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PubMed1988

Diethylpyrocarbonate and permanganate provide evidence for an unusual DNA conformation induced by binding of the antitumour antibiotics bleomycin and phleomycin.

DNA structural changes induced by bleomycin have been investigated using diethylpyrocarbonate and permanganate as probes under conditions in which the antibiotic binds to, but does not cut the DNA. Diethyl-pyrocarbonate shows an enhanced reaction with adenines in the presence of the antibiotic in the sequences GTA greater than GCA greater than GAA, on the 3' side of the drug cutting site (GPy). Permanganate ions display an enhanced reactivity at the second pyrimidine of the sequence GPyPy. The results are consistent with a model in which bleomycin distorts the structure of the base pair on the 3' side of its binding site.

Base Sequence↗
PubMed1992

The role of lysine, histidine and carboxyl residues in biological activity of equinatoxin II, a pore forming polypeptide from the sea anemone Actinia equina L.

Equinatoxin II, a pore forming polypeptide from the sea anemone Actinia equina L. was subjected to chemical modifications with group specific reagents. Lysine residues were modified with pyridoxal-5'-phosphate, histidine residues with diethyl pyrocarbonate and carboxyl groups with the use of a water soluble carbodiimide. Modification of charged residues had no significant influence on the toxin interaction with serum lipoproteins. Lysine 5'-phosphopyridoxylated and histidine carbethoxylated derivatives of the toxin retained lethal and hemolytic activities, but the pH profile of hemolytic activity of 5'-phospho-pyridoxylequinatoxin II was markedly altered. Modification of the toxin carboxyl groups impaired both hemolytic and lethal activities, the latter, however, to the greater extent.

Animals↗
PubMed2005

Spacer-based selectivity in the binding of "two-prong" ligands to recombinant human carbonic anhydrase I.

Benzenesulfonamide and iminodiacetate (IDA)-conjugated Cu(2+) independently interact at the active site and a peripheral site of carbonic anhydrases, respectively [Banerjee, A. L., Swanson, M., Roy, B. C., Jia, X., Haldar, M. K., Mallik, S., and Srivastava, D. K. (2004) J. Am. Chem. Soc. 126, 10875-10883]. By attaching IDA-bound Cu(2+) to benzenesulfonamide via different chain length spacers, we synthesized two "two-prong" ligands, L1 and L2, in which the distances between Cu(2+) and NH(2) group of sulfonamide were 29 and 22 A, respectively. We compared the binding affinities of L1 and L2, vis-a-vis their parent compound, benzenesulfonamide, for recombinant human carbonic anhydrase I (hCA-I) by performing the fluorescence titration and steady-state kinetic experiments. The experimental data revealed that whereas the binding affinity of L1 for hCA-I was similar to that of benzenesulfonamide, the binding affinity of L2 was approximately 2 orders of magnitude higher, making L2 one of the most potent ligands or inhibitors of hCA-I. Since the enhanced binding or inhibitory potency of L2 is diminished (to the level of benzenesulfonamide) either in the presence of EDTA or upon treatment of the enzyme with diethyl pyrocarbonate, it is proposed that Cu(2+) of L2 interacts with one of the surface-exposed histidine residues of the enzyme. A cumulative account of the experimental data leads to the suggestion that the differential binding of L1 versus L2 to hCA-I is encoded in the chain length of the spacer moiety.

Acetazolamide↗
PubMed1997

Evidence for the essential histidine residues in geranylgeranyl transferase type I from bovine testis.

Geranylgeranyl transferase was purified 30,000-fold by sequential use of 30-50% ammonium sulfate fractionation, Q-Sepharose anion exchange chromatography, Phenyl Superose hydrophobic interaction chromatography, Sephacryl S-300 gel filtration chromatography, and peptide (YREKKFFCAIL) affinity chromatography. Geranylgeranyl transferase, when incubated with diethyl pyrocarbonate (DEPC), a histidine-specific reagent, shows time-dependent inactivation, and the activity is restored by the addition of neutral hydroxylamine. The inactivation follows pseudo-first order kinetics with a second order rate constant of 0.319 M-1min-1. The overall results thus provide evidence that a histidine residue in the active site is involved in the catalytic mechanism of the geranylgeranyl transferase reaction.

Alkyl and Aryl Transferases↗
PubMed1994

Translation through an uncDC mRNA secondary structure governs the level of uncC expression in Escherichia coli.

