The inhibition of mitochondrial energized processes by fluorescein mercuric acetate

Fluorescein mercuric acetate (FMA) has been shown to be a potent inhibitor of energized processes in both beef heart mitochondria and ETP_H particles. FMA reacts preferentially with a small number of specific sulfur atoms and inhibits the phosphate-dependent configurational transition. FMA enhances the anaerobic to aerobic pH changes observed in intact mitochondria and submitochondrial particles, and also enhances nonenergized swelling in 0·15 M sodium or potassium chloride. The results are interpreted in terms of a model whereby FMA, in reacting with the mitochondrion, modifies its conformation. The resulting conformational changes which occur upon energization are therefore different from those conformational changes which would occur in the absence of FMA. The net result of this process is the inhibition of some processes (e.g., oxidative phosphorylation, ATP-³²P_i exchange, etc.) and the enhancement of other processes (the proton shift and nonenergized swelling in chloride salts).This work was supported in part by U.S. Public Health Service Program Project Grant GM-12847 and by a training grant GM-88, both from the National Institute of General Medical Sciences. Meat by-products were generously furnished by Oscar Mayer and Co., Madison, Wisconsin.     

On the stabilization by fixation of configurational states in beef heart mitochondria

The energized configuration of the cristal membrane of beef heart mitochondria can be maintained only as long as oxygen is available for electron transfer. When the oxygen supply is exhausted, the membrane undergoes a transition to the nonenergized configuration. Since the exhaustion of the available oxygen supply is complete in 5–20 sec, it is impossible to apply the method of sedimenting the mitochondria prior to fixation for studying the energized configurational states of mitochondria. The direct addition of glutaraldehyde followed by osmium tetroxide to the mitochondrial suspension is the most effective way of freezing the configurational state of the cristal membrane. Fixation with glutaraldehyde appears to be complete within 1–2 sec even at 0°. Osmium tetroxide alone can also freeze the energized configuration by fixation but the concentration of the fixative is critical. The problem of capturing the configurational state applies not only to energized transitions (nonenergized to energized) but also to nonenergized transitions (orthodox to aggregated). The freezing by fixation of the cristal membrane in the aggregated configuration is best accomplished by the sequential use of glutaraldehyde and osmium tetroxide. When the levels of glutaraldehyde and osmium tetroxide are respectively too low or too high, the mitochondrion will undergo a transition from the aggregated to the orthodox configuration before fixation is complete. Light-scattering studies provide an independent method for monitoring configurational changes in mitochondria; these light-scattering measurements confirm that the conditions for fixation which lead to stabilization of the energized state as judged by electron microscopy, also show maintenance of configuration as judged by absence of light-scattering changes after the fixatives are introduced. Reagents used in negative staining will induce the geometrical form of the energized configuration of the mitochondrion even under nonenergizing conditions. These reagents are thus unsuitable for use in studies of configurational transitions in mitochondria.     

Specific inhibition of phosphate transport in mitochondria by N-ethylmaleimide

N-ethylmaleimide (NEM), a reagent that alkylates free sulfhydryl groups, was shown to be a highly effective inhibitor of the following coupled mitochondrial processes: oxidative phosphorylation, ATP-³²Pi exchange, Pi-induced light scattering and configurational changes, State III respiration, valinomycin-induced translocation of potassium with Pi as the anion, and calcium accumulation in presence of Pi. However, NEM was less effective or ineffective in inhibiting some processes that do not require inorganic Pi, namely electron transfer and ATPase activity, ADP binding, energized light scattering changes induced by arsenate and nonenergized light scattering changes induced by acetate. The rate of oxidative phosphorylation and of ATP-³²Pi exchange was normal in ETP_H particles prepared from NEM-treated mitochondria. Also NEM, even et levels 2–3 times greater than those required to inhibit oxidative phosphorylation in intact mitochondria, did not inhibit coupled processes in submitochondrial particles. We are proposing that NEM alkylates sulfhydryl groups in the mitochondrion that modulate Pi translocation, and that the suppression of Pi translocation blocks oxidative phosphorylation, the Pi-dependent energized configurational change in mitochondria and Pi-dependent transport processes.On leave of absence from the Department of Biochemistry, Cancer Institute Okayama University Medical School, Okayama, Japan.On leave of absence from the Department of Pathology, Nagoya University Medical School, Nagoya, Japan.     

