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PubMed1980

Affinity labeling of the Escherichia coli aspartate-beta-semialdehyde dehydrogenase with an alkylating coenzyme analogue. Half-site reactivity and competition with the substrate alkylating analogue.

3-Chloroacetylpyridine-adenine dinucleotide phosphate (clac3PdADP+, a NADP+ alkylating analogue, irreversibly inactivates aspartate-beta-semialdehyde dehydrogenase with pseudo-first-order kinetics. NADP+ and NADPH, but not the substrate, protected the enzyme from inactivation. The pH dependence of the inactivation kinetics was determined. Incorporation of 1 mol cl[14C]-ac3PdADP+/dimer totally inactivates the enzyme. Successive alkylation by the coenzyme analogue and by the substrate analogue, L-2-amino-4-oxo-5-chloropentanoic acid, was studied. After inactivation with the coenzyme analogue, no incorporation of the substrate analogue was detected. However, when the enzyme was first inactivated with the substrate analogue, the protein could subsequently be alkylated with the coenzyme analogue. The binding of NADP+ and NADPH to aspartate-beta-semialdehyde dehydrogenase was determined by fluorescence.

Affinity Labels↗
PubMed1987

Affinity labeling of the sialoglycopeptide antimitogen receptor.

An 18-kDa 125I-sialoglycopeptide growth inhibitor was covalently cross-linked to its binding site on intact cultured Swiss 3T3 cells by three bifunctional cross-linkers with short (dimethyl adipimate), medium (disuccinimidyl suberate), and long (bis(2-succinimidooxycarbonyloxyethyl)sulfone) chain lengths. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography demonstrated a band of Mr approximately 168,000 regardless of which cross-linker was used. The labeling of this band was specific in that it was prevented by excess unlabeled inhibitor and the apparent molecular weight of the cross-linked receptor-ligand complex was unchanged by treatment with reducing agent. The efficiency of the cross-linking was increased by increasing pH, and the extent of covalent cross-linking was dependent on the concentration of the bifunctional reagent. Octyl glucoside and sodium dodecyl sulfate were effective in solubilizing the receptor while Triton X-100 did not extract the receptor from the plasma membrane. These observations suggest that the 168-kDa binding species represents the 125I-sialoglycopeptide cross-linked to a specific plasma membrane receptor and that the receptor does not appear to contain interchain disulfide bonds.

Affinity Labels↗
PubMed1990

Interaction of ATP with acetyl-CoA carboxylase from rat liver. The role of the polyphosphate chain. Affinity labelling with alkylating amides of ATP and ADP.

The interaction of a number of ATP analogs with a modified triphosphate moiety as well as 2-chloro-ethyl-amino derivatives of nucleotides, 4(N-2-chloroethyl-N-methylamino)-benzyl-gamma-amide of ATP and the corresponding ADP beta-amide with acetyl-CoA carboxylase (EC 6.4.1.2.) from rat liver has been studied. Halophosphonate derivatives of ATP have been synthesized from bromomethylene diphosphonic acid and found to be reversible inhibitors of the enzyme. ATP and ADP alkylating amides have proved to form a reversible complex with the ATP-binding site and to modify a group in the acetyl-CoA-binding site. The bicarbonate ion accelerates the process of inactivation. The estimate of the distance between the ATP-binding site and the acetyl-CoA-binding site ranges within 0.8-1.2 nm.

Acetyl-CoA Carboxylase↗
PubMed1989

Affinity labeling of cofactor-binding region of human placental estradiol 17 beta-dehydrogenase by periodate-oxidized NADP+ (o-NADP+).

Periodate-oxidized NADP+ (o-NADP+), an analogue of the cofactors, is a reversible inhibitor of estradiol 17 beta-dehydrogenase in human placenta. Mode of the inhibition by o-NADP+ appeared to be competitive type (Ki = 0.84 microM) against NAD+ and non-competitive type (Ki = 1.13 microM) against estradiol, respectively. Treatment of the estradiol 17 beta-dehydrogenase with o-NADP+ resulted in time-dependent loss of the enzyme activity. The inactivation exhibited pseudo-first order kinetics (t1/2 = 15 min) and was protected by NAD+ and NADP+. On the other hand, periodate-oxidized ATP inactivated slightly the estradiol 17 beta-dehydrogenase. These results indicate that the residue(s) of lysines is located near the cofactor-binding region of estradiol 17 beta-dehydrogenase of human placenta.

