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PubMed2001

Stimulation of cardiac glucose transport by inhibitors of oxidative phosphorylation.

Previously we showed that hypoxia in heart stimulates glucose transport via translocation of glucose transporters from intracellular membranes to the plasma membrane. We later showed that rotenone, an inhibitor of oxidative phosphorylation, also decreased intracellular transporters. Here, using another membrane fractionation technique, we show that rotenone increases plasma membrane transporters, and that another respiratory chain inhibitor, azide, acts similarly. Thus, they likely activate the same signaling pathway as hypoxia. Genistein, a tyrosine kinase inhibitor, inhibited insulin- and azide-stimulated 3-O-methylglucose transport similarly in cardiac myocytes. It also increased glucose transporters in the plasma membranes of perfused hearts even though it inhibited glucose uptake, suggesting effects on membrane trafficking. Another tyrosine kinase inhibitor, lavendustin A, and the cyclic nucleotide-dependent protein kinase inhibitors H-8 and H-7 had little effect on basal or azide-stimulated transport. Polymyxin B was a weak inhibitor of basal, insulin-stimulated, and azide-stimulated transport. A nitric oxide donor and a nitric oxide synthase inhibitor had no effect on basal and azide-stimulated transport. The results indicate that tyrosine kinases; protein kinases A, G, and C; and nitric oxide are not involved in the hypoxic activation of cardiac glucose transport.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1979

Effect of thiourea on microsomal oxidation of alcohols and associated microsomal functions.

Thiourea and diethylthiourea, two compounds which react with hydroxyl radicals, inhibited NADPH-dependent microsomal oxidation of ethanol and 1-butanol. Inhibition by both compounds was more effective in the presence of the catalase inhibitor, azide. Inhibition by thiourea was noncompetitive with respect to ethanol in the absence of azide but was competitive in the presence of azide. Urea, a compound which does not react with hydroxyl radicals or H2O2, was without effect. Thiourea had no effect on NADH- and NADH-cytochrome c reductase, NADPH oxidase, and NADH- and NADPH-dependent oxygen uptake. Thiourea inhibited the activities of aniline hydroxylase and aminopyrine demethylase. Thiourea, but no other hydroxyl radical scavengers, e.g., dimethyl sulfoxide, mannitol, and benzoate, reacted directly with H202 and decreased H2O2 accumulation in the presence of azide. Therefore the actions of thiourea are complex because it can react with both hydroxyl radicals and H2O2. Differences between the actions of thiourea and those previously reported for dimethyl sulfoxide, mannitol, and benzoate, e.g., effects on drug metabolism, effectiveness of inhibition in the absence of azide, or kinetics of the inhibition, probably reflect the fact that thiourea reacts directly with H2O2 whereas the other agents do not. The current results remain consistent with the concept that microsomal oxidation of alcohols involves interactions of the alcohols with hydroxyl radicals generated from microsomal electron transfer.

Aminopyrine N-Demethylase↗

PubMed1977

A spectroscopic and kinetic investigation of anion binding to ascorbate oxidase.

The binding of azide, fluoride, and cyanide to ascorbate oxidase has been investigated in detail. Both azide and fluorid inhibit the enzyme competitively with respect to ascorbic acid and noncompetitively with respect to oxygen. Cyanide inhibition is much more complex and also results in inactivation of the enzyme. The binding of azide and fluoride to the resting enzyme is partially competitive. Fluoride binds more strongly to the resting enzyme, while azide binds more strongly to the functioning enzyme. It is proposed that both azide and fluoride bind to type 2 copper and that this copper is also part of an ascorbate binding site. It seems likely that type 2 copper is a reductant binding site in all of the "blue" oxidases. This proposal is used to explain the effect of fluoride on the enzymes and also to suggest a mechanism for the internal electron transfer which is necessary for the reduction of oxygen to water.

Ascorbate Oxidase↗

PubMed1999

Models for molybdenum coordination during the catalytic cycle of periplasmic nitrate reductase from Paracoccus denitrificans derived from EPR and EXAFS spectroscopy.

