The antifertility agent,gossypol, changes several mitochondrial functions in the presence of Mg2+

1. The effect of gossypol in the presence of K⁺ or Mg²⁺, or both, was studied on ATPase activity and respiration of rat liver mitochondria.2. Respiration was uncoupled in the presence of gossypol, Mg²⁺, and K⁺, whereas in the presence of gossypol and Mg²⁺ a partial inhibition was observed.3. Gossypol stimulated ATPase activity in the presence of K⁺ or Mg²⁺, but maximal activity was observed when both cations were in the incubation medium.4. Stimulation of ATPase activity in the presence of Mg²⁺ was dose related.5. EDTA reverted the stimulation produced by gossypol on ATPase activity.6. Gossypol had no effect on the ATPase activity of submitochondrial particles, which suggests an indirect action of gossypol on the enzyme.7. Mitochondrial membrane potential showed a higher collapse in the presence of gossypol and 1mM MgCl₂.8. The observed effects of gossypol could be explained by the collapse of the mitochondrial membrane potential.     

Effects of iodine upon the structure and function of mitochondria

The influence of iodine in its positive and negative monovalent form upon the oxygen consumption in euthyroid and thyroidectomized rats and the oxidative phosphorylation in liver mitochondria isolated from both groups of animals, as well as the spontaneous swelling and total ATPase activity of mitochondria have been studied.It was established that the administration of ICI increased the oxygen consumption of normal and thyroidectomized rats while under the same conditions no effect was found with NaI. IBr stimulated the oxygen consumptionin vitro in liver mitochondria isolated both from normal and thyroidectomized rats and decreased the P/O ratio while NaI had no effect. I₂ and IBr increased the swelling and inhibited the ATPase activity of isolated rat liver mitochondria, while these effects were not observed when KI was used. The thyroidstatic 1-methyl-2-mercaptoimidazol decreased the stimulating effect of iodine upon the swelling of mitochondria and to a certain extent lowered its inhibiting effect upon the ATPase activity.It is concluded that iodine in its positive monovalent form has a thyroxine-like effect upon the structure and function of isolated rat liver mitochondria, as well asin vivo upon the respiration of euthyroid and thyroidectomized rats.     

Biochemical mechanisms of temperature adaptation in the chick embryo

The effect of a short-term cooling of the incubated eggs has been investigated on the intensity of oxidative phosphorylation and the activity of ATPase in mitochondria from muscles and liver of chick embryos and chicks. It was found that the decrease of temperature increases oxygen consumption in muscle mitochondria decreasing esterification of inorganic phosphate. As a consequence, the value of P/O decreases. The activity of ATPase significantly increases. Uncoupling between oxidation and phosphorylation in liver mitochondria takes place more slowly. It is suggested that these changes account for realization of thermoregulation.     

Bioflavonoid regulation of ATPase and hexokinase activity in Ehrlich ascites cell mitochondria.

(1) The mitochondrial ATPase (EC 3.6.1.3) Ehrlich ascites cell mitochondria, was inhibited by D-glucose under physiological concentrations of ATP. The generation of ADP by the mitochondrial bound hexokinase, seems to be the reason for the D-glucose inhibitory effect. Reversal of the inhibitory effect of ADP on Ehrlich ascites cell mitochondria ATPase by an ATP-regenerating system was achieved. (2) Dissociation of mitochondrial bound hexokinase from the mitochondria eliminated the inhibitory effect of D-glucose. Rebinding of the hexokinase to the mitochondria regenerated the D-glucose inhibitory effect on Ehrlich ascites cell mitochondria ATPase. (3) Bioflavonoids such as quercetin inhibit the mitochondrial hexokinase activity, but do not change the mitochondrial ATPase activity of isolated Ehrlich ascites tumor cell mitochondria. (4) The inhibitory effect of bioflavonoids on mitochondrial bound hexokinase activity is shown to be dissociable from the ascites tumor cell mitochondria and seems to be associated with regulatory rather than catalitic sites of the enzyme.     

