The effect of endogenous phosphate on the H+/Mn2+ ratio and the state of Mn2+ in the mitochondrial matrix.

1. Kinetics and stoichiometry of H+ extrusion and reuptake and of Mn2+ uptake and release have been measured in respiring liver mitochondria in the absence of external added Pi. H+ and Mn2+ fluxes are parallel during aerobic cation uptake but not during uncoupler induced cation release. The H+/Mn2+ is 1.24. Addition of SH reagents, in concentrations inhibiting the Pi carrier, modifies the kinetics of H+ extrusion and of Mn2+ uptake and release. The slow phase of uncoupler induced Mn2+ release is diminished. The H+/Mn2+ is increased to 1.72. Addition of SH reagents, after the phase of aerobic uptake is completed, results in a significant reduction of the extent of uncoupler-induced Mn2+ release. The extent of reuptake of endogenous Pi during aerobic uptake of Mn2+ is about 8 nmol x mg protein-1. 2. Aerobic uptake of Mn2+ in the absence of external Pi results in an electron spin resonance spectrum which is the sum of two components. One, denoted as S, corresponds to Mn(H2O)2+(6). Another denoted as E, reflects spin exchange narrowing. In contrast to previous claims the following evidence suggests that the spin exchange component is due to Mn3(PO4)2 precipitate: (a) the dimension of the spin exchange spectrum is markedly reduced by abolition of Pi transport; (b) the spin exchange spectrum is released very slowly by addition of uncouplers under conditions where uncouplers cause a rapid deenergization of mitochondria, reuptake of H+ and release of cations; (c) the free matrix Mn2+ is released slowly after addition of uncoupler if there is a large spin exchange signal; howeover the free matrix Mn2+ is abolished rapidly by uncoupler when formation of the spin exchange signal is prevented by pretreatment with Ca2+; (d) the band width of the spin exchange fraction is independent of the Mn2+/protein ratio either under kinetic or steady state conditions; (e) the experimental spectrum recalls closely that obtained by computer simulation by assuming it as a combination of Mn(H2O)2+(6) and Mn3(PO4)2. 3. It is concluded that endogenous Pi affects the process of aerobic divalent cation uptake. A part of Mn2+ uptake in the absence of externally added anions, consists of a Mn3(PO4)2 precipitate. This accounts for a H+/Mn2+ ratio lower than 2.     

A 31P NMR study of mitochondrial inorganic phosphate visibility: effects of Ca2+, Mn2+, and the pH gradient.

The effects of external pH, temperature, and Ca2+ and Mn2+ concentrations on the compartmentation and NMR visibility of inorganic phosphate (Pi) were studied in isolated rat liver mitochondria respiring on succinate and glutamate. Mitochondrial matrix Pi is totally visible by NMR at 8 degrees C and at low external concentrations of Pi. However, when the external Pi concentration is increased above 7 mM, the pH gradient decreases, the amount of matrix Pi increases, and the fraction not observed by NMR increases. Raising the temperature to 25 degrees C also decreases the pH gradient and the Pi fraction observed by NMR. At physiologically relevant concentrations, Ca2+ and Mn2+ do not seem to play a major role in matrix Pi NMR invisibility. For Ca2+ concentrations above 30 nmol/mg of protein, formation of insoluble complexes will cause loss of Pi signal intensity. For Mn2+ concentrations above 2 nmol/mg of protein, the Pi peak can be broadened sufficiently to preclude detection of a high-resolution signal. The results indicate that mitochondrial matrix Pi should be mostly observable up to 25 degrees C by high-resolution NMR. While the exact nature of the NMR-invisible phosphate in perfused or in vivo liver is yet to be determined, better success at detecting and resolving both Pi pools by NMR is indicated at high field, low temperature, and optimized pulsing conditions.     