The periplasmic nitrate reductase from Paracoccus denitrificans is a soluble two-subunit enzyme which binds two hemes (c-type), a [4Fe-4S] center, and a bis molybdopterin guanine dinucleotide cofactor (bis-MGD). A catalytic cycle for this enzyme is presented based on a study of these redox centers using electron paramagnetic resonance (EPR) and extended X-ray absorption fine structure (EXAFS) spectroscopies. The Mo(V) EPR signal of resting NAP (High g [resting]) has g(av) = 1.9898 is rhombic, exhibits low anisotropy, and is split by two weakly interacting protons which are not solvent-exchangeable. Addition of exogenous ligands to this resting state (e.g., nitrate, nitrite, azide) did not change the form of the signal. A distinct form of the High g Mo(V) signal, which has slightly lower anisotropy and higher rhombicity, was trapped during turnover of nitrate and may represent a catalytically relevant Mo(V) intermediate (High g [nitrate]). Mo K-edge EXAFS analysis was undertaken on the ferricyanide oxidized enzyme, a reduced sample frozen within 10 min of dithionite addition, and a nitrate-reoxidized form of the enzyme. The oxidized enzyme was fitted best as a di-oxo Mo(VI) species with 5 sulfur ligands (4 at 2. 43 A and 1 at 2.82 A), and the reduced form was fitted best as a mono-oxo Mo(IV) species with 3 sulfur ligands at 2.35 A. The addition of nitrate to the reduced enzyme resulted in reoxidation to a di-oxo Mo(VI) species similar to the resting enzyme. Prolonged incubation of NAP with dithionite in the absence of nitrate (i.e., nonturnover conditions) resulted in the formation of a species with a Mo(V) EPR signal that is quite distinct from the High g family and which has a g(av) = 1.973 (Low g [unsplit]). This signal resembles those of the mono-MGD xanthine oxidase family and is proposed to arise from an inactive form of the nitrate reductase in which the Mo(V) form is only coordinated by the dithiolene of one MGD. In samples of NAP that had been reduced with dithionite, treated with azide or cyanide, and then reoxidized with ferricyanide, two Mo(V) signals were detected with g(av) elevated compared to the High g signals. Kinetic analysis demonstrated that azide and cyanide displayed competitive and noncompetitive inhibition, respectively. EXAFS analysis of azide-treated samples show improvement to the fit when two nitrogens are included in the molybdenum coordination sphere at 2.52 A, suggesting that azide binds directly to Mo(IV). Based on these spectroscopic and kinetic data, models for Mo coordination during turnover have been proposed.

Azides↗

PubMed2004

Steric and hydrogen-bonding effects on the stability of copper complexes with small molecules.

A series of the copper(II) complexes with tripodal tetradentate tris(pyridyl 2-methyl)amine-based ligands possessing the hydrogen-bonding 6-aminopyridine units (tapa, three amino groups; bapa, two amino groups; mapa, one amino group) have been synthesized, and their copper(II) complexes with a small molecule such as dioxygen and azide have been studied spectroscopically and structurally. The reaction of their Cu(II) complexes with NaN(3) have given the mononuclear copper complexes with azide in an end-on mode, [Cu(tapa)(N(3))]ClO(4) (1a), [Cu(bapa)(N(3))]ClO(4) (2a), [Cu(mapa)(N(3))]ClO(4) (3a), and [Cu(tpa)(N(3))]ClO(4) (4a) (tpa, no amino group). The crystal structures have revealed that the coordination geometries around the metal centers are almost a trigonal-bipyramidal rather than a square-planar except for 1a with an intermediate between them. The UV-vis and ESR spectral data indicate that the increase of NH(2) groups of ligands causes the structural change from trigonal-bipyramidal to square-pyramidal geometry, which is regulated by a combination of steric repulsion and hydrogen bond. The steric repulsion of amino groups with the azide nitrogen gives rise to elongation of the Cu-N(py) bonds, which leads to the positive shift of the redox potentials of the complexes. The hydrogen bonds between the coordinated azide and amino nitrogens (2.84-3.05 A) contribute clearly to the fixation of azide. The Cu(I) complexes with bapa and mapa ligands have been obtained as a precipitate, although that with tapa was not isolated. The reactions of the Cu(I) complexes with dioxygen in MeOH at -75 degrees C have given the trans-micro-1,2 peroxo dinuclear Cu(II) complexes formulated as [((tapa)Cu)(2)(O(2))](2+) (1c), [((bapa)Cu)(2)(O(2))](2+) (2c), and [((mapa)Cu)(2)(O(2))](2+) (3c), whose characterizations were confirmed by UV-vis, ESR, and resonance Raman spectroscopies. UV-vis spectra of 1c, 2c, and 3c exhibited intense bands assignable to pi(O(2)(2)(-))-to-d(Cu) charge transfer (CT) transitions at lambda(max)/nm (epsilon/M(-1)cm(-1)) = 449 (4620), 474 (6860), and 500 (9680), respectively. The series of the peroxo adducts generated was ESR silent. The resonance Raman spectra exhibited the enhanced features assignable to two stretching vibrations nu((16)O-(16)O/(18)O-(18)O)/cm(-1) and nu(Cu-(16)O/Cu-(18)O)/cm(-1) at 853/807 (1c), 858/812 (2c), 847/800 (3c), and at 547/522 (2c), 544/518 (3c), respectively. The thermal stability of the peroxo-copper species has increased with increase in the number of the hydrogen-bonding interactions between the peroxide and amino groups.

