Changes in the proportions of two mitochondrial populations during the development of embryonic chick liver

1. Two populations of morphologically intact mitochondria were isolated from embryonic, neonatal and adult chick liver by isopycnic centrifugation. 2. The protein/phospholipid ratio of the total mitochondrial fraction, the low-density mitochondria (B2, d1.176) and the high-density mitochondria (B3, d1.206) did not differ significantly. 3. During development there is a marked increase in the B2 fraction in relation to the B3 fraction. 4. Cytochrome oxidase and malate dehydrogenase activities as well as respiratory control increased during the embryonic development of the chick, though their rates of increase were not correlated. 5. In the three different embryonic stages that were investigated, as well as in the neonatal and adult chick, the protein/lipid as well as the protein/phospholipid ratio stayed constant and showed no progressive increase, as had been previously reported. 6. It was shown that forces greater than 18400gav. for 2h have to be used before chick liver mitochondria reach isopycnic equilibrium. 7. As for rat liver mitochondria, the constant protein/phospholipid ratio of the B2 and B3 fractions and their apparent morphological intactness leads one to conclude that the matrix space of B2 mitochondria is inaccessible to sucrose, whereas B3 mitochondria possess an inner membrane that is permeable to sucrose.

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Rat liver mitochondrial ADP-ribose pyrophosphatase in the matrix space with low Km for free ADP-ribose

Biochemical Journal ◽

10.1042/bj2990679 ◽

1994 ◽

Vol 299 (3) ◽

pp. 679-682 ◽

Cited By ~ 19

Author(s):

D Bernet ◽

R M Pinto ◽

M J Costas ◽

J Canales ◽

J C Cameselle

Keyword(s):

Rat Liver ◽

Gradient Centrifugation ◽

Liver Mitochondria ◽

Rat Liver Mitochondria ◽

Matrix Space ◽

The Matrix ◽

Submitochondrial Fractions

A study involving markers of subcellular and submitochondrial fractions, gradient centrifugation, latency measurements and extraction with digitonin, demonstrates the association of a specific ADP-ribose pyrophosphatase with rat liver mitochondria and its localization in the matrix space. The enzyme hydrolyses ADP-ribose to AMP, with a Km of 2-3 microM. The results support the occurrence of a specific turnover pathway for free ADP-ribose and its relevance in mitochondria.

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Stable enhancement of calcium retention in mitochondria isolated from rat liver after the administration of glucagon to the intact animal

Biochemical Journal ◽

10.1042/bj1760705 ◽

1978 ◽

Vol 176 (3) ◽

pp. 705-714 ◽

Cited By ~ 62

Author(s):

Veronica Prpić ◽

Terry L. Spencer ◽

Fyfe L. Bygrave

Keyword(s):

Rat Liver ◽

Molecular Mechanisms ◽

Adenine Nucleotide ◽

Adenine Nucleotides ◽

Mitochondrial Protein ◽

Liver Mitochondria ◽

Rat Liver Mitochondria ◽

Matrix Space ◽

The Matrix

1. Mitochondria isolated from rat liver by centrifugation of the homogenate in buffered iso-osmotic sucrose at between 4000 and 8000g-min, 1h after the administration in vivo of 30μg of glucagon/100g body wt., retain Ca2+ for over 45min after its addition at 100nmol/mg of mitochondrial protein in the presence of 2mm-Pi. In similar experiments, but after the administration of saline (0.9% NaCl) in place of glucagon, Ca2+ is retained for 6–8min. The ability of glucagon to enhance Ca2+ retention is completely prevented by co-administration of 4.2mg of puromycin/100g body wt. 2. The resting rate of respiration after Ca2+ accumulation by mitochondria from glucagon-treated rats remains low by contrast with that from saline-treated rats. Respiration in the latter mitochondria increased markedly after the Ca2+ accumulation, reflecting the uncoupling action of the ion. 3. Concomitant with the enhanced retention of Ca2+ and low rates of resting respiration by mitochondria from glucagon-treated rats was an increased ability to retain endogenous adenine nucleotides. 4. An investigation of properties of mitochondria known to influence Ca2+ transport revealed a significantly higher concentration of adenine nucleotides but not of Pi in those from glucagon-treated rats. The membrane potential remained unchanged, but the transmembrane pH gradient increased by approx. 10mV, indicating increased alkalinity of the matrix space. 5. Depletion of endogenous adenine nucleotides by Pi treatment in mitochondria from both glucagon-treated and saline-treated rats led to a marked diminution in ability to retain Ca2+. The activity of the adenine nucleotide translocase was unaffected by glucagon treatment of rats in vivo. 6. Although the data are consistent with the argument that the Ca2+-translocation cycle in rat liver mitochondria is a target for glucagon action in vivo, they do not permit conclusions to be drawn about the molecular mechanisms involved in the glucagon-induced alteration to this cycle.

