Spermidine stimulation of foot-and-mouth disease virus RNA-dependent RNA polymerase activity

Lazarus, Lawrence H.; Popescu, M.; Barzilai, R.; Goldblum, Natan

doi:10.1007/bf01249861

Cited by 6 publications

(4 citation statements)

References 24 publications

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“…At 180 min after infection, the time of maximum synthesis of both viral specific RNA and replicase (Fig.l), the cell layer was harvested [16]. Cell lysates were prepared in buffer (0.01 M Tris-HC1 pH 7.8, 1 mM MgCl,, 1 mM dithiothreitol, 0.1 mM EDTA) containing l.Oo/, nonidet P-40 on ice.…”

Section: Induction Of Foot-and-mouth Disease Virusmentioning

confidence: 99%

“…Induction of foot-and-mouth disease virus replicase was carried out a t 37 "C [16]. At 180 min after infection, the time of maximum synthesis of both viral specific RNA and replicase (Fig.l), the cell layer was harvested [16].…”

Section: Induction Of Foot-and-mouth Disease Virusmentioning

confidence: 99%

“…Our previous investigations on the effect of the polyamine spermidine in vitro [16] has led us to define specific ionic interactions involved with this replicase. I n this communication we present the results of that undertaking. …”

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confidence: 99%

“…As a comparative model for the synthesis of RNA on an RNA template, the replicase induced by foot-and-mouth disease virus is ideally suited. Our previous investigations on the effect of the polyamine spermidine in vitro [16] has led us to define specific ionic interactions involved with this replicase. I n this communication we present the results of that undertaking.…”

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Mono- and Divalent Cationic Parameters of Foot-and-Mouth Disease Virus Replicase

et al. 1972

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Replicase induced by foot-and-mouth disease virus has an absolute requirement for Mg2+ that cannot be replaced by other divalent cations. Mn2+ and Ca2+ are effective non-competitive inhibitors. At low ionic strength, Mg2+ directly influence the V of the reaction. High ionic strength (0.2 M KCI), produces a shift in the optimum Mg2+ concentration toward lower values which enhances the V and K,. KCI is more active in stimulating activity than NaC1, LiCl or CsCl but similar to NH,Cl. (NH,),SO,, however, is less stimulatory than the chloride salt. K+ appear to interact specifically with replicase so as to affect initiation : once the reaction has been initiated it becomes refractory to the addition of K+ and shows typical plateau kinetics under all conditions. Divalent cations are essential cofactors for the activity of enzymes catalyzing the transfer of phosphoryl groups. The distinct optimal requirement of specific cations by nucleosidetriphosphate nucleotidyltransferase (DNA-or RNA-dependent polymerase) has resulted in their general classification [1,2]. Usually required in catalytic amounts, divalent cations also form complexes with nucleoside triphosphates which serve as the substrate [1,3] during polymerization. I n contrast to the specificity of divalent cations, monovalent cations influence the activity of a wide variety of unrelated enzyme reactions [4]. Their mode of action on transcriptases is complex, effecting numerous interrelated phenomena : they increase template efficiency through dissociation of nucleoproteins [1,5-71 and allow reinitiation [8] with the release of nascent RNA from the DNA template [S-lo]. I n this latter capacity, monovalent cations mimic the function of the rho factor 11 11. Monovalent cations also influence the rate of enzyme-substrate interactions during catalysis [3,4,12] with a possible binding site on the enzyme [12], the physical conformation of the enzyme [5,13] and its stability [13-151 and possibly inhibit nucleotic activity [5,6].For the most part, studies concerning ionic interactions with polymerases have utilized DNA-dependent enzymes from bacterial [8][9][10]14,15] and mammalian sources [1,2,5-71. As a comparative model for the synthesis of RNA on an RNA template, the replicase induced by foot-and-mouth disease virus is ideally suited. Our previous investigations on the effect of the polyamine spermidine in vitro [16] has led us to define specific ionic interactions involved with this replicase. I n this communication we present the results of that undertaking. MATERIALS AND METHODS Induction of Foot-and-Mouth Disease VirusReplicase A baby hamster kidney cell line was grown in a modified Eagle's medium [I71 with loo/, calf serum to a density of 2 x lo8 cells/Z-l rolling bottle a t 37 ".Foot-and-mouth disease virus strain SAT-1 (Veterinary Institute, Beth Dagan, Israel) grown in this cell line was concentrated 100-fold [18] and dialyzed for 2 h a t 4 "C against medium before infection.Induction of foot-and-mouth disease virus replicase was carried out a t 37 "C [16]....

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Section: Induction Of Foot-and-mouth Disease Virusmentioning

confidence: 99%

Section: Induction Of Foot-and-mouth Disease Virusmentioning

confidence: 99%

mentioning

confidence: 99%

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confidence: 99%

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