Differences in unwinding of supercoiled DNA induced by the two enantiomers of anti-benzo[a]pyrene diol epoxide.

The unwinding of supercoiled phi X174 RFI DNA induced by the tumorigenic (+) and non-tumorigenic (-) enantiomers of trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) has been investigated by agarose slab-gel and ethidium titration tube gel electrophoresis. The differences in adduct conformations were verified by flow linear dichroism techniques. Both enantiomers cause a reversible unwinding by the formation of noncovalent intercalative complexes. The effects of covalently bound BPDE residues on the electrophoretic mobilities of the RF I DNA form in agarose gels were investigated in detail in the range of binding ratios rb approximately 0.0-0.06 (covalently bound BPDE residues/nucleotide). In this range of rb values, there is a striking difference in the mobilities of (+)-BPDE- and (-)-BPDE-adducted phi X174 DNA in agarose slab-gels, the covalently bound (+)-BPDE residues causing a significantly greater retardation than (-)-BPDE residues. Increasing the level of covalent adducts beyond rb approximately 0.06 in the case of the (+)-BPDE enantiomer, leads to further unwinding and a minimum in the mobilities (corresponding to comigration of the nicked form and the covalently closed relaxed modified form) at rb 0.10 +/- 0.01; at still higher rb values, rewinding of the modified DNA in the opposite sense is observed. From the minimum in the mobility, a mean unwinding angle (per BPDE residue) of theta = 12 +/- 1.5 degrees is determined, which is in good agreement the value of theta = 11 +/- 1.8 degrees obtained by the tube gel titration method. Using this latter method, values of theta = 6.8 +/- 1.7 degrees for (-)-BPDE-phi X174 adducts are observed. It is concluded that agarose slab gel techniques are not suitable for determining unwinding angles for (-)-BPDE-modified phi X174 DNA because the alterations in the tertiary structures for rb < 0.06 are too small to cause sufficiently large changes in the electrophoretic mobilities. The major trans (+)-BPDE-N2-guanosine covalent adduct is situated at external binding sites and the mechanisms of unwinding are therefore different from those relevant to noncovalent intercalative BPDE-DNA complexes or to classical intercalating drug molecules; a flexible hinge joint and a widening of the minor groove at the site of the lesion may account for the observed unwinding effects. The more heterogeneous (-)-BPDE-nucleoside adducts (involving cis and trans N2-guanosine, and adenosine adducts) are less effective in causing unwinding of supercoiled DNA for reasons which remain to be elucidated.     

Extent of helix perturbation associated with DNA modification by the o-acetyl derivative of the carcinogen 4-hydroxyaminoquinoline-1-oxide

Duplex unwinding associated with DNA modification by 4-acetoxyaminoquinoline-1-oxide, a model ultimate carcinogen of 4-nitroquinoline-1-oxide, has been determined by the agarose gel electrophoresis band-shift method. An average unwinding angle per stable adduct of -15.1 degrees +/- 1.5 degrees for negatively supercoiled topoisomers and -6.5 degrees +/- 1.4 degrees for positively supercoiled topoisomers was obtained. Because of the different proportion of stable adducts (dGuo-N2-AQO, dGuo-C8-AQO, dAdo-N6-AQO) between negatively (8:1.5:0.5) and positively (5:2.5:1) supercoiled topoisomers, the difference in unwinding angles is suggestive of a diverse contribution of the various adducts to the overall conformational change. Since the largest unwinding angle was coupled with the highest proportion of dGuo-N2-AQO adduct, it is likely that this adduct is the most distortive lesion. A contribution of sites of base loss to DNA unwinding was also observed.     

Synthesis and DNA nicking studies of a novel cyclic peptide: cyclo[Lys-Trp-Lys-Ahx-].

