Protein Synthesis in Semliki Forest Virus-infected Cells is Not Controlled by Permeability Changes

Gray, Michael A.; Austin, Sara; Clemens, Michael J.; Rodrigues, L. M.; Pasternak, C. A.

doi:10.1099/0022-1317-64-12-2631

Cited by 17 publications

(4 citation statements)

References 36 publications

(32 reference statements)

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“…Gray et al (163)(164)(165) confirmed that there are alterations in the concentration of monovalent cations in cells infected by SF. From studies with drugs, including the monovalent cation ionophore nigericin, they concluded that these changes could not account for the extent of inhibition of protein synthesis seen in infected cells, although they could contribute to inhibition, consistent with the results for SIN.…”

Section: Early Events In Establishment Of Infectionmentioning

confidence: 96%

The alphaviruses: gene expression, replication, and evolution.

Strauss¹,

Strauss²

1994

Microbiological Reviews

1,388

1,135

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Section: Early Events In Establishment Of Infectionmentioning

confidence: 96%

The alphaviruses: gene expression, replication, and evolution.

Strauss¹,

Strauss²

1994

Microbiological Reviews

1,388

1,135

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“…As a result, GMPPN generated by heat extraction of nucleotides from the Ras protein can be quantified by HPLC-UV and tracked back to the starting, bound GMPPNP prior to heating. In contrast, GTP-γ-S and GppCH 2 p are hydrolyzed primarily to GDP and GMP respectively, which cannot be tracked back to the starting nucleotide [56]. Turning to functional studies of the inactive state, we chose GDP as the stabilizing nucleotide since it is the native ligand of the off-state, is highly resistant to further hydrolysis by the G protein or heat and is readily quantifiable by HPLC-UV.…”

Section: Discussionmentioning

confidence: 99%

HPLC Method to Resolve, Identify and Quantify Guanine Nucleotides Bound to the GTPase Ras

Hannan

Swisher

Martyr

et al. 2021

Preprint

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The Ras superfamily of small G proteins play central roles in diverse signaling pathways. Superfamily members act as molecular on-off switches defined by their occupancy with GTP or GDP, respectively. In vitro functional studies require loading with a hydrolysis-resistant GTP analogue to increase the on-state lifetime, as well as knowledge of fractional loading with activating and inactivating nucleotides. The present study describes a method combining elements of previous approaches with new, optimized features to analyze the bound nucleotide composition of a G protein loaded with activating (GMPPNP) or inactivating (GDP) nucleotide. After nucleotide loading, the complex is washed to remove unbound nucleotides then bound nucleotides are heat-extracted and subjected to ion-paired, reverse-phase HPLC-UV to resolve, identify and quantify the individual nucleotide components. These data enable back-calculation to the nucleotide composition and fractional activation of the original, washed G protein population prior to heat extraction. The method is highly reproducible. Application to multiple HRas preparations and mutants confirms its ability to fully extract and analyze bound nucleotides, and to resolve the fractional on- and off-state populations. Furthermore, the findings yield a novel hypothesis for the molecular disease mechanism of Ras mutations at the E63 and Y64 positions.

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“…Miller, Castellot & Pardee, 1978, 1979Dunn & Holz, 1983;Wilson & Kirshner, 1983), and more recently Sendai virus and S. aureus s-toxin have been employed to bring Ca 2+ chelates (Gomperts, Baldwin & Micklem, 1983; Ahnert-Hillger, Bhakdi & Gratzl, 1985), GTP analogues (Gray et al, 1983;Barrowman, Cockroft & Gomperts, 1986) or antibiotics (Cameron et al, 1986) into metabolically competent cells to study processes such as exocytosis. Pore-forming agents therefore have a role as biological tools, in addition to their relevance to diseases of viral (Pasternak, 1987a), microbial (McCartney & Arbuthnott, 1978;Arbuthnott, 1983), animal (e.g.…”

Section: Introductionmentioning

confidence: 97%

Ion modulation of membrane permeability: Effect of cations on intact cells and on cells and phospholipid bilayers treated with pore-forming agents

et al. 1988

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Leakage of ions (Na+, K+) and phosphorylated metabolites (phosphorylcholine, 2-deoxyglucose 6-phosphate) through membrane lesions in intact cells or in cells modified by 'pore-forming' agent has been studied. Leakage from intact cells is induced by protons and by divalent cations such as Cu2+, Cd2+ or Zn2+. Leakage from agent-modified cells--or across phospholipid bilayers modified by agent--is prevented by low concentrations of the same cations and by higher concentrations of Ca2+, Mn2+ or Ba2+; Mg2+, dimethonium, spermine, or spermidine are virtually ineffective. The relative efficacy of a particular cation (e.g. Ca2+) depends more on cell type than on the nature of the pore-forming agent. The predominant effect is on binding of cation to specific sites, not on surface charge. Surface charge, on the other hand, does affect leakage from agent-modified cells in that suspension in nonionic media reduces leakage, which can be restored by increasing the ionic strength: univalent (Na+, K+, Rb+, NH4+) and divalent (Mg2+, dimethonium) cations are equally effective; addition of protons or divalent cations such as Zn2+ to this system inhibits leakage. From this and other evidence here presented it is concluded that leakage across membranes is modulated by the presence of endogenous anionic components: when these are in the ionized state, leakage is favored; when unionized (as a result of protonation) or chelated (by binding to divalent cation), leakage is prevented. It is suggested that such groups are exposed at the extracellular face of the plasma membrane.

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Protein Synthesis in Semliki Forest Virus-infected Cells is Not Controlled by Permeability Changes

Cited by 17 publications

References 36 publications

The alphaviruses: gene expression, replication, and evolution.

The alphaviruses: gene expression, replication, and evolution.

HPLC Method to Resolve, Identify and Quantify Guanine Nucleotides Bound to the GTPase Ras

Ion modulation of membrane permeability: Effect of cations on intact cells and on cells and phospholipid bilayers treated with pore-forming agents

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