The (Na + K)-dependent ATPase: mode of inhibition of ADP/ATP exchange activity by MgCl2

Na⁺-dependent ADP/ATP exchange activity, of a (Na⁺ + K⁺)-dependent ATPase preparation from eel electric organ, was measured in terms of the incorporation of ¹⁴C into ATP during incubations with unlabeled ATP and [¹⁴C]ADP. Estimates of initial rates of exchange were possible by keeping changes in nucleotide concentrations, from both exchange and extraneous hydrolytic processes, to less than 10%. Under these conditions, increases in MgCl₂ concentration, from 0.2 to 3 mM, generally inhibited this exchange activity. The concentrations of free Mg²⁺, Mg · ATP, and Mg · ADP present, with a range of MgCl₂, ATP, and ADP concentrations, were calculated from measured dissociation constants. Inhibition was associated with Mg · ATP as well as with Mg²⁺, at concentrations from 0.4 to 1 mM (Mg · ADP, in the same concentration range, probably inhibited also). The affinity of the enzyme for these inhibitors is in fair correspondence with demonstrated affinities for Mg²⁺, Mg · ATP, and Mg · ADP at low affinity substrate sites, measured kinetically. These observations are considered in terms of a dimeric enzyme with high and low affinity substrates sites: ADP/ATP exchange being catalyzed at the high affinity sites, with inhibition occurring through occupancy by Mg²⁺, Mg · ATP, or Mg · ADP, of the low affinity sites, thereby pulling the reaction process away from those steps involved in exchange.     

Some properties of, and the effect of temperature on the (Mg + Ca) ATPase from immature and adult rat brain

1. 1. (Mg²⁺ + Ca²⁺) ATPases of microsomal and synaptic membrane preparations from immature and adult rat brain were activated by calcium (0.1–10 μM), maximal activation was found at 3 μM. The increase in (Mg²⁺ + Ca²⁺) ATPase seen during development was greatest in the synaptic membrane preparations.

2. 2. At 37°C both Na⁺ or K⁺ at concentrations higher than 30 mM inhibited the microsomal Mg²⁺ ATPase, but the (Mg²⁺ + Ca²⁺) ATPase was stimulated by both Na⁺ and K⁺. Synaptic membrane Mg²⁺ ATPase was inhibited by concentrations higher than 100 mM K⁺; Na⁺ however stimulated this enzyme at all concentrations. Much of this Na⁺ stimulated activity was ouabain sensitive. Synaptic membrane (Mg²⁺ + Ca²⁺) ATPase was stimulated by Na⁺ or K⁺, this stimulation follows approximate saturation kinetics with an apparent K_m of 18.8 mM Na⁺ or K⁺.

3. 3. Arrhenius plots of microsomal (Mg²⁺ + Ca²⁺) ATPase were curvilinear, but two intersecting lines with a break at 20°C could be fitted. The calculated energies of activation from these lines were very similar in immature and adult preparations. The synaptic membrane preparation (adult) also gave a curvilinear plot; but two intersecting lines with a break at 25°C could be fitted to the data. These lines had slopes of 21 and 28 Kcal mole⁻¹ above and below the break, respectively. The immature preparation when made using EDTA gave a Arrhenius plot of very similar form to the adult preparation. Without EDTA however the Arrhenius plot was complex with a plateau at 25–32°C. Pretreatment with EDTA activated the synaptic membrane (Mg²⁺ + Ca²⁺) ATPase from both immature and adult brain.

Properties and function of clostridial membrane ATPase

ATPase (ATP phosphohydrolase, EC 3.6.1.3) was detected in the membrane fraction of the strict anaerobic bacterium, Clostridium pasteurianum. About 70% of the total activity was found in the particulate fraction. The enzyme was Mg²⁺ dependent; Co²⁺ and Mn²⁺ but not Ca²⁺ could replace Mg²⁺ to some extent; the activation by Mg²⁺ was slightly antagonized by Ca²⁺. Even in the presence of Mg²⁺, Na⁺ or K⁺ had no stimulatory effect. The ATPase reaction was effectively inhibited by one of its products, ADP, and only slightly by the other product, inorganic phosphate. Of the nucleoside triphosphates tested ATP was hydrolyzed with highest affinity ([S]_{0.5 V} = 1.3 mM) and maximal activity (120 U/g). The ATPase activity could be nearly completely solubilized by treatment of the membranes with 2 M LiCl in the absence of Mg²⁺. Solubilization, however, led to instability of the enzyme.

