PubMed Health⌕ Search

Biomedical subjects

R F Colman

Publications and source records attributed to R F Colman.

At least 37 records · Page 2Linked to original sources
PubMed1997

Identification of the nonsubstrate steroid binding site of rat liver glutathione S-transferase, isozyme 1-1, by the steroid affinity label, 3beta-(iodoacetoxy)dehydroisoandrosterone.

3beta-(Iodoacetoxy)dehydroisoandrosterone (3beta-IDA), an analogue of the electrophilic substrate, Delta5-androstene-3,17-dione, as well as an analogue of several other steroid inhibitors of glutathione S-transferase, was tested as an affinity label of rat liver glutathione S-transferase, isozyme 1-1. A time-dependent loss of enzyme activity is observed upon incubation of 3beta-IDA with the enzyme. The rate of enzyme inactivation exhibits a nonlinear dependence on 3beta-IDA concentration, yielding an apparent Ki of 21 microM. Upon complete inactivation of the enzyme, a reagent incorporation of approximately 1 mol/mol of enzyme subunit or 2 mol/mol of enzyme dimer is observed. Protection against inactivation and incorporation is afforded by alkyl glutathione derivatives and nonsubstrate steroid ligands such as 17beta-estradiol-3,17-disulfate but, surprisingly, not by Delta5-androstene-3,17-dione or any other electrophilic substrate analogues tested. These results suggest that the site of reaction is within the nonsubstrate steroid binding site of the enzyme, which is distinguishable from the electrophilic substrate binding site, near the active site of the enzyme. Two cysteine residues, Cys17 and Cys111, are modified in nearly equal amounts, despite an average reagent incorporation of 1 mol/mol enzyme subunit. Isolation of enzyme subunits indicates the presence of unmodified, singly labeled, and doubly labeled subunits, consistent with mutually exclusive modification of cysteine residues across enzyme subunits; i.e., modification of Cys111 on subunit A prevents modification of Cys111 on subunit B and similarly for Cys17. Molecular modeling analysis suggests that Cys17 and Cys111 are located in the nonsubstrate steroid binding site, within the cleft between the subunits of the dimeric enzyme.

Affinity Labels↗
PubMed1997

Modulation of platelet responses by 2-[3-(bromo-2-oxopropylthio)]adenosine-5'-diphosphate involves its binding to as well as covalent modification of an ADP-receptor, aggregin.

The 2-substituted ADP derivatives are known to activate human blood platelets with varying degrees of potency. For example, 2-(4-bromo-2,3-dioxobutylthio)adenosine-5'-diphosphate [2-BDB-TADP], an ADP-affinity analog, was previously shown by us to be 50% as potent as ADP in inducing human blood platelet responses [Puri, R. N., Colman, R. F., and Colman, R. W. (1996) Eur. J. Biochem. 236, 862-870]. 2-Methylthio-ADP (2-MeS-ADP) has been known to be a far more potent agonist than ADP. However, the molecular basis for defining the rank order of potency of the 2-substituted ADP derivatives as agonists of platelet responses have been incompletely understood. We now report that 2-BOP-TADP (a one carbon atom lower homolog of 2-BDB-TADP) at equimolar concentration is as potent as ADP in inducing platelet responses. Prolonged incubation of platelets with 2-BOP-TADP abolished its ability to elicit cellular responses. An autoradiogram of the gel obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized platelets labeled by incubating the platelets with 2-BOP-TADP for 1 h followed by reduction by NaB[3H]4 showed the presence of a single covalently radiolabeled protein band at 100 kDa. Preincubation of platelets with either ADP or ATP reduced the intensity of the band corresponding to the 100-kDa protein radiolabeled by 2-BOP-TADP and NaB[3H]4. The results show that (i) 2-BOP-TADP modulates ADP-induced platelet responses by interacting with aggregin and (ii) 2-BOP-TADP was twice as potent as 2-BDB-TADP, and (iii) the chain length of the substituent in a homologous series has an important bearing on the potency of a 2-substituted ADP analog.

Adenosine Diphosphate↗
PubMed1997

Resonance energy transfer between sites in rat liver glutathione S-transferase, 1-1, selectively modified at cysteine-17 and cysteine-111.

