Genetically defined lysosomal acetylesterase EC 3.1.1.6 in the cauda epididymidis of mouse,rat, and man

After inhibition by bis-p-nitrophenyl phosphate and subsequent staining for esterase using naphthol AS-D acetate as the substrate, a strong lysosomal esterase was demonstrated in the cauda epididymidis of mouse, rat, and man. Owing to its behaviour towards the classifying inhibitors eserine, diisopropyl fluorophosphate, bis-p-nitrophenyl phosphate, and p-chloromercuriphenylsulphonate, this lysosomal esterase was shown to be an acetylesterase (EC 3.1.1.6). Control experiments involving isoelectric focusing revealed that this acetylesterase was identical with the genetically defined homologues ES-17, ES-6, and ES-A4 in mouse, rat, and man, respectively.     

Esterase-18 (ES-18) of the house mouse (Mus musculus): Biochemical characterization and genetics of an allozyme system linked to chromosome 19

This study describes the biochemical characterization, genetic variation, and linkage of a codominantly inherited murine esterase, termed ES-18. The enzyme was identified by isoelectric focusing of supernatants obtained after centrifugation of tissue homogenates and subsequent staining for esterase using either alpha-naphthyl acetate or 4-methylumbelliferyl elaidate as substrate. ES-18 exhibited an organ-specific variation of the intensity pattern of bands as seen in kidney, spleen, and macrophages, respectively. Its activity was highly sensitive to inhibition by 1 mmol.liter-1 p-chloromercuriphenylsulfonate but was resistant to bis-p-nitrophenyl phosphate. Four allozymes could be distinguished in kidney supernatants obtained from the inbred strains C57BL/10Sn (ES-18A), MOLF/Ei (ES-18B), WLL/BrA (ES-18C), and CAST/Ei (ES-18D). The enzyme is shown to be controlled by a structural locus, Es-18, which resides on chromosome 19. The gene order Ly-1 - Got-1 - 4.7 +/- 1.6 - Es-18 is suggested.     

Egasyn, a protein which determines the subcellular distribution of beta-glucuronidase, has esterase activity

The glycoprotein egasyn complexes with and stabilizes precursor beta-glucuronidase in microsomes of several mouse organs. Several observations indicate egasyn is, in addition, an esterase. Liver homogenates of egasyn-positive strains have specific electrophoretically separable esterases which are absent in egasyn-negative mice. These esterases react with anti-egasyn serum. A specific esterase was likewise complexed with immunopurified microsomal beta-glucuronidase. The esterases were, like egasyn and microsomal beta-glucuronidase, concentrated in the microsomal subcellular fraction. Egasyn which is not bound to beta-glucuronidase, which represents 80-90% of total liver egasyn, is not complexed with other liver proteins. Egasyn, therefore, specifically stabilizes beta-glucuronidase in microsomes. The esterase activity is inhibited by bis-p-nitrophenyl phosphate indicating it is a carboxyl esterase. Several possible functions of egasyn-esterase activity are discussed.     

Genetic variation as a tool for histochemical localization of a nonspecific esterase

A prominent esterase activity was demonstrated histochemically in the straight portion of the proximal tubules in kidney of the mouse strain DBA/2J after inhibition with bis-p-nitrophenyl phosphate and subsequent staining, using 5-bromoindoxyl acetate as substrate. In the strain PUC/1Fre, the corresponding esterase was only weakly expressed. By comparing data from the literature (von Deimling et al. 1981) with the characteristic features of this kidney esterase including substrate preference, sensitivity to inhibitors, solubility, histochemical location, and strain differences, it was concluded that it was identical with the previously electrophoretically defined esterase-16.     

Lymph esterases of the house mouse (Mus musculus)--I. Identification and possible origins of abdominal lymph esterases

1. Abdominal lymph was obtained from Mus musculus by cannulation of the thoracic duct: lymph esterases were identified by polyacrylamide gel electrophoresis. Seven known esterases (ES-1, ES-2, ES-5, ES-27, SE-I, SE-II and SE-III) and a newly described activity (SE-IV) were demonstrated, all of which were also present in serum. 2. Electrophoretic staining intensities indicated that the lymph esterases were less concentrated than the corresponding activities in serum, with the single exception of ES-2. This finding was supported by quantitative immunoelectrophoresis of ES-1 and ES-2 (two allozymes each). 3. The jejunum appeared to be the origin of lymph ES-2 by a comparison of organ distribution of the allozymes ES-2B and ES-2D and by monitoring the re-appearance of ES-2 in several organs, serum and lymph after total inhibition in vivo by bis-p-nitrophenyl phosphate.     

