Effects of magnesium and ATP on pre-steady-state phosphorylation kinetics of the Na+,K(+)-ATPase.

The aim of the present work was to elucidate the role played by ATP and Mg2+ ions in the early steps of the Na+,K(+)-ATPase cycle. The approach was to follow pre-steady-state phosphorylation kinetics in Na(+)-containing K(+)-free solutions under variable ATP and MgCl2 concentrations. The experiments were performed with a rapid mixing apparatus at 20 +/- 2 degrees C. The concentrations of free and complexes species of Mg2+ and ATP were calculated on the basis of a dissociation constant of 0.091 +/- 0.004 mM, estimated with Arsenazo III under identical conditions. A simplified scheme were ATP binds to the ENa enzyme, which is phosphorylated to MgEPNa and consequently dephosphorylated returning to the ENa form, was used. In the absence of ADP and phosphate four rate constants are relevant: k1 and k-1, the on and off rate constants for ATP binding; k2, the transphosphorylation rate constant and k3, the constant that governs the dephosphorylation rate. The values obtained were: k1 = 0.025 +/- 0.003 microM-1 ms-1 for both free ATP and ATPMg; k-1 = 0.038 +/- 0.004 ms-1 for free ATP and 0.009 +/- 0.002 ms-1 for ATPMg; k2 = 0.199 +/- 0.005 ms-1; k3 = 0.0019 +/- 0.0002 ms-1. The model that seems best to explain the data is one where (i) the role of true substrate can be played equally well by free ATP or ATPMg, and (ii) free Mg2+, an essential activator, acts by binding to a specific Mg2+ site on the enzyme molecule.     

Stoichiometry of the reaction of oxyhemoglobin with nitrite.

During the reaction of oxyhemoglobin (HbO2) with nitrite, the concentration of residual nitrite, nitrate, oxygen, and methemoglobin (Hb+) was determined successively. The results obtained at various pH values indicate the following stoichiometry for the overall reaction: 4HbO2 + 4NO2- 4H+ leads to 4Hb+ + 4NO3- + O2 + 2H2 O (Hb denotes hemoglobin monomer). NO2- binds with methemoglobin noncooperatively with a binding constant of 340 M-1 at pH 7.4 and 25 degrees C. Thus, the major part of Hb+ produced is aquomethemoglobin, not methemoglobin nitrite, when less than 2 equivalents of nitrite is used for the oxidation.     

Effects of p-hydroxymercuribenzoate binding on the visible absorption spectrum of methemoglobin.

The binding of p-hydroxymercuribenzoate to human methemoglobin causes a perturbation of the visible heme abosrption spectrum which is expressed by an increase in absorbance in the high spin band regions, 480 to 510 nm and 590 to 640 nm, concomitant with a decrease in absorbance in the alpha- and beta-band absorption regions. The pH dependence of the p-hydroxymercuribenzoate-induced difference spectrum can be accounted for quantitatively by a 5% shift toward higher spin of the aquo form of methemoglobin, a 15% shift toward higher spin of the hydroxide form, and a shift in the apparent pKa for the water to hydroxide transition from 7.92 to 8.04 when mercurial is bound. The rate of these heme abosrbance changes is consistent with the rapid second order formation of the beta93 cysteine, mercury-mercaptide bond and does not represent a change due to the dissociation of methemoglobin tetramers into dimers, even though the latter, slow process does follow mercurial binding. The observation of an increase in spin produced by the binding of a reagent which also promotes dimer formation argues strongly against any direct correlation between an increase in spin and the appearance of deoxyhemoglobin-like conformations.     

A novel method of measuring reduction of nitrite-induced methemoglobin applied to fetal and adult blood of humans and sheep.

