Analytical Study of Microsomes and Isolated Subcellular Membranes From Rat Liver

Beaufay, Henri; Amar-Costesec, A; Thinessempoux, D.; Wibo, Maurice; Robbi, Mariette; Berthet, J

doi:10.1083/jcb.61.1.213

Cited by 237 publications

(77 citation statements)

References 35 publications

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“…Alpha 1,2-mannosidase activity was separated in two peaks, with 32% of the activity associated with densities in the range reported for Golgi (1.122-1.142 g cm -3 ) and 68% of the activity associated with densities reported for ER membranes (1.178-1.182 g cm -3 ) (Beaufay et al 1974, Chrispeels et al 1982, Harris & Waters 1996, Mora-Montes et al 2008a, consistent with distribution of the enzyme between the Golgi complex and ER. Monensin blocks the vesicle transport within the Golgi complex (Rosa et al 1992) and, as a consequence, proteins are accumulated in the ER.…”

Section: Discussionsupporting

confidence: 56%

Purification and biochemica characterisation of endoplasmic reticulum α 1,2-mannosidase from Sporothrix schenckiil

Mora-Montes

Robledo-Ortiz

Gonzalez-Sanchez

et al. 2010

Mem. Inst. Oswaldo Cruz

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N-glycosylation of proteins is a common post-translational modification in eukaryotic cells. Following the translocation of nascent proteins into the endoplasmic reticulum (ER), the Glc 3 Man 9 GlcNAc 2 oligosaccharide is transferred to asparagine residues and sequentially processed by ER α-glucosidases, which remove the three glucose residues and by α-mannosidases that trim at least one mannose residue (Kornfeld & Kornfeld 1985, Herscovics 1999a.Alpha-mannosidases are grouped within families 38 and 47 of the glycosyl hydrolase classification (Henrissat 1991, Henrissat & Davies 1997. Members of family 38 are less specific than enzymes from family 47, removing α1,2, α1,3 and α1,6-linked mannose units (Daniel et al. 1994, Herscovics 1999a. Enzymes from family 47 are α1,2-mannosidases that participate in the trimming of N-glycans and in ER-associated degradation (Herscovics 1999a, b, 2001, Helenius & Aebi 2004. Two subgroups of enzymes comprise family 47: the ER α1,2-mannosidases that trim only one mannose residue from Man 9 GlcNAc 2 (M 9 ) generating Man 8 GlcNAc isomer B (M 8 B) (Ziegler & Trimble 1991, Tremblay & Herscovics 1999, Mora-Montes et al. 2004, Movsichoff et al. 2005; and Golgi α1,2-mannosidases from mammalian cells and filamentous fungi, which act after the ER enzyme to remove three α1,2-linked mannose units, generating Man 5 GlcNAc 2 , an intermediate required for the biosynthesis of complex and hybrid N-glycans (Yoshida et al. 1993, Lal et al. 1998, Ichishima et al. 1999, Eades & Hintz 2000, Tremblay & Herscovics 2000, Lobsanov et al. 2002, Akao et al. 2006. Lower eukaryotes, such as Saccharomyces cerevisiae, lack Golgi α1,2-mannosidases and synthesise only high-mannose N-glycans (Herscovics 1999b).Sporothrix schenckii, the etiological agent of sporotrichosis, is a dimorphic fungus that grows as filamentous and yeast cells during saprophytic and parasitic phases, respectively. This organism is closely related to Ophiostoma stenoceras, a non-pathogenic fungus that grows on sapwood from coniferous and hardwood trees (de Beer et al. 2003). Sporotrichosis is a subcutaneous mycosis caused by traumatic inoculation of colonised materials (soil, wood, decomposed vegetable matter) or inhalation of the conidia (Ramos-e-Silva et al. 2007). The disease has been reported as an emerging mycosis in HIV-infected humans (Durden & Elewski 1997). The cell wall of S. schenckii is being actively researched, as this structure has an important role in adhesion to host tissues and is the main source of antigens that can be exploited in the immuno-diagnosis of the disease (LopesBezerra et al. 2006). The cell wall of S. schenckii contains β1,3, β1,6 and β1,4-glucans (Lopes-Bezerra et al. In order to gain insight into the processing steps of Nglycan biosynthesis, a membrane-bound α-mannosidase from S. schenckii was targeted for purification and characterisation. Biochemical characterisation and intracellular localisation studies indicated that this enzyme is an ER α1,2-mannosidase and therefore a new member of glycosyl hydrol...

