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The dodecadeoxynucleotide duplex d(GGTTAATGCGGT).d(ACCGCATTAACC) and its 1:1 complex with the minor groove binding drug SN-6999 have been prepared and studied by two-dimensional 1H nuclear magnetic resonance spectroscopy. Complete sequence-specific assignments have been obtained for the free duplex by standard methods. The line widths of the resonances in the complex are greater than those observed for the free duplex, which complicates the assignment process. Extensive use of two-quantum spectroscopy was required to determine the scalar correlations for identifying all of the base proton and most of the 1'H-2'H-2''H spin subsystems for the complex. This permitted unambiguous sequence-specific resonance assignments for the complex, which provides the necessary background for a detailed comparison of the structure of the duplex, with and without bound drug. A series of intermolecular NOEs between drug and DNA were identified, providing sufficient structural constraints to position the drug in the minor groove of the duplex. However, the combination of NOEs observed can only be rationalized by a model wherein the drug binds in the minor groove of the DNA in both orientations relative to the long helix axis and exchanges rapidly between the two orientations. The drug binds primarily in the segment of five consecutive dA-dT base pairs d(T3T4A5A6T7).d(A18T19T20A21A22), but surprisingly strong interactions are found to extend one residue in the 3' direction along each strand to G8 and C23. The observation of intermolecular contacts to residues neighboring the AT-rich region demonstrates that the stabilization of the bis(quaternary ammonium) heterocycle family of AT-specific, minor groove binding drugs is not based exclusively on interactions with dA-dT base pairs.
A method to obtain uniformly isotopically labeled (15N and 15N/13C) protein from mammalian cells is described. The method involves preparation of isotopically labeled media consisting of amino acids isolated from bacterial and algal extracts supplemented with cysteine and enzymatically synthesized glutamine. The approach is demonstrated by producing 15N-labeled and 15N/13C-labeled urokinase from Sp2/0 cells and successfully growing Chinese hamster ovary (CHO) cells on the labeled media. Thus, using the procedures described, isotopically labeled proteins that have been expressed in mammalian cells can be prepared, allowing them to be studied by heteronuclear multidimensional NMR techniques.
The interaction between fibrinogen gamma-peptide 392-411, LTIGEGQQHHLGGAKQAGDV, and monoclonal antibody 4A5, an antibody with a high affinity for both for the peptide and native fibrinogen, is being studied as a model for peptide-antibody interaction. Two-dimensional NMR studies of the free peptide at pH 5.2 indicated the presence of a significant population, about 60%, of type II beta-turn, spanning residues Gln407-Asp410. At pH 2.7, little, if any, turn structure is present. The D-Ala409 analog, which, for steric reasons, would be expected to preserve the beta-turn, and the L-Ala409 analog, which would not be expected to have this conformational feature, were synthesized, and NMR studies confirmed the respective structural predictions. The affinity of the D-Ala analog for antibody 4A5 is even greater than that displayed by native gamma 392-411, while the affinity of the L-Ala analog is less than one-tenth that of the native peptide. Both conformational and steric effects involving residues 407-410 may be important in recognition by antibody 4A5. Since gamma 392-411 includes a platelet receptor binding locus of fibrinogen, and this and related peptides are inhibitors of platelet aggregation, the D-Ala409 and L-Ala409 analogs were tested for platelet binding. Neither of the analogs displays any measurable platelet binding, indicating that the recognition requirements for the platelet receptor differ considerably from those for antibody 4A5.
