Characterization of lecithin-cholesterol acyltransferase from human plasma. III. Chemical properties of the enzyme

K. S. Chong, M. Jahani, S. Hara, Andras G. Lacko

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Abstract

The polypeptide molecular weight of lecithin-cholesterol acyltransferase (LCAT) (45000) was obtained by deducting the weight of carbohydrate moiety (25%, w/w) from the total molecular weight of 60,000. LCAT was found to have a relatively high content of glutamic acid, aspartic acid, glycine, and leucine residues and four half-cystines. The carbohydrate content was found to be about 25% (w/w): hexoses, 13%; hexosamines, 6.2%; and sialic acid, 5.4%. The total number of 408 amino acid residues per mole and the mean residue weight of 110.3 were found. From fluorescence spectroscopy analysis 6-7 mol of tryptophan were found per mole of LCAT in 10 mM phosphate (pH 7.4). However, when LCAT was digested by the mixture of chymotrypsin and pronase the tryptophan residues increased to 10-11 mol/mol of LCAT, which agrees well with data obtained previously by ultraviolet absorption spectroscopy. A partial specific volume of 0.707 mL/g was determined by compositional analysis. Human LCAT was found to have a relatively high extinction coefficient (E(1cm)(1%)) of 21 at 280 nm and neutral pH. Two residues of cysteine per mole of LCAT were estimated both in the presence or absence of sodium dodecyl sulfate by titration with 5,5'-dithiobis-2-nitrobenzoic acid. The enzyme showed a lower tendency to staining with Coomassie blue R-250 than bovine serum albumin. The enzyme was rapidly inactivated by diisopropyl fluorophosphate (DFP), regardless of whether the free sulfhydryl were blocked or not. The enzyme was also irreversibly inhibited by cysteine above concentrations of 1 mM. Neither the high density lipoprotein nor liposome substrate was able to protect against the inhibition by cysteine or DFP.

Original languageEnglish
Pages (from-to)875-881
Number of pages7
JournalCanadian Journal of Biochemistry and Cell Biology
Volume61
Issue number8
DOIs
StatePublished - 1 Jan 1983

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Phosphatidylcholine-Sterol O-Acyltransferase
fluorophosphate
Enzymes
Cysteine
Tryptophan
Molecular Weight
Carbohydrates
Dithionitrobenzoic Acid
Hexosamines
Weights and Measures
Pronase
Hexoses
Fluorescence Spectrometry
Chymotrypsin
N-Acetylneuraminic Acid
HDL Lipoproteins
Bovine Serum Albumin
Aspartic Acid
Liposomes
Leucine

Cite this

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title = "Characterization of lecithin-cholesterol acyltransferase from human plasma. III. Chemical properties of the enzyme",
abstract = "The polypeptide molecular weight of lecithin-cholesterol acyltransferase (LCAT) (45000) was obtained by deducting the weight of carbohydrate moiety (25{\%}, w/w) from the total molecular weight of 60,000. LCAT was found to have a relatively high content of glutamic acid, aspartic acid, glycine, and leucine residues and four half-cystines. The carbohydrate content was found to be about 25{\%} (w/w): hexoses, 13{\%}; hexosamines, 6.2{\%}; and sialic acid, 5.4{\%}. The total number of 408 amino acid residues per mole and the mean residue weight of 110.3 were found. From fluorescence spectroscopy analysis 6-7 mol of tryptophan were found per mole of LCAT in 10 mM phosphate (pH 7.4). However, when LCAT was digested by the mixture of chymotrypsin and pronase the tryptophan residues increased to 10-11 mol/mol of LCAT, which agrees well with data obtained previously by ultraviolet absorption spectroscopy. A partial specific volume of 0.707 mL/g was determined by compositional analysis. Human LCAT was found to have a relatively high extinction coefficient (E(1cm)(1{\%})) of 21 at 280 nm and neutral pH. Two residues of cysteine per mole of LCAT were estimated both in the presence or absence of sodium dodecyl sulfate by titration with 5,5'-dithiobis-2-nitrobenzoic acid. The enzyme showed a lower tendency to staining with Coomassie blue R-250 than bovine serum albumin. The enzyme was rapidly inactivated by diisopropyl fluorophosphate (DFP), regardless of whether the free sulfhydryl were blocked or not. The enzyme was also irreversibly inhibited by cysteine above concentrations of 1 mM. Neither the high density lipoprotein nor liposome substrate was able to protect against the inhibition by cysteine or DFP.",
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Characterization of lecithin-cholesterol acyltransferase from human plasma. III. Chemical properties of the enzyme. / Chong, K. S.; Jahani, M.; Hara, S.; Lacko, Andras G.

