Biomedical subjects

R F Colman

Publications and source records attributed to R F Colman.

At least 109 records · Page 6Linked to original sources

PubMed1987

Direct evidence for the interaction of the nucleotide affinity analog 5'-p-fluorosulfonylbenzoyl adenosine with a platelet ADP receptor.

Previous reports have indicated that the nucleotide affinity analog 5'-p-fluorosulfonylbenzoyl adenosine (FSBA) at concentrations between 40 and 100 mumol/L and at times greater than 20 minutes covalently modifies a single protein component on the external platelet membrane surface and that adenosine diphosphate (ADP) protects against this reaction. That this protein is an ADP receptor linked to platelet activation is shown by FSBA inhibition of ADP-mediated platelet shape change, aggregation, and fibrinogen receptor exposure. In this report, further evidence for the interaction of FSBA with the ADP receptor on platelets is provided by the observation that FSBA at high concentrations (100 to 500 mumol/L) behaves as a weak agonist to produce platelet shape change within one minute as detected by spectroscopic assay and scanning electron microscopy with concomitant phosphorylation of the light chain of platelet myosin. The specificity of FSBA as an agonist is demonstrated by inhibition of FSBA-induced shape change by ATP and the covalent incorporation of SBA as well as the failure of 5'-fluorosulfonylbenozoyl guanosine (FSBG) to cause shape change. In contrast, incubation of platelets with low concentrations of [3H]-FSBA (40 mol/L) is not associated with stimulation of platelet shape change or myosin light chain phosphorylation.

Adenosine↗

PubMed1987

Inhibition of thrombin-induced platelet aggregation by high molecular weight kininogen.

1. Thrombin-induced platelet aggregation and cleavage of [3H]FSBA-labeled aggregin is mediated by platelet calpain. 2. Plasma HMWK inhibits platelet aggregation and cleavage of aggregin in intact platelets, indirectly by thrombin and directly by calpain. 3. Inhibition of thrombin-induced platelet aggregation by HMWK is specific for this proteolytic enzyme. 4. HMWK is the major inhibitor of thrombin-induced activation of platelets in plasma.

Fibrinogen↗

PubMed1986

Affinity labeling of an allosteric ADP site of glutamate dehydrogenase by 2-(4-bromo-2,3-dioxobutylthio)adenosine 5'-monophosphate.

Bovine liver glutamate dehydrogenase reacts covalently with the adenine nucleotide analogue 2-(4-bromo-2,3-dioxobutylthio)adenosine 5'-monophosphate (2-BDB-TAMP) with incorporation of about 1 mol of reagent/mol of enzyme subunit. The modified enzyme is not inactivated by this reaction as measured in the absence of allosteric effectors. Native glutamate dehydrogenase is activated by ADP and inhibited by high concentrations of NADH; both of these effects are irreversibly decreased upon reaction of the enzyme with 2-BDB-TAMP. The decrease in activation by ADP was used to determine the rate constant for reaction with 2-BDB-TAMP. The rate constant (kobs) for loss of ADP activation exhibits a nonlinear dependence on 2-BDB-TAMP concentration, suggesting a reversible binding of reagent (KR = 0.74 mM) prior to irreversible modification. At 1.2 mM 2-BDB-TAMP, kobs = 0.060 min-1 and is not affected by alpha-ketoglutarate or GTP, but is decreased to 0.020 min-1 by 5 mM NADH and to zero by 5 mM ADP. Incorporation after incubation with 1.2 mM 2-BDB-TAMP for 1 h at pH 7.1 is 1.02 mol/mol enzyme subunit in the absence but only 0.09 mol/subunit in the presence of ADP. The enzyme protected with 5 mM ADP behaves like native enzyme in its activation by ADP and in its inhibition by NADH. Native enzyme binds reversibly 2 mol of [14C]ADP/subunit, whereas modified enzyme binds only 1 mol of ADP/peptide chain. These results indicate that incorporation of 1 mol of 2-BDB-TAMP causes elimination of one of the ADP sites of the native enzyme. 2-BDB-TAMP acts as an affinity label of an ADP site of glutamate dehydrogenase and indirectly influences the NADH inhibitory site.