17-Hydroxysteroid Dehydrogenases↗
PubMed1995

Affinity labeling of recombinant ricin A chain with Procion blue MX-R.

Recombinant ricin A chain was irreversibly modified by Procion blue MX-R, a dichlorotriazinyl analogue of Cibacron blue F3G-A, at pH 7.5 and 4 degrees C in 90 h with over 95% loss of activity in an in vitro translation assay. The presence of total yeast RNA reduced the covalent attachment of Procion blue MX-R to ricin A chain. Quantitatively modified ricin A chain contained 2 mol Procion blue MX-R/mol 29-kDa subunit. Tryptic digestion and resolution of the peptides by reverse-phase high-performance liquid chromatography yields a blue peptide corresponding to Gln5-Arg26 of ricin A chain. Thus, a likely dye-binding site on recombinant ricin A was identified. This region is removed from the active-site cleft of recombinant ricin A but may be involved in its substrate binding.

Affinity Labels↗
PubMed2002

The product of the natural reaction catalyzed by 4-oxalocrotonate tautomerase becomes an affinity label of its mutant.

4-Oxalocrotonate tautomerase (4-OT) catalyzes the isomerization of 4-oxalocrotonate, 1, to 2-oxo-3E-hexenedioate, 3, using a general acid/base mechanism that involves a conserved N-terminal proline residue. The P1A and P1G mutants have been shown to catalyze this isomerization but at reduced rates. Analysis of these mutants by mass spectrometry demonstrated that P1A is susceptible to a 1,4-addition of the N-terminal primary amine across the double bond of enone 3 to form a covalent adduct. Although slower than the isomerization reaction, the addition is fast, with 50% of the active sites being alkylated within 12 min. By contrast, the wt4-OT shows no detectable modification over 24 h. These results support the hypothesis that avoidance of nucleophilic reactions, such as the irreversible Michael addition to the product, could be a contributing factor in the evolutionary conservation of N-terminal proline residues in 4OT.

Affinity Labels↗
PubMed1975

Affinity labelling to - SH groups in adenosine - triphosphate - phosphoribosyl transferase with the dinitrophenyl group from S-dinitrophenyl-6-mercaptopurine-riboside 5'-phosphate.

Adenosine-triphosphate-phosphoribosyl transferase from Escherichia coli reacts with S-dinitrophenyl-6-mercaptopurine-riboside 5'-phosphate. In this reaction the dinitrophenyl group becomes attached to the enzyme, while the nucleotide is split off. Most aliphatic high and low-molecular-weight-SH compounds react with the thioether in the opposite way, i.e. bind the nucleotide and split off dinitrothiophenol. It appears that the dinitrophenyl moiety of the thioether interacts with the enzyme in a specific way, and that this interaction activates the bond between the dinitrophenyl group and the sulfur atom. In support of this it was found that dinitrophenol inhibits the transferase reaction with half maximal effect at 0.4 mM. The inhibition is competitive with ATP. Dinitrophenol also competes with ATP in binding studies.

ATP Phosphoribosyltransferase↗
PubMed1988

Affinity labeling of the GDP/GTP binding site in Thermus thermophilus elongation factor Tu.

Elongation factor Tu from Thermus thermophilus was treated successively with periodate-oxidized GDP or GTP and cyanoborohydride. Covalently modified cyanogen bromide or trypsin fragments of the protein were isolated, and the position of their modification was determined. Lysine residues 52 and 137 were heavily labeled, lysine-137 being considerably more reactive in the GTP form as compared to the GDP form of the protein. These residues are in the proximity of the GDP/GTP binding site. Lys-325 was also labeled, but to a lower extent. The part of the EF-Tu containing residue 52 is missing in crystallized EF-Tu.GDP from Escherichia coli [Jurnak, F. (1985) Science (Washington, D.C.) 230, 32-36]. These results place the part of T. thermophilus EF-Tu corresponding to the missing fragment in E. coli EF-Tu in the vicinity of the nucleotide binding site and allow its role in the interaction with aminoacyl-tRNA and elongation factor Ts to be evaluated. Cross-linking of EF-Tu.GDP by irradiation at 257 nm showed that a sequence of 10 amino acids residues which is found in the Thermus thermophilus elongation factor Tu but not in other homologous bacterial proteins is located in the vicinity of the GDP/GTP binding site.

