Subcellular Distribution of Sulfite Cytochrome <i>c</i> Reductase in Rat Liver Tissue

Coninck, Simone Wattiaux‐De; Wattiaux, Robert

doi:10.1111/j.1432-1033.1971.tb01348.x

Cited by 63 publications

(29 citation statements)

References 15 publications

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“…The reaction was stopped by adding 1.3 ml of 50 mM glycine buffer (pH 10.5) containing 5 mM EDTA and 0.5 % Triton X-100. In each determination, the released fluorogenic group (aminomethyl-coumarin, 4-methyl umbelliferone or naphthylamine) was determined with a Versa Fluor fluorimeter (Bio-Rad, Nazareth-Eke, Belgium), excitation filter 170-2420 and emission filter 170-2421. β-Galactosidase and cathepsin C were measured as described by Lecocq et al [21], sulfite cytochrome c reductase by the method of Wattiaux-De Coninck and Wattiaux [15] and cytochrome oxidase as described by de Duve et al [20].…”

Section: Enzyme Measurementsmentioning

confidence: 99%

“…Hepatocyte apoptosis was assessed by measuring the activity of the major executioner caspase, caspase 3, with a site-specific tetrapeptide substrate (DEVDase activity) and by determining the percentage of sulfite oxidase released in the high-speed supernatant of the homogenate. This enzyme is located in the intermembrane space of mitochondria [15] and is co-released with cytochrome c into the cytosol during apoptosis [16]. The measurement of this enzymatic activity with cytochrome c as electron acceptor (sulfite cytochrome c reductase) allows quantification of the proportion of mitochondria whose outer membrane is permeabilized.…”

Section: Introductionmentioning

confidence: 99%

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Lysosomes and Fas-mediated liver cell death

Wattiaux

Coninck

Thirion

et al. 2007

Biochemical Journal

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A number of studies, mostly performed ex vivo, suggest that lysosomes are involved in apoptosis as a result of a release of their cathepsins into the cytosol. These enzymes could then contribute to the permeabilization of the outer mitochondrial membrane; they could also activate effector caspases. The present study aims at testing whether the membrane of liver lysosomes is disrupted during Fas-mediated cell death of hepatocytes in vivo, a process implicated in several liver pathologies. Apoptosis was induced by injecting mice with aFas (anti-Fas antibody). The state of lysosomes was assessed by determining the proportion of lysosomal enzymes (β-galactosidase, β-glucuronidase, cathepsin C and cathepsin B) present in homogenate supernatants, devoid of intact lysosomes, and by analysing the behaviour in differential and isopycnic centrifugation of β-galactosidase. Apoptosis was monitored by measuring caspase 3 activity (DEVDase) and the release of sulfite cytochrome c reductase, an enzyme located in the mitochondrial intermembrane space. Results show that an injection of 10 µg of aFas causes a rapid and large increase in DEVDase activity and in unsedimentable sulfite cytochrome c reductase. This modifies neither the proportion of unsedimentable lysosomal enzyme in the homogenates nor the behaviour of lysosomes in centrifugation. Experiments performed with a lower dose of aFas (5 µg) indicate that unsedimentable lysosomal hydrolase activity increases in the homogenate after injection but with a marked delay with respect to the increase in DEVDase activity and in unsedimentable sulfite cytochrome c reductase.Comparative experiments ex vivo performed with Jurkat cells show an increase in unsedimentable lysosomal hydrolases, but much later than caspase 3 activation, and a release of dipeptidyl peptidase III and DEVDase into culture medium. It is proposed that the weakening of lysosomes observed after aFas treatment in vivo and ex vivo results from a necrotic process that takes place late after initiation of apoptosis.

