Nature of O2 and CO binding to metalloporphyrins and heme proteins.
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The O2 vibration of dioxygen adducts of Fe and Co model complexes of alpha,alpha,alpha,alpha-tetrapivalamidophenylporphyrin ("picket fence" porphyrin, TpivPP) with 1-methylimidazole and 1-tritylimidazole as axial bases are reported, obtained with difference techniques between 16O2, 18O2, 169-18O, and NO with a Fourier transform infrared spectrometer. Assignments of upsilono2 are (O2)Fe(TpivPP) 1-methylimidazole, 1159 cm-1 in Nujol; (O2)Fe(TpivPP) 1-tritylimidazole, 1163 in benzene; (O2)Co(TpivPP) 1-methylimidazole, 1150 in Nujol; (O2)Co(TpivPP) 1-tritylimidazole, 1153 in benzene. Comparisons with other known Fe, Co, Cr, and Ti dioxygen complexes are made, and it is concluded that the bent dioxygen ligand is best viewed as bound superoxide, O2-. The CO affinities of various hemoproteins and model systems are discussed. A correlation between the CO stretching frequency and its binding constant is described. The drastically lowered affinity of hemoproteins for CO compared with unencumbered models is attributed to steric hindrance in the distal binding site, which allows discrimination between the already bent FeIII-O2- and the normally linear FeII-CO systems. If the affinity of hemoproteins in living systems for CO relative to O2 were not decreased, then massive poisoning would result from endogenous CO. (+info)
Glycosylation of hemoglobin in vitro: affinity labeling of hemoglobin by glucose-6-phosphate.
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To determine the mechanism for the formation of hemoglobin A1c (Hb A1c) in vivo, we incubated human hemoglobin with glucose and metabolites of glucose. [14C]Glucose-6-phosphate (G6P) reacted readily with deoxyhemoglobin, and formed a covalent linkage. The reaction rate was considerably reduced in the presence of carbon monoxide or 2,3-diphosphoglycerate (2,3-DPG). Purified G6P hemoglobin had a lowered oxygen affinity and decreased reactivity with 2,3-DPG compared to Hb A. G6P behaved as a 2,3-DPG analog and reacted specifically at the NH2-terminal amino group of the beta chain. In contrast, the interaction of hemoglobin with glucose was much slower, and was unaffected by carbon monoxide or 2,3-DPG. Neither glucose-1-phosphate, fructose-6-phosphate, nor fructose-1,6-diphosphate formed a reaction product with hemoglobin. G6P behaves as an affinity label with the phosphate group forming electrostatic bonds at the 2,3-DPG binding site and the aldehvde group reacting with the NH2-terminal amino group of the beta chain. Thus, G6P hemoglobin may be an intermediate in the conversion of Hb A to Hb A1c. (+info)
Identification of organic phosphorus covalently bound to collagen and non-collagenous proteins of chicken-bone matrix. The presence of O-phosphoserine and O-phosphothreonine in non-collagenous proteins, and their absence from phosporylated collagen.
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Non-collagenous phosphoproteins, almost all of which can be extracted in EDTA at neutral pH in the presence of proteinase inhibitors, are identified in the matrix of chicken bone, and are therefore not covalently bound to collagen. Similarly, all the peptides containing gamma-carboxyglutamic acid are present in the EDTA extract and none in the insoluble residue, confirming that none is covalently linked to chicken bone collagen. However, organic phosphorus is also found to be present in chicken bone collagen, principally in the alpha2-chains. Of the total protein-bound organic phosphorus present in chicken bone matrix, approx. 80% is associated with the non-collagenous proteins and 20% with collagen. The soluble non-collagenous proteins contain both O-phosphoserine and O-phosphothreonine and these account for essentially of their organic phosphorus content. In contrast, collagen contains neither O-phosphoserine nor O-phosphothreonine. Indeed, no phosphorylated hydroxy amino acid, phosphoamidated amino acid or phosphorylated sugar could be identified in purified components of collagen, which contain approximately four to five atoms of organic phosphorus per molecule of collagen. Peptides containing organic phosphorus were isolated from partial acid hydrolysates and enzymic digests of purified collagen components, which contain an as-yet-unidentified cationic amino acid. These data, the very high concentrations of glutamic acid in the phosphorylated peptides, and the pH-stability of the organic phosphorus moiety in intact collagen chains strongly suggest that at least part of the organic phosphorus in collagen is present as phosphorylated glutamic acid. This would indicate that the two major chemically different protein fractions in chicken bone matrix that contain organic phosphorus may represent two distinct metabolic pools of organic phosphorus under separate biological control. (+info)
DNA methylation in adenovirus, adenovirus-transformed cells, and host cells.