Monobromobimane (mBBr) can label both Cys111 and Cys17 of rat liver glutathione S-transferase, 1-1 (GST 1-1). However, selective modification of Cys111 was achieved by the maleimide-based sulfhydryl reagents N-ethylmaleimide (NEM) and fluorescein 5-maleimide (NFM). Incubation of GST 1-1 with 5 mM NEM for 30 min at pH 7.5 and 25 degrees C leads to the formation of modified enzyme with 92% residual activity toward 1-chloro-2,4-dinitrobenzene and completely blocks Cys111 from subsequent reaction with either NFM or mBBr. Reaction of GST 1-1 with 0.2 mM NFM under the same conditions affords a modified enzyme with only 14% residual activity even though NFM and NEM target the same Cys111. The results indicate that when the bulky fluorescein is covalently bound to Cys111, the ligand projects into both the xenobiotic binding site and the glutathione site. After NEM or NFM modification of GST 1-1, the enzyme was further modified by monobromobimane at Cys17 with loss of activity. Together with the only tryptophan (Trp20), fluorescein linked to Cys111 and bimane to Cys17 provide three fluorescent probes to study the solution structure of GST 1-1. Fluorescence spectral analysis suggests that Trp20 and bimane linked to Cys17 are located in a relatively hydrophobic environment, while fluorescein linked to Cys111 is located in a charged environment. These fluorescent probes constitute three sets of donor-acceptor pairs for the measurement of fluorescence energy transfer, and distances calculated from such measurements are 20 A between Trp20 and bimane at Cys17, 19 A between Trp20 and fluorescein at Cys111, and < 22 A between bimane at Cys17 and fluorescein at Cys111. Molecular modeling studies indicate that fluorescein lies between the two subunits, is surrounded by charged residues, and is extended into the xenobiotic binding site. They also suggest that mBBr must approach from the dimer interface in order to reach the reaction site at Cys17.

Amino Acid Sequence↗
PubMed1997

Inactivation of recombinant monocyte cAMP-specific phosphodiesterase by cAMP analog, 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic monophosphate.

Two cAMP analogs, 8- and 2- [(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic monophosphate (8- and 2-BDB-TcAMP) have been used in probing the catalytic site of recombinant monocyte cAMP-specific phosphodiesterase (PDE4a). 2-BDB-TcAMP is a reversible and competitive inhibitor (Ki = 5.5 mumol/L) of cAMP hydrolysis by PDE4a, 8-BDB-TcAMP irreversibly inactivates the enzyme in a time- and concentration-dependent manner with a second order rate constant of 0.022 mmol/L-1 min-1. The rate of inactivation of PDE4a is reduced by the presence of the substrate cAMP and specific inhibitors, rolipram and denbufylline, but not by cGMP or AMP. Reduction of the enzyme-inhibitor complex with sodium [3H]borohydride shows that 1.2 mol of the affinity label/mol of enzyme was incorporated. The radiolabeled peptide is composed of 10 amino acid residues (697 to 706) located near the carboxyl end of the proposed catalytic domain. The peptide (GPGHPPLPDK) has seven nonpolar and aliphatic residues, of which four are proline, giving the peptide a highly structured conformation. This peptide is the first to be identified in the putative catalytic domain involved in substrate recognition.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1997

Identification of His141 in the active site of Bacillus subtilis adenylosuccinate lyase by affinity labeling with 6-(4-bromo-2,3-dioxobutyl)thioadenosine 5'-monophosphate.

Adenylosuccinate lyase of Bacillus subtilis is inactivated by 25-400 microM 6-(4-bromo-2,3-dioxobutyl)thioadenosine 5'-monophosphate (6-BDB-TAMP) at pH 7.0 and 25 degrees C. The initial inactivation rate constant exhibits nonlinear dependence on the concentration of 6-BDB-TAMP, implying there is reversible formation of enzyme-reagent complex (K(I) = 30 +/- 4 microM) prior to irreversible modification (kmax = 0.139 +/- 0.005 min(-1)). The tetrameric enzyme incorporates about 1 mol of 6-BDB-[32P]TAMP per mol of enzyme subunit concomitant with complete inactivation. Protection against inactivation and incorporation of [32P]reagent is provided by adenylosuccinate or a combination of AMP and fumarate, whereas either AMP or fumarate alone is much less effective. These observations suggest that 6-BDB-TAMP targets the adenylosuccinate-binding site. Hydrolyzed 6-BDB-TAMP is a competitive inhibitor with respect to adenylosuccinate in the catalytic reaction and also decreases the rate of inactivation by 6-BDB-TAMP. These results account for the decrease in the inactivation rate as the reaction of 6-BDB-TAMP with the enzyme proceeds. Purification by chromatography on dihydroxyboryl-agarose and high performance liquid chromatography of the tryptic digest of inactivated enzyme yields a single radioactive peptide, Thr140-Phe150, as determined by gas-phase sequencing. Modified His141 is the reaction product of 6-BDB-TAMP and adenylosuccinate lyase. We conclude that 6-BDB-TAMP functions as a reactive adenylosuccinate analog in modifying His141 in the substrate-binding site of adenylosuccinate lyase, where it may serve as a general base accepting a proton from the succinyl group during catalysis.

