Enantiotopic Selectivity of-Pig Liver Esterase Isoenzymes

Pig liver esterase was separated into isoenzyme fractions with known subunit compositions. The fractions showed differences in enantiotopic ester group selectivity in hydrolysis of two substrates of synthetic value, benzylmethylpropanedioic acid dimethyl ester and cis-N-benzyl-2,5-bismethoxy-carbonylpyrrolidine. A difference in aliphatic chain length specificity between the isoenzyme fractions was also observed. The results indicate that pig liver esterase cannot be regarded as homogeneous when used in organic synthesis.     

Diurnal variations of rat liver enzymes catalyzing cholesterol ester hydrolysis

Cholesterol ester hydrolase activity was determined at 3 h time intervals over 24 h in lysosomes, cytosol and microsomes from ad libitum-fed and 24 h food-deprived female rat liver. Diurnal rhythms were identified for the acid and neutral esterases, which were strikingly changed by fasting. In fed animals, lysosomal esterase specific activity exhibited a peak at noon and a sustained medium rate at early darkness, whereas total esterase was maximal at midnight. The circadian patterns of the cytosolic and the microsomal esterases paralleled each other, though the amplitude of rhythms differed, showing higher activities around midnight. After fasting, cholesterol esterase activity from all cell fractions reached a maximum near dark onset. These results are the first to indicate that cholesteryl ester hydrolysis may play a role in generating the diurnal rhythm of hepatic cholesterol.     

Topological studies on rat liver microsomal cholesterol ester hydrolase

Lateral and transversal distribution of cholesterol ester hydrolase activity in rat liver microsomal membranes has been studied. Total cholesterol ester hydrolase activity was found predominantly (75%) in rough microsomes though specific esterase activities were similar in rough and smooth microsomal fractions. The transversal asymmetry of the enzyme was examined using the criteria of protease sensitivity and latency of mannose-6-phosphate phosphatase. Cholesterol ester hydrolase resulted drastically inhibited by proteolysis with trypsin when microsomal integrity had been previously disrupted with sodium deoxycholate or sodium taurocholate. Under these conditions, most lumenal mannose-6-phosphate phosphatase activity was destroyed. However, cholesterol esterase was unaffected by preincubating microsomes with the detergent alone, which led to the complete expression of latent mannose-6-phosphate phosphatase or by preincubating them with trypsin, where less than a 15% of the lumenal mannose-6-phosphate phosphatase was lost. These findings suggest that cholesterol ester hydrolase activity is located on the lumenal surface of the hepatic microsomal vesicles.     

Some properties of site-specific mutants of human carbonic anhydrase II having active-site residues characterizing carbonic anhydrase III

Four amino acid residues, His64, Asn67, Leu198 and Val207, in the active site of human carbonic anhydrase II, have been replaced by Lys64, Arg67, Phe198 and Ile207, which are characteristic for the muscle-specific, low-activity isoenzyme form, carbonic anhydrase III. The aim of the investigation has been to test if any of these residues, or a combination of them, is important for the low CO2 hydration activity, low esterase activity, low pKa for the pH/rate profile and low affinity for sulfonamide inhibitors characterizing carbonic anhydrases III. However, no evidence for such critical roles was found. A combination of Lys64 and Arg67 appears to result in a decrease in CO2 hydration activity, but even the quadruple mutant having all four changes is only eight times less active (kcat/Km) than unmodified isoenzyme II, in contrast to isoenzyme III which is nearly 300 times less active than isoenzyme II. The 4-nitrophenyl acetate hydrolase activity of the quadruple mutant is sevenfold lower than that of unmodified isoenzyme II, while the active site of isoenzyme III hardly catalyzes the hydrolysis of this ester at all. The pKa controlling the esterase activity of the quadruple mutant is 6.2, which should be compared to a value of 6.8 for unmodified isoenzyme II, and about 5 for isoenzyme III. While isoenzyme III binds sulfonamide inhibitors 10(3)-10(4) times less strongly than isoenzyme II, only [Asn-67----Arg]isoenzyme II shows a weaker binding of the investigated sulfonamide, dansylamide, but only by a factor of two. Some of the other mutants show enhanced affinities, up to nearly fourfold for the double mutant with Phe198 and Ile207. It is speculated that additional differences between the active sites of isoenzyme II and III might be important for the precise orientations and interactions of the side chains of isoenzyme-III-specific amino acid residues.     

