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.     

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.     

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.     

Rat carboxylesterase ES-4 enzyme functions as a major hepatic neutral cholesteryl ester hydrolase

Although esterification of free cholesterol to cholesteryl ester in the liver is known to be catalyzed by the enzyme acyl-coenzyme A:cholesterol acyltransferase, ACAT, the neutral cholesteryl ester hydrolase (nCEH) that catalyzes the reverse reaction has remained elusive. Because cholesterol undergoes continuous cycling between free and esterified forms, the steady-state concentrations in the liver of the two species and their metabolic availability for pathways, such as lipoprotein assembly and bile acid synthesis, depend upon nCEH activity. On the basis of the general characteristics of the family of rat carboxylesterases, we hypothesized that one member, ES-4, was a promising candidate as a hepatic nCEH. Using under- and overexpression approaches, we provide multiple lines of evidence that establish ES-4 as a bona fide endogenous nCEH that can account for the majority of cholesteryl ester hydrolysis in transformed rat hepatic cells and primary rat hepatocytes.     

Simultaneous purification and comparative characterization of six serine hydrolases from rat liver microsomes

Rat liver microsomes contain many serine hydrolases, which can be demonstrated in electropherograms with carboxylesterase stain and with an active-site-directed radioactive organophosphate. Five of the most prominent of these enzymes plus dipeptidyl aminopeptidase IV, a microsomal serine hydrolase without activity against simple esters, have been highly purified with a simultaneous procedure after solubilization with saponin. The five carboxylesterases belong to at least three groups of chemically different proteins. Terminal amino acids, amino acid composition, and substrate specificity are different, while the subunit molecular weight of all esterases is very similar (about 60,000). All purified carboxylesterases have monooleylglycerol-cleaving capacity. The subunit weight (84,000) and the N-terminal amino acid (serine) of the peptidase differ from those of all isolated carboxylesterases. The data are correlated to other reports on individual serine hydrolases from rat liver.     

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.     

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.     

Biochemical genetics of Es-14 (formerly Es-Si) and a new esterase variation,Es-15, of the laboratory rat (Rattus norvegicus): Biochemistry,tissue expression,and linkage to Es-1 in linkage group V

The segregation of rat esterases controlled by loci residing in linkage group V (LGV) has been studied in two backcross series, (LEW/Han × BN/Han)F₁ × LEW/Han and (LEW/Han × LE/Han)F₁ × LEW/Han. Es-14 (formerly Es-Si) was shown to be closely linked to Es-1. A new esterase locus, Es-15, was described which codes for a liver isozyme. The distribution pattern of three alleles at the Es-15 locus is presented for 52 independent inbred strains. Close linkage of Es-15 to Es-14 and to Es-1 was established, proposing the following gene order: [Es-2, Es-10]—[ES-1, ES-14, ES-15]. The esterase loci on LGV are thus separated into two gene clusters, cluster 1 and cluster 2. These conclusions are supported by the strain distribution patterns of the two RI strain series, LXB and DXE.Otto von Deimling was supported by the Deutsche Forschungsgemeinschaft (De 315/2-1, communication No. 56).     

The tumor promoter 12-O-tetradecanoyl phorbol 13-acetate and regulatory diacylglycerols are substrates for the same carboxylesterase

Rat liver homogenate or cell fractions deacylate 12-O-tetradecanoyl phorbol 13-acetate (TPA) in vitro mainly by conversion to phorbol 13-acetate. The highest specific activity is located in the microsomal fraction. The deacylation is inhibited by bis-(4-nitrophenyl) phosphate, a selective inhibitor of nonspecific carboxylesterases. Only two of five purified esterases from rat liver endoplasmic reticulum deacylate TPA. These two esterases have formerly been characterized as acylcarnitine hydrolases and the more active one is also a potent diacylglycerol lipase. Its TPA-hydrolyzing activity is inhibited by other substrates like 1-naphthylacetate, lauroylcarnitine, or dioleoyl glycerol. The results support the view that phorbol esters act like structural analogs of diacylglycerols, not only with respect to their activating effect on protein kinase C, but also as substrates for the same lipases.     

Heterogeneity and heat resistance of esterases of the pancreas and liver of rats]

A higher heat resistance of loosely-attached microsomal esterases as compared with that of free esterases of the rat's pancreas is due to a higher proportion of heat resistant slow carboxylesterases in the former. More significant differences in the heat resistance of pancreas and liver is caused not only by different heterogeneity of esterases, but also by their qualitative differences.     

