Intracellular distribution of 5′-ribonuclease and 5′-phosphodiesterase in rat liver

Lamirande, Gaston de; Morais, Réjean; Blackstein, Martin E.

doi:10.1016/0003-9861(67)90359-1

Cited by 31 publications

(9 citation statements)

References 7 publications

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“…White Leghorn chickens, 3 to 6 weeks old, were fasted overnight and decapitated. A liver homogenate was prepared as previously described for rat liver (8). Mitochondria were isolated by differential centrifugation (17), and mtDNA was extracted and centrifuged through a two-step CsCI-EtdBr gradient as indicated above.…”

Section: Methodsmentioning

confidence: 99%

Ethidium bromide-induced loss of mitochondrial DNA from primary chicken embryo fibroblasts.

Desjardins¹,

Frost²,

Morais³

1985

Mol. Cell. Biol.

156

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Chicken embryo fibroblasts in uridine-containing medium are inherently resistant to the growth-inhibitory effect of ethidium bromide. The drug was found to inhibit the incorporation of [3lHlthymidine into mitochondrial DNA circular molecules. Mitochondrial DNA was quantitated by DNA-DNA reassociation kinetics with a probe of chicken liver mitochondrial DNA. A mean number of 604 copies of mitochondrial DNA per cell was found. This number decreased progressively in cells exposed to ethidium bromide, and by day 13 ca. one copy of mitochondrial DNA was detected per cell. When the cells were then transferred to drug-free medium, the number of copies increased very slowly as a function of time. On the other hand, analyses of DNA extracted from cell populations exposed to ethidium bromide for 20 or more days, with or without subsequent transfer to drug-free medium, revealed very little or no mitochondrial DNA by reassociation kinetics or by Southern blot hybridization of AvaI-or HindIII-digested total cellular DNA. As a result of the elimination of mitochondrial DNA molecules, the establishment of cell populations with a respiration-deficient phenotype was confirmed by measuring cytochrome c oxidase activity as a function of the number of cell generations and the absorption spectrum of mitochondrial cytochromes.

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Section: Methodsmentioning

confidence: 99%

Ethidium bromide-induced loss of mitochondrial DNA from primary chicken embryo fibroblasts.

Desjardins¹,

Frost²,

Morais³

1985

Mol. Cell. Biol.

156

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“…Subsequently deLamirande et al [8,9] reported the presence of bhe enzyme in the nuclei and the microsomal membranes, and the absence of any activity in the ribosomes. The microsoma1 localization of phosphodiesterase I was subsequently supported by Brightwell and Tappel [lo].…”

mentioning

confidence: 99%

Intracellular Localization of Phosphodiesterases I and II in Rat Liver

Erecińska

Sierakowska

Shugar

1969

European Journal of Biochemistry

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Differential and density gradient centrifugation techniques have been employed to study the intracellular distribution of phosphodiesterases I and I1 in rat liver, using as specific substrates the p-nitrophenyl esters of thymidine-5'-phosphate and thymidine-3'-phosphate. Some of the results for phosphodiesterase I were also examined with the use of a 5'-terminated DNA core as substrate.Phosphodiesterase I was found to be largely (> 80°/,) localized in the plasma membrane, whereas purified nuclei, rough microsomes, and the inner mitochondria1 membrane are essentially free of this enzyme. The possible sources of apparent phosphodiesterase I activity of the outer mitochondrial membrane, the lysosomes and the smooth microsomal membrane are discussed.Phosphodiesterase I1 is localized largely in the soluble fraction of the lysosomes, and exhibits the latency typical of lysosomal hydrolases. The overall findings are examined in relation to those of other investigators. Possible reservations regarding the absolute specificities of the substrates employed, and their bearing on the localization results, are discussed.The delineation of the specificities of phosphodiesterases I and I1 [1,2], and the preparation o f relatively specific and convenient substrates for the assay of these enzymes [3-61, has paved the way for studies on their intracellular distribution [S].With p-nitrophenyl thymidine-5'-phosphate as substrate, Razzell [7] first demonstrated the localization of phosphodiesterase I largely in the microsomal fraction of rat liver. Subsequently deLamirande et al. [8,9] reported the presence of bhe enzyme in the nuclei and the microsomal membranes, and the absence of any activity in the ribosomes. The microsoma1 localization of phosphodiesterase I was subsequently supported by Brightwell and Tappel [lo]. On the other hand, cytochemical investigations on phosphodiesterase I localization in rat liver, with the aid of cc-napht*hyl thymidine-5'-phosphate as substrate, revealed most of the enzyme activity a t the border of the bile canaliculi [4].

