Glycerol allows low-temperature phase separation of membrane proteins solubilized in triton X-114: Application to the purification of plant cytochromes P-450 and b5

Werck‐Reichhart, Danièle; Benveniste, Irène; Teutsch, H.; Durst, Francis; Gabriac, B

doi:10.1016/0003-2697(91)90367-3

Cited by 54 publications

(43 citation statements)

References 22 publications

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“…Samples were incubated for 10 min at 37°C, followed by centrifugation (500 ϫ g, 5 min, room temperature) to induce separation into detergent-poor and -rich phases. Inclusion of glycerol in the partitioning mixture allowed flotation, rather than sedimentation, of the detergent-rich phase (71), thereby removing the concern that the detergent-rich fraction contained particles merely due to pelleting during centrifugation. The bottom of each centrifuge tube was punctured with a needle, and both detergent-poor and -rich phases were harvested by collecting drops.…”

Section: Methodsmentioning

confidence: 99%

Strategy for Nonenveloped Virus Entry: a Hydrophobic Conformer of the Reovirus Membrane Penetration Protein μ1 Mediates Membrane Disruption

Chandran

Farsetta

Nibert

2002

J Virol

166

335

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The mechanisms employed by nonenveloped animal viruses to penetrate the membranes of their host cells remain enigmatic. Membrane penetration by the nonenveloped mammalian reoviruses is believed to deliver a partially uncoated, but still large (ϳ70-nm), particle with active transcriptases for viral mRNA synthesis directly into the cytoplasm. This process is likely initiated by a particle form that resembles infectious subvirion particles (ISVPs), disassembly intermediates produced from virions by proteolytic uncoating. Consistent with that idea, ISVPs, but not virions, can induce disruption of membranes in vitro. Both activities ascribed to ISVP-like particles, membrane disruption in vitro and membrane penetration within cells, are linked to N-myristoylated outer-capsid protein 1, present in 600 copies at the surfaces of ISVPs. To understand how 1 fulfills its role as the reovirus penetration protein, we monitored changes in ISVPs during the permeabilization of red blood cells induced by these particles. Hemolysis was preceded by a major structural transition in ISVPs, characterized by conformational change in 1 and elution of fibrous attachment protein 1. The altered conformer of 1 was required for hemolysis and was markedly hydrophobic. The structural transition in ISVPs was further accompanied by derepression of genome-dependent mRNA synthesis by the particle-associated transcriptases. We propose a model for reovirus entry in which (i) primed and triggered conformational changes, analogous to those in enveloped-virus fusion proteins, generate a hydrophobic 1 conformer capable of inserting into and disrupting cell membranes and (ii) activation of the viral particles for membrane interaction and mRNA synthesis are concurrent events. Reoviruses provide an opportune system for defining the molecular details of membrane penetration by a large nonenveloped animal virus.

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Section: Methodsmentioning

confidence: 99%

Strategy for Nonenveloped Virus Entry: a Hydrophobic Conformer of the Reovirus Membrane Penetration Protein μ1 Mediates Membrane Disruption

Chandran

Farsetta

Nibert

2002

J Virol

166

335

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“…The spectra were obtained without and with co-transformation with pSBET, but in all cases the Cyt P450 content was too low to permit quantification. To obtain an accurate determination, the Cyt P450s were enriched by isolation of E. coli spheroblasts, followed by temperature-induced Triton X-114 phase partitioning (Werck-Reichart et al, 1991;. The highest expression level (JM109 cells, 48 h) was 56 nmol/L culture and was obtained using CYP79E2 lacZ(24aa) (Fig.…”

Section: Expression Of Cyp79e1 and Cyp79e2 In E Colimentioning

confidence: 99%

“…For in vitro studies, the recombinant Cyt P450s obtained by expression of CYP79E1 na , CYP79E1⌬ (1-52) 2E1(10aa) , and CYP79E2 lacZ(24aa) were partially purified by temperatureinduced Triton X-114 phase partitioning (Werck-Reichart et al, 1991; and reconstituted using S. bicolor NADPH-Cyt P450 oxidoreductase and DLPC (Fig. 5).…”

Section: Determination Of the Enzymatic Activity Of Cyp79e1 And Cyp79e2mentioning

confidence: 99%

Cloning and Expression of Cytochrome P450 Enzymes Catalyzing the Conversion of Tyrosine to p-Hydroxyphenylacetaldoxime in the Biosynthesis of Cyanogenic Glucosides in Triglochin maritima

Nielsen¹,

Møller²

2000

Plant Physiology

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“…Recombinant CYP79D1 was isolated using P. pastoris microsomes as the starting material and TX-114 phase partitioning (29,30) as the first purification step. The phase partitioning mixture contained microsomal protein (4 mg/ml), 50 mM KP i , pH 7.9, 1 mM DTT, 30% glycerol, and 1% TX-114.…”

Section: P Pastoris Cell Cultures and Preparation Of Microsomes-singlementioning

confidence: 99%

Cytochromes P-450 from Cassava (Manihot esculentaCrantz) Catalyzing the First Steps in the Biosynthesis of the Cyanogenic Glucosides Linamarin and Lotaustralin

Andersen¹,

Busk²,

Svendsen³

et al. 2000

Journal of Biological Chemistry

186

140

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The first committed steps in the biosynthesis of the two cyanogenic glucosides linamarin and lotaustralin in cassava are the conversion of L-valine and L-isoleucine, respectively, to the corresponding oximes. Two fulllength cDNA clones that encode cytochromes P-450 catalyzing these reactions have been isolated. The two cassava cytochromes P-450 are 85% identical, share 54% sequence identity to CYP79A1 from sorghum, and have been assigned CYP79D1 and CYP79D2. Functional expression has been achieved using the methylotrophic yeast, Pichia pastoris. The amount of CYP79D1 isolated from 1 liter of P. pastoris culture exceeds the amounts that putatively could be isolated from 22,000 grown-up cassava plants. Each cytochrome P-450 metabolizes Lvaline as well as L-isoleucine consistent with the cooccurrence of linamarin and lotaustralin in cassava. CYP79D1 was isolated from P. pastoris. Reconstitution in lipid micelles showed that CYP79D1 has a higher k c value with L-valine as substrate than with L-isoleucine, which is consistent with linamarin being the major cyanogenic glucoside in cassava. Both CYP79D1 and CYP79D2 are present in the genome of cassava cultivar MCol22 in agreement with cassava being allotetraploid. CYP79D1 and CYP79D2 are actively transcribed, and production of acyanogenic cassava plants would therefore require down-regulation of both genes.

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Glycerol allows low-temperature phase separation of membrane proteins solubilized in triton X-114: Application to the purification of plant cytochromes P-450 and b5

Cited by 54 publications

References 22 publications

Strategy for Nonenveloped Virus Entry: a Hydrophobic Conformer of the Reovirus Membrane Penetration Protein μ1 Mediates Membrane Disruption

Strategy for Nonenveloped Virus Entry: a Hydrophobic Conformer of the Reovirus Membrane Penetration Protein μ1 Mediates Membrane Disruption

Cloning and Expression of Cytochrome P450 Enzymes Catalyzing the Conversion of Tyrosine to p-Hydroxyphenylacetaldoxime in the Biosynthesis of Cyanogenic Glucosides in Triglochin maritima

Cytochromes P-450 from Cassava (Manihot esculentaCrantz) Catalyzing the First Steps in the Biosynthesis of the Cyanogenic Glucosides Linamarin and Lotaustralin

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