Inorganic phosphate stimulates the toxic effects of Tl+ in rat liver mitochondria

We studied action of inorganic phosphate (P(i)) on toxic effects of Tl+ in isolated rat liver mitochondria. This is a convenient model to study the toxicity of heavy metals. P(i) markedly retarded contraction of energized mitochondria swollen in the TlNO3 medium and even stronger stimulated swelling and state 4 of succinate-energized mitochondria in the TlNO3 medium. A valinomycin-induced decrease of K+-diffusion potential was also accelerated by Tl+ in the presence of P(i). The mitochondrial permeability transition pore in the medium containing Ca2+, TlNO3, and nitrates of univalent cations was distinctly stimulated by P(i). However, P(i) did not affect both the Tl+-stimulated swelling of nonenergized mitochondria in the TlNO3 medium and swelling of energized mitochondria in the Tl acetate medium. Respiration stimulated by 2,4-dinitrophenol and monoamine oxidase activity of energized mitochondria were not affected by Tl+ regardless of the presence of P(i). We suggested that stimulation by P(i) of toxic action of Tl+ in mitochondria and cells could be due to even greater enhancement of uncoupling of mitochondria as shown by an additional increase of swelling and state 4, and in the greater probability of opening of MPTP in the presence of P(i) and Ca2+.     

The mechanism of mitochondrial swelling. VIII. Permeability of mitochondria to alkali metal acetates

The capacity of beef heart mitochondria to undergo osmotically induced volume changes in decimolar M⁺-acetate or other weak acid anion media is characterized by the following features: (1) mitochondria resist swelling when suspended in potassium or rubidium acetate media in the presence of respiratory inhibitors; (2) mitochondria swell extensively when suspended in ammonium or sodium acetate media in the presence of respiratory inhibitors; and (3) actively respiring mitochondria swell extensively whether suspended in ammonium, sodium, potassium, or rubidium acetate media. These findings have been interpreted to mean that (1) the nonenergized mitochondrial inner membrane is permeable to acetate anions, (2) the nonenergized mitochondrial inner membrane is permeable to ammonium and sodium ions in the presence of acetate or other weak acid anions, (3) the nonenergized mitochondrial inner membrane is relatively impermeable to potassium and rubidium ions in the presence of acetate or other weak acid anions, and (4) energized mitochondria are considerably more permeable to potassium and rubidium (acetate) ions than are non-energized mitochondria. The experiments described in this communication which provide the evidence for these interpretations involve methods which are independent of volume changes. The results confirm the first three of the above interpretations but are inconsistent with the fourth. A general theory for passive ion movements in mitochondria is presented and the results are discussed in terms of the development of an energy dependent ion gradient as the key to energized swelling in potassium or rubidium acetate.     

Adenine nucleotide translocase as a site of regulation by ADP of the rat liver mitochondria permeability to H+ and K+ lons

In the presence of oligomycin ADP inhibits the osmotic swelling of the nonenergized rat liver mitochondria in the NH₄NO₃ medium. With the energized mitochondria ADP enhances contraction of the mitochondria swollen in the NH₄NO₃ medium. Carboxyatractyloside and atractyloside abolish or prevent the effects of ADP. The direct measurements of the proton conductance of rat liver mitochondria shows that the inhibitory action of ADP + oligomycin on the H⁺ permeability does not depend on the energization of mitochondria. In these experiments the local anesthetic nupercaine and ADP additively inhibit the inner membrane conductance for protons, but carboxyatractyloside abolishes only the effect of ADP. In the presence of oligomycin ADP also inhibits the osmotic swelling of the nonenergized liver mitochondria in the KNO₃ medium, and the energy-dependent swelling of rat liver mitochondria in the medium with K⁺ ions and P_i. The inhibition by ADP of the membrane passive permeability for K⁺ is also sensitive to carboxyatractyloside. It is concluded that rat liver mitochondria possess an ADP-regulated channel for H⁺ and K⁺. The properties of this pathway for protons and potassium ions favor the idea that ADP regulates the mitochondrial permeability via adenine nucleotide translocase. It is assumed that the adenine nucleotides carrier should operate according to the “gated pore” mechanism.     