In: Canadian Journal of Biochemistry and Cell Biology, Vol. 61, No. 8, 01.01.1983, p. 875-881.

Research output: Contribution to journalArticle

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T1 - Characterization of lecithin-cholesterol acyltransferase from human plasma. III. Chemical properties of the enzyme

AU - Chong, K. S.

AU - Jahani, M.

AU - Hara, S.

AU - Lacko, Andras G.

PY - 1983/1/1

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N2 - The polypeptide molecular weight of lecithin-cholesterol acyltransferase (LCAT) (45000) was obtained by deducting the weight of carbohydrate moiety (25%, w/w) from the total molecular weight of 60,000. LCAT was found to have a relatively high content of glutamic acid, aspartic acid, glycine, and leucine residues and four half-cystines. The carbohydrate content was found to be about 25% (w/w): hexoses, 13%; hexosamines, 6.2%; and sialic acid, 5.4%. The total number of 408 amino acid residues per mole and the mean residue weight of 110.3 were found. From fluorescence spectroscopy analysis 6-7 mol of tryptophan were found per mole of LCAT in 10 mM phosphate (pH 7.4). However, when LCAT was digested by the mixture of chymotrypsin and pronase the tryptophan residues increased to 10-11 mol/mol of LCAT, which agrees well with data obtained previously by ultraviolet absorption spectroscopy. A partial specific volume of 0.707 mL/g was determined by compositional analysis. Human LCAT was found to have a relatively high extinction coefficient (E(1cm)(1%)) of 21 at 280 nm and neutral pH. Two residues of cysteine per mole of LCAT were estimated both in the presence or absence of sodium dodecyl sulfate by titration with 5,5'-dithiobis-2-nitrobenzoic acid. The enzyme showed a lower tendency to staining with Coomassie blue R-250 than bovine serum albumin. The enzyme was rapidly inactivated by diisopropyl fluorophosphate (DFP), regardless of whether the free sulfhydryl were blocked or not. The enzyme was also irreversibly inhibited by cysteine above concentrations of 1 mM. Neither the high density lipoprotein nor liposome substrate was able to protect against the inhibition by cysteine or DFP.

AB - The polypeptide molecular weight of lecithin-cholesterol acyltransferase (LCAT) (45000) was obtained by deducting the weight of carbohydrate moiety (25%, w/w) from the total molecular weight of 60,000. LCAT was found to have a relatively high content of glutamic acid, aspartic acid, glycine, and leucine residues and four half-cystines. The carbohydrate content was found to be about 25% (w/w): hexoses, 13%; hexosamines, 6.2%; and sialic acid, 5.4%. The total number of 408 amino acid residues per mole and the mean residue weight of 110.3 were found. From fluorescence spectroscopy analysis 6-7 mol of tryptophan were found per mole of LCAT in 10 mM phosphate (pH 7.4). However, when LCAT was digested by the mixture of chymotrypsin and pronase the tryptophan residues increased to 10-11 mol/mol of LCAT, which agrees well with data obtained previously by ultraviolet absorption spectroscopy. A partial specific volume of 0.707 mL/g was determined by compositional analysis. Human LCAT was found to have a relatively high extinction coefficient (E(1cm)(1%)) of 21 at 280 nm and neutral pH. Two residues of cysteine per mole of LCAT were estimated both in the presence or absence of sodium dodecyl sulfate by titration with 5,5'-dithiobis-2-nitrobenzoic acid. The enzyme showed a lower tendency to staining with Coomassie blue R-250 than bovine serum albumin. The enzyme was rapidly inactivated by diisopropyl fluorophosphate (DFP), regardless of whether the free sulfhydryl were blocked or not. The enzyme was also irreversibly inhibited by cysteine above concentrations of 1 mM. Neither the high density lipoprotein nor liposome substrate was able to protect against the inhibition by cysteine or DFP.

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