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Section: Discussionsupporting

confidence: 56%

Purification and biochemica characterisation of endoplasmic reticulum α 1,2-mannosidase from Sporothrix schenckiil

Mora-Montes

Robledo-Ortiz

Gonzalez-Sanchez

et al. 2010

Mem. Inst. Oswaldo Cruz

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“…2 (15,23), and catalase is situated in peroxisomes (5). NADPH cytochrome c reductase is an endoplasmic reticulum enzyme (8), and alkaline phosphodiesterase is located in the plasma membrane (8). As ascertained by the amount of acid phosphatase and /3-galactosidase and of proteins recovered in the L fraction, lysosomes are purified between 8 and 10 times in this fraction.…”

Section: Density Gradient Of Metrizamidementioning

confidence: 99%

“…Indeed, acid hydrolases located in lysosomes may still be endowed with a slight but significant activity at alkaline pH on substrates used to determine plasma membrane enzymes. In some cases, one can remedy this situation, by making use of a selective inhibitor for the acid Calculations were performed as described in the text; percentages of total liver protein constituted by different membrane components were taken from published results; mitochondria, endoplasmic reticulum, and peroxisomes from Leighton et al (18), outer mitochondrial membrane from Schnaitman and Greenawalt (22), plasma membrane and Golgi membranes from Beaufay et al (8).…”

Section: Purification Of L Ysosomes By Centrifugation Of the L Fractimentioning

confidence: 99%

“…Marker enzymes of the main subcellular membranes were assayed according to the following references: monoamine oxidase (32), cytochrome oxidase (1), acid pbosphatase (1), ~galactosidase (27), N-acetylglucosaminidase (27), catalase (5), glucose-6-phosphatase (15), NADPH cytochrome c reductase (8), galactosyltransferase (8), alkaline phosphodiesterase (8) alkaline pbosphatase (8), and 5'AMPase (31). To test a possible effect of metrizamide on enzyme activity, preliminary experiments were performed.…”

Section: Enzyme Assaysmentioning

confidence: 99%

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Isolation of rat liver lysosomes by isopycnic centrifugation in a metrizamide gradient.

