The separation of rat liver mitochondria into two morphologically different fractions by density-gradient centrifugation

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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Intramitochondrial localization of δ-aminolaevulate synthetase and ferrochelatase in rat liver

Biochemical Journal ◽

10.1042/bj1140455 ◽

1969 ◽

Vol 114 (3) ◽

pp. 455-461 ◽

Cited By ~ 69

Author(s):

Roxane McKay ◽

R. Druyan ◽

G. S. Getz ◽

M. Rabinowitz

Keyword(s):

Glutamate Dehydrogenase ◽

Density Gradient ◽

Malate Dehydrogenase ◽

Rat Liver ◽

Mitochondrial Biogenesis ◽

Mitochondrial Membrane ◽

Density Gradient Centrifugation ◽

Gradient Centrifugation ◽

Differential Centrifugation ◽

Inner Mitochondrial Membrane

Intramitochondrial loci for δ-aminolaevulate synthetase and ferrochelatase, the initial and final enzymes in haem synthesis, have been found in rat liver. Two different methods of fractionation were applied to mitochondria: (a) sonication and density-gradient centrifugation; (b) treatment with digitonin and differential centrifugation. Similar results were obtained with each technique. δ-Aminolaevulate synthetase is distributed similarly to two known matrix enzymes, malate dehydrogenase and glutamate dehydrogenase. Ferrochelatase is firmly bound to the the inner mitochondrial membrane. These results are considered in terms of the regulation of haem synthesis and in relation to mitochondrial biogenesis.

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Purification, characterization and identification of rat liver histidine-pyruvate aminotransferase isoenzymes

Biochemical Journal ◽

10.1042/bj1550107 ◽

1976 ◽

Vol 155 (1) ◽

pp. 107-115 ◽

Cited By ~ 23

Author(s):

T Noguchi ◽

E Okuno ◽

Y Minatogawa ◽

R Kido

Keyword(s):

Density Gradient ◽

Rat Liver ◽

Density Gradient Centrifugation ◽

Gradient Centrifugation ◽

Polyacrylamide Gel Electrophoresis ◽

Gel Filtration ◽

Sucrose Density Gradient ◽

Aminotransferase Activity ◽

Sucrose Density Gradient Centrifugation ◽

Ph Optimum

1. Histidine-pyruvate aminotransferase (isoenzyme 1) was purified to homogeneity from the mitochondrial and supernatant fractions of rat liver, as judged by polyacrylamide-gel electrophoresis and isolectric focusing. Both enzyme preparations were remarkably similar in physical and enzymic properties. Isoenzyme 1 had pI8.0 and a pH optimum of 9.0. The enzyme was active with pyruvate as amino acceptor but not with 2-oxoglutarate, and utilized various aromatic amino acids as amino donors in the following order of activity: phenylalanine greater than tyrosine greater than histidine. Very little activity was found with tryptophan and 5-hydroxytryptophan. The apparent Km values were about 2.6mM for histidine and 2.7 mM for phenylalanine. Km values for pyruvate were about 5.2mM with phenylalanine as amino donor and 1.1mM with histidine. The aminotransferase activity of the enzyme towards phenylalanine was inhibited by the addition of histidine. The mol.wt. determined by gel filtration and sucrose-density-gradient centrifugation was approx. 70000. The mitochondrial and supernatant isoenzyme 1 activities increased approximately 25-fold and 3.2-fold respectively in rats repeatedly injected with glucagon for 2 days. 2. An additional histidine-pyruvate aminotransferase (isoenzyme 2) was partially purified from both the mitochondrial and supernatant fractions of rat liver. Nearly identical properties were observed with both preparations. Isoenzyme 2 had pI5.2 and a pH optimum of 9.3. The enzyme was specific for pyruvate and did not function with 2-oxoglutarate. The order of effectiveness of amino donors was tyrosine = phenylalanine greater than histidine greater than tryptophan greater than 5-hydroxytryptophan. The apparent Km values for histidine and phenylalanine were about 0.51 and 1.8 mM respectively. Km values for pyruvate were about 3.5mM with phenylalanine and 4.7mM with histidine as amino donors. Histidine inhibited phenylalanine aminotransferase activity of the enzyme. Gel filtration and sucrose-density-gradient centrifugation yielded a mol.wt. of approx. 90000. Neither the mitochondrial nor the supernatant isoenzyme 2 activity was elevated by glucagon injection.

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Separation of different cell populations of rat liver by density gradient centrifugation in a vertical rotor with self-generated Percoll gradients

Acta Physiologica Scandinavica ◽

10.1111/j.1748-1716.1983.tb07218.x ◽

1983 ◽

Vol 117 (4) ◽

pp. 497-505 ◽

Cited By ~ 11

Author(s):

B. SINGH ◽

B. BORREBAEK ◽

H. OSMUNDSEN

Keyword(s):

Density Gradient ◽

Rat Liver ◽

Density Gradient Centrifugation ◽

Gradient Centrifugation ◽

Cell Populations ◽

Vertical Rotor ◽

Percoll Gradients

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Isolation of the 67Gallium Accumulating Fraction in Normal Rat Liver

Nuklearmedizin ◽

10.1055/s-0038-1624845 ◽

1974 ◽

Vol 13 (01) ◽

pp. 72-84

Author(s):

K. Hierholzer ◽

K. zum Winkel ◽

U. Haubold ◽

E. Aulbert

Keyword(s):

Electron Microscopy ◽

Density Gradient ◽

Intravenous Injection ◽

Rat Liver ◽

Soluble Fraction ◽

Density Gradient Centrifugation ◽

Gradient Centrifugation ◽

Differential Centrifugation ◽

Normal Rat

SummarySubcellular 67Gallium distribution was investigated in normal rat liver after intravenous injection. By differential centrifugation and density gradient centrifugation 67Gallium accumulating bodies were isolated and identified as lysosomes by enzyme determination and electron microscopy. 67Gallium enrichment in this fraction was 23-fold. Using the isolated 67Gallium accumulating lysosomes the binding state of the isotope inside the lysosomes was studied. 67Gallium was found to be associated with the soluble fraction of lysosomes.

