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PubMed1994

Guanosine 5'-O-[S-(4-bromo-2,3-dioxobutyl)]thiophosphate and adenosine 5'-O-[S-(4-bromo-2,3-dioxobutyl)]thiophosphate. New nucleotide affinity labels which react with rabbit muscle pyruvate kinase.

Three new reactive nucleotide analogues with bromo-keto substituents adjacent to a thiophosphate have been synthesized. Guanosine 5'-O-[S-(4-bromo-2,3-dioxobutyl)]thiophosphate (GMPS-BDB), reacts covalently with rabbit muscle pyruvate kinase with complete inactivation and incorporation of 1.8 mol of reagent/mol of enzyme subunit. By contrast, the mono-keto compound, guanosine 5'-O-[S-(3-bromo-2-oxopropyl)]thiophosphate (GMPS-BOP), causes no loss of pyruvate kinase activity. When the analogous adenosyl nucleotide derivatives are incubated with pyruvate kinase, the di-keto compound, adenosine 5'-O-[S-(4-bromo-2,3-dioxobutyl)]thiophosphate (AMPS-BDB), rapidly effects inactivation, whereas the mono-keto compound, adenosine 5'-O-[S-(3-bromo-2-oxopropyl)]thiophosphate (AMPS-BOP), causes no loss of activity. Complete protection against inactivation by GMPS-BDB is provided by phosphoenolpyruvate in the presence of K+ and Mn2+ and the amount of reagent incorporated (0.9 mol/reagent/mol subunit) is reduced to half that observed in the absence of protectants. Gas-phase sequencing of the tryptic peptides purified from inactive GMPS-BDB or AMPS-BDB-modified enzyme gave the cysteine-labeled peptides: C151DENILWLDYK161, and N162IC164K165 as the two major peptide products, with a smaller amount of N43TGIIC48TIGPASR55. Reaction in the presence of the protectants PEP, K+, and Mn2+ yielded Cys164 as the only labeled residue, indicating that inactivation is primarily due to modification of Cys151. We propose that GMPS-BDB (or AMPS-BDB), which may exist in enolized form in aqueous solution, functions as a reactive analogue of phosphoenolpyruvate and GDP (ADP) to target Cys151 in the active site of pyruvate kinase.

Adenosine Diphosphate↗
PubMedDate not supplied

INAKT--an interactive non-linear regression program for enzyme inactivation and affinity labelling studies.

An interactive program for analysing enzyme activity-time data using non-linear regression analysis is described. Protection studies can also be dealt with. The program computes inactivation rates, dissociation constants and promotion or inhibition parameters with their standard errors. It can also be used to distinguish different inactivation models. The program is written in SIMULA and is menu-oriented for refining or correcting data at the different levels of computing.

Affinity Labels↗
PubMed1987

Affinity labeling of adenosine A1 binding sites.

The adenosine A1 receptors of sheep brain membranes have been identified by the specific binding of radiolabeled cyclohexyl[3H]adenosine ([3H]CHA). Pretreatment of membranes with periodate-oxidized CHA causes a dose- and time-dependent decrease in the number of binding sites. No decrease occurs when membranes are pretreated with CHA. Binding of [3H]CHA to the remaining sites occurs with the same characteristics as binding to the untreated receptor population.

Adenosine↗
PubMed1984

[Photochemical affinity labeling of the macrolide binding site on the 70S E. coli ribosome].

Photoactivation of the alpha, beta-unsaturated ketone-epoxide system of [3H] dihydrorosaramycin at a wavelength above 300 nm allows the covalent attachment of the antibiotic to its receptor site. The radioactivity is mainly associated to proteins L1, L5, L6, S1; as a consequence, the binding site of this type of drug could be located at the peptidyltransferase center and in between both subunits.

Affinity Labels↗
PubMed1991

Affinity labeling of folate transport proteins with the N-hydroxysuccinimide ester of the gamma-isomer of fluorescein-methotrexate.