Characterization of the ribulosebisphosphate carboxylase-carbon dioxide-divalent cation-carboxypentitol bisphosphate complex

Ribulosebisphosphate carboxylase forms a stable quaternary complex with CO2, divalent cation, and carboxypentitol bisphosphate. Incorporation of nonexchangeable CO2 into the complex requires the presence of a divalent cation. MG2+, Mn2+, or Co2+ supports stoichiometric binding of CO2 activator. When the quaternary complex is formed in the presence of saturating CO2, stoichiometric amounts of cation are bound in a nonexchangeable fashion. Incorporation of Mn2+ into an enzyme-CO2-Mn2+-carboxypentitol bisphosphate complex permitted investigation of cation environment by electron spin resonance (ESR) techniques. Measurements at 9 and 35 GHz suggest rhombic distortion of the coordination sphere of bound Mn2+. A complex inner sphere liganding of the cation bound in the quaternary complex would account for both the ESR spectra and the marked stability of the complex with respect to cation exchange.     

Vanadyl-induced Fenton-like reaction in RNA. An ESR and spin trapping study.

Vanadyl (VO2+) complexed to RNA reacts with hydrogen peroxide in a Fenton-like manner producing hydroxyl radicals (.OH). The hydroxyl radicals can be spin trapped with 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) forming the DMPO-OH spin adduct. In addition, in the presence of ethanol the formation of the hydroxyethyl radical adduct of DMPO (DMPO-ETOH) confirms the production of hydroxyl radicals by the RNA/VO2+ complex. When the reaction between the RNA/VO2+ complex and H2O2 is carried out in the presence of the spin trap 2-methyl-2-nitrosopropane (MNP), radicals produced in the reaction of .OH with RNA are trapped. Base hydrolysis of the MNP-RNA adducts (pH 12) followed by a reduction in the pH to pH 7 after hydrolysis is complete, yields an MNP adduct with a well-resolved ESR spectrum identical to the ESR spectrum obtained from analogous experiments with poly U. The ESR spectrum consists of a triplet of sextets (aN = 1.48 mT, a beta N = 0.25 mT and a beta H = 0.14 mT), indicating that the unpaired nitroxide electron interacts with the nuclei of a beta-nitrogen and beta-hydrogen. The results suggest that the .OH generated in the RNA/VO2+ reaction with H2O2 add to the C(5) carbon of uracil forming a C(6) carbon centered radical. This radical is subsequently spin trapped by MNP.     

Regulation of the monovalent cation permeability of brain mitochondria

The Na+ and K+ conductances of rat brain mitochondria were estimated from rates of metabolically dependent swelling and uncoupling of respiration. These were maximal in the presence of EDTA plus Pi. Pi could not be replaced with acetate. Na+ conductance was greater than that of K+ and was therefore examined in greater detail. According to the influences of N-ethylmaleimide, internal Pi (exogenous and perhaps endogenous) promoted Na+ permeability. Treatment with the ionophore A23187 obviated the Pi requirement although EDTA was still necessary. The stimulation by EDTA with Pi or A23187 and inhibition by exogenous Mg2+ suggested endogenous polyvalent cations could also regulate Na+ conductance. The influence of these substances upon endogenous Mg2+ (and Ca2+) levels is consistent with such a role of membrane-bound Mg2+. Low levels of ruthenium red (150 pmol/mg) inhibit Na+ permeation, indicating that the number of 'sites' or 'channels' involved may be small. The Ca2+ uniport is not directly involved in Na+ flow according to its greater sensitivity to inhibition by ruthenium red.     

On the relationship between the uncoupler-induced efflux of K+ from heart mitochondria and the oxidation-reduction state of pyridine nucleotides