Adenosine Diphosphate↗

PubMed1986

Inactivation of NAD-dependent isocitrate dehydrogenase by affinity labeling of the allosteric ADP site by 2-(4-bromo-2,3-dioxobutylthio)adenosine 5'-diphosphate.

A new reactive ADP analogue has been synthesized: 2-(4-bromo-2,3-dioxobutylthio)adenosine 5'-diphosphate (2-BDB-TADP). Reaction of ADP with m-chloroperoxybenzoic acid gave ADP 1-oxide, which was treated with NaOH, followed by reaction with carbon disulfide to yield 2-thioadenosine 5'-diphosphate. The final product was synthesized by condensation of 2-thioadenosine 5'-diphosphate with 1,4-dibromobutanedione. Reaction of pig heart NAD-specific isocitrate dehydrogenase with this nucleotide analogue (0.4 mM) causes a time-dependent loss of activity to a limiting value of 75% inactivation. The rate constant for inactivation exhibits a nonlinear dependence on the concentration of 2-BDB-TADP, with kmax = 0.021 min-1 and KI = 0.067 mM. Complete protection against inactivation by 0.2 mM 2-BDB-TADP is provided by ADP + Mn2+, but not by Mn2+ alone, isocitrate, alpha-ketoglutarate, or NAD. Incorporation of 2-BDB-TADP is proportional to the extent of inactivation, reaching 1 mol of reagent/mol of enzyme subunit when the enzyme is maximally inactivated. However, when inactivation is totally prevented by incubation with 2-BDB-TADP in the presence of ADP and Mn2+, 0.5 mol of reagent/mol of subunit is still incorporated, suggesting that inactivation may be attributed to 0.5 mol of reagent/mol of average subunit. In the native enzyme, the Km for total isocitrate is 1.8 mM and is decreased 6-fold to 0.3 mM in the presence of 1 mM ADP, whereas in the modified enzyme, with 25% residual activity, the Km for total isocitrate is about the same in the absence (2.0 mM) or presence (1.8 mM) of ADP. These results indicate that 2-BDB-TADP acts as an affinity label of the ADP allosteric site of NAD-dependent isocitrate dehydrogenase.

Adenosine Diphosphate↗

PubMed1986

Reaction of the 2',3'-dialdehyde derivative of NADPH at a nucleotide site of bovine liver glutamate dehydrogenase.

The 2',3'-dialdehyde derivative of NADPH (oNADPH) acts as a coenzyme for the reaction catalyzed by bovine liver glutamate dehydrogenase. Incubation of 250 microM oNADPH with enzyme for 300 min at 30 degrees C and pH 8.0 yields covalent incorporation of 1.0 mol of oNADPH/mol of enzyme subunit. The modified enzyme has a functional catalytic site and is activated by ADP, but is no longer inhibited by high NADH concentrations and exhibits decreased sensitivity to GTP inhibition. Using the change in inhibition by 600 microM NADH or 1 microM GTP to monitor the reaction leads to rate constants of 44.0 and 41.5 min-1 M-1, respectively, suggesting that loss of inhibition by the two regulatory compounds results from reaction by oNADPH at a single location. The oNADPH incorporation is proportional to the decreased inhibition by 600 microM NADH or 1 microM GTP, extrapolating to less than 1 mol of oNADPH/mol of subunit when the maximum change in NADH or GTP inhibition has occurred. Modified enzyme is still 93% inhibited at saturating levels of GTP, although its K1 is increased 20-fold to 4.6 microM. The kinetic effects caused by oNADPH are not prevented by alpha-ketoglutarate, ADP, 5 mM NADH, or 200 microM GTP alone, but are prevented by 5 mM NADH with 200 microM GTP. Incorporation of oNADPH into enzyme at 255 min is 0.94 mol/mol of peptide chain in the absence of ligands but only 0.53 mol/mol of peptide chain in the presence of the protectants 5 mM NADH plus 200 microM GTP. These results indicate that oNADPH modifies specifically about 0.4-0.5 sites/enzyme subunit or about 3 sites/enzyme hexamer and that reaction occurs at a GTP-dependent inhibitory NADH site of glutamate dehydrogenase.