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The entrapment of the Ca2+ indicator arsenazo III in the matrix space of rat liver mitochondria by permeabilization and resealing. Na+-dependent and -independent effluxes of Ca2+ in arsenazo III-loaded mitochondria

Biochemical Journal ◽

10.1042/bj2390031 ◽

1986 ◽

Vol 239 (1) ◽

pp. 31-40 ◽

Cited By ~ 29

Author(s):

I Al-Nasser ◽

M Crompton

Keyword(s):

Rat Liver ◽

Liver Mitochondria ◽

Ruthenium Red ◽

Arsenazo Iii ◽

Rat Liver Mitochondria ◽

Matrix Space ◽

The Matrix ◽

Matrix Compartment ◽

Matrix Free ◽

Regulatory Sites

The permeabilization-resealing technique [Al-Nasser & Crompton, Biochem. J. (1986) 239, 19-29] has been applied to the entrapment of arsenazo III in the matrix compartment of rat liver mitochondria. The addition of 10 mM-arsenazo III to mitochondria permeabilized with Ca2+ partially restores the inner-membrane potential (delta psi) and leads to the recovery of 3.9 nmol of arsenazo III/mg of protein in the matrix when the mitochondria are washed three times. The recovery of entrapped arsenazo III is increased 2-fold by 4 mM-Mg2+, which also promotes repolarization. ATP with or without Mg2+ decreased arsenazo III recovery. Under all conditions, less arsenazo III than [14C]sucrose is entrapped, in particular in the presence of ATP. The amount of arsenazo III entrapped is proportional to the concentration of arsenazo III used as resealant, and is equally distributed between heavy and light mitochondria. Arsenazo III-loaded permeabilized and resealed (PR) mitochondria develop delta psi values of 141 +/- 3 mV. PR mitochondria retain arsenazo III and [14C]sucrose for more than 2 h at 0 degrees C. At 25 degrees C, and in the presence of Ruthenium Red, PR mitochondria lose arsenazo III and [14C]sucrose at equal rates, but Ca2+ efflux is more rapid; this indicates that Ca2+ is released by an Na+-independent carrier in addition to permeabilization. The Na+/Ca2+ carrier of PR mitochondria is partially (60%) inhibited by extramitochondrial free Ca2+ stabilized with Ca2+ buffers; maximal inhibition is attained with 2 microM free Ca2+. A similar inhibition occurs in normal mitochondria with 3.5 nmol of matrix Ca2+/mg of protein, but the inhibition is decreased by increased matrix Ca2+. The data suggest the presence of Ca2+ regulatory sites on the Na+/Ca2+ carrier that change the affinity for matrix free Ca2+.

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The reversible Ca2+-induced permeabilization of rat liver mitochondria

Biochemical Journal ◽

10.1042/bj2390019 ◽

1986 ◽

Vol 239 (1) ◽

pp. 19-29 ◽

Cited By ~ 129

Author(s):

I Al-Nasser ◽

M Crompton

Keyword(s):

Rat Liver ◽

Liver Mitochondria ◽

Ruthenium Red ◽

Rat Liver Mitochondria ◽

Internal Space ◽

Matrix Space ◽

Acute Dependence ◽

The Matrix ◽

Mitochondrial Suspension ◽

Time Courses

Rat liver mitochondria became permeabilized to sucrose according to an apparent first-order process after accumulating 35 nmol of Ca2+/mg of protein in the presence of 2.5 mM-Pi, but not in its absence. A fraction (24-32%) of the internal space remains sucrose-inaccessible. The rate constant for permeabilization to sucrose decreases slightly when the pH is decreased from 7.5 to 6.5, whereas the rate of inner-membrane potential (delta psi) dissipation is markedly increased, which indicates that H+ permeation precedes sucrose permeation. Permeabilization does not release mitochondrial proteins. [14C]Sucrose appears to enter permeabilized mitochondria instantaneously. Chelation of Ca2+ with EGTA restores delta psi and entraps sucrose in the matrix space. With 20 mM-sucrose at the instant of resealing, about 21 nmol of sucrose/mg of protein becomes entrapped. The amount of sucrose entrapped is proportional to the degree of permeabilization. Entrapped sucrose is not removed by dilution of the mitochondrial suspension. Resealed mitochondria washed three times retain about 74% of the entrapped sucrose. In the presence of Ruthenium Red and Ca2+ buffers permeabilized mitochondria reseal only partially with free [Ca2+] greater than 3 microM. [14C]Sucrose enters partially resealed mitochondria continuously with time, despite maintenance of delta psi, in accordance with continued interconversion of permeable and impermeable forms. Kinetic analyses of [14C]sucrose entry indicate two Ca2+-sensitive reactions in permeabilization. This conclusion is supported by the biphasic time courses of resealing and repolarization of permeabilized mitochondria and the acute dependence of the rapid repolarization on the free [Ca2+]. A hypothetical model of permeabilization and resealing is suggested and the potential of the procedure for matrix entrapment of substances is discussed.