Affinity Labels↗
PubMed1979

Phosphoenolpyruvate carboxylase of Escherichia coli. Affinity labeling with bromopyruvate.

Phosphoenolpyruvate carboxylase [EC 4.1.1.31] from Escherichia coli W was alkylated by incubation with bromopyruvate, substrate analog, leading to irreversible inactivation. The reaction followed pseudo-first-order kinetics. Mg2+, an essential cofactor for catalysis, enhanced the inactivation, and the enhancing effect increased as the pH increased. The inactivation rate showed a tendency to saturate with increasing concentrations of bromopyruvate, indicating that an enzyme-bromopyruvate complex was formed prior to the alkylation. DL-Phospholactate, a potent competitive inhibitor with respect to phosphoenolpyruvate, protected the enzyme from inactivation in a competitive manner. Examination of the acid hydrolysate of the enzyme modified with [14C]bromopyruvate by paper chromatography showed that radioactivity was solely incorporated into carboxyhydroxyethyl cysteine. In addition, determination of sulfhydryl groups of the native and modified enzymes with 5,5'-dithiobis(2-nitrobenzoate) showed that inactivation occurred concomitant with the modification of one cysteinyl residue per subunit. The results indicate that bromopyruvate reacted with the enzyme as an active-site-directed reagent.

Affinity Labels↗
PubMed1995

A new class of photoactivatable and cleavable derivatives of neurotoxin II from Naja naja oxiana. Synthesis, characterisation, and application for affinity labelling of the nicotinic acetylcholine receptor from Torpedo californica.

A new series of photoactivatable and cleavable derivatives of neurotoxin II from the cobra Naja naja oxiana is investigated which can be used for mapping the surface topology of the nicotinic acetylcholine receptor from Torpedo electric tissue. The preparation and characterisation of five toxin derivatives, each with a radioactive 125I-azidosalicylamidoethyl-1,3'-dithiopropyl group in a defined position within the primary structure, are described. The photoinduced cross-linking reaction of the toxin derivatives with membrane-bound receptor is investigated. The photoactivatable group located at position K25 reacts almost exclusively with the delta subunit of the receptor, whereas the K15 derivative reacts with the alpha and beta subunits. The other derivatives did not react with the receptor to any significant extent. It is shown that, with respect to the receptor subunits, the cross-linking pattern depends on the length and chemical nature of the cross-linking group.

Affinity Labels↗
PubMed1985

Decrease of metastatogenic potential by pregraft treatment of Lewis lung carcinoma cells with proteinase and protein kinase affinity labels.

Three synthetic irreversible enzyme inhibitors (75 microM di-iso-propylphosphorofluoridate (DFP), 310 microM N alpha-p-tosyl-L-lysine (TLCK) and 240 microM L-1-tosylamide-2-phenylethyl (TPCK) chloromethyl ketone), as well as the transition state analogue chymostatin, inhibit the development of Lewis lung adenocarcinoma (3LL) in C57 BI/6 mice, when 3LL cells are treated once and for a limited period (60 min) prior to grafting. These compounds demonstrate divergent protease specificity and, in the case of TLCK and TPCK, convergent reactivity toward the highly conserved protein kinase catalytic subunit. Using 200 microM chymostatin and low doses (25-40 microM) of the irreversible enzyme inhibitors, the antimetastatogenic effect is revealed to be specific, as primary tumor development is not affected. Although no direct experimental evidence can be forwarded, our results fit with the concept that the motile metastatogenic 3LL cells may constitute a phenotype which, in contrast to the resident cells from the primaries, responds to these enzyme inhibitors in a highly sensitive manner.

