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The Journal of Cell Biology May 1984We studied retinal photoreceptors of Rana pipiens by using techniques designed to investigate calcium localization. Particularly useful were methods in which...
We studied retinal photoreceptors of Rana pipiens by using techniques designed to investigate calcium localization. Particularly useful were methods in which intracellular sites of calcium uptake were detected by incubation of saponin-treated isolated retinas in calcium-containing media, with oxalate present as a trapping agent. With these procedures, cell compartments accumulate deposits, which can be shown to contain calcium by x-ray microanalysis. Calcium accumulation was prominent in the rough endoplasmic reticulum in the myoid region. In addition, deposits were observed in agranular reticulum and in certain Golgi-associated compartments of the myoid region, in mitochondria, in axonal reticulum, and in agranular reticulum of presynaptic terminals. Calcium was also detected in the endoplasmic reticulum of retinas fixed directly upon isolation, by a freeze-substitution method. The factors influencing accumulation of calcium in the endoplasmic reticulum were evaluated by a semiquantitative approach based on determining the relative frequency of calcium oxalate crystals under varying conditions. Calcium accumulation was markedly enhanced by ATP. Studies with a nonhydrolyzable ATP analogue (adenylyl- imidodiphosphate ) and with inhibitors of the sarcoplasmic reticulum Ca2+-Mg2+ ATPase (mersalyl and tetracaine) indicated that this ATP-dependent calcium uptake reflects an energy-dependent process roughly comparable to that in the sarcoplasmic reticulum.
Topics: Adenosine Triphosphate; Adenylyl Imidodiphosphate; Animals; Biological Transport, Active; Calcium; Calcium Oxalate; Cell Compartmentation; Endoplasmic Reticulum; Golgi Apparatus; Mersalyl; Microscopy, Electron; Photoreceptor Cells; Rana pipiens; Tetracaine
PubMed: 6609924
DOI: 10.1083/jcb.98.5.1645 -
The Journal of Physiology Jun 19721. In the perfused cat submandibular gland efflux and influx of (45)Ca, and concentrations of K, (40)Ca and Mg in the effluent from the gland were measured under...
1. In the perfused cat submandibular gland efflux and influx of (45)Ca, and concentrations of K, (40)Ca and Mg in the effluent from the gland were measured under different experimental conditions.2. When the standard perfusion fluid was shifted to a high Mg (5 mM) or a low Ca (0.25 mM) solution the efflux of (45)Ca from the pre-labelled gland declined. The magnitude and the duration of the effect of the high Mg concentration was more marked at a low external Ca concentration and was abolished by Mersalyl (1 mM). When the standard perfusion fluid was shifted to a Mg-free solution the efflux of (45)Ca from the pre-labelled gland increased.3. After shift of (45)Ca containing perfusion fluid from normal to a high Mg (5 mM) solution the influx of (45)Ca to the gland increased rapidly.4. Both acetylcholine (ACh) and adrenaline caused a marked increase in the efflux of (45)Ca from the pre-labelled gland. This increase in efflux was also seen under conditions where the gland was unable to secrete, i.e. during perfusion with Ca-free and Na-free tetraethylammonium Locke solutions.5. Stimulation with ACh failed to reveal any rapidly occurring increase in influx of (45)Ca.6. Stimulation with ACh evoked a small temporary increase in the concentration of (40)Ca. and Mg in the effluent.7. It is suggested that Ca uptake by intracellular Ca-accumulating systems of the submandibular gland depends on the external Mg concentration and that ACh and adrenaline cause a release of Ca bound intracellularly.
Topics: Acetylcholine; Animals; Biological Transport, Active; Calcium; Calcium Isotopes; Cats; Epinephrine; Magnesium; Perfusion; Potassium; Spectrophotometry, Atomic; Stimulation, Chemical; Submandibular Gland
PubMed: 5045737
DOI: 10.1113/jphysiol.1972.sp009869 -
The Biochemical Journal Dec 1978Diamide is reduced by mitochondria utilizing endogenous substrates with Vmax. 20nmol/min per mg of protein and Km 75micrometer. The reaction is inhibited by: (a)...