Escherichia coli expresses the beta and epsilon subunits of F1F0-ATP synthase at relative levels consistent with the 3:1 (beta/epsilon) stoichiometry in the holoenzyme. The mechanism of translational control of expression of the uncC gene (epsilon subunit) relative to the immediately 5' uncD gene (beta subunit) was examined. Previous expression studies and a computer analysis suggested the presence of an RNA secondary structure including the 3' end of uncD, the uncDC intergenic region, and the uncC Shine-Dalgarno sequence (S. D. Dunn and H. G. Dallmann, J. Bacteriol. 172:2782-2784, 1990). Analysis of in vitro-transcribed RNA by cleavage with RNases T1, V1, and CL3 and by chemical modification with dimethyl sulfate and diethyl pyrocarbonate confirmed a predicted structure. Introduction of premature uncD stop codons inserted 5' of the secondary structure strongly reduced epsilon expression, whereas stop codons inserted at positions within the secondary structure showed smaller effects, indicating that translational control of epsilon synthesis involves partial coupling to beta synthesis. Possible mechanisms by which the RNA secondary structure and the unfolding of this structure by translation of uncD may govern the level of uncC expression are discussed.

Base Sequence↗
PubMed1992

Active-site characterization of S1 nuclease. II. Involvement of histidine in catalysis.

Modification of the histidine residues of purified S1 nuclease resulted in loss of its single-stranded (ss)DNAase, RNAase and phosphomonoesterase activities. Kinetics of inactivation indicated the involvement of a single histidine residue in the catalytic activity of the enzyme. Furthermore, histidine modification was accompanied by the concomitant loss of all the activities of the enzyme, indicating the presence of a common catalytic site responsible for the hydrolysis of ssDNA, RNA and 3'-AMP. Substrate protection was not observed against Methylene Blue- and diethyl pyrocarbonate (DEP)-mediated inactivation. The histidine (DEP)-modified enzyme could effectively bind 5'-AMP, a competitive inhibitor of S1 nuclease, whereas the lysine (2,4,6-trinitrobenzenesulphonic acid)-modified enzyme showed a significant decrease in its ability to bind 5'-AMP. The inability of the substrates to protect the enzyme against DEP-mediated inactivation, coupled with the ability of the modified enzyme to bind 5'-AMP effectively, suggests the involvement of histidine in catalysis.

Aspergillus oryzae↗
PubMed1989

Supercoil-stabilized left-handed DNA in the plasmid (dA-dT)16 insert formed in the presence of Ni2+.

The (dA-dT)16 insert of the plasmid pAT32 was probed with diethyl pyrocarbonate (DEPC) and nuclease Bal3l in the presence of Ni2+ known to be able to induce transition to left-handed conformation in the synthetic poly(dA-dT).poly(dA-T). It has been shown that this insert in a supercoiled plasmid displays a DEPC modification pattern characteristic of left-handed DNA under conditions not sufficient to induce a left-handed structure in the linear plasmid and poly(dA-dT).poly(dA-T).

DNA Transposable Elements↗
PubMed1990

Structural relationship between the enzymatic and streptococcal binding sites of human salivary alpha-amylase.

Previous studies have demonstrated that human salivary alpha-amylase specifically binds to the oral bacterium Streptococcus gordonii. This interaction is inhibited by substrates such as starch and maltotriose suggesting that bacterial binding may involve the enzymatic site of amylase. Experiments were performed to determine if amylase bound to the bacterial surface possessed enzymatic activity. It was found that over one-half of the bound amylase was enzymatically active. In addition, bacterial-bound amylase hydrolyzed starch to glucose which was then metabolized to lactic acid by the bacteria. In further studies, the role of amylase's histidine residues in streptococcal binding and enzymatic function was assessed after their selective modification with diethyl pyrocarbonate. DEP-modified amylase showed a marked reduction in both enzymatic and streptococcal binding activities. These effects were diminished when DEP modification occurred in the presence of maltotriose. DEP-modified amylase had a significantly altered secondary structure when compared with native enzyme or amylase modified in the presence of maltotriose. Collectively, these results suggest that human salivary alpha-amylase may possess multiple sites for bacterial binding and enzymatic activity which share structural similarities.

Acids↗
PubMed1983

Nucleotides in 16S rRNA that are required in unmodified form for features recognized by ribosomal protein S8.

