The Effect of The Modification of The Acidic and Zwitterionic Phospholipid Composition on The Enzymatic Activity of D-Beta-Hydroxybutyrate Dehydrogenase
Clancy, Robert Michael
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https://hdl.handle.net/2142/70503
Description
Title
The Effect of The Modification of The Acidic and Zwitterionic Phospholipid Composition on The Enzymatic Activity of D-Beta-Hydroxybutyrate Dehydrogenase
Author(s)
Clancy, Robert Michael
Issue Date
1982
Department of Study
Biochemistry
Discipline
Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Biochemistry
Abstract
The effect of the modification of the phospholipid composition on the enzymatic activity of D-beta-hydroxybutyrate dehydrogenase (BDH) was examined. Phospholipase D was used to increase the acidic phospholipid composition. This enzyme hydrolyzed primarily zwitterionic phospholipids to phosphatidic acid, an acidic phospholipid. The accumulation of phosphatidic acid increases the electrostatic potential at the surface of the membrane. The apparent K(,m) for the negatively charged substrates of BDH increased as the membranes were hydrolyzed with phospholipase D. There was a 10-fold increase in the apparent K(,m) for NADH when the content of acidic phospholipids was increased by 24%. The addition of monovalent or divalent cations, which reduced the electrostatic potential, largely reversed the apparent K(,m) changes. At the same ionic strength divalent cations had a substantially larger effect than monovalent cations. Similar results were obtained when the purified apoenzyme was reconstituted in unilamellar vesicles containing different ratios of phosphatidylcholine and acidic phospholipids. When the apoenzyme was reconstituted into phosphatidylcholine vesicles containing increasing amounts of phosphatidylethanolamine, the apparent K(,m) increased but to a smaller extent and increasing the ionic strength did not reverse this effect. In another study, analogues of choline were placed in the media of primary rat liver hepatocytes. The unnatural choline analogue 1-2-amino-1-butanol was found to be incorporated to the largest extent into the phospholipid. However, when the hepatocytes were placed in media without serine or methionine, N-methylethanolamine was incorporated to the largest extent into the phospholipid. The altered phospholipid composition had an effect on the kinetic parameters of BDH. When BDH was assayed in submitochondrial membranes containing either phosphatidylbutanolamine or phosphatidylmethylethanolamine, the apparent K(,m) for NADH increased relative to choline supplemented cells while the K(,m) for acetoacetate remained unaffected. The data suggest that the enzymatic activity of BDH is sensitive to the phosphatidylcholine content in the submitochondrial membrane. Thus, the results of this study show that the apparent K(,m) of BDH can be significantly altered by an electrostatic potential as well as other properties of the phospholipid polar head group.
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