Solubilization and purification of aldolase from bovine sperm and muscle

The spermatozoan is a highly compartmentalized structure consisting of head, midpiece and tail regions. Various structural components and numerous enzymes occupy these three compartments. Evidence for the organization of glycolytic enzymes into a macroenzyme complex has previously been presented for both skeletal muscle and Escherichia coli. A multienzyme glycolytic complex would benefit the spermatozoan by increasing fructolytic efficiency by decreasing diffusion of intermediary metabolites and by directly providing ATP for sperm cell motility and other functions. Data from the present work suggests the existence of a multiprotein complex in spermatozoa. Aldolase from bovine muscle (BMA) and sperm (BSpA) have been characterized with respect to the nature of their subcellular associations. Solubilization properties of BMA and BSpA were investigated. Deoxycholate, when included in homogenization buffer, solubilized approximately 81%, of the BMA per gram of tissue. Triton X100 in combination with phosphate buffer solubilized only 10% of the total 3SpA per cell. BMA was purified to greater than 90% homogeneity by salt fractionation, gel filtration, and phosphocellulose chromatography. Aldolase from bovine muscle was a tetramer with a molecular weight per subunit of 41,000. BSpA was partially purified using the above methods. Aldolases from bovine muscle and spermatozoa were compared with regard to their activity toward FBP and F-1-P. BMA exhibited an FBP to F-1-P activity ratio of 9.62, which is typical of type A aldolases. BSpA showed an FBP to F-1-P activity ratio of 0.038 which is not characteristic of the previously identified type A, B or C aldolases. BMA and BSpA were different with respect to solubilization, purification and kinetic properties.

Name thesis:degree_name

Master of Science

Level thesis:degree_level

masters

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

1982

Author dc:creator

• Gillis, Beth A.

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/10919/80126

OAI identifier oai:identifier

oai:vtechworks.lib.vt.edu:10919/80126