Use of dodecyl-beta-D-maltoside in the purification and stabilization of RNA polymerase from brome mosaic virus-infected barley.
The activity and specificity of RNA-dependent RNA polymerase (replicase) isolated from brome mosaic virus-infected barley was enhanced by extraction with the nonionic detergent dodecyl-beta-d-maltoside. The enzyme was stable for at least 8 weeks when stored at -70 degrees . A further 100-fold purification was obtained by centrifugation through sucrose in the presence of detergent. The polymerase activity was associated with the pellet fraction; the template dependence and specificity were similar to those of the enzyme before sucrose purification. SDS-PAGE analysis revealed a 110-kd protein in the purified pellet fraction from infected leaves that was absent from a similar fraction from healthy leaves. The protein had an identical electrophoretic mobility to that of protein la, the product of brome mosaic virus RNA 1 translation in vitro, and the profile of its tryptic polypeptides was very similar to that of protein 1a. These results support data obtained by inoculation of protoplasts with separated BMV RNA components (Kiberstis, et al. (1981), Virology 112, 804-808) that are consistent with the notion that RNA 1 codes for the viral replicase, or a subunit thereof.
Bujarski
J J
JJ
Department of Horticulture, University of Wisconsin, Madison, Wisconsin 53706, USA.
Hardy
S F
SF
Miller
W A
WA
Hall
T C
TC