Two novel cyclic tetrapeptides: cyclo[Lys-Tyr-Lys-Ahx-] 7a and cyclo[Lys-Trp-Lys-Ahx-] 7b were synthesized by coupling protected amino acid in solution and the subsequent cyclization effected by the pentafluorophenyl ester method as described in previous papers. These cyclic peptides were designed and synthesized to study their interaction with DNA, based on previous reports that linear peptides Lys-Tyr-Lys and Lys-Trp-Lys could bind to various forms of DNA and cleaved supercoiled DNA at apurinic sites. Ethidium bromide displacement assay showed that the apparent DNA binding constant of linear Lys-Tyr-Lys and cyclic peptide 7a are far below 1 x 10(3) M(-1), whereas those of cyclic peptide 7b and linear Lys-Trp-Lys are 1.9 x 10(4) M(-1) and 9.5 x 10(3) M(-1), respectively. Kinetic studies using agarose gel electrophoresis showed that cyclic peptide 7b and Lys-Trp-Lys possessed DNA nicking activity on natural supercoiled phi X174 DNA with nicking rate of 50.7 and 75.6 pM min(-1) at 65 degrees C, respectively, whereas cyclic peptide 7a and linear Lys-Tyr-Lys were devoid of the corresponding activity. The DNA nicking rate increased significantly with increase in reaction temperature. At reaction temperatures lower than 65 degrees C, the DNA nicking rate of cyclic peptide 7b exceeded that of linear Lys-Trp-Lys. The addition of 1 microM ferrous ion did not give significant enhancement effect on the DNA nicking rate by the peptides. UV irradiation gave a marked rate enhancement on the DNA nicking rate of linear Lys-Trp-Lys and a moderate enhancement on the DNA nicking rate of cyclic peptide 7b.     

Interactions of norharman and harman with DNA.

The interactions of norharman (9H-pyrido [3,4-b] indole) and harman (1-methyl-9H-pyrido [3,4-b] indole) with DNA were studied. DNA caused remarkable fluorescence quenching and change in the absorption spectra of the dyes. Scatchard plots obtained by optical titration gave Kd values of 2.2 X 10(-5)M and 7.7 X 10(-6)M, and apparent numbers of binding sites of 0.13/base and 0.12/base for norharman and harman, respectively. Agarose gel electrophoresis of circular DNA, closed in the presence or absence of norharman revealed that the dye intercalates DNA, thereby causing 17 +/- 3 degrees unwinding of the double helix.     

Effects of temperature and ATP on the kinetic mechanism and kinetic step-size for E.coli RecBCD helicase-catalyzed DNA unwinding

The kinetic mechanism by which Escherichia coli RecBCD helicase unwinds duplex DNA was studied using a fluorescence stopped-flow method. Single turnover DNA unwinding experiments were performed using a series of fluorescently labeled DNA substrates containing duplex DNA regions ranging from 24 bp to 60 bp. All or no DNA unwinding time courses were obtained by monitoring the changes in fluorescence resonance energy transfer between a Cy3 donor and Cy5 acceptor fluorescent pair placed on opposite sides of a nick in the duplex DNA. From these experiments one can determine the average rates of DNA unwinding as well as a kinetic step-size, defined as the average number of base-pairs unwound between two successive rate-limiting steps repeated during DNA unwinding. In order to probe how the kinetic step-size might relate to a mechanical step-size, we performed single turnover experiments as a function of [ATP] and temperature. The apparent unwinding rate constant, kUapp, decreases with decreasing [ATP], exhibiting a hyperbolic dependence on [ATP] (K1/2=176(+/-30) microM) and a maximum rate of kUapp=204(+/-4) steps s(-1) (mkUapp=709(+/-14) bp s(-1)) (10 mM MgCl2, 30 mM NaCl (pH 7.0), 5% (v/v) glycerol, 25.0 degrees C). kUapp also increases with increasing temperature (10-25 degrees C), with Ea=19(+/-1) kcal mol(-1). However, the average kinetic step-size, m=3.9(+/-0.5) bp step(-1), remains independent of [ATP] and temperature. This indicates that even though the values of the rate constants change, the same elementary kinetic step in the unwinding cycle remains rate-limiting over this range of conditions and this kinetic step remains coupled to ATP binding. The implications of the constancy of the measured kinetic step-size for the mechanism of RecBCD-catalyzed DNA unwinding are discussed.     

Protein-induced unwinding of DNA: measurement by gel electrophoresis of complexes with DNA minicircles. Application to restriction endonuclease EcoRI, catabolite gene activator protein and lac repressor.