In vitro effects of gossypol and lactic acid on rat uterus and ovary during implantation and antiimplantation

ATPase activity of uterus and ovary was markedly elevated in presence of gossypol and decreased in presence of lactic acid indicating activation and inhibition of energy metabolism by gossypol and lactic acid respectively. The elevated levels of glycogen in uterus indicate inhibition of glycogenolysis as supported by phosphorylase activity. Whereas in ovary the glycogen depletion indicates activation of glycogenolysis supported by phosphorylase activity. The activity levels of aldolase and G-6-PDH decreased in the uterus in presence of gossypol and increased in presence of lactic acid. The same were elevated in ovary indicating the activation of hexose mono and diphosphate pathways. Lactic acid accumulated in presence of both gossypol and lactic acid with a depletion in level of pyruvic acid in both the tissues. This situation in the uterus indicates the condition of anti-implantation in presence of both gossypol and lactic acid. The NAD-LDH activity was inhibited in presence of gossypol and activated in presence of lactic acid in both tissues.     

Purification and properties of ATPase inhibitor from rat liver mitochondria.

(1) The ATPase inhibitior 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 Pi-ATP exchange activity in sonicated submitochondrial particles. However, unexpectedly the inhibitor greatly stimules Pi-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.     

The interaction of glucagon treatment and uncoupler concentration on ATPase activity of rat liver mitochondria

Mitochondria isolated from livers of rats treated briefly with glucagon show an increased ATPase activity in the presence of appropriate concentrations of protonophoric uncouplers (Yamazaki, R. K., Sax, R.D., and Hauser, M.A. (1977) FEBS Lett. 75, 295-299). With the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) the effect of glucagon treatment was most evident at concentrations of uncoupler higher than required for maximal stimulation of ATPase in control mitochondria. In this range of FCCP concentrations that produced the greatest contrast in ATPase activity of control and hormone-stimulated mitochondria, there were no significant differences in delta pH, delta psi, or delta p between the two groups. The presence of added succinate in the ATPase assay system mimicked the effect of glucagon treatment, permitting greater activity at high concentrations of uncoupler without significantly affecting delta p. No significant effect of glucagon treatment or uncoupler concentrations on mitochondrial volumes was observed. Following treatment with glucagon, the mitochondria retained a greater content of Mg+ and K+ throughout the range of FCCP concentrations tested. In the upper range of FCCP concentrations there was appreciable loss of K+ from the mitochondria which was greater in control mitochondria than in mitochondria from glucagon-treated rats or in mitochondria assayed in the presence of succinate. The activity of the uncoupler-dependent ATPase was greatly stimulated by increased concentrations of potassium chloride in the assay medium without significantly diminishing the hormone effect. It is proposed that the intrinsic peptide inhibitor of ATPase is dissociated from the enzyme to an increased degree following glucagon treatment and that high levels of uncoupler inhibit by causing an increased association of the enzyme and its inhibitor.     

The adenosine triphosphatase-inhibitor content of bovine heart submitochondrial particles. Influence of the inhibitor on adenosine triphosphate-dependent reactions.

1. The activity of the ATPase (adenosine triphosphatase) of phosphorylating particles prepared by sonication of bovine heart mitochondria in the presence of MgCl2 and ATP is influenced by the isolation method for the mitochondria used in the preparation of particles. Type-I particles, made from mitochondria isolated in a medium lacking succinate, have a lower ATPase activity than to Type-II particles, which are prepared from mitochondria isolated in a medium containing succinate. 2. Centrifugation under appropriate energized conditions increases the ATPase activity of Type-I particles almost to that of the Type-II particles. The ATPase activity of Type-II particles was only slightly stimulated by this procedure. These data are interpreted as indicating a higher content of the ATPase-inhibitor protein in the Type-I particles. 3. A comparison was made of the ATP-driven enhancement of 8-anilinonaphthalene-1-sulphonate fluorescence and the exchange of the endogenous tightly bound nucleotides of the ATPase in Type-I and Type-II particles. The effect of exogenous inhibitor protein on both these reactions was also studied. 4. The time-scale on which the inhibitor protein can exchange between ATPase molecules is discussed.     