Subcellular localization of non-specific carboxylesterases, acylcarnitine hydrolase, monoacylglycerol lipase and palmitoyl-CoA hydrolase in rat liver

The subcellular distribution and sidedness on the membranes of four chemically and genetically distinct esterases (esterases ES-3, ES-4, ES-8, ES-15) in rat liver was investigated using selective substrates. (1) Rat liver homogenate was divided into nine subcellular fractions by differential centrifugation techniques. The cell fractions were assayed for the enzymatic hydrolysis of acetanilide (ES-3), propanidid, palmitoyl-CoA and monopalmitoylglycerol (ES-4), methyl butyrate and octanoylglycerol (ES-8), and decanoylcarnitine (ES-15). With all substrates, the highest specific activities were found in the rough and smooth endoplasmic reticulum fractions. This localization of the esterases was confirmed by labelling the cell fractions with the specific, covalently binding inhibitor bis(4-nitro[14C]phenyl) phosphate. The enzymatic hydrolysis of the palmitoyl esters in differing cell fractions did not completely parallel that of propanidid. This confirms the well-known existence of palmitoyl-CoA hydrolases other than esterase ES-4. (2) Density gradient fractionations with crude mitochondria indicated that a low amount of at least one of these carboxylesterases was an integral part of these organelles too. (3) Proteinase treatment reduced the non-specific esterase activities as well as lipase activities versus dioctanoylglycerol, acylcarnitines and palmitoyl-CoA only in detergent-disrupted microsomal vesicles. This might indicate a lumenal orientation of these enzymes. However, of the charged substrates palmitoylcarnitine and palmitoyl-CoA only the latter one showed the typical latency to be expected for a hydrolysis in the lumen of the endoplasmic reticulum.     

Esterase-16 (ES-16) of the rat (Rattus norvegicus): Identification and genetic characterization

Esterase-16, an esterase present in lung and other tissues of the laboratory rat, has been characterized by its biochemical properties (electrophoretic mobility, substrate pattern, sensitivity to inhibitors) and genetic variation in 107 inbred strains and substrains including 14 RI strains. It was classified as a carboxylesterase (EC 3.1.1.1). The phenotype ES-16A (BN/Han and 63 other strains) was defined as a narrow electrophoretic band migrating between ES-1A and ES-13A, ES-16B (LEW/Han and 42 other strains) exhibited the same electrophoretic mobility as ES-16A but was distinguished by its extremely weak activity. Segregation of ES-16 in RI strains and backcrosses indicated linkage to linkage group V (LGV). The Es-16 locus was tentatively placed into esterase cluster 2 and homology with Es-7 of the house mouse is proposed.     

Esterase-13 of the rat (Rattus norvegicus) and its homology with esterase-3 of the house mouse (Mus musculus)

A new allele of esterase-13 was detected in various laboratory inbred strains of Rattus norvegicus and designated Es-13c. The activity of ES-13 towards a range of chromogenic substrates, inhibitor profile, isoelectric points and retardation coefficients on polyacrylamide gel electrophoresis were determined. The organ specific expression of ES-13 alleles was investigated and it was shown that kidney homogenates contained a factor which modified the liver enzyme banding pattern in vitro. The features of ES-13 from the rat indicated homology between this esterase and ES-3 from the house mouse, Mus musculus domesticus.     

Genetic identification of non-specific esterases of the mouse cauda epididymidis and description of esterase-28, a new carboxylesterase isoenzyme (EC 3.1.1.1)

Twenty-five (25) electrophoretic bands with esterase activity were distinguished in supernatants of cauda epididymidis of DBA/2J mice. Twenty (20) of these were assigned to 10 genetically defined esterases (ES-1, ES-2, ES-3, ES-6, ES-7, ES-11, ES-14, ES-17, ES-19, ES-22) which were already known from investigations of other mouse tissues. Furthermore, ES-10 was identified in cauda supernatants after isoelectric focussing. A hitherto genetically undefined esterase was assigned to locus Es-28 which was expressed solely in the epididymis. Three phenotypes were distinguished: ES-28A was present in the majority of the inbred strains examined. ES-28B was observed in AKR/Han mice and ES-28C was found in SEG/1 mice.     

Hydrolysis of retinyl esters by non-specific carboxylesterases from rat liver endoplasmic reticulum.