Diamide is reduced by mitochondria utilizing endogenous substrates with Vmax. 20nmol/min per mg of protein and Km 75micrometer. The reaction is inhibited by: (a) thiol-blocking reagents (N-ethylmaleimide, p-hydroxymercuribenzoate, mersalyl and 2,6-dichlorophenol-indophenol);(b) respiratory inhibitors (arsenicals, malonate and antimycin, but not cyanide or oligomycin; inhibition by antimycin is reversed by ATP); (c) uncouplers (carbonyl cyanide p-trifluoromethoxyphenylhydrazone, 2,4-dinitrophenol and valinomycin with K+; inhibition by the first of these uncouplers is not reversed by cyanide); (d) reagents affecting energy conservation (Ca2+, increasing pH, phosphate; phosphate inhibition is augmented by catalytic ADP or ATP and augmentation is abolished by respiratory inhibitors). Concentrations of mitochondrial glutathione are high when diamide reduction is uninhibited, but low after adding one of the above inhibitors such that the reduction rate is roughly proportional to the glutathione concentration. Endogenous ATP concentrations are lower in the presence of diamide than without, but the difference is abolished by respiratory inhibitors. With oligomycin added, however, ATP concentrations are higher in the presence of diamide and this positive increment is decreased by antimycin, N-ethylmaleimide and malonate. In the presence of diamide and an uncoupler, the mitochondrial glutathione content does not fall if various reducible substrates are present, although the inhibition of diamide reduction is not relieved. Some of these substrates prevent the fall in reduced glutathione concentration found with diamide and phosphate. They also relieve the inhibition of diamide reduction and the relief is sensitive to butylmalonate. The inhibition of diamide reduction by N-ethylmaleimide, mersalyl or p-hydroxymercuribenzoate is not relieved by reducible substrates, but the latter mitigate the fall in the concentration of glutathione. Inhibitors of carriers of tricarboxylic acid-cycle intermediates also inhibit reduction of diamide. The reduced glutathione concentration remains high when they are added singly, but falls when two of them are combined. It is proposed that diamide may enter the matrix as a protonated adduct formed with the thiol groups of mitochondrial carriers and then be reduced in the matrix by glutathione, which is regenerated via NADH, energy-dependent transhydrogenase and NADP+-specific glutathione reductase. Some of the high-energy equivalents required for the transhydrogeneration may be generated by the substrate phosphorylation step of the tricarboxylic acid cycle.
Topics: Adenosine Triphosphate; Animals; Azo Compounds; Diamide; Female; Glutathione; In Vitro Techniques; Kinetics; Mitochondria, Liver; Oxidation-Reduction; Phosphates; Rats; Sulfhydryl Reagents; Uncoupling Agents
PubMed: 747642
DOI: 10.1042/bj1760649 -
The Journal of Biological Chemistry Jun 1975MgATP-dependent calcium sequestering activity of rat liver microsomes has been characterized. This activity is linear over a 90-min period and specifically requires...
MgATP-dependent calcium sequestering activity of rat liver microsomes has been characterized. This activity is linear over a 90-min period and specifically requires MgATP. Substitution of CTP, UTP, GTP, or ADP will not support calcium uptake. Oxalate, which serves as a trapping agent in calcium uptake of skeletal muscle microsomes, is required to maintain net accumulation of calcium. The reaction is temperature-dependent and has an apparent Vmax of 11.2 nmol/mg of protein/min. The apparent Km for calcium is 23.2 muM calculated from total calcium concentration, and approximately 4.6 muM based on free calcium concentration, and apparent Km for ATP is 1.8 mM. Calcium uptake activity normally measured in presence of 100 mM KCl is only slightly depressed if 100 mM NaCl is substituted and is considerably depressed when 200 mM sucrose replaces KCl. An appropriate hydrolysis of ATP is associated with the calcium uptake. Separation of smooth and rough endoplasmic reticulum on sucrose gradients indicates a considerably lower specific activity per mg of protein in the fraction enriched with rough endoplasmic reticulum. Azide, at a level which completely inhibits liver mitochondrial calcium sequestration, has no effect on the liver microsomal system. Oligomycin, which inhibits ATP-dependent calcium uptake of liver mitochondria, has a considerably lesser effect on calcium uptake of liver microsomes. p-Chloromercuribenzoate and mersalyl inhibit the liver microsomal calcium pump at levels as low as 10- minus 7 M. Calcium uptake activity is considerably reduced in adult female rats. Weanling rats, both male and female, have calcium uptake activities like that of the adult males. Because of the higher activity in the male rat, the fatty acid composition of the liver microsomal phospholipids was analyzed. The male rat had a higher percentage of linoleic and palmitic acids in the microsomal phospholipids. Endoplasmic reticulum and plasma membrane are postulated to play a role in regulation of the levels of free cytoplasmic calcium in the mammalian liver.