Azides↗

PubMed2002

Second-sphere contributions to substrate-analogue binding in iron(III) superoxide dismutase.

A combination of spectroscopic and computational methods has been employed to explore the nature of the yellow and pink low-temperature azide adducts of iron(III) superoxide dismutase (N(3)-FeSOD), which have been known for more than two decades. Variable-temperature variable-field magnetic circular dichroism (MCD) data suggest that both species possess similar ferric centers with a single azide ligand bound, contradicting previous proposals invoking two azide ligands in the pink form. Complementary data obtained on the azide complex of the Q69E FeSOD mutant reveal that relatively minor perturbations in the metal-center environment are sufficient to produce significant spectral changes; the Q69E N(3)-FeSOD species is red in color at all temperatures. Resonance Raman (RR) spectra of the wild-type and Q69E mutant N(3)-FeSOD complexes are consistent with similar Fe-N(3) units in all three species; however, variations in energies and relative intensities of the RR features associated with this unit reveal subtle differences in (N(3)(-))-Fe(3+) bonding. To understand these differences on a quantitative level, density functional theory and semiempirical INDO/S-CI calculations have been performed on N(3)-FeSOD models. These computations support our model that a single azide ligand is present in all three N(3)-FeSOD adducts and suggest that their different appearances reflect differences in the Fe-N-N bond angle. A 10 degrees increase in the Fe-N-N bond angle is sufficient to account for the spectral differences between the yellow and pink wild-type N(3)-FeSOD species. We show that this bond angle is strongly affected by the second coordination sphere, which therefore might also play an important role in orienting incoming substrate for reaction with the FeSOD active site.

Azides↗

PubMed2006

In-situ synthesis of a tacrine-triazole-based inhibitor of acetylcholinesterase: configurational selection imposed by steric interactions.

Recently, researchers have used acetylcholinesterase (AChE) as a reaction vessel to synthesize its own inhibitors. Thus, 1 (syn-TZ2PA6), a femtomolar AChE inhibitor, which is formed in a 1:1 mixture with its anti-isomer by solution phase reaction from 3 (TZ2) and 4 (PA6), can be synthesized exclusively inside the AChE gorge. Our computational approach based on quantum mechanical/molecular mechanical (QM/MM) calculations, molecular dynamics (MD), and targeted molecular dynamics (TMD) studies answers why 1 is the sole product in the AChE environment. Ab initio QM/MM results show that the reaction in the AChE gorge occurs when 3/azide and 4/acetylene are extended in a parallel orientation. An MD simulation started from the final structure of QM/MM calculations keeps the azide's and acetylene's parallel orientations intact for 10 ns of simulation time. A TMD simulation applied on an antiparallel azide-acetylene conformation flips the acetylene easily to bring it to a position that is parallel to azide. A second set of QM/MM calculations performed on this flipped structure generates a similar minimum-energy path as obtained previously. Even a TMD simulation carried out on a parallel azide-acetylene conformation could not deform their parallel arrangement. All of these results, thus, imply that inside the AChE gorge, the azide group of 3 and the acetylene group of 4 always remain parallel, with the consequence that 1 is the only product. The architecture of the gorge plays an important role in this selective formation of 1.