An electrophoretic procedure for the measurement of the helix unwinding induced by a sequence-specific protein is described. The method, which was applied here to EcoR I, CAP and lac repressor, involved the migration of the complexes with positively and negatively supercoiled DNA minicircles carrying a single protein binding site. Mobility shifts of complexes relative to naked DNAs appeared to be a result of i) the unwinding; of ii) an increase in the molecular frictional coefficient, which led to a retardation; of iii) bending, in the particular case of CAP, which induced an acceleration; and of iv) looping, in the case of lac repressor, which also resulted in an acceleration. Under conditions where the migration of the naked topoisomers was V-like (topoisomer mobility showed the same linear increase with both negative and positive supercoilings; Zivanovic et al. (1986) J. Mol. Biol., 192, 645-660), the protein unwinding contribution to mobility was assumed to be identical to that experimentally observed in the case of a thermal unwinding: all negatively supercoiled topoisomers were retarded and all positively supercoiled topoisomers were accelerated to the same extent. In contrast, the mobility contribution of the frictional term, as well as those of bending and looping, appeared to vary strongly with the magnitude of the supercoiling, but only weakly with its polarity. As a consequence, these latter contributions may approximately cancel when one is measuring the difference between the shifts observed for two comigrating, negatively and positively supercoiled, topoisomers, allowing the unwinding to be calculated. While estimates obtained for EcoR I, 23 +/- 3 degrees, and CAP, about 29 degrees, were in good agreement with previous measurements using topoisomerase I, the value found for lac repressor, 13 to 16 degrees, was significantly smaller.     

Resistance to alloxan of tumoral insulin-producing cells

Rat pancreatic islets and insulin-producing cells of the RINm5F line were incubated for 5 min at 7 or 23 degrees C in media containing 3H2O and either L-[1-14C]glucose or [2-14C]alloxan. In the islets the intracellular distribution space of [2-14C]alloxan represented, at 7 and 23 degrees C respectively, 11.4 +/- 1.0 and 25.5 +/- 2.3% of the intracellular 3H2O space. In the RINm5F cells, the distribution space of [2-14C]alloxan failed to be affected by the ambient temperature and represented, after correction for extracellular contamination, no more than 5.2 +/- 0.5% of the intracellular 3H2O space. Preincubation for 30 min at 7 degrees C in the presence of alloxan (10 mM) failed to affect subsequent D-[U-14C]glucose oxidation in the tumoral cells, whilst causing a 70% inhibition of glucose oxidation in the islets. It is proposed that RINm5F cells are resistant to the cytotoxic action of alloxan, this being attributable, in part at least, to poor uptake of the diabetogenic agent.     

A study of the interactions of some polypyridylruthenium (II) complexes with DNA using fluorescence spectroscopy, topoisomerisation and thermal denaturation.

The nature of binding of Ru(phen) 2+ (I), Ru(bipy) 2+ (II), Ru(terpy) 2+ (III) (phen = 1,10-phenanthroline, bipy 3 = 2,2'-bipyridyl, 3 terpy = 2,2'2," - 2 terpyridyl) to DNA, poly[d(G-C)] and poly[d(A-T)] has been compared by absorption, fluorescence, DNA melting and DNA unwinding techniques. I binds intercalatively to DNA in low ionic strength solutions. Topoisomerisation shows that it unwinds DNA by 22 degrees +/- 1 per residue and that it thermally stabilizes poly[d(A-T)] in a manner closely resembling ethidium. Poly[d(A-T)] induces greater spectral changes on I than poly[d(G-C)] and a preference for A-T rich regions is indicated. I binding is very sensitive to Mg2+ concentration. In contrast to I the binding of II and III appears to be mainly electrostatic in nature, and causes no unwinding. There is no evidence for the binding of the neutral Ru(phen)2 (CN)2 or Ru(bipy)2 (CN)2 complexes. DNA is cleaved, upon visible irradiation of aerated solutions, in the presence of either I or II.     

