[en] The photoaffinity probes [γ-³²P]2-azidoATP (2-N₃ATP) and [α-³²P]8-azido-ATP (8-N₃ATP) were used to investigate the binding of ATP to highly purified 2-5A synthetase. 2-N₃APT and 8-N₃ATP are substrates for 2-5A synthetase. In this study the authors show that 2- and 8-N₃ATP are competitive inhibitors of the enzymatic conversion of ATP to 2-5A. Ultraviolet irradiation results in the photoinsertion of 2-N₃ATP and 8-N₃ATP into the enzyme. The covalent photoinsertion of [α-³²P]8-N₃ATP into the 2-5A synthetase is proportional to the inactivation of the enzyme as UV irradiation is increased. Photolabeling of 2-5A synthetase is saturated at 1.5 mM 2-N₃ATP and 2.0 mM 8-N₃ATP. Computer analysis of the curvilinear Scatchard plots of the 2-5A synthetase suggest the presence of high-affinity and low-affinity binding sites that may correspond to the acceptor and the 2'-adenylation sites of the enzyme. The competition of nucleotides for the covalent photoinsertion of 8-N₃ATP into the binding site(s) of the synthetase was determined. Photoinsertion of 8-N₃ATP into 2-5A synthetase increases with the addition of poly(rI)/times/poly(rC). Without the addition of poly(rI)/times/poly(rC) to the synthetase, the [α-³²P]8-N₃ATP is photoinserted into the enzyme; however, in the absence of dsRNA and in the absence of UV irradiation, the synthetase cannot convert ATP to 2-5A. The finding suggest that the formation of the enzyme/substrate complex can occur in the absence of dsRNA but dsRNA is essential to activate the 2-5A synthetase to form the productive complex needed for synthesis of 2-5A from ATP

[en] It has been reported that ACA sequences in DNA are mutagenic hot spots in UV-induced mutagenesis and are sites of an alkali-sensitive lesion produced by UV irradiation. In order to characterize the UV-induced lesion of an ACA site, chemically synthesized trideoxyribonucleotide d(ApCpA) was irradiated with UV light and the alkali-sensitive photoproduct was isolated. The structure of this photoproduct was characterized as the trinucleotide containing 2-deoxyribonolactone at the internal residue by 2D DQF-COSY, FT-IR, FAB-MS, and chemical properties. It is known that this lesion is also produced by gamma-irradiation, neocarzinostatin, the 1,10-phenanthroline-copper complex, and hydrogen peroxide and is highly mutagenic because of its resistance to cleavage by certain apurinic/apyrimidinic (AP) endonucleases. Thus, 2-deoxyribonolactone may be one of the lethal DNA lesions induced by UV irradiation to organisms and one of the intermediates of UV-induced DNA strand breaks because the DNA strand is cleaved at this site with beta- and subsequent delta-elimination mechanisms

[en] A method combining spin trapping, ESR, and HPLC was employed to obtain evidence for the formation of sugar radicals in OH-attacked TMP with special emphasis on the detection of strand-break precursors of DNA. OH radicals were produced by irradiating an N2O-saturated aqueous solution with X-rays. When an N2O-saturated aqueous solution containing TMP and a spin trapping reagent, MNP, was irradiated with X-rays, it was estimated on the basis of theoretical calculations using rate constants that 94% of the TMP radicals were induced by OH radicals. Since several spin adducts between TMP radicals and MNP, as well as the byproducts of the spin trapping reagent itself, were produced, reverse-phase HPLC was used to separate them. The presence of six spin adducts was confirmed by ESR examination. Further examination of these spin adducts by UV absorbance spectrophotometry showed the presence of a chromophore at 260 nm in three adducts. Since a gradual increase in the release of unaltered base from these adducts was observed when they were allowed to stand for 0-22 h at room temperature, they could be regarded as the spin adducts of sugar radicals and MNP. ESR spectra from the spin adducts were consistent with hydrogen abstraction radicals at the C1', C4', and C5' positions of the sugar moiety. These radicals appeared to be precursors of AP sites and strand breaks. In addition to these spin adducts, ESR spectra that were consistent with the spin adducts of base radicals (the C5 and C6 radicals) and MNP were observed