Fluorescein-methotrexate, a derivative in which the fluorophore is linked via a diaminopentane spacer to either the alpha- or gamma-carboxyl group of the glutamate moiety in the drug [Gapski et al. (1975) J. Med. Chem. 18, 526-528], has been synthesized by an improved procedure and separated by DEAE-Trisacryl chromatography into the alpha- and gamma-isomers (alpha-F-MTX and gamma-F-MTX). Each isomer was characterized by mass spectrometry, elemental analysis, absorbance spectrum, TLC, and reversed-phase HPLC. Identity of the isomers was established by the following enzymatic criteria: (a) gamma-F-MTX (but not the alpha-isomer) was hydrolyzed at the pteroate-glutamate bond by carboxypeptidase G2 to yield 4-amino-4-deoxy-10-methylpteroate and gamma-glutamyldiaminopentane-fluorescein; and (b) gamma-F-MTX was a much better inhibitor of human dihydrofolate reductase than the alpha-isomer (Ki values of 0.079 and 4.6 nM). alpha- and gamma-F-MTX were comparable as inhibitors (Ki values of 1.6 and 0.6 microM) of the transport system for reduced folates and MTX in L1210 cells, but the transporter in Lactobacillus casei was inhibited only by the gamma-isomer (Ki = 4.3 microM). The gamma-isomer, therefore, was selected for covalent labeling of proteins. When L. casei folate transport protein (18 kDa) was treated with gamma-F-MTX that had been activated with N-hydroxysuccinimide (NHS), the protein was readily visualized as a fluorescent band on SDS-PAGE electrophoretograms. The probe was also able to detect the transporter in membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1995

Identification of the active sites of human and schistosomal hypoxanthine-guanine phosphoribosyltransferases by GMP-2',3'-dialdehyde affinity labeling.

Labeling of human and schistosomal hypoxanthine-guanine phosphoribosyltransferases (HGPRTases) with GMP-2',3'-dialdehyde (ox-GMP) results in nearly complete inactivation of the enzymes. Digestion of the [3H]ox-GMP-modified HGPRTases with trypsin followed by high-performance liquid chromatographic fractionation, partial amino acid sequencing, and mass spectral analysis of the labeled peptides revealed that four peptides from each of the two HGPRTases were labeled with ox-GMP. The conclusion from these studies indicates that two segments of the human enzyme protein, Ser 4-Arg 47 and Ser 91-Arg 100, and one region in the schistosomal enzyme, Gly 95-Lys 133, were labeled by ox-GMP. Since the ox-GMP labeling of human HGPRTase was effectively blocked by either GMP or PRibPP, whereas that of schistosomal HGPRTase was inhibited only by GMP [Kanaaneh, J., Craig, S. P., III, & Wang, C. C. (1994) Eur. J. Biochem. 223, 595-601], the two labeled peptides in human enzyme may be involved in binding to both GMP and PRibPP while the one peptide in schistosomal enzyme may be implicated only in GMP binding. We have also confirmed a previous observation [Keough, D. T., Emmerson, B. T., & de Jersey, J. (1991) Biochim. Biophys. Acta 1096, 95-100] that carboxymethylation of Cys 22 in the human HGPRTase by iodoacetate was inhibited by PRibPP. We also demonstrated that the carboxymethylation of Cys 25 in schistosomal HGPRTase by iodoacetate was specifically blocked by PRibPP. Apparently, the N-terminal regions in both enzymes are involved in PRibPP binding. The fact that ox-GMP labels the N-terminal region in human enzyme but not in schistosomal enzyme and that PRibPP protects against ox-GMP labeling in human enzyme but not in schistosomal enzyme both suggest that the amino-terminal PRibPP-binding site may be in close proximity to the GMP-binding site in human HGPRTase but not in schistosomal HGPRTase. This clear distinction between the active sites of human and schistosomal HGPRTases could be further exploited for potential opportunities for antischistosomal chemotherapy.

