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PMID: 2831385 Published · ppublish English Journal Article

Purification and partial characterization of poliovirus protease 2A by means of a functional assay.

Journal of virology ·Vol. 62 ·No. 4 ·1988-04-00 ·Pages 1243-50

König H, Rosenwirth B

Abstract

The purification of poliovirus protease 2A from infected cells by a functional assay is described. A small synthetic peptide was cleaved specifically by an esterase present in poliovirus-infected cells. Since the enzyme proved extremely unstable in crude extracts a rapid and efficient purification procedure had to be developed. By treatment with different detergents followed by high-speed centrifugation, the esterase activity was separated from inactivating cellular enzymes and was solubilized. Purification to more than 90% homogeneity could be achieved by a single chromatography step, namely, by gel filtration through Superose 12 under fast-protein liquid chromatography conditions. The esterase activity was associated with a protein of 17,000 daltons and copurified with poliovirus protein 2A. Furthermore, antibodies to 2A specifically precipitated the esterase activity. Thus, the esterase was identified as poliovirus protease 2A. Inhibition studies with known protease inhibitors revealed that 2A is probably a sulfhydryl protease. Of the metal ions tested, only zinc exerted significant inhibitory effects. The esterase activity was optimal near neutral pH and had an extremely short half-life at physiological temperatures.

MeSH Terms
Centrifugation Chromatography, Gel Chromatography, Thin Layer Electrophoresis, Polyacrylamide Gel HeLa Cells Humans Immunoassay Peptide Hydrolases/analysis,isolation & purification Poliovirus/enzymology
Chemicals
Peptide Hydrolases
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
König H
Sandoz Forschungsinstitut, Vienna, Austria.
Rosenwirth B
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Article Info
Journal
Journal of virology
Abbr.
J Virol
ISSN
0022-538X
DOI
10.1128/JVI.62.4.1243-1250.1988
Published
1988-04-00
Pages
1243-50
Language
English
Region
United States
NLM ID
0113724
PMCID
PMC253133
Subset
IM
External Links
PubMed source DOI: 10.1128/JVI.62.4.1243-1250.1988 PMC Full Text View journal details
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