Topics: Adenosine Triphosphate; Animals; Antimycin A; Azides; Biological Transport, Active; Calcium; Edetic Acid; Endoplasmic Reticulum; Female; Kinetics; Male; Microsomes, Liver; Oligomycins; Oxalates; Phospholipids; Rats; Sex Factors; Time Factors
PubMed: 806589
DOI: No ID Found -
The Journal of Biological Chemistry Nov 1994In a reconstituted system, the participation of the ATP/ADP carrier (AAC) in the free fatty acid (FFA)-induced proton transport was demonstrated (i) by direct measuring...
In a reconstituted system, the participation of the ATP/ADP carrier (AAC) in the free fatty acid (FFA)-induced proton transport was demonstrated (i) by direct measuring of the proton transport through the membranes of AAC proteoliposomes and (ii) by monitoring of the transmembrane potential delta psi in AAC-cytochrome-c oxidase (COX)-coreconstituted proteoliposomes. FFA increased the initial rate of proton transport in AAC proteoliposomes and decreased delta psi in AAC-COX proteoliposomes. Inhibitors of AAC suppressed the effects of FFA. Without AAC or with inactive AAC, FFA cannot maintain proton leakage through the membrane. In these cases, even a small increase of delta psi was induced by FFA. These results demonstrate for the first time with purified components a participation of AAC in FFA-induced proton transport supporting an earlier suggestion (Skulachev, V.P. (1991) FEBS Lett. 294, 158-162). Mersalyl treatment of the AAC-COX proteoliposomes resulted in an increase of the AAC-mediated protonophoric action of FFA. Mersalyl also sensitized the protonophoric action of the FFA against nucleotides so that even guanine nucleotides, which are inactive in transport, become inhibitory. The effect of mersalyl is rationalized in terms of a specific interaction with cysteine 159 being attracted as anion by surrounding positive charges. This might open a gate similarly as suggested for eosin 5-maleimide interaction (Majima, E., Koike, H., Hong, Y.-M., Shinohara, Y., and Terada, H. (1993) J. Biol. Chem. 268, 22181-22187) and, thus, transform the AAC into undirectional transport mode.
Topics: Adenosine Triphosphate; Animals; Biological Transport; Cattle; Electron Transport Complex IV; Fatty Acids, Nonesterified; Guanine Nucleotides; Hydrogen-Ion Concentration; In Vitro Techniques; Ligands; Mersalyl; Mitochondria, Heart; Mitochondrial ADP, ATP Translocases; Proteolipids; Uncoupling Agents
PubMed: 7961643
DOI: No ID Found -
The Journal of Biological Chemistry Aug 1975Bovine spermatozoa accumulated a small amount of 32Pi during aerobic incubation in vitro. At least 50% of the acquired isotope rapidly entered cellular nucleotides. Both...
Bovine spermatozoa accumulated a small amount of 32Pi during aerobic incubation in vitro. At least 50% of the acquired isotope rapidly entered cellular nucleotides. Both adenosine and guanosine di- and triphosphates were labeled, but contrary to expectations, the specific activity of ADP exceeded that of ATP. The uptake of phosphate and its incorporation into nucleotides were suppressed by respiratory inhibitors and were abolished by treatment with sulfhydryl-directed reagents at 10 to 20 nmol/mg of sperm protein. With fructose as an energy source for motility, glycolysis did not support phosphate uptake. Nucleotide labeling was increased 60 to 80-fold when the cells were treated with the polyene antibiotic filipin, and filipin was able to reverse the inhibition of phosphate (and succinate) entry produced by N-ethylmaleimide or mersalyl. Since filipin interacts specifically with the cholesterol-containing plasma membrane of bovine spermatozoa and increases its permeability, it is probable that the plasma membrane normally limits phosphate and succinate transport into these cells. This contention is further supported by the observation that high concentrations of extracellular Pi, the penetration of which was extremely limited under these conditions, protected against inactivation by N-ethylmaleimide. Phosphate uptake was increased 10 to 20-fold, but nucleotide labeling was inhibited, when calcium was present in the incubation medium. Ruthenium red, presumably acting extracellularly, prevented these effects of calcium. Thus, the entry of phosphate and succinate into spermatozoa is controlled by plasma membrane components that resemble the phosphate and succinate exchangers and calcium carrier found in mitochondria isolated from other sources.