[en] Rate constants for the hydrolysis of acetyl-TPP were measured pH values of 2.5 and 7.5 and plotted as log k_obs versus pH. The pH-rate profile defined two legs, each with a slope of +1 but separated by a region of decreased slope between pH 4 and pH 6. The rates were insensitive to buffer concentrations. Each leg of the profile reflected specific-base-catalyzed hydrolysis of acetyl-TPP, analogous to the hydrolysis of 2-acetyl-3,4-dimethylthiazolium ion. The separation of the two legs of this profile has been shown to be caused by the ionization of a group exhibiting a pK_a of 4.73 within acetyl-TPP that is remote from the acetyl group, the aminopyrimidine ring, which is promoted below pH 4.73. The protonation level of this ring has been shown to control the equilibrium partitioning of acetyl-TPP among its carbinolamine, keto, and hydrate forms. The differential partitioning of these species is a major factor causing the separation between the two legs of the pH-rate profile. The characteristic pH-rate profile and the availability of synthetic acetyl-TPP have facilitated the isolation and identification of [1-¹⁴C]acetyl-TPP from acid-quenched enymatic reaction mixtures at steady states. [1-¹⁴C]Acetyl-TPP was identified as a transient species in reactions catalyzed by the PDH complex or the pyruvate dehydrogenase component of the complex (E₁). The pH-rate profile for hydrolysis of [1-¹⁴C]-acetyl-TPP, isolated from enzymatic reactions was found to be indistinguishable from that for authentic acetyl-TPP, which constituted positive identification of the ¹⁴C-labeled enzymic species

[en] A nitroxide spin-labeled analogue of thymidine (1a), in which the methyl group is replaced by an acetylene-tethered nitroxide, was evaluated as a probe for structural and dynamics studies of sequence specifically spin-labeled DNA. Residue 1a was incorporated into synthetic deoxyoligonucleotides by using automated phosphite triester methods. ¹H NMR, CD, and thermal denaturation studies indicate that 1a (T) does not significantly alter the structure of 5'-d(CGCGAATT*CGCG) from that of the native dodecamer. EPR studies on monomer, single-stranded, and duplexed DNA show that 1a readily distinguishes environments of different rigidity. Comparison of the general line-shape features of the observed EPR spectra of several small duplexes (12-mer, 24-mer) with simulated EPR spectra assuming isotropic motion suggests that probe 1a monitors global tumbling of small duplexes. Increasing the length of the DNA oligomers results in significant deviation from isotropic motion, with line-shape features similar to those of calculated spectra of objects with isotropic rotational correlation times of 20-100 ns. EPR spectra of a spin-labeled GT mismatch and a T bulge in long DNAs are distinct from those of spin-labeled Watson-Crick paired DNAs, further demonstrating the value of EPR as a tool in the evaluation of local dynamic and structural features in macromolecules

[en] The pyruvate kinase reaction occurs in separate phosphate- and proton-transfer stages. K⁺, Mg²⁺, and Mg-ADP are known to be required for the phosphoryl transfer step, and K⁺ and Mg²⁺ with allosteric stimulation by MgATP are important for proton transfer. This paper uses the isotope trapping method with ³H-labeled water to identify the proton donor and determine when in the sequence of the catalytic cycle it is generated. When the enzyme was allowed to exchange briefly with ³H₂O (pulse phase) and then diluted into a mixture containing PEP, ADP, and the cofactor K⁺, Mg²⁺, or Co²⁺ in D₂O (chase phase), an amount of [³H]pyruvate was formed in great excess of the amount expected from steady-state catalysis in the diluted ³H-labeled water. With K⁺, Mg²⁺, and ADP at pH 6-9.5 in the pulse phase, a limit of 1.25 enzyme equiv of ³H were trapped. The concentration of PEP required for half-maximum trapping was 14-fold greater than its steady-state K_m. Therefore, the rate constant for dissociation of the donor proton is estimated to be 14 times the steady-state rate of [³H]pyruvate formation. With Co²⁺ and ADP in both the pulse and chase, 3 enzyme equiv of ³H were trapped. For the pulse and chase pHs to be as high as 9.5 without a decrease in the amount of ³H that could be trapped a lysine of very high pK_a requires catalysis. To achieve proton-exchange rate >10³ s^-1 from a group of such high pK_a requires catalysis. Buffer catalysis could be ruled out. Therefore, internal exchange probably occurs with a residue of low pK_a with good access to the medium. Upon addition of PEP this circuit would be broken as the Glu-271 serves some important function in the catalyses or is otherwise diverted

[en] A polypeptide was identified in the venom of the scorpion Leiurus quinquestriatus hebraeus by its potency to inhibit the high affinity binding of the radiolabeled snake venom toxin dendrotoxin I (¹²⁵I-DTX_I) to its receptor site. It has been purified, and its properties investigated by different techniques were found to be similar to those of MCD and DTX_I, two polypeptide toxins active on a voltage-dependent K⁺ channel. However, its amino acid sequence was determined, and it was shown that this toxin is in fact charybdotoxin (ChTX), a toxin classically used as a specific tool to block one class of Ca²⁺-activated K⁺ channels. ChTX, DTX_I, and MCD are potent convulsants and are highly toxic when injected intracerebroventricularly in mice. Their toxicities correlate well with their affinities for their receptors in rat brain. These three structurally different toxins release [³H]GABA from preloaded synaptosomes, the efficiency order being DTX_I > ChTX > MCD. Both binding and cross-linking experiments of ChTX to rat brain membranes and to the purified MCD/DTX_I binding protein have shown that the α-subunit of the MCD/DTX_I-sensitive K⁺ channel protein also contains the ChTX binding sites. Binding sites for DTX_I, MCD, and ChTX are in negative allosteric interaction. The results show that charybdotoxin belongs to the family of toxins which already includes the dendrotoxins and MCD, which are blockers of voltage-sensitive K⁺ channels. ChTX is clearly not selective for Ca²⁺-activated K⁺ channel