The reaction of nitrite with deoxyhemoglobin results in the production of nitric oxide and methemoglobin, a reaction recently proposed as an important oxygen-sensitive source of vasoactive nitric oxide during hypoxic and anoxic stress, with several animal studies suggesting that nitrite may have therapeutic potential. Accumulation of toxic levels of methemoglobin is suppressed by reductase enzymes present within the erythrocyte. Using a novel method of measuring methemoglobin reductase activity in intact erythrocytes, we compared fetal and adult sheep and human blood. After nitrite-induced production of 20% methemoglobin, the blood was equilibrated with carbon monoxide, which effectively stopped further production. Methemoglobin disappearance was first order in nature with specific rate constants (k x 1,000) of 12.9 +/- 1.3 min(-1) for fetal sheep, 5.88 +/- 0.26 min(-1) for adult sheep, 4.27 +/- 0.34 for adult humans, and 3.30 +/- 0.15 for newborn cord blood, all statistically different from one another. The effects of oxygen tensions, pH, hemolysis, and methylene blue are reported. Studies of temperature dependence indicated an activation energy of 8,620 +/- 1,060 calories/mol (2.06 kJ/mol), appreciably higher than would be characteristic of processes limited by passive membrane diffusion. In conclusion, the novel methodology permits absolute quantification of the reduction of nitrite-induced methemoglobin in whole blood.     

Binding of inositol hexaphosphate to human methemoglobin.

The observed static difference spectrum produced by inositol hexaphosphate binding to methemoglobin is the sum of a very fast and a slow spectral transition. The more rapid absorbance change is too fast to be measured by stopped flow techniques, whereas the slow change exhibits a half-time in the range 1 to 6 s. From the pH dependence of the rapidly formed difference spectrum and from a series of heme ligand binding studies, the rapid phase is interpreted to reflect a localized tertiary conformational change which immediately accompanies inositol hexaphosphate binding and results in a selective increase in spin and reactivity of the beta chain heme groups. In contrast, the slow phase appears to reflect a first order isomerization process which involves only a small portion (less than 10%) of the hemoglobin molecules and results primarily in a marked alteration of the spectral properties of the alpha chains with little change in spin. While the rapid spectral transition cannot be directly related to the overall quaternary transition which occurs during oxygen binding to ferrous deoxyhemoglobin, the slow spectral transition may represent the abortive formation of a deoxyhemoglobin A-like conformation which is inhibited in both rate and extent by the presence of water molecules bound to the heme iron atoms.     

Structure of azide methemoglobin

We have compared the structures of horse azide methemoglobin and methemoglobin (MetHb) at 2.8 Å resolution by X-ray difference Fourier analysis. Of four low-spin liganded Hb derivatives (nitric oxide Hb, carbon monoxide Hb, cyanide MetHb, and azide MetHb), azide MetHb is closest in structure to MetHb. In azide MetHb the ligands are co-ordinated end-on at angles of about 125 ° to the heme axes, which is similar to the stereochemistry assumed by azide in binding to free heme. Because of its bent binding geometry, azide encounters less interference in binding and perturbs the protein structure less than carbon monoxide and cyanide, which are smaller, but prefer linear axial co-ordination to heme. Steric interactions between ligand and protein are greater on the β chain, where the E helix is pushed away from the heme relative to MetHb, than on the α chain. Iron position is the same and heme stereochemistry and position are very similar in azide MetHb and MetHb.     

Pulse radiolytic investigation of single heme group reduction in hum an methemoglobin.

Reduction of one of the four heme groups of human aquomethemoglobin A has been investigated by the pulse radiolysis method. The reactivity of e-a-q, the hydrated electron, with methemoglobin was determined by observing this species directly. The separate reactions of the hydroxy yl radical and hydrogen atom, as well as of e-a-q, were studied by observing absorbance changes in the protein spectrum over the wavelength range 290 to 600nm, with appropriate scavengers in solution...     

External cadmium and internal calcium block of single calcium channels in smooth muscle cells from rabbit mesenteric artery.