Adenosine Monophosphate↗
PubMed1997

Probing the active site of alpha-class rat liver glutathione S-transferases using affinity labeling by monobromobimane.

Monobromobimane (mBBr) is a substrate of both mu- and alpha-class rat liver glutathione S-transferases, with Km values of 0.63 microM and 4.9 microM for the mu-class isozymes 3-3 and 4-4, respectively, and 26 microM for the alpha-class isozymes 1-1 and 2-2. In the absence of substrate glutathione, mBBr acts as an affinity label of the 1-1 as well as mu-class isozymes, but not of the alpha-class 2-2 isozyme. Incubation of rat liver isozyme 1-1 with mBBr at pH 7.5 and 25 degrees C results in a time-dependent inactivation of the enzyme but at a slower (threefold) rate than for reactions with the mu-class isozyme 3-3 and 4-4. The rate of inactivation of 1-1 isozyme by mBBr is not decreased but, rather, is slightly enhanced by S-methyl glutathione. In contrast, 17 beta-estradiol-3,17-disulfate (500 microM) gives a 12.5-fold decrease in the observed rate constant of inactivation by 4 mM mBBr. When incubated for 60 min with 4 mM mBBr, the 1-1 isozyme loses 60% of its activity and incorporates 1.7 mol reagent/mol subunit. Peptide analysis after thermolysin digestion indicates that mBBr modification is equally distributed between two cysteine residues at positions 17 and 111. Modification at these two sites is reduced equally in the presence of the added protectant, 17 beta-estradiol-3,17-disulfate, suggesting that Cys 17 and Cys 111 reside within or near the enzyme's steroid binding sites. In contrast to the 1-1 isozyme, the other alpha-class isozyme (2-2) is not inactivated by mBBr at concentrations as high as 15 mM. The different reaction kinetics and modification sites by mBBr suggest that distinct binding site structures are responsible for the characteristic substrate specificities of glutathione S-transferase isozymes.

Affinity Labels↗
PubMed1997

Chemical arrows for enzymatic targets.

Reactive analogs of substrates or allosteric regulators can be designed to bind reversibly to particular ligand sites of enzymes. Subsequently, these compounds can react covalently with amino acids accessible from the ligand site, thereby functioning as chemical arrows aimed at specific enzymatic target sites. The approach of affinity labeling can be used to identify amino acid participants in active or regulatory sites, to provide a rational choice of targets for site-directed mutagenesis experiments, or to monitor conformational changes in the region of a particular enzyme site. Illustrations of these approaches include: 1) the use of reactive nucleotide analogs directed to substrate sites in adenylosuccinate synthetase and adenylosuccinate lyase and to regulatory sites of glutamate dehydrogenase, 2) the use of affinity cleavage by Fe2(+)-isocitrate to locate the metal-substrate site of isocitrate dehydrogenase, and 3) the use of reactive peptides and aromatic compounds to target the glutathione and xenobiotic sites of glutathione S-transferases.

Adenosine↗
PubMed1996

Crystallization and preliminary diffraction analysis of porcine heart mitochondrial NADP(+)-dependent isocitrate dehydrogenase.

Isocitrate dehydrogenases [isocitrate:NAD(P)(+) oxidoreductase (decarboxylating), E.C. 1.1.1.42] are ubiquitous metabolic enzymes which occur in all living organisms. The NADP(+)- dependent mitochondrial isocitrate dehydrogenase from pig heart has been crystallized from polyethylene glycol/sodium sulfate mixtures in the presence of Mg(2+) and isocitrate. The crystals belong to space group C2 with a = 137.0, b = 113.4, c = 65.0 A and beta = 98.5 degrees, and diffract to at least 2.4 A resolution. There are two protein monomers per asymmetric unit which are related by non-crystallographic twofold symmetry.