Respiring heart mitochondria exchange matrix 42K+ with extramitochondrial K+ at a rapid rate in the presence of Pi (Chávez, E., Jung, D. W., and Brierley, G. P. (1977) Arch. Biochem. Biophys. 183, 460-470, 1977). This exchange reaction is strongly inhibited by uncouplers. However, under two rather similar sets of conditions, the addition of an uncoupler results in a rapid, transient increase in the exchange of matrix 42K+ with external K+ when the mitochondria are suspended in KCl or, alternatively, in a net loss of matrix 42K+ from mitochondria suspended in K+-free media. These conditions are: (a) the addition of an uncoupler to respiring mitochondria after the accumulation of a small amount of phosphate salt, and (b) the presence of a Ca2+-chelator or ruthenium red with uncoupler. Loss of 42K+ under these conditions occurs with all substrates tested, is completely blocked by rotenone, and is accompanied by an almost complete oxidation of both NADH and NADPH. In the presence of rotenone and acetoacetate, only NADH is oxidized and 42K+ efflux does not occur. It is concluded that simply dissipating the mitochondrial protonmotive force by addition of an uncoupler is not sufficient to induce release of mitochondrial K+. Uncoupler-induced oxidation of mitochondrial NADPH, in conjunction with elevated internal Pi, opens a rather nonspecific pathway for K+ loss which can be inhibited by ADP and enhanced by Ca2+. The more specific loss of K+ which occurs in the absence of elevated internal Pi when uncoupler and EGTA or ruthenium red are present suggests that K+ efflux is related to the Ca2+-uniporter. Loss of K+ by either of these pathways can be differentiated from efflux of K+ on the endogenous K+/H+ exchanger which functions without dissipation of the mitochondrial membrane potential.     

Calcium-induced cooperativity of manganese binding to concanavalin A.

Titrations employing electron spin resonance spectroscopy and equilibrium dialysis studies have revealed that Mn2+ binding to concanavalin A is cooperative in the presence and noncooperative in the absence of Ca2+. The degree of cooperativity increases with increasing pH. Hill coefficients range from 1.4 at pH 5.0 to 1.8 at pH 6.85. In addition to inducing cooperativity in Mn2+ binding, Ca2+ influences the pH dependence and increases the affinity of Mn2+ binding. In contrast to previous suggestions based mostly on work conducted near pH 5, demetallized concanavalin A does bind Ca2+ with an appreciable binding constant. These observations indicate that at physiological pH the role of metal ions in determining functional properties of concanavalin A is different from that suggested by metal binding studies conducted at lower pH values.     

Absence of generation of oxygen-containing free radicals with 4'-deoxydoxorubicin, a non-cardiotoxic anthracycline drug

In vitro ESR studies of doxorubicin and 4'-deoxydoxorubicin have been conducted using the spin traps PBN and DMPO. Solutions prepared in the presence of atmospheric oxygen at pH = 12 exhibited a typical semiquinone signal preceded by a short-lived signal attributed to perhydroxyl radical, HO2. At physiological pH the perhydroxyl signal was observed in the doxorubicin solution but not in 4'-deoxydoxorubicin. In the absence of oxygen, at pH = 7.5, neither drug exhibited a signal. A relationship is proposed between the perhydroxyl radical and the cardiotoxicity observed in doxorubicin but not in 4'-deoxydoxorubicin.     

Intramitochondrial phospholipase activity and the effects of Ca2+ plus N-ethylmaleimide on mitochondrial function.

