Does the conformation of Mg-ATPase (spectrin-dependent ATPase) influence the passive permeability to K+?

The processing of human erythrocytes disclosed changes in Mg-ATPase activity following action of Pb2+ and Nile blue, and changes of permeability of K+ after treatment with Nile blue. The obtained results and those from previous papers can be summarized as follows : Substances decreasing the activity of stimulated membrane Mg-ATPase (spectrin-dependent ATPase) in red blood cells increase the passive permeability to K+, and substances increasing the stimulated Mg-ATPase activity decrease the passive permeability to K+. A hypothesis is proposed that the conformation of Mg-ATPase is secondarily reflected in the state of the proper path for K+ transport through the membrane; thus the rate of passive permeability to K+ is influenced.     

Effect of freezing-thawing rates on the functional state and ionic permeability of rat liver mitochondria

The effects of various rats of freezing-thawing reactions on the functional state and ionic permeability of rat liver mitochondria were studied. The degree of mitochondrial damage during the freezing -- thawing process depended on the rate of thawing rather than on that of freezing. The mitochondria which were slowly or rapidly frozen down to --196 degrees and subsequently slowly thawed revealed a higher membrane permeability for K+ Na+ and H+ and a more than 2-fold increase of the ATPase activity and the maximal rate of NADH oxidation via the antimycin-insensitive pathway in the presence of cytochrome c. This was concomitant with a complete inhibition of the ATP-synthetase activity and a marked inhibition of the respiratory chain function due to the efflux of cytochrome c from the inner mitochondrial membrane. After freezing and rapid thawing the functional activity of mitochondria changed insignificantly. A comparison of different cryoeffects demonstrated that the minimal damaging effects were exerted by rapid freezing -- rapid thawing, when the mitochondria partly restored their ability for oxidative phosphorylation.     

Orientation of membrane vesicles fromEscherichia coli prepared by different procedures

Summary The orientation of membrane vesicles prepared fromEscherichia coli by either French press, sonication or ethylenediamine tetraacetate (EDTA)-lysozyme was examined. The following procedures were used to determine orientation: (1) accessibility of the impermeable ferricyanide ion to the respiratory chain; (2) inhibition of membranal ATPase by specific antiserum; (3) binding of ATPase to the membrane. Data with spheroplasts indicated that ATPase, ATPase binding sites and ferricyanide reductase activities were localized on the inner part of the cytoplasmic membrane. Thus, there was no demonstrable NADH-ferricyanide reductase activity, low ATPase activity, no inhibition of ATPase by antiserum and no binding of purified ATPase by spheroplasts. In the case of membrane vesicles prepared by French press or sonication, the ATPase activity, the ATPase binding site and the site where ferricyanide takes electrons from the respiratory chain all appeared to be on the outside of the vesicles, suggesting that they are inverted. In the case of EDTA-lysozyme vesicles, which are widely used for transport studies, about half of the ATPase binding sites and ferricyanide reactive sites were exposed to the outside. Sixty percent of the ATPase activity was sensitive to antiserum. The two most probable explanations for these data are: (1) partial inversion of EDTA-lysozyme vesicles in the course of preparation; (2) movement of marker enzymes within the membrane vesicles during their isolation.     

运动性疲劳状态下大鼠心肌线粒体内膜变化的研究

采用递增负荷力竭性运动模型,观察了Ｓｐｒａｇｕｅ－Ｄａｗｌｅｙ大鼠急性运动至力竭后心肌线粒体内膜流动性、ＮＡＤＨ－ＣｏＱ还原酶及ＡＴＰ酶活性的变化。结果表明,大鼠心肌线粒体内膜荧光偏振值较安静时显著增高（Ｐ＜０．０１）,示膜流动性降低。线粒体内膜ＮＡＤＨ－ＣｏＱ还原酶和肌线粒体内膜功能改变,其膜流动性和呼吸链酶活性变化,可能是运动性疲劳的重要膜分子制之一。     

Mechanism of action of some quinoline alkaloids on respiratory chain of mitochondria]

The mechanism of action of some quinoline alkaloids and their derivatives on respiratory chain of rat liver and Candida lipolytica yeast mitochondria was studied. The alkaloids were shown to inhibit electron transfer in the respiratory chain. The site of their action is localized between b and c cytochromes. Besides their ability to inhibit electron transfer in the respiratory chain, alkaloids are shown to be specific inhibitors of "exogenous" NADH-dehydrogenase of C. lipolytica yeast mitochondria. In addition to their inhibiting properties alkaloids can stimulate ATPase activity of mitochondria. O-alkylation of pseudane-IX permits to differentiate the inhibiting and uncoupling properties of this alkaloid.     