Journal Article↗
PubMed1996

Involvement of arginine 143 in nucleotide substrate binding at the active site of adenylosuccinate synthetase from Escherichia coli.

Adenylosuccinate synthetase from Escherichia coli is inactivated in a biphasic reaction by guanosine 5'-O-[S-(4-bromo-2,3-dioxobutyl)thio]phosphate (GMPSBDB) at pH 7.1 and 25 degrees C. Reaction of the enzyme with [8-3H]GMPSBDB results in the incorporation of 2 mol of the reagent/mol of subunit; in the presence of active site ligands the incorporation is reduced to 1 mol of reagent/mol of subunit. GMPSBDB reacts with Cys-291 in the initial rapid reaction which is accompanied by loss of 50% of the enzymatic activity; this reaction is not affected by the presence of active site ligands. In the slower reaction, GMPSBDB inactivates the enzyme by reacting with Arg-143. The inactivation kinetics of the slower phase are consistent with the formation of an enzyme--GMPSBDB complex having a Kd of 42 microM. Active site nucleotides, either adenylosuccinate or IMP + GTP, prevent both slower phase inactivation and labeling of Arg-143. Replacement of Arg-143 with a Leu by site-directed mutagenesis does not change the catalytic constant or the K(m) for aspartate but does significantly impair nucleotide binding: the Michaelis constants for IMP and GTP increase by 60-fold and 10-fold, respectively, in the R143L mutant. The crystal structure of the E. coli enzyme [Poland, B.W., Silva, M.M., Serra, M.A., Cho, Y., Kim, K. H., Harris, E.M.S., & Honzatko, R.B. (1993) J. Biol. Chem. 268, 25334--25342] shows that Arg-143 from one subunit projects into the putative active site of the other subunit. These results indicate that both subunits of dimeric adenylosuccinate synthetase contribute to each active site and that Arg-143 plays an important role in nucleotide binding.

Adenylosuccinate Synthase↗
PubMed1996

Platelet activation by 2-(4-bromo-2,3-dioxobutylthio)adenosine 5'-diphosphate is mediated by its binding to a putative ADP receptor, aggregin.

Platelet responses induced by ADP are mediated by a unique P21-purinergic receptor. Although a variety of ADP analogs, substituted at C2, have been used to delineate pharmacological properties of the ADP-binding site(s), the identity of the receptor protein has not been firmly established. 2-(4-Bromo-2,3-dioxobutylthio)- ADP [2-BrCH2(CO)2CH2-S-ADP], a well-characterized ADP analog, has been previously used as an affinity label to examine the structure/function relationship of ADP-requiring enzymes [Kapetanovic, E., Bailey, J.B. & Colman, R.F. (1985) Biochemistry 24, 7586-7593]. We found that it induced platelet shape change, aggregation, exposure of fibrinogen binding sites, secretion and mobilization of intracellular calcium, but was less potent than ADP. Under non-stirring conditions, incubation of platelets with this analog for longer time periods blocked ADP-induced shape change, aggregation, and the ability to ADP to antagonize the rise in intracellular levels of cAMP induced by iloprost (a prostaglandin I2 analog). Of a variety of agonists examined, only ADP-induced aggregation was almost completely inhibited in platelets irreversibly modified by the analog. An autoradiogram of the gel obtained by SDS/PAGE of solubilized platelets modified by the ADP analog followed by reduction of the dioxo group by NaB[3H], showed the presence of a single radiolabeled protein band at 100 kDa. Platelets incubated first with either ADP, ATP, or 2-methylthio-ADP were not labeled by 2-BrCH2(CO)2CH2S-ADP and NaB[3H]4-8-BrCH2(CO)2CH2-S-ADP was previously shown by us to irreversibly antagonize ADP-induced platelet responses by selectively modifying aggregin. Incubation of platelets with 2-BrCH2(CO)2CH2S-ADP completely blocked labeling of aggregin in platelets by 8-BrCH2(CO)2CH2S-[32P]ADP. These results show that 2-BrCH2(CO)2CH2S-ADP initially interacts reversibly with aggregin (100kDa), a putative ADP receptor, and induces platelet shape change and aggregation, and at longer periods of incubation reacts irreversibly to block the ability of ADP to antagonize stimulated adenylate cyclase activity. In contrast, 6-BrCH2(CO)2CH2S-ADP was found to be a weak and reversible inhibitor of ADP-induced platelet aggregation. Prior incubation of platelets with the latter analog reduced labeling of aggregin by 8-BrCH2(CO)2CH2S-[32P]ADP. Taken together, the results further show that substitution by the BrCH2(CO)2CH2 group at the C2 and C8 positions is tolerated, while the presence of a free amino function at the C6 position is essential for its interaction with aggregin.