Affinity Labels↗
PubMed1975

The interaction of N-acetylglucosamine and an affinity-label analogue with alpha-lactalbumin and lactose synthetase.

We have attempted to detect binding of N-acetylglucosamine (NAG) to alpha-lactalbumin, the B protein of lactose synthetase, under conditions in which binding of NAG to lysozyme, a protein to which alpha-lactalbumin has a significant sequence homology, is observed. Using 1H nuclear magnetic resonance spectroscopy, uv difference spectroscopy, competition of NAG with N-methylnicotinamide chloride, and fluorescence spectroscopy, no binding was detected. The synthesis of a NAG analogue, N-diazoacetyl-glucosamine (diazoNAG), was carried out, and the molecule was demonstrated to be an active galactose acceptor in the lactose synthetase reaction. Use of this molecule in photochemical labeling experiments resulted in a large amount of nonspecific labeling of alpha-lactalbumin, lactose synthetase A protein, ribonuclease, and lysozyme, but competition experiments in the presence of an excess of NAG revealed some specific labeling in the case of A protein and lysozyme, but not with alpha-lactalbumin or a ribonuclease control. Thus, it is highly questionable that a NAG binding site is retained in alpha-lactalbumin; furthermore, it appears that the galacyosyl acceptor makes significant contacts with the A protein rather than alpha-lactalbumin in the lactose synthetase complex.

Acetylglucosamine↗
PubMed1996

GLUT1 transmembrane glucose pathway. Affinity labeling with a transportable D-glucose diazirine.

We synthesized a transportable diazirine derivative of D-glucose,3-deoxy-3,3-azi-D-glucopyranose (3-DAG), and studied its interaction with purified human erythrocyte facilitative glucose transporter, GLUT1. 3-DAG was rapidly transported into human erythrocytes and their resealed ghosts in the dark via a mercuric chloride-inhibitable mechanism and with a speed comparable with that of 3-O-methyl-D-glucose (3-OMG). The rate of 3-DAG transport in resealed ghosts was a saturable function of 3-DAG concentration with an apparent Km of 3.2 mM and the Vmax of 3.2 micromol/s/ml. D-Glucose inhibited the 3-DAG flux competitively with an apparent KI of 11 mM. Cytochalasin B inhibited this 3-DAG flux in a dose-dependent manner with an estimated KI of 2.4 x 10(-7) M. Cytochalasin E had no effect. These findings clearly establish that 3-DAG is a good substrate of GLUT1. UV irradiation of purified GLUT1 in liposomes in the presence of 3-DAG produced a significant covalent incorporation of 3-DAG into glut1, and 200 mM D-glucose abolished this 3-dag incorporation. Analyses of trypsin and endoproteinase Lys-C digestion of 3-DAG-photolabeled GLUT1 revealed that the cleavage products corresponding to the residues 115 183, 256 300, and 301 451 of the GLUT1 sequence were labeled by 3-DAG, demonstrating that not only the C-terminal half but also the N-terminal half of the transmembrane domain participate in the putative substrate channel formation. 3-DAG may be useful in further identification of the amino acid residues that form the substrate channel of this and other members of the facilitative glucose transporter family.

Affinity Labels↗
PubMed1990

Identification of an active-site residue in yeast invertase by affinity labeling and site-directed mutagenesis.

Deglycosylated yeast invertase is irreversibly inactivated by conduritol B epoxide (CBE), an active-site-directed reagent. The inactivated enzyme contained 0.8 mol of CBE/mol of invertase monomer suggesting that the inactivation results from the modification of a single amino acid residue. Peptic digestion of [3H]CBE-labeled invertase followed by reverse phase column chromatography yielded two labeled peptides, both located at the amino-terminal end of the enzyme. Sequence analyses of these peptides revealed that Asp-23 is the modified residue. The role of Asp-23 in the catalytic process was investigated by changing it to Asn using site-directed mutagenesis of the SCU2 gene. The mutant enzyme was basically inactive, confirming a role for Asp-23 in the catalytic process.

