Date of Award

Summer 1976

Document Type


Degree Name

Master of Science (MS)


Chemistry & Biochemistry


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


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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