Wattiaux¹,

Coninck²,

Ronveaux-Dupal³

et al. 1978

The Journal of Cell Biology

327

138

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A preparation, similar to the light mitochondrial fraction of rat liver (L fraction of de Duve et al. [1955, Biochem. J. 60: 604-617]), was subfractionated by isopycnic centrifugation in a metrizamide gradient and the distribution of several marker enzymes was established. The granules were layered at the top or bottom of the gradient. In both cases, as ascertained by the enzyme distributions, the lysosomes are well separated from the peroxisomes. A good separation from mitochondria is obtained only when the L fraction is set down underneath the gradient. Taking into account the analytical centrifugation results, a procedure was devised to purify lysosomes from several grams of liver by centrifugation of an L fraction in a discontinuous metrizamide gradient. By this method, a fraction containing 10-12% of the whole liver lysosomes can be prepared. As inferred from the relative specific activity of marker enzymes, it can be estimated that lysosomes are purified between 66 and 80 times in this fraction. As ascertained by plasma membrane marker enzyme activity, the main contaminant could be the plasma membrane components. However, cytochemical tests for 5'AMPase and for acid phosphatase suggest that a large part of the plasma membrane marker enzyme activity present in the purified lysosome preparation could be associated with the lysosomal membrane.The procedure for the isolation of rat liver lysosomes described in this paper is compared with the already existing methods.KEY WORDS metrizamide centrifugation rat liver lysosomes 9 plasma membrane gradient Isopycnic centrifugation in a metrizamide gradient of a total mitochondrial fraction of rat liver (M + L according to de Duve et al. [15]), allows a good separation of peroxisomes from the other major constituents of the fraction, mitochondria, and lysosomes (13). On the other hand, lysosomes are poorly separated from mitochondria as ascertained by the distribution curves of their reference enzymes (13). In the present paper, we show that by using a light mitochondrial fraction (L fraction of de Duve et al. [15]), in certain conditions it is possible to purify rat liver lysosomes extensively by centrifugation in a metrizamide gradient, with satisfactory yield. MATERIALS AND METHODS Tissue FractionationExperiments were performed on male Wistar rats weighing 200-250 g. The animals were decapitated after J. CELL BmLO~V 9 The Rockefeller University Press 9 0021-9525/78/0801-034951.00 349 being starved for 20 h. The livers were removed, chilled in ice-cold 0.25 M sucrose, and homogenized in the same medium by means of a smooth glass tube fitted with a Teflon pestle (Arthur H. Thomas Co., Philadelphia, Pa.) rotating at 3,000 rpm. The homogenate was made up to a volume of 7 ml/g of liver. After filtration on two layers of cheesecloth, a small sample was kept for enzyme and protein determination; the remainder was centrifuged at an integrated force of 30,000 g. min in the N ~ 40 rotor (ravg 5.9 cm) of the Spinco preparative ultracentrifuge (Beckman Instruments, Inc....

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“…Subcellular marker enzymes were assayed according to the following methods: succinate dehydrogenase (King, 1967) for mitochondria; palmitoyl-CoA oxidase (Lazarow and De Duve, 1976) for peroxisomes; NADPH-cytochrome c reductase (Beaufay et al, 1974) for microsomes and glyceraldehyde 3-phosphate dehydrogenase (Serrano et al, 1991) for cytosol.…”

Section: Enzyme Assaysmentioning

confidence: 99%

Prehibernation and hibernation effects on the d-3-hydroxybutyrate dehydrogenase of the heavy and light mitochondria from liver jerboa (Jaculus orientalis) and related metabolism

Mountassif¹,

Kabine²,

Latruffe³

et al. 2007

Biochimie

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Kebbaj To cite this version:Driss Mountassif, Mostafa Kabine, Norbert Latruffe, M'Hammed Saïd El Kebbaj. Prehibernation and hibernation effects on the D-3-hydroxybutyrate dehydrogenase of the heavy and light mitochondria from liver jerboa (Jaculus orientalis) and related metabolism.. Biochimie, Elsevier, 2007, 89 (8) Abstract:The D-3-hydroxybutyrate dehydrogenase (BDH) (EC 1.1.1.30) from liver jerboa (Jaculus orientalis), a ketone body converting enzyme in mitochondria, in two populations of mitochondria (heavy and light) has been studied in different jerboa states (euthermic, prehibernatingand hibernating). The results reveal: (1) important variations between states in terms of ketones bodies, glucose and lipid levels; (2) significant differences between the BDH of the two mitochondrial populations in term of protein expression and kinetic properties. These results suggest that BDH leads an important conformational change depending on the physiological state of jerboa. This BDH structural change could be the consequence of the lipid composition modifications in inner mitochondrial membrane leading to changes in BDH catalytic properties.

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Analytical Study of Microsomes and Isolated Subcellular Membranes From Rat Liver

Cited by 237 publications

References 35 publications

Purification and biochemica characterisation of endoplasmic reticulum α 1,2-mannosidase from Sporothrix schenckiil

Purification and biochemica characterisation of endoplasmic reticulum α 1,2-mannosidase from Sporothrix schenckiil

Isolation of rat liver lysosomes by isopycnic centrifugation in a metrizamide gradient.

Prehibernation and hibernation effects on the d-3-hydroxybutyrate dehydrogenase of the heavy and light mitochondria from liver jerboa (Jaculus orientalis) and related metabolism

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