The patch clamp technique was used to record unitary currents through single calcium channels from smooth muscle cells of rabbit mesenteric arteries. The effects of external cadmium and cobalt and internal calcium, barium, cadmium, and magnesium on single channel currents were investigated with 80 mM barium as the charge carrier and Bay K 8644 to prolong openings. External cadmium shortened the mean open time of single Ca channels. Cadmium blocking and unblocking rate constants of 16.5 mM-1 ms-1 and 0.6 ms-1, respectively, were determined, corresponding to dissociation constant Kd of 36 microM at -20 mV. These results are very similar to those reported for cardiac muscle Ca channels (Lansman, J. B., P. Hess, and R. W. Tsien. 1986. J. Gen. Physiol. 88:321-347). In contrast, Cd2+ (01-10 mM), when applied to the internal surface of Ca channels in inside-out patches, did not affect the mean open time, mean unitary current, or the variance of the open channel current. Internal calcium induced a flickery block, with a Kd of 5.8 mM. Mean blocking and unblocking rate constants for calcium of 0.56 mM-1 ms-1 and 3.22 ms-1, respectively, were determined. Internal barium (8 mM) reduced the mean unitary current by 36%. We conclude that under our experimental conditions, the Ca channel is not symmetrical with respect to inorganic ion block and that intracellular calcium can modulate Ca channel currents via a low-affinity binding site.     

Kinetic analysis of cooperative interactions induced by Mn2+ binding to the chloroplast H(+)-ATPase

The kinetics of Mn2+ binding to three cooperatively interacting sites in chloroplast H(+)-ATPase (CF1) were measured by EPR following rapid mixing of the enzyme with MnCl2 with a time resolution of 8 ms. Mixing of the enzyme-bound Mn2+ with MgCl2 gave a measure of the rate of exchange. The data could be best fitted to a kinetic model assuming three sequential, positively cooperative binding sites. (1) In the latent CF1, the binding to all three sites had a similar on-rate constants of (1.1 +/- 0.04) X 10(4) M-1s-1. (2) Site segregation was found in the release of ions with off-rate constants of 0.69 +/- 0.04 s-1 for the first two and 0.055 +/- 0.003 s-1 for the third. (3) Addition of one ADP per CF1 caused a decrease in the off-rate constants to 0.31 +/- 0.02 and 0.033 +/- 0.008 s-1 for the first two and the third sites, respectively. (4) Heat activation of CF1 increased the on-rate constant to (4.2 +/- 0.92) X 10(4) M-1s-1 and the off-rate constants of the first two and the third site to 1.34 +/- 0.08 and 0.16 +/- 0.07 s-1, respectively. (5) The calculated thermodynamic dissociation constants were similar to those previously obtained from equilibrium binding studies. These findings were correlated to the rate constants obtained from studies of the catalysis and regulation of the H(+)-ATPase. The data support the suggestion that regulation induces sequential progress of catalysis through the three active sites of the enzyme.     

Structure of imidazole methemoglobin

Crystals of horse methemoglobin shatter when soaked in crystallization buffer containing high concentrations of imidazole. By using less than saturating concentrations of imidazole, a stable imidazole derivative of crystalline methemoglobin was prepared and analyzed by X-ray difference Fourier techniques. Both subunits of imidazole methemoglobin show extensive, but different, changes in tertiary structure. Many of the tertiary structural changes observed in the transition from deoxyhemoglobin to methemoglobin are amplified in the transition from methemoglobin to imidazole methemoglobin. Unlike all other ligands that have been examined, imidazole only partially enters the ligand pocket and does not occupy the usual ligand site distal to pyrrole II. The position of the imidazole is on a possible pathway for entrance of smaller diatomic ligands from the solvent into the heme pocket. The extent of imidazole binding of the α-hemes and β-hemes is about 25% and 45%, respectively. An explanation for this difference in occupancy is suggested, involving steric interaction of the distal histidine and phenylalanine CD4 in each subunit. This structural hypothesis may have implications for the kinetics of ligand binding.     

The role of divalent cations in the activation of the NADP+-specific isocitrate dehydrogenase from Pisum sativum L.