Acetylcholinesterase↗

PubMed1994

Vibrational circular dichroism measurements of ligand vibrations in haem and non-haem metalloenzymes.

We have measured the vibrational circular dichroism (VCD) spectra of the stretching vibrations of azide and cyanide ligated to the Fe3+ atoms of haemoglobin (Hb) and myoglobin (Mb). The antisymmetric azide-stretch of the low-spin haems have an anomalously large g-value of ca. -1 x 10(-3). In contrast, CN- has a g-value of ca. +2.4 x 10(-3). We also show, for the first time, that a significant VCD occurs for the azide ligand antisymmetric stretches of non-haem proteins; we measure a g-value of ca. -1 x 10(-4) for azide bound to haemerythrin. We have examined the mechanism of the VCD phenomenon by: (1) reconstituting Mb with haems substituted such that they insert differently in the haem pocket; (2) replacing the Fe3+ with Mn3+; (3) examining proteins where replacements occur for E-7 His and E-11 Val distal amino acids close to the haem and (4) examining an Mb mutant where the proximal F-8 His is replaced by Gly, and where an imidazole ligand inserts into the resulting crevice and binds to the haem in a way similar to that of the proximal histidine in the native protein. The VCD anisotropy appears insensitive to the haem substituent replacements used in this study. Exchange of the E-7 distal His or the E-11 Val has a dramatic effect on the g-value. Exchange of the F-8 proximal His reverses the sign of the g-value for the azide complex, but not for the cyanide complex. The work to date indicates that VCD has the potential to become a sensitive technique for examining the structure of metalloenzymes. Work is needed to determine the mechanism giving rise to the large g-values and to correlate the VCD spectrum with the metalloenzyme structure at the active site.

Azides↗

PubMed1994

Two sites of glucose control of insulin release with distinct dependence on the energy state in pancreatic B-cells.

The energy state of pancreatic B-cells may influence insulin release at several steps of stimulus-secretion coupling. By closing ATP-sensitive K+ channels (K(+)-ATP channels), a rise in the ATP/ADP ratio may regulate the membrane potential, and hence Ca2+ influx. It may also modulate the effectiveness of Ca2+ on its intracellular targets. To assess the existence of these two roles and determine their relative importance for insulin release, we tested the effects of azide, a mitochondrial poison, on mouse B-cell function under various conditions. During stimulation by glucose alone, when K(+)-ATP channels are controlled by cellular metabolism, azide caused parallel, concentration-dependent (0.5-5 mM), membrane repolarization, decrease in cytosolic Ca2+ concentration [Ca2+]i and inhibition of insulin release. When K(+)-ATP channels were closed pharmacologically (by tolbutamide in high glucose), azide did not repolarize the membrane or decrease [Ca2+]i, and was much less effective in inhibiting insulin release. A similar resistance to azide was observed when K(+)-ATP channels were opened by diazoxide, and high K+ was used to depolarize the membrane and raise [Ca2+]i. In contrast, azide similarly decreased ATP levels and increased ADP levels, thereby lowering the ATP/ADP ratio under all conditions. In conclusion, lowering the ATP/ADP ratio in B-cells can inhibit insulin release even when [Ca2+]i remains high. However, this distal step is much more resistant to a decrease in the energy state of B-cells than is the control of membrane potential by K(+)-ATP channels. Generation of the signal triggering insulin release, high [Ca2+]i, through metabolic control of membrane potential requires a higher global ATP/ADP ratio than does activation of the secretory process itself.

Adenosine Diphosphate↗

PubMed1989

Photooxidation of cell membranes using eosin derivatives that locate in lipid or protein to study the role of diffusible intermediates.