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.     

Detoxication of paraoxon by rat liver homogenate and serum carboxylesterases and A-esterases.

Paraoxon, the active metabolite of parathion, can be detoxified through a noncatalytic pathway by carboxylesterases and a catalytic pathway by calcium-dependent A-esterases, producing p-nitrophenol as a common metabolite. The detoxication patterns of carboxylesterases and A-esterases were investigated in vitro in the present study with a high tissue concentration (75 mg/mL rat liver homogenate or 50% rat serum solution) to more closely reflect enzyme concentrations in intact tissues. A final paraoxon concentration of 3.75 microM was used to incubate with liver homogenates or serum solutions for 5 seconds or 3, 5, 15, or 25 minutes; also 0.625, 1.25, 2.5, 3.125, 3.75, or 5.0 microM paraoxon (final concentration) was incubated with liver homogenates or serum solutions for 15 minutes. Phenyl saligenin cyclic phosphate and EDTA were used to inhibit carboxylesterases and A-esterases, respectively. Significant amounts of p-nitrophenol were generated with or without either inhibitor during a 15 minute incubation with paraoxon from low (0.625 microM) to high (5.0 microM) concentrations. The amount of p-nitrophenol generated via carboxylesterase phosphorylation was greater than via A-esterase-mediated hydrolysis in the initial period of incubation or when incubating with a low concentration of paraoxon. Plateau shape curves of p-nitrophenol concentration versus time or paraoxon concentration indicated that carboxylesterase phosphorylation was saturable. When incubated for long time intervals or with high concentrations of paraoxon, more p-nitrophenol was generated via A-esterase-mediated hydrolysis than from carboxylesterase phosphorylation. The ratio of paraoxon concentration to tissue amount used in in vitro assays of this study was equivalent to dosing a rat with toxicologically relevant dosages. These in vitro data suggest that both carboxylesterases and A-esterases detoxify paraoxon in vivo; carboxylesterases may be an important mode of paraoxon detoxication in initial exposures to paraoxon or parathion before they become saturated, whereas A-esterases may contribute to paraoxon detoxication in repeated exposures to paraoxon or parathion because they will not become inhibited and will remain catalytically active unlike the carboxylesterases. The importance of carboxylesterases in detoxication of paraoxon was verified by an in vivo study. In rats pretreated with tri-o-tolyl phosphate, an in vivo carboxylesterase inhibitor, brain acetylcholinesterase was significantly inhibited after intravenous exposure to parathion. No significant inhibition of brain acetylcholinesterase was observed in rats pretreated with corn oil.     

Physicochemical characterization and tissue distribution of esterases in two salamandridae species (Mertensiella luschani and Salamandra salamandra)

1. Tissue- and species-specificity of the electrophoretic patterns of the multiple molecular forms of esterases were observed in the urodele amphibians Mertensiella luschani luschani, M.l. helverseni and Salamandra salamandra. All esterases--distributed into two electrophoretic mobility areas in gonads, muscles and brain and into four areas in liver, stomach and intestine--were characterized as carboxylesterases. 2. M. l. luschani and S. salamandra liver esterases were electrofocused into nine and eleven major bands with pIs ranging from 4.60 to 5.65 and from 4.40 to 6.20, respectively. 3. Two size groups of esterases were observed in liver extracts of the above three subspecies by Sephadex G-200 gel filtration. The mean values of their apparent molecular weights were 70,000 and 230,000 respectively.     

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. This work was supported by the Deutsche Forschungsgemeinschaft. This is communication No. 72 of a research program devoted to the cellular distribution, genetics, and regulation of nonspecific esterases.     

Physicochemical characterization and tissue distribution of multiple molecular forms of fish (Trachurus trachurus) esterases

1. Tissue-specific electrophoretic patterns of multiple molecular forms of esterases were observed in fish Trachurus trachurus. They were composed of three (in intestine) to nine (in liver) bands characterized mainly as carboxylesterases and acetylesterases. 2. Two major esterase activity bands of pI 4.60 and 4.77 were accompanied by minor ones of higher pH in tissue extracts. 3. Under optimum assay conditions, liver was the richest source of esterase activity followed by intestine, stomach, brain, red muscles, heart, white muscles and gills. 4. Esterases of 70,000 and 420,000 mol. wt were resolved by gel-filtration in liver, intestine and brain. Low size esterases prevailed in liver while the opposite was the case in brain.     