Nucleotides in 16S rRNA which are required in unmodified form for specific recognition of ribosomal protein S8 from Escherichia coli were identified using a damage-selection experimental approach. Prior to complex formation with S8, 16S rRNA was treated under fully denaturing conditions with either diethyl pyrocarbonate or 25% hydrazine. Following separation of bound from unbound fragments of RNA, those associated with S8 were analyzed for their content of modified bases by treatment with aniline. Nucleotides found to be consistently unmodified in such fragments were located near the base of a stable helix (encompassing bases 581-656) or near the apex of the helix on the 3' proximal side. A minor S8 ribonucleoprotein particle was found to contain fragments which extended in the 3' direction to position 671.

Base Sequence↗
PubMed1978

Crystalline reduced nicotinamide adenine dinucleotide phosphate-adrenodoxin reductase from pig adrenocortical mitochondria. Essential histidyl and cysteinyl residues of the NADPH-binding site and environment of the adrenodoxin-binding site.

Pig NADPH-adrenodoxin reductase was crystallized from pig adrenocortical mitochondria and its physicochemical properties were investigated. Pig NADPH-adrenodoxin reductase is a typical flavoprotein. Its optical absorption spectrum showed peaks at 272, 377, and 450 nm in the oxidized form. The adrenodoxin reductase contained one FAD per mol. The molecular weight was 49,000. The isoelectric points of the adrenodoxin reductase and its complex with adrenodoxin were 5.3 and 4.6, respectively. Pig NADPH-adrenodoxin reductase, unlike bovine NADPH-adrenodoxin reductase, was found to be free of carbohydrate. The fluorescences of tryptophanyl residues and FAD of the adrenodoxin reductase were quenched by holo- and apo-adrenodoxins. The NADPH-binding site of the adrenodoxin reductase was examined by photooxidation and selective chemical modifications with diethyl pyrocarbonate and sulfhydryl reagents. The results indicate that a histidyl and a cysteinyl residue of the adrenodoxin reductase are essential for the NADPH-binding site. The circular dichroism spectrum of the adrenodoxin reductase showed negative ellipticity in the visible region. Spur formation was observed between pig and bovine NADPH-adrenodoxin reductases against the antibody to bovine NADPH-adrenodoxin reductase in Ouchterlony double-diffusion agar plates. The antibody did not interact with spinach ferredoxin-NADP+ reductase.

Adrenal Cortex↗
PubMed1996

Biosynthesis of N-acetylsphingosine by platelet-activating factor: sphingosine CoA-independent transacetylase in HL-60 cels.

We have previously identified a novel CoA-independent transacetylase in the membrane fraction of HL-60 cells that transfers the acetate group from platelet activating factor (PAF) to a variety of lysophospholipid acceptors (Lee, T.-c., Uemura, Y., and Snyder, F. (1992) J. Biol. Chem. 267, 19992-20001). In the present study, we demonstrate that a similar transacetylase can transfer the acetate group from PAF to sphingosine forming N-acetylsphingosine (C2-ceramide). The chemical structure of the reaction product, C3-ceramide, was established by its identical Rf value with authentic C2-ceramide standard on thin-layer plate, sensitivity to acid treatment, resistance to alkaline hydrolysis, and ability to form the C2-ceramide dibenzoate derivative. Nonspecific transfer of the acetate from PAF to sphingosine in the absence of enzyme and nonlinearity of the reaction rates were rectified by complexing sphingosine to bovine serum albumin in a 1:1 molar ratio. Under these conditions, the apparent Km for PAF is 5.4 microM, which is in the same range as the Km (12.0 microM) when lysoplasmalogen is the acetate acceptor. PAF:sphingosine transacetylase has a narrow substrate specificity and strict stereochemical configuration requirements. Ceramide, sphingosylphosphocholine, stearylamine, sphingosine 1-phosphate, or sphingomyelin are not substrates, whereas sphinganine has a limited capacity to accept the acetate from PAF. Also, only the naturally synthesized D-erythroisomer but not the synthetic L-erythro-, D-threo-, or L-threosiomers of sphingosine can serve as a substrate. PAF transacetylase activity is widely distributed among several tissues and may involve histidine and cysteine for its catalytic activity due to inhibitory effects to the enzyme by diethyl pyrocarbonate and N-ethylmaleimide, respectively. C2-ceramide is produced via PAF:sphingosine transacetylase, and physiological levels of C2-ceramide are detected in both undifferentiated and differentiated intact HL-60 cells. Collectively, because C2-ceramide has many biological activities that differ from that of PAF and sphingosine, the CoA-independent, PAF-dependent transacetylase serves as a modifier of PAF, and sphingosine functions by generating a variant lipid mediator, C2-ceramide.

