Dissertation - Open Access
Doctor of Philosophy (PhD)
The differences in the response to KCl of the RNA-degrading enzymes of two barley cultivars, “Dicktoo” (a hardy type) and "Tennessee Winter” (a non-hardy type), were investigated. Two types of enzymes, ribonuclease (RNase) and nuclease, were separated from crude extracts by disc electrophoresis. The electrophoretic gels were assayed for RNA-degrading activity in the presence and absence of EDTA (EDTA is a nuclease inhibitor). By means of density gradient isoelectric focusing, the nuclease preparation from Tennessee Winter was shown to have an isoelectric point between 5.3 and 5.5, while the nuclease preparation from Dicktoo has three isoelectric points of 4.9, 5.5 and 6.0. The RNase preparation from Tennessee Winter has isoelectric points of 4.5, 5.8 and a trace of activity at 5.3, while the RNase from Dicktoo has isoelectric points of 4.9, 5.8 and 6.1. In the presence of KCl, nuclease activity obtained from both cultivars was diminished, while RNase activity from Tennessee Winter showed increased activity, when compared with the RNase from Dicktoo. Preparative electrophoresis was employed in order to separate larger quantities of enzymes from the two cultivars. In the RNase preparation from Dicktoo, two distinct activity peaks were found and they were processed as two separate samples for further purification. The RNase preparation from Tennessee Winter was processed as one sample. A purification scheme consisting of Sephadex G-25, Sephadex DEAE A-50, preparative electrophoresis and Bio-Gel P-100 was employed. The contaminating acid phosphomonoesterase activity was removed from the RNase in the final step of purification using Bio-Gel P-100. The purified enzyme from Tennessee Winter and the two enzymes from Dicktoo had the same pH optima of 5.8 and showed lowered pH optima in the presence of KCl. For all three enzymes, maximum RNA-degrading activity was obtained at pH 5.4 with 0.19 M KCl. Substrate specificities of the three purified enzymes were determined by noting the rates at which they cleaved 14 dinucleoside monophosphates. The two enzymes from Dicktoo were not significantly different with respect to cleavage of the dinucleoside monophosphates in this experiment. The major difference between the enzymes from Tennessee Winter and Dicktoo was their cleavage rates for ApC and ApU. Tennessee Winter RNase had a faster rate of cleavage of the phosphodiester bond between adenosine and a pyrimidine. The RNases from the two cultivars were inferred as being the plant RNase I type of enzyme.
Library of Congress Subject Headings
South Dakota State University
Wong, Raymond Sze-Chung, "A Comparative Study of Ribonuclease From Two Winter Barley Cultivars Based Upon Separation, Purification and Partial Characterization of RNA-Degrading Enzymes" (1975). Electronic Theses and Dissertations. 5532.