Conversion of esterified fura-2 and indo-1 to Ca2+-sensitive forms by mitochondria

1988 ◽  
Vol 255 (3) ◽  
pp. C304-C310 ◽  
Author(s):  
T. E. Gunter ◽  
D. Restrepo ◽  
K. K. Gunter
Keyword(s):  
Liver Mitochondria ◽  
Systematic Investigation ◽  
Rat Liver Mitochondria ◽  
Mitochondrial Matrix ◽  
Acid Form ◽  
Conversion Product ◽  
Acetoxymethyl Ester ◽  
Fura 2 ◽  
Before And After ◽  
Hepatocyte Suspension

Rat liver mitochondria are shown to convert the acetoxymethyl ester forms of fura-2 and indo-1 into Ca2+-dependent forms of these indicators. The excitation spectrum of the Ca2+-dependent conversion product of fura-2 acetoxymethyl ester is shown to be similar to that of the pentacarboxylic acid form of fura-2. A systematic investigation of the ionic strength and pH dependences of the fluorescence of the pentacarboxylic acid forms of these indicators shows small changes within the ranges thought to obtain within the mitochondrial matrix after Ca2+ uptake. Intramitochondrial free Ca2+ levels are studied both before and after Ca2+ sequestration by mitochondria, and a rough estimate is made of the mitochondrial contribution to the Ca2+-dependent fura-2 fluorescence of a hepatocyte suspension.

scholarly journals MORPHOLOGY AND ATP-ASE OF ISOLATED MITOCHONDRIA

1955 ◽  
Vol 1 (2) ◽  
pp. 127-138 ◽  
Author(s):  
Robert F. Witter ◽  
Michael L. Watson ◽  
Mary A. Cottone
Keyword(s):  
Electron Microscope ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Substantial Part ◽  
Mitochondrial Matrix ◽  
Isolated Mitochondria ◽  
The Matrix ◽  
First Time ◽  
Methods Of Isolation

Changes in the morphology of rat liver mitochondria brought about by different methods of isolation and the concomitant changes in ATP-ase activity were studied. The morphology was investigated with the electron microscope. It was found that the ATP-ase activity of the isolated mitochondria cannot be readily correlated with the morphology of the mitochondria. The ATP-ase found in these preparations was latent, resembling the enzyme described in mitochondria prepared in 0.25 M sucrose. In confirmation of earlier results the use of 0.88 M sucrose yielded preparations with a higher initial ATP-ase than did other methods. Preparation in 0.25 M sucrose resulted in round, swollen mitochondria of which 30 to 40 per cent appeared to have lost a substantial part of the mitochondrial matrix. Preparations in 0.44 to 0.88 M sucrose contained mainly rod-shaped mitochondria plus a small amount of another type of swollen mitochondria. The matrix of mitochondria isolated in 0.88 M sucrose was highly condensed. By the use of 0.44 M sucrose adjusted to pH 6.2 with citric acid, it was possible to isolate, for the first time, mitochondria closely resembling those in situ and containing latent ATP-ase.

scholarly journals Stimulation of the respiration rate of rat liver mitochondria by sub-micromolar concentrations of extramitochondrial Ca2+

1987 ◽  
Vol 245 (1) ◽  
pp. 217-222 ◽  
Author(s):  
J D Johnston ◽  
M D Brand
Keyword(s):  
Respiration Rate ◽  
Rat Liver ◽  
Oxidative Metabolism ◽  
Mitochondrial Respiration ◽  
Liver Mitochondria ◽  
Ruthenium Red ◽  
Rat Liver Mitochondria ◽  
Mitochondrial Matrix ◽  
Stimulation Of