Biochemical variation between non-embryogenic and embryogenic calli of silver fir

Comparative study on SDS-protein profiles and isoenzyme composition of non-embryogenic and embryogenic calli in two callus lines of silver fir (Abies alba Mill.) revealed the presence of abundant polypeptide fractions and an increased number of isoperoxidases in non-embryogenic calli. Non-specific esterase, on the other hand, exhibited an opposite tendency, while glutamate dehydrogenase was the only enzyme system consisting uniformly of one isoenzyme band in both types of calli investigated. This revised version was published online in July 2006 with corrections to the Cover Date.     

白细胞酯酶同工酶对胃癌诊断价值的探讨

本文应用薄平板等电聚焦电泳法对15例未经手术治疗的胃癌患者进行了外周血白细胞酯酶同工酶的分离.结果:正常人外周血白细胞酯酶同工酶显示12条带,等电点(PI)为3.73～7.80.胃癌患者显示11条带,与正常相比第十二条带缺如,PI为3.80～7.90.同工酶各分带与正常相比,酶活性发生明显异常(P<0.01).提示;胃癌患者酯酶同工酶的改变可能是胃癌早期诊断的酶学指标.     

Purification of a membrane-associated serine esterase from murine cytotoxic T lymphocytes by a single reverse-phase column

The plasma and organelle membranes of a cytotoxic T lymphocyte line, CTLL-R8, were isolated by subcellular fractionation. After dissolving in detergent-containing buffer, the membrane proteins were isolated by high-performance liquid chromatography on a single reverse-phase column. The serine esterase activity in the fractions was detected by measuring hydrolysis of the ester compound N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester. A major band was revealed in the fraction with highest serine esterase activity. Under sodium dodecyl sulfate-polyacrylamide gel electrophoresis, this band assumes a molecular weight of about 30 kDa. The amino-terminal sequence of the protein was analyzed and shows 100% identity with that of MCSP-3/granzyme F, a soluble serine esterase previously identified in the cytoplasmic granules of cytotoxic T lymphocytes. Modifications of this reverse-phase column method would thus represent a simple, convenient strategy for obtaining high yields of all the lymphocyte surface proteases, which could then be further characterized for function.     

Oncogenic transformation of 3T3 cells is associated with conversion from an ‘adult’ to an ‘embryonic’ esterase isoenzyme pattern

Soluble esterases from virus-transformed sublines of 3T3 Swiss mouse fibroblasts exhibit an isoenzyme pattern in polyacrylamide gel electrophoresis similar to the pattern exhibited by primary mouse embryo cells but distinct from that exhibited by 3T3 cells. The soluble esterase isoenzyme pattern exhibited by 3T3 cells is similar to that exhibited by primary and secondary fibroblastoid cells derived from adult Swiss mouse kidney, suggesting that, despite its embryonic origin, 3T3 is an ‘adult’ cell line selected and maintained in that state by the requirement that it exhibits a low saturation density and a characteristic morphology in culture. The pattern of soluble esterase isoenzymes is similar in growing and non-growing 3T3 cells, although the specific activity is higher in preparations from non-growing cells. Sparse 3T3 cells contain at least three detergent-soluble esterase isoenzymes present at much lower levels in denser cultures.The esterase and amidase enzyme activities measured in solution with the fluorogenic substrates fluorescein diacetate and rhodamine diacetate, respectively, are substantially higher in three subcellular fractions from virus-transformed 3T3 mouse fibroblasts than in the corresponding fractions from 3T3 mouse fibroblasts or from primary mouse embryo cells. The largest increases in activity associated with viral transformation were observed in membrane-associated esterases.     

ON THE ASSOCIATION OF NON-SPECIFIC ESTERASE ACTIVITY WITH CENTRAL NERVE MYELIN PREPARATIONS

Abstract— Isolated bovine central nerve myelin sheath preparations showed non-specific esterase activity towards naphthyl ester substrates of increasing chain length from acetate to palmitate. Short chain esters were hydrolysed much faster than long chain substrates by myelin, the specific activity for the hydrolysis of β-naphthyl acetate being the highest. Micro-somal fractions from brain white matter were much higher in esterase activity to all naphthyl ester substrates. NADPH-cytochrome c reductase activity was absent from isolated myelin samples. Distilled water and salt and buffer solutions of different ionic strengths and pH were ineffective in releasing non-specific esterase activity from myelin although tri-potassium citrate caused marked inhibition of the membrane-bound esterase activity. The detergent Triton X-100 released esterase activity from the myelin preparations but at a concentration of 0.1 per cent was also inhibitory.     