Acetyltransferases↗
PubMed1998

Involvement of aspartate/glutamate antiporter in uncoupling effect of fatty acids in heart mitochondria.

Earlier it was shown that fatty acid-induced uncoupling in liver mitochondria is suppressed by the substrates of the aspartate/glutamate antiporter (V. N. Samartsev, A. V. Smirnov, I. P. Zeldi, O. V. Markova, E. N. Mokhova, and V. P. Skulachev (1997) Biochim. Biophys. Acta, 1319, 251-257). In this study it is shown that in heart mitochondria aspartate, glutamate, and diethyl pyrocarbonate do not affect oxygen consumption and membrane potential in the presence of laurate at pH 7.4. These compounds have a weak (versus carboxyatractylate) coupling effect at pH 7.0. This effect is manifested only in the presence of carboxyatractylate, magnesium, and phosphate in the incubation medium. It is suggested that these tissue-specific effects are due not only to the specific characteristics of aspartate/glutamate antiporter, but also to the differences in the content of endogenous metabolites in heart mitochondria.

Animals↗
PubMed1982

Direct measurement of lactose/proton symport in Escherichia coli membrane vesicles: further evidence for the involvement of histidine residue(s).

Addition of lactose to Escherichia coli ML 308-225 membrane vesicles under nonenergized conditions induces transient alkalinization of the medium, and the initial rate of proton influx is stimulated by valinomycin and abolished by nigericin or carbonyl cyanide m-chlorophenylhydrazone. A functional lac y gene product is absolutely required as the effect is not observed in ML 308-225 vesicles treated with N-ethylmaleimide nor with vesicles from uninduced Escherichia coli ML 30. Furthermore, the magnitude of the phenomenon is enhanced about 3-fold in vesicles from Escherichia coli T206, which contain amplified levels of the lac carrier protein. Kinetic parameters for lactose-induced proton influx are the same as those determined for lactose-facilitated diffusion, and quantitative comparison of the initial rates of the two fluxes indicates that the stoichiometry between protons and lactose is 1:1. Treatment of ML 308-225 vesicles with diethyl pyrocarbonate causes inactivation of lactose-induced proton influx. Remarkably, however, treatment with the histidine reagent enhances the rate of lactose-facilitated diffusion in a manner suggesting that the altered lac carrier catalyzes lactose influx without the symport of protons. The results are consistent with the hypothesis that acylation of a histidyl residue(s) in the lac carrier protein dissociates lactose influx from proton influx and indicate that this residue(s) play(s) an important role in the pathway of proton translocation.

Biological Transport↗
PubMed1976

Properties of crystalline reduced nicotinamide adenine dinucleotide phosphate-adrenodoxin reductase from bovine adrenocortical mitochondria. II. Essential histidyl and cysteinyl residues at the NADPH binding site of NADPH-adrenodoxin reductase.

The binding site of NADPH in NADPH-adrenodoxin reductase was examined using crystalline enzyme from bovine adrenocortical mitochondria by studies on the effects of photooxidation and chemical modifications of amino acid residues in the reductase. (1) Photoxication decreased the enzymatic activity of NADPH-adrenodoxin reductase. Photooxidation of the reductase was prevented by NADP+, adrenodoxin, or reduced glutathione, but not NAD+. Photoinactivation caused loss of a histidyl residue, but not of tyrosyl, tryptophanyl, cysteinyl, or methionyl residues of the reductase. It did not affect the circular dichroism spectrum of the reductase appreciably. (2) NADPH-adrenodoxin reductase activity was inhibited by diethyl pyrocarbonate and the inhibition was partially reversed by addition of hydroxylamine. The inhibition was prevented by NADP+, but not NAD+. (3) NADPH-adrenodoxin reductase activity was inhibited by 5,5'-dithiobis(2-nitrobenzoate) and the inhibition was reversed by reduced glutathione. It was also protected by NADP+, but not NAD+. The results indicate that a histidyl residue and a cysteinyl residue of NADPH-adrenodoxin reductase are essential for the binding of NADPH by the reductase.