Death by apoptosis in salivary glands of females of the tick Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae)

The salivary glands of females of the tick Rhipicephalus sanguineus at three feeding stages: unfed, engorged, and at day three post-engorgement, were subjected to cytochemical methods of enzymatic analysis and cell viability. Comparing glands at these stages, was observed distinct staining patterns in cells of different types of acini, specially in degenerating types III, II, I, which were affected in this sequence by cell death. This study also revealed changes in: nuclei, staining intensity for acid phosphatase and ATPase activities, and permeability of the plasma membrane. Acid phosphatase activity was inversely proportional to that of ATPase, while ATPase activity was always proportional to membrane integrity. The glands of unfed females exhibited high metabolic activity and cells with intact nucleus and plasma membrane, suggesting that the presence of acid phosphatase detected in these individuals may participate in the normal physiology of some acini, as they were not undergoing degeneration. In acini I and II of engorged females, we observed cells with intact membranes, as well as changes characterized by nuclear changes, decrease in ATPase activity, and stronger acid phosphatase activity. At day three post-engorgement, degeneration progressed to more advanced stages, loss of membrane integrity was observed in most cells (of some type I acini, most type II acini, and all type III acini), as well as prominent nuclear changes, decrease in ATPase activity, and intense acid phosphatase activity, resulting in apoptotic bodies. During the death of cells nuclear changes preceded cytoplasmic ones in the following sequence: nuclear changes, loss of ATPase activity, loss of integrity of the plasma membrane, increase in acid phosphatase activity, and formation of apoptotic bodies. The presence of acid phosphatase with a secondary role (late) during cell death, degrading final cell remnants, characterized this process in the glands of R. sanguineus females as atypical or non-classic apoptosis.     

Protonophores as inducers of energy-dependent changes of ultrastructure of mitochondria in wheat root cells

The membrane potential of plasmalemma, the release of K⁺ ions into incubation medium, respiratory gas exchange, the ATP content, and changes in the ultrastructure in cells of excised roots of wheat seedlings have been studied under the effect of protonophores 2,4-DNP (2,4-dinitrophenol) and CCCP (carbonyl cyanide m-chlorophenylhydrazone). After 1–4 h, a drop occurred in the plasmalemma membrane potential, as well as the release of K⁺ ions into incubation solution and the suppression of the intensity of oxygen absorption by cells. Mitochondria were of ovoid shape and had numerous and clearly outlined, slightly swollen cristae, which corresponds to the condensed type of the organelles. Additionally, a peculiar spatial arrangement of cristae in mitochondria has been revealed (as piles parallel to each other, as well as in the form of fans and of propellers) under the effects of protonophores. After 5 h of the action of protonophores against a background of the significant stimulation of oxygen consumption, low membrane potential, and a decrease in the functional activity of mitochondria, the destruction of the cell ultrastructure began. It is suggested that the revealed conformational transitions of mitochondria reflect gradual changes in their functional activity and the functional state of the cells under the long action of protonophores.     

Electron microscope study of lens fibers

The in vitro swelling action of L-thyroxine on rat liver mitochondria as examined photometrically represents an acceleration of a process which the mitochondria are already inherently capable of undergoing spontaneously, as indicated by the identical kinetic characteristics and the extent of thyroxine-induced and spontaneous swelling, the nearly identical pH dependence, and the fact that sucrose has a specific inhibitory action on both types of swelling. However, thyroxine does not appear to be a "catalyst" or coenzyme since it does not decrease the temperature coefficient of spontaneous swelling. The temperature coefficient is very high, approximately 6.0 near 20 degrees . Aging of mitochondria at 0 degrees causes loss of thyroxine sensitivity which correlates closely with the loss of bound DPN from the mitochondria, but not with loss of activity of the respiratory chain or with the efficiency of oxidative phosphorylation. Tests with various respiratory chain inhibitors showed that the oxidation state of bound DPN may be a major determinant of thyroxine sensitivity; the oxidation state of the other respiratory carriers does not appear to influence sensitivity to thyroxine. These facts and other considerations suggest that a bound form of mitochondrial DPN is the "target" of the action of thyroxine. The thyroxine-induced swelling is not reversed by increasing the osmolar concentration of external sucrose, but can be "passively" or osmotically reversed by adding the high-particle weight solute polyvinylpyrrolidone. The mitochondrial membrane becomes more permeable to sucrose during the swelling reaction. On the other hand, thyroxine-induced swelling can be "actively" reversed by ATP in a medium of 0.15 M KCl or NaCl but not in a 0.30 M sucrose medium. The action of ATP is specific; ADP, Mn(++), and ethylenediaminetetraacetate are not active. It is concluded that sucrose is an inhibitor of the enzymatic relationship between oxidative phosphorylation and the contractility and permeability properties of the mitochondrial membrane. Occurrence of different types of mitochondrial swelling, the intracellular factors affecting the swelling and shrinking of mitochondria, as well as the physiological significance of thyroxine-induced swelling are discussed.     