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.     

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

1. The uncoupler-stimulated ATPase activity of castor bean endosperm mitochondria and submitchondrial 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. ATP-ase 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.     

Investigations on the mitochondria of the housefly,Musca domestica L. III. Requirements for oxidative phosphorylation

It has been found that mitochondria isolated from the flight muscle of the housefly, Musca domestica, are capable of effecting oxidative phosphorylation. A systematic investigation of the factors which regulate this coupling was undertaken. It was found: 1. The molarity of the isolation medium had considerable influence on the morphology of the mitochondria. These physical alterations were associated with changes in oxidation, phosphorylation, and ATPase activity. 2. In addition to an optimum isolation medium, the normal morphology of the mitochondria needed to be further stabilized by serum albumin. 3. A "latent" ATPase activity in insect mitochondria was demonstrated. An inverse relationship was found between oxidative phosphorylation and ATPase activity. 4. Oxygen consumption and the uptake of phosphate were linear with respect to time. 5. A respiratory substrate was necessary for phosphorylation and for maintenance of spatially organized mitochondria. 6. No differences in oxygen uptake were found in the presence or absence of inorganic phosphate. 7. Magnesium was required for optimal oxidative phosphorylation. Calcium and manganese inhibited both respiration and phosphorylation. 8. The addition of cytochrome c had no effect on either oxygen or phosphate uptake. 9. ATP, ADP, or AMP were capable of participating in oxidative phosphorylation, but the glucose-hexokinase trapping system was necessary. 10. Fluoride inhibited the phosphorylation of AMP, but increased P/O when ATP was used. This stimulation was not due to the inhibition of ATPase. 11. Neither arginine nor creatine was phosphorylated. 12. The addition of other isolated fractions of flight muscle to the mitochondrial system had no appreciable effect on respiration or phosphorylation.     

Cell-free synthesis of mitochondrial ATPase inhibitor precursor and its transport into yeast mitochondria

ATPase inhibitor protein, which blocks mitochondrial ATPase activity by forming an enzyme-inhibitor complex, was found to be synthesized as a larger precursor in a cell-free translation system directed by yeast mRNA. Other protein factors, which stabilize latent ATPase by binding to the enzyme-inhibitor complex, were also found to be formed as larger precursors. The precursor of ATPase inhibitor protein was transported into isolated yeast mitochondria and was cleaved to the mature peptide in the mitochondria. Impaired mitochondria lacking phosphorylation activity could not convert the precursor to the mature form. Neither antimycin A nor oligomycin alone exhibited a marked effect on the transport-processing of the precursor by intact mitochondria. However, when antimycin A was added with oligomycin, the transport-processing was markedly inhibited. The processing was also strongly inhibited by an uncoupler, carbonylcyanide p-trifluoro-methoxyphenyl hydrazone. The inhibition by the uncoupler was not relieved by ATP added externally. It is concluded that the transport-processing of precursor proteins requires intact mitochondria with a potential difference across the inner membrane.     

Differential effects of 2,4-dinitrophenol and valinomycin (+K+) on uncoupler-stimulated ATPase of human tumor mitochondria

