The physico-chemical properties of lecithin:cholesterol acyltransferase were investigated. The amino acid composition analysis showed a relatively high content of glutamic acid, aspartic acid, glycine and leucine. The spectrophotometric titration of phenolic groups in the enzyme showed a large increase in absorbance at 295 nm with an apparent pK of about 12.0. The largest change in molar ellipticity at 222 nm was also observed above pH 11. Circular dichroism studies revealed that human lecithin:cholesterol acyltransferase has a relatively high content of β-pleated sheet structure (48%) with 20% α-helix, and 32% remaining structure. Human lecithin:cholesterol acyltransferase has a high extinction coefficient at neutral pH. Microsequencing of the amino terminal residues of the enzyme revealed a hydrophobic character. Inactivation of lecithin:cholesterol acyltransferase activity was observed using diisopropylfluorophosphate with a stoichiometry of 1 mole of diisopropylphosphate incorporated per mole of enzyme. This suggests the involvement of a serine residue in the active site of the enzyme, possibly for the formation of an acyl-intermediate. A new quicker assay method for lecithin:cholesterol acyltransferase was developed. This assay involved coupling reaction with acyl CoA synthetase, ΡΡᵢ-dependent phosphofructokinase, aldolase, triosephosphate isomerase and α-glycerol-3-phosphate dehydrogenase monitoring a change in the absorbance or fluorescence intensity due to the oxidation of NADH. The activity of each coupling enzyme was accurately determined to establish the optimum assay condition for lecithin:cholesterol acyltransferase. The coupled enzyme assay for lecithin:cholesterol acyltransferase by spectrofluorometry showed a significant change in relative fluorescence intensity whereas a UV absorption spectroscopy method showed no significant absorbance change for the initial rate of lecithin:cholesterol acyltransferase reaction.
| Identifer | oai:union.ndltd.org:unt.edu/info:ark/67531/metadc331696 |
| Date | 12 1900 |
| Creators | Hara, Shinichi |
| Contributors | Lacko, Andras G., Cook, Paul F., Harris, Ben G., Wu, Edward Ming-chi, 1938-, Norton, S. J. |
| Publisher | North Texas State University |
| Source Sets | University of North Texas |
| Language | English |
| Detected Language | English |
| Type | Thesis or Dissertation |
| Format | x, 128 leaves: ill., Text |
| Rights | Public, Hara, Shinichi, Copyright, Copyright is held by the author, unless otherwise noted. All rights reserved. |
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