The clostridial solubilized and membrane-bound ATPase showed different properties similar to the “allotopic” properties of mitochondrial and other bacterial ATPases. The membrane-bound ATPase in contrast to the soluble ATPase was sensitive to the ATPase inhibitor dicyclohexylcarbodiimide (DCCD). DCCD, at 10^-4 M, led to 80% inhibition of the membrane-bound enzyme; oligomycin, ouabain, or NaN₃ had no effect. The membrane-bound ATPase could not be stimulated by trypsin pretreatment.

Since none of the mono- or divalent cations had any truly stimulatory effect, and since a pH gradient (interior alkaline), which was sensitive to the ATPase inhibitor DCCD, was maintained during growth of C. pasteurianum, it was concluded that the function of the clostridial ATPase was the same as that of the rather similar mitochondrial enzyme, namely H⁺ translocation. A H⁺-translocating, ATP-consuming ATPase appears to be intrinsic equipment of all prokaryotic cells and as such to be phylogenetically very old; in the course of evolution the enzyme might have been developed to a H⁺-(re)translocating, ATP-forming ATPase as probably realized in aerobic bacteria, mitochondria and chloroplasts.     

Effect of trinitrophenylation on myosin ATPase

1. The enzymic and actin binding properties of myosins trinitrophenylated to different extents in the presence or absence of ATP have been studied.

2. The enzymic properties of myosin trinitrophenylated in the absence of ATP are different from those of myosin treated in the presence of ATP even on trinitrophenylating an equal number of lysyl residues. On trinitrophenylation in the absence of ATP the EDTA-(K⁺-)activated ATPase and Ca²⁺-activated ATPase decrease while the Mg²⁺-activated ATPase considerably increases. In the presence of ATP the enzymic properties of myosin are much less affected by trinitrophenylation.

3. The actin binding capacity of trinitrophenylated myosin does not change, although its enzymic properties may be greatly altered, and even if its property to be activated by actin is completely lost.     

Association and dissociation of the thick and thin filaments within myofibrils in conditions of “contraction” and “relaxation”

1. The current assumption that the low ATPase activity of relaxed myofibrils is represented by the ATPase activity of myosin which has been set free during the dissociation of actomyosin was investigated. For this purpose, the ATPase activity of relaxed skeletal myofibrils of the rabbit and of the crab Maia squinado has been compared with the activity of contracted fibrils and of purified rabbit myosin in conditions of varying ionic strength, pH and concentrations of MgATP (i.e. MgATP²⁻ + MgHATP⁻) and Mg²⁺.

2. Contraction and relaxation of the fibrils was induced by changing the concentration of Ca²⁺ from about 5×10⁻⁵ to below 1×10⁻⁸ M.

3. In all conditions studied, the ATPase activity of relaxed fibrils was about 6–8 times less than that of the contracted fibrils, but it remained a typical actomyosin ATPase.

4. Quantitatively and qualitatively, this ATPase differs from the ATPase of myosin. For instance, its dependence on pH is the reverse of that of the myosin ATPase.

5. Calculation showed that the fibrils are dissociated by 90% in conditions of relaxation. Since the ATPase activity of myosin was merely some 2% of the actomyosin activity, the major part of the ATPase of fibrils, even at a dissociation of 90%, is bound to show the properties of the ATPase of actomyosin.

6. However, a dissociation of 90% cannot be distinguished from a dissociation of 100% by means of physical methods (viscosity, superprecipitation, resistance to stretch, etc.). This explains why physical methods indicate a “full” dissociation of actomyosin although, enzymatically, the ATPase is still of the actomyosin type.

7. The possible reasons are discussed for the discrepancy between the 100-fold increase in the ATP turnover and the 1000-fold increase in energy turnover of the living muscle during the transition from relaxed to active state. The most probable explanation seems to be an ATPase activity of myosin which is too high by a factor of ten as compared to the energy turnover of living muscle at the resting state. This high activity cannot be caused by a contamination of the myosin by Ca²⁺-insensitive actomyosin.     