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DNAs of adenovirus type 2 and type 12 contain low amounts of methylated bases (0.01 and 0.02% N6-methyl-adenine per adenine, if any, and 0.04 and 0.06% 5-methylcytosine per cytosine for type 2 and type 12, respectively), whereas the DNA of the mammalian host cells contains much more 5-methylcytosine (3.57% for human KB cells). The DNA of hamster cells transformed by adenovirus type 12 contains 3.11 and 3.14% 5-methycytosine (HA12/7 and T627 cells, respectively), whereas the DNA from untransformed hamster cells (BHK21 cells) contains 2.22% 5-methylcytosine. In the DNA of human and hamster cells, little, if any, N6-methyladenine was detected. Methylation of DNA was determined by a sensitive method based on two consecutive steps of two-dimensional thin-layer chromatography of the radioactively labeled DNA bases. By this procedure the detection limits of 5-methylcytosine and N6-methyladenine could be lowered to 0.01% per main base. (+info)
Free acid, anion, alkali, and alkaline earth complexes of lasalocid a (X537A) in methanol: structural and kinetic studies at the monomer level.
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We report below on nuclear magnetic resonance investigations of the structure and exchange kinetics for the free acid, anion, sodium complex, and barium complex of the ionophore lasalocid A (X537A) in methanol solution. A comparison between the proton and carbon longitudinal relaxation times of lasalocid in nonpolar and polar solvents demonstrates that the free acid (HX) is a monomer in methanol solution. Parallel proton and carbon relaxation measurements demonstrate that the anion (X-), sodium complex (NaX), and barium complex (BaX+) are also monomeric in methanol solution. These results are in contrast to the Na2X2 dimer and the BaX2-H20 dimer observed in crystals and in nonpolar (cyclohexane and methylene chloride) solutions. Large downfield shifts on complex formation (X- to NaX and BaX+) are detected for protons located on the polar face of the ionophore with their C-H bonds directed towards and proximal to the metal ion. The exchange of lasalocid anion between free (X-) and complexed (BaX+) states in methanol can be monitored from the temperature-dependent line shapes of the proton resonances at superconducting fields. The exchange rates are independent of the reactant concentrations and are characteristic of a rate-determining dissociation of BaX+ in methanol solution with activation parameters delta H++ = 6.5 kcal mol-1 (25 degrees) and delta S++ = -20.0 cal mol-1 degree -1 (1 cal = 4.184 J). The rate constants for dissociation and formation of BaX+ complex in methanol, 25 degrees, are 5.2 X 10(3) sec-1 and 1.5 X 10(10) M-1 sec-1, respectively. These studies were extended to derive the activation parameters for the exchange of lasalocid anion between BaX+ and NaX and between BaX+ and HX in methanol, while the exchange among HX, X-, and NaX is too rapid to be monitored on the time scale of nuclear magnetic resonance. (+info)
Structural characterization of a bridged 99Tc-Sn-dimethylglyoxime complex: implications for the chemistry of 99mTc-radiopharmaceuticals prepared by the Sn (II) reduction of pertechnetate.
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Reduction of pertechnetate by tin(II) in the presence of dimethylglyoxime is shown, by single crystal x-ray analysis, to yield a technetium-tin-dimethylglyoxime complex in which tin and technetium are intimately connected by a triple bridging arrangement. One bridge consists of a single oxygen atom and it is hypothesized that this bridge arises from the inner sphere reduction of technetium by tin(II), the electrons being transferred through a technetium "yl" oxygen which eventually becomes the bridging atom. Two additional bridges arise from two dimethylglyoxime ligands that function as bidentate nitrogen donors towards Tc and monodentate oxygen donors towards Sn. The tin atom can thus be viewed as providing a three-pronged "cap" on one end of the Tc-dimethylglyoxime complex. The additional coordination sites around Tc are occupied by the two nitrogens of a third dimethylglyoxime ligand, making the Tc seven-coordinate. The additional coordination sites around Sn are occupied by three chloride anions, giving the Sn a fac octahedral coordination environment. From indirect evidence the oxidation states of tin and technetium are tentatively assigned to be IV and V, respectively. Since most 99mTc-radiopharmaceuticals are synthesized by the tin(II) reduction of pertechnetate, it is likely that the Sn-O-Tc linkage described in this work is an important feature of the chemistry of these species. This linkage also provides a ready rationale for the close association of tin and technetium observed in many 99mTc-radiopharmaceuticals. (+info)
Purine nucleoside phosphorylase cleaves the C--O bond of ribose 1-phosphate. Evidence from the 18O shift in 31P NMR.