Liver mitochondria treated with N-ethylmaleimide can accumulate Ca2+ but cannot retain it. Ca2+ loss following uptake occurs in parallel with a proton uptake and collapse of the membrane potential. Respiration is not activated during Ca2+ release and cannot be stimulated by uncoupler. After Ca2+ release and accompanying phenomena are nearly complete, the mitochondria undergo a large amplitude swelling. Nupercaine inhibits the premature release of Ca2+, proton uptake, decline in membrane potential, inhibition of uncoupler-stimulated respiration, and large amplitude swelling. Ruthenium red also prevents these effects. Neither Sr2+ or Mn2+ will substitute for Ca2+ to induce these effects in N-ethylmaleimide-treated mitochondria. The effects of N-ethylmaleimide plus Ca2+ on mitochondria are not accompanied by a significant alteration in the content or composition of phospholipids but are accompanied by small increases in the mitochondrial content of free fatty acids. Free fatty acids accumulate more rapidly in response to limited Ca2+ loading in the absence of N-ethylmaleimide than they do in its presence. In the absence of N-ethylmaleimide, polyunsaturated fatty acids and saturated plus monounsaturated fatty acids accumulate at nearly equal rates. In the presence of N-ethylmaleimide, polyunsaturated fatty acids accumulate more rapidly than saturated plus monounsaturated fatty acids. Any condition or agent tested which inhibited swelling and the other effects produced by Ca2+ plus N-ethylmaleimide also prevented the more rapid accumulation of polyunsaturated, compared to saturated plus monounsaturated, fatty acids. In the light of a positional analysis of phospholipid acyl moieties, these data suggest that 1-acyllysophospholipids accumulate in swelling mitochondria but not in response to noraml Ca2+ loading or when swelling is blocked by other agents. The free fatty acid accumulation, per se, is not responsible for swelling, but levels of exogenous palmitic acid as low as 1 nmol/mg of protein dramatically alter the dependence of swelling velocity on Ca2+ concentration, producing a shift from a sigmoidal- to a hyperbolic-like relationship. This same alteration is brought about by aging the mitochondrial preparation at 0 degrees C. Either pyruvate or DL-carnitine prevents the effect of exogenous palmitate and restores the Aa2+ swelling dependence of aged N-ethylmaleimide-treated mitochondria to that of fresh N-ethylmaleimide-treated mitochondria. Intramitochondrial acylcoenzyme A or acylcarnitine, or both, therefore, to be the modulator of Ca2+ sensitivity rather than free fatty acid. The findings are discussed in terms of the role of intramitochondrial phospholipase and other phospholipid metabolizing enzymes in the mechanisms of N-ethylmaleimide plus Ca2+ effects on mitochondria.     

Divalent cation binding to wheat germ calmodulin

The Ca2+ binding to plant (wheat germ) calmodulin was measured in 0.1 M NaCl by a flow-dialysis method. The four macroscopic binding constants best fitted to the data were 0.20, 0.25, 0.025, and 0.0024 microM-1. The cysteine residue of this calmodulin is located at the 27th position from the NH2-terminal (Yazawa, M. et al. (1982) Abstr. 33th Conf. Protein Structure pp. 9-12, Osaka). According to the quantitative analysis of the reaction of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) with Cys 27, the calmodulin which binds 3 Ca2+ showed the minimum reactivity with DTNB. This suggests that the site for the third Ca2+ binding is located close to Cys 27. Cys 27 was spin-labeled with N-(2,2,6,6-tetramethyl-4-piperidine-1-oxyl)maleimide, and its ESR spectrum was measured in the presence of Mn2+ and/or Ca2+. The rotational relaxation time of the label (1.2 ns) was increased by about one-tenth with 1 to 2 mol of bound Ca2+, but was unchanged with Mn2+. On the other hand, Mn2+ induced a remarkable quenching of the spectrum. From the decrease in the peak heights of the ESR spectrum, the distance between the label and the first bound Mn2+ was estimated to be 0.8 nm. It is concluded that the first Mn2+ binds to a domain near the NH2-terminal. The difference UV absorption spectrum induced by Mn2+ was similar to that induced by Ca2+. However, the amount of Mn2+ needed to saturate the difference spectrum was 1 mol more than the amount of Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electron spin resonance study of chloroplast photosynthetic activity in the presence of amphiphilic amines

Electron spin resonance spectroscopy (ESR) was used to study the effects of amphiphilic amines of the carbamate, amide, and ester type and amine oxide on the photosynthetic system of spinach chloroplasts. The ESR signal II connected to the photosynthetic center PS II donor side was observed to diminish in the presence of amines, whereas that of PS I remained unchanged. The inhibition of PS II increased with the increasing of amine concentration. In the presence of amines, the light: dark chloroplast ESR signals ratio as well as the intensity of the ESR signal of unbound Mn2+ increased. It is suggested that the amphiphilic amines affect the structure of PS II and the electron transfer to PS I. The effects of the amines tested on the photosynthetic system correlate with their potency to perturb the lipid membrane structure.     