1. The respiration rate of rat liver mitochondria was stimulated by up to 70% when the extramitochondrial Ca2+ concentration was raised from 103 to 820 nM. This occurred when pyruvate, 2-oxoglutarate, or threo-(Ds)-isocitrate was employed as substrate, but not when succinate was used. 2. Ruthenium Red prevented the stimulation of mitochondrial respiration by extramitochondrial Ca2+, showing that the effect required Ca2+ uptake into the mitochondrial matrix. 3. Starvation of rats for 48 h abolished the stimulation of mitochondrial respiration by extramitochondrial Ca2+ when pyruvate was used as substrate, but did not affect the stimulation of 2-oxoglutarate oxidation by extramitochondrial Ca2+. 4. Our findings are in accord with proposals that oxidative metabolism in liver mitochondria may be stimulated by Ca2+ activation of intramitochondrial dehydrogenases.

scholarly journals Inhibition in vitro of acyl-CoA dehydrogenases by 2-mercaptoacetate in rat liver mitochondria

1983 ◽  
Vol 215 (3) ◽  
pp. 457-464 ◽  
Author(s):  
F Bauché ◽  
D Sabourault ◽  
Y Giudicelli ◽  
J Nordmann ◽  
R Nordmann
Keyword(s):  
Rat Liver ◽  
Conformational Changes ◽  
Reaction Medium ◽  
Liver Mitochondria ◽  
Fatty Acyl ◽  
Rat Liver Mitochondria ◽  
Mitochondrial Matrix ◽  
Main Effect ◽  
Acyl Coa Dehydrogenases

In rat liver hypo-osmotically treated mitochondria, 2-mercaptoacetate inhibits respiration induced by palmitoyl-CoA, octanoate or butyryl-CoA only when the reaction medium is supplemented with ATP. Under this condition, NADH-stimulated respiration is not affected. In liver mitochondrial matrix, the presence of ATP is also required to observe a 2-mercaptoacetate-induced inhibition of acyl-CoA dehydrogenases tested with palmitoyl-CoA, butyryl-CoA or isovaleryl-CoA as substrate. As the oxidation of these substrates is also inhibited by the incubation medium resulting from the reaction of 2-mercaptoacetate with acetyl-CoA synthase, with conditions under which 2-mercaptoacetate has no effect, 2-mercaptoacetyl-CoA seems to be the likely inhibitory metabolite responsible for the effects of 2-mercaptoacetate. Kinetic experiments show that the main effect of the 2-mercaptoacetate-active metabolite is to decrease the affinities of fatty acyl-CoA dehydrogenases towards palmitoyl-CoA or butyryl-CoA and of isovaleryl-CoA dehydrogenase towards isovaleryl-CoA. Addition of N-ethylmaleimide to mitochondrial matrix pre-exposed to 2-mercaptoacetate results in the immediate reversion of the inhibitions of palmitoyl-CoA and isovaleryl-CoA dehydrogenations and in a delayed reversion of butyryl-CoA dehydrogenation. These results led us to conclude that (i) the ATP-dependent conversion of 2-mercaptoacetate into an inhibitory metabolite takes place in the liver mitochondrial matrix and (ii) the three fatty acyl-CoA dehydrogenases and isovaleryl-CoA dehydrogenase are mainly competitively inhibited by this compound. Finally, the present study also suggests that the inhibitory metabolite of 2-mercaptoacetate may bind non-specifically to, or induce conformational changes at, the acyl-CoA binding sites of these dehydrogenases.

scholarly journals ROLE OF EDTA AND METALS IN MITOCHONDRIAL CONTRACTION

1964 ◽  
Vol 23 (1) ◽  
pp. 9-19 ◽  
Author(s):  
William S. Lynn ◽  
Sydney Fortney ◽  
Rose H. Brown
Keyword(s):  
Atp Hydrolysis ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Mitochondrial Matrix ◽  
Mitochondrial Membranes ◽  
Semipermeable Membranes ◽  
Microscopic Appearance ◽  
Hydration And Dehydration ◽  
Mitochondrial Contraction