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An equilibrium mixture of highly enriched [18(O)]Pi (represents the mixture of [[18(O)4]Pi, [[18(O)3]Pi, [18(O)2]Pi as represented in the figures, unless otherwise specified), alpha-D-ribose 1-[16(O)]phosphate, and hypoxanthine plus inosine was equilibrated with calf spleen purine-nucleoside phosphorylase (EC 2.4.2.1). The 31P NMR spectrum clearly indicated the formation of alpha-D-ribose 1-[18(O)4]-phosphate and of [16(O)]Pi. Incubation for the same time span in the absence of alpha-D-ribose 1-phosphate left the [18(O)4]Pi isotopic distribution unchanged. The results clearly demonstrated that the C--O bond of alpha-D-ribose 1-phosphate is cleaved in the enzymatic reaction. It is unlikely that the enzyme catalyzes the exchange of oxygen between Pi and H2O. Several possible mechanistic pathways are ruled out by the results, which demand attack by a phosphate oxygen at the anomeric C-1' atom. (+info)
Potassium channel openers as potential therapeutic weapons in ion channel disease.
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The opening of potassium (K+) channels, causing hyperpolarization of the cell membrane, is a physiological means of decreasing cell excitability. Thus, drugs with this property will demonstrate a broad clinical potential. The identification of synthetic molecules that evoke physiological responses (for example smooth muscle relaxation) by the opening of K+ channels led to a new direction in the pharmacology of ion channels. The term "potassium channel openers" was initially associated with a group of chemically diverse agents (for example, cromakalim, pinacidil, nicorandil) that evoke K+ efflux through adenosine 5'-triphosphate (ATP)-sensitive K+ channels (KATP). This finding initiated a search to identify molecules that specifically open other K+ channel subtypes (for example large conductance calcium-activated K+ channels [BKCa]). K+ channel opening properties have been demonstrated in a diverse range of synthetic chemical structures and endogenous substances. Second generation KATP channel openers (KATPCOs) demonstrate heterogeneous pharmacology indicative of independent sites of action for the different agents. Successful cloning of the KATP channel has shed light on the heterogeneity of the structure targeted by KATPCOs. Expression of the actions of KATPCOs involves three isoforms of the sulfonylurea (SUR) receptor (which forms the beta subunit of the KATP channel). The distribution of the SUR isoforms (and potential of identifying new isoforms) provides unique targets for the development of selective KATPCOs giving focused therapeutic approaches to clinical conditions for example cardiac ischemia, urinary incontinence, neurodegeneration, obesity and autoimmune diseases. BKCa channels are found in a diverse array of tissues and due to voltage and Ca sensitivity may work as a negative feedback process. A variety of small synthetic molecules (for example, NS004, fenamates) and natural product-derived compounds (DHS-I, maxikdiol) have been identified as selective BKCa channel openers which should have a profound impact in controlling diseases. The discovery of numerous variants of the alpha subunit (ion conductance pore) and beta subunit (contributes biophysical and pharmacological properties) complex of the BKCa channel gives potential to target specific tissues with selective openers. Little is known, however, about the site(s) of interaction of openers of these channels. The discovery of K+ channel subtype-specific openers and their evaluation in different diseases will determine the degree to which these channels (KATP, BKCa), or their isoforms, represent realistic therapeutic targets. Drugs already marketed that open K+ channels were discovered empirically, and most have serious safety and efficacy problems. New scientific methods, utilizing molecular insight, are implicating K+ channel dysfunction in numerous disease states and are identifying new targets for the future generation of K+ channel opening drugs. (+info)