Histidine-specific Reagent Research Articles

Histidine-15: an important role in the cytotoxic activity of human tumor necrosis factor

The amino acids that are required for the cytotoxic activity of recombinant human tumor necrosis factor-alpha (TNF) were investigated by chemical modification and oligonucleotide-directed site-specific mutagenesis. TNF contains three histidine residues, located at positions 15, 73 and 78. The histidine-specific reagent diethylpyrocarbonate (DEP) was used to chemically modify TNF. The chemical inactivation of the in vitro cytotoxic activity of this lymphokine (using murine L929 target cells) was found to be time- and dose-dependent. Inactivated TNF failed to compete with fully bioactive [125I]TNF for human MCF-7 target cell receptors. Mutant polypeptides of TNF were genetically engineered by oligonucoleotide-directed site-specific mutagenesis. The cytotoxicity of a double histidine mutant, in which histidine-73 and histidine-78 were replaced with glutamine, was not altered and was chemically inactivated by DEP. Substituting glutamine for histidine-15 resulted in 10-15% of the wild-type bioactivity. Replacing histidine-15 with either asparagine, lysine or glycine resulted in a biologically inactive molecule. The data show that the histidine residue at position 15 is an amino acid that is required for the cytotoxic activity of TNF.

Chemical modification of A1 adenosine receptors in rat brain membranes. Evidence for histidine in different domains of the ligand binding site.

Chemical modification of amino acid residues was used to probe the ligand recognition site of A1 adenosine receptors from rat brain membranes. The effect of treatment with group-specific reagents on agonist and antagonist radioligand binding was investigated. The histidine-specific reagent diethylpyrocarbonate (DEP) induced a loss of binding of the agonist R-N6-[3H] phenylisopropyladenosine ([3H]PIA), which could be prevented in part by agonists, but not by antagonists. DEP treatment induced also a loss of binding of the antagonist [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX). Antagonists protected A1 receptors from this inactivation while agonists did not. This result provided evidence for the existence of at least 2 different histidine residues involved in ligand binding. Consistent with a modification of the binding site, DEP did not alter the affinity of [3H]DPCPX, but reduced receptor number. From the selective protection of [3H] PIA and [3H]DPCPX binding from inactivation, it is concluded that agonists and antagonists occupy different domains at the binding site. Sulfhydryl modifying reagents did not influence antagonist binding, but inhibited agonist binding. This effect is explained by modification of the inhibitory guanine nucleotide binding protein. Pyridoxal 5-phosphate inactivated both [3H]PIA and [3H]DPCPX binding, but the receptors could not be protected from inactivation by ligands. Therefore, no amino group seems to be located at the ligand binding site. In addition, it was shown that no further amino acids with polar side chains are present. The absence of hydrophilic amino acids from the recognition site of the receptor apart from histidine suggests an explanation for the lack of hydrophilic ligands with high affinity for A1 receptors.

Chemical modification of the actin binding site of rabbit muscle aldolase by diethylpyrocarbonate.

To extend the available information on the significance of the interactions between glycolytic enzymes and the actin component of the cellular ultrastructure, investigations into the compositional characteristics of the actin binding site on one of the major glycolytic enzymes, aldolase, have been undertaken. As the electrostatic nature of the association has been previously reported indicative of a cationic region on the enzyme involved in the binding, these studies have investigated the possibility of the involvement of histidine residues in this binding region. By the use of the histidine specific reagent, diethylpyrocarbonate, we have been able to establish a difference in nature of an actin binding domain and the active site domain which does contain an essential histidine. The results have been discussed in relation to the significance of this finding with respect to the binding of aldolase to subcellular structure.

