Adam Ronai

Genetic Identification of Rat-liver Carboxylesterases Isolated in Different Laboratories

Six carboxylesterases previously isolated from rat liver microsomes, characterized in Brussels and in Kiel, were compared with genetically defined liver esterases of various reference strains using polyacrylamide gel electrophoresis and isoelectric focusing. The six liver carboxylesterases were identified as alloenzymic forms of ES-3, ES-4, ES-8/ES-10 and ES-15 according to the genetic nomenclature recommended by van Zutphen (Van Zutphen, L.F.M. (1983) Transplant. Proceed. 15, 1687-1688). The genetic and biochemical characteristics of the four isoenzymes are summarized, and their identity with several other drug-metabolizing esterases/amidases and lipases of rat liver endoplasmic reticulum is discussed.

Esterase-7, a common constituent of numerous mouse tissues

1. Electrophoretic and staining techniques for the relatively specific demonstration of esterase-7 in mouse tissues are described. Esterase-7 was found to be a common constituent of most mouse tissues. 2. The electrophoretic polymorphisms determined by three alleles a, b and c were studied in nine mouse strains. 3. The banding pattern of esterase-7 is being described and the isoelectric points of the allelic bands are being estimated. 4. Recombination studies on a further 129 animals confirmed that the Es-7 locus is tightly linked to Es-2 and Es-11. 5. Esterase-7 precipitates with antiesterase-2 from the rabbit. 6. It differs from esterase-2 by its individual substrate preference and by its susceptibility to inhibition by acylating agents.

Esterase XXVII. Purification and characterization of esterase-9A of mouse kidney

Esterase-9A, which appears electrophoretically as a triplet of the bands III-50, III-40 and III-30, was isolated from the kidneys of male NMRI-mice by isoelectrofocusing and refocusing followed by repeated molecular sieve chromography. The overall purification was approx. 250 fold and each of the three bands was isolated separately. The band of the triplet nearest to the cathode, III-50, changed in vitro into the satellite bands III-40 and III-30 and, further, into the band III-22 not observed before in the homogenate. It is assumed that the band III-50 represents the original gene product. The molecular weight (45 000) of the band III-50 is identical with those of III-40 and III-30, as measured by analytical electrophoresis, whereas the molecular weight obtained by thin-layer chromatography was 51 000. There were no obvious signs that esterase-9 was composed of subunits. The Km constant for 4-nitrophenyl proprionate was identical for each of three bands. The esterase-9A is the first testosterone-dependent isozyme of the mouse carboxylesterase (carboxylicester hydrolase, EC 3.1.1.1) system which has been isolated.

Nonspecific esterases of mammalian testis

SummaryTen different nonspecific esterases in both mouse (Mus musculus) and rat (Rattus norvegicus) testis were identified following the analysis of electrophoretic patterns using genetic, developmental, and biochemical criteria. None of the enzymes were unique to testis, although the pattern of activity was testis specific. The enzymes comprised, in each species, six carboxylesterases (EC 3.1.1.1), one arylesterase (EC 3.1.1.2), one acetylesterase (EC 3.1.1.6), and two butyrylesterases (tentative designation). Cholinesterase (EC 3.1.1.8) was not detected. Individual homology relationships were recognized between the two species for all of these activities, except three of the carboxylesterases; however, these were coded for by homologous gene clusters. Similarities between the two species extended to the developmental course of expression and the modulation of the pattern of activity by the testicular feminization (Tfm) mutation. We describe the effects of the sex reversal (Sxr) mutation in the mouse, as well as the distribution of individual activities between Leydig cells and seminiferous tubules. The results of earlier histochemical studies are interpreted in the light of the present investigation. The correspondence between mouse- and rat-testis esterases suggests that the results could serve as a basis for mammalian testis esterase systems in general.

Immunochemical interrelationships between carboxylesterase isozymes (EC 3.1.1.1) of the house mouse, Mus musculus

Abstract 1. 1. Fourteen carboxylesterase isozymes of Mus musculus fell into two immunologically distinct groups. Six isozymes, coded for by esterase gene cluster 1 (ES-1, ES-6, ES-9, ES-20, ES-22, ES-24) all formed immunoprecipitates with antisera raised against purified ES-1, ES-6, ES-9 and ES-20. Eight cluster 2-coded isozymes (ES-2, ES-5, ES-7, ES-11, ES-23 and three hitherto genetically undefined serum esterases, SE-I, SE-II and SE-III) formed immunoprecipitates with anti-ES-2. 2. 2. The isozymes within each cluster were thus apparently more closely related to one another phylogenetically than to isozymes from the other cluster—a property not evident from their biochemical characteristics. 3. 3. It is proposed that the two carboxylesterase gene clusters on chromosome 8 each formed a multigene family.

Organ specific expression of esterase-6 in the house mouse, Mus musculus

SummaryEsterase-6 in fresh homogenates of heart muscle and testis of the house mouse shows a two band (C allele) or three band (A allele) pattern in disc electrophoresis. These primary bands generated a series of secondary bands upon lowering the pH of the homogenates, and the secondary pattern, possibly resulting from partial proteolysis, was seen in varying degrees in fresh homogenates from a range of organs. Interrelationships between the primary and secondary bands were demonstrated by isoelectric focusing. The esterase-6 content of twenty different organ homogenates was estimated from electrophoretic gels, and a high level of this enzyme was observed in those organs most actively involved in fat metabolism. The possible participation of esterase-6 in fatty acid utilization is discussed. Similarities between esterase-6 of the house mouse and esterase-4 of the rat were demonstrated, further strengthening the view that these enzymes are homologous.