Bodily heat resistance and polymorphism of liver esterases of grass frogs]

The determination of organismal heat resistance and qualitative composition of polymorphous liver esterases during heat acclimation (25 degrees) has been made on frogs Rana temporaria. During hybernation the most heat resistant frogs possess the homozygous allele of A2 esterase. Heat acclimation and the summer rise in temperature in nature lead to an increase in heat resistance of frogs and to the disappearance of selective advantage of animals possessing the isoenzyme of the A2A2 esterase. The functional homeostasis of populations can maintain biochemical polymorphism regardless of the selective advantage of individuals possessing one of the homozygous alleles of the isoenzyme.     

Protein complex and esterase isoenzyme patterns ofAllium sativum L. cultivars and clones-regenerants

Protein complex patterns of cloves and esterase isoenzyme patterns of apical buds of cloves were studied with Czechoslovak virus-free cultivars ofAllium sativum L. and the wild speciesA. longicuspis Regel, Similarly, four clones-regenerants obtained using explant culture techniques from A.sativum L. cv. Bzenecky paličák (two somaclones and two clones derived from plants regenerated from meristem cultures treatedin vitro with colchicine) differing in their ploidy, morphology, and yields were studied. Immunophoreograms of protein complexes of theA. sativum L. cultivars under investigation differed from one another in the number and mobility of protein fractions in both the cathodic and anodic regions and thus these cultivars can be distinguished. On the basis of esterase isoenzyme patterns, the Czechoslovak cultivars of A. sativum L. can be arranged into three groups - bolting winter cultivars with broad leaves, non-bolting winter cultivars with broad leaves, and non-bolting spring cultivars with narrow leaves. All the clones-regenerants showed the same protein complex and esterase isoenzyme patterns as their original cultivar.A. longicuspis Regel markedly differed in its protein complex and esterase isoenzyme patterns from all the other genotypes studied.Received May 17, 1989: accepted January 5,1990     

Characteristics of acid esterase in Wolman's disease

The characteristics of acid esterase from the patient with Wolman's disease, a rare familial lipidosis, were studied. Enzymatic analysis as well as mineral analysis were performed on the patient's liver, spleen, and adrenal glands. Acid esterase was low in the patient's leucocytes and other affected tissues. Further enzymatic study with subcellular fractions of the liver in both patient and control subject revealed that acid esterase was mostly localized in the membrane of lysosomes. The lysosomal esterase was unaffected by Ca2+, Mg2+, EDTA, E600 (microsomal esterase inhibitor), and it was less inhibited by NaCl than other fractions. Studies with those inhibitors showed that acid esterase has different properties compared to other lipases, such as lipoprotein lipase, adipose tissue lipase, and hepatic microsomal lipase. Studies with inhibitors also gave a negative view on a possible suppressive interaction of the high content of calcium in the target organs with acid esterase in Wolman's disease.     

菜粉蝶不同发育期酯酶同工酶的比较研究

利用垂直板型聚丙烯酰胺凝胶电泳法研究菜粉蝶Pieris rapae不同发育期的酯酶同工酶,并探讨在个体发育过程中的作用。     

Coenzyme A- and NADH-dependent esterase activity of methylmalonate semialdehyde dehydrogenase.

Methylmalonate semialdehyde dehydrogenase purified to homogeneity from rat liver possesses, in addition to its coupled aldehyde dehydrogenase and CoA ester synthetic activity, the ability to hydrolyze p-nitrophenyl acetate. The following observations suggest that this activity is an active site phenomenon: (a) p-nitrophenyl acetate hydrolysis was inhibited by malonate semialdehyde, substrate for the dehydrogenase reaction; (b) p-nitrophenyl acetate was a strong competitive inhibitor of the dehydrogenase activity; (c) NAD+ and NADH activated the esterase activity; (d) coenzyme A, acceptor of acyl groups in the dehydrogenase reaction, accelerated the esterase activity; and (e) the product of the esterase reaction proceeding in the presence of coenzyme A was acetyl-CoA. These findings suggest that an S-acyl enzyme (thioester intermediate) is likely common to both the esterase reaction and the aldehyde dehydrogenase/CoA ester synthetic reaction.     