ATP-dependent proton translocation and quenching of 9-aminoacridine fluorescence in inside-out membrane vesicles of a cytochrome-deficient mutant of Escherichia coli,.

1. ATP-dependent proton translocation and ATP-dependent quenching of the fluorescence of 9-aminoacridine were measured in inside-out vesicles derived from a cytochrome-deficient mutant of Escherichia coli. 2. ATP-dependent quenching of fluorescence was inhibited by nigericin gramicidin, NH4Cl, and carbonylcyanide-m-chlorophenylhydrazone. Inhibition was also produced by the ATPase inhibitors N,N'-dicyclohexylcarbodimide (DCCD) and diphenyl phosphorazidate (DPA), and by the respiratory chain inhibitors piericidin A, 2-heptyl-4-hydroxyquinoline N-oxide, and An2+. The inhibition of ATP-dependent fluorescence quenching by the ionophores, uncouplers, and respiratory chain inhibitors was not due to an effect on ATPase activity which was insensitive to these agents. 3. By use of the ATPase inhibitors DCCD and DPA, or by replacing ATP with GTP, ITP and CTP, a correlation between the ATPase activity and the rate of ATP-dependent membrane energization, as measured by fluorescence quenching, was obtained.     

查尔酮衍生物对黑曲霉线粒体结构及功能的影响

本研究旨在探讨查尔酮衍生物对黑曲霉线粒体结构和功能的影响,评估查尔酮衍生物对黑曲霉的抗真菌效果.采用不同浓度查尔酮衍生物处理黑曲霉菌丝体,通过透射电子显微镜观察线粒体结构;并进一步对黑曲霉线粒体的活性氧、丙二醛水平、三羧酸循环相关酶活性和线粒体膜电位的变化进行测定.结果 表明,查尔酮衍生物以剂量依赖的方式诱导黑曲霉线粒...     

Relationship between configuration, function, and permeability in calcium-treated mitochondria.

Low levels of calcium (100 nmol/mg) added to beef heart mitochondria induced a configurational transition from the aggregated to the orthodox state and a simultaneous uncoupling of oxidative phosphorylation. The primary effect of calcium was to cause a nonspecific increase in the permeability of the inner membrane, resulting in entry of sucrose into the matrix space and the observed configurational transition. The uncoupling and permeability change induced by calcium could readily be reversed by lowering the calcium:magnesium ratio in the presence of either substrate or ATP. The configurational state, however, remained orthodox. This, along with studies of hypotonically induced orthodox mitochondria in which the membrane remained coupled and impermeable until after the addition of calcium, led to the conclusion that coupling was related to the permeability state of the inner membrane rather than the configurational state. Phosphate, arsenate, or oleic acid was found to cause a transition similar to that induced by calcium. Studies with the specific calcium transport inhibitors, EGTA, ruthenium red, and lanthanum revealed that endogenous calcium is required for the anion-induced transitions. A single mechanism was further indicated by a common sensitivity to N-ethylmaleimide. Strontium was ineffective as an inducer of the transition, even though it is transported by the same mechanism as calcium. This indicates that there are additional calcium-binding sites responsible for triggering the transition. Magnesium and calcium appeared to compete for these additional sites, since magnesium competitively inhibited the calcium-induced transition, but had no effect on calcium uptake. Calcium was found to potently inhibit the respiration of all NAD+-requiring substrates prior to the transition. Strontium also produced this inhibition without a subsequent transition. ATPase activity was induced at the exact time of transition with calcium and was not induced by strontium. This suggests that calcium-induced ATPase uniquely required the transition for activity, in contrast to the ATPase induced by uncoupler or valinomycin. The results of this work indicate that mitochondria have a built-in mechanism which responds to low levels of calcium, phosphate, and fatty acids, resulting in simultaneous changes, including increased permeability, inducation of ATPase, uncoupling of oxidative phosphorylation, and loss of respiratory control.     