Topics: Animals; Antimycin A; Biological Transport, Active; Calcium; Cattle; Cell Membrane; Dinitrophenols; Egtazic Acid; Ethylmaleimide; Filipin; Fructose; Kinetics; Lactates; Male; Mersalyl; Oxygen Consumption; Phosphates; Rotenone; Rutamycin; Spermatozoa; Succinates
PubMed: 808544
DOI: No ID Found -
Plant Physiology Feb 1992Mitochondria from some plant tissues possess the ability to take up Ca(2+) by a phosphate-dependent mechanism associated with a decrease in membrane potential, H(+)...
Mitochondria from some plant tissues possess the ability to take up Ca(2+) by a phosphate-dependent mechanism associated with a decrease in membrane potential, H(+) extrusion, and increase in the rate of respiration (AE Vercesi, L Pereira da Silva, IS Martins, CF Bernardes, EGS Carnieri, MM Fagian [1989] In G Fiskum, ed, Cell Calcium Metabolism. Plenum Press, New York, pp 103-111). The present study reexamined the nature of the phosphate requirement in this process. The main observations are: (a) Respiration-coupled Ca(2+) uptake by isolated corn (Zea mays var Maya Normal) mitochondria or carbonyl cyanide p-trifluoromethoxyphenylhydrazone-induced efflux of the cation from such mitochondria are sensitive to mersalyl and cannot be dissociated from the silmultaneous movement of phosphate in the same direction. (b) Ruthenium red-induced efflux is not affected by mersalyl and can occur in the absence of phosphate movement. (c) In Ca(2+)-loaded corn mitochondria, mersalyl causes net Ca(2+) release unrelated to a decrease in membrane potential, probably due to an inhibition of Ca(2+) cycling at the level of the influx pathway. It is concluded that corn mitochondria (and probably other plant mitochondria) do possess an electrophoretic influx pathway that appears to be a mersalyl-sensitive Ca(2+)/inorganic phosphate-symporter and a phosphate-independent efflux pathway possibly similar to the Na(2+)-independent Ca(2+) efflux mechanism of vertebrate mitochondria, because it is not stimulated by Na(+).
PubMed: 16668661
DOI: 10.1104/pp.98.2.452 -
European Journal of Biochemistry Dec 1991Both the external oxidation of NADH and NADPH in intact potato (Solanum tuberosum L. cv. Bintje) tuber mitochondria and the rotenone-insensitive internal oxidation of...
Both the external oxidation of NADH and NADPH in intact potato (Solanum tuberosum L. cv. Bintje) tuber mitochondria and the rotenone-insensitive internal oxidation of NADPH by inside-out submitochondrial particles were dependent on Ca2+. The stimulation was not due to increased permeability of the inner mitochondrial membrane. Neither the membrane potential nor the latencies of NAD(+)-dependent and NADP(+)-dependent malate dehydrogenases were affected by the addition of Ca2+. The pH dependence and kinetics of Ca(2+)-dependent NADPH oxidation by inside-out submitochondrial particles were studied using three different electron acceptors: O2, duroquinone and ferricyanide. Ca2+ increased the activity with all acceptors with a maximum at neutral pH and an additional minor peak at pH 5.8 with O2 and duroquinone. Without Ca2+, the activity was maximal around pH 6. The Km for NADPH was decreased fourfold with ferricyanide and duroquinone, and twofold with O2 as acceptor, upon addition of Ca2+. The Vmax was not changed with ferricyanide as acceptor, but increased twofold with both duroquinone and O2. Half-maximal stimulation of the NADPH oxidation was found at 3 microM free Ca2+ with both O2 and duroquinone as acceptors. This is the first report of a membrane-bound enzyme inside the inner mitochondrial membrane which is directly dependent on micromolar concentrations of Ca2+. Mersalyl and dicumarol, two potent inhibitors of the external NADH dehydrogenase in plant mitochondria, were found to inhibit internal rotenone-insensitive NAD(P)H oxidation, at the same concentrations and in manners very similar to their effects on the external NAD(P)H oxidation.
Topics: Calcium; Cations, Divalent; Cell Membrane; Cell Membrane Permeability; Dicumarol; Hydrogen-Ion Concentration; Intracellular Membranes; Kinetics; Membrane Potentials; Mersalyl; Mitochondria; NADP; NADPH Dehydrogenase; Oxidation-Reduction; Rotenone; Solanum tuberosum; Submitochondrial Particles
PubMed: 1722151
DOI: 10.1111/j.1432-1033.1991.tb16415.x -
The Journal of Biological Chemistry Aug 1997Yeast mitochondria (Saccharomyces cerevisiae) contain a permeability transition pore which is regulated differently than the pore in mammalian mitochondria. In a...