The role of histidyl residues in zinc-induced precipitation of α 2-macroglobulin-proteinase complexes

When α 2 -macroglobulin ( α 2M ) is reacted with proteinases including trypsin, plasmin, α-thrombin, or with CH 3NH 2, each resulting α 2M derivative is precipitated by Zn 2+ in a similar manner. By contrast, unreacted α 2M is not precipitated over the same Zn 2+ concentration range. Zn 2+-induced precipitation of α 2M -CH 3NH 2 or α 2M -trypsin is prevented by acylation of the protein employing the histidine-specific reagent diethylpyrocarbonate (DEP). The Zn 2+-induced precipitation of α 2M -trypsin is prevented by acylation of the preformed α 2M -trypsin complex or by the reaction of acylated native α 2M with trypsin. Acylation of α 2M by treatment with DEP results in the modification of 13.5 histidyl residues per subunit of either native α 2M or α 2M -CH 3NH 2. Subsequent treatment with hydroxylamine reverses the modification of 10.5 histidyl residues per subunit in each protein preparation. These results indicate that histidyl residues are involved in the Zn 2+-induced precipitation of α 2M -proteinase or α 2M -CH 3NH 2 complexes, and that these residues are accessible to extensive protein-metal interactions only after α 2M has undergone a major conformational change. These appear to be the same histidyl residues which, upon acylation by DEP, are responsible for recognition of α 2M -proteinase complexes by the acyl-low-density-lipoprotein cell surface receptor ( S. V. Pizzo, P. A. Roche, S. R. Feldman, and S. L. Gonias (1986) Biochem. J. 238, 217–225).

Amino acid residues complexed with eosin 5-isothiocyanate in band 3 protein of the human erythrocyte

The amino-acid residues of band 3 protein taking part in the ionic interaction with an anion-transport inhibitor, eosin 5-isothiocyanate (EITC), were determined by pH titration. The plots of the absorbance of EITC-ghost system against pH reveal five equilibria, at pH 3.7, 6.4, 8.0, 11.0 and 13.1. Since the three equilibria, 3.7, 8.0 and 11.0, are representative of the EITC molecule, the others, 6.4 and 13.1, may be due to the interaction of EITC molecules with histidine and arginine residues, respectively. The same experiment using a reconstituted system of band 3-lipid vesicles gave results in good agreement with the EITC-ghost system. The intensity of the induced CD band at 530 nm of EITC molecules bound to ghosts was decreased by preincubation with arginine-specific reagents, phenylglyoxal and 1,2-cyclohexanedione, or histidine-specific reagents, diethylpyrocarbonate (DEPC) and p-diazobenzene sulfonate. The repression effects by these chemical modifiers were evaluated by measuring the concentrations which elicit 50% reduction. The histidine-specific reagents repressed the CD of EITC more effectively than the arginine-specific reagents. Furthermore, it was found that DEPC effectively inhibited the sulfate efflux from intact erythrocytes. These results suggest that the histidine residues participate in the anion-transport system of human red cells.

Aluminum ions are required for stabilization and inhibition of hepatic microsomal glucose-6-phosphatase by sodium fluoride.

Stabilization and inhibition of hepatic microsomal glucose-6-P phosphohydrolase (EC 3.1.3.9) by F- requires the presence of Al3+ ions. At millimolar concentrations, reagent grade NaF inhibited glucose-6-P hydrolysis and protected the enzyme against inactivation induced by heat in the presence of 0.025% (w/v) Triton X-100 or by reaction of the catalytic site with the histidine-specific reagent, diethyl pyrocarbonate. The presence of millimolar EDTA in all test systems abolished the effectiveness of NaF, yet EDTA by itself was without significant influence on the kinetics of phosphohydrolase reaction, the thermal stability of the enzyme or its reactivity with diethyl pyrocarbonate. Although ultrapure NaF was ineffectual in all test systems, its potency as a competitive inhibitor or protective agent was markedly increased by micromolar AlCl3 or when assays were carried out in flint glass test tubes. The latter response is explained by the well documented ability of fluoride solutions to extract Al3+ from glass at neutral pH. Our analysis indicates that the effectiveness of fluoride in all test systems derives from the formation of a specific complex with Al3+, most likely Al(F)4-. The apparent dissociation constant for interaction of the enzyme and Al(F)4- is 0.1 microM. The combination of NaF and AlCl3 holds promise as an unusually effective and versatile means to stabilize this notoriously labile enzyme during efforts to purify it.

Inactivation of the renal microvillus membrane Na+-H+ exchanger by histidine-specific reagents.