2-Ethynylnaphthalene (2EN) had previously been demonstrated to be a mechanism-based inactivator of rat cytochrome P450 (P450) 1A2 [Hammons, G.J., Alworth, W.L., Hopkins, N.E., Guengerich, F. P., & Kadlubar, F. F. (1989) Chem. Res. Toxicol. 2, 367-374]. In this work 2EN was also demonstrated to be a useful inactivator of rabbit P450 1A2 (k(inactivation) 0.094 min-1, K(i) 11 microM) but it did not inactivate human P450 1A2, although the sequences of the three proteins are approximately 80% identical. Rat and rabbit P450 1A2 were modified by incubation with NADPH-P450 reductase, NADPH, and [3H]2EN to levels of 0.35 and 0.47 nmol of adduct (nmol of P450)-1, respectively. In each case only a single tryptic peptide was labeled; recovery of labeled peptides was low under the acidic HPLC conditions. The rabbit P450 1A2 peptide FQELMAAVGR (positions 175-184) and the rat P450 1A2 peptide L(S)QQYGDVLQIR (positions 67-78) were identified. 4-Azidobiphenyl (4-N3BP) was developed as a photoaffinity label for P-450 1A2 proteins because of its similarity to 4-aminobiphenyl, a known substrate for the enzymes. 4-N3BP was shown to be photolyzed with 350-nm light and radioactive label could be incorporated into rat P450 1A2. Labeling of the protein was found to be saturable with increasing concentrations of 4-N3BP and up to 0.59 nmol of label could be incorporated (nmol P450 1A2)-1. The substrate 4-aminobiphenyl and the competitive inhibitor 7,8-benzoflavone blocked photolabeling of P450 1A2 with 4-N3BP, and 4-N3BP inhibited N-hydroxylation of 4-aminobiphenyl by P450 1A2 in the usual enzyme assay.(ABSTRACT TRUNCATED AT 250 WORDS)
Treatment of prostaglandin endoperoxide (PGH) synthase apoprotein with a 100- or 1000-fold excess of N-acetylimidazole (NAI) led to time-dependent inactivation of both cyclooxygenase and peroxide activities. Reconstitution of apoprotein with heme prior to incubation with NAI substantially protected the enzyme from inactivation. Pretreatment of the protein with either acetylsalicylic acid (aspirin) or (+/-)-2-fluoro-alpha-methyl-4-biphenylacetic acid (flurbiprofen), which inhibit cyclooxygenase activity, did not alter the time course of peroxidase inactivation by NAI. Treatment of NAI-inactivated apoPGH synthase with hydroxylamine led to substantial regeneration of both cyclooxygenase and peroxidase activities. Quantitation of radioactivity following incubation of PGH synthase with [3H-acetyl]NAI indicated incorporation of 1.7 +/- 0.9 acetyl groups/70-kDa subunit. Cleavage of acetylated protein with trypsin under nondenaturing conditions followed by high-performance liquid chromatography analysis demonstrated that most of the radioactivity was incorporated into the 33-kDa fragment although significant radioactivity was also detectable in the 38-kDa fragment. Chymotryptic peptide mapping of acetylated protein revealed numerous potential sites of acetylation distributed in widely divergent regions of the protein. No apparent differences were observed between the chymotryptic maps of apo- and holoenzyme, suggesting that the adduct responsible for loss of catalytic activity is unstable to the chromatographic conditions. The different biochemical properties of PGH synthase acetylated by NAI or aspirin suggest that a major determinant of the specificity of aspirin for Ser530 is binding of the salicylate moiety to this region of the PGH synthase protein.
To study the effect of chelation of iron ions by quinones on the generation of OH radicals in biological redox systems, we have synthesized quinones that can form complexes with Fe(III) ions: 2-phenyl-4-(butylamino)naphtho[2,3-h]quinoline-7,12-dione (Qbc) and 2-phenyl-4-(octylamino)naphtho[2,3-h]quinoline-7,12-dione (Qoc). A quinone with a similar structure without chelating group was synthesized as a control sample: 2-phenyl-5-nitronaphtho[2,3-g]indole-6,11-dione (Qn). Using optical spectroscopy, we determined the stability constant of Qbc with Fe(III) [Ks = (7 +/- 1) x 10(18) M-3] and the stoichiometry of the complex Fe(Qbc)3 in chloroform solutions. One-electron reduction potentials of Qbc, Qn, and adriamycin in dimethyl sulfoxide were measured by cyclic voltammetry. In the presence of Fe(III) the one-electron reduction potentials shifted toward positive values by 0.16 and 0.1 V for Qbc and adriamycin, respectively. Using the spin trap 5,5'-dimethyl-1-pyroline N-oxide (DMPO) and EPR, it was found that Qbc in the Fe(III) complex stimulated the formation of OH radicals in the enzymatic system consisting of NADPH and NADPH-cytochrome P-450 reductase more efficiently than adriamycin and quinone Qn. This is indicated by the absence of a lag period in the spin adduct appearance for Qbc and by a significantly higher rate of the spin adduct production, as well as by a larger absolute concentration of the spin adduct obtained for Qbc in comparison with Qn in the presence of Fe(III).(ABSTRACT TRUNCATED AT 250 WORDS)