Animals↗

PubMed1986

Isolation and identification of cysteinyl peptide labeled by 6- [( 4-bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-diphosphate in the reduced diphosphopyridine nucleotide inhibitory site of glutamate dehydrogenase.

6-[(4-Bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-diphosphate (6-BDB-TADP) has been shown to react at the reduced diphosphopyridine nucleotide (DPNH) inhibitory site of bovine liver glutamate dehydrogenase with incorporation of 1 mol of reagent/mol of enzyme subunit [Batra, S. P., & Colman, R. F. (1984) Biochemistry 23, 4940-4946]. The modified enzyme had lost one of the six free sulfhydryl groups per enzyme subunit as detected by 5,5'-dithiobis(2-nitrobenzoate). In the unmodified enzyme digested with trypsin, six cysteinyl peptides labeled with [14C]iodoacetic acid were detected by high-performance liquid chromatography (HPLC), whereas only five were observed in the 6-BDB-TADP-modified enzyme. A cysteinyl peptide has been isolated from modified enzyme digested with trypsin and chymotrypsin. Purification of the nucleotidyl peptide was accomplished by chromatography on phenyl boronate-agarose, followed by gel filtration on Sephadex G-25 and Bio-Gel P-4 in 50 mM ammonium bicarbonate, pH 8.0. The modified peptides were finally purified by HPLC on a C18 column using 0.1% trifluoroacetic acid with an acetonitrile gradient. By comparison of the amino acid composition and N-terminal residue of the isolated peptide with the known amino acid sequence of the enzyme, the peptide in the DPNH inhibitory site labeled by 6-BDB-TADP has been identified as the 19-membered fragment from Glu-311 to Lys-329. A unique residue, Cys-319, was identified as the reactive amino acid within the DPNH inhibitory site.

Adenosine Diphosphate↗

PubMed1986

Platelet ADP receptor and alpha 2-adrenoreceptor interaction. Evidence for an ADP requirement for epinephrine-induced platelet activation and an influence of epinephrine on ADP binding.

The nucleotide affinity analog 5'-p-fluorosulfonylbenzoyl adenosine (FSBA) is a potent irreversible inhibitor of ADP-mediated platelet activation. Utilizing this compound, the role of ADP in epinephrine-mediated platelet activation was evaluated. Pretreatment of platelets with FSBA under conditions producing covalent incorporation was able to completely block epinephrine-stimulated aggregation of human platelets. In addition, the exposure of latent fibrinogen-binding sites by epinephrine was also inhibited in platelets modified by FSBA. The inhibition of epinephrine-mediated activation of the cells was time dependent, reflecting the need for covalent modification of the ADP receptor by FSBA. The inhibitory effect of FSBA was not due to effects on the affinity of binding methyl [3H]yohimbine or the number of platelet alpha 2-adrenergic receptors. Studies of the effect of epinephrine on the ability of ADP to protect against FSBA incorporation demonstrated that epinephrine can increase the affinity of ADP for its receptor 10-fold without affecting the total amount of FSBA covalently bound. This effect of epinephrine is mediated through the alpha 2-adrenoreceptor since the effect can be reversed by the competitive antagonist, methyl yohimbine. These results suggest that promotion of platelet aggregation and the exposure of fibrinogen receptors by epinephrine is dependent on ADP. The mechanism by which epinephrine renders low concentrations of ADP effective appears to be mediated by an increased avidity of the ADP receptor for the nucleotide.

Adenosine↗

PubMed1986

Identification of tyrosine and lysine peptides labeled by 5'-p-fluorosulfonylbenzoyl adenosine in the active site of pyruvate kinase.