Acetylglucosaminidase↗
PubMed1982

Platelet-derived growth factor. III. Identification of a platelet-derived growth factor receptor by affinity labeling.

Two homobifunctional cross-linking reagents have been used to cross-link 125I-platelet-derived growth factor (PDGF) to a cell surface component with an approximate Mr = 164,000 that has many characteristics of a specific PDGF receptor. Excess unlabeled PDGF competed for labeling of this component, while high concentrations of fibroblast growth factor, insulin, epidermal growth factor, low density lipoprotein or acetylated low density lipoprotein had no effect. Preincubation of cells with 125I-PDGF at 37 degrees C reduced specific 125I-PDGF binding (down regulation) and produced a parallel decrease in the amount of the 164,000-dalton receptor available for labeling. The 164,000-dalton component contains at least some protein that is accessible to trypsin in the extracellular medium. A complex of comparable Mr is seen on all PDGF-responsive cell types examined, but not on a nonresponsive cell type. 125I-PDGF does not become covalently cross-linked to this component in the absence of a cross-linking reagent.

Affinity Labels↗
PubMed1994

Active site mapping of affinity-labeled rat oxidosqualene cyclase.

Rat liver oxidosqualene cyclase (OSC), a 78-kDa membrane-bound enzyme, was purified and labeled with the mechanism-based irreversible inhibitor, [3H]29-methylidene-2,3-oxidosqualene (Abe, I., Bai, M., Xiao, X.-Y., and Prestwich, G. D. (1992) Biochem. Biophys. Res. Commun. 187, 32-38). A 6-kDa CNBr peptide was separated by Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted to a polyvinylidene difluoride membrane. The sequence of the first 30 amino acids of this peptide were determined by Edman degradation and showed unexpectedly high similarity to the fungal OSC from Candida albicans (50% identity with Arg413-Val442) and to the bacterial squalene cyclase from Alicyclobacillus (formerly Bacillus) acidocaldarius (37% identity with Lys356-Leu385). Further, radioanalysis clearly established that the two adjacent Asp residues in the highly conserved region (Asp-Asp-Thr-Ala-Glu-Ala or DDTAEA) were equally labeled by the irreversible inhibitor. This result provides the first information on the structural details of the active site of OSC and shows for the first time the ancient lineage of this vertebrate enzyme to ancestral eukaryotic and prokaryotic cyclases. Interestingly, the covalently modified DDXX(D/E) sequence of rat liver OSC showed surprising similarity to the putative allylic diphosphate binding site sequence of sesquiterpene cyclases and prenyl transferases.

Affinity Labels↗
PubMed1988

Characterization of a benzyladenine binding-site peptide isolated from a wheat cytokinin-binding protein: sequence analysis and identification of a single affinity-labeled histidine residue by mass spectrometry.

A wheat embryo cytokinin-binding protein was covalently modified with the radiolabeled photoaffinity ligand 2-azido-N6-[14C]benzyladenine. A single labeled peptide was obtained after proteolytic digestion and isolation by reversed-phase and anion-exchange HPLC. Sequencing by classical Edman degradation identified 11 of the 12 residues but failed to identify the labeled amino acid. Analysis by laser photodissociation Fourier-transform mass spectrometry of 10 pmol of the peptide independently confirmed the Edman data and also demonstrated that the histidine residue nearest the C terminus (underlined) was modified by the reagent in the sequence Ala-Phe-Leu-Gln-Pro-Ser-His-His-Asp-Ala-Asp-Glu.

Adenine↗
PubMed1990

Characterization of the ATP binding site on Escherichia coli DNA gyrase. Affinity labeling of Lys-103 and Lys-110 of the B subunit by pyridoxal 5'-diphospho-5'-adenosine.