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Section: Enzyme Measurementsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Lysosomes and Fas-mediated liver cell death

Wattiaux

Coninck

Thirion

et al. 2007

Biochemical Journal

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“…Marker enzymes of the main subcellular membranes were assayed according to the following references ; cytochrome oxidase [12], monoamine oxidase [19], glutamate dehydrogenase [14], malate dehydrogenase [15], sulfite cytochrome c reductase [16], acid phosphatase [9], P-galactosidase [17], catalase [18], urate oxidase [9], galactosyltransferase [19], 5'-nucleotidase [20]. Proteins were measured according to Lowry et al [21].…”

Section: Enzyme Assaysmentioning

confidence: 99%

A Centrifugation Study of Rat‐Liver Mitochondria, Lysosomes and Peroxisomes during the Perinatal Period

Mertens-Strijthagen

Schrijver

Coninck

et al. 1979

European Journal of Biochemistry

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We have investigated the intracellular distribution of several enzymes on homogenates of late foetal, early postnatal and adult rat livers. Homogenates were subjected to differential centrifugations in 0.25 M sucrose and four fractions were isolated which corresponded to the N (nuclear) ML (total mitochondrial) P (microsomal) and S (soluble) fractions of de . In general the age of the animal did not significantly affect the distribution pattern. Reference enzymes of mitochondria, lysosomes and peroxisomes were mainly recovered in the total mitochondrial fraction (ML). Glucose-6-phosphatase and esterase, both located in the endoplasmic reticulum, were chiefly associated with the microsomal fraction P together with galactosyltransferase (a reference enzyme of the Golgi apparatus). 5'-Nucleotidase, (a plasma membrane enzyme) exhibits a bimodal distribution and is mainly recovered in the N and the P fractions. Such results indicate that the membrane composition of the fractions isolated by the fractionation scheme we used, does not appreciably differ for the late foetal, early postnatal and adult rat livers.An analytical fractionation of the mitochondrial (ML) fraction of livers at different stages of development was performed by isopycnic centrifugation in sucrose gradients and in glycogen gradients using sucrose solutions of various concentrations as the solvents. The distributions of mitochondria, lysosomes and peroxisomes were assessed by establishing the distribution of their reference enzymes. Some physical characteristics of the particles were deduced from the manner in which the distributions were influenced by the sucrose concentration of the centrifugation medium. The distribution of liver mitochondrial enzymes one day prenatal differs strikingly from that of enzymes one day postnatal: foetal mitochondria seem characterized by a high osmotic space and a high hydrated matrix density; neonatal mitochondria seem devoid of an osmotic space and the density of their hydrated matrix is markedly lower than that of the foetal mitochondria. As ascertained by the distribution of mitochondrial enzymes in a sucrose 2H20 gradient, the high density of a foetal mitochondria matrix does not mainly originate from a lower amount of hydration water. The behavior of lysosomal enzymes in media with increasing concentrations of sucrose suggests that lysosomes originating from late foetal rat liver are endowed with a very small osmotic space. As for the peroxisomes, our results do not display significant behavior differences in centrifugations that would indicate physicochemical changes of these particles during the perinatal period.

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“…Fig. 3 shows a comparison of the free activity of DNA polymerase, two intermembrane space enzymes: (sulfite cytochrome c reductase [6], alkaline DNAse [ 141) and a soluble enzyme of the organelle matrix, malate dehydrogenase [ 151 when mitochondria are exposed to hypotonic media ( fig. 3A) or subjected to high speed centrifugation in isoosmotic sucrose ( fig.…”

Section: Structure Linked Latency Of Mitochondrial Dna Polymerasementioning

confidence: 99%

“…Cytochrome oxidase, acid phosphatase and glucose-6-phosphatase were measured according to de Duve et al [2], monoamine oxidase according to Schnaitman et al [4], alkaline DNAase according to Beaufay et al [S], sulfite cytochrome c reductase according to Wattiaux-De Coninck and Wattiaux [6], malate dehydrogenase according to Ochoa [7]. DNA polymerase was assayed in a volume of 0.2 ml containing 0.05 M Tris buffer pH 7.5,0.05 M phosphate buffer pH 7.5, 20 mM succinate, 125 mM KCl, 8 mM MgCl,, 0.5 mM ATP, 4 mM EDTA, 5 mM mercaptoethanol, 0.25 mg/ ml heat denatured DNA, 0.25 M sucrose, 0.15% Triton X-100, 15 PM dCTP, dGTP, dATP, 2.5 PM tritiated dTTP.…”

Section: Enzyme Assaysmentioning

confidence: 99%