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Title:Purification and Properties of Lecithin-Cholesterol Acyltransferase
Author(s):Doi, Yukio
Department / Program:Food Science
Discipline:Food Science
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Chemistry, Biochemistry
Abstract:A simple and convenient method for the purification of human plasma lecithin-cholesterol acyltransferase was developed. The method involves dialysis of plasma followed by dextran sulfate treatment, 1-butanol treatment in the presence of ammonium sulfate, DEAE-Sephadex chromatography, rechromatography on DEAE-Sephadex, and hydroxylapatite chromatography. The final purification of the enzyme is 20,000 fold over the starting plasma with about 12% yield. The purified enzyme showed a single main band on both disc and sodium dodecyl sulfate polacrylamide gel electrophoreses. The enzyme is stable for over two months when it is kept at 4(DEGREES)C under nitrogen in a buffer of low ionic strength, e.g. 0.4 mM phosphate buffer, pH 6.8, containing 4 mM 2-mercaptoethanol.
The purified enzyme was used to study its physical and chemical properties. The amino acid and carbohydrate compositions of the enzyme were determined. The enzyme is a glycoprotein with about 18% carbohydrate including 7% sialic acid. The molecular weights determined by sedimentation equilibrium and sodium dodecyl sulfate polacrylamide gel electrophoresis are 63,000 amd 65,000 daltons, respectively. However, the molecular weights obtained by Sephadex G-150 column chromatography and pore gradient gel electrophoresis are considerably higher, the values being 95,000 and 83,000 daltons, respectively. These high molecular weights reflect a non-ideal behavior of highly hydrated glycoproteins. The frictional coefficient ratio of 1.53 was estimated from the Stoke's radius of the enzyme (40 (ANGSTROM)) determined by Sephadex G-150 chromatography. These hydrodynamic properties lead to the conclusion that LCAT is a globular protein with considerable asymmetry, probably due to its high carbohydrate content, especially, sialic acid.
In spite of its apparent homogeneity, the enzyme showed six or more bands on isoelectric focusing on polyacrylamide gel with pI's ranging from 3.9 to 4.2. This microheterogeneity was observed consistently on the enzyme fractions obtained from pooled plasma and from the plasma of individual subjects. When the enzyme was treated with neuraminidase, those multiple bands shifted towards a higher pH region with concomitant increase in heterogeneity. However, all bands converged into a single band after the extensive treatment with neuraminidase. This single band corresponded to the enzyme entirely devoid of sialic acid residue. As a whole, 17 different bands including six or seven original bands can be produced on polyacrylamide gels upon isoelectric focusing of the neuraminidase-treated and untreated enzyme preparations. Each consecutive band appeared to differ from the neighboring bands by a single sialic acid residue, implying that the maximum 16 sialic acid residues can be attached to a common core of a polypeptide chain. The reduction in the sialic acid content by the neuraminidase treatment was accompanied by a progressive increase in the transferase activity of the enzyme.
The secondary structure of lecithin-cholesterol acyltransferase was estimated by circular dichroic measurements. The enzyme consists of 17% (alpha)-helical, 27% (beta)-sheet, and 56% disordered structure. Little change in the circular dichroic spectrum was observed upon binding of the enzyme to lecithin vesicles or upon alteration of the medium ionic strength at the N-terminal end of the enzyme determined by the dansylation-Edman degradation method is as follows: Phe-Trp-Leu-Leu-X-Val-Leu-Phe-Pro. Strong hydrophobicity of this segment suggests the participation of the N-terminal region in the binding of the enzyme to substrate particles.
Issue Date:1981
Description:106 p.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1981.
Other Identifier(s):(UMI)AAI8203445
Date Available in IDEALS:2015-05-13
Date Deposited:1981

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