The bovine adrenodoxin gene gives rise to two species of mRNA differing only at their 5'-ends. The synthesis of these two types of mRNA in bovine adrenal cortical cells is regulated transcriptionally in part by ACTH via the action of cAMP. Examination of the 5'-end of the adrenodoxin gene revealed that each mRNA contains sequences derived from a different exon encoding the mitochondrial leader sequence. To define the sequences necessary for the synthesis of these two types of mRNA and to determine if the synthesis of each is driven by a separate promoter, 5'-regions of the adrenodoxin gene were inserted upstream from two different reporter genes and tested for promoter/enhancer regulatory activity by transient transfection into mouse adrenocortical Y1 tumor cells. The results clearly demonstrated that the bovine adrenodoxin gene contains two functional promoters; one, ADXP1, located in the 5'-flanking region gives rise to the minor form of mRNA, and a second, stronger promoter, ADXP2, which maps within intron 1 gives rise to the major form of mRNA. Unique cAMP-responsive sequences were found upstream from each promoter which share no sequence homology to the consensus CRE (cAMP-responsive element). Upon transient expression, the cAMP-responsive sequence associated with the ADXP2 promoter, termed CRS2, confers the cAMP responsiveness to stimulate the transcription of the linked beta-globin reporter gene regardless of whether the adrenodoxin ADXP2 promoter or the beta-globin promoter was utilized.(ABSTRACT TRUNCATED AT 250 WORDS)
The class III DNA dependent RNA polymerases (nucleoside triphosphate:RNA nucleotidyltransferase EC 18.104.22.168 from HeLa cells have been solubilized and characterized as to function and properties. Two chromatographically distinct forms of enzyme III, designated polymerases IIIA and IIIB, can be resolved when cell extracts are chromatographed on DEAE-Sephadex columns. Enzymes IIIA and IIIB exhibit nearly identical catalytic properties such as divalent cation stimulation, broad biphasic ammonium sulfate optima, and characteristic alpha-amanitin sensitivities which clearly distinguish them from the homologous enzymes, forms I and II. Polymerases IIIA and IIIB are both primarily localized in the nucleus (greater than 60%). The most notable characteristic of the class III enzymes is a unique sensitivity to inhibition by alpha-amanitin (50% inhibition at 15 mug/ml). HeLa cell enzyme I is not inhibited by the mushroom toxin even at very high concentrations (greater than 400 mug/ml), while HeLa cell polymerase II is inhibited by very low concentrations of amanitin (50% inhibition at 0.003 mug/ml). The three major classes of enzyme (I, II, III) exhibit characteristic sensitivities to alpha-amanitin whether assayed in nuclei, crude homogenates, or in a chromatographically purified state. Using a nuclear in vitro RNA synthesizing system to investigate the alpha-amanitin sensitivities of the synthesis of tRNA precursor (4.5S pre-tRNA) and 5S ribosomal RNA, it was found that the synthesis of these RNA species was inhibited 50% at 15 mug/ml of alpha-amanitin. The alpha-amanitin inhibition curves for the synthesis of pre-tRNA-5S ribosomal RNA in nuclei and the alpha-amanitin titration curves for the partially purified class III enzymes (IIIA and IIIB) are identical. These data, therefore, show that the in vivo functional role of the class III RNA polymerases (IIIA-IIIB) is the transcription of the genes coding for transfer RNA and 5S ribosomal RNA.
DNA-dependent RNA polymerase III (nucleosidetriphosphate: RNA nucleotidyltransferase, EC 2.7.-7.6) has been isolated and partially purified from calf thymus tissue. Significant amounts of enzyme III are present in this tissue (up to 15% of the total activity of thymus homogenates). This enzyme has been characterized with respect to its chromatographic properties, broad ammonium sulfate optimum (0.04-0.2 M), template requirements, divalent metal optima, and its unique alpha-amanitin sensitivity (50% inhibition of activity occurring at an alpha-amanitin concentration of 10 mug/ml).
The structure of rabbit transferrin was investigated with regard to number, size, and composition of the heteropolysaccharide units and their relative location on the polypeptide chain. The composition and molecular weight of the Pronase glycopeptides revealed that rabbit transferrin contains two heteropolysaccharide units, each composed of 2 sialic acid residues, 2 galactose residues, 3 mannose residues, and 4-N-acetylglucosamine residues. The composition and molecular weight of the tryptic glycopeptides further substantiated the existence of two identical heteropolysaccharide units and revealed that both units have identical amino acid residues in the immediate vicinity of the carbohydrate attachment sites to the polypeptide chain, suggesting a sequence homology surrounding the two glycosylation sites. Characterization of the cyanogen bromide fragments from rabbit transferrin indicated that both heteropolysaccharide units are located within a single polypeptide fragment representing approximately one-third of the molecule.