The nucleotide affinity label 5'-p-fluorosulfonylbenzoyl adenosine reacts at the active site of rabbit muscle pyruvate kinase, with irreversible inactivation occurring concomitant with incorporation of about 1 mol of reagent/mol of enzyme subunit (Annamalai, A. E., and Colman, R. F. (1981) J. Biol. Chem. 256, 10276-10283). Purified peptides have now been isolated from 70% inactivated enzyme containing 0.7 mol of reagent/mol of enzyme subunit. Rabbit muscle enzyme labeled with radioactive 5'-p-fluorosulfonylbenzoyl adenosine was digested with thermolysin. Nucleosidyl peptides were purified by chromatography on phenylboronate-agarose and reverse-phase high performance liquid chromatography. After amino acid and N-terminal analysis, the peptides were identified by comparison with the primary sequences of chicken and cat muscle enzyme. About 75% of the reagent incorporated was distributed equally among three O-(4-carboxybenzenesulfonyl)tyrosine-containing peptides: Leu-Asp-CBS-Tyr-Lys-Asn, Val-CBS-Tyr, and Leu-Asp-Asn-Ala-CBS-Tyr. These tyrosines are located in a 28-residue segment of the 530-amino acid sequence. The remainder of the incorporation was found in two N epsilon-(4-carboxybenzenesulfonyl)lysine-containing peptides. Leu-CBS-Lys and Ala-CBS-Lys-Gly-Asp-Tyr-Pro. Modification in the presence of MnATP or MnADP resulted in a marked decrease in labeling of these peptides in proportion to the decreased inactivation. It is suggested that these modified residues are located in the region of the catalytically functional nucleotide binding site of pyruvate kinase.

Adenosine↗

PubMed1986

Separation of the 100-kDa membrane protein mediating ADP-induced platelet shape change and activation from glycoprotein IIIa.

The following lines of evidence suggest that MP100, a putative ADP receptor, and GPIIIa are distinct proteins. [3H]FSBA incorporated equally into normal and thrombasthenic platelets (less than 5% GPIIIa), quantitatively as well as qualitatively. The dose-dependent inhibition of ADP-induced platelet shape change by FSBA is identical for normal and thrombasthenic platelets. Polyclonal rabbit antibodies precipitate MP100 and GPIIIa, but monoclonal antibodies directed against GPIIb/GPIIIa complex GPIIIa and P1A1 fail to precipitate the ADP receptor protein. A monoclonal antibody which inhibits ADP-induced platelet aggregation and fibrinogen binding fails to inhibit ADP-induced shape change. Thus, both functional and immunochemical evidence clearly indicates the distinct character of the ADP and fibrinogen receptors. We hypothesize that conformational changes in an ADP receptor on binding ADP or proteolytic changes as with chymotrypsin digestion may be responsible for exposure of the normally latent fibrinogen receptor (GPIIb/GPIIIa complex).

Adenosine↗

PubMed1985

2-[(4-Bromo-2,3-dioxobutyl)thio]adenosine 5'-monophosphate, a new nucleotide analogue that acts as an affinity label of pyruvate kinase.

