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PMID: 1255853 Published · ppublish English Journal Article

In vitro replication of cowpea mosaic virus RNA. II. Solubilization of membrane-bound replicase and the partial purification of the solubilized enzyme.

Journal of virology ·Vol. 17 ·No. 3 ·1976-03-00 ·Pages 679-85

Zabel P, Jongen-Neven I, Van Krammen A

Abstract

A method for the solubilization of membrane-bound Cowpea mosaic virus RNA replicase has been developed by bypassing the use of detergents. Solubilization has been achieved by washing the 31,000 x g-pellet containing the bound replicase with a Mg2+-deficient buffer. This procedure had several advantages as compared to treatments with nonionic or ionic detergents: (i) the solubilized enzyme was stable at 4 C, (ii) more than 80% of the replicase could be solubilized without loss of total enzyme activity, (iii) the replicase was rather selectively released resulting in a two- to threefold increase in specific activity per se, and (iv) most of the green color from chloroplast fragments present in the crude replicase fraction remained membrane bound resulting in only slightly colored preparations of solubilized enzyme. The solubilized replicase has been further purified by DEAE-Bio Gel column chromatography. RNA synthesis directed by the DEAE-purified enzyme was template dependent and proceeded at a linear rate for at least 9 h.

MeSH Terms
Cell-Free System Magnesium Mosaic Viruses/enzymology,growth & development,metabolism Plant Viruses/metabolism RNA Nucleotidyltransferases/metabolism RNA, Viral/biosynthesis RNA-Dependent RNA Polymerase/isolation & purification,metabolism Solubility Surface-Active Agents Virus Replication
Chemicals
RNA, Viral Surface-Active Agents RNA Nucleotidyltransferases RNA-Dependent RNA Polymerase Magnesium
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Zabel P
Jongen-Neven I
Van Krammen A
References (34)
34 references, click to expand
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Article Info
Journal
Journal of virology
Abbr.
J Virol
ISSN
0022-538X
DOI
10.1128/JVI.17.3.679-685.1976
Published
1976-03-00
Pages
679-85
Language
English
Region
United States
NLM ID
0113724
PMCID
PMC515465
Subset
IM
External Links
PubMed source DOI: 10.1128/JVI.17.3.679-685.1976 PMC Full Text View journal details
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