A divalent cation electrode was used to measure the stability constants (association constants) for the magnesium and manganese complexes of the substrates for the NADP+-specific isocitrate dehydrogenase (EC 1.1.1.42) from pea stems. At an ionic strength of 26.5 mM and at pH 7.4 the stability constants for the Mg2+-isocitrate and Mg2+-NADP+ complexes were 0.85 +/- 0.2 and 0.43 +/- 0.04 mM-1 respectively and for the Mn2+-isocitrate and Mn2+-NADP+ complexes they were 1.25 +/- 0.07 and 0.75 +/- 0.09 mM-1 respectively. At the same ionic strength but at pH 6.0 the Mg2+-NADPH and Mn2+-NADPH complexes had stability constants of 0.95 +/- 0.23 and 1.79 +/- 0.34 mM-1 respectively. Oxalosuccinate and alpha-ketoglutarate do not form measureable complexes under these conditions. Saturation kinetics of the enzyme with respect to isocitrate and metal ions are consistent with the metal-isocitrate complex being the substrate for the enzyme. NADP+ binds to the enzyme in the free form. Saturation kinetics of NADPH and Mn2+ indicate that the metal-NADPH complex is the substrate in the reverse reaction. In contrast the pig heart enzyme appears to bind free NADPH and Mn2+. A scheme for the reaction mechanism is presented and the difference between the reversibility of the NAD+ and NADP+ enzyme is discussed in relation to the stability of the NADH and NADPH metal complexes.     

Electrochemical reduction of methemoglobin either directly or with flavin mononucleotide as a mediator

Electrochemical reduction of methemoglobin on a platinum electrode is studied by means of thin layer spectroelectrochemistry. For methemoglobin alone in solution, direct reduction is very slow even for potentials close to those of the reduction of the solvent. The reduction of a methemoglobin-oxyhemoglobin mixture with an imposed potential causes the electrochemical reduction of oxygen, the conversion of oxyhemoglobin into deoxyhemoglobin, and a simultaneous transformation of part of the molecules into methemoglobin. When fixed oxygen has disappeared, reduction of methemoglobin takes place. The reduction of methemoglobin and deoxyhemoglobin is catalyzed by the presence of flavin mononucleotide (FMN). For the oxyhemoglobin-methemoglobin mixture, flavin makes a fast deoxygenation of oxyhemoglobin without a change in the oxidation state of the iron. It also allows the rapid reduction of methemoglobin. In each case, the resulting deoxyhemoglobin solutions do not show any electrolysis-induced modification of the equilibrium curves for oxygen binding.     

Quaternary equilibrium analysis of the imidazole methemoglobin bound with inositol hexaphosphate

The visible and proton NMR spectral responses of imidazole methemoglobin by the binding of inositol hexaphosphate were examined in the 2-40 degrees C range. The magnitude of the +/- (inositol hexaphosphate) visible difference spectrum increased and the intensity of the 33 ppm NMR peak decreased with lowering of the temperature. The NMR results were quantitatively analyzed with a simple two-state allosteric model. The results show that the T conformer fraction is 0.6 at 20 degrees C and that the equilibrium shifts toward the T state at lower temperature. The large changes in delta H and delta S associated with the equilibrium suggest participation of numerous factors in the determination of the equilibrium position. The increase in the T conformer population of imidazole methemoglobin, which is pure low-spin, suggests that the appearance of the T state with decreasing temperature is not directly coupled to an increase in spin of the heme iron.     

Reduction of ferricytochrome c, methemoglobin and metmyoglobin by hydroxyl and alcohol radicals.

We have studied the reaction of ferricytochrome c, methemoglobin and metmyoglobin with OH and alcohol radicals (methanol, ethanol, ethylene glycol and glycerol). These radicals can be divided into three groups: 1. The OH radicals which reduce the ferricytochrome c with a yield of (30 +/- 10)% and methemoglobin with a yield of (40 +/- 10)%. They do not reduce metmyoglobin. The reduction is not a normal bimolecular reaction but is most probably an intramolecular electron transfer of a protein radical. 2. Methanol and ethanol radicals which reduce all three hemoproteins with a yield of (100 +/- 5)%. This reduction is a normal bimolecular reaction. 3. Glycerol radicals which do not reduce the ferrihemoproteins under our experimental conditions. Ethylene glycol radicals do not reduce ferricytochrome c and metmyoglobin but they do reduce methemoglobin with a yield of (30 +/- 10)%.     