We examined the effect of histidine-specific reagents on the transport activity of the Na+-H+ exchanger in microvillus (brush-border) membrane vesicles isolated from the rabbit renal cortex. Rose bengal-catalyzed photo-oxidation caused irreversible inhibition of the rate of Na+-H+ exchange but also caused significant loss of vesicle integrity. Treatment of the membrane vesicles with diethylpyrocarbonate caused inactivation of Na+-H+ exchange that could not be attributed to vesicle disruption or collapse of transmembrane H+ gradients. Inactivation of Na+-H+ exchange by diethylpyrocarbonate followed pseudo-first order kinetics to below 10% residual activity, could be reversed by hydroxylamine, was reflected by a decreased Vmax with no change in the Km for Na+, was dependent on external pH but not internal pH, was blocked by amiloride, and was enhanced by Na+. These data are consistent with the hypothesis that a diethylpyrocarbonate-sensitive imidazolium residue is the titratable group found in kinetic studies to bind H+ at the external transport site of the Na+-H+ exchanger.

Four serine proteases from the latex of Euphorbia tirucalli

Four proteolytic enzymes, euphorbains t1–t4, were isolated from the latex of the succulent Euphorbia tirucalli ("milk bush") and purified to homogeneity. The enzymes all have molecular weights of 74 000. Each enzyme has several different charged forms; t1, for example, has four with isoelectric points in the range of 5.0–5.5. The four proteases examined are of similar amino acid compositions but yield differing two-dimensional maps of tryptic digests. The euphorbain t's are not closely related in composition to their counterparts isolated from Euphorbia lathyris, Euphorbia pulcherrima, and Euphorbia cyparissias. The euphorbains from E. tirucalli were inhibited by phenylmethylsulphonyl fluoride and by histidine-specific reagents, and so are trypsin-like proteases.

Interaction between the mitochondrial ATP synthetase and ATPase inhibitor protein: Active/inactive slow pH-dependent transitions of the inhibitor protein

The rate of mitochondrial ATPase inactivation by the naturally occurring inhibitor protein in the presence of saturating ATP and Mg 2+ at pH 8.0 depends hyperbolically on the amount of inhibitor added; the upper limit of an apparent first-order constant for the inactivation process is 1.0 −1 at 25°C. A dramatic difference in the inactivation rate is observed when the protein inhibitor is added to the same assay system from either acidic (pH 4.8) or alkaline (pH 8.2) solutions. The slow reversible transition of the inhibitor from its rapidly reacting ‘acidic’ form to the slow reacting ‘alkaline’ form occurs when the solution of the protein inhibitor is subjected to a pH-jump from 4.8 to 8.2 ( t 1 2 ~ 30 s at 25°C). The pH-profile of the inhibitor active/inactive equilibrium suggests that a group with p K a ~ 6.5 is involved in the transition. Treatment of the inhibitor protein with a histidine-specific reagent (e.g. diethyl pyrocarbonate) abolishes its inactivating effect on the ATPase activity. It is concluded that the protonation/deprotonation of the inhibitor protein followed by its slow conformational changes is the rate-limiting step in the inhibitor-ATP synthetase interaction.

Chemical modification of the Na +/H + exchanger of thymic lymphocytes. Inhibition by [formula omitted

A Na +/H + exchanger is involved in the regulation of cytoplasmic pH and cellular volume in a variety of cells. Little is known about the molecular nature of this exchanger. The purpose of this study was to survey a variety of group-specific covalent reagents as potential inhibitors of the exchanger. Na +/H + countertransport activity was assayed as the amiloride-sensitive rate of Na +-induced alkalinization in acid-loaded lymphocytes, or as the rate of swelling in cells suspended in sodium propionate medium. Activity was not affected by proteinases or by carboxyl-group and amino-group specific reagents. A significant inhibition was produced by diethylpyrocarbonate, a histidine-specific reagent and by N- ethylmaleimide , a sulfhydryl group reagent. A similarly reactive but nonpermeating sulfhydryl agent, glutathione-maleimide, failed to inhibit Na +-H + exchange. Moreover, the reaction with N- ethylmaleimide was sensitive to changes in the cytoplasmic pH. The data suggest that the chemically reactive groups of the Na +/H + exchanger of lymphocytes have limited exposure to the extracellular medium but that an internally located sulfhydryl group is critical for the cation-exchange activity.