Inhibition of oxidative phosphorylation by Ca or Sr: A competition with Mg for the formation of adenine nucleotide complexes

Intramitochondrial Sr²⁺, similar to Ca²⁺, inhibits oxidative phosphorylation in intact rat-liver mitochondria. Both Ca²⁺ and Sr²⁺ also inhibit the hydrolytic activity of the ATPase in submitochondrial particles. Half-maximal inhibition of ATPase activity was attained at a concentration of 2.5 mM Ca²⁺ or 5.0 mM Sr²⁺ when the concentration of Mg²⁺ in the medium was 1.0 mM. The inhibition of ATPase activity by both cations was strongly decreased by increasing the Mg²⁺ concentration in the reaction medium. In addition, kinetical data and the determination of the concentration of MgATP, the substrate of the ATPase, in the presence of different concentrations of Ca²⁺ or Sr²⁺ strongly indicate that these cations inhibit ATP hydrolysis by competing with Mg²⁺ for the formation of MgATP. On the basis of a good agreement between these results with submitochondrial particles and the results of titrations of oxidative phosphorylation with carboxyatractyloside or oligomycin in mitochondria loaded with Sr²⁺ it can be concluded that intramitochondrial Ca²⁺ or Sr²⁺ inhibits oxidative phosphorylation in intact mitochondria by decreasing the availability of adenine nucleotides to both the ADP/ATP carrier and the ATP synthase.     

Studies on the exchange of G-actin-bound calcium with bivalent cations

1. The possibility of the replacement of G-actin-bound calcium by various bivalent cations has been investigated. After the reaction with all cations studied, with the exception of Cu²⁺, action remains active, i.e., contains bound ATP and polymerizes in 0.1 M KCl.

2. The amount of G-actin-bound calcium, as well as the sum of bivalent cation after replacement, not removable by short-time Dowex-50 treatment, accounts to about 1 mole per 50000 g of G-actin.

3. The rate of exchange is of the same order for bivalent cations studied, including calcium.

4. G-actin-bound Ca²⁺ is fully replaced, besides free Ca²⁺, by free Mn²⁺ and Cd²⁺. The replacement with Mg²⁺, Co²⁺, Ni²⁺ and Zn²⁺ is not complete, and there is practically no reaction with Ba²⁺ and Sr²⁺.

5. Assuming the affinity constant of Ca²⁺ as 1, the following affinity constants for other bivalent cations were obtained: Mn²⁺, 0.90; Cd²⁺, 1.07; Mg²⁺, 0.27; Zn²⁺, 0.22; Co²⁺, 0.18; Ni²⁺, 0.08.

6. The results obtained show that there exists a close correlation between the ionic radius of a particular bivalent cation, and its ability to replace bound Ca²⁺.     

Purification and properties of ATPase inhibitor from rat liver mitochondria

(1) The ATPase inhibitor protein has been isolated from rat liver mitochondria in purified form. The molecular weight determined by sodium dodecyl sulfate gel electrophoresis is approximately 9500, and the isoelectric point is 8.9.

(2) The protein inhibits both the soluble ATPase and the particle-bound ATPase from rat liver mitochondria. It also inhibits ATPase activities of soluble F₁, and inhibitor-depleted submitochondrial particles derived from bovine heart mitochondria.

(3) On particle-bound ATPase the inhibitor has its maximal effect if incubated in the presence of Mg²⁺. ATP at slightly acidic pH.

(4) The inhibitor has a minimal effect on P_i-ATP exchange activity in sonicated submitochondrial particles. However, unexpectedly the inhibitor greatly stimulates P_i-ATP exchange activity in whole mitochondria while the low ATPase activity of the mitochondria is not affected. The possible mechanism of action of the inhibitor on intact mitochondria is offered.     

Modulation of ouabain-insensitive Na(+)-ATPase activity in the renal proximal tubule by Mg(2+), MgATP and furosemide