We have labeled the adenosine triphosphate binding site of Escherichia coli DNA gyrase with the ATP affinity analog, [3H]pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP). PLP-AMP strongly inhibits the ATP-ase and DNA supercoiling activities of DNA gyrase, with 50% inhibition occurring at 7.5 microM inhibitor. ATP and ADP compete with PLP-AMP for binding and protect the enzyme against inhibition. The labeling appears to proceed by a Schiff base complex between the 4-formyl group of the pyridoxyl moiety of PLP-AMP and a protein primary amino group, since the inhibition and reagent labeling are reversible unless the complex is treated with NaBH4. Complete inactivation is estimated to occur upon the covalent incorporation of 2 mol of inhibitor/mol of gyrase. The Km for ATP was found to be unchanged for partially inhibited enzyme samples, suggesting an all-or-none type of inhibition. A 3H-labeled peptide spanning residues 93-131 of the B protein was isolated from a V-8 protease digest. Radioactive peaks corresponding to Lys-103 and Lys-110 were found during the Edman degradation, suggesting that these amino acids form part of the ATP binding site. A comparison of the amino acid sequence in this region with the sequences of other type II topoisomerases indicates the possible location of a common ATP binding domain.

Adenosine Diphosphate↗
PubMed1978

Aminoacyl-tRNA synthetases: affinity labeling of the ATP binding site by 2', 3' -ribose oxidized ATP.

Homogeneous Escherichia coli methionyl-, isoleucyl-, tryptophanyl-, and phenylalanyl-tRNA synthetases and Bacillus stearothermophilus methionyl- and tyrosyl-tRNA synthetases are irreversibly inactivated by reaction of their active ATP sites with the 2',3'-dialdehyde derivative of ATP obtained by periodate oxidation. In each case, the amount of 14C-labeled dialdehyde derivative incorporated per molecule of inactivated enzyme appears consistent with the expected active stoichiometry of the synthetase. These results strongly support the presence, at the active site of the studied aminoacyl-tRNA synthetases, of a common residue, probably a lysine whose epsilon-NH2 group is known, from the work of others, to form a Schiff's base specifically with the 2',3'-dialdehyde derivatives of ribonucleotides.

Adenosine Triphosphate↗
PubMed1989

Affinity labeling of chicken liver fatty acid synthase with chloroacetyl-CoA and bromopyruvate.

Fatty acid synthase of chicken liver is inactivated rapidly and irreversibly by incubation with chloroacetyl-CoA or with bromopyruvate. Inactivation by both reagents follows saturation kinetics, indicating the formation of an E ... I complex (dissociation constants of 0.36 microM for chloroacetyl-CoA and 31 microM for bromopyruvate) prior to alkylation. The limiting rate constants are 0.15 s-1 for bromopyruvate and 0.041 s-1 for chloroacetyl-CoA. Inactivation by both reagents is protected by NADPH and 200 mM KCl, and by saturating amounts of thioester substrates which reduced the limiting rate constants 6.5-30-fold. Active-site-directed reaction of chloroacetyl-CoA is supported by the ability of this compound to form a kinetically viable complex with the enzyme as competitive inhibitor of acetyl-CoA. Chloroacetyl-CoA interacts initially at the CoA binding pocket, since the nucleotide afforded competitive protection of inactivation and caused a large decrease in its affinity. Subsequently, the phosphopantetheine prosthetic group is alkylated. Evidence is presented to show that bromopyruvate competes with chloroacetyl-CoA for the same target site.

Acetyl Coenzyme A↗
PubMed2002

Activation of chymotrypsin-like serine protease(s) during apoptosis detected by affinity-labeling of the enzymatic center with fluoresceinated inhibitor.