Studies comparing the state of hydration and dehydration of rat liver mitochondria to their content of ATP, Ca, and fatty acid, along with the rate of ATP hydrolysis, as well as microscopic appearance of mitochondria, have led to the following generalizations: 1. The competition between cationic translocations and water translocation for the available chemical energy (ATP) determines under many circumstances the water content of mitochondria. 2. Swelling of mitochondria by electron transport substrates is an example of the activation of the cationic translocations at the expense of water translocation. 3. Electron micrographic studies are interpreted to indicate that EDTA alone can cause condensation and dehydration of the mitochondrial matrix. However, both EDTA and substrate are necessary to remove appreciable quantities of water from mitochondrial intramembranous spaces. 4. Since the data in the accompanying report indicated that EDTA, in the absence of energy, decreased the permeability of mitochondrial membranes, it appears likely that ballooning of intramembranous spaces, following addition of EDTA, represents trapping of water between two semipermeable membranes following dehydration of mitochondrial matrix.

scholarly journals Leaky β-Oxidation of atrans-Fatty Acid

2004 ◽  
Vol 279 (50) ◽  
pp. 52160-52167 ◽  
Author(s):  
Wenfeng Yu ◽  
Xiquan Liang ◽  
Regina E. Ensenauer ◽  
Jerry Vockley ◽  
Lawrence Sweetman ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Fatty Acid ◽  
Double Bond ◽  
Octadecenoic Acid ◽  
Liver Mitochondria ◽  
Gas Chromatography Mass Spectrometry ◽  
Rat Liver Mitochondria ◽  
Mitochondrial Matrix ◽  
Chain Acyl ◽  
Long Chain

The degradation of elaidic acid (9-trans-octadecenoic acid), oleic acid, and stearic acid by rat mitochondria was studied to determine whether the presence of atransdouble bond in place of acisdouble bond or no double bond affects β-oxidation. Rat mitochondria from liver or heart effectively degraded the coenzyme A derivatives of all three fatty acids. However, with elaidoyl-CoA as a substrate, a major metabolite accumulated in the mitochondrial matrix. This metabolite was isolated and identified as 5-trans-tetradecenoyl-CoA. In contrast, little or none of the corresponding metabolites were detected with oleoyl-CoA or stearoyl-CoA as substrates. A kinetic study of long-chain acyl-CoA dehydrogenase (LCAD) and very long-chain acyl-CoA dehydrogenase revealed that 5-trans-tetradecenoyl-CoA is a poorer substrate of LCAD than is 5-cis-tetradecenoyl-CoA, while both unsaturated acyl-CoAs are poor substrates of very long-chain acyl-CoA dehydrogenase when compared with myristoyl-CoA. Tetradecenoic acid and tetradecenoylcarnitine were detected by gas chromatography/mass spectrometry and tandem mass spectrometry, respectively, when rat liver mitochondria were incubated with elaidoyl-CoA but not when oleoyl-CoA was the substrate. These observations support the conclusion that 5-trans-tetradecenoyl-CoA accumulates in the mitochondrial matrix, because it is less efficiently dehydrogenated by LCAD than is itscisisomer and that the accumulation of this β-oxidation intermediate facilitates its hydrolysis and conversion to 5-trans-tetradecenoylcarnitine thereby permitting a partially degraded fatty acid to escape from mitochondria. Analysis of this compromised but functional process provides insight into the operation of β-oxidation in intact mitochondria.

scholarly journals Levels of carbamoyl phosphate synthetase I in livers of young and old rats assessed by activity and immunoassays and by electron microscopic immunogold procedures.