Specific inactivation of the phosphohydrolase component of the hepatic microsomal glucose-6-phosphatase system by diethyl pyrocarbonate

We have examined the interactions of the histidine-specific reagent diethyl pyrocarbonate (DEPC) with the components of the rat hepatic glucose-6-phosphatase system (EC 3.1.3.9). DEPC is the first known reagent that satisfies the criteria of an active-site-specific label for the phosphohydrolase component. (a) It inactivates through formation of a stable covalent bond. (b) It is effective at reasonably low concentrations (2-4 mM) under relatively mild conditions (e.g. 30 degrees C at neutral pH). (c) Inactivation is substantially blocked by glucose 6-phosphate, Pi and NaF, compounds which are known to interact quite specifically with the phosphohydrolase. (d) Under conditions where glucose 6-phosphate and NaF protect the enzyme, no protection is provided against DEPC-mediated inactivation of two other functional components of the membrane, the glucose 6-phosphate translocase and UDP-glucuronyltransferase. DEPC also shows potential for use at 0 degree C as a label for UDP-glucuronyltransferase.

Determination of a histidine residue at the yeast orotate phosphoribosyltransferase active-site.

A purified preparation of orotate phosphoribosyltransferase(0-PRTase) from yeast has been shown to catalyze the formation of orotidine monophosphate (OMP) from orotate and 5-phosphoribosyl α-1-pyrophosphate (PRibPP) through the use of a ping pong kinetic mechanism (1) in the presence of an optimum concentration of Mgll(2). The present investigation was initiated to search for nucleo-philic active-site residues of 0-PRTase which would stabilize (in an SN1 elimination) or react with (in a triple SN2 displacement) the enzyme-phosphoribosyl intermediate, formed as a consequence of this mechanism. Analysis of the amino acid composition, pH dependency of the 0-PRTase activity in the forward and reverse directions, and chemical modification of 0-PRTase using histidine-specific reagents have been accomplished.

Metabolism of glycerate-2,3-P 2—V. Histidine-specific reagents inactivate the phosphoglycerate mutase, glycerate-2,3-P 2 synthase and glycerate-2,3-P 2 phosphatase activities of rabbit muscle phosphoglycerate mutase

1. 1. Both treatment with diethylpyrocarbonate and photo-oxidation with rose bengal produces the loss of the three enzymatic activities of rabbit muscle phosphoglycerate mutase: phosphoglycerate mutase, glycerate-2,3-P 2 synthase and glycerate-2,3-P 2 phosphatase. 2. 2. The synthase and the phosphatase activities are less affected than the mutase activity. 3. 3. Glycerate-2,3-P 2 and glycerate-3-P protect against diethylpyrocarbonate inactivation, but do not protect against inactivation produced by photo-oxidation. 4. 4. Hydroxylamine reactivates the diethylpyrocarbonate-treated enzyme. 5. 5. Chemical modification of phosphoglycerate mutase markedly reduces its ability to form the functionally active phosphoenzyme. 6. 6. These results provide evidence of the intrinsic character of the three enzymatic activities of phosphoglycerate mutase. In addition, they support the existence of two separate binding sites for monophosphoglycerates and for bisphosphoglycerates.

Modification of the insulin receptor by diethyl pyrocarbonate: effect on insulin binding and action.