There is evidence in the literature that serine (Ser) proteases, like caspases, are activated during apoptosis. Little is known, however, about individual Ser proteases and the mechanism of their activation. In the present study, we employed a new type of cell permeant reagent to detect activation of chymotrypsin-like proteases in human leukemic HL-60 cells induced to undergo apoptosis. The reagent, 5(6)-carboxyfluoresceinyl-L-phenylalanyl-chloromethyl ketone (FFCK), is a fluorochrome-labeled analog of N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), the inhibitor known to specifically and covalently bind to the active center of chymotrypsin-like enzymes. In cultures treated with the DNA topoisomerase I inhibitor, camptothecin (CPT), or tumor necrosis factor (TNFalpha), populations of cells appeared that had the capability to bind FFCK. Most FFCK-binding cells were identified by fluorescence microscopy and laser scanning cytometry (LSC) as the cells undergoing apoptosis. Frequency of cells binding FFCK strongly correlated with frequency of cells having activated caspases (r=0.98 in CPT-treated, and r=0.99 in TNFalpha-treated cultures). The observed induction of FFCK binding we interpret as representing the activation of a chymotrypsin-like apoptotic Ser protease(s). Pretreatment of cells with the poly-caspase inhibitor, Z-VAD-FMK, prevented the activation of these Ser protease(s). Pretreatment with TPCK, however, had a less pronounced, although distinct and reproducible suppressive effect, on caspase activation. The data, thus, suggest that activation of caspases is an upstream event required for activation of Ser protease(s). Activation of the latter, however, appears to additionally amplify, in a cascade-like mode, caspases activation. Differential color fluorochrome-labeling allowed us to discriminate, within the same cells, between the activation of active caspases and Ser protease(s). Despite a certain degree of co-localization, the inter- and intra-cellular pattern of caspase- vs. Ser-protease(s) was different. Our approach makes it possible to simultaneously monitor activation of caspases and Ser proteases in the same live cells that are induced to apoptosis.

Affinity Labels↗
PubMed1989

Novel ligands for the affinity labelling of luteinizing hormone releasing hormone receptors.

A number of novel luteinizing hormone releasing hormone (LHRH) analogues incorporating biotin together with potential covalent attachment sites have been synthesized. Those based on the des-Gly10-[D-Lys6]-LHRH ethylamide peptide backbone resulted in the most useful characteristics of binding to the LHRH receptor in rat anterior pituitary gland membranes. Of these, des-Gly10-[biotinyl-aminoethylglycyl-D-Lys6]-LHRH ethylamide (XBAL) gave the best specific: non-specific binding ratio, with 44 +/- 6% (+/- S.E.M.) of total binding being specific with a Kd of 131 +/- 16 pM (+/- S.E.M., n = 4) as determined by Scatchard analysis. Two methods have been used to covalently crosslink these analogues with the LHRH receptor; photoaffinity labelling and the use of homobifunctional N-hydroxysuccinimide ester crosslinkers. The photoaffinity analogues gave poor specific: non-specific binding ratios. Of the chemical crosslinkers tested, ethylene glycolbis(succinimidylsuccinate) (EGS) was found to be the most efficient at covalently linking the 125I-XBAL bound to the LHRH receptor site. At an EGS concentration of 5 mM, 23 +/- 3% (+/- S.E.M.) of the specific binding of 125I-XBAL was covalently crosslinked.

Affinity Labels↗
PubMed1976

The use of 1-fluoro-2,4-dinitrobenzene as an affinity label for the antigen receptor of delayed hypersensitivity.

The passive transfer of delayed contact sensitivity to dinitrophenyl can be specifically inhibited by brief treatment of lymph node cells (LNC) from sensitized guinea-pigs with 1-fluoro-2,4-dinitrobenzene (DNFB). Analysis of KC1 extracts of LNC previously treated with (3H)DNFB, using polyacrylamide gel electrophoresis, revealed a peak of radioactivity present in both sensitive and normal cell extracts as well as a peak found only in sensitive cell extracts. Cell-free extracts appeared capable of transmitting delayed-onset skin reactivity to non-sensitized recipients.

Affinity Labels↗