Adrenal Cortex↗
PubMed1999

Structural properties of Friedreich's ataxia d(GAA) repeats.

The expansion of trinucleotide repeat sequences is the underlying cause of a growing number of inherited human disorders. To provide correlations between DNA structure and mechanisms of trinucleotide repeat expansion, we investigated potential secondary structures formed from the complementary strands of d(GAA.TTC)n, a sequence whose expansion is associated with Friedreich's ataxia. In 50 mM NaCl, pH 7.5, d(GAA)15 exhibited a cooperative and reversible decrease in large circular dichroism bands at 248 and 272-274 nm over the temperature range of 5-50 degrees C, providing evidence for a base-paired structure at reduced temperatures. Ultraviolet absorbance melting profiles indicated that the melting temperature (Tm) of d(GAA)15 was 40 degrees C. At 5 degrees C, the central portion of d(GAA)15 was hypersensitive to single-strand-specific P1 nuclease degradation and diethyl pyrocarbonate modification, providing evidence for a hairpin conformation. At temperatures between 25 and 35 degrees C in 50 mM NaCl, the triplet repeat region of d(GAA)15 was uniformly resistant to degradation by P1 nuclease, including the central portion of the sequence. Our results indicate that the structure of d(GAA)15 is a hairpin at 5 degrees C, unknown but partially base-paired at 37 degrees C, and an approximately random coil above 65 degrees C.

Circular Dichroism↗
PubMed1990

[Chemical modification of cholesterol-hydroxylating cytochrome P-450 from adrenal cortex mitochondria with diethylpyrocarbonate].

Chemical modification of adrenocortical cytochrome P-450scc with diethyl pyrocarbonate has been carried out. The histidine residues and the protein amino groups were shown to undergo modification. Carbethoxylation was accompanied by the hemoprotein inactivation and the loss of enzymatic activity. Neither of the high spin effectors (i.e., substrate and adrenodoxin) protected cytochrome P-450scc either from inactivation or from the loss of enzymatic activity. The data obtained are discussed in terms of the functional role of histidine residues in the cytochrome P-450scc molecule.

Adrenal Cortex↗
PubMed1986

Microtubule assembly is dependent on a cluster of basic residues in alpha-tubulin.

Previous studies have shown that tubulin, a major protein component of the microtubule, is rendered assembly incompetent when a highly reactive lysine residue (HRL) in the alpha polypeptide of tubulin dimer is reductively methylated [cf. Sherman, G., Rosenberry, T. L., & Sternlicht, H. (1983) J. Biol. Chem. 258, 2148-2156]. In this study we demonstrate that the HRL in bovine brain tubulin is Lys-394, a residue proximal in the alpha-tubulin sequence to the highly negatively charged carboxy-terminus region (residues 412-450) previously implicated in assembly. pH studies were undertaken to probe the local environment of Lys-394. These studies indicated that Lys-394 reactivity toward HCHO is sensitive to the titration of a pKa 6.3 group presumed to be a histidine residue. This assignment is supported by our finding that histidine modification via diethyl pyrocarbonate strongly affects Lys-394 reactivity toward HCHO as well as microtubule assembly. We propose on the basis of secondary structure considerations and published sequence data for a variety of tubulins that Lys-394 is part of an evolutionarily conserved cluster of basic residues (effective charge: 2+ to 2.5+ at neutral pH) composed of Lys-394, His-393, and Arg-390, which is important for tubulin function and which renders Lys-394 reactive as a nucleophile.

Amino Acid Sequence↗
PubMed1980

Chemical probes for higher-order structure in RNA.

Three chemical reactions can probe the secondary and tertiary interactions of RNA molecules in solution. Dimethyl sulfate monitors the N-7 of guanosines and senses tertiary interactions there, diethyl pyrocarbonate detects stacking of adenosines, and an alternate dimethyl sulfate reaction examines the N-3 of cytidines and thus probes base pairing. The reactions work between 0 degrees C and 90 degrees C and at pH 4.5--8.5 in a variety of buffers. As an example we follow the progressive denaturation of yeast tRNAPhe terminally labeled with 32P as the tertiary and secondary structures sequentially melt out. A single autoradiograph of a terminally labeled molecule locates regions of higher-order structure and identifies the bases involved.

Alkylating Agents↗