Determination of mitochondrial/cytosolic metabolite gradients in isolated rat liver cells by cell disruption

Tischler, Marc E.; Hecht, Pat; Williamson, John

doi:10.1016/0003-9861(77)90506-9

Cited by 161 publications

(51 citation statements)

References 33 publications

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“…4, in which the carbonic anhydrase activity, expressed as kct/mg of protein (std kct), is plotted for intact and broken mitochondria as a function of the pH of the suspending medium. A matrix pH that is 0.2-0.5 unit more alkaline is calculated for guinea pig liver mitochondria respiring with endogenous substrate, in agreement with the cytosol/ matrix ApH value of 0.4 (matrix alkaline) obtained with isolated rat hepatocytes (30).…”

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confidence: 76%

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Mitochondrial carbonic anhydrase.

Dodgson¹,

Forster²,

Storey³

et al. 1980

Proc. Natl. Acad. Sci. U.S.A.

130

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We have assayed carbonic anhydrase activity (carbonate dehydratase, carbonate hydro-lyase, EC 4.2.1.1) and icarbonate permeability in suspensions of broken and intact guinea pig mitochondria by monitoring the disappearance of C16'080. We found significant activity in preparations from liver and skeletal muscle, but not in preparations from heart muscle, brain, and kidney. Intact mitochondria containing carbonic anhydrase produce a two-phase acceleration of the disappearance of the labeled CO2, which indicates that the enzyme is located in a region more accessible to CO2 than to HCOj-. Acetazolamide inhibits the enzyme activity instantly in broken mitochondria but only after a delay in intact mitochondria, indicating that the enzyme is in a region not immediately accessible to the inhibitor. Sonication of mitochondria containing carbonic anhydrase activity releases the enzyme, which remains in the supernatant after sedimentation of the submitochondrial particles. This shows that mitochondrial carbonic anhydrase is in the matrix compartment and not in, or bound to, the inner membrane. The activity of the enzyme increases markedly with increasing pH. The enzyme activity of intact mitochondria is greater than that of the broken mitochondria at the same pH of the suspending fluid, corresponding to an intramitochondrial pH that is 0.2-0.5 unit more alkaline.CO2 plays an important role in mitochondrial metabolism. The enzymes of the tricarboxylic acid cycle that produce CO2 (1) are located within the mitochondrial matrix (2), as are those enzymes that fix CO2 in the pathways of gluconeogenesis and urea production (3, 4). Prior to 1972, the few studies of the interaction of CO2 and HCO% with mitochondria dealt with the permeability of the inner mitochondrial membrane to these two species and with the possible existence of a mitochondrial carbonic anhydrase. Chappell and Crofts (5) showed that the inner mitochondrial membrane was essentially impermeable to HC0% but readily permeable to C02, a finding now widely accepted (6). Early reports of mitochondrial carbonic anhydrase (7-9) were cautious because of the problem of contamination, with the exception of that of Karler and Woodbury (10). Rossi (11) found 4% of the carbonic anhydrase of rat liver homogenate in the mitochondrial fraction and showed that it was an intramitochondrial enzyme. Mitochondria incubated with 10 ,gM acetazolamide showed no activity. Holton (12) found carbonic anhydrase activity in isolated rat liver mitochondria with both penetrant CO2 and nonpenetrant HCO as substrates. In contrast to these two positive findings with liver mitochondria, Deprez and Francois (13) found no carbonic anhydrase activity in mitochondrial preparations from various tissues, including liver. In all the above studies, carbonic anhydrase activity was measured by change in pH.A functional role for C02/HC0% in providing a counteranion for energy-linked mitochondrial Ca2+ uptake was proposed by Elder (14). Elder and Lehninger (15,16) showed that HCO3 as such cannot se...

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confidence: 76%

“…Mitochondria were prepared from skeletal muscle (mixed types) excised from the hind legs of two guinea pigs, exactly following the procedure for rabbit leg muscle described by Storey et al (26). The final mitochondrial suspension contained 30 …”

Section: Methodsmentioning

confidence: 99%

Mitochondrial carbonic anhydrase.

Dodgson¹,

Forster²,

Storey³

et al. 1980

Proc. Natl. Acad. Sci. U.S.A.