Ultrastructural transformations in bean inner mitochondrial membranes

The ultrastructure of inner membrane-matrix mitochondria isolated from bean (Phaseolus vulgaris) shoots was examined in different metabolic states. Gross ultrastructural transformations analogous to the condensed-to-orthodox configurational changes reported in mammalian mitochondria are observed on transistion from nonrespiring to respiring metabolism. With the induction of oxidative phosphorylation, the particles remain in the orthodox configurational state. The reverse orthodox-to-condensed configurational changes observed in mammalian preparations does not occur. Optically monitored absorbancy studies with bean particles show a substrate-supported P_i-induced swelling under the same conditions that induce the condensed-to-orthodox ultrastructural transformation. The swelling is associated with the net uptake of K⁺ and P_i as well as a small P_i-induced respiratory stimulation. When phosphorylation is initiated with these swollen particles, the optically monitored volume remains unchanged. Thus a positive correlation exists between the ultrastructural configuration and the osmotic volume changes, which supports the conclusion that configurational changes reflect internal osmotic adjustments.     

Quantitation of mitochondrial injury in cultured rat heart endothelioid cells with nitroblue tetrazolium

Synopsis A sensitive method is presented for measurement of changes in the permeability of mitchondria in cultured cells. Rat heart endothelioid cells were used to determine the penetration rate of nitroblue tetrazolium (NitroBT) or other reactants into mitochondriain situ. Nitroblue formazan, produced as a consequence of succinate dehydrogenase activity in the mitochondria, was eluted and measured with a spectrophotometer. Prior injury of cells with hypo-osmolar solutions increased the rate of formazan production. Several methods are described or suggested for the statistical analysis of the data.     

Effect of high sucrose concentrations on mitochondria: Analysis of mitochondrial populations by density-gradient centrifugation after fixation with glutaraldehyde

It has been shown previously that intact rat liver mitochondria can be separated into two populations (designated B2 and B3) with mean buoyant densities of 1·184 and 1·216 respectively, by isopycnic sucrose density gradient centrifugation. A comparison has been made of some properties of these mitochondrial fractions from density gradients with non-fractionated mitochondria. Use was made of density gradient centrifugation for analysis of preparations fixed with appropriate concentrations of glutaraldehyde. The permeability of the membranes of non-fractionated mitochondria to sucrose was increased by exposure to hypoosmotic sucrose solutions. The B3 mitochondria differed from the non-fractionated mitochondria in their response to changes in osmotic pressure of the suspending medium while the B2 mitochondria showed essentially identical behaviour with the controls. However, under conditions of energized swelling the B2 mitochondria were markedly different to the controls. This difference, which is attributed to reduced permeability of the mitochondrial membranes to metabolites brought about by exposure to the high concentrations of sucrose encountered in the density gradient, was reversed by incubation in hypo-osmotic sucrose solutions in the presence of oxidizable substrate and permeant ions.Died December, 1969.     

Formation and degradation of mitochondria in the cell

Summary Prolonged deaeration ofSaccharomyces cerevisiae cells results in degenerative changes in mitochondria which can be revealed when measuring the enzymic activities of the respiratory chain in isolated organelles and by electron microscope examination of the cells. The same changes are observed after a 3-h incubation of the cells with cyanide or carbonyl cyanide, m-chlorophenyl hydrazone in aerobic conditions. These results suggest the important role of oxidative phosphorylation in the maintenance of the integrity of mitochondria in the cell.The sensitivity of yeast mitochondria to anaerobiosis and cyanide changes as the culture grows. Mitochondria are especially labile during the early exponential growth phase when their respiratory system and structure are not fully formed. Possible reasons for and the mechanism of degradation of mitochondriain vivo are discussed.     

不同悬浮介质对激光衍射法测得的变形指数—渗透压曲线的影响

本文用PBS和PVP两种介质作为激光衍射仪的悬浮介质,系统地观测了悬浮介质物化性能不同对红细胞变形指数(DI)—渗透压曲线的影响,发现:1.在不同悬浮介质中的变形指数—渗透压曲线有显著差别,在高渗区这种差别更明显.2.同一红细胞试样,在相同高渗压下,在不同的悬浮介质中进行交叉实验表明、PBS悬浮介质能使得红细胞变形性得到恢复,而PVP悬浮介质却使得红细胞变形性显著降低.3.在高渗情况下,首次观察到红细胞变形性随时间变化,一开始DI增大,约3小时后趋于稳定,无论PBS还是PVP其趋势皆相似.     

pH对紫膜表面电位的影响

用荧光标记物1,8-AMS与紫膜结合,测量了能化态和非能化态下紫膜表面电位随介质pH的变化.在pH5.5以下,紫膜表面电位随pH的降低而下降,但囊泡中的紫膜表面电位变化幅度较大;在pH5.5以上,处于非能化态的紫膜(无论是紫膜碎片还是处于囊泡中)的表面电位都没有明显变化,处于能化态时,紫膜碎片的表面电位在pH9.2出现一个峰.     