The uncoupler-stimulated mitochondrial ATPase of four human tumors, mouse kidney, brain and fetal liver exhibited a characteristic behavior when preincubated with the H⁺-conducting uncouplers, dinitrophenol, CCCP, S-13 and gramicidin. The ATPase activity was considerably lower with preincubation than without. Preincubation with valinomycin (+K⁺), on the other hand, did not result in a significant decrease of the ATPase activity. These results may be contrasted with those obtained with liver or heart mitochondria, the ATPase activity of which did not suffer any loss when preincubated with dinitrophenol. The effect of preincubation with dinitrophenol on the tumor mitochondria could not be accounted for by dinitrophenol-induced Mg²⁺ efflux, since the differential effects of dinitrophenol and valinomycin (+K⁺) remained even when ATPase activity was determined in presence of Mg²⁺. Small amounts of ATP and ADP in the preincubation mixture containing dinitrophenol protected against the decay of the ATPase activity, implicating the exchangeable adenine nucleotides in the tumor mitochondria. In a model system where liver mitochondria were depleted of their adenine nucleotides, a lower ATPase activity was indeed obtained. However, direct determination of the concentations of adenine nucleotides in dinitrophenol- and valinomycin-treated tumor mitochondria revealed only slight differences.     

Impaired cardiac mitochondrial membrane potential and respiration in copper-deficient rats

Cardiac mitochondrial respiration, ATP synthase activity, and membrane potential and intactness were evaluated in copper-deficient rats. In the presence of NADH, both copper-deficient and copper-adequate mitochondria had very low oxygen consumption rates, indicating membrane intactness. However copper-deficient mitochondria had significantly lower oxygen consumption rates with NADH than did copper-adequate mitochondria. Copper-deficient mitochondria had significantly lower membrane potential than did copper-adequate mitochondria using fluorescent dyes. Copper-deficient mitochondria had significantly lower state 3 oxygen consumption rates and were less sensitive to inhibition by oligomycin, an ATP synthase inhibitor. Copper-deficient and copper-adequate mitochondria responded similiarly to CCCP. No difference was observed in mitochondrial ATPase activity between copper-deficient and copper-adequate rats using submitochondrial particles. We conclude that cardiac mitochondrial respiration is compromised in copper-deficient rats, and may be related to an altered ATP synthase complex and/or a decreased mitochondrial membrane potential.     

CYTOCHEMICAL STUDIES OF ADENOSINE TRIPHOSPHATASES IN SKELETAL MUSCLE FIBERS

Mitochondrial ATPase and myosin ATPase have been localized in the muscle fibers of the rat diaphragm. The principal fiber type possesses a structure favorable for making this cytochemical separation with the light microscope. This small red fiber has numerous large, nearly spherical, mitochondria (ca. 1.5 µ) which are aggregated beneath the sarcolemma. In the interior of the fiber, smaller paired filamentous mitochondria (ca. 0.2 µ diameter) are aligned with the I band. Distribution of mitochondria was determined by sudanophilia, succinic dehydrogenase activity, and by direct examination with the electron microscope. ATPase activity at pH 7.2 is located in the large peripheral mitochondria and in the smaller mitochondria associated with the I band. The alignment of the small mitochondria results in a discrete cross-striated appearance in fibers stained for this enzymic activity. This mitochondrial ATPase does not cleave adenosine diphosphate or adenosine monophosphate; it is not sulfhydryl dependent and, in fact, is enhanced by the mercurial, p-hydroxymercuribenzoate. It requires magnesium ion and is stimulated by dinitrophenol. It is inhibited after formol-calcium fixation, but the residual activity is demonstrable by lengthening the incubation time. At pH 9.4 the ATPase is myofibrillar in origin and is located in the A bands. This myosin ATPase activity is sulfhydryl-dependent. Mercurial at this high pH has an interesting dual effect: it suppresses myosin ATPase but evokes mitochondrial ATPase activity. A third type of ATPase activity can be demonstrated, especially in the large white fibers. This activity occurs at pH 7.2 in the presence of cysteine. Its position is manifested cytochemically as a fine reticular pattern which surrounds individual myofibrils. The distribution suggests that it may originate in the sarcoplasmic reticulum.     

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.     

Curcumin restores mitochondrial functions and decreases lipid peroxidation in liver and kidneys of diabetic db/db mice

Background

Nitrosative and oxidative stress play a key role in obesity and diabetes-related mitochondrial dysfunction. The objective was to investigate the effect of curcumin treatment on state 3 and 4 oxygen consumption, nitric oxide (NO) synthesis, ATPase activity and lipid oxidation in mitochondria isolated from liver and kidneys of diabetic db/db mice.