In addition to the (Na⁺+K⁺)ATPase another P-ATPase, the ouabain-insensitive Na⁺-ATPase has been observed in several tissues. In the present paper, the effects of ligands, such as Mg²⁺, MgATP and furosemide on the Na⁺-ATPase and its modulation by pH were studied in the proximal renal tubule of pig. The principal kinetics parameters of the Na⁺-ATPase at pH 7.0 are: (a) K_0.5 for Na⁺=8.9±2.2 mM; (b) K_0.5 for MgATP=1.8±0.4 mM; (c) two sites for free Mg²⁺: one stimulatory (K_0.5=0.20±0.06 mM) and other inhibitory (I_0.5=1.1±0.4 mM); and (d) I_0.5 for furosemide=1.1±0.2 mM. Acidification of the reaction medium to pH 6.2 decreases the apparent affinity for Na⁺ (K_0.5=19.5±0.4) and MgATP (K_0.5=3.4±0.3 mM) but increases the apparent affinity for furosemide (0.18±0.02 mM) and Mg²⁺ (0.05±0.02 mM). Alkalization of the reaction medium to pH 7.8 decreases the apparent affinity for Na⁺ (K_0.5=18.7±1.5 mM) and furosemide (I_0.5=3.04±0.57 mM) but does not change the apparent affinity to MgATP and Mg²⁺. The data presented in this paper indicate that the modulation of the Na⁺-ATPase by pH is the result of different modifications in several steps of its catalytical cycle. Furthermore, they suggest that changes in the concentration of natural ligands such as Mg²⁺ and MgATP complex may play an important role in the Na⁺-ATPase physiological regulatory mechanisms.     

The number and localisation of adenine nucleotide-binding sites in beef-heart mitochondrial ATPase (F1) determined by photolabelling with 8-azido-ATP and 8-azido-ADP

1. When irradiated 8-azido-ATP becomes covalently bound (as the nitreno compound) to beef-heart mitochondrial ATPase (F₁) as the triphosphate, either in the absence or presence of Mg²⁺, label covalently bound is not hydrolysed.

2. In the presence of Mg²⁺ the nitreno-ATP is bound to both the and β subunits, mainly (63%) to the subunits.

3. After successive photolabelling of F₁ with 8-azido-ATP (no Mg²⁺) and 8-azido-ADP (with Mg²⁺) 4 mol label is bound to F₁, 2 mol to the and 2 mol to the β subunits.

4. When the order of photolabelling is reversed, much less 8-nitreno-ATP is bound to F₁ previously labelled with 8-nitreno-ADP. It is concluded that binding to the -subunits hinders binding to the β subunits.

5. F₁ that has been photolabelled with up to 4 mol label still contains 2 mol firmly bound adenine nucleotides per mol F₁.

6. It is concluded that at least 6 sites for adenine nucleotides are present in isolated F₁.     

Oxidative phosphorylation in yeast VI. ATPase activity and protein synthesis in mitochondria isolated from nuclear mutants deficient in cytochromes

1. The oligomycin-sensitive Mg²⁺-dependent ATPase activity of mitochondria isolated from wild-type yeast Saccharomyces cerevisiae was only slightly inhibited by atractyloside at concentrations which entirely prevented oxidative phosphorylation. This indicated that most of the ATPase in these mitochondrial preparations was located outside the atractyloside-sensitive barrier and did not participate in the energy-transfer process.

2. ATPase activity of mitochondria isolated from nuclear gene mutants deficient in a single cytochrome, a, b, or c, respectively, was strongly inhibited by oligomycin. The mitochondria from these mutants, like those from the wild-type strain, were able to incorporate amino acids into protein.

3. Mitochondrial ATPase activity of single nuclear gene mutants deficient in both cytochromes a and b was only slightly inhibited by oligomycin. These mitochondria were incapable of incorporating amino acids into protein. The mitochondria from these nuclear mutants thus resembled mitochondria of cytoplasmic respiration-deficient mutants.

4. The results suggest that mitochondrial cytochromes may be coded by nuclear genes and that product(s) of mitochondrial protein synthesis may be required for integrating the cytochromes a and b and the components of the oligomycin-sensitive ATPase complex into the mitochondrial membranes.     

Oxidative phosphorylation in Azotobacter vinelandii. Effect of inhibitors and uncouplers on P/O ratio, trypsin-induced ATPase and ADP-stimulated respiration

1. The effect of various inhibitors and uncouplers of mitochondrial oxidative phosphorylation on phosphorylation by Azotobacter particles is reported.

2. The ATPase activity of Azotobacter particles and of the soluble factor involved in oxidative phosphorylation is stimulated 10–20-fold by incubation with trypsin. The trypsin-induced ATPase is Mg²⁺ or Ca²⁺ dependent, Mg²⁺ being the more effective cation. The ATPase is stimulated somewhat by 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole, desaspidin and carbonyl cyandie m-chlorophenylhydrazone, and inhibited by atebrin and Dio-9.