The four most important non-specific carboxylesterases from rat liver were assayed for their ability to hydrolyse retinyl esters. Only the esterases with pI 6.2 and 6.4 (= esterase ES-4) are able to hydrolyse retinyl palmitate. Their specific activities strongly depend on the emulsifier used (maximum rate: 440 nmol of retinol liberated/h per mg of esterase). Beside retinyl palmitate, these esterases cleave palmitoyl-CoA and monoacylglycerols with much higher rates, as well as certain drugs (e.g. aspirin and propanidid). However, no transacylation between palmitoyl-CoA and retinol occurs. Retinyl acetate also is a substrate for the above esterases and for another one with pI 5.6 (= esterase ES-3). Again the emulsifier influences the hydrolysis by these esterases (maximum rates: 475 nmol/h per mg for ES-4 and 200 nmol/h per mg for ES-3). Differential centrifugation of rat liver homogenate reveals that retinyl palmitate hydrolase activity is highly enriched in the plasma membranes, but only moderately so in the endoplasmic reticulum, where the investigated esterases are located. Since the latter activity can be largely inhibited with the selective esterase inhibitor bis-(4-nitrophenyl) phosphate, it is concluded that the esterases with pI 6.2 and 6.4 (ES-4) represent the main retinyl palmitate hydrolase of rat liver endoplasmic reticulum. In view of this cellular localization, the enzyme could possibly be involved in the mobilization of retinol from the vitamin A esters stored in the liver. However, preliminary experiments in vivo have failed to demonstrate such a biological function.     

Purification and molecular properties of rabbit liver esterase ES-1A

1. The isolation of esterase ES-1A from rabbit liver microsomes/lysosomes is reported. The purification as measured by methylbutyrate-hydrolysing activity, was about 27-fold with a recovery of 2.4%. 2. The resulting product is apparently homogeneous by polyacrylamide (gradient) gel electrophoresis and sodium dodecyl sulphate/polyacrylamide gradient gel electrophoresis after protein staining. The enzyme exhibits heterogeneity after staining for esterase activity and in isoelectric focusing. 3. The molecular mass of the native protein was found to be about 183 kDa (determined by gel filtration and polyacrylamide gel electrophoresis) with a subunit mass of about 63 kDa, indicating a trimeric structure of the enzyme, with subunits of equal size. 4. ES-1A is a glycoprotein and is classified as a carboxylesterase (EC 3.1.1.1). 5. The high degree of similarity of the properties of rabbit ES-1A with those of mouse ES-6A and rat ES-10 suggests that these three esterases may have a common evolutionary origin.     

Identification and development of a genetically closely-linked carboxylesterase family of the mouse liver

Six carboxylesterase isozymes (viz. ES-1, ES-6, ES-9, ES-20, ES-22 and ES-24), governed by esterase gene cluster 1 on chromosome 8 of the house mouse, were identified electrophoretically in liver supernatants using their biochemical, genetic and developmental characteristics. ES-1 and ES-20 were expressed as liver-specific forms. The peri- and postnatal development of the six isozymes indicated that they were individually regulated at the genetic level, although the isozymes were regulated as a group when compared to genetically unrelated esterases. The concept of evolutionary divergence following repeated gene duplication of an ancestral esterase structural gene was extended to cover divergence of the temporal (regulatory) genes associated with the multigene family. Allelic variation of the temporal genes was more limited than that of the corresponding structural genes.     

A new variant of the Es-4 locus in the rat

A new variant of kidney esterase in the DK/Nac rat strain is reported. The new esterase was tentatively named ES-4C determined by a third allele of the Es-4 locus of Linkage Group V (LGV). Strain distribution was surveyed using 17 inbred strains, but no strain except for the DK/Nac strain possessed the ES-4C type. Although we surveyed outbred stocks (Jcl: Wistar and Jcl: SD) we could not find rats carrying the ES-4C type. Genetic analysis of the ES-4C type was carried out using mating experiments between DK/Nac and BUF/Nac (ES-4B). The results indicated that the new variant was controlled by the Es-4 locus and it was named the Es-4c allele.     

Biochemistry and genetics of esterase-20 (ES-20), a second trimeric carboxylesterase of the house mouse (mus musculus). II. A unique recombination reveals ES-20 as a hybrid enzyme

A unique recombination is described between (Es-1, Es-6) and (Es-9, Es-22) within gene cluster 1 of the esterase gene region on chromosome 8 of the house mouse. This recombination established the gene order Es-1--Es-6--(Es-9, Es-22)--Got-2. A further 73 recombinations, from a total of 911 backcrosses, had taken place between cluster 1 and cluster 2. A distance between the clusters of 8.01 +/- 0.90% was calculated; the genes within the clusters appeared more tightly linked than previously assumed. ES-20 behaved anomalously following the recombination within cluster 1: homozygous descendants of the recombinant expressed a new form of ES-20. In vitro incubation of purified ES-6A3 and ES-9A generated ES-20A, revealing this esterase to be a hybrid of different cluster 1 gene products, Es-9 and possibly Es-6. This result satisfactorily accounted for the genetic finding, as well as a range of biochemical properties of ES-20.     

