Comparison of metabolism-mediated binding to DNA of 7-hydroxymethyl-12-methylbenz(a)anthracene and 7, 12-dimethylbenz(a)anthracene.
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Comparison of the binding to DNA of 7-hydroxymethyl-12-methylbenza(a)anthracene and 7, 12-dimethylbenz(a)anthracene (DMBA) catalyzed by mouse embryo cells in culture or by rat liver microsomes indicates that the products formed are different for the two hydrocarbons. Thus, the hydroxy compound is not an intermediate in the binding of DMBA to DNA in these systems. Binding of the hydroxy compound to DNA in mouse embryo cells is less efficient than for DMBA and is inhibited by 1,1,1-trichloropropylene 2,3-oxide, an inhibitor of epoxide hydrase. This and the fluorescence spectra of the hydroxy compound-DNA adducts indicate that the hydroxy compound is activated for DNA binding through the formation of a diol-epoxide in the 1,2,3,4-ring. As previously found for DMBA, this is consistent with the activation of this compound through a bay-region diol-epoxide. (+info)
Intramolecular crosslinking of tropomyosin via disulfide bond formation: evidence for chain register.
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Rabbit skeletal muscle tropomyosin can be crosslinked in the native state by the use of 5,5'-dithiobis(2-nitrobenzoate), which forms disulfide bonds between the two subunits. Using polyacrylamide gel electrophoresis in sodium dodecyl sulfate we have shown that this crosslinking goes to completion over a wide range of protein concentration, ionic strength, and reagent concentration. Crosslinks are not formed in denaturing solvents such as sodium dodecyl sulfate and guanidine hydrochloride despite the fact that the same number of SH groups react as in the native state (2.3 +/- 0.2). The sedimentation coefficients of crosslinked and non-crosslinked samples are identical. Thus, crosslinks are formed between corresponding cysteines on different chains of the same molecule. This provides strong evidence for a model of chain interaction with both chains in register. Evidence has also been obtained that rabbit skeletal tropomyosin is composed only of alphaalpha and alphabeta subunits rather than a random mixture of chains. (+info)
Identification of a novel RNA molecule in a new RNA processing mutant of Escherichia coli which contains 5 S rRNA sequences.
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A temperature-sensitive mutant of Escherichia coli at the nonpermissive temperature fails to produce normal levels of 5 S rRNA. Instead, a number of larger RNA molecules are accumulated. One of these molecules, a 9 S RNA, contains 5 S rRNA sequences. When the strain is shifted from a nonpermissive to a permissive temperature, radioactive label is lost from the 9 S RNA and appears in 5 S rRNA. The identification of this 5 S rRNA-containing molecule indicates the participation of a new processing ribonuclease (RNase E) in the maturation of rRNA in E. coli. The 9 S RNA was not detected in a wild type strain, indicating that the processing step(s) involved in the formation of 5 S rRNA might be performed before the growing rRNA transcript is terminated. (+info)
Chemical reactivities of catalytic and noncatalytic zinc or cobalt atoms of horse liver alcohol dehydrogenase: differentiation by their thermodynamic and kinetic properties.
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Horse liver alcohol dehydrogenase (EC 1.1.1.1) contains one catalytic and one noncatalytic pair of zinc atoms that can be replaced selectively with cobalt and/or 65zince. We have now prepared "hybird" metalloenzymes by specifically replacing one or both pairs of zinc atoms with 65zinc and/or cobalt. Their differential chemical reactivities serve to characterize the metal atoms at either site. The spectral and kinetic properties of the resultant 65zinc, cobalt, and hybrid enzymes, as well as those of their complexes with 1,10-phenanthroline, identify the metal atoms that are at the catalytic sites and differentiate them from those at the noncatalytic sites. All data are in complete agreement with the results of the x-ray crystal structure analysis. Remarkably, under the conditions used, chemical reactivity, as gauged by thermodynamic methods under equilibrium conditions, identifies the catalytic metal atoms as those which are reactive to 1,10-phenanthroline, while this reagnet does not affect the noncatalytic pair. Under dynamic conditions the kinetics of the metal-metal exchange reveals the converse to be true: the chemical reactivity of the noncatalytic atoms is much higher and, hence, they exchange more rapidly. The results are examined in terms of thermodynamic and kinetic properties of metal complex ions which serve as the basis of possible mechanisms underlying these observations. (+info)
Reconstitution of Qbeta RNA replicase from a covalently bonded elongation factor Tu-Ts complex.