Characterization of the interaction of the radioprotector 1-methyl-2-[2-(methylthio)-2-piperidinovinyl]quinolinium iodide with supercoiled DNA

The interaction of the radioprotector 1-methyl-2-[2-(methylthio)-2-piperidinovinyl]quinolinium iodide (VQ) with linear and supercoiled pIBI30 DNA was studied by flow linear dichroism spectroscopy, equilibrium dialysis, circular dichroism, and UV absorption spectroscopy. The negative linear dichroism spectra of VQ-DNA complexes throughout the 220-500 nm wavelength region, a red shift in the VQ main absorption band (at 452 nm) of 1-2 nm upon binding to DNA, and a concentration-dependent unwinding of supercoiled DNA suggest that the primary mode of interaction of VQ with DNA (at least at low concentrations) is intercalative in nature. A least-squares analysis of the equilibrium dialysis binding of VQ to supercoiled DNA using the McGhee-von Hippel equation gives an association constant K = 7300 +/- 300 M-1, and an exclusion number n in the range of 3.3-5.3. The lower value of n is obtained when effects of polyelectrolytes are also taken into account. Because quinolinium iodide derivatives with different substituents and DNA binding affinities can be synthesized, this family of compounds could be employed to probe relationships, if any, between radioprotective efficacy and DNA binding affinity.     

Molecular recognition between oligopeptides and nucleic acids. Sequence specific binding of (4S)-(+)- and (4R)-(-)-dihydrokikumycin B to DNA deduced from 1H NMR, footprinting studies and thermodynamic data

The sequence specific binding of the antibiotic (4S)-(+)-dihydrokikumycin B and its (4R)-(-) enantiomer, [(S)-1 and (R)-1, respectively] to DNA were characterized by DNase I and MPE footprinting, calorimetry, UV spectroscopy, circular dichroism, and 1H NMR studies. Footprinting analyses showed that both enantiomers [(S)-1 and (R)-1] bind to AT-rich regions of DNA. 1H NMR studies (ligand induced chemical shift changes and NOE differences) of the dihydrkikumycins with d-[CGCAATTGCG]2 show unambiguously that the N to C termini of the ligands are bound to 5'-A5T6T7-3' reading from left to right. From quantitative 1D-NOE studies, the AH2(5)-ligand H7 distance of complex A [(S)-1 plus decamer (which is bound more strongly)] and complex B [(R)-1 and decamer] are estimated to be 3.8 +/- 0.3 A and 4.9 +/- 0.4 A, respectively. This difference in binding properties is reflected in the thermodynamic profiles of the two enantiomeric ligands determined by a combination of spectroscopic and calorimetric techniques. The binding free energies (delta G degrees) of (S)-1 and (R)-1 to poly d(AT).poly d(AT) at 25 degrees C are -31.8 and -29.3 kJ mol-1, respectively while the corresponding binding enthalpies (delta H degrees) are -11.3 and -0.8 kJ mol-1. These data permit the construction of models for the binding of the enantiomeric dihydrokikumycins to DNA and account for the more efficient binding of the natural (S) isomer to DNA.     

Structures, spectra, and DNA-binding properties of mixed ligand copper(II) complexes of iminodiacetic acid: the novel role of diimine co-ligands on DNA conformation and hydrolytic and oxidative double strand DNA cleavage

The coordination geometry around copper(II) in [Cu(imda)(phen)(H2O)] (1) (H2imda = iminodiacetic acid, phen = 1,10-phenanthroline) is described as distorted octahedral while those in [Cu(imda)(5,6-dmp)] (2) (5,6-dmp = 5,6-dimethyl-1,10-phenanthroline) and [Cu(imda)(dpq)] (3) (dpq = dipyrido-[3,2-d:2',3'-f]-quinoxaline) as trigonal bipyramidal distorted square-based pyramidal with the imda anion facially coordinated to copper(II). Absorption spectral (Kb: 1, 0.60+/-0.04x10(3); 2, 3.9+/-0.3x10(3); 3, 1.7+/-0.5x10(4) M(-1)) and thermal denaturation studies (deltaTm: 1, 5.70+/-0.05; 2, 5.5+/-10; 3, 10.6+/-10 degrees C) and viscosity measurements indicate that 3 interacts with calf thymus DNA more strongly than 1 and 2. The relative viscosities of DNA bound to 1 and 3 increase while that of DNA bound to 2 decreases indicating formation of kinks or bends and/or conversion of B to A conformation as revealed by the decrease in intensity of the helicity band in the circular dichroism spectrum of DNA. While 1 and 3 are bound to DNA through partial intercalation, respectively, of phen ring and the extended planar ring of dpq with DNA base stack, the complex 2 is involved in groove binding. All the complexes show cleavage of pBR322 supercoiled DNA in the presence of ascorbic acid with the cleavage efficiency varying in the order 3 > 1 > 2. The highest oxidative DNA cleavage of dpq complex is ascribed to its highest Cu(II)/Cu(I) redox potential. Oxidative cleavage studies using distamycin reveal minor groove binding for the dpq complex but a major groove binding for the phen and 5,6-dmp complexes. Also, all the complexes show hydrolytic DNA cleavage activity in the absence of light or a reducing agent with cleavage efficiency varying in the order 1 > 3 > 2.     

