Date of Award
Fall 1994
Document Type
Thesis
Degree Name
Master of Science (MS)
Department
Biological Sciences
Program/Concentration
Biotechnology
Committee Director
Richard P. Ciavarra
Committee Member
Laura K. Moen
Committee Member
Lloyd Wolfinbarger
Call Number for Print
Special Collections LD4331.B52 W46
Abstract
Previous studies (Ciavarra et al., 1994) demonstrated that the constitutive 70 kDa heat shock protein (hsc70) protected purified topoisomerase I from thermal injury. In addition, hsc70 was capable of regenerating catalytic activity of heat-denatured topoisomerase I. A whole cell lysate was also active in this reaction assay. The present study demonstrates that heat-denatured topoisomerase I is reactivated by a cytosolic fraction and that this activity is dependent on the presence of cytosolic hsc70. The efficacy of hsc70-mediated refolding of heat-denatured topoisomerase I is greatly enhanced by a cytosolic cofactor(s). In all these refolding reactions, exogenous ATP is not required. Size exclusion chromatography of nuclear extracts indicates that topoisomerase I forms complexes with other proteins and that these complexes possess enzymatic activity. Furthermore, topoisomerase I activity in isolated nuclei is inhibited immediately after thermal injury and does not increase during a recovery period at 37 °C. These data suggest that a cytosolic protein(s) is required for protection and refolding of nuclear topoisomerase I. Since the nuclear membrane may not have been intact during the isolation of clean nuclei, protection and refolding assays were also performed in permeabilized cells following thermal injury. The results with permeabilized cells demonstrate that the cytosol is active in both the protection and refolding assays. Hsc70, but not other cytosolic proteins, is able to refold heat-inactivated topoisomerase I. These in situ reactions are not ATP-dependent.
Rights
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DOI
10.25777/vyvw-mn91
Recommended Citation
Wen, Kuo-Kuang.
"Role of Heat Shock Protein 70 kDa Cognate in Limiting Thermal Inactivation and Refolding of Heat-Denatured Nuclear Type I Topoisomerase"
(1994). Master of Science (MS), Thesis, Biological Sciences, Old Dominion University, DOI: 10.25777/vyvw-mn91
https://digitalcommons.odu.edu/biomedicalsciences_etds/147
Comments
A Thesis Submitted to the Faculties of Old Dominion University and Eastern Virginia Medical School in Partial Fulfillment of the Degree of Master of Science in Biotechnology.