[en] The transfer kinetics of [³H]-1-palmitoyl-2-oleoylphosphatidylcholine ([³H]POPC) and 1-palmitoyl-2-(pyrenyldecanoyl)phosphatdylcholine (PyrPC) from POPC small unilamellar vesicles were examined at 37 degree C with lipid concentrations ranging from 0.1 to 40 mM. The rate of [³H]POPC transfer was determined by analyzing the movement of this lipid from charged donor to neutral acceptor vesicles. The rate of decay of the ratio of the intensity of pyrene excimer fluorescence to that from the pyrene monomer (E/M) upon addition of an unlabeled vesicle population to a population containing PyrPC was used to evaluate PyrPC transfer. For both lipids, the kinetic data are best described by a model which assumes that transfer occurs by vesicle collisions as well as by desorption from the bilayer. For [³H]POPC, the off-rate constant is 0.014 h^-1 while the collisional rate constant is 0.0016 mM^-1 h^-1. PyrPC has an off-rate constant of 0.023 h^-1 and a collisional constant of 0.0015 mM^-1 h^-1. These numbers were calculated by assuming the rate of interbilayer transfer to be negligible relative to that of intervesicular transfer. The large transfer fluxes in the high vesicle concentration range where the collisional process dominates suggest that spontaneous transfer may be of importance in membrane biogenesis

[en] Protein kinase C (PKC) from bovine neutrophils was purified 1,420-fold. Subcellular fractionation analysis of bovine neutrophil homogenate in the presence of EGTA indicated that more than 95% of the PKC activity was present in the soluble fraction. Whereas bovine brain PKC could be resolved into four isoenzymatic forms by chromatography on a hydroxylapatite column, bovine neutrophil PKC was eluted in a single peak, suggesting that it corresponded to a single isoform. The apparent molecular weight of bovine neutrophil PKC was 82,000, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Bovine neutrophil PKC was autophosphorylated in the presence of [γ-³²P]ATP, provided that the medium was supplemented with Mg²⁺, Ca²⁺, phosphatidylserine, and diacylglycerol; phorbol myristate acetate could substitute for diacylglycerol. Autophosphorylated PKC could be cleaved by trypsin to generate two radiolabeled peptides of M_r 48,000 and 39,000. The labeled amino acids were serine and threonine. During the course of the purification procedure of bovine neutrophil PKC, a protein of M_r 23,000 was found to exhibit a strong propensity to PKC-dependent phosphorylation in the presence of [γ-³²P]ATP, Mg²⁺, Ca²⁺, phosphatidylserine, and diacylglycerol. This protein was recovered together with PKC in one of the two active peaks eluted from the Mono Q column at the second step of PKC purification. It is suggested that the M_r 23,000 protein might be a natural substrate for bovine neutrophil PKC

[en] Lima bean agglutinin-fluorescein 5-isothiocyanate conjugate (FluNCS-lima bean lectin) interacts with specific receptor molecules on membranes both from the rod outer segment (ROS) of the frog retina and from S49 mouse lymphoma cells. When [125I]-5-iodonaphthyl 1-azide (125I-INA), which freely and randomly partitions into the lipid bilayer, is added to membranes and the suspension is irradiated at 480 nm, the FluNCS-conjugated lectin photosensitizes the [125I]INA but only at discrete sites. This results in the selective labeling of specific proteins: an 88-kDa protein on ROS membranes and a 56-kDa protein on S49 plasma membranes. Labeling is dependent upon the interaction of the FluNCS-lectin with glycosylated receptor sites, since N-acetylgalactosamine, but not methyl alpha-mannoside, blocked labeling of the 56-kDa protein on S49 membranes. In contrast, a random labeling pattern of membrane proteins was observed upon irradiation at 480 nm using other fluorescein conjugates, such as FluNCS-bovine serum albumin (FluNCS-BSA) or FluNCS-soybean trypsin inhibitor (FluNCS-STI), which interact with cell membranes in a nonselective manner, or with N-(fluorescein-5-thiocarbamoyl)-n-undecyclamine (FluNCS-NHC11), which is freely miscible in the membrane lipid. Random labeling was also obtained by direct photoexcitation of [125I]INA at 314 nm, with no distinct labeling of the 88- and 56-kDa proteins in the respective membranes. These results suggest that protein ligands can be used to guide sensitizers to discrete receptor sites and lead to their selective labeling by photosensitized activation of [125I]INA