Results

Hyperglycaemia increased oxygen consumption and decreased NO synthesis in liver mitochondria isolated from diabetic mice relative to the control mice. In kidney mitochondria, hyperglycaemia increased state 3 oxygen consumption and thiobarbituric acid-reactive substances (TBARS) levels in diabetic mice relative to control mice. Interestingly, treating db/db mice with curcumin improved or restored these parameters to normal levels; also curcumin increased liver mitochondrial ATPase activity in db/db mice relative to untreated db/db mice.

Conclusions

These findings suggest that hyperglycaemia modifies oxygen consumption rate, NO synthesis and increases TBARS levels in mitochondria from the liver and kidneys of diabetic mice, whereas curcumin may have a protective role against these alterations.     

Increased content of natural ATPase inhibitor in tumor mitochondria

The ATPase activity of Zajdela hepatoma and Yoshida sarcoma submitochondrial particles was several times lower than the enzyme activity in rat heart and rat liver submitochondrial particles. The content of F1-ATPase in the tumor mitochondria was found not to be very different from that in mitochondria of rat liver. Immunochemical determination of the amount of the natural ATPase inhibitor revealed that the tumor mitochondria contain 2-3-times more ATPase inhibitor than control mitochondria. It is concluded that the low ATPase activity of the tumor mitochondria results from the inhibition of the enzyme activity by the natural ATPase inhibitor.     

Response of mitochondrial ATPase activity to uncouplers in isolated organelles and whole cells of Zajdela hepatoma

ATPase activity of coupled Zajdela hepatoma mitochondria was rendered uncoupler-sensitive by decreasing free fatty acids content in mitochondria or by preincubation of mitochondria with ATP prior to the addition of an uncoupler. The latter treatment resulted in an accelerated transport of ATP into the organelles. The effect of carbonylcyanide-m-chlorophenylhydrazone and oligomycin on the decrease of the ATP content in whole Zajdela hepatoma cells indicated that the hepatoma mitochondrial ATPase is stimulated by uncouplers $\text{in}\text{vivo}$. The conclusion is that the uncoupler-insensitive ATPase activity of coupled Zajdela hepatoma mitochondria is exhibited only by isolated organelles and results from a reduced $\text{ATP}\text{ADP}$ translocase activity.     

An oligomycin-resistant Mg2+-dependent ATPase in rabbit bone marrow mitochondria

Summary Rabbit bone marrow mitochondria isolated by differential centrifugation showed typical oxypolarographic tracings with glutamate oxidation with ADP:O ratio of 2.9. Similar results were obtained with liver mitochondria of the same animal. When marrow mitochondria were oxydizing a substrate such as glutamate, added MgCl₂ markedly stimulated state-4 respiration giving a respiratory rate identical to that of state-3. In contrast, no Mg²⁺-stimulation was observed with liver mitochondria. Oligomycin completely blocked the stimulation by Mg²⁺ but further addition of 2,4-dinitrophenol reactivated the oxygen consumption by uncoupling. Further purification of marrow mitochondria by density gradient centrifugation in Percoll provided identical oxypolarographic results. Moreover, when marrow mitochondria were incubated without Mg²⁺, they showed a low ATPase activity that was stimulated by 2,4-dinitrophenol and blocked by oligomycin. The presence of Mg²⁺ in the incubation medium uncovered an additional ATPase activity which was resistant to oligomycin and apparently unaffected by 2,4-dinitrophenol. It is concluded that bone marrow mitochondria possess two types of ATPase activity distinguished on the basis of their reactivity with oligomycin, 2,4-dinitrophenol and Mg²⁺.Abbreviations EDTA ethylenediamine tetraacetate - DNP 2,4-dinitrophenol - BSA bovine serum albumin - BMM bone marrow mitochondria - LM liver mitochondria - Oligo. oligomycin - Anti A antimycin A Howard Hughes Investigator.