Publication Date

5-1989

Advisor(s) - Committee Chair

Valgene Dunham, Frank Toman, Martin Houston

Degree Program

Department of Biology

Degree Type

Master of Science

Abstract

Topoisomerases are enzymes of critical biological significance. Despite this fact, little data specific to the topoisomerases of higher plants has been published. This research was undertaken to isolate and characterize a higher plant topoisomerase from soybean hypocotyls to further the understanding of the structure and function of these enzymes.

Nuclei were isolated from frozen hypocotyls of 4 day old etiolated soybeans by homogenization and centrifugation, then lysed by gentle stirring in the presence of 1.5 mM ammonium sulfate. The resultant extract was desalted and purified by column chromatography on DEAF sepharose, 5-200 sephacryl and CM cellulose. The components of the purified fraction were separated by electrophoresis on non-denaturing polyacrylamide and recovered by electro-elution.

The molecular weight of the native enzyme was determined to be 225 kilodaltons (Kd) by gel filtration and 300 Kd by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Three smaller molecular sub-species, possibly resulting from dissociation of the native enzyme in the presence of beta mercaptoethanol (BME), were isolated. These smaller molecules had approximate molecular weights of 155 Kd, 29 Kd and 25 Kd. The two smaller polypeptides appeared to re-associate to form a 68 Kd aggregate in the absence of BME. This aggregate was subsequently resistant to denaturation by sodium dodecyl sulfate (SDS).

The purified native enzyme was found to be adenosine triphosphate (ATP) and Mg++ (divalent magnesium ion) independent. Although not required, the presence of magnesium (Mg) stimulated enzyme activity. Manganese (Mn++) stimulated activity to a lesser degree. Enzymatic activity was inhibited by ATP, potassium chloride (KC1) and N-ethylmaleimide (NEM) but not inhibited by novobiocin.

The results indicate that a type I topoisomerase was purified from the nuclei of soybeans (Glycine max). The soybean topoisomerase I has a native molecular weight similar to that of cauliflower (200 Kd) and like cauliflower topoisomerase was inhibited by NEM but not by novobiocin and was stimulated by Mg++. It is similar to vaccinia virus topoisomerase I in being slightly inhibited by ATP. The data suggests that the native enzyme may be composed of enzymatically active domains as small as 27 Kd and thus is similar to vaccinia virus and Ustilaqo maydis topoisomerase I.

Disciplines

Agricultural Science | Agronomy and Crop Sciences | Biology | Life Sciences | Plant Sciences

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