Date of Award

Summer 1976

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Chemistry & Biochemistry

Program/Concentration

Chemical Sciences

Committee Director

James Yuan

Committee Member

Thomas O. Sitz

Committee Member

Frank E. Scully, Jr.

Call Number for Print

Special Collections LD4331.C45 B36

Abstract

Purification of Yeast NAD+-dependent isocitrate dehydrogenase was partially successful when utilizing an NAD+-ribosyl-hydrazide Sepharose affinity chromatography column. Although no specific elution system was found, a maximum purification of sixty-five fold was achieved with 0.5M KCl elution. A 38-fold purification was obtained when 20mM NAD+ was employed for elution.

Yeast and calf liver NADP+-dependent isocitrate dehydrogenase were also found to bind to NAD+-ribosyl-hydrazide Sepharose as well as the NAD+-dependent enzyme. Again, elution was achieved with either o,5M KCl or 20 mM NADP+.

Ammonium sulfate fractionation decreased the yield by 60 to 80% and specific activity by 50% in both forms of isocitrate dehydrogenase. Sepharose 4B proved to be a mild purification step, resulting in up to 100% recovery and a 2 to 3.5-fold increase in specific activity.

Separation of Yeast NAD+-dependent isocitrate dehydrogenase from other dehydrogenases was not successful when utilizing Sepharose 4B, or the affinity column when eluting with a 0 to 30mM NAD+ gradient.

NAD+-dependent isocitrate dehydrogenase activity in bull seminal plasma was observed to be stimulated by either ADP or AMP with maximum stimulation obtained at 0.2mM and 0.7mM respectively.

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DOI

10.25777/c53t-qw43

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