Identification of the persistently bound form of the carcinogen N-acetyl-2-aminofluorene to rat liver DNA in vivo

In this article the structural analysis of the persistently bound form of the carcinogen N-acetyl-2-aminofluorene (AAF) to rat liver DNA in vivo is described. This compound appears to result from the formation of a covalent bond between carbon-3 of the aromatic ring and the amino group of guanine. Experimental evidence from three different approaches has led to the identification of the structure of the persistently DNA-bound AAF moity. First, [3-³H, 9-¹⁴C]N-acetoxy-AAF was reacted with DNA in vitro. As reported previously, a minor product was isolated from enzymatic digests of the reacted DNA, which had chemical and chromatographic properties identical to those of the persistent—AAF moiety in DNA in vivo. The ration ³H/¹⁴C of this product had diminished to the same extent as 3-CH₃S-AAF resulting from the reaction of methionine with [3-³H, 9-¹⁴C]N-acetoxy-AAF.Secondly, reaction of [9-¹⁴C]N-acetoxy-AAF with DNA, which was tritiated in the C-8 positions of the purines, did not result in removal of tritium in the persistent fraction obtained after acid hydrolysis, thus excluding substitution at C-8 and N-7 of guanine. Finally, by reacting N-OSO₃-K-AAF with deoxyguanosine in dimethylsulfoxide-triethylamine, a compound could be isolated, which was identified as 3-(deoxyguanosin-N²-yl)-AAF based on its NMR spectrum and on the mass spectrum of the corresponding guanine derivative obtained after removing deoxyribose by acid hydrolysis. This compound appeared to be identical with the persistently bound form present in DNA hydrolysates from rat liver after injection of [2′-³H]N-hydroxy-AAF.     

High mobility group protein 1 preferentially conserves torsion in negatively supercoiled DNA

HMG 1 is known to bind to a variety of DNAs and to unwind nicked and closed circular DNA. We now report evidence that it has a significantly higher unwinding angle on negatively supercoiled DNA than on the other torsional forms. The degree of unwinding observed on nicked circular DNA depends on the purity of the HMG 1 preparation used. HMG 1 from CM-Sephadex has an unwinding angle of 28.8 degrees, compared to 7.2 degrees for the purer preparation obtained from Mono S, suggesting that contaminating strand-separating activity is removed by the additional purification step. The subsequent studies on closed circular forms of DNA were all performed using the purer HMG 1. After preincubation of highly negatively supercoiled DNA (sigma = -0.040) with HMG 1, the DNA-protein mixture was relaxed with Escherichia coli topoisomerase I. At molar ratios of less than 100:1 (HMG 1 to DNA), negatively supercoiled DNA displays a dose-dependent change in the linking number, indicating an unwinding angle of 57.6 degrees. HMG 1 protects 50% of highly negatively supercoiled DNA from E. coli topoisomerase I at a molar ratio of 100:1, and protects all supercoils at a molar ratio of 200:1, indicating saturation of the DNA at this concentration. HMG 1 also protects highly negatively supercoiled DNA from calf thymus topoisomerase I, with an apparent unwinding angle of 57.6 degrees. Moderately negatively supercoiled DNA (sigma = -0.018), but not moderately positively supercoiled DNA (sigma = +0.011), competes for the protective effect of HMG 1 on highly negatively supercoiled DNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of pleural pressure and abdominal pressure on diaphragmatic blood flow