Adenosine Diphosphate↗
PubMed1996

Affinity Labeling of the active site of rabbit muscle adenylosuccinate lyase by 2-[(4-bromo-2.3-dioxobutyl)thio] adenosine 5'-monophosphate.

Rabbit muscle adenylosuccinate lyase upon incubation with 7.5-50 muM 2 -[(4-bromo-2.3-dioxobutyl)thio]adenosine 5'-monophosphate (2-BDB-TAMP) in 0.05 M PIPES buffer, ph 7.0 and 10 degrees C, gives a time dependent biphasic inactivation. The rate of inactivation exhibits a nonlinear dependence on the concentration 2-BDB-TAMP, which can be described by reversible binding of reagent to the enzyme (K1=8.5 microM. 5.2 microM) prior to the irreversible reaction, with maximum rate constants of 0.319 and 0.027 min-1 for the fast and slow phases, respectively. The enzyme is a tetramer, with subunits of 50 000 Da. When the enzyme was 90% inactivated, 0.84 mol of reagent/mol of subunit was incorporated as measured by protein-bound phosphate analysis; similar results were obtained using 2-BDB-[14C]TAMP. Complete protection against inactivation and incorporation was afforded by 1 mM 5'-AMP and by 0.1 mM 5'-AMP + 5 mM fumarate (the natural products of adenylosuccinate hydrolysis) but not by 0.1 mM 5'-AMP alone, 5 mM fumarate alone, or 0.1 mM 5'-AMP + 5 mM maleate or 5 mM succinate. These studies suggest that 2-BDB-TAMP inactivates adenylosuccinate lyase by specific reaction at the substrate binding site, with negative cooperativity between subunits accounting for the appearance of two phases of inactivation. Cleavage of 2-BDB-TAMP-modified enzyme with cyanogen bromide and subsequent separation of peptides by reverse phase HPLC gave only one radioactive peak. This radioactive peptide was further digested with papain and the target site of the 2-BDB-TAMP reaction was identified as Arg112. We conclude that Arg112 is located in the substrate binding site of rabbit muscle adenylosuccinate lyase.

Adenosine Monophosphate↗
PubMed1996

Tyrosine 8 contributes to catalysis but is not required for activity of rat liver glutathione S-transferase, 1-1.

Reaction of rat liver glutathione S-transferase, isozyme 1-1, with 4-(fluorosulfonyl)benzoic acid (4-FSB), a xenobiotic substrate analogue, results in a time-dependent inactivation of the enzyme to a final value of 35% of its original activity when assayed at pH 6.5 with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. The rate of inactivation exhibits a nonlinear dependence on the concentration of 4-FSB from 0.25 mM to 9 mM, characterized by a KI of 0.78 mM and kmax of 0.011 min-1. S-Hexylglutathione or the xenobiotic substrate analogue, 2,4-dinitrophenol, protects against inactivation of the enzyme by 4-FSB, whereas S-methylglutathione has little effect on the reaction. These experiments indicate that reaction occurs within the active site of the enzyme, probably in the binding site of the xenobiotic substrate, close to the glutathione binding site. Incorporation of [3,5-3H]-4-FSB into the enzyme in the absence and presence of S-hexylglutathione suggests that modification of one residue is responsible for the partial loss of enzyme activity. Tyr 8 and Cys 17 are shown to be the reaction targets of 4-FSB, but only Tyr 8 is protected against 4-FSB by S-hexylglutathione. DTT regenerates cysteine from the reaction product of cysteine and 4-FSB, but does not reactivate the enzyme. These results show that modification of Tyr 8 by 4-FSB causes the partial inactivation of the enzyme. The Michaelis constants for various substrates are not changed by the modification of the enzyme. The pH dependence of the enzyme-catalyzed reaction of glutathione with CDNB for the modified enzyme, as compared with the native enzyme, reveals an increase of about 0.9 in the apparent pKa, which has been interpreted as representing the ionization of enzyme-bound glutathione; however, this pKa of about 7.4 for modified enzyme remains far below the pK of 9.1 for the -SH of free glutathione. Previously, it was considered that Tyr 8 was essential for GST catalysis. In contrast, we conclude that Tyr 8 facilitates the ionization of the thiol group of glutathione bound to glutathione S-transferase, but is not required for enzyme activity.