A new reactive adenine nucleotide has been synthesized: 2-[(4-bromo-2,3-dioxobutyl)thio]-adenosine 5'-monophosphate (2-BDB-TAMP). Adenosine 5'-monophosphate 1-oxide was synthesized by reaction of AMP with m-chloroperoxybenzoic acid. Treatment with NaOH followed by reaction with carbon disulfide yielded 2-thioadenosine 5'-monophosphate (TAMP). The final product was generated by reaction of TAMP with 1,4-dibromobutanedione. The structure of 2-BDB-TAMP was determined by UV, 1H NMR, and 13C NMR spectroscopy as well as by bromide and phosphorus analysis. Rabbit muscle pyruvate kinase is inactivated by 2-BDB-TAMP at pH 7.0 and 25 degrees C. The inactivation rate exhibits a nonlinear dependence on the reagent concentration with KI = 0.57 mM. Protection against inactivation is provided by ADP and ATP, in the presence of Mn2+, as well as by phosphoenolpyruvate, in the presence of K+; in addition, partial protection is provided by AMP plus Mn2+. Incubation of pyruvate kinase with 0.075 mM 2-BDB-TAMP for 70 min in the absence of protective ligands leads to incorporation of 1.55 mol of reagent/mol of enzyme subunit when the enzyme is 53% inactive. In the presence of ADP and Mn2+, only 0.96 mol of reagent/mol of subunit is incorporated at 70 min, while the enzyme retains 100% activity. Similar results were obtained in the presence of ATP plus Mn2+. Assuming that the groups modified in the absence of ligands include those modified in the presence of the nucleotides, the 53% inactivation can be attributed to the modification of 0.59 (1.55-0.96) group per enzyme subunit.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenine Nucleotides↗

PubMed1985

Affinity labeling of NADP+-specific isocitrate dehydrogenase by a new fluorescent nucleotide analogue, 2-[(4-bromo-2,3-dioxobutyl)thio]-1,N6-ethenoadenosine 2',5'-bisphosphate.

A new reactive fluorescent adenine nucleotide analogue has been synthesized and characterized: 2-[(4-bromo-2,3-dioxobutyl)thio]-1,N6-ethenoadenosine 2',5'-bisphosphate (BDB-T epsilon ADP). This compound reacts irreversibly with NADP+-specific isocitrate dehydrogenase. Biphasic kinetics of inactivation are observed that can be described in terms of a fast initial phase of inactivation resulting in partially active enzyme of 8-10% residual activity, followed by a slower phase leading to total inactivation. NADPH protects completely against the fast phase of the reaction, indicating that modification occurs at the coenzyme binding site, whereas isocitrate with MnSO4 protects totally against the slow phase of reaction. The inactivation rate constants for both phases exhibit nonlinear dependence on BDB-T epsilon ADP concentration, consistent with the formation of a reversible complex with the enzyme prior to irreversible modification. Covalent incorporation of BDB-T epsilon ADP is limited and specific; only 0.99 mol of reagent/mol of subunit is incorporated when the enzyme is 98% inactivated in the absence of ligands. A maximum incorporation of 0.5 mol of reagent/mol of subunit is obtained in the presence of isocitrate and MnSO4, while incorporation in the presence of NADPH equals the difference between the incorporation in the absence of ligands and that measured in the presence of isocitrate and MnSO4. It appears that 0.5 mol of reagent/mol of subunit is responsible for the fast phase of inactivation and the remaining incorporation causes the slow phase. Under all conditions used in this study, isocitrate dehydrogenase has been shown to exist as a dimer by analytical ultracentrifugation and by cross-linking with dimethyl suberimidate followed by analysis on polyacrylamide gels in the presence of sodium dodecyl sulfate. It is proposed that, in the fast phase of inactivation, 2-[(4-bromo-2,3-dioxobutyl)thio]-1,N6-ethenoadenosine 2',5'-bisphosphate reacts at the coenzyme binding site of one subunit of dimeric isocitrate dehydrogenase, causing complete inactivation of the modified subunit and substantial inactivation of the other subunit. This new reagent is likely to have general applicability as an affinity label for other NADP+ binding enzymes.

Adenosine Diphosphate↗

PubMed1985

1H nuclear magnetic resonance studies of the conformation and environment of nucleotides bound to pig heart NADP+-dependent isocitrate dehydrogenase.