Esterases in inbred strains of mice with differential cholesterolemic responses to a high-cholesterol diet.

Specific esterase isoenzyme patterns in plasma may be associated with responsiveness of serum cholesterol to dietary cholesterol. In rabbits and rats the presence and absence of a high-mobility, anodal esterase band on electrophoresis have been shown to be associated with hypo- and hyperresponsiveness, respectively. We fed for 28 days male mice of 7 inbred strains either a low-cholesterol, commercial diet or a diet containing 2% (w/w) cholesterol, 0.5% cholic acid and 5% olive oil. Feeding the high-cholesterol diet revealed marked inter-strain differences in the responses of plasma and liver cholesterol; the increases ranged from 21 to 129% and from 10 to 80-fold, respectively. There was no association between esterase isoenzyme patterns in plasma and the sensitivity to the high-cholesterol diet. The mean baseline plasma total esterase activity tended to be positively associated with the absolute response of plasma cholesterol to the high-cholesterol diet (r = 0.56; n = 7), but the positive relationship between the baseline concentration of the ES-1 component in plasma and the cholesterolemic response was stronger (r = 0.84; n = 7; P less than 0.05). The high-cholesterol diet caused a significant increase in plasma total esterase activities in 6 out of the 7 strains. Evidence is presented that the increase in plasma total esterase activity, which was associated with an increase in the activity and concentration of the so-called ES-2 isoenzyme, is the result of an enhanced release of esterases from the intestine, rather than from the liver. A significant, positive correlation was found between the baseline intestinal esterase activity and the cholesterolemic response after cholesterol feeding (r = 0.83; n = 7; P less than 0.05).

Biochemistry and genetics of esterase-20 (ES-20), a second trimeric carboxylesterase of the house mouse (Mus musculus). I. Purification and characterization of ES-20C1 from male kidney

ES-201 was isolated from male mouse kidney and purified 350-fold by ion-exchange chromatography, isoelectric focusing, and gel filtration. The resultant product was apparently homogeneous by the criteria of polyacrylamide gel electrophoresis and immunodiffusion and represented a major fraction of male mouse kidney esterase. Sodium dodecyl sulfate gel electrophoresis revealed the presence of a single subunit band, molecular weight 59,500; the molecular weight of the native protein was found to be 179,000. Titration of the active site yielded an equivalent weight of about 175,000. The enzyme was further characterized by its kinetic parameters for the hydrolysis of a series of 4-nitrophenyl esters and was classified as a carboxylesterase (EC 3.1.1.1). ES-20C1 bound to concanavalin A, indicating that it was a high-mannose-type glycoprotein; the role of terminal β-N-acetylglucosamine residues in the carbohydrate side chains for stabilization of the quaternary structure of the trimer was revealed. Extensive biochemical and immunological similarities to ES-9C supported an earlier suggestion that theEs-9c gene product is a component of the ES-20C1 trimer.

Lymph esterases of the house mouse (Mus musculus) — II. The role of esterase-2 in fat resorption☆

1. Intralipid infusion into the duodenum of Mus musculus was accompanied by changes in lymph and serum concentrations of two esterase isozymes, ES-1 and ES-2. Whereas ES-1 levels declined in both lymph and serum, ES-2 levels increased 5-fold in lymph within 120 min, and fell to a plateau 3- to 4-fold the fasting level; serum levels of ES-2 increased continually. 2. The changes in lymph ES-2 concentrations were paralleled by lymph triglyceride concentration during Intralipid infusion. Genetically determined differences in the concentration of two allozymes, ES-2B and ES-2D, were reflected in differences in lymph triglyceride levels. The lymph triglyceride concentration was strongly correlated with approximately the cube root of the lymph ES-2 concentration for both allozymes. 3. The source of lymph ES-2 during fat resorption was probably an intracellular jejunal pool; serum ES-2 also re-entered the lymph but this fraction was not influenced by fat resorption. 4. Purified chylomicrons possessed no esterase activity; however, it was postulated that ES-2 plays an essential role in fat resorption and is extruded with the primary chylomicrons from the enterocyte.

Esterase-30 (ES-30) of the house mouse: Biochemical characterization and genetics of a new carboxylesterase isozyme linked to cluster-2 loci on chromosome 8

A new carboxylesterase isozyme (EC 3.1.1.1), designated ES-30, is described in mouse liver. Two phenotypes were distinguished, ES-30A, a possible null type, was found in SPE/Pas and in other lines derived fromMus spretus, and ES-30B was found in BALB/cJ and other laboratory inbred strains. ES-30B is characterized by a distinct electrophoretic band when stained using 5-bromoindoxyl acetate as the substrate. After isolation and purification from other esterases by ion-exchange chromatography and molecular sieving, the molecular mass was estimated by two independent methods to be 62 and 64 kDa, respectively. The activity of ES-30B is higher in adult males than in females and can be stimulatedin vivo by testosterone. The distribution of phenotypes on the progeny of a backcross series suggests a separate locus,Es-30, with the allele a for absence andb for presence of the isozyme. LocusEs-30 is shown to be closely linked toEs-2 and toEs-7 of cluster-2 on chromosome 8. The gene orderEs-9—Got-2—(Es-2, Es-7, Es-30) is suggested.