Affinity Labels↗
PubMed1985

Affinity-labelling of the thyrotropin receptor. Characterization of the photoactive ligand.

Thyrotropin (TSH) has been coupled to the photoactive heterobifunctional reagent N-hydroxysuccinimidyl 4-azidobenzoate (HSAB) and the properties of the product (HSAB-TSH) investigated. Preparations of HSAB-TSH containing two molecules of HSAB per molecule of TSH were used in most experiments and these preparations retained about 40% of the original receptor-binding activity of the TSH. HSAB-TSH could be labelled with 125I and cross-linked to porcine and human TSH receptors. Analysis of the cross-linked complexes indicated that the receptors consisted of two subunits (designated A and B) linked by a disulphide bridge. In the case of the human TSH receptor, the A- and B-subunits had approximate Mr values of 50 000 and 30 000 respectively, whereas the Mr values for porcine TSH-receptor A- and B-subunits were approx. 45 000 and 25 000 respectively. Only the A subunit was cross-linked to TSH. Comparison of the effects of trypsin and mercaptoethanol on the TSH-TSH-receptor complexes suggested that the trypsin cleavage point on the A-subunit was at a point close to the disulphide bridge.

Affinity Labels↗
PubMed1988

Photo-affinity labelling at the peptidyl transferase centre reveals two different positions for the A- and P-sites in domain V of 23S rRNA.

Photo-reactive 3-(4'-benzoylphenyl)propionyl-Phe-tRNA bound to the A- or the P-site was crosslinked to 23S RNA upon irradiation at 320 nm. The sites of reaction were identified as U-2584 and U-2585 at the A-site and A-2451 and C-2452 at the P-site. Minor crosslinks from both sites were observed at nucleotides A-2503 to U-2506. All sites identified lie in close proximity according to the secondary structure model and constitute part of the highly conserved loop region of domain V. Antibiotics known to inhibit peptidyl transferase activity had a pronounced effect on photo-crosslinking. In addition, tetracycline was also shown to photo-crosslink to this region. These experiments permit a dissection of the peptidyl transferase region on the 23S RNA into two distinct areas for the A- and P-site.

Acyltransferases↗
PubMed1985

Affinity labeling of Escherichia coli glutamine synthetase by beta, gamma-Cr(III)(H2O)4ATP.

The interaction of Escherichia coli glutamine synthetase with beta, gamma-Cr(III)(H2O)4ATP (CrATP) has been studied. This substitution inert nucleotide functioned as an active site directed irreversible inhibitor of glutamine synthetase in solutions containing 15 mM MgCl2, 100 mM KCl, and 10 mM Pipes (pH 6.6). The inactivation reaction followed pseudo-first order saturation kinetics which demonstrated reversible binding of CrATP prior to the formation of inactive enzyme. CrATP was shown to be a competitive inhibitor versus MgATP. Also, significant protection was afforded by MgATP indicating that CrATP inactivates at the active site. Partial protection was afforded by glutamate or inorganic phosphate while inactivation was enhanced by Mn(II). The stoichiometry of CrATP incorporation was approximately one molecule per enzyme subunit, determined spectrophotometrically. Both the delta and lambda isomers of CrATP bound to glutamine synthetase, but only the lambda isomer was an active site directed irreversible inhibitor.

Adenosine Triphosphate↗
PubMed1988

Biochemistry of terminal deoxynucleotidyltransferase. Affinity labeling and identification of the deoxynucleoside triphosphate binding domain of terminal deoxynucleotidyltransferase.