Insulin binding to rat liver plasma membranes is inhibited in a time- and dose-dependent fashion by prior treatment of membranes with the histidine-specific reagent diethyl pyrocarbonate. If all receptors are occupied by unlabeled hormone during diethyl pyrocarbonate treatment, no inhibition of 125I-labeled insulin binding is observed folowing washout of unlabeled hormone and unreacted reagent. Scatchard analysis of the binding inhibtion due to diethyl pyrocarbonate reveals a loss in receptor number rather than a change in receptor affinity for hormone. Fat cells treated with diethyl pyrocarbonate exhibit a rightward shift in the dose-response relationship for insulin-stimulated glucose oxidation consistent with a loss in receptor number due to the reagent. The pH profile for inhibition of insulin binding by diethyl pyrocarbonate and the partial reversibility of this inhibition by hydroxylamine are consistent with modification of a histidine residue. These results suggest that a histidine residue at or near the receptor binding site is required for formation of the biologically relevant insulin - receptor complex.

Phylogeny and ontogeny of the phosphoglycerate mutases—v. inactivation of phosphoglycerate mutase isozymes by histidine-specific reagents

1. 1. The three isozymes of glycerate-2,3-P 2 dependent phosphoglycerate mutase present in tissues of mammals and reptiles were inactivated by both treatment with diethylpyrocarbonate and photooxidation with rose bengal. 2. 2. Inactivation of type M isozyme purified from rabbit muscle was complete when two histidine residues per enzyme subunit were carboethoxylated. Hydroxylamine removed the carboethoxy groups, with partial recovery of the enzymatic activity. The cofactor protected the enzyme against inactivation. 3. 3. The inactivation of rabbit muscle phosphoglycerate mutase by photooxidation with methylene blue and rose bengal was sharply pH dependent. The pH profile of enzyme inactivation followed the titration curve of histidine, suggesting that this amino acid was critical for enzyme activity. Glycerate-2,3-P 2 did not protect phosphoglycerate mutase against photoinactivation.

Effect of diethylpyrocarbonate on lactose/proton symport in Escherichia coli membrane vesicles.

Exposure of Escherichia coli ML 308-225 membrane vesicles to the histidine-specific reagent diethylpyrocarbonate (DEPC) led to concentration- and time-dependent inactivation of active lactose transport, and the sensitivity of the system to inactivation was enhanced when an electrochemical proton gradient (delta- muH+, interior negative and alkaline) was generated across the vesicle membrane. Although beta-D-galactopyranosyl 1-thio-beta-D-galactopyranoside blocked DEPC inactivation, binding of p-nitrophenyl alpha-D-galactopyranoside was not significantly altered, indicating that DEPC does not react at the binding sites of the lac carrier protein. Strikingly, vesicles treated with DEPC exhibited an increased apparent Km for delta- muH+-driven lactose transport and counterflow but no change in the Vmax of these reactions and no change in the apparent Km or Vmax of facilitated diffusion. Moreover, DEPC treatment increased the apparent Km observed for delta- muH+-driven proline and D-lactate transport with no change in Vmax. Finally, the lactose counterflow activity of DEPC-treated vesicles was regenerated by subsequent exposure to hydroxylamine. It is suggested that a histidyl residue(s) in the lac carrier or another protein in the translocation complex is involved either in the binding and translocation of protons or in a conformational change that may occur upon protonation of the lac carrier protein.

Proton magnetic resonance studies on Escherichia coli dihydrofolate reductase. Assignment of histidine C-2 protons in binary complexes with folates on the basis of the crystal structure with methotrexate and on chemical modifications.

