Selective purification of sex‐influenced esterase from rat serum by immunoaffinity chromatographies

Abstract

Sex-influenced esterase (ES-SI) is a female-specific serum esterase of rats. There are two alleles at the Es-Si locus, Es-Si" coding for ES-SI and Es-Sib, a silent allele. ES-SI was purified to electrophoretic homogeneity from the serum of adult female S13 rats by a four-step purification. First, albumin was removed from the serum by Blue-Sepharose CL-6B affinity chromatography. Sera of rats carrying Es-Si" and Es-Sib were utilized to make two novel immunoaffinity columns. Rat anti-(ES-SI) alloantibody was obtained after the immunization of rats carrying Es-Sib with rat ES-SI-positive serum. ES-SI bound to the gel in this antibody-conjugated affinity chromatography. Simultaneously, rabbit anti-(rat serum protein lacking ES-SI) antibody was raised against ES-SI-negative rat serum and used to exclude other serum proteins. ES-SI was recovered in the flow-through fractions in this antibody-conjugated immunoaffinity chromatography, whereas the other serum proteins bound to the gel. ES-SI was selectively purified about 240-fold by combining these immunoaffinity chromatographies. This combination is a very powerful technique for purification of the protein. Finally, ES-SI was purified by preparative polyacrylamide gel electrophoresis after which it appeared as a single protein band (using silver staining) on SDS/ polyacrylamide gel electrophoresis with a molecular mass of 72 kDa. In the ordinary estimation of enzyme purification, the purification of ES-SI was 30-fold at most, because the specific activity of the enzyme was calculated from the bulky esterase activity. However, ES-SI was selectively purified 2280-fold comparing its recovery to that of total protein. ES-SI exhibited a PI of 4.6 and optimum pH of 6.7. The activity of ES-SI was completely inhibited by diisopropyl fluorophosphate at 10 nM but not at all by eserine.A variety of esterases is distributed in various tissues of rats. All carboxylesterase genes are mapped in linkage group V and form two gene clusters: cluster 1 includes Es-2, ; cluster 2 includes Es-I, Es-Si (also called , . Three genes (Es-I, Es-2 and Ex-Si) are known to code for serum esterases. The multigene family of these carboxylesterases resembles molecular evolution by gene duplication [2]. In spite of the importance of these genes as a model system for gene evolution, there are no reports on the purification of the enzyme protein or molecular cloning of genes for these isozymes to our knowledge.ES-SI was reported as a female-specific serum esterase found in some inbred strains of rat [3]. There are two alleles at the Es-Silocus, Es-Si" coding for the sex-influenced esterase (ES-SI) and Es-Sib, a silent allele [3 -51. In mature male rat strains which carry the Es-Si" allele, ES-SI decreased in their serum after puberty [6] which suggests that testosterone plays an important role in the regulation of ES-SI [6]. ES-SI is not detected in the serum of male and female rat strains which carry the Es-Sib allele throughout their lifetime.Isozyme patterns of various esterases resolved by electropho...

Dimethoate, fenvalerate and their mixture affects Hylyphantes graminicola (Araneae: Linyphiidae) adults and their unexposed offspring

“…AChE and CarE concentrations are expressed per mass of protein (Ellman et al , 1961; Hirose et al , 1990). Protein concentrations in the spiders were estimated using the colourimetric method of Bradford (1976).…”

“…CarE activity was assayed using the method of Hirose et al (1990). In brief, the assay uses ρ-nitrophenyl acetate (PNPA) as substrate.…”

Classification, Localization, and Some Physiological Roles of Hydrolytic Enzymes

“…In the rat, for example, the esterase ES1 found in the plasma, intestinal lymph, and small intestine seems to be involved in the uptake of fatty acid esters [81]. Esterases ES12 and ES13 have been described as butyrylesterases, and ES14 (also called ES-SI) is a female-specific serum esterase, the concentration of which is regulated by levels of both testosterone and estradiol [83]. The enzyme ES6 appears to be an acetylesterase (EC 3.1.1.6), and the liver microsomal ES10 hydrolyzes medium-chain acylglycerols.…”