Eosin derivatives that bind primarily to lipid or protein sites in erythrocyte membranes were studied in solution and as sensitizers of erythrocyte membranes. In 50% ethanol-water mixtures eosin maleimide (EYMA) and 5-N-hexadecanoyl amino eosin (E16) had nearly identical absorption spectra. Higher ethanol concentrations did not change peak absorbances. In the presence of neutral detergent both sensitizers had equivalent absorbance at all ethanol concentrations. In water, EYMA was more effective than E16 at bleaching RNO, probably because of E16 aggregation into micelles, while in ethanol-water mixtures E16 was slightly more effective at bleaching DPBF, indicating equivalent singlet oxygen generation when the sensitizers are in monomeric form. In water with neutral detergent, azide in the 20 microM range inhibited the majority of RNO bleaching with both sensitizers; in 50% ethanol-water mixtures azide at 1 mM showed a 50% inhibition of DPBF bleaching with both sensitizers. Iodide in the 30 mM range reduced DPBF bleaching by 50% in 50% ethanol-water mixtures. When matched for amount loaded in erythrocyte membranes these sensitizers were about equally effective at sensitizing induction of cation permeability, assayed as rate of delayed photohemolysis, while E16 was slightly more effective at sensitizing loss of cholinesterase (AchE) activity. The relation of lysis rate to load was somewhat steeper for E16 than EYMA. For both sensitizers lysis rate increased at about the 1.5 power of light dose. Deoxygenation of the reaction media with argon totally blocked detectable photomodification. Ghost membranes made from sensitizer-treated cells were effective generators of singlet oxygen, assayed by RNO bleaching. However, when mixtures of EYMA-treated and untreated cells were illuminated together, only the EYMA-treated cells showed evidence of photomodification. Azide at 5 mM slowed the initial rate of AchE loss by about 75% with E16 and EYMA. Azide partially slowed photohemolysis. Azide decreased RNO bleaching by sensitizer-treated ghosts as it did in water with detergent micelles. A deuterium oxide solvent increased photohemolysis rate with E16 by 41%, but did not increase photohemolysis rate with EYMA. Deuterium oxide had a positive, but statistically insignificant effect on loss of AchE with both sensitizers. Deuterium oxide following illumination slowed lysis sensitized by both sensitizers more than 50%. Iodide exerted a modest inhibition of photohemolysis and loss of AchE sensitized by E16, but had virtually no influence on sensitization by EYMA. The results in solution indicate that EYMA and E16 have nearly identical photochemical properties when in monome

Azides↗

PubMed1976

Chemiluminescence and superoxide production by myeloperoxidase-deficient leukocytes.

The role of superoxide anion- and myeloperoxidase-dependent reactions in the light emission by phagocytosing polymorphonuclear leukocytes has been investigated using leukocytes that lack myeloperoxidase, inhibitors (azide, superoxide dismutase), and model systems. Our earlier finding that oxygen consumption, glucose C-1 oxidation, and formate oxidation are greater in polymorphonuclear leukocytes that lack myeloperoxidase than in normal cells during phagocytosis has been confirmed with leukocytes from two newly described myeloperoxidase-deficient siblings. Although the maximal rate of superoxide anion production by myeloperoxidase-deficient leukocytes is not significantly different from that of normal cells, superoxide production falls off less rapidly with time so that with prolonged incubation, it is greater in myeloperoxidase-deficient than in normal cells. Chemiluminescence by myeloperoxidase-deficient leukocytes during the early postphagocytic period however is decreased. Light emission by normal leukocytes is strongly inhibited by both superoxide dismutase and azide, whereas that of myeloperoxidase-deficient leukocytes, while still strongly inhibited by superoxide dismutase is considerably less sensitive to azide. Zymosan, the phagocytic particle employed in the intact cell system, considerably increased the chemiluminescence of a cell-free superoxide-H2O2 generating system (xanthine-xanthine oxidase) and a system containing myeloperoxidase, H2O2, and chloride. Light emission by the xanthine oxidase model system is strongly inhibited by superoxide dismutase and is not inhibited by azide, whereas the myeloperoxidase-dependent model system is strongly inhibited by azide but only slightly inhibited by superoxide dismutase. These findings suggest that light emission by phagocytosing polymorphonuclear leukocytes is dependent on both myeloperoxidase-catalyzed reactions and the superoxide anion, and involves in part the excitation of the ingested particle. These studies are discussed in relation to the role of the superoxide anion and chemiluminescence in the microbicidal activity of the polymorphonuclear leukocyte.

Anions↗

PubMed1990

Adenosine triphosphatase and nucleotide binding activity of isolated beta-subunit preparations from Escherichia coli F1F0-ATP synthase.