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Escherichia coli phage Qbeta RNA replicase, an RNA-dependent RNA polymerase (RNA-dependent RNA nucleotidyltransferase), is a tetramer composed of one phage-coded polypeptide and three host-supplied polypeptides which are known to function in the biosynthesis of proteins in the uninfected host. Two of these polypeptides, protein synthesis elongation factors EF-Tu and EF-Ts, can be covalently crosslinked with dimethyl suberimidate to form a complex which lacks the ability to catalyze the known host functions catalyzed by the individual elongation factors. Using a previously developed reconstitution system we have examined the effects of crosslinking the EF-Tu-Ts complex on reconstituted replicase activity. Renaturation is significantly more efficient when exogenously added native EF-Tu-Ts is crosslinked than when it is not. Crosslinked EF-Tu-Ts can be purified from a crude crosslinked postribosomal supernatant by its ability to replace EF-Tu and EF-Ts in the renaturation of denatured Qbeta replicase. A sample of Qbeta replicase with crosslinked EF-Tu-Ts replacing the individual elongation factors was prepared. Although it lacked EF-Tu and EF-Ts activities, it could initiate transcription of both poly(C) and Qbeta RNA normally and had approximately the same specific activity as control enzyme. Denatured Qbeta replicase formed with crosslinked EF-Tu-Ts was found to renature much more rapidly than untreated enzyme and, in contrast to normal replicase, its renaturation was not inhibited by GDP. The results demonstrate that EF-Tu and EF-Ts function as complex in Qbeta replicase and do not perform their known protein biosynthetic function in the RNA synthetic reaction. (+info)
2-diazo-3,3,3-trifluoropropionyl chloride: reagent for photoaffinity labeling.
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2-Diazo-3,3,3-trifluoropropionyl chloride has been synthesized from trifluorodiazoethane and phosgene. Its derivatives are acid stable, can be used to label enzymes, and undergo photolysis with substantially less rearrangement than do derivatives of other known diazoacyl reagents designed for photoaffinity labeling. In particular, the diazotrifluoropropionyl thioester of methyl N-acetylcysteine undergoes photolysis in methanol with about 40% insertion into the - OH bond of the solvent; by contrast, photolysis of other diazoacyl thioesters gives substantially quantitative Wolff rearrangement. The trifluoro compounds hold promise for the photoaffinity labeling of thiols. (+info)
Elucidation of hydrocarbon structure in an enzyme-catalyzed benzo[a]pyrene-poly (G) covalent complex.
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The carcinogen, benzo[a]pyrene, was covalently attached to poly (G) by liver microsomes from rats pretreated with 3-methylcholanthrene. The complex was hydrolyzed with enzymes or base and products were isolated by Sephadex chromatography. Absorbance and fluorescence spectra of the products fit that of red-shifted pyrene aromatic system and suggest that metabolism has occurred at the 7-, 8-, 9-, and 10-positions of the hydrocarbon. Benzanthracene or chrysene fluorescence were not observed in these preparations. Benzo[a]pyrene derivatives were synthesized and purified by high-pressure liquid chromatography. Dehydration of 7,8-dihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene resulted in the formation of small amounts of 7-oxo-7,8,9,10-tetrahydrobenzoa[a]pyrene. A 7-keto species was also observed after similar treatment of the hydrocarbon-poly(G) hydrolysis products. Evidence of dehydration at the 9,10-positions was not observed. The hydrocarbon covalently bound to poly(G) is, therefore, a derivative of 7,8-dihydroxy-7,8,9,10-tetrahydrobenzol[a]pyrene with nucleic acid substitution at C-10 or 9. (+info)
Soluble dextran-hemoglobin complex as a potential blood substitute.
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A complex between soluble dextran and human hemoglobin has been synthesized by two different methods. In the alkylation method, hemoglobin was allowed to react with bromoacetyl groups incorporated into the dextran; the yield of the complex was about 80% in terms of the hemoglobin used. In the dialdehyde method, hemoglobin was allowed to react with dialdehyde groups on the dextran generated by periodate oxidation; the yield of the complex was about 60%. Both soluble dextran-hemoglobin complexes could bind and release oxygen reversibly, but the oxygen-binding curves were shifted to the left relative to that of free hemoglobin. In the rabbit, the complex obtained by the alkylation method was excreted by the kidneys and cleared from the circulation much more slowly than free hemoglobin. (+info)