Using the technique of UV-mediated cross-linking of nucleotides to their acceptor sites (Modak, M. J., and Gillerman-Cox, E. (1982) J. Biol. Chem. 257, 15105-15109), we have labeled calf terminal deoxynucleotidyltransferase (TdT) with [32P]dTTP. The specificity of dTTP cross-linking at the substrate binding site in TdT is demonstrated by the competitive inhibition of the cross-linking reaction by other deoxynucleoside triphosphates, and ATP and its analogues, requiring concentrations consistent with their kinetic constants. Tryptic peptide mapping of the [32P]dTTP-labeled enzyme showed the presence of a single radioactive peptide fraction that contained the site of dTTP cross-linking. The amino acid composition and sequence analysis of the radioactive peptide fraction revealed it to contain two tryptic peptides, spanning residues 221-231 and 234-249. Since these two peptides were covalently linked to dTTP, the region encompassed by them constitutes a substrate binding domain in TdT. Further proteolytic digestion of the tryptic peptide-dTTP complex, using V8 protease, yielded a smaller peptide, and its analysis narrowed the substrate binding domain to 14 amino acids corresponding to residues 224-237 in the primary amino acid sequence of TdT. Furthermore, 2 cysteine residues, Cys-227 and Cys-234, within this domain were found to be involved in the cross-linking of dTTP, suggesting their participation in the process of substrate binding in TdT.

Affinity Labels↗
PubMed1977

Affinity labeling of gamma-glutamyl transpeptidase and location of the gamma-glutamyl binding site on the light subunit.

Gamma-Glutamyl transpeptidase, which consists of two nonidentical subunits, is rapidly inactivated with respect to its transpeptidase and hydrolase activities by the gamma-glutamyl analogs 6-diazo-5-oxo-L-norleucine and L-azaserine. Inactivation, which is prevented by gamma-glutamyl substrates (but not by acceptor substrates), is accelerated by maleate, which was previously shown to enhance utilization of glutamine by transpeptidase. 6-Diazo-5-oxo--norleucine reacts specifically, covalently, and stoichiometrically at the gamma-glutamyl site of the enzyme, which was localized through studies with 6-diazo-5-OXO-[14C]norleucine to the light subunits of both the transpeptidase of rat kidney (which has subunits of molecular weights 22,000 and 46,000) and the transpeptidase of human kidney (which has subunits of molecular weights 22,000 and 62,000). The findings, which indicate that these enzymes have similar gamma-glutamyl binding subunits, are relevant to the structure-function relationships of this membrane-bound enzyme and its physiological role.

Affinity Labels↗
PubMed1989

Ribosomal proteins S7 and L1 are located close to the decoding site of E. coli ribosome--affinity labeling studies with modified tRNAs carrying photoreactive probes attached adjacent to the 3'-end of the anticodon.

Two photoreactive azidonitrophenyl probes have been attached to Yeast methionine elongator tRNA by chemical modification of the N6-(threoninocarbonyl)adenosine located next to the 3'-end of the anticodon. The maximum distance between the purine ring and the azido group estimated for the two probes is 16-17 and 23-24A, respectively. Binding and cross-linking of the uncharged, modified tRNAs to E. coli ribosomes have been studied with and without poly(A,U,G) as a message, under conditions directing uncharged tRNAs preferentially to the P-site. The modified tRNAs retain their binding activity and upon irradiation bind covalently to the ribosome with very high yields. Protein S7 is the major cross-linking target for both modified tRNAs, in the presence or absence of poly(A,U,G). Protein L1 and to a lesser extent proteins L33 and L27 have been found to be cross-linked with the short probe. Cross-linking to 168 rRNA reaches significant levels only in the absence of the message.

Affinity Labels↗
PubMed1982

Affinity labels for membrane components involved in the uptake of bile acids and of phallotoxins by hepatocytes. Development of covalently binding derivatives of bile acids and of compounds related to cholecystographic agents.

A series of covalently binding derivatives of bile acids, fusidic acid and of compounds similar to cholecystographic agents were synthesized. Nearly all of them inhibited the development of protrusions on the surface of isolated hepatocytes regularly seen after treatment with phalloidin. The same compounds inhibited the uptake of demethylphalloin and of cholate in a concentration dependent manner. Two kinds of effects could be distinguished: The irreversible part of the inhibition depended on the incubation period and could not be removed by washing procedures. The reversible one was independent on the duration of the preincubation. Final results indicated that the tested derivatives inhibited either both transports, and the phalloidin response of liver cells to the same degree and in the same manner, or were found to be ineffective in all tests. The above parallelism supports the hypothesis that phallotoxins may be translocated by a carrier system normally responsible for the uptake of bile acids from the portal blood.

Affinity Labels↗