Hydroperoxide-induced radical production in liver mitochondria

When isolated rat liver mitochondria are treated with tert-butyl hydroperoxide in the presence of the spin trap 5,5-dimethyl-1-pyrroline-N-oxide, a six-line ESR signal is observed with parameters characteristic of a carbon-centered radical. The radical is shown to be CH3. using 2-methyl-2-nitrosopropane as the spin trap. Inhibition of radical production by EDTA and N-ethylmaleimide provides evidence for participation by metals and reduced sulfhydryl groups in the radical-generating reaction. It is proposed that radicals are formed through the reaction between a reducing agent, a metal and the hydroperoxide.     

Formation of artifactual DMPO-OH spin adduct in acid solutions containing nitrite ions

We investigated aqueous solutions containing nitrite ions and DMPO (5,5-dimethyl-1-pyrroline-N-oxide) by electron spin resonance (ESR) in the pH range from 1 to 6. A DMPO-OH signal was observed below pH 3.0 in the presence of nitrite ions, whereas in the absence of nitrite ion, an extremely weak signal was observed below pH 1.5. Addition of methanol, a hydroxyl radical scavenger, to this system did not lead to the appearance of a detectable DMPO-CH₂OH signal. The possibility of this DMPO-OH signal being due to a genuine spin trapping process with hydroxyl radical was, therefore, ruled out. The reactivities of reactive nitrogen species (RNS) in this system with DMPO have also been investigated by density functional theory (DFT) at the IEFPCM (water)/B3LYP/6–311?+?G ** level of theory. On the basis of the pH dependence of the signal intensity and the redox potential E° (versus SHE) calculated by DFT theory, we propose that the origin of this signal is “inverted spin trapping” via one-electron oxidation of DMPO by H₂ONO⁺, followed by the nucleophilic addition of water. Prevention of these false-positive results when detecting hydroxyl radical using ESR spin trapping requires an awareness of both the presence of nitrite ions in the solution and the solution pH.     

Study of the binding of substrates and paramagnetic Mn2+ ions with phosphoglycerate kinase by saturating ESR spectra of a spin-labelled protein]

A single SH-group of phosphoglycerate kinase from yeast was modified by mercury-containing spin label. The saturation curves of ESR spectra of the spin-labeled enzyme were studied. The paramagnetic ions of Mn2+ bound to the centre of ion nonspecific binding or active centre in the complex with ATP can influence the saturation of the spin-labeled enzyme. The saturation curves of the ESR signal of the spin-labeled enzyme in the presence of paramagnetic complex of CrATP were studied. It has been demonstrated that the second nonspecific centre of ATP binding is located at the active site of the enzyme (3-phosphoglycerate binding centre).     

Evidence for monovalent phosphate transport in Ehrlich ascites tumor cells

In an effort to determine whether the Na+-dependent Pi transport system of Ehrlich ascites tumor cells exhibits specificity for H2PO4- or HPO4(-2), Pi fluxes were determined by measuring 32Pi-Pi self-exchange. Three experimental approaches were employed. First, the effect of pH on steady-state Pi transport at 0.5 and 5 mM was studied. Second, the relationship between Pi transport and Pi concentration (0.25-9.2 mM) at pH 5.6 and 7.9 was determined. Third, the dependence of Pi transport on [H2PO4-] (0.05-4.2 mM) at constant [HPO4(-2)] (0.5 mM), and the converse, [HPO4(-2)] (0.06-4.5 mM) at constant [H2PO4-] (0.5 mM), was evaluated. Ks (apparent half-saturation constant) and Jmax (maximal transport rate) were calculated by two methods: weighted linear regression (WLR) and a nonparametric procedure. The dependence of Pi flux on pH indicates that optimum transport occurs at pH 6.9. Pi transport decreases as pH is reduced when extracellular Pi is either 0.5 or 5 mM. However, at pH 7.9, Pi flux is reduced only in 0.5 mM Pi. At pH 5.6, H2PO4- comprises 93% of the total Pi present, and the calculated Ks is 0.055 +/- 0.026 mM (WLR). This is the same as the Ks determined from the initial phase of the flux vs. [H2PO4-] relationship (0.056 +/- 0.020 mM). However, at pH 7.9 (where 94% of Pi is HPO4(-2)), the measured Ks is 0.58 +/- 0.11 mM (WLR), which is ten times higher than at pH 5.6. This value is also five times greater than the Ks calculated from the flux vs. [HPO4(-20)] curve (0.106 +/- 0.16 mM). Kinetic parameters calculated by the nonparametric method, though somewhat different, gave similar relative results. Taken together, these results support two conclusions: (1) H2PO4- is the substrate for the Na+-dependent Pi transport system of the Ehrlich cell, and (2) H+ can inhibit Pi transport.     