Chromatographic study on the specificity of bis-p-nitrophenylphosphate in vivo. Identification of labelled proteins of rat liver after intravenous injection of bis-p-nitro[14C]phenylphosphate as carboxylesterases and amidases

The procedure established to isolate the carboxylesterases E1, E2 and EA from rat liver (Arndt, R. and Krisch, K. (1972) Hoppe-Seyler's Z. Physiol. Chem. 353, 589-598) was applied to characterize in vivo bis-p-nitro[14C]-phenyl-P-labelled proteins. The peaks of radioactivity and of residual enzyme activities (hydrolysing methylbutyrate, p-nitrophenylacetate and acetanilide) were found in the same peaks after column chromatography and could be related to the well-defined esterases E1, E2 and EA. There is no indication of a nonspecific binding of bis-p-nitrophenyl-P or of one of its metabolites. The relative quantitative amounts of E1, E2 and EA were calculated to represent 40, 14 and 46%, respectively, of the total carboxylesterase content of rat liver. The relative amount of bound (not dialysable) radioactivity in rat liver depended on the survival time. During purification, the yield of enzyme activities corresponded to that of bound radioactivity, confirming the specificity of bis-p-nitrophenyl-P in vivo. Hence the radioactive metabolites of the inhibitor obviously do not possess binding affinities of quantitative importance to the rat liver proteins.     

Nonspecific esterases of mammalian testis

Summary Ten 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.This is communication no. 47 of a research program devoted to the cellular distribution, genetics, and regulation of nonspeific esterases. This work was supported by the Dcutsche Forschungsgemeinschaft     

Is histidine involved in the catalytic mechanism of unspecific carboxylesterases?

The reaction between the liver carboxylesterases from ox and pig and the inhibitor α-bromoacetophenone was studied by amino acid analysis. A significant modification of histidine in pig liver esterase was not found, but there was a slight loss of some other residues. In ox liver esterase the total inhibition correlated with the loss of about 1.7 histidine residues. However, in contrast to previous results with chicken and ox esterases the specific active-site-directed inhibitor E 600 did not prevent the modification of the reactive histidine. It is concluded that an earlier report on the involvement of histidine in the action of liver esterases (1) is partly incorrect or perhaps applicable only to chicken liver esterase.     

Enantioselective hydrolysis of oxazepam 3-acetate by esterases in human and rat liver microsomes and rat brain S9 fraction

Rates of hydrolysis of racemic and enantiomeric oxazepam 3-acetates (OXA) by esterases in human and rat liver microsomes and rat brain S9 fraction were compared. When rac-OXA was the substrate, esterases in human and rat liver microsomes were highly enantioselective toward (R)-OXA. In contrast, esterases in rat brain S9 fraction were highly enantioselective toward (S)-OXA. Hydrolysis rates of rac-OXA were highly dependent on the amount of esterases used. At 0.05 mg protein equivalent of esterases and 150 nmol of rac-OXA per ml of incubation mixture, the (R)-OXA was hydrolyzed 3.6-fold and 18.5-fold faster than (S)-OXA by rat and human liver microsomes, respectively. The specific activities (nmol of OXA hydrolyzed/mg microsomal protein/min) of liver microsomes in the hydrolysis of enantiomerically pure (R)-OXA were approximately 120 (rat) and 1,980 (human), and in the hydrolysis of enantiomerically pure (S)-OXA were 4 (rat) and 7 (human), respectively. In the incubation of rac-OXA with rat brain S9 fraction, (S)-OXA was hydrolyzed approximately 6-fold faster than (R)-OXA. Results also indicated an enantiomeric interaction in the hydrolysis of rac-OXA by esterases in rat and human liver microsomes; the presence of (R)-OXA stimulated the hydrolysis of (S)-OXA, whereas the presence of (S)-OXA inhibited the hydrolysis of (R)-OXA. In rat brain S9 fraction, the presence of (R)-OXA inhibited the hydrolysis of (S)-OXA, whereas the presence of (S)-OXA appeared to have stimulated the hydrolysis of (R)-OXA.