3. In previous work ADP was shown to stimulate the oxidation of NADH but not of the Site-II substrates malate, lactate or succinate. Stimulation by ADP of oxidation of malate has now been observed by inhibiting electron transport by 2-heptyl-4-hydroxyquinoline-N-oxide or by increasing the electron flow by adding lactate. The stimulation is, therefore, not confined to phosphorylation Site I.     

The activation by Ca of the ATPase of extracted muscle fibrils with variation of ionic strength, pH and concentration of MgATP

1. The alteration of the Ca²⁺ requirements of the ATPase activity of fibrils from rabbits and crabs at varying ionic strength, pH and concentration of MgATP (i.e. MgATP²⁻ + MgHATP⁻) was investigated.

2. Under physiological conditions, it was found that the ATPase activity of rabbit and crab fibrils after an initial increase decreased steeply when the Ca²⁺ concentration is raised above 1×10⁻⁴ M. This is a primary effect of the over-optimal Ca²⁺ concentration and not a secondary one caused by the influence of accompanying ions.

3. The Ca²⁺ requirements for ATP splitting by rabbit fibrils remain constant at an ionic strength from 0.1 to 0.2 and for a MgATP concentration in the range from 0.5 to 10 mM. At I = 0.05 it is about 5 times smaller than at 0.1. When the pH is decreased from 8 to 7, the Ca²⁺ requirements are increased some 10 times but only 3 times when the pH is varied between 7 and 6.

4. In crab fibrils, there is no alteration of the Ca²⁺ requirements when the ionic strength is varied between 0.05 and 0.2, but a reduction of the pH from 8.0 to 6.0 raises the Ca²⁺ requirements for half activation and for threshold by a factor of 10. Changing the MgATP concentration increases the Ca²⁺ requirements only in the range from 1 to 5 mM, while the concentration required in 0.5 mM is identical with that at 1 mM, and 10 mM corresponds to 5 mM.

5. It can be deduced from the experimental results that at a pH above 6.0 maximal activation is always obtained if the Ca²⁺ concentration is 5×10⁻⁵ M. By contrast, relaxation is only achieved when the Ca²⁺ concentration is below 1×10⁻⁷ M for pH 7.0 and I > 0.1 or below 1×10⁻⁸ for pH > 7.0 or I < 0.1.

6. To achieve complete relaxation, an ethyleneglycoldiaminotetraacetate (EGTA) concentration of 1 mM is sufficient, even when there is a large degree of contamination by Ca²⁺ as long as the pH stays above 6.5.     

The effects of phosphate and electron transport on the carbonyl cyanide m-chlorophenylhydrazone-induced ATPase of rat-liver mitochondria

1. The effects of phosphate and electron transport on the ATPase induced in ratliver mitochondria by the uncoupler carbonyl cyanide m-chlorophenylhydrazone have been measured at different uncoupler concentrations and compared with those of ATP, oligomycin and aurovertin.

2. The inhibitory action of respiratory-chain inhibitors on the ATPase activity, which is independent of the actual inhibitor used, is greatly delayed or prevented by the presence of uncoupler, and, in the case of rotenone, can be reversed completely by the subsequent addition of succinate (in the absence of uncoupler). These results can be explained on the basis of the proposal previously made by others that coupled electron transfer causes a structural change in the ATPase complex that results in a decreased affinity of the ATPase inhibitor for the mitochondrial ATPase.

3. Inorganic phosphate specifically stimulates the ATPase activity at high uncoupler concentrations (> 0.2 μM), but has no effect at low concentrations. The stimulation is prevented or abolished by sufficiently high concentrations of aurovertin.

4. Aurovertin prevents the inhibition of the uncoupler-induced ATPase by high uncoupler concentrations.

5. It is proposed that the steady-state concentration of endogenous P_i may be an important regulator of the turnover of the ATPase in intact mitochondria and that the inhibition of ATPase activity by high concentrations of uncoupler is at least partially mediated via changes in the concentration of endogenous P_i.     

Ion transport in heart mitochondria. XIII. The effect of ethylenediaminetetraacetate on monovalent ion uptake

1. Ethylenediaminetetraacetate (EDTA) markedly activates the accumulation of Na⁺ and Li⁺ and the swelling which accompanies the ion uptake by isolated heart mitochondria. This activation is reflected in the removal of limited amounts of endogenous Mg²⁺ and extensive loss of K⁺. The removal of these cations requires the presence of Na⁺, a source of energy, and a permeant anion as well as EDTA. The effects of EDTA on the activation of Na⁺ uptake and cation removal are duplicated by chelators with a high affinity for Mg²⁺, but not by ethyleneglycol-bis-(β-aminoethylether)-N, N′-tetraacetic acid. Mg²⁺ at concentrations 5 to 6 times less than EDTA prevents both activation of Na⁺ uptake and cation removal.

