Date of Award:

5-1-1992

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Biology

Department name when degree awarded

Biology (Entomology)

Committee Chair(s)

William A. Brindley

Committee

William A. Brindley

Committee

Howard M. Deer

Committee

Thomas A. Grover

Committee

Ting H. Hsiao

Committee

Frank J. Messina

Committee

Raghubir P. Sharma

Abstract

Acetylcholinesterase (AChE, EC 3.1.1.7) was purified from Lygus hesperus Knight by affinity chromatography following extraction by Triton X-100. The degree of purification ranged from 616- to 900-fold and the yield ranged from 42 to 56% (n = 5). The optimal pH and temperature for purified AChE were 7.5-8.0 and 35-40 °C, respectively, for hydrolyzing selected substrates. The efficiency for hydrolyzing various substrates was acetylthiocholine > acetyl-(β-methyl) thiocholine > propionylthiocholine > S-butyrylthiocholine. The inhibitor and substrate specificities, and substrate inhibition confirmed that the enzyme activity was due to AChE rather than butyrylcholinesterase. The susceptibility of AChE to inhibition by various organophosphrous compounds was mainly determined by affinity between the compounds and enzyme. AChE was a globular enzyme with three distinct molecular forms. The major form, accounting for about 89% of total AChE activity, was a hydrophilic dimer (150 kDa) with a sedimentation coefficient of 7.3. Two other minor forms, accounting for about 4 and 7% of total AChE activity, were a hydrophilic dimer (199 kDa) and an amphiphilic monomer (82 kDa), respectively. All three molecular forms had a very similar isoelectric point (7.4), and responded similarly to inhibition by paraoxon. The affinity of the major molecular form of AChE to the selected substrates and organophosphorus compounds was higher than that of the unresolved AChE. The turnover numbers for the native protein of the major form were 7,000 for acetylthiocholine, 4,800 for acetyl-(β-methyl) thiocholine, 3,000 for propionylthiocholine, and 390 min-1 for S-butyrylthiocholine, while the turnover numbers for the subunit or active site were about half of those for the native AChE. Thus, each native AChE possessed two active sites or each subunit possessed one active site. The sensitivity of AChE to various organophosphorus compounds in five populations of L. hesperus was Logan > Roswell > Caldwell > Kuna > Star. The insensitivity spectrum of AChE to different organophosphorus compounds was rather broad. However, trichlorfon resistance was mainly established by carboxylesterase (CarE, EC 3.1.1.1) activity rather than the insensitivity of AChE. High CarEs could significantly diminish the inhibition success of certain organophosphorus compounds to AChE, and enhance the significance of the less-sensitive AChE contributing to insecticide resistance in L. hesperus.

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