Alveolar transfer of prostaglandin E2 (PGE2) was characterized in isolated perfused guinea pig lungs (n = 19) by measuring radioactivity appearing in the venous effluent during 30 min after intratracheal instillation of [3H]PGE2, [14C]-mannitol, and [125I]iodoantipyrine. Recovery of lipid-soluble [125I]iodoantipyrine [91 +/- 3% (SE)] after 30 min was used to estimate total 3H and 14C delivered to the exchanging region of lung at time 0. In seven control lungs, 58 +/- 4% of [14C]mannitol and 16 +/- 4% of [3H]PGE2 was retained 10 min after instillation. Neither perfusion with diphloretin phosphate (10 micrograms/ml; n = 4) nor hypothermia (5 degrees C; n = 5) significantly affected the amount of [14C]mannitol retained; however, [3H]PGE2 remaining in these lungs increased significantly to 36 +/- 4 and 53 +/- 2%, respectively. Addition of unlabeled PGE2 (200 micrograms) to the instilled solution (n = 3) increased retention of both [14C]mannitol (80 +/- 3%) and [3H]PGE2 (65 +/- 4%). Alveolar transfer of [3H]PGE2 was calculated as the difference in percent retention of [14C]mannitol and [3H]PGE2 and normalized to that of [14C]mannitol. After 10 min, alveolar transfer of [3H]PGE2 was 71 +/- 8% in control lungs but was decreased to 26 +/- 7, 10 +/- 5, and 19 +/- 6% by diphloretin phosphate, hypothermia, or unlabeled PGE2, respectively. These data suggest that alveolar clearance of PGE2 involves a saturable drug- and temperature-sensitive process.     

Unwinding of supercoiled DNA by cis- and trans-diamminedichloroplatinum(II): influence of the torsional strain on DNA unwinding.

The effective unwinding angle, phi, for cis-diamminedichloroplatinum(II) (cis-DDP) and trans-DDP was determined by utilizing high resolution gel electrophoresis and supercoiled phi X174 RF DNA as a substrate. The effective unwinding angle was calculated by equating the reduction in mobility of the DDP-modified DNA to the removal of a number of superhelical turns. The value of the effective unwinding angle for both DDP isomers was greatest at the low levels of DDP bound and decreased with increasing amounts of unwinding agent. The cis-isomer is a better unwinding agent than is the trans-isomer, being nearly twice as effective in unwinding the supercoiled DNA at the DDP levels investigated. A comparison of the magnitude of phi below rb values of 0.005 and those at high levels of binding reveals that the extent of torsional strain in the supercoiled DNA influences the magnitude of the unwinding of the DNA by these complexes. When this method is used in the analysis of the unwinding angle for a covalently bound species on supercoiled DNA, it may provide a more reliable estimate of the magnitude of phi at high degrees of supercoiling and at low levels of modification.     

Temperature and Ca2+ dependence of [3H]ryanodine binding in the burbot (Lota lota L.) heart

Opening and closing of the cardiac ryanodine (Ry) receptor (RyR) are coordinated by the free intracellular Ca2+ concentration, thus making the Ca2+ binding properties of the RyR important for excitation-contraction coupling. Unlike mammalian cardiac RyRs, which lose their normal function at low temperatures, RyRs of ectothermic vertebrates remain operative at 2-4 degrees C, as indicated by Ry sensitivity of contractile force. To investigate the mechanisms of low temperature adaptation of ectothermic RyRs, we compared Ca2+-dependent kinetics of [3H]ryanodine binding in cardiac preparations of a fish (burbot, Lota lota) and a mammal (rat). The number of ventricular [3H]ryanodine binding sites determined at 20 degrees C was 1.54 times higher in rat than burbot heart (0.401 +/- 0.039 and 0.264 +/- 0.019 pmol/mg protein, respectively) (P < 0.02), while the binding affinity (Kd) for [3H]ryanodine was similar (3.38 +/- 0.63 and 4.38 +/- 1.14 nM for rat and burbot, respectively) (P = 0.47). The high-affinity [3H]ryanodine binding to burbot and rat cardiac preparations was tightly coordinated by the free Ca2+ concentration at both 20 degrees C and 2 degrees C and did not differ between the two species. Half-maximal [3H]ryanodine binding occurred at 0.191 +/- 0.027 microM and 0.164 +/- 0.034 microM Ca2+ for rat and at 0.212 +/- 0.035 microM and 0.188 +/- 0.039 microM Ca2+ for burbot (P = 0.65), at 2 degrees C and 20 degrees C, respectively. In two other fish species, rainbow trout (Oncorhynchus mykiss) and crucian carp (Carassius carassius), the Ca2+-binding affinity at 20 degrees C was 4.4 and 5.9 times lower, respectively, than in the burbot. At 20 degrees C, the rate of [3H]ryanodine binding to the high-affinity binding site was similar in rat and burbot but was drastically slowed in rat at 2 degrees C. At 2 degrees C, [3H]ryanodine failed to dissociate from rat cardiac RyRs, and at 10 degrees C and 20 degrees C, the rate of dissociation was two to three times slower in rat than burbot preparations. The latter finding is compatible with a channel gating mechanism, where the closing of the Ca2+ release channel is impaired or severely retarded by low temperature in rat but less so in burbot preparations. The stronger effect of low temperature on association and dissociation rate of [3H]ryanodine binding in rat compared with burbot suggests that RyRs of the ectothermic fish, unlike those of endothermic rat, are better able to open and close at low temperatures.     