Pi in equilibrium ATP exchange in the absence of proton gradient by the H+-ATPase from yeast plasma membranes

Purified soluble H+-ATPase from Schizosaccharomyces pombe catalyzes a Pi in equilibrium ATP exchange in the absence of a H+ gradient. When the pH of the assay medium is raised from 5.5 to 8.0 there is a decrease of the ATPase activity and an increase of the rate of Pi in equilibrium ATP exchange. At pH 7.0 the addition of the organic solvent dimethyl sulfoxide (20%, v/v) promotes a decrease of ATPase activity and an increase of the Pi in equilibrium ATP exchange reaction. The effect of the organic solvent on the Pi in equilibrium ATP exchange is related to a decrease of the apparent Km for Pi.     

The formation of enzyme-bound and medium pyrophosphate and the molecular basis of the oxygen exchange reaction of yeast inorganic pyrophosphatase.

Yeast inorganic pyrophosphatase, with 10 mM 32Pi and 10 mM Mg2+ present at pH 7.3 TO 7.6, rapidly forms enzyme-bound pyrophosphate equivalent to about 5% of the total catalytic sties on the two enzyme subunits. The enzyme thus appears to bind PPi so as to favor thermodynamically its formation from Pi. The enzyme catalyzes a measurable equilibrium formation of free PPi at a much slower rate. Under similar conditions, the enzyme catalyzes a rapid exchange of oxygen atoms between Pi and water with the relative activation by metals being Mg2+ greater than Zn2+ greater than Co2+ greater than Mn2+. Millisecond mixing and quenching experiments demonstrate that the rate of formation and cleavage of the enzyme-bound PPi is rapid enough to explain most or all of the oxygen exchange reaction.     

Effects of hormones and N6O2''-dibutyryl-adenosine 3'' :5''-cyclic monophosphate, administered in vivo, on phosphate transport and metabolism in isolated rat liver mitochondria.

1. The administration of glucagon or N6O2'-dibutyryl cyclic AMP to fed rats by intraperitoneal injection was associated with a 2-fold increase in the amounts of endogenous Pi and ATP, and an increase in the rate and extent of transport of exogenous Pi (measured in either the presence or the absence of Ca2+) in mitochondria subsequently isolated from the liver. No change was observed in either the maximum rate of transport of exogenous Pi or in the rate of 32Pi exchange. 2. The changes induced by glucagon and dibutyryl cyclic AMP were markedly decreased by the co-administration of cycloheximide. 3. The administration of insulin to rats resulted in an increase of about 1.3-fold in the concentration of endogenous mitochondrial Pi 4. The amounts of endogenous Pi in mitochondrial isolated from the livers of starved rats were 3 times those in mitochondria isolated from fed animals. 5. It is concluded that the liver mitochondrial phosphatetransport system may be an important site of hormone action. 6. In the course of these experiments, it was shown that Ca2+ markedly stimulates mitochondrial phosphate transports.     