Activation of plasma membrane ATPase of Saccharomyces cerevisiae by octanoic acid.

Plasma membrane ATPase activity of Saccharomyces cerevisiae IGC 3507III grown in the presence of the lipophilic acid octanoic acid [4-50 mg l-1 (0.03-0.35 mM), pH 4.0] was 1.5-fold higher than that in cells grown in its absence. The Km for ATP, the pH profile and the sensitivity to orthovanadate of the basal and the activated forms of the membrane ATPase were identical. This activation was closely associated with a decrease in the biomass yield and an increase in the ethanol yield, and was rapidly reversed in vivo after removal of the acid. However, the activated level was preserved when membranes were extracted and subjected to manipulations which eliminated or decreased octanoic acid incorporation in the plasma membrane. The activity of the basal plasma membrane ATPase in the total membrane fraction was slightly increased by incubation at pH 6.5 with octanoic acid at 100 mg l-1 or less (2.4 mg acid form plus 97.6 mg octanoate ion l-1). However, destruction of the permeability barrier between the enzyme and its substrate could not explain the in vivo activation. A role for plasma membrane ATPase activation in the regulation of the intracellular pH (pHi) of cells grown with octanoic acid was not proven.     

ABA对ZT对小麦叶细胞质膜某些生理特性的影响

激素的原初作用一般与细胞膜的生理变化密切相关,ABA对细胞膜的透性增加,ZT对细胞膜的影响较小;二者对于膜的离子外渗的影响同透性一样,对于叶绿体膜上的Ca^ 2-ATPase,Mg^ 2-ATPase,ABA抑制其活性,ZT则促进其活性的增加。因此,二者可影响叶绿体膜内外离子的交换,改变膜内外质子的平衡,ABA降低光下叶绿体悬浮介质的pH,降低膜的电热值,ZT则增加其电势。两种激素对于膜生理变化的影响是其影响植物细胞衰老,叶绿体光合作用的机制之一。     

Energization of membrane vesicles from the cells of glycolyzing bacterium Streptococcus faecalis]

Membrane fractions were isolated from Streptococcus faecalis cells of a glycolyzing microorganism, devoid of the respiratory chain, using the methods of osmotic shock of the protoplasts, ultrasonic treatment of the cells and ultrasonic treatment of the protoplasts. All fractions possessed the ATPase activity, the highest activity being observed in the fraction isolated by ultrasonication of the protoplasts. All preparations were estimated with respect to the presence of vesicles, formed by the "inside-out" and "inside-in" membranes, using ATPase as a marker of the membrane orientation. In the membrane fractions obtained by ultrasonication of the protoplasts, the "inside-out" vesicles were prevalent. ATP-dependent energization of the membranes, sensitive to the action of dicyclohexylcarbodiimide and tetrachlorotrifluoromethyl benzimidazole, was demonstrated by measuring the transport of the lipophylic anion of phenyldicarbaundecaborane and aniline naphthalene sulfonate fluorescence.     

Deterioration of rat liver mitochondria under conditions of metabolite deprivation.

In a previous study [Parce, Cunningham & Waite (1978) Biochemistry 17, 1634-1639] changes in mitochondrial phospholipid metabolism and energy-linked functions were monitored as coupled mitochondria were aged in iso-osmotic sucrose solution at 18 degrees C. The sequence of events that occur in mitochondrial deterioration under the above conditions have been established more completely. Total adenine nucleotides are depleted early in the aging process, and their loss parallels the decline in respiratory control. Related to the loss of total adenine nucleotides is a dramatic decrease in ADP and ATP translocation (uptake). The decline of respiratory control is due primarily to a decrease in State-3 respiration; loss of this respiratory activity can be related to the decline in ADP translocation. Mitochondrial ATPase activity does not increase significantly until State-4 respiration has increased appreciably. At the time of loss of respiratory control the ATPase activity increases to equal the uncoupler-stimulated activity. The H+/O ratio and P/O ratios do not decrease appreciably until respiratory control is lost. Similarly, permeability of the membrane to the passive diffusion of protons increases only after respiratory control is lost. There observations reinforce our earlier conclusion that there are two main phases in mitochondrial aging. The first phase is characterized by loss of the ability to translocate adenine nucleotides. The second phase is characterized by a decline in the ability of the mitochondrion to conserve energy (i.e. maintain a respiration-driven proton gradient) and to synthesize ATP.     