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“…The activity of 5'-endonuclease, succinate dehydrogenase, acid phosphatase and NADPH-cytochrome c reductase was determined according to published methods [8,12,18,19]. Cytochrome c oxidase was measured in the following incubation medium: 2.0 ml of 0.15 M phosphate buffer, pH 7.5 ; 0.03 ml of reduced cytochrome c, 0.008 M [18]; and 0.1 ml of a suitable amount of homogenate or tissue fractions proteins.…”

Section: Enzymic and Chemical Analysismentioning

confidence: 99%

“…The enzyme involved in this particular cytoplasmic process is still unknown but could well be cellular poly(A) hydrolase [5-71, which catalyzes the hydrolysis of poly(A) to oligonucleotides with 3'-hydroxyl and 5'-monophosphoester termini [5,6]. More than 60 % of poly(A) hydrolase activity of rat liver homogenate is recovered in the mitochondrial fraction [8]; the activity present in the nuclear fraction (around 20 %) has been mainly accounted for by mitochondrial contamination [8,9] and a nuclear exxibonuclease which has been identified in several tissues [lo, 11 1. The mitochondrial poly(A) hydrolase, which has been tentatively termed polyadenylase by others [7], also catalyzes the hydrolysis of single but not double-stranded RNA and DNA molecules [5,6] and is localized in the intermembrane space [7,12,13].…”

mentioning

confidence: 99%

Poly(A) Hydrolase of Chick‐Embryo Fibroblasts

Leblond-Larouche

Dupuis

Morais

1976

European Journal of Biochemistry

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1. Homogenates of cultured chick embryo fibroblasts have been quantitatively fractionated by differential centrifugation. Using cytochrome c oxidase, succinate dehydrogenase, acid phosphatase and NADPH-cytochrome c reductase as marker enzymes, poly(A) hydrolase has been shown to be a mitochondrial enzyme.2. To test the biosynthetic origin of mitochondrial poly(A) hydrolase and to demonstrate its cytoplasmic site of synthesis, we have treated the cells with ethidium bromide, inhibitor of mitochondrial transcription, and chloramphenicol and cycloheximide, inhibitors of mitochondrial and cytoplasmic translations respectively. The activity of poly(A) hydrolase has been compared to that of succinate dehydrogenase, an enzyme coded for by the nuclear genome and that of cytochrome c oxidase, an enzyme coded for partly by the nuclear genome and partly by the mitochondrial genome. The results obtained indicate that in chick embryo fibroblasts poly(A) hydrolase is an enzyme coded for by the nuclear genome. Further, the hydrolase is synthesized on cytoplasmic ribosomes and has a half-life much shorter than succinate dehydrogenase and cytochrome c oxidase.Sheiness et a/. [l] have recently reported that the poly(A) attached to heterogeneous RNA appears to have the same size as the poly(A) that is initially found as messenger RNA. Soon after cytoplasmic appearance, however, the poly(A) begins to diminish in size, decreasing from about 200 nucleotides to 50 nucleotides or less [2-41. The enzyme involved in this particular cytoplasmic process is still unknown but could well be cellular poly(A) hydrolase [5-71, which catalyzes the hydrolysis of poly(A) to oligonucleotides with 3'-hydroxyl and 5'-monophosphoester termini which has been tentatively termed polyadenylase by others [7], also catalyzes the hydrolysis of single but not double-stranded RNA and DNA molecules [5,6] and is localized in the intermembrane space [7,12,13].

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Intracellular distribution of 5′-ribonuclease and 5′-phosphodiesterase in rat liver

Cited by 31 publications

References 7 publications

Ethidium bromide-induced loss of mitochondrial DNA from primary chicken embryo fibroblasts.

Ethidium bromide-induced loss of mitochondrial DNA from primary chicken embryo fibroblasts.

Intracellular Localization of Phosphodiesterases I and II in Rat Liver

Poly(A) Hydrolase of Chick‐Embryo Fibroblasts

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