Affinity Labels↗
PubMed1996

Expression of pig heart mitochondrial NADP-dependent isocitrate dehydrogenase in Escherichia coli.

Pig heart mitochondrial NADP-specific isocitrate dehydrogenase is the most extensively studied among the mammalian isocitrate dehydrogenases. The 1.2-kbp cDNA encoding this porcine mitochondrial NADP-specific enzyme has now been inserted into an expression vector, pMAL-c2, to be expressed as a fusion protein with maltose binding protein. Initially, the vector was constructed with a cleavage site for protease Factor Xa between the maltose binding protein and isocitrate dehydrogenase; however, since Factor Xa was also found to digest isocitrate dehydrogenase, a thrombin recognition site was substituted. The fusion protein was expressed in Escherichia coli by IPTG induction at 25 degrees C, and was separated from the endogenous E. coli isocitrate dehydrogenase by affinity chromatography on an amylose resin which adsorbs maltose binding protein and its fusion products. Cleavage of the fusion protein with thrombin generated pig heart NADP-specific isocitrate dehydrogenase, which was purified to homogeneity by affinity chromatography on Matrex Gel Red-A resin and gel filtration by FPLC. A 41-fold increase in specific activity to 37 enzyme units/mg with an approximate yield of 34% for the expressed enzyme was achieved by this purification procedure. This enzyme exhibits a single band (M(r) = 46,600) on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and, under standard assay conditions, has a Km for DL-isocitrate of 7.74 +/- 0.18 microM and a Km for NADP+ of 6.63 +/- 1.34 microM. These values are similar to the Kms measured for the enzyme purified from pig heart. The amino-terminal sequence of the expressed enzyme is identical with that of authentic porcine enzyme and distinguishable from the E. coli enzyme at 17 of the 18 residues determined. We conclude that this expression and purification system yields pure pig heart mitochondrial NADP-specific isocitrate dehydrogenase and should allow generation of wild-type and mutant enzymes in amounts suitable for their biochemical characterization and comparison.

ATP-Binding Cassette Transporters↗
PubMed1995

Inhibition of ADP-induced platelet responses by covalent modification of aggregin, a putative ADP receptor, by 8-(4-bromo-2,3-dioxobutylthio)ADP.

ADP is an important platelet agonist which initiates platelet shape change, aggregation, exposure of fibrinogen receptors, and calcium mobilization. Because of the limitations of previously used affinity analogs and photo-labeling studies as well as controversies surrounding the identity of an ADP receptor on platelets, we have used an affinity label capable of alkylating a putative exofacial receptor on platelets. We now report that 8-(4-bromo-2,3-dioxobutylthio)adenosine-5'-diphosphate (8-BDB-TADP), which is an analog of the natural ligand ADP, blocked ADP-induced platelet shape change, aggregation, exposure of fibrinogen-binding sites, secretion, and calcium mobilization. Following modification by 8-BDB-TADP, the rates of aggregation of platelets induced by thrombin, a calcium ionophore (A23187) or a stimulator of protein kinase C (phorbol myristate acetate) were minimally affected. However, the 8-BDB-TADP-modified platelets exhibited decreased rates of aggregation in response to ADP, as well as collagen and a thromboxane mimetic (U46619), both of which partially require ADP. Autoradiograms of the gels obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized platelets modified by either [beta-32P]8-BDB-TADP, or 8-BDB-TADP and NaB[3H]4 showed the presence of a single radiolabeled protein band at 100 kDa. The intensity of this band was reduced when platelets were preincubated with ADP, ATP, and 8-bromo-ADP prior to labeling by the radioactive 8-BDB-TADP. The results show that 8-BDB-TADP selectively and covalently labeled aggregin (100 kDa), a putative ADP receptor, resulting in a loss of ADP-induced platelet responses.