2. EDTA does not appear to be bound by heart mitochondria. At neutral pH the chelator penetrates into the mitochondrial water volume to the same extent as sucrose and mannitol. At pH 8.1 where the removal of mitochondrial Mg²⁺ by EDTA is more effective, EDTA penetrates virtually the entire water volume. This penetration requires the presence of a source of energy, a transported cation such as Na⁺, and a permeant anion. It appears possible that the oscillations in ion uptake and swelling observed in the presence of EDTA at pH 8.1 may be related to the presence of the chelator in the interior compartment under these conditions.     

The relationship between the bovine heart mitochondrial adenosine triphosphatase, lipophilic compounds, and oligomycin.

The lipid-free particulate preparations of the mitochondrial ATPase require phospholipid for activity and can be inhibited by oligomycin, as has been demonstrated previously. In this communication a steady state analysis of the activation of a particulate preparation of the ATPase by phospholipids and its subsequent inhibition by oligomycin has been carried out. The relative affinity of the ATPase for purified phospholipids has been determined by measuring the Km for activation (Ka) for several phospholipids. The Ka values varied from 30 to 100 mum. The Vmax in the presence of phosphatides varies from 0.29 to 1.11 mumol ATP hydrolyzed/min/mg of protein; no correlation is noted between the relative affinity of the enzyme for a phospholipid and the V max value. Higher V max values are noted with the more acidic phospholipids, however. Sodium dodecyl sulfate and monoolein also activate with Ka values of 25 and 800 mum, respectively. Diglycerides, however, do not activate. With all lipids the ATPase activity stimulated is oligomycin-sensitive. The Ki values for oligomycin range from 0.1 to 0.6 mum. Oligomycin is a competitive inhibitor with respect to all the phospholipids tested except phosphatidylethanolamine and phosphatidyglycerol. It is also competitive with respect to sodium dodecyl sulfate (k-i equals 0.94 mum). In reciprocal plots of activity versus ATP concentration, with and without oligomycin, an intercept consistent with either mixed or partial noncompetitive inhibition kinetics is noted. Comparable K-i values for oligomycin are obtained when calculated assuming either mixed or partial noncompetitive inhibition. The Km for ATP is the same in the unactivated and the lipid activated particulate ATPase; the value obtained is slightly lower than the Km for ATP in the solubilized, purified ATPase. Using a spectrophotometric assay the time required for activation with phospholipid and inhibition with oligomycin has also been determined. This investigation suggests the possibility that activation of the ATPase is due a position to interact with the water-soluble substrate. Consistent with the above suggestion is the supposition that the lipids do not necessarily confer inhibitor sensitivity to the ATPase, but rather allow an oligomycin-sensitive activity to be expressed.     

Actomyosin-like protein from brain separation and characterization of the actin-like component

1. Actin-like protein (neurin) has been separated from actomyosin-like protein (neurostenin) isolated from bovine brain. This was accomplished by gel filtration chromatography (Sephadex G-200) and by ultracentrifugation in a continuous sucrose gradient containing 0.6 M KI.

2. The actin-like protein stimulated the Mg²⁺-ATPase activity of muscle myosin.

3. It contained bound nucleotide which exchanged with free [¹⁴C]ATP.

4. It polymerized in the presence of 0.1 M KCl and 0.1 mM Mg²⁺ with release of P_i; increase in viscosity occurred upon dilution of the 0.6 M KI to 0.1 M.

5. The neurin reacted immunologically to form a single band with antiserum to neurostenin.

6. The neurin, similar to muscle actin, contained 3-methylhistidine.

7. The sedimentation constant of the protein was 2.8 S.     

The binding of aurovertin to mitochondria, and its effect on mitochondrial respiration

1. The fluorescence of aurovertin increases about 100-fold on binding to sub-mitochondrial particles.

2. The mitochondrial ATPase (F₁) binds one mole aurovertin/mole F₁ with a dissociation constant of 6·10⁻⁸ M.

3. The fluorescence of mitochondrion-bound aurovertin is maximal during State-3 respiration and is partially quenched on anaerobiosis, addition of respiratory inhibitor, oligomycin or uncoupler, or transition to State 4. This quenching is still present when the binding site is saturated with aurovertin, showing that the quantum yield of fluorescence is lowered.

