Use of immunological procedures to measure rate of accumulation and degradation of inducible NADP-specific glutamate dehydrogenase during cell cycle of synchronous Chlorella

A new five-step purification procedure was developed which could be completed in five days with an 80 to 85 percent yield of electrophoretically pure nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase from Chlorella sorokiniana. A monospecific, highly purified antibody against the enzyme was prepared from the antisera of immunized rabbits. The antibody was purified on a stable antigen affinity column which was prepared by covalent-coupling of the holoenzyme to CNBr activated Sepharose 4B followed by cross-linking of the enzyme subunits together with dimethylsuberimidate. With ³H-leucine and ³⁵S-sulfate, as labeling agents of whole cells, the reproducibility and efficiency of both direct- and indirect-immunoprecipitation procedures were tested with pure labeled-enzyme as an internal standard. For these studies, a partially purified sheep anti-rabbit immunoglobulin G fraction was employed as a secondary antibody. A modified indirect immunoprecipitation procedure was tested in a cell cycle experiment to determine whether it would be feasible to use as a tool for further cell cycle experiments on enzyme turnover. Although the method was somewhat lengthy, it appeared to be reliable and was used to reveal that during the first one-third of the cell cycle of fully-induced cells, the NADPGDH has a maximum half-life of 1.6 h. Rocket immunoelectrophoresis was used to show that enzyme catalytic activity and antigen increased in a parallel fashion during the cell cycle of fully-induced cells. These data suggest that the cell cycle catalytic activity pattern reflects de novo synthesis of new molecules of enzyme. Since the enzyme in fully-induced cells undergoes rapid in vivo degradation, changes in the rate of enzyme synthesis probably determine the rate of enzyme accumulation during the Chlorella cell cycle in the continuous presence of ammonium.

Name thesis:degree_name

Ph. D.

Level thesis:degree_level

doctoral

Discipline thesis:degree_discipline

Biochemistry and Nutrition

Department dc:contributor.department

Biochemistry and Nutrition

Grantor dc:publisher

Virginia Polytechnic Institute and State University

Year dc:date.issued

1979

Author dc:creator

• Yeung, Anthony Tung

Statement dc:rights

• In Copyright

Licence dc:rights.uri

• http://rightsstatements.org/vocab/InC/1.0/

Language dc:language.iso

en_US

Handle dc:identifier.uri

http://hdl.handle.net/10919/54732

OAI identifier oai:identifier

oai:vtechworks.lib.vt.edu:10919/54732