Immunochemical study of role of chargerin II, a product of URFA6L of mitochondrial DNA in energy transduction of rat liver mitochondria

Antibody against chargerin II [product of the unidentified reading frame A6L (URFA6L) of mitochondrial DNA] inhibited the ATP-Pi exchange reaction and reversed electron flow from succinate to NAD in mitoplasts (inner membrane plus matrix). The antibody against chargerin II caused greater inhibition on incubation with mitoplasts in the energized than the nonenergized state, suggesting that redox reactions are coupled with a conformational change of chargerin II. The present findings showed that chargerin II, the URFA6L product, may have a key role in energy transduction of mitochondrial oxidative phosphorylation.     

The induction of energized configurational changes in plant mitochondria,in vivo

Configurational changes in the mitochondrial membranes of the salt gland ofTamarix aphylla, which are dependent on the biochemical state of the mitochondria, are demonstrated. In the energized state the cristae expand and become closely associated. There is also an increase in the density of the matrix and a formation of strands of material in the matrix and between the closely associated cristae membranes. The energized condition can be discharged by incubation in a medium containing 2,4-dinitrophenol and the mitochondria are comparable to those observed in glands fixed by typical methods for electron microscopy.These studies were supported in part by Grant GB-8199 (W.W.T.) from the National Science Foundation.     

The effect of water stress on the activities of key regulatory enzymes of the sucrose to starch pathway in wheat

Developmental changes in the starch and sucrose content of grains andthe activities of enzymes of starch synthesis in wheat were studied under waterstress conditions. Water stress caused a marked reduction in the sucrose andstarch content of the grains. Sucrose synthase (SS) and UDP-glucosepyrophosphorylase (UDP-Gppase), showed higher catalytic activity and moreresistance to water stress compared with amyloplastic enzymes. ADP-glucosepyrophosphorylase (ADP-Gppase) activity was reduced to a low level under bothin situ and osmotic stress conditions in which grainsfailed to accumulate dry matter in vivo. Granule-boundstarch synthase (GBSS) also responded rapidly to in situwater stress treatments as did ADP-Gppase. Reduction in GBSS activity at thetime of growth cessation in situ was less than that ofADP-Gppase and the enzyme did not respond to severe osmotic stress. Solublestarch synthase (SSS) was the enzyme most sensitive to water stress in that itresponded earlier, and to a greater extent, than the other enzymes. However,under severe dehydration conditions, leading to cessation of growth, thedeclinein SSS activity was less than that for ADP-Gppase. SSS showed the lowestin vitro activity followed by GBSS. These results suggestthat SSS is the site of response to water stress by which the rate of graingrowth can be affected, whereas growth cessation is due mainly to theinactivation of ADP-Gppase.     

Transport of alpha-p-nitrophenylgalactoside by the lactose carrier of Escherichia coli.

alpha-p-Nitrophenylgalactoside was found to be accumulated by the lactose transport-system of Escherichia coli. This fact may help to resolve the differences in the reported number of sugar binding sites of the lactose transport protein in nonenergized and energized membrane vesicles.     

Paracrystalline arrays in mitochondria following ageing of mitochondriain situ

Paracrystalline arrays of helical configuration were observed in the mitochondrial intracrystal spaces following prolonged ageing of mitochondriain situ. The occurrence of these mitochondria with the paracrystalline arrays, average diameter of about 70 Å, appeared to increase following an increase in the time of ageingin situ.The exact function of the mitochondria containing the paracrystalline arrays is unknown. These mitochondria could not possibly be responsible for the overall decline in the State 3 respiration, respiratory control index and the ADP/O ratio observed with intact mitochondria isolated after prolonged ageingin situ [21].     