The effect of heme-linked ionizable groups on cyanide binding to methemoglobin

The pH dependence of the kinetics of the binding of cyanide ion to methemoglobins A and S and to guinea pig and pigeon methemoglobins appears to be not directly correlated with the net charges on the proteins. The kinetics can, however, be adequately explained in terms of three sets of heme-linked ionizable groups with pK1 ranging between 4.9 and 5.3, pK2 between 6.2 and 7.9, and pK3 between 8.0 and 8.5 at 20 degrees C. pK1 is assigned to carboxylic acid groups, pK2 to histidines and terminal amino groups, and pK3 to the acid-alkaline methemoglobin transition. Kinetic second order rate constants have also been determined for the binding of cyanide ion by the four sets of methemoglobin species present in solution. The pKi values and the rate constants of methemoglobin S are strikingly different from those of methemoglobin A. This result is explained in terms of different electrostatic contributions to the free energy of heme linkage arising from differences in the environments of ionizable groups at the surfaces of the two molecules.     

Purification and partial characterization of the b-type cytochrome from human polymorphonuclear leukocytes

Polymorphonuclear leukocytes contain an oxidase system that can be activated to produce superoxide radicals and hydrogen peroxide. A nonmitochondrial b cytochrome, functioning in the generation of these oxygen species, has been purified to apparent homogeneity from human polymorphonuclear phagocytes. After solubilization of the cytochrome with Triton X-100, the cell extract was subsequently chromatographed on Blue Sepharose and Sephacryl S-300. The final preparation was maximally purified 170-fold with a specific content of 5.33 +/- 2.03 nmol mg-1 of protein (mean +/- S.D.; n = 7) and a yield of 21 +/- 13% (n = 5). The apparent molecular mass of the nondenatured cytochrome was estimated by gel filtration to be 235 kDa. Upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, a single polypeptide was found with a molecular mass of 127 kDa. From the pyridine hemochrome spectrum 1 protoheme IX/polypeptide was calculated. The light absorbance bands of the dithionite-reduced cytochrome were found to be at 558.5 (alpha), 529 (beta), and 426 nm (Soret), and that of the oxidized cytochrome at 413.5 nm. The difference absorbance coefficients are delta epsilon (426.5 - 440 nm) = 160.6 +/- 11 mM-1 cm-1 and delta epsilon (558.5 - 542 nm) = 29.3 +/- 2 mM-1 cm-1 (mean +/- S.D.; n = 5). Carbon monoxide binds to the cytochrome in a time-dependent fashion (maximum binding after 50-60 min). The midpoint potential of the solubilized nonpurified cytochrome is identical to the cytochrome in situ (Em7.0 = -218 +/- 7 mV (mean +/- S.D.; n = 5)). However, purified cytochrome b shows a significantly decreased midpoint potential, estimated at -407 +/- 18 mV (n = 4). The protein does not contain noncovalently bound FAD or FMN, and no spectral evidence was obtained for the presence of covalently bound flavin. Preliminary amino acid analysis of the cytochrome shows a high content of hydrophilic residues.     

Reduction of methemoglobin by cobaltocytochrome c catalyzed by mediators.

The reduction of methemoglobin by cobaltocytochrome c (Cocyt c) has been measured using nine mediators of different half-reduction potentials, Em, 7. The rate increases with the increase of Em, 7 for the mediator but dropped precipitously when it becomes more positive than the Em, 7 for the methemoglobin/hemoglobin couple. The reaction is most efficient with phenzaine methosulfate, therefore it was studied in detail. The reaction is first order in the concentrations of Cocyt c and phenazine methosulfate. The average second-order rate constant for Cocyt c + phenazine methosulfate (M) k1 leads to Cocyt c+ M-. is 2.9 x 10(4) M-1 s-1 at 25 degrees C, 0.1 M phosphate pH 7.0. There is a slight negative temperature dependence of k1 at low temperature; at higher temperatures the process has deltaH not equal to approximately 27 kJ mol-1 and deltaS not equal to approxmately - 75 J mol-1 K-1. The effect of anions reflects the dependence of Em, 7 for the methemoglobin/hemoglobin couple with various anions. There is no significant effect on k1 by the addition of inositol hexakisphosphate. The variation of k1 with pH is complicated. The experimental rate constants are compared with values calculated with the theory of nonadiabatic multiphonon process of electron tunneling.     

The mechanism of reduction of cytochrome c as studied by pulse radiolysis.