Electron-paramagnetic-resonance studies of manganese(II) complexes with elongation factor Tu from Bacillus stearothermophilus. Observation of a GTP hydrolysis intermediate state complex

Changes in the coordination of Mn2+ to nucleotide, water and protein at the active site of elongation factor Tu (EF-Tu) have been studied by electron paramagnetic resonance (EPR) spectroscopy. From the time dependence of the Mn2+ spectrum after addition of GTP to EF-Tu X Mn, it was apparent that three complexes with different EPR linewidths could be detected. Using additional information from the kinetics of 32Pi production and release from EF-Tu X Mn X [gamma-32P]GTP these were identified as EF-Tu X Mn X GTP (linewidth 4.2 mT), EF-Tu X Mn X GDP X Pi (1.20 mT) and EF-Tu X Mn X GDP (1.29 mT). The linewidth for EF-Tu X Mn was 1.51 mT. The rate constant for GTP cleavage on EF-Tu was 0.01 min-1 at 24 C, for Pi release from the EF-Tu X GDP X Pi complex 0.0033 min-1. The corresponding rate constants in the presence of Mg2+ were 0.003 min-1 and 0.0065 min-1. The rate constant for reversal of the cleavage step was found to be much smaller than that for the rate of Pi release (and consequently much smaller than in the forward direction), as shown by 31P-NMR experiments on the incorporation of 18O into Pi from GTP hydrolyzed in the presence of H2 18O. EPR experiments using specifically 17O-labelled GTPs demonstrated an interaction of Mn2+ with the beta-phosphate in both the EF-Tu X GDP X Pi and EF-Tu X GDP complexes. Inorganic phosphate in the EF-Tu X GDP X Pi complex was found not to interact with the metal ion. From EPR experiments in H2 17O, it was concluded that the most probable number of water molecules in the different complexes was 4 (EF-Tu X Mn), 5 (EF-Tu X Mn X GDP X Pi) and 3 (EF-Tu X Mn X GDP), with 2, 0 and 2 metal-protein interactions respectively.     

Studies on an activator of the (Ca2+ plus Mg2+)-ATPase of human erythrocyte membranes.

1. An activator of the (Ca2+ plus Mg2+)-stimulated ATPase present in the human erythrocytes (membrane) has been isolated in soluble form from hemolysates of these cells. Partial purification has been achieved through use of carboxymethyl-Sephadex chromatography. The resulting activator fraction contained no hemoglobin and only 0.3% of the total adenylate kinase activity of the cell. 2. Whereas the activator was released from erythrocytes subjected to hemolysis in 20 miosM buffer at pH 7.6 or at pH 5.8, only the membranes prepared at pH 7.6 were affected by it. 2. Whereas the activator was released from erythrocytes subjected to hemolysis in 20 miosM buffer at pH 7.6 or at pH 5.8, only the membranes prepared at pH 7.6 were affected by it. 3. When (Ca2+ plus Mg2+)-ATPase activity was measured by 32Pi release from (gamma-32P)ATP, freeze-thawed erythrocytes, as well as membranes prepared at pH 5.8 and at pH 7.6, expressed lower values than noted by assay for total Pi release. When ADP instead of ATP was used as substrate, significant amount of Pi were released by these erythrocyte preparations. Further study revealed (a) production of ATP and AMP from ADP with membranes and hemolysate alone, and (b) exchange of the gamma-and B-position phosphate on (gama-32P)ATP in the presence of membranes plus hemolysates. These observations established the presence of adenylate kinase activity in the (membrane-free) hemolysates and in membranes. It further supports the conclusion that Pi release from ADP by human erythrocytes (freeze-thawed) and by their isolated membranes is due to formation of ATP by adenylate kinase and hydrolysis of this generated ATP by (Ca2+ plus Mg2+)-ATPase. 4. The following points were also established: (a) absence of an ADPase in human erythrocytes; (b) the (Ca2+ plus Mg2+)-ATPase activator enhanced cleavage only of the gama-position of ATP and (c) the (Ca2+ plus Mg2+)-ATPase activator is neither adenylate kinase nor hemoglobin.