Functional changes in mitochondrial properties as a result of their membrane cryodestruction. II. Influence of freezing and thawing on ATP complex activity of intact liver mitochondria

The influence of the freeze-thawing rates on ATP synthetase (ATPase) complex of intact liver mitochondria was investigated. It was shown that the increase in latent ATPase activity and decrease in ATP synthetase activity resulted from an influence on the inner mitochondrial membrane. An increase in freeze-thawing rates led to the preservation of ATP synthetase activity and ATP hydrolysis reduction. Kinetic parameter changes of the ATP synthetase reaction resulted from an insignificant nonspecific increase in the inner mitochondrial membrane permeability and changes in its electrochemical potential level.     

耗竭性运动对大鼠骨骼肌线粒体内膜的影响

观察SD大鼠一次急性运动至力竭后骨骼肌线粒体内膜流动性、NADH-CoQ还原酶及ATP酶活性变化．结果显示,大鼠骨骼肌线粒体内膜微粘度较安静时显著增高,线粒体内膜NADH-CoQ还原酶和ATP酶活性分别较安静时下降34．2％和46．2％．研究提示,耗竭性运动后大鼠骨骼肌线粒体呼吸链内膜分子动力学和呼吸链酶组分活性变化,可能是运动性疲劳重要的膜分子特征．     

Localization of ATPase activity, respiration and ultrastructure of wheat root cells with modulated ion conductivity of plasma membrane

Changes in the localization of ATPase activity, respiration and ultrastructure of wheat root cells with modulated ion conductivity of plasma membrane were studied. A 2 h treatment of excised root with valinomycin (20 microM), N,N-dicyclohexylcarbodiimid (100 microM), gramicidin S (20 microM) and chlorpromazine (100 microM) caused an increased loss of potassium by cells, a decreased respiration and changes in the localization of ATPase activity and in cell ultrastructure. Differences in the observed changes may be conditioned by different mechanisms of action of the membrane active compounds used. It is concluded that changes in the localization of ATPase activity and ultrastructure may indicate some early specific responses of root cells, whereas the increase in the ion conductivity and decrease in respiration under disruption of ion homeostasis caused by membrane active compounds indicate unspecific responses of cells.     

Cytotoxic action of Pseudomonas aeruginosa hemolysin]

The cytopathic action of haemolysin of Pseudomonas aeruginosa has been studied in mouse and human leucocytes. The morphological changes suggest that haemolysin affects the molecular architecture of the cell membrane, whose permeability is increased. It does not induce non-specific stimulation of peripheral lymphocytes. Normal sera and albumin neutralize the hemolytic activity of haemolysin; this inhibition is also observed, to a les extent, on the lytic action on leucocytes. This raises the possibility that the two activities are probably associated with the same molecule.     

Parallel effects of freezing and osmotic stress on the ATPase activity and protein composition of the plasma membrane of winter rye seedlings

The objective of this study was to determine the influence of freezing versus hypertonic stress on the ATPase activity and polypeptide profile of the plasma membrane of nonacclimated winter rye leaves (Secale cereale L. cv Puma). Exposure of leaves to hypertonic sorbitol solutions resulted in a similar extent of injury as did freezing to subzero temperatures that resulted in equivalent osmotic stresses. When isolated with a two-phase partition system of aqueous polymers, the plasma membrane fractions of control, frozen, or hypertonically stressed leaves were of similar purity as judged by the distribution of marker enzyme activities. When assayed in the presence of Triton X-100 (0.05% w/w), ATPase activity was decreased only slightly in plasma membrane fractions isolated from either frozen or hypertonically stressed leaves. In contrast, the specific ATPase activity of the plasma membrane fractions assayed in the absence of Triton X-100 increased following freezing or hypertonic stress. As a result, the Triton X-100 stimulation of the ATPase activity decreased significantly from sixfold in control leaves to threefold in lethally stressed leaves and reflects an increase in the permeability of the plasma membrane vesicles. The increased permeability was also manifested as a decrease in H⁺-transport following exposure to freezing or hypertonic stress. Both freezing and hypertonic exposure at subzero temperatures altered the polypeptide profile of the plasma membrane, but with the exception of one polypeptide, there was no difference between the two treatments.