Yeast mitochondria (Saccharomyces cerevisiae) contain a permeability transition pore which is regulated differently than the pore in mammalian mitochondria. In a mannitol medium containing 10 mM Pi and ethanol (oxidizable substrate), yeast mitochondria accumulate large amounts of Ca2+ (>400 nmol/mg of protein) upon the addition of an electrophoretic Ca2+ ionophore (ETH129). Pore opening does not occur following Ca2+ uptake, even though ruthenium red-inhibited rat liver mitochondria undergo rapid pore opening under analogous conditions. However, a pore does arise in yeast mitochondria when Ca2+ and Pi are not present, as monitored by swelling, ultrastructure, and matrix solute release. Pore opening is slow unless a respiratory substrate is provided (ethanol or NADH) but also occurs rapidly in response to ATP (2 mM) when oligomycin is present. Pi and ADP inhibit pore opening (EC50 approximately 1 and 4 mM, respectively), however, cyclosporin A (7 microg/ml), oligomycin (20 microg/ml), or carboxyatractyloside (25 microM) have no effect. The pore arising during respiration is also inhibited by nigericin or uncoupler, indicating that an acidic matrix pH antagonizes the process. Pi also inhibits pore opening by lowering the matrix pH (Pi/OH- antiport). However, inhibition of the ATP-induced pore by Pi is seen in the presence of mersalyl, suggesting a second mechanism of action. Since pore induction by ATP is not sensitive to carboxyatractyloside, ATP appears to act at an external site and Pi may antagonize the interaction. Isoosmotic polyethylene glycol-induced contraction of yeast mitochondria swollen during respiration, or in the presence of ATP, is 50% effective at a solute size of 1.0-1.1 kDa. This suggests that the same pore is induced in both cases and is comparable in size with the permeability transition pore of heart and liver mitochondria.
Topics: Adenosine Triphosphate; Animals; Biological Transport; Calcium; Cyclosporine; Ethanol; Hydrogen-Ion Concentration; Mitochondria; Mitochondria, Liver; Mitochondrial Swelling; Permeability; Phosphates; Rats; Saccharomyces cerevisiae
PubMed: 9261114
DOI: 10.1074/jbc.272.34.21104 -
The Journal of Biological Chemistry Oct 1975Mitochondria from dog renal cortex were incubated with L-[14Cglutamine. Glutamate metabolism was prevented by inhibitors so that glutamate accumulated either in the...
Mitochondria from dog renal cortex were incubated with L-[14Cglutamine. Glutamate metabolism was prevented by inhibitors so that glutamate accumulated either in the mitochondrial matrix space or in the medium. The formation and accumuation of glutamate formed from glutamine and the distribution of glutamine in the mitochondrial fluid spaces were studied. In the matrix space glutamate rapidly reaches levels over 5 times that of glutamine in the medium. A more gradual accumulation occurs in the medium as glutamate is transported out of the mitochondria. Addition of an energy source such as succinate to the medium accelerates glutamate formation. A Km of 0.6 mM appears to govern the reaction at low concentrations of glutamine; at about 4 mM an abrupt change kinetics occurs with a Km of 5 mM above that level. Both NH4+ and glutamate inhibit glutamine metabolism and phosphate stimulates it, but little effect glutamate or phosphate occurs at low levels of these substances. The pH optimum of the reaction is between 7.4 and 7.8. Mersalyl and p-chloromercuribenzoate strongly inhibit glutamate formation; N-ethylmaleimide and bromcresol green have weaker inhibitory actions, and borate increases the reaction rate. In the presence of mersalyl, glutamine is striclly confined to the outer space of mitochondria and none is detectable in the matrix space. Similarly at ) degrees glutamine is confined to the simultaneously determined sucrose or mannitol spaces...
Topics: Acidosis; Alkalosis; Ammonia; Animals; Antimycin A; Biological Transport, Active; Dogs; Glutamates; Glutamine; Kidney Cortex; Kinetics; Mitochondria; Oxidative Phosphorylation; Oxygen Consumption; Phosphates; Succinates
PubMed: 1181338
DOI: No ID Found