Pressure Dissociation and Temperature Stability of Lactate Dehydrogenase (Oligomeric, Proteins, Conformational, Drift, Activity)
King, Lan
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https://hdl.handle.net/2142/70713
Description
Title
Pressure Dissociation and Temperature Stability of Lactate Dehydrogenase (Oligomeric, Proteins, Conformational, Drift, Activity)
Author(s)
King, Lan
Issue Date
1985
Department of Study
Physiology and Biophysics
Discipline
Biophysics
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Biochemistry
Abstract
The deactivation of lactate dehydrogenase by pressure and its subsequent reactivation have been studied by Jaenicke et al. Although they did not carry out direct measurements of the molecular size they ascribe the slow reactivation at atmospheric pressure to the reassociation of the separated monomers. We have used fluorescence polarization methods (Paladini and Weber, 1982) to monitor the changes in molecular volume under pressure and our results clearly show the dissociation of the enzyme at pressures of 1.0 - 2.5 kbars. The change in volume upon dissociation is ca. 200-300 ml/mol. The dissociated product is shown to be the monomer by its 30 ns rotational relaxation time and by the uniform appearance in time of the three mixed isozymes when the H(,4) and M(,4) isozymes of lactate dehydrogenase are jointly subjected to high pressure. The dissociated monomers undergo a progressive change in conformation with time, i.e., a "conformational drift", the extent of which depends upon the pressure applied and the duration of high-pressure incubation. The drifted monomer is proven to reassociate into tetramers immediately after release of pressure. The newly associated tetramers have an average conformation different from the native enzyme as shown by both diminished catalytic activity and subunit affinity. The original active conformation of the enzyme gradually returns after a time that increases with the magnitude and time of pressure incubation. In addition to the apparent dissociation of the enzyme at the pressures at which the average molecular volume is clearly reduced, we recognized the existence of a microscopic association-dissociation cycle in the relatively low pressure range (<lkbar), in which dissociation is not yet measurable but both slow inactivation and hybridization of mixed M(,4) and H(,4) are seen after long incubation (3 to 63 hours). The microscopic association-dissociation cycle was also recognized at 4(DEGREES)C by the same methods. Decreasing temperature plays a role equivalent to that of increasing pressure. We finally were able to demonstrate the existence of the microscopic cycle at room temperature and atmospheric pressure by the hybridization of active and inactive enzyme and by the separation of monomer, native tetramer and conformational altered tetramer by HPLC gel filtration.
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