Retinyl esterase activity of purified rat liver retinyl ester lipoprotein complex.

Retinyl ester lipoprotein complex from rat liver was shown to possess a retinyl esterase activity toward its own ligand complement. In the presence of serum albumin the retinyl esterase activity at 30 °C was about fivefold larger than the activity at 4 °C, while higher temperatures than 30 °C led to some degradation of retinyl compounds. The pH optimum was 7.8. The esterase activity was markedly enhanced by serum albumin although the serum albumin as such had no retinyl esterase activity. In the presence of serum albumin and under optimal conditions, some 75 to 80% of the total retinyl ester complement of the lipoprotein was hydrolyzed in 24 h. The retinyl esterase activity was totally abolished by treatment with the serine esterase inhibitor diisopropyl fluorophosphate (1.4 × 10^?4 M), by treatment with sulfhydryl reagents, and by detergents (0.2% of Tween 80 and sodium deoxycholate). From this series of experiments it was concluded that the retinyl ester lipoprotein complex possesses the additional physiological function of hydrolyzing its own retinyl ester complement to unesterified retinol which may then combine with serum retinol-binding protein.     

Subcellular distribution and properties of aldehyde dehydrogenase in the rat liver

The subcellular distribution and certain properties of rat liver aldehyde dehydrogenase are investigated. The enzyme is shown to be localized in fractions of mitochondria and microsomes. Optimal conditions are chosen for detecting the aldehyde dehydrogenase activity in the mentioned fractions. The enzyme of mitochondrial fraction shows the activity at low (0,03-0.05 mM; isoenzyme I) and high (5 mM; isoenzyme II) concentrations of the substrate. The seeming Km and V of aldehyde dehydrogenase from fractions of mitochondria and microsomes of rat liver are calculated, the acetaldehyde and NAD+ reaction being used as a substrate.     

Enzyme catalyzed hydrolysis of the diesters of cis- and trans-cyclohexanedicarboxylic acids

Summary Pig liver esterase (EC 3.1.1.1) catalyzed hydrolysis of the dimetrhy ester of meso-cis-1,2-cyclohexanedicarboxylic acid yielded the optically pure (1S,2R)-monoester. The corresponding diethyl ester yielded racemic monoester.The diethyl ester of racemic trans-1,2-cyclohexanedicarboxylic acid was kinetically resolved by partial hydrolysis with subtilisin (EC 3.4.21.14) or pig liver esterase. The (1R,2R)-monoester had an enantiomeric excess of 45% and was obtained in an enantiomerically pure form through recrystallisation. The remaining (1S,2S)-diester exhibited an enantiomeric excess of 83%. The nature of the ester function (methyl, ethyl, and propyl esters) had a great influence on the enantiomeric excess obtained and on the kinetic parameters.     

Lipase and Esterase Activities of Propionibacterium freudenreichii subsp. freudenreichii

The lipase and esterase activities of eight strains of dairy Propionibacterium freudenreichii subsp. freudenreichii were studied. A lipase activity was detected on whole cells and in the culture supernatant. The highest activity was expressed at 45°C and pH 6.8. An esterase activity was also detected in the culture medium. The electrophoresis of the intracellular fractions of the cells revealed from three to six different esterase activities. Two esterases were common to all the strains. The substrate specificity was dependent on each esterase, but no activity was revealed, in our experimental conditions, on ester substrates with a chain length longer than that of butyrate.     

Polyacrylamide gel immobilization of porcine liver esterase for the enantioselective production of levofloxacin

Porcine liver esterase was immobilized in polyacrylamide gel for the enantioselective production of levofloxacin from ofloxacin butyl ester. The initial activity of immobilized esterase was found to be significantly affected by the polyacrylamide gel composition. The optimum concentrations of monomer and crosslinker were determined to be 20% and 8.3%, respectively. The activity of immobilized esterase was 55.4% compared to a free enzyme. Enantiomeric excess was maintained at 60%, almost the same level as that of free enzyme. In addition, the immobilized esterase could be used repeatedly up to 10 times without experiencing any severe loss of activity and enantioselectivity.