- Collection:
- Atlanta University and Clark Atlanta University Theses and Dissertations
- Title:
- The chemical modification and immobilization of fructose-1, 6-bisphosphatase, 1991
- Creator:
- Hunter, Edward
- Date of Original:
- 1991-05-01
- Subject:
- Degrees, Academic
Dissertations, Academic - Location:
- United States, Georgia, Fulton County, Atlanta, 33.749, -84.38798
- Medium:
- theses
- Type:
- Text
- Format:
- application/pdf
- Description:
- Fructose-1,6-bisphosphatase was purified to homogeneous state from chicken livers. The functional consequences of treating the enzyme with oxidized glutathione (GSSG), diethylpyrocarbonate, and butanedione were investigated. Activation of the enzyme with GSSG, as previously reported from this laboratory, was found to be accompanied by alteration of other properties, such as reduced sensitivity to fructose 2,6- P2 inhibition and loss of activation by K+. Treatment with butanedione leads to desensitization of the enzyme to AMP inhibiton, indicating the presence of arginyl residue(s) at the AMP allosteric site. Modification with diethylpyrocarbonate results in rapid and almost total loss of the catalytic activity, suggesting the presence of essential histidine residue at the catalytic site. Fructose 1,6-bisphosphatase was bound to cysteamine -Sepharose by thiol/disulfide exchange with cystamine- Sepharose. The immobilized enzyme retained about 80% of the specific activity of the free enzyme. The activity of the immobilized enzyme could further be enhanced by iodoacetamide and cystamine. The optimum pH remained essentially unchanged but the pH activity curve became much broader. The bound enzyme could be released from cysteamine-Sepharose by reduction with dithiothreitol. The immobilized enzyme lost about 50% of its activity after storage for 3 weeks in 0.5 M NaCl at 4C. Albumin significantly enhanced the stability. The immobilized enzyme is more effective than the free form in selective hydrolysis of fructose 1,6-bisphosphate in the process of fructose 2,6-bisphosphate synthesis. The enzyme was also bound to pyridoxal-P-Sepharose through formation of Schiff base linkage using the epsilon amino group of lysyl residue(s) at the AMP allosteric site. Stable and highly active immobilized enzyme was obtained by reduction of the Schiff base with NaBH4.
- External Identifiers:
- Metadata URL:
- http://hdl.handle.net/20.500.12322/cau.td:1991_hunterjr_edward.pdf
- Rights Holder:
- Clark Atlanta University
- Holding Institution:
- Atlanta University Center Robert W. Woodruff Library
- Rights:
-