4. Aurovertin is bound co-operatively to State-3 mitochondria.

5. The curve relating inhibition of State-3 respiration to aurovertin concentration is more sharply sigmoidal than the binding curve.

6. An analysis of the binding and inhibition data leads to the conclusion that aurovertin induces a conformation change in the binding site on F₁ in two ways: (i) directly by acting as an allosteric effector of an oligomeric system, (ii) indirectly by inhibiting State-3 respiration which changes the allosteric constant of the oligomeric system.

7. The concentration of the aurovertin-binding site in both rat-liver and rat-heart mitochondria is about the same as that of the antimycin-binding and oligomycin-binding sites.     

Light-dependent changes of the Mg concentration in the stroma in relation to the Mg dependency of CO2 fixation in intact chloroplasts

(1) Light-dependent changes of the Mg²⁺ content of thylakoid membranes were measured at pH 8.0 and compared with earlier measurements at pH 6.6. In a NaCl and KCl medium, the light-dependent decrease in the Mg²⁺ content of the thylakoid membranes at pH 8.0 is found to be 23 nmol Mg²⁺ per mg chlorophyll, whereas in a sorbitol medium it is 83 nmol Mg₂₊ per mg chlorophyll.

(2) A light dependent increase in the Mg²⁺ content of the stroma was detected when chloroplasts were subjected to osmotic shock, amounting to 26 nmol/mg chlorophyll. Furthermore, a rapid and reversible light-dependent efflux of Mg²⁺ has been observed in intact chloroplasts when the divalent cation ionophore A 23 187 was added, indicating a light-dependent transfer of about 60 nmol of Mg²⁺ per mg chlorophyll from the thylakoid membranes to the stroma.

(3) CO₂ fixation, but not phosphoglycerate reduction, could be completely inhibited when A 23 187 was added to intact chloroplasts in the absence of external Mg²⁺. If Mg²⁺ was then added to the medium, CO₂ fixation was restored. Half of the maximal restoration was achieved with about 0.2 mM Mg²⁺, which is calculated to reflect a Mg²⁺ concentration in the stroma of 1.2 mM. The further addition of Ca²⁺ strongly inhibits CO₂ fixation.

(4) The results suggest that illumination of intact chloroplasts causes an increase in the Mg²⁺ concentration of 1–3 mM in the stroma. Compared to the total Mg²⁺ content of chloroplasts, this increase is very low, but it appears to be high enough to have a possible function in the light regulation of CO₂ fixation.     

Respiration-dependent uncoupler-stimulated ATPase activity in castor bean endosperm mitochondria and submitochondrial particles

1. The uncoupler-stimulated ATPase activity of castor bean endosperm mitochondria and submitochondrial particles has been studied. The rate of ATP hydrolysis catalyzed by intact mitochondria was slow and little enhanced by addition of uncouplers at the concentration required for uncoupling the oxidative phosphorylation. ATPase activity was stimulated at higher concentrations of uncouplers.

2. 1-Anilinonaphthalene 8-sulfonate fluorescence was decreased when the mitochondria were oxidizing succinate. Carbonylcyanide-p-trifluoromethoxyphenylhydrazone and antimycin reversed the succinate-induced fluorescence diminution. ATP did not induce the fluorescence response.

3. The addition of succinate, NADH or ascorbate/N,N,N′,N′-tetramethyl-p-phenylenediamine as electron donor induced high ATPase activity in the presence of low concentrations of uncouplers. Stimulating effect of uncouplers was completely abolished by further addition of antimycin.

4. Submitochondrial particles were prepared by sonication. The particles catalyzed a rapid hydrolysis of ATP and carbonylcyanide-p-trifluoromethoxyphenylhydrazone at 10^-8 M did not stimulate the ATPase activity. Addition of succinate induced uncoupler-stimulated ATPase activity. The effect of succinate was completely abolished by further addition of antimycin.

5. The treatment of submitochondrial particles by trypsin or high pH also induced uncoupler-stimulated ATPase activity.

6. The above results were interpreted to indicate that ATPase inhibitor regulated the back-flow reaction of mitochondrial oxidative phosphorylation.