Date sent:      	Fri, 27 Apr 2001 

Characterization of a 2-5A dependent 37-kDa RNase L.
2. Azido photoaffinity labeling and 2-5A Dependent activation.

J Biol Chem 2001, Apr 25

Shetzline SE, Suhadolnik RJ.

Biochemistry, Temple University School of Medicine,
Philadelphia, PA 19140.

PMID: 11323422

The preceding paper in this issue described the characterization of the
molecular structure of the 37-kDa RNase L identified in peripheral blood
mononuclear cell (PBMC) extracts from individuals with chronic fatigue
syndrome (CFS) [Shetzline, S., et al., (2001) J. Biol. Chem. (preceding
paper in this issue)].

In this study, analysis of 2-5A binding and activation of the 80-kDa and
the 37-kDa forms of RNase L has been completed utilizing
photolabeling/immunoprecipitation and affinity assays, respectively.
Saturation of photolabeling of the 80-kDa and the 37-kDa RNase L with the
2-5A azido photoprobe, [32P]pApAp(8-azidoA), was achieved.

Half-maximal photoinsertion of [32P]pApAp(8-azidoA) occurred at 3.7 x
10-8 M for the 80-kDa RNase L and at 6.3 x 10-8 M for the 37-kDa RNase L.
Competition experiments using 100-fold excess unlabeled 2-5A
photoaffinity probe, pApAp(8-azidoA), and authentic 2-5A (p3A3) resulted
in complete protection against photolabeling, demonstrating that
[32P]pApAp(8-azidoA) binds specifically to the 2-5A binding site of the
80-kDa and the 37-kDa RNase L. The rate of RNA hydrolysis by the 37-kDa
RNase L was three times faster than the 80-kDa RNase L.

The data obtained from these 2-5A binding and 2-5A-dependent activation
studies demonstrate the utility of [32P]pApAp(8-azidoA) for the detection
of the 37-kDa RNase L in PBMC extracts.

[Please note that the accepted manuscript version is available for free
in PDF format at
http://www.jbc.org/cgi/reprint/M101243200v1 ]