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Location of dinucleoside triphosphatase in the matrix space of rat liver mitochondria

FEBS Letters ◽

10.1016/0014-5793(91)80609-7 ◽

1991 ◽

Vol 283 (2) ◽

pp. 286-288 ◽

Cited By ~ 7

Author(s):

Diego Bernet ◽

Rosa Maria Pinto ◽

Antonio Sillero ◽

José Carlos Cameselle

Keyword(s):

Rat Liver ◽

Liver Mitochondria ◽

Rat Liver Mitochondria ◽

Matrix Space ◽

The Matrix

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The membrane permeability transition in liver mitochondria of the great green goby Zosterisessor ophiocephalus (Pallas)

Journal of Experimental Biology ◽

10.1242/jeb.203.22.3425 ◽

2000 ◽

Vol 203 (22) ◽

pp. 3425-3434 ◽

Cited By ~ 1

Author(s):

A. Toninello ◽

M. Salvi ◽

L. Colombo

Keyword(s):

Membrane Potential ◽

Rat Liver ◽

Adenine Nucleotide ◽

Respiratory Control ◽

Permeability Transition ◽

Liver Mitochondria ◽

Transport Systems ◽

Rat Liver Mitochondria ◽

The Matrix ◽

Zosterisessor Ophiocephalus

Liver mitochondria from the great green goby Zosterisessor ophiocephalus (Pallas) normally exhibit bioenergetic variables (membrane potential 165+/−7 mV; respiratory control ratio 6.6+/−0.4; ADP/O ratio 1.85+/−0.8; means +/− s.e.m., N=6) and activities of physiological transport systems (phosphate/proton symporter, adenine nucleotide antiporter, Ca(2+) electrophoretic uniporter) comparable with those of rat liver mitochondria. When incubated in the presence of Ca(2+) and an inducer agent such as phosphate, these mitochondria undergo a complete collapse of membrane potential accompanied by a large-amplitude swelling of the matrix, influx of sucrose from the incubation medium, release of endogenous Mg(2+) and K(+) (approximately 90% of the total) and of preaccumulated Ca(2+) and oxidation of endogenous pyridine nucleotides. All these phenomena, which are completely eliminated by cyclosporin A and inhibited with different efficacies by Mg(2+) and spermine, demonstrate that the induction of the permeability transition in this type of mitochondria has characteristics similar to those described in rat liver mitochondria. In contrast, the requirement for very high Ca(2+) concentrations (greater than 100 micromol l(−1) for the induction of the permeability transition represents a very important difference that distinguishes this phenomenon in fish and mammalian mitochondria.

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Changes in intramitochondrial adenine nucleotides in blowfly flight-muscle mitochondria

Biochemical Journal ◽

10.1042/bj1420353 ◽

1974 ◽

Vol 142 (2) ◽

pp. 353-358 ◽

Cited By ~ 7

Author(s):

Susan M. Danks ◽

J. B. Chappell

Keyword(s):

Citrate Synthase ◽

Adenine Nucleotide ◽

Adenine Nucleotides ◽

Liver Mitochondria ◽

Rat Liver Mitochondria ◽

Glycerol Phosphate ◽

Matrix Space ◽

Carbonyl Cyanide ◽

The Matrix ◽

Metabolic Conditions

1. With freshly isolated blowfly mitochondria 38% of the intramitochondrial adenine nucleotide was present as AMP. 2. On incubation with oxidizable substrates the AMP and ADP concentrations fell and that of ATP rose; with pyruvate together with proline the ATP concentration reached its maximum value at 6min; with glycerol phosphate the phosphorylation of endogenous nucleotide was more rapid. 3. Addition of the uncoupling agent carbonyl cyanide phenylhydrazone caused a rapid fall of ATP and a parallel rise in ADP, then ADP was converted into AMP. 4. This was in contrast with rat liver mitochondria endogenous AMP concentrations, which were always lower than those of blowfly mitochondria and changed little under different metabolic conditions. 5. Evidence is presented that adenylate kinase (EC 2.7.4.3) has a dual distribution in blowfly mitochondria, a part being located in the matrix space and a part in the space between the outer and inner mitochondrial membranes, as in liver and other mitochondria. 6. The possible regulatory role of changing AMP concentrations in the mitochondrial matrix was investigated. Partially purified pyruvate carboxylase (EC 6.4.1.1) and citrate synthase (EC 4.1.3.7) were inhibited 30% by 2mm-AMP, whereas pyruvate dehydrogenase (EC 1.2.4.1) was unaffected. 7. AMP activated the NAD+-linked isocitrate dehydrogenase (EC 1.1.1.41) activity of blowfly mitochondria in the absence of ADP, but in the presence of ADP, AMP caused inhibition. 8. It is suggested that AMP may exert a controlling effect on the oxidative activity of blowfly mitochondria.

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