1990 ◽  
Vol 38 (3) ◽  
pp. 371-376 ◽  
Author(s):  
A Martinez-Ramon ◽  
E Knecht ◽  
V Rubio ◽  
S Grisolia
Keyword(s):  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Mitochondrial Matrix ◽  
Carbamoyl Phosphate Synthetase ◽  
Enzyme Protein ◽  
Carbamoyl Phosphate ◽  
Electron Microscopic ◽  
Liver Functions ◽  
Old Rats ◽  
Liver Homogenates

Carbamoyl phosphate synthetase I, the most abundant protein of rat liver mitochondria, plays a key role in synthesis of urea. Because aging affects some liver functions, and because there is no information on the levels of carbamoyl phosphate synthetase I during aging, we assayed the activity of this enzyme and determined immunologically the level of carbamoyl phosphate synthetase I in liver homogenates from young (4 months) and old (18 or 26 months) rats. In addition, we used electron microscopic immunogold procedures to locate and measure the amount of the enzyme in the mitochondrial matrix. There is no significant change in enzyme activity or enzyme protein content with age, although there is a higher concentration of the enzyme in the mitochondria (c. 1.5 times greater) from old rats, which is compensated by a decrease in the fractional volume of the mitochondrial compartment during aging.

scholarly journals Regulation of glycine catabolism in rat liver mitochondria

1992 ◽  
Vol 283 (2) ◽  
pp. 435-439 ◽  
Author(s):  
M Jois ◽  
H S Ewart ◽  
J T Brosnan
Keyword(s):  
Rat Liver ◽  
Fold Increase ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Mitochondrial Matrix ◽  
Isolated Rat Liver ◽  
Matrix Volume ◽  
Isolated Rat ◽  
Stimulation Of

1. The catabolism of glycine was studied in isolated rat liver mitochondria by measuring release of 14CO2 from [1-14C]-glycine. Incubation of mitochondria in a medium containing 0.5 microM free Ca2+ resulted in an 8-fold increase in the rate of degradation of glycine. Intraperitoneal injection of glucagon (33 or 100 micrograms/100 g body wt.) 25 min before killing of rats also resulted in a 3-fold or 10-fold (depending on dosage) increase in the rate of catabolism of glycine. 2. Both the stimulation by free Ca2+ and that by injection of glucagon in vivo were dependent on phosphate in the incubation medium. This requirement for phosphate was specific, as replacement of phosphate by other permeant anions such as thiocyanate and acetate did not permit the stimulation. The phosphate-dependent stimulation of glycine catabolism by Ca2+ was also evident when mitochondria were incubated in the absence of K+. 3. Mitochondria isolated from rats previously injected with glucagon showed elevated rates of degradation of glycine even in the presence of rotenone, provided that regeneration of NAD+ was affected by providing acetoacetate. 4. Hypo-osmolarity of the medium markedly stimulated the rate of degradation of glycine by mitochondria. Although hypo-osmolarity-induced stimulation of glycine degradation was accompanied by parallel changes in mitochondrial matrix volume, no measurable changes in matrix volume were observed in mitochondria stimulated either by free Ca2+ (0.5 microM) or by injection of glucagon in vivo. Furthermore, Ca2+ stimulated glycine decarboxylation in mitochondria exposed to either hyper-osmolar (410 mosmol) or hypo-osmolar (210 mosmol) conditions. Although hyper-osmolarity decreased and hypo-osmolarity increased matrix volume, stimulation of glycine degradation by Ca2+ was not associated with any further changes in matrix volume. 5. These data demonstrate that the regulation of hepatic glycine oxidation by glucagon and by free Ca2+ is largely independent of changes in mitochondrial matrix volume.

scholarly journals Factors influencing the inactivation of phosphate-dependent glutaminase in the matrix fraction of rat liver mitochondria

1991 ◽  
Vol 274 (1) ◽  
pp. 109-114
Author(s):  
J D McGivan ◽  
F A Doyle ◽  
K Boon
Keyword(s):  
Liver Mitochondria ◽  
Polyclonal Antiserum ◽  
Rat Liver Mitochondria ◽  
Mitochondrial Matrix ◽  
Factors Influencing ◽  
Low Concentrations ◽  
The Matrix ◽  
High Concentrations ◽  
Matrix Fraction ◽  
Mitochondrial Extract