Adenosine triphosphatase activity and nucleotide binding affinity of isolated beta-subunit preparations from Escherichia coli F1F0-ATP synthase were studied. The aim was to find out whether isolated beta-subunit would provide an experimental model in which effects of mutations on catalysis per se, unencumbered by complications due to their effects on positive catalytic cooperativity, could be studied. Three types of purified, isolated beta-subunit preparations were studied. Type I-beta was from a strain lacking all F1F0 subunits except beta and epsilon. Type II-beta was from F1 carrying the alpha S375F mutation which blocks positive catalytic cooperativity. Type III-beta was from normal F1. Type I- and II-beta had very low ATPase activity (less than 10(-4) s-1) which was azide-insensitive, aurovertin-insensitive, and unaffected by anti-beta antibody. Type I-beta activity was EDTA-insensitive. We conclude that isolated beta-subunit from E. coli F1F0 has zero or at most very low intrinsic ATPase activity. Type III-beta had low ATPase activity (8.4 x 10(-5) s-1 to 1.1 x 10(-3) s-1 in seven different preparations). This activity was aurovertin-sensitive, but varied in azide sensitivity from 0 to 34% inhibited. The azide-sensitive component, like F1 and alpha 3 beta 3 gamma oligomer, was inhibited by anti-beta and anti-alpha antibodies. The azide-insensitive component was stimulated by anti-beta and unaffected by anti-alpha. We show here that (alpha beta)-oligomer has ATPase activity which is azide-insensitive, aurovertin-sensitive, stimulated by anti-beta, and unaffected by anti-alpha. The intrinsic ATPase activity of Type III-beta could be due to contaminating (alpha beta)-oligomer plus alpha 3 beta 3 gamma-oligomer. Isolated beta had very low affinity for nucleotide as compared to the first catalytic site on F1. Taken together with the very low ATPase activity of isolated beta (even if real), the work shows that isolated beta is not a good experimental model of F1 catalysis.

Adenosine Diphosphate↗

PubMed1984

Metal site cooperativity within cytochrome oxidase.

Low temperature (9-15 K) EPR of isolated bovine heart cytochrome oxidase titrated potentiometrically in the presence of azide reveals the formation of two distinct species of low-spin cytochrome a3(III)-azide which differ in redox properties and g values. Both species are formed with characteristic midpoint potentials during the course of oxidative titration and disappear at higher potentials. The signal appearing at lower potential has principal g values 2.88, 2.19, and 1.64; that appearing at higher potential has g values 2.77, 2.18, and 1.74. A good fit to the experimental data (per cent of cytochrome present in a given paramagnetic state versus oxidation potential) was obtained with a model whereby the gz = 2.88 species arises from cytochrome a3(III)-azide with cytochrome a reduced, which is converted to the gz = 2.77 species upon oxidation of cytochrome a. Potentiometric titration of cytochrome oxidase in the presence of cyanide produces two low-spin heme EPR signals attributable to cytochrome a3(III)-cyanide which are incompletely resolved, but are distinguishable nonetheless. The low-potential signal has peak amplitude at gz = 3.63 and a long high-field tail; this resonance has been seen by other workers in the partially reduced enzyme (DerVartanian, D. V., Lee, I. Y., Slater, E. C., and van Gelder, B. F. (1974) Biochim. Biophys. Acta 347, 321-327). The high-potential signal is much more symmetric about its peak amplitude, which is at approximately 10 G higher field with gz = 3.61. As with the azide complex, the titration behavior in the presence of 2 mM KCN is adequately simulated by assuming that the appearance of the two species is a function of the oxidation state of cytochrome a. Like the a3-azide signals, the a3-cyanide signals disappear upon further oxidation with some characteristic midpoint potential. If the disappearance of the a3-ligand signals with increasing potential is assumed to be the result of antiferromagnetic (or ferromagnetic) coupling of a3(III) (S = 1/2) to CuB(II) (S = 1/2), then cooperativity between cytochrome a and CuB is implied. The data are consistent with the hypothesis that oxidation of cytochrome a raises the midpoint potential of CuB by 55 +/- 10 mV.

