affinity binding site on the nuclear envelope is the glucocorticoid receptor. Affinity labeling revealed the presence of a doublet of peptides at 85 and 110 kDa, in the same mol wt range as that reported for the glucocorticoid receptor. Furthermore, these affinity-labeled peptides responded to hormonal manipulation like nuclear glucocorticoid receptors. The monoclonal antibody identified a doublet of peptides, a major component of 92-94 kDa and a minor component of 98 kDa. Again, both peptides responded to hormonal manipulation like nuclear glucocorticoid receptors. The nuclear envelope-associated glucocorticoid receptor is not extracted by 0.1 M NaCl or 1% Triton X-100. These results show that glucocorticoid hormone interacts with the nuclear envelope via binding to the transformed glucocorticoid receptor, lending support to the two-step model of steroid hormone action.

Affinity Labels↗
PubMed1984

Alkylboronic acids accelerate affinity labelling of acetylcholinesterase with N,N-dimethyl-2-phenylaziridinium ion.

The kinetics of acetylcholinesterase alkylation with N,N-dimethyl-2-phenylaziridinium ion, the anionic-site-directed affinity label, has been investigated in the presence of alkylboronic acids, which are known as the esteratic-site-directed reversible inhibitors of the enzyme. The ternary complex of the enzyme, the aziridinium ion and alkylboronic acid, are formed in this reaction. In the case of propylboronic acid, for which the complete kinetic analysis of the acceleration effect has been carried out, the 85-fold increase in the rate of the enzyme alkylation reaction has been found. This acceleration effect was connected with the alkylation step, whereas the non-covalent binding of the aziridinium ion in the enzyme active centre was even hindered by the alkylboronic acid. The possible mechanism of this kinetic acceleration phenomenon is discussed with special reference to the kinetic data for the spontaneous solvolysis reaction of the aziridinium ion in water and organic solvents.

Acetylcholinesterase↗
PubMed1985

Affinity labeling the ribosome with eukaryotic-specific antibiotics: (bromoacetyl)trichodermin.

Trichodermin, a eukaryotic-specific antibiotic, inhibits protein synthesis in Drosophila cells. We have synthesized a 14C-labeled bromoacetyl derivative of trichodermin that binds to Drosophila 80S ribosomes and once bound reacts covalently with ribosomal proteins. It does not react with rRNA. Three large-subunit proteins (L1, L3, and L24) and three small-subunit proteins (S3/S5, 2/3S, and S8) are labeled by [14C] (bromoacetyl)trichodermin. Reaction with each of these proteins can be competed by an excess of unmodified trichodermin, indicating that the labeling has occurred from the native binding site of the parent drug. One of the (bromoacetyl)trichodermin-labeled proteins (S8) is also labeled by photoactivated puromycin in the A site. A second protein (S3/S5) is found to be labeled by a P-site affinity reagent. The results suggest that the trichodermin binding site spans both the small and large subunits and portions of both the A and P sites. These data combined with previous studies on the A and P sites of Drosophila ribosomes have allowed us to construct a model of the protein locations in this important active site.

Affinity Labels↗
PubMed1974

Affinity labelling of proteinases with tryptic specificity by peptides with C-terminal lysine chloromethyl ketone.

Methods are described for the synthesis of peptides terminating in Lys-CH(2)Cl. The products were examined as affinity labels for several enzymes of trypsin-like specificity which are resistant to Tos-Lys-CH(2)Cl. In part, the inertness of the latter may be due to the sulphonamide group, since Z-Lys-CH(2)Cl was more effective. However, a number of tripeptides with C-terminal Lys-CH(2)Cl were superior in their ability to inactivate subtilisin, thrombin and plasma kallikrein. The possibility of developing enzyme-specific reagents selective for members within the trypsin-like group is demonstrated by Ala-Phe-Lys-CH(2)Cl, which readily inactivates plasma kallikrein but not thrombin.

Amino Acid Chloromethyl Ketones↗
PubMed1970

Photo-affinity labeling of specific acetylcholine-binding sites on membranes.

Acetylcholinesterase of intact red blood cell membranes and the acetylcholine receptor at the neuromuscular junction of whole-frog sartorius muscle have been irreversibly inactivated by photo-affinity labeling with two quaternary ammonium aryl azides. The inactivation requires that the azides, at the time of their photolytic conversion to highly reactive nitrenes, are reversibly bound to the specific acetylcholine-binding sites.

Acetylcholine↗
PubMed1985

Vasoactive intestinal peptide effects on GH3 pituitary tumor cells: high affinity binding, affinity labeling, and adenylate cyclase stimulation. Comparison with peptide histidine isoleucine and growth hormone-releasing factor.

The vasoactive intestinal polypeptide (VIP) receptor was characterized on the GH3 rat pituitary tumor cell line using competitive binding studies with peptides having sequence homology with VIP. Further studies investigated receptor coupling to the adenylate cyclase complex by measurement of cAMP levels. Finally, the molecular weight of the receptor was estimated by affinity labeling techniques. Studies using 125I-VIP and unlabeled competing peptides revealed a single class of high affinity binding sites with a dissociation constant (KD) of 17 +/- 2 nM (mean +/- S.E.M.) for VIP, 275 +/- 46 nM for peptide histidine isoleucine (PHI), and 1380 +/- 800 nM for human pancreatic growth hormone releasing factor (GHRF). VIP and PHI each stimulated intracellular cAMP accumulation in a dose-dependent manner; both peptides demonstrated synergism with forskolin. In contrast, GHRF neither stimulated accumulation of cAMP nor demonstrated synergism with forskolin. VIP plus PHI (1 microM each) caused no significant increase in cAMP over either VIP or PHI alone, implying that the two peptides act through the same receptor. Covalent crosslinking of 125I-VIP to its binding site using either disuccinimidyl suberate (DSS) or ethylene glycol bis(succinimidyl succinate) (EGS) was followed by SDS-PAGE and autoradiography. The result is consistent with an Mr 47 000 VIP-binding subunit comprising or being associated with the VIP receptor of GH3 pituitary tumor cells.

Adenylyl Cyclases↗