Adenosine Diphosphate↗
PubMed1995

Monobromobimane as an affinity label of the xenobiotic binding site of rat glutathione S-transferase 3-3.

Monobromobimane (mBBr), besides being a substrate in the presence of glutathione, inactivates rat liver glutathione S-transferase 3-3 at pH 7.5 and 25 degrees C as assayed using 1-chloro-2,4-dinitrobenzene (CDNB). The rate of inactivation is enhanced about 5-fold by S-methylglutathione. Substrate analogs bromosulfophthalein and 2,4-dinitrophenol decrease the rate of inactivation at least 20-fold. Upon incubation for 60 min with 0.25 mM mBBr and S-methylglutathione, the enzyme loses 91% of its activity toward CDNB and incorporates 2.14 mol of reagent/mol of subunit, whereas incubation under the same conditions but with added protectant 2,4-dinitrophenol yields an enzyme that is catalytically active and contains only 0.89 mol of reagent/mol of subunit. mBBR-modified enzyme is fluorescent, and fluorescence energy transfer occurs between intrinsic tryptophan and covalently bound bimane in modified enzyme. Both Tyr115 and Cys114 are modified, but Tyr115 is the initial reaction target and its modification correlates with loss of activity toward CDNB. The fact that the activity toward mBBr is retained by the enzyme after modification suggests that rat isozyme 3-3 has two binding sites for mBBr.

Affinity Labels↗
PubMed1995

Cadmium-113 and magnesium-25 NMR study of the divalent metal binding sites of isocitrate dehydrogenases from pig heart.

The metal activator sites of NAD(+)-dependent and NADP(+)-dependent isocitrate dehydrogenases from pig heart have been probed using 113Cd- and 25Mg-NMR. In the presence of isocitrate and ADP, a broad resonance for cadmium bound to NAD-dependent isocitrate dehydrogenase was observed (-8 ppm) arising from exchange with isocitrate (-20 ppm) and/or ADP (27 ppm) complexes. The Cd shift with ADP suggests interaction of the metal with the nucleotide ring nitrogen. Increasing shifts with excess ADP are indicative of macrochelate formation. 25Mg-NMR demonstrates that, unlike manganese, magnesium has a similar dissociation constant (1.8 mM) from NADP-dependent isocitrate dehydrogenase as from the enzyme-isocitrate complex (1.1 mM). The extrapolated line width of bound magnesium increases from 674 Hz in the binary complex to 10,200 Hz in the ternary complex. The quadrupole coupling constant, calculated from relaxation rates, is larger in the ternary complex, indicative of greater distortion in the magnesium coordination sphere. The line widths of magnesium complexed to NAD-dependent isocitrate dehydrogenase are broader, as expected for the larger octamer. 113Cd- and 25Mg-NMR both show that the metal sites have anisotropic octahedral symmetry. 25Mg relaxation rates yield correlation times corresponding to motions of a domain with motion independent of the enzyme multimers.

Animals↗
PubMed1995

N-ethylmaleimide profiling of yeast NADP-dependent isocitrate dehydrogenase.

Yeast NADP-dependent isocitrate dehydrogenase is inactivated by N-ethyl-maleimide (NEM) at pH 7.7 and 30 degrees C. Reaction with cysteine382 occurs most rapidly and is accompanied by loss of about 50% of the enzymatic activity. A slower phase of inactivation ensues during which lysine343 is the major target of NEM, while minor products result from reaction at cysteine73 and cysteine354. Protection against the second phase of inactivation is provided by NADP, NAPH, or manganous-isocitrate. Comparison of the time-dependence of inactivation and the products of reaction with N-ethyl-ethylmaleimide (NEM profiling) of the pig heart (G. E. Smyth and R. F. Colman, 1991, J. Biol. Chem. 266, 14918-14925) and yeast NADP-specific isocitrate dehydrogenases have been coupled with an examination of the crystal structure of the Escherichia coli isocitrate dehydrogenase. The following conclusions have been reached: while no cysteine is essential for activity, yeast Cys382/pig Cys379 is close to the adenine portion of the NADP binding site, and pig Cys269 is located in the region of the metal-isocitrate binding site.

Amino Acid Sequence↗