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SEDIMENTATION PROPERTIES OF RAT LIVER MITOCHONDRIA

The Journal of Cell Biology ◽

10.1083/jcb.46.1.17 ◽

1970 ◽

Vol 46 (1) ◽

pp. 17-26 ◽

Cited By ~ 3

Author(s):

John N. Loeb ◽

Daniel V. Kimberg

Keyword(s):

Rat Liver ◽

Gradient Centrifugation ◽

Sucrose Density Gradient ◽

Liver Mitochondria ◽

Rat Liver Mitochondria ◽

Electron Microscopic ◽

Sucrose Density Gradient Centrifugation ◽

Sedimentation Behavior ◽

Sucrose Medium ◽

Mitochondrial Volume

A prediction of the velocity of sedimentation of rat liver mitochondria in sucrose gradients is made on the basis of recent measurements of the size of isolated mitochondria suspended in sucrose medium and the model proposed by Bentzel and Solomon to describe the osmotic behavior of mitochondria. The experimentally observed velocity is extremely close to the predicted value and confirms by a different approach the estimate of mitochondrial volume made by Baudhuin and Berthet on the basis of electron microscopic measurements. Because cortisone treatment of rats is known to result in a marked increase in mitochondrial size as observed under the electron microscope, mitochondria were co-isolated from livers of control and cortisone-treated animals, and the sedimentation behavior of the mixtures was examined by sucrose density gradient centrifugation. Mitochondria from cortisone-treated animals were found to sediment 1.4 times as rapidly as those from control animals, indicating that their increased size cannot entirely be due to an increased imbibition of fluid from the surrounding sucrose medium, and that the change in size must at least in part be due to a change in content of nondiffusible mitochondrial components. Although the increase in sedimentation velocity of mitochondria from cortisone-treated animals is striking, it is less than that predicted solely on the basis of their size relative to that of control mitochondria. It is concluded that the increases in mitochondrial size and content of nondiffusible components produced by cortisone treatment are accompanied by alterations in mitochondrial composition as well.

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Heterogeneous distribution of glucose 6-phosphatase in rat liver microsomal fractions as shown by adaptation of a cytochemical technique

Biochemical Journal ◽

10.1042/bj1340069 ◽

1973 ◽

Vol 134 (1) ◽

pp. 69-78 ◽

Cited By ~ 20

Author(s):

John A. Lewis ◽

Jamshed R. Tata

Keyword(s):

Phosphatase Activity ◽

Density Gradient ◽

Rat Liver ◽

Density Gradient Centrifugation ◽

Gradient Centrifugation ◽

Lead Phosphate ◽

Sucrose Density Gradient Centrifugation ◽

General Applicability ◽

Heterogeneous Distribution ◽

Low Concentrations

1. A novel technique for the subfractionation of rat liver smooth and rough microsomal fractions according to their content of glucose 6-phosphatase is described. This technique, based on the Gomori lead histochemical procedure, involves incubation of smooth and rough microsomal fractions with low concentrations of Pb(NO3)2 and glucose 6-phosphate. Control experiments, in which enzyme was assayed in the presence of various amounts of Pb(NO3)2 or in which microsomal fractions were reisolated after incubation with low concentrations of Pb(NO3)2 and glucose 6-phosphate, showed that lead does not interfere with glucose 6-phosphatase activity. 2. Discontinuous sucrose-density-gradient centrifugation of microsomal fractions which had previously been incubated with various amounts of Pb(NO3)2 and glucose 6-phosphate showed that it is possible to subfractionate both smooth- and rough-microsomal fractions into several bands, owing to a differential modification of the density of the microsomal vesicles by the trapping of lead phosphate within them. 3. When the material in the bands obtained by density-gradient centrifugation of incubated microsomal fractions was assayed for glucose 6-phosphatase activity, it was found that the modification of the density of the microsomal fractions was directly related to their relative enrichment in glucose 6-phosphatase activity. Control experiments, in which microsomal fractions were incubated with Pb(NO3)2 and glucose 6-phosphate and then treated with EDTA, showed that the subfractionation was not due to aggregation of microsomal vesicles, lead and glucose 6-phosphate. Thus the resolution of microsomal preparations into subfractions with different glucose 6-phosphatase activities is interpreted as indicating heterogeneity of glucose 6-phosphatase distribution in the microsomal vesicles. 4. Electron micrographs of both smooth- and rough-microsomal subfractions show deposits of lead phosphate within the microsomal vesicles. The frequency and extent of these deposits correlate with the different amounts of glucose 6-phosphatase activity measured biochemically. 5. The nature of the heterogeneous distribution of glucose 6-phosphatase is discussed and the more general applicability of the technique for studying membrane fractions containing a heterogeneous distribution of phosphatases is indicated.

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