Camel Liver Esterase （LE） was isolated through five consecutive steps： Extraction with Tris/HCl buffer, pH 8, precipitation with ammonium sulfate, affinity chromatography on Affi-gel-butyric acid and on Affi-gel-ol...Camel Liver Esterase （LE） was isolated through five consecutive steps： Extraction with Tris/HCl buffer, pH 8, precipitation with ammonium sulfate, affinity chromatography on Affi-gel-butyric acid and on Affi-gel-oleic acid, and preparative polyacrylamide gel electrophoresis （PAGE）. The Km values were found as： methyl butyrate （8.3 mM）, α-naphthyl acetate （3.65 mM）, 13-naphthyl myristate （66.7 mM）, p-nitrophenyl acetate （0.29 mM）, and phenyl acetate （5.26 mM）. The Ki of LE inhibition by bis（4-nitrophenyl） phosphate was 7.9 p.M, and 58 μM by phenyl-methyl-sulfonyl fluoride. The inhibition by these inhibitors is irreversible. p-Hydroxymercuribenzoate or ethylenediamine-tetra-acetic acid did not inhibit the enzyme. LE showed a dimeric structure with molecular weight of 129 kD. The energy of activation of LE was 15.0, 5.5 and 10.75 Kcal, using the substrates： a-naphthyl acetate, p-nitrophenyl acetate, and methyl butyrate, respectively. The optimal pH for LE was between 8 and 10. The N-terminus was found as aspartic acid. The percentage of glycine residues （13.3%） was the highest whereas the percentage of cysteine residues （0.68%） was the lowest in LE. Amino acid composition shows that LE has -50% of its histidine residues as N-methylhistidines.展开更多
文摘Camel Liver Esterase （LE） was isolated through five consecutive steps： Extraction with Tris/HCl buffer, pH 8, precipitation with ammonium sulfate, affinity chromatography on Affi-gel-butyric acid and on Affi-gel-oleic acid, and preparative polyacrylamide gel electrophoresis （PAGE）. The Km values were found as： methyl butyrate （8.3 mM）, α-naphthyl acetate （3.65 mM）, 13-naphthyl myristate （66.7 mM）, p-nitrophenyl acetate （0.29 mM）, and phenyl acetate （5.26 mM）. The Ki of LE inhibition by bis（4-nitrophenyl） phosphate was 7.9 p.M, and 58 μM by phenyl-methyl-sulfonyl fluoride. The inhibition by these inhibitors is irreversible. p-Hydroxymercuribenzoate or ethylenediamine-tetra-acetic acid did not inhibit the enzyme. LE showed a dimeric structure with molecular weight of 129 kD. The energy of activation of LE was 15.0, 5.5 and 10.75 Kcal, using the substrates： a-naphthyl acetate, p-nitrophenyl acetate, and methyl butyrate, respectively. The optimal pH for LE was between 8 and 10. The N-terminus was found as aspartic acid. The percentage of glycine residues （13.3%） was the highest whereas the percentage of cysteine residues （0.68%） was the lowest in LE. Amino acid composition shows that LE has -50% of its histidine residues as N-methylhistidines.