130

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“…If ADP-binding proteins have been lost owing to the chromatography on Ap5A-agarose, this ratio would be even higher. The ratio of 19 calculated on the basis of the present results is considerably higher than the cytosolic ratio of ATPtota1/ADPtotal of 2.6-10 found by various fractionation techniques (Siess & Wieland, 1976;Tischler et al, 1977;Akerboom et al, 1978;Soboll et al, 1978). However, the present results agree well with previous findings showing that a ratio of at least 20 is a prerequisite for obtaining physiological rates of gluconeogenesis in cell-free systems (Morikofer-Zwez et al, 1982;Stoecklin et al, 1986).…”

Section: Discussionsupporting

confidence: 80%

“…The total concentration of ATP in rat liver cytosol has been reported to be 2.8-3.4mm (Tischler et al, 1977;Akerboom et al, 1978;Siess et al, 1982). Since little, if any, ATP is bound to cytosolic proteins (Gankema et al, 1983), the concentration of free ATP in rat liver cytosol can be assumed to be about 3 mm.…”

Section: Discussionmentioning

confidence: 98%

“…Physiological rates of hexose 6-phosphate formation from different substrates were obtained at an ATP/ADP ratio of at least 20. However, cytosolic ratios of ATP/ADP reported for rat liver and rat hepatocytes are not higher than 10 (Siess & Wieland, 1976;Tischler et al, 1977;Akerboom et al, 1978;Soboll et al, 1978). This apparent discrepancy may stem from the fact that in cell-free, highly diluted, systems the adenine nucleotides are essentially present as free compounds (Stoecklin et al, 1986), whereas in the cell they may be bound in part to cytosolic proteins.…”

Section: Introductionmentioning

confidence: 92%

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Binding of ADP to rat liver cytosolic proteins and its influence on the ratio of free ATP/free ADP

Mörikofer‐Zwez

Walter

1989

Biochemical Journal

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In a cytosolic extract from rat liver, the number and the concentration of ADP-binding sites as well as their dissociation constants were determined by using the rate-of-dialysis technique. Interfering cytosolic adenylate kinase was extracted from the cytosol by affinity chromatography on Ap5A-agarose, and remaining traces of enzyme activity were inhibited with (+ )-catechin. Binding of ADP to cytosolic proteins was increased by poly(ethylene glycol) and decreased by EDTA. The effect of 0.1 mM-EDTA could be reversed by addition of equimolar concentrations of Mn2+ or Mg2+. In presence of 5 % poly(ethylene glycol), added to increase local protein concentration, two binding sites for ADP were observed, with KD values of 1.9 /tM (site I) and 10.8 ,UM (site II). The concentration of these binding sites, when extrapolated to cellular protein concentrations, were 30 /LM (site I) and 114,M (site II). It is concluded that a minimum of about 50 % of total cytosolic ADP is bound to proteins, and that the ratio of free ATP/free ADP is at least twice that of total ATP/total ADP.

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Renal Metabolism: Integrated Responses

Klahr

Hamm

Hammerman

et al. 1992

Comprehensive Physiology

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The sections in this article are: Methodological Considerations Coupling of Metabolism to Transport Oxidative vs. Glycolytic Metabolism in the Mammalian Kidney Stoichiometry of Transepithelial Na + Transport to QO 2 Transport as the Pacemaker of Respiration Control of Transport by Metabolism Metabolic Substrate Utilization by the Kidney Metabolic Heterogeneity of the Kidney Metabolism of Glucose Lactate Metabolism Pyruvate Metabolism Renal Lipid Metabolism Amino Acid Metabolism Citrate Metabolism Ketone Metabolism Synthetic Functions of the Kidney Gluconeogenesis Alanine Serine Renal Phospholipid Metabolism Phospholipid Composition of Kidney Metabolism of Specific Renal Phospholipids Metabolism of Membrane Proteins in Kidney Phosphorylation of Membrane Proteins ADP ‐Ribosylation

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Determination of mitochondrial/cytosolic metabolite gradients in isolated rat liver cells by cell disruption

Cited by 161 publications

References 33 publications

Mitochondrial carbonic anhydrase.

Mitochondrial carbonic anhydrase.

Binding of ADP to rat liver cytosolic proteins and its influence on the ratio of free ATP/free ADP

Renal Metabolism: Integrated Responses

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