Esterase-25(Es-25): Identification and characterization of a new kidney arylesterase of the house mouse,genetically linked toLy-18 on chromosome 12

Genetic variation of a codominantly inherited kidney esterase, designated ES-25, has been discovered in the house mouse using disc electrophoresis. The ES-25A phenotype was found in A strains, AKR, and BALB/c. ES-25B was found in C57BL strains and several other laboratory strains. The enzyme was shown to be controlled by a presumed structural locus, Es-25. The high concordance in 48 RI strains of Es-25 with Ly-18 indicated the location of Es-25 on chromosome 12. The gene order Es-25-Ly-18-D12Nyul-Pre-1 was proposed.     

Polymorphism of esterase 11 in Mus musculus,a further esterase locus on chromosome 8

A further esterase, esterase 11, which exhibits a polymorphism detectable by electrophoresis, has been observed in the house mouse, Mus musculus. In 15 inbred strains and two outbred strains, the ES-11A phenotype has been found, composed of two bands of enzyme activity of greater anodal electrophoretic mobility than the two bands of the ES-11B phenotype found in one inbred strain, one wild stock, and 101 wild mice. In F₁ hybrids (IS/Cam×C57 BL/Gr), the phenotype shown corresponds to a mixture of the two parental phenotypes. In backcrosses, ES-11 segregates as an autosomal gene, designated Es-11, closely linked to Es-2 and Es-5 on chromosome 8.This work was supported by the Medical Research Council.     

Esterase-23 (ES-23): Characterization of a new carboxylesterase isozyme (EC 3.1.1.1) of the house mouse,genetically linked to ES-2 on chromosome 8

Genetic variation of a carboxylesterase isozyme (EC 3.1.1.1) of the house mouse, designated ES-23, is described. ES-23 was found in kidney, liver, and intestine. The isozyme was resistant to inhibition by 10(-3) mol/liter eserine and was stained using alpha-naphthyl butyrate or 5-bromoindoxyl acetate as substrate. Five different phenotypes, ES-23A to ES-23E, could be distinguished by disc electrophoresis and by isoelectric focusing. ES-23 is controlled by a structural locus situated within the esterase gene cluster 2 on chromosome 8. An analysis of allele distribution among different strains suggested a separate structural locus for the isozyme, Es-23e, which is closely linked to the loci Es-2, Es-5, Es-7, and Es-11. Of the five phenotypes, only ES-23B was expressed in lung. This variation is apparently controlled by a cis-acting regulatory element, presumably a temporal locus, Es-23t, closely linked to the presumed structural locus Es-23e.     

Esterase-26 (ES-26): Characterization and genetic location on chromosome 3 of an eserine-sensitive esterase of the house mouse (Mus musculus)

Genetic variation of a codominantly inherited esterase, designated ES-26, has been discovered in the house mouse using isoelectric focusing in polyacrylamide gels. The ES-26A phenotype (pI 8.2) was found in C57BL/10Sn. A/J showed the ES-26B phenotype (pI 7.8-7.9). A third phenotype, ES-26C (double-banded: pI's 8.1 and 8.3), was observed in SJL/J. ES-26 was detected only in liver, stomach, and small intestine. The enzyme was shown to be controlled by the presumed structural locus Es-26, located on chromosome 3. From a four-point cross, the gene order Car-2--6.2 +/- 2.7--Es-16--21.0 +/- 4.5--Es-26--13.6 +/- 3.8--Amy-1 was established.     

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.     

Polymorphism of esterase-10 in Mus musculus

An esterase, esterase-10, in the house mouse, Mus musculus, is specific for esters of 4-methylumbelliferone and exhibits a polymorphism detectable by electrophoresis. Fifteen inbred strains and two outbred strains have been examined for this polymorphism, and two phenotypes, ES-10A and ES-10B, have been observed. Each phenotype manifests itself as a single band of enzyme activity, but under the electrophoretic conditions used the ES-10A phenotype has less anodal electrophoretic mobility than the ES-10B phenotype. In F₁ hybrids (C3H/He/Lac×C57BL/Gr) a third phenotype was observed, ES-10AB, consisting of three bands of enzyme activity, two of which correspond to the parental forms and the third with intermediate mobility. The triple-band pattern in the F₁ hybrids indicates that esterase-10 is a dimeric enzyme protein.This work was supported by the Medical Research Council.