The binding of coenzymes, NADP+ and NADPH, and coenzyme fragments, 2'-phosphoadenosine 5'-(diphosphoribose), adenosine 2',5'-bisphosphate, and 2'-AMP, to pig heart NADP+-dependent isocitrate dehydrogenase has been studied by proton NMR. Transferred nuclear Overhauser enhancement (NOE) between the nicotinamide 1'-ribose proton and the 2-nicotinamide ring proton indicates that the nicotinamide-ribose bond assumes an anti conformation. For all nucleotides, a nuclear Overhauser effect between the adenine 1'-ribose proton and 8-adenine ring proton is observed, suggesting a predominantly syn adenine--ribose bond conformation for the enzyme-bound nucleotides. Transferred NOE between the protons at A2 and N6 is observed for NADPH (but not NADP+), implying proximity between adenine and nicotinamide rings in a folded enzyme-bound form of NADPH. Line-width measurements on the resonances of free nucleotides exchanging with bound species indicate dissociation rates ranging from less than 7 s-1 for NADPH to approximately 1600 s-1 for adenosine 2',5'-bisphosphate. Substrate, magnesium isocitrate, increases the dissociation rate for NADPH about 10-fold but decreases the corresponding rate for phosphoadenosine diphosphoribose and adenosine 2',5'-bisphosphate about 10-fold. These effects are consistent with changes in equilibrium dissociation constants measured under similar conditions. The 1H NMR spectrum of isocitrate dehydrogenase at pH 7.5 has three narrow peaks between delta 7.85 and 7.69 that shift with changes in pH and hence arise from C-4 protons of histidines. One of those, with pK = 5.35, is perturbed by NADP+ and NADPH but not by nucleotide fragments, indicating that this histidine is in the region of the nicotinamide binding site. Observation of nuclear Overhauser effects arising from selective irradiation at delta 7.55 indicates proximity of either a nontitrating histidine or an aromatic residue to the adenine ring of all nucleotides. In addition, selective irradiation of the methyl region of the enzyme spectrum demonstrates that the adenine ring is close to methyl side chains. The substrate magnesium isocitrate produces no observable differences in these protein--nucleotide interactions. The alterations in enzyme--nucleotide conformation that result in changes in affinity in the presence of substrate must involve either small shifts in the positions of amino acid side chains or changes in groups not visible in the proton NMR spectrum.

Adenine Nucleotides↗

PubMed1985

Two mechanisms for inhibition of ADP-induced platelet shape change by 5'-p-fluorosulfonylbenzoyladenosine. Conversion to adenosine, and covalent modification at an ADP binding site distinct from that which inhibits adenylate cyclase.

The interaction of ADP with platelets leads to shape change, exposure of fibrinogen binding sites, and aggregation, all of which have been shown to be inhibited by 5'-p-fluorosulfonylbenzoyladenosine (FSBA), an alkylating analogue of adenine nucleotides which binds covalently to a 100-kDa polypeptide in intact platelet membranes (Figures, W. R., Niewiarowski, S., Morinelli, T., Colman, R. F., and Colman, R. W. (1981) J. Biol. Chem. 256, 7789-7795). In plasma, FSBA can break down to adenosine which stimulates adenylate cyclase. To distinguish between direct effects of FSBA and the actions of adenosine, we have used washed platelet suspensions and adenosine deaminase. We studied the effects of FSBA on shape change and cyclic AMP metabolism, and on the binding of 2-methylthio-ADP, which mimics the effects of ADP on cyclic AMP metabolism at concentrations too low to activate platelets. Inhibition of ADP-induced shape change of platelets incubated with FSBA for 2 min in platelet-rich plasma was greatly reduced by adenosine deaminase. In the presence of a phosphodiesterase inhibitor, 100 microM FSBA increased platelet cyclic AMP to the same extent as did 10 microM adenosine. These effects were inhibited by theophylline, an adenosine receptor antagonist, and by adenosine deaminase. Incubation of washed platelets for 60 min with FSBA and adenosine deaminase caused a concentration-dependent inhibition of ADP-induced shape change. Inhibition closely paralleled the covalent incorporation of 3H from tritiated FSBA into platelet membranes. Under these conditions, FSBA did not block inhibition of cyclic AMP accumulation by ADP, nor did it block the binding of 2-methylthio-ADP. We conclude that part of the inhibition of shape change caused by brief exposure to FSBA is due to adenosine, but at longer times shape change is inhibited in association with covalent incorporation of sulfonylbenzoyladenosine. This effect of FSBA is independent of adenosine and occurs at a site distinct from that at which ADP inhibits adenylate cyclase.