Animals↗

PubMed1981

Interaction of methemoglobin with inositol hexaphosphate. Presence of the T state in human adult methemoglobin in the low spin state.

The interaction of human methemoglobin with inositol hexaphosphate (IHP) was examined by proton NMR spectroscopy. Upon addition of IHP, new proton peaks which are distinguishable from those observed in the absence of IHP appeared irrespective of the spin state of the ferric heme iron. the IHP-induced NMR peaks of low spin azide methemoglobin were not observed when azide was titrated to stripped aquomethemoglobin. Therefore, it was indicated that the IHP-induced peaks are not due to the slight dissociation of azide. Azide titration to aquomethemoglobin in the presence of IHP further revealed that the IHP-induced peaks are not due to a localized structural change but rather due to the presence of an IHP-induced new conformer. The NMR spectral intensity calibration for the 5-methylimidazole complex of methemoglobin showed that the intensity of the spectrum of stripped methemoglobin decreased by about 80% upon addition of IHP, suggesting the structural alterations in both of the alpha and beta subunits. The intensity of the IHP-induced peak at 31.1 ppm in azide methemoglobin spectrum decreased at both higher and lower pD values with its optimal around pD 6.7. The pD dependence of this peak was closely similar to that of the exchangeable proton NMR peak which has been used as a measure of the T quaternary conformation for human aquomethemoglobin (Huang, T.-H. (1979) J. Biol. Chem. 254, 11467-11474). From all of the above results, it was concluded that human low spin methemoglobin can be switched from the R to T quaternary structures by the binding of IHP. The allosteric constant L4 = [T4]/[R4] for the fully ligated low spin methemoglobin increased with decreasing temperature and was estimated to be 1 to 5 at room temperature.

Adult↗

PubMed1979

Photoaffinity labeling of procaine-binding sites in normal and sickle cell membranes.

A photoaffinity probe, procaine azide, was employed to determine the sites of interaction of procaine in normal and sickle cell erythrocytes. Studies show that the number of binding sites and affinity of procaine to membranes derived from normal and sickled cell erythrocytes were similar, although procaine retards the in vitro formation of irreversibly sickled cells from cells, The results show that procaine azide, a photoaffinity analogue of procaine, is covalently incorporated into both protein (60--70%) and lipid (40--30%) components of the membrane. Sodium dodecyl sulfate-gel electrophoresis of the labeled ghosts show that procaine binds specifically to band 3 and periodic acid Schiff staining bands in membranes derived from labeled erythrocytes. Binding of procaine or covalent incorporation of procaine azide into membrane proteins does not affect the phosphate transport. Moreover, pre treatment of intact erythrocytes with 4,4-'diisothiocyano-2,2'-stilbene disulfonate, an anion transport inhibitor, did not affect either the binding or covalent incorporationof procaine azide into erythrocytes. These results indicate that the binding of procaine azide to Band 3 protein occurs at a locus different than that involved in anion translocation process.

Affinity Labels↗

PubMed1985

Subcellular fractionation of pig coronary artery smooth muscle.

A detailed procedure for subcellular fractionation of the smooth muscle from pig coronary arteries based on dissection of the proper tissue, homogenization, differential centrifugation and sucrose density gradient centrifugation is described. A number of marker enzymes and Ca2+ uptake in presence or absence of oxalate, ruthenium red and azide were studied. The ATP-dependent oxalate-independent azide- or ruthenium red-insensitive Ca2+ uptake, and the plasma membrane markers K+-activated ouabain-sensitive p-nitrophenylphosphatase, 5'-nucleotidase and Mg2+-ATPase showed maximum enrichment in the F2 fraction (15-28% sucrose) which was also contaminated with the endoplasmic reticulum marker NADPH: cytochrome c reductase, and to a small extent with the inner mitochondrial marker cytochrome c reductase, and also showed a small degree of oxalate stimulation of the Ca2+ uptake. F3 fraction (28-40% sucrose) was maximally enriched in the ATP- and oxalate-dependent azide-insensitive Ca2+ uptake and the endoplasmic reticulum marker NADPH: cytochrome c reductase but was heavily contaminated with the plasma membrane and the inner mitochondrial markers. The mitochondrial fraction was enriched in cytochrome c oxidase and azide- or ruthenium red-sensitive ATP-dependent Ca2+ uptake but was heavily contaminated with other membranes. Electron microscopy showed that F2 contained predominantly smooth surface vesicles and F3 contained smooth surface vesicles, rough endoplasmic reticulum and mitochondria. The ATP-dependent azide-insensitive oxalate-independent and oxalate-stimulated Ca2+ uptake comigrated with the plasma membrane and the endoplasmic reticulum markers, respectively, and were preferentially inhibited by digitonin and phosphatidylserine, respectively. This study establishes a basis for studies on receptor distribution and further Ca2+ uptake studies to understand the physiology of coronary artery vasodilation.