3-Hydroxy-3-methylglutaryl coenzyme A synthase. Evidence for an acetyl-S-enzyme intermediate and identification of a cysteinyl sulfhydryl as the site of acetylation.

Homogeneous liver 3-hydroxy-3-methylglutaryl coenzyme A synthase, which catalyzes the condensation of acetyl-CoA with acetoacetyl-CoA to form 3-hydroxy-3-methylglutaryl-CoA, also carries out: (a) a rapid transacetylation from acetyl-CoA to 31-dephospho-CoA and (b) a slow hydrolysis of acetyl-CoA to acetate and CoA. Transacetylation and hydrolysis occur at 50 and 1 percent, respectively, the rate of the synthasecatalyzed condensation reaction. It appears that an acetyl-enzyme intermediate is involved in the transacetylase and hydrolase reactions of 3-hydroxy-3-methylglutaryl-CoA synthase, as well as in the over-all condensation process. Covalent binding to the enzyme of a [14C]acetyl group contributed by [1(-14)C]acetyl-CoA is indicated by migration of the [14C]acetyl group with the dissociated synthase upon electrophoresis in dodecyl sulfate-urea and by precipitation of [14C]acetyl-enzyme with trichloroacetic acid. At 0 degrees and a saturating level of acetyl-CoA, the synthase is rapidly (less than 20 s) acetylated yielding 0.6 acetyl group/enzyme dimer. Performic acid oxidation completely deacetylates the enzyme, suggesting the site of acetylation to be a cysteinyl sulfhydryl group. Proteolytic digestion of [14C]acetyl-S-enzyme under conditions favorable for intramolecular S to N acetyl group transfer quantitatively liberates a labeled derivative with a [14C]acetyl group stable to performic acid oxidation. The labeled oxidation product is identified as N-[14C]acetylcysteic acid, thus demonstrating a cysteinyl sulfhydryl group as the original site of acetylation. The ability of the acetylated enzyme, upon addition of acetoacetyl-CoA, to form 3-hydroxy-3-methylglutaryl-CoA indicates that the acetylated cysteine residue is at the catalytic site.     

Comparative studies on the 7-iodo and 7-fluoro derivatives of N-acetoxy-N-2-acetylaminofluorene: binding sites on DNA and conformational change of modified deoxytrinucleotides.

GMP and native DNA were reacted with 7-iodo and 7-fluoro derivatives of N-acetoxy-N-2-acetylaminofluorene. It was shown that the 7-halogeno derivatives react on C-8 of guanine. Furthermore the respective amount of arylamidation (covalent linkage on the C-8 of guanine) and arylation (covalent linkage on 2-NH2 groups of C3 of guanine) addition products was determined in both native and denatured DNA-[14C]AAIF. Two G containing deoxytrinucleotides modified by either AAFF or AAIF were studied comparatively by means of circular dichroism, and as a function of several parameters known to affect the conformation of the deoxytrinucleotides. The induced optical activity in fluorofluorene ring seemed to be very sensible to the conformational changes of the deoxytrinucleotides. On the other hand, the AAIF residue exhibit a lower induced optical activity which remained unchanged when the deoxytrinucleotides conformation was affected. The results presented in this paper led us to conclude that the AAFF and AAIF modified deoxytrinucleotides adopt a conformation which nicely fits with the insertion-denaturation and outside-binding model, respectively.