Date of Award:
5-1977
Document Type:
Dissertation
Degree Name:
Doctor of Philosophy (PhD)
Department:
Plants, Soils, and Climate
Committee Chair(s)
G. W. Cochran
Committee
G. W. Cochran
Committee
G. W. Miller
Committee
F. B. Salisbury
Committee
N. N. Youssef
Abstract
Ribonucleic acid polymerases (E.C. 2.7.7.6) have been solubilized form whole green leaves of barley (Hordeum vulgare L.). Three major forms of the solubilized enzyme have been isolated by sonication in high ionic strength buffers, cation and anion exchange chromatography, and agarose gel permeation chromatography and designated I, II, and III. The enzyme fractions appear analogous to the Class I, II, and III ribonucleic acid polymerases defined in other eucaryotic systems. The ionic strength optima, amatoxin sensitivity, and relative activities with denatured calf thymus deoxyribonucleic acid were different for Class I, II, and III polymerases.
A simple, rapid, reproducible method was developed for purification and characterization of the Class II ribonucleic acid polymerase enzyme. The method involved batchwise treatments with phosphocellulose and DEAE-cellulose, sievorptive chromatography on DEAE-Sepharose CL-6B. Analysis of purified ribonucleic acid polymerase II by polyacrylamide gel electrophoresis under nondenaturing conditions revealed heterogeneity (two protein brands, designated IIA and IIB, in order of electrophoretic mobility). The heterogeneity of this became even more pronounced after further purification of either the phosphocellulose or DEAE-Sepharose CL-6B.
The subunit composition of DEAE-Sepahrose CL-6B fraction was analyzed by electrophoresis under denaturing conditions. The enzyme II form contained three high molecular weight polypeptides (180,000; 170,000; 85,000) and six low molecular weight polypeptides. The enzyme has a provisional molecular weight between 650,000 to 700,000. A chelating agent, such as 1,10-phenanthroline, inhibited enzyme activity, indicating that it is probably a zinc metalloenzyme.
Recommended Citation
Javali, Sachchidananda, "Multiple Forms of Deoxyribonucleic Acid-Dependent Ribonucleic Acid Polymerase in Barley Leaves (Hordeum vulgare L.)" (1977). All Graduate Theses and Dissertations, Spring 1920 to Summer 2023. 9096.
https://digitalcommons.usu.edu/etd/9096
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