Mechanisms of bactericidal action of cinnamaldehyde against Listeria monocytogenes and of eugenol against L. monocytogenes and Lactobacillus sakei

The spice oil components eugenol and cinnamaldehyde possess activity against both gram-positive and gram-negative bacteria, but the mechanisms of action remain obscure. In broth media at 20 degrees C, 5 mM eugenol or 30 mM cinnamaldehyde was bactericidal (>1-log reduction in the number of CFU per milliliter in 1 h) to Listeria monocytogenes. At a concentration of 6 mM eugenol was bactericidal to Lactobacillus sakei, but treatment with 0.5 M cinnamaldehyde had no significant effect. To investigate the role of interference with energy generation in the mechanism of action, the cellular and extracellular ATP levels of cells in HEPES buffer at 20 degrees C were measured. Treatment of nonenergized L. monocytogenes with 5 mM eugenol, 40 mM cinnamaldehyde, or 10 microM carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 5 min prevented an increase in the cellular ATP concentration upon addition of glucose. Treatment of energized L. monocytogenes with 40 mM cinnamaldehyde or 10 microM CCCP caused a rapid decline in cellular ATP levels, but 5 mM eugenol had no effect on cellular ATP. Treatment of L. sakei with 10 mM eugenol prevented ATP generation by nonenergized cells and had no effect on the cellular ATP of energized cells. CCCP at a concentration of 100 microM had no significant effect on the cellular ATP of L. sakei. No significant changes in extracellular ATP were observed. Due to their rapidity, effects on energy generation clearly play a major role in the activity of eugenol and cinnamaldehyde at bactericidal concentrations. The possible mechanisms of inhibition of energy generation are inhibition of glucose uptake or utilization of glucose and effects on membrane permeability.     

Zeaxanthin Formation and Energy-Dependent Fluorescence Quenching in Pea Chloroplasts under Artificially Mediated Linear and Cyclic Electron Transport

Artificially mediated linear (methylviologen) and cyclic (phenazine methosulfate) electron transport induced zeaxanthin-dependent and independent (constitutive) nonphotochemical quenching in osmotically shocked chloroplasts of pea (Pisum sativum L. cv Oregon). Nonphotochemical quenching was quantitated as Stern-Volmer quenching (SV_N) calculated as (F_m/F′_m)-1 where F_m is the fluorescence intensity with all PSII reaction centers closed in a nonenergized, dark-adapted state and F′_m is the fluorescence intensity with all PSII reaction centers closed in an energized state. Reversal of quenching by nigericin and electron-transport inhibitors showed that both quenching types were energy-dependent SV_N. Under light-induced saturating ΔpH, constitutive-SV_N reached steady-state in about 1 minute whereas zeaxanthin-SV_N continued to develop for several minutes in parallel with the slow kinetics of violaxanthin deepoxidation. SV_N above the constitutive level and relative zeaxanthin concentration showed high linear correlations at steady-state and during induction. Furthermore, F_o quenching, also treated as Stern-Volmer quenching (SV_O) and calculated as (F_o/F′_o)-1, showed high correlation with zeaxanthin and consequently with SV_N (F_o and F′_o are fluorescence intensities with all PSII reaction centers in nonenergized and energized states, respectively). These results support the view that zeaxanthin increases SV_N above the constitutive level in a concentration-dependent manner and that zeaxanthin-dependent SV_N occurs in the pigment bed. Preforming zeaxanthin increased the rate and extent of SV_N, indicating that slow events other than the amount of zeaxanthin also affect final zeaxanthin-SV_N expression. The redox state of the primary electron acceptor of photosystem II did not appear to determine SV_N. Antimycin, when added while chloroplasts were in a dark-adapted or nonenergized state, inhibited both zeaxanthin-SV_N and constitutive-SV_N induced by linear and cyclic electron transport. These similarities, including possible constitutive F_o quenching, suggest that zeaxanthin-dependent and constitutive SV_N are mechanistically related.     

Homology model of the multidrug transporter LmrA from Lactococcus lactis

LmrA is an ATP dependent multidrug transporter from Lactococcus lactis conferring antibiotic resistance to 17 out of 21 most frequently administered antibiotics. Starting from the dimeric crystal structure of Vc-MsbA, we built two homology models, with NBD:NBD interfaces reflecting the nonenergized and energized state, respectively. The TMD:TMD topology of the dimer is consistent with the previously obtained substrate photoaffinity labeling pattern suggesting binding of substrates at the TMD:TMD interface involving helix 3 of one monomer and helices 5 and 6 of the other monomer.