1. The reaction of hydrated electrons with ferricytochrome c was studied using the pulse-radiolysis technique. 2. In 3.3 mM phosphate-buffer (pH 7.2), 100 mM methanol and at a concentration of cytochrome c of less than 20 muM the reduction kinetics of ferricytochrome c by hydrated electrons is a bimolecular process with a rate constant of 4.5-10-10 M-1-S-1 (21 degrees C). 3. At a concentration of cytochrome c of more than 20 muM the apparent order of the reaction of hydrated electrons with ferricytochrome c measured at 650 nm decreases due to the occurrence of a rate-determining first-order process with an estimated rate constant of 5-10-6s-1 (pH 7.2, 21 degrees C). 4. At high concentration of cytochrome c the reaction-time courses measured at 580 and 695 nm appear to be biphasic. A rapid initial phase (75% and 30% of total absorbance change at 580 and 695 nm, respectively), corresponding to the reduction reaction, is followed by a first-order change in absorbance with a rate constant of 1.3-10-5 S-1 (pH 7.2, 21 degrees C). 5. The results are interpreted in a scheme in which first a transient complex between cytochrome c and the hydrated electron is formed, after which the heme iron is reduced and followed by relaxation of the protein from its oxidized to its reduced conformation. 6. It is calculated that one of each three encounters of the hydrated electron and ferricytochrome c results in a reduction of the heme iron. This high reaction probability is discussed in terms of charge and solvent interactions. 7. A reduction mechanism for cytochrome c is favored in which the reduction equivalent from the hydrated electron is transmitted through a specific pathway from the surface of the molecule to the heme iron.     

Effect of organic phosphates on methemoglobin reduction by ascorbic acid.

The rate of methemoglobin reduction by ascorbic acid was accelerated in the presence of ATP,2,3-diphosphoglycerate (2,3-DPG), and inositol hexaphosphate (IHP). The acceleration was as much as three times, four times, and ten times in the presence of ATP, 2.3-DPG, and IHP at pH 7.0, respectively. The changes of the concentrations of methemoglobin and ascorbic acid during the methemoglobin reduction were determined, and the reaction was found to proceed stoichiometrically in the presence of IHP. The reduction rate of methemoglobin by ascorbic acid was compared at different concentrations of organic phosphates (ATP,2,3-DPG, and IHP) at various pH values (6.3, 7.0, 7.7). From the changes in the reduction rate under different concentrations of organic phosphates, the dissociation constants of ATP, 2,3-DPG, and IHP to methemoglobin could be determined and were estimated to be 3.3 X 10(-4) M, 2 X 10(-3) M, and 8 X 10(-6) M at pH 7.0, respectively. On the basis of these results, the acceleration mechanism of methemoglobin reduction by ascorbic acid due to the presence of organic phosphates was described. The physiological role of 2,3-DPG in human red cells was discussed in relation to the reduction of methemoglobin by ascorbic acid.     

The uptake of protons by heme-linked ionizable groups on azide binding to methemoglobin

When azide ion reacts with methemoglobin in unbuffered solution the pH of the solution increases. This phenomenon is associated with increases in the pK values of heme-linked ionizable groups on the protein which give rise to an uptake of protons from solution. We have determined as a functional of pH the proton uptake, delta h+, on azide binding to methemoglobin at 20 degrees C. Data for methemoglobins A (human), guinea pig and pigeon are fitted to a theoretical expression based on the electrostatic effect of these sets of heme-linked ionizable groups on the binding of the ligand. From these fits the pK values of heme-linked ionizable groups are obtained for liganded and unliganded methemoglobins. In unliganded methemoglobin pK1, which is associated with carboxylic acid groups, ranges between 4.0 and 5.5 for the three methemoglobins; pK2, which is associated with histidines and terminal amino groups, ranges from 6.2 to 6.7. In liganded methemoglobin pK1 lies between 5.8 and 6.3 and pK2 varies from 8.1 to 8.5. The pH dependences of the apparent equilibrium constants for azide binding to the three methemoglobins at 20 degrees C are well accounted for with the pK values calculated from the variation of delta h+ with pH.