4-Nitrophenylphosphatase↗

PubMed1977

Induction of respiratory deficient mutants in Saccharomyces cerevisiae by mono- and diazido analogs of ethidium.

Mono- and diazido analogs of ethidium when photolyzed with yeast cells were highly effective in inducing respiratory deficient (RD) mutants. The monoazide was more mutagenic, though slightly less photosensitive, and under the concentrations and conditions used, both required photolysis to be significantly mutagenic. Ethidium bromide (EB) competed with either its mono- or diazide analog for RD induction when applied before, but not after, the photolysis step. This suggested that the initial mutagenic binding sites for azides were identical with those of EB. There was no self-rescue or recovery in azide mutagenesis in contrast to EB. Furthermore, recovery from azide mutagenesis could not be provoked by EB. This confirmed a simple competition between binding of EB and its azide analogs to account for the prevention by EB of the azide induced mutations.

Binding Sites↗

PubMed1989

Structure-function relationships of mitochondrial ATPase-ATPsynthase using Schizosaccharomyces pombe yeast mutants with altered F1 subunits.

Phenotypic revertants have been selected from mutants of the yeast Schizosaccharomyces pombe devoid of either alpha or beta subunits of mitochondrial ATPase-ATPsynthase. In contrast to parental mutants, phenotypic revertants are able to grow on glycerol respiratory medium and show immunodetectable alpha and beta subunits. However, growth and cellular respiration are only partially restored as compared to the wild strain, indicating that the recovered subunits are mutated. ATPase activity of revertant submitochondrial particles shows markedly different parameters: more acidic optimal pH, absence of bicarbonate activation and decreased sensitivity to azide inhibition in the alpha subunit-modified R3.51. Opposite differences are observed in the beta subunit-modified R4.3: more alkaline optimal pH, much higher bicarbonate activation, and increased sensitivity to azide. The ITPase activity of R4.3 submitochondrial particles is also more sensitive to azide as compared to the wild strain. ATPase activity of purified F1 also exhibits marked differences: loss of bicarbonate-sensitive negative cooperativity, decreased sensitivity to both ADP and azide inhibitions in the R3.51 revertant. On the contrary, increased negative cooperativity and increased sensitivity to both ADP and azide inhibitions are observed for the R4.3 revertant enzyme which in addition exhibits a much lower maximal rate. The beta subunit-mutation of R4.3 also increases the sensitivity of ITPase activity to tripolyphosphate inhibition, whereas the alpha subunit-mutation of R3.51 is without any effect. Soluble F1 with beta subunit-mutation is very sensitive to high ammonium sulfate concentrations required for enzyme precipitation and concentration and known to partially deplete the enzyme from its endogenous nucleotides. On the contrary, poly(ethylene)glycol is very efficient for preparing from any strain a pure and very stable enzyme retain-ing high amounts of endogenous nucleotides. The R4.3 revertant F1 retains even more nucleotides than the wild-strain F1 and is much less sensitive to high iodide concentrations which favor enzyme dissociation and precipitation. The tryptophan intrinsic fluorescence of F1 is modified by both mutations that increase the maximal emission intensity. The most important effect is produced by beta subunit-mutation which decreases the quenchable fraction, one-third to one-half tryptophans being no longer accessible to iodide. The overall results suggest that both mutations modify enzyme-nucleotide interactions: the alpha subunit-mutation of R3.51 would favor ADP release by lowering interactions with the adenine moiety, whereas the beta subunit-mutation of R4.3 would lower ADP release by strengthening interactions with the phosphate chain moiety.

Adenosine Triphosphatases↗