1. The activity of phosphate-dependent glutaminase was measured in a matrix extract of essentially lysosome-free liver mitochondria. 2. ATP, GTP or a non-hydrolysable analogue of ATP stimulated the rapid inactivation of glutaminase, but not of other matrix enzymes. 3. Glutaminase was protected against inactivation if high concentrations of glutamine or low concentrations of NH3 were present. 4. Inactivation of glutaminase in the presence of ATP did not markedly affect the reaction of the enzyme with a specific polyclonal antiserum. 5. These results in a mitochondrial extract are similar to the characteristics of glutaminase inactivation in intact hepatocytes, suggesting a similar mechanism in each case. 6. The presence of a specific ATP-activated protease in the mitochondrial matrix is suggested to be responsible for glutaminase inactivation.

Effects of 5-5'-Diphenyl-2-thiohydantoin (DPTH) and Thyroxine on the Ultrastructure and Chemical Composition of Rat Liver

1971 ◽  
Vol 29 ◽  
pp. 570-571
Author(s):  
E. A. Elfont ◽  
R. B. Tobin ◽  
D. G. Colton ◽  
M. A. Mehlman
Keyword(s):  
Chemical Composition ◽  
Rat Liver ◽  
Future Study ◽  
Mode Of Action ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Effective Inhibitor ◽  
Biochemical Effects ◽  
Glycerophosphate Dehydrogenase ◽  
Stimulation Of

Summary5,-5'-diphenyl-2-thiohydantoin (DPTH) is an effective inhibitor of thyroxine (T4) stimulation of α-glycerophosphate dehydrogenase in rat liver mitochondria. Because this finding indicated a possible tool for future study of the mode of action of thyroxine, the ultrastructural and biochemical effects of DPTH and/or thyroxine on rat liver mere investigated.Rats were fed either standard or DPTH (0.06%) diet for 30 days before T4 (250 ug/kg/day) was injected. Injection of T4 occurred daily for 10 days prior to sacrifice. After removal of the liver and kidneys, part of the tissue was frozen at -50°C for later biocheailcal analyses, while the rest was prefixed in buffered 3.5X glutaraldehyde (390 mOs) and post-fixed in buffered 1Z OsO4 (376 mOs). Tissues were embedded in Araldlte 502 and the sections examined in a Zeiss EM 9S.Hepatocytes from hyperthyroid rats (Fig. 2) demonstrated enlarged and more numerous mitochondria than those of controls (Fig. 1). Glycogen was almost totally absent from the cytoplasm of the T4-treated rats.

A new family of fluorescent calcium indicators designed to resist leakage or for measuring calcium near membranes

1994 ◽  
Vol 52 ◽  
pp. 168-169
Author(s):  
Martin Poenie ◽  
Akwasi Minta ◽  
Charles Vorndran
Keyword(s):  
Metal Binding ◽  
Acetoxymethyl Ester ◽  
Binding Properties ◽  
Fura 2 ◽  
New Family ◽  
Anion Transporter ◽  
Calcium Indicator ◽  
Calcium Indicators ◽  
High Calcium ◽  
Intracellular Compartmentalization

The use of fura-2 as an intracellular calcium indicator is complicated by problems of rapid dye leakage and intracellular compartmentalization which is due to a probenecid sensitive anion transporter. In addition there is increasing evidence for localized microdomains of high calcium signals which may not be faithfully reported by fura-2.We have developed a new family of fura-2 analogs aimed at addressing some of these problems. These new indicators are based on a modified bapta which can be readily derivatized to produce fura-2 analogs with a variety of new properties. The modifications do not affect the chromophore and have little impact on the spectral and metal binding properties of the indicator. One of these new derivatives known as FPE3 is a zwitterionic analog of fura-2 that can be loaded into cells as an acetoxymethyl ester and whose retention in cells is much improved. The improved retention of FPE3 is important for both cuvettebased measurements of cell suspensions and for calcium imaging.

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