Adenosine↗

PubMed1985

Spectroscopic studies of the interactions of coenzymes and coenzyme fragments with pig heart, oxidized triphosphopyridine nucleotide specific isocitrate dehydrogenase.

Spectroscopic, ultrafiltration, and kinetic studies have been used to characterize interactions of reduced and oxidized triphosphopyridine nucleotides (TPNH and TPN), 2'-phosphoadenosine 5'-diphosphoribose (Rib-P2-Ado-P), and adenosine 2',5'-bisphosphate [Ado(2',5')P2] with with TPN-specific isocitrate dehydrogenase. Close similarity of the UV difference spectra and of the protein fluorescence changes accompanying the formation of the binary complexes provides evidence for the binding of these nucleotides to the same site on the enzyme. From the pH dependence of the dissociation constants for TPNH binding to TPN-specific isocitrate dehydrogenase in the absence and in the presence of Mn2+, over the pH range 5.8-7.6, it has been demonstrated that the nucleotide binds to the enzyme in its unprotonated, metal-free form. The involvement of positively charged residues, protonated over the pH range studied, has been postulated. One TPNH binding site per enzyme subunit has been measured by fluorescence and difference absorption titrations. A dramatic effect of ionic strength on binding has been demonstrated: about a 1000-fold decrease in the dissociation constant for TPNH has been observed at pH 7.6 upon decreasing ionic strength from 0.336 (Kd = 1.2 +/- 0.2 microM) to 0.036 M (Kd = 0.4 +/- 0.1 nM) in the presence and in the absence of 100 mM Na2SO4, respectively. Weak competition of sulfate ions for the nucleotide binding site has been observed (KI = 57 +/- 3 mM). The binding of TPN in the presence of 100 mM Na2SO4 at pH 7.6 is about 100-fold weaker (Kd = 110 +/- 22 microM) than the binding of the reduced coenzyme and is similarly affected by ionic strength. These results demonstrate the importance of electrostatic interactions in the binding of the coenzyme to TPN-specific isocitrate dehydrogenase. The large enhancement of protein fluorescence caused by binding of TPN and Rib-P2-Ado-P (delta Fmax = 50%) and of Ado(2',5')P2 (delta Fmax = 41%) has been ascribed to a local conformational change of the enzyme. An apparent stoichiometry of 0.5 nucleotide binding site per peptide chain was determined for TPN, Rib-P2-Ado-P, and Ado(2',5')P2 from fluorescence titrations, in contrast to one binding site per enzyme subunit determined from UV difference spectral titration and ultrafiltration experiments. Thus, the binding of one molecule of the nucleotide per dimeric enzyme molecule is responsible for the total increase in protein fluorescence, while binding to the second subunit does not cause further change.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Diphosphate↗

PubMed1985

Reaction of 5'-p-fluorosulfonylbenzoyl-1,N6-ethenoadenosine with histidine and cysteine residues in the active site of rabbit muscle pyruvate kinase.

The inactivation of rabbit muscle pyruvate kinase by 0.3 mM 5'-p-fluorosulfonylbenzoyl-1,N6-ethenoadenosine at pH 7.8 is biphasic. The first phase proceeds rapidly to yield a partially active enzyme (46% residual activity) followed by a slower rate which leads to total inactivation. The inactivation of the first phase can be reversed by addition of 20 mM dithiothreitol, whereas the second phase is unaffected. These two phases have second-order rate constants of 250 M-1 X min-1 (dithiothreitol-sensitive reaction) and 52 M-1 X min-1 (dithiothreitol-insensitive reaction), respectively. Marked protection against inactivation is afforded by phosphoenolpyruvate and by metal-nucleotide complexes in the presence of free metal, indicating that reaction occurs in the region of the active site. Loss of approximately two sulfhydryls per enzyme subunit correlates well with the dithiothreitol-sensitive inactivation, suggesting that this phase of the inactivation may be attributable to disulfide formation. Incorporation of about one mole of fluorescent reagent per enzyme subunit correlates closely with the dithiothreitol-insensitive phase of inactivation, yielding a modified histidine residue. The quantum yield of the fluorescent sulfonylbenzoyl-1,N6-ethenoadenosine-pyruvate kinase is only 0.007, as compared to 0.54 for the parent nucleoside 1,N6-ethenoadenosine. The quenched fluorescence is consistent with stacking of the sulfonylbenzoyl moiety on the purine ring in the modified enzyme, which suggests that the altered histidine may be located in the adenine region of the metal-nucleotide binding site.