The effects of pH upon the C-2 resonances of the 5 histidine residues of Escherichia coli MB 1428 dihydrofolate reductase in binary complexes with methotrexate, aminopterin, folate, methopterin, and trimethoprim were studied by 300-MHz 1H nmr spectroscopy. Three of the five histidine residues, labeled 1, 2, and 3, exhibited similar pK' values and chemical shifts for their C-2 protons in the five binary complexes. One histidine, 4, was quite different in the folate complex and the last histidine, 5 was quite different in the trimethoprim complex. For all five binary complexes, each histidine had a pK' which was significantly different from the other 4 histidines of that complex. Titration of the binary methotrexate complex of a 5,5'-dithiobis(2-nitrobenzoate)-modified enzyme showed that 2 histidines were not perturbed by this modification of Cys 152, and that the alkaline form of histidine 2, the acid form of histidine 4, and, to a lesser extent, the acid form of histidine 3 were slightly perturbed. Titration of the binary methotrexate complex of a N-bromosuccinimide-modified enzyme demonstrated that this modification slightly affected all of the histidines and drastically affected histidine 5. Histidines 3 and 5 of the binary methotrexate complex reacted rapidly with the histidine-specific reagent, ethoxyformic anhydride, while histidines 2 and 4 reacted at a moderate rate and histidine 1 reacted slowly if at all. The local electrostatic environments of the 5 histidine residues as deduced from the crystal structure of the binary complex of the enzyme with methotrexate (Matthews, D.A., Alden, R.A., Bolin, J.T., Freer, S.T., Hamlin, R., Xuong, N., Kraut, J., Poe, M., Williams, M.N., and Hoogsteen, K. (1977) Science 197, 594-597) were used as the basis for proposed assignments of the five histidine C-2 nmr resonances. The assignments were: 1, pK' 7.9 to 8.2, His 124; 2, pK' 7.2 to 7.4, His 141; 3, pK' 6.5 to 6.7, His 149; 4, pK' 5.7 to 6.3, His 114; and 5, pK' 5.2 to 5.9, His 45. The effect of the chemical modifications upon the enzyme's histidine residues were consistent with the assignments, but no direct chemical evidence in support of the assignments was obtained. It was proposed that, since the crystallographic data provided consistent assignments of the histidine nmr data for both native and chemically modified enzyme, the local environment of each of the 5 histidine residues was similar in the crystal and in solution.

Modification of phenylalanyl-tRNA synthetase from Escherichia coli by histidine-specific reagents. Effects on structure and function.

Phenylalanyl‐tRNA synthetase from Escherichia coli was subjected to chemical modification with diethylpyrocarbonate at pH 6.0 and to photochemical oxidation in the presence of rose Bengal, both methods giving rise to loss of enzymic activity. The highly sensitive reaction of the enzyme with either reagent is pseudo‐first order. The specificity of the modification methods employed has been investigated.All substrates were studied for their ability to protect the enzyme against inactivation. Presence of phenylalanine, ATP and Mg2+ provides the most pronounced effect. Under this condition the tRNA‐aminoacylation activity may be completely abolished, whereas the pyrophosphate–ATP exchange activity is only partly affected. However, such enzyme is still able to form a specific complex with tRNAPhe. The results presented therefore suggest that histidine residues of phenylalanyl‐tRNA synthetase accessible to modification by diethylpyrocarbonate or to photooxidation do not seem to be involved in the binding of tRNA but might possibly take part in the esterification reaction.Quantitative analysis showed that a total of 50 histidine residues are accessible to diethylpyrocarbonate in the unprotected enzyme under non‐denaturing conditions. In the presence of phenylalanine, ATP and Mg2+ the loss of aminoacylation activity is correlated with the modification of 2–4 residues.During extensive modification of the enzyme a dissociation process takes place. It was found that the observed loss of quaternary structure is not correlated with the decrease of the aminoacylation activity of the enzyme upon modification.

Studies on glucose dehydrogenase of Aspergillus oryzae: IV. Histidyl residue as an active site

1. 1.|Glucose dehydrogenase from Aspergillus oryzae was inhibited by Ag +, Hg 2+ and Cu 2+, but was insensitive to sulfhydryl reagents. 2. 2.|The involvement of a histidyl residue (or residues) in the activity was suggested from photoinactivation in the presence of methylene blue, the pH dependence of K m for glucose, and titration with the histidine-specific reagent, diazo- i-H-tetrazole (DHT). 3. 3.|The inhibiton by Ag + was reversed by dialysis and by addition of a high concentration of glucose. The activity of DHT-inhibited enzyme was not restored by dialysis, and high concentrations of glucose partly protected the enzyme from DHT inhibition only when added prior to the addition of DHT. 4. 4.|The reducton of the enzyme-bound FAD by glucose was prevented in the Ag +- or DHT-treated enzyme, but the reoxidation of the reduced flavin by vitamin K 3 was not impaired in the trested enzyme. 5. 5.|It was concluded that a histidyl residue is involved in the reduction of the flavin by glucose, acting probably as the glucose-binding site.