Interferon-Induced Enzymes: Microassays and Their Applications; Purification and Assay of (2′-5′)-Oligoadenylate Synthetase and Assay of 2′-Phosphodiesterase

M. Revel, D. Wallach, G. Merlin, A. Schattner, A. Schmidt, D. Wolf, L. Shulman, A. Kimchi

Research output: Contribution to journalArticlepeer-review

Abstract

There are three enzymic activities elevated in cells that have been exposed to interferon: a double-stranded (ds) RNA-activated protein kinase (PK-i) phosphorylating initiation factor eIF-2's small subunit, the dsRNA-dependent (2'-5')-oligoadenylate synthetase (E), and a 2'- phosphodiesterase (2'-PDi), which splits the (2'-5')ppp(A2'p)nA oligonucleotide formed by the synthetase. The chapter discusses several applications for these enzyme measurements with a detailed description of the assay procedures used. The experiments described are concerned with the (2'-5')- oligoadenylate synthetase, which shows the largest variations, and with the 2'-phosphodiesterase. The (2'-5')-oligoadenylate produced by this enzyme inhibits protein synthesis in cell-free systems by activating a specific ribonuclease, F. This property can be used to assay the synthetase. The incorporation of [a-32P]ATP into pppA2'p5'A or longer chains, is measured. The enzyme in crude cell extracts is subjected to a one-step purification by adsorption to poly(rI)· poly(rC)-agarose beads.

Original languageEnglish
Pages (from-to)149-161
Number of pages13
JournalMethods in Enzymology
Volume79
Issue numberC
DOIs
StatePublished - 1 Jan 1981

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