Adenosine↗

PubMed1984

Isolation and identification of a tyrosyl peptide labeled by 5'-[p-(fluorosulfonyl)benzoyl]-1,N6-ethenoadenosine at a GTP site of glutamate dehydrogenase.

The fluorescent nucleotide analogue 5'-[p-(fluorosulfonyl)benzoyl]-1,N6-ethenoadenosine (5'-FSB epsilon A) was shown previously to react at a GTP inhibitory site on bovine liver glutamate dehydrogenase. The incorporation was limited to 1.28 mol of reagent/mol of subunit and was attributed to 0.95 mol of modified tyrosine/mol of subunit and 0.33 mol of modified lysine/mol of subunit, quantitatively accounting for the total incorporation prior to acid hydrolysis [Jacobson, M. A., & Colman, R. F. (1983) Biochemistry 22, 4247-4257]. The specific tyrosyl peptide modified by 5'-FSB epsilon A has been isolated from a tryptic and chymotryptic digest of modified enzyme by gel filtration and reverse-phase high-performance liquid chromatography and characterized by amino acid and amino-terminal analysis. A unique residue, tyrosine-262, was identified as an essential amino acid within the GTP binding site. The stacked conformation of the fluorescent analogue when enzyme bound suggests that tyrosine-262 may be located in the region of the GTP site which binds the purine ring.

Adenosine↗

PubMed1984

Identification of the lysine and tyrosine peptides labeled by 5'-p-fluorosulfonylbenzoyladenosine in the NADH inhibitory site of glutamate dehydrogenase.

5'-p-Fluorosulfonylbenzoyladenosine (5'-FSBA) is a specific affinity label for the inhibitory NADH site of bovine liver glutamate dehydrogenase. Reaction of the enzyme with 5'-FSBA results in the loss of inhibition by high concentrations of NADH with covalent attachment of 0.53 sulfonylbenozyladenosine/subunit, i.e. modification of three subunits of the hexameric enzyme. Equal amounts of N epsilon-(4-carboxybenzenesulfonyl)lysine (Lys-(CBS] and O-(4-carboxybenzenesulfonyl)tyrosine (Tyr-(CBS] are found throughout the course of the reaction (Saradambal, K. V., Bednar, R. A., and Colman, R. F. (1981) J. Biol. Chem. 256, 11866-11872). Modified enzyme, prepared by incubating 2 mg/ml glutamate dehydrogenase with 0.3 mM 3H-labeled 5'-FSBA at pH 8 for 1 h, was carboxymethylated and digested with thermolysin. Two nucleosidyl peptides were isolated by a combination of chromatography on phenyl boronate-agarose, high-performance liquid chromatography in ammonium bicarbonate and high-performance liquid chromatography in trifluoroacetic acid. By comparison of the amino acid analysis and NH2-terminal residue of each isolated peptide with the known amino acid sequence of the enzyme, the peptides were identified as Leu-Gly-Arg-Lys(CBS) and Ile-Gly-His-Tyr(CBS)-Asp. These sequences correspond to residues 417-420 and 187-191, respectively. Lys-420 and Tyr-190 of glutamate dehydrogenase react with 5'-FSBA, and both are apparently located in the NADH inhibitory site.

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↗