Isoelectric focusing studies of human red cell esterase D: Evidence for polymorphic occurrence of a new allele EsD 7 in Japanese

Summary The isoelectric focusing study of esterase D in Japanese revealed evidence of a new polymorphic allele (EsD ⁷) which is difficult to find by conventional starch gel electrophoresis only. A comparison with the occurrence of a subdivision of EsD ² in Caucasians (EsD ⁵) suggests a remarkable difference in allele distribution of esterase D among races. Quantitative analysis showed a relatively low value of enzyme activity for this new allele. It is therefore emphasized that in addition to conventional electrophoresis, enzyme assay and further detection by isoelectric focusing are essential in analyzing the esterase D system.     

The effect of phenylacetic acid on cholinesterase activity and on some isoenzymes of esterases and cholinesterases of peain vitro

Phenylacetic acid at 1.5 × 10^-3 M inhibits the activity of some esterase isoenzymes from pea leaves separated by means of polyacrylamide gel electrophoresis. Some of the inhibited esterases show cholinesterase activity. Inhibition of the total activity has been demonstrated with a partially purified protein fraction from pea leaves containing choline esterase. The inhibition constant established after Dixon was 7.9 × 10^-3 M and the type of inhibition was competitive.     

鸭血清胆碱酯酶的纯化及性质研究

首次采用新技术双水相萃取方法作为鸭血清胆碱酯酶(EC.3.1.1.8 CHE) 纯化的第一步,后经 DEAE-Sephadex A50,sephadex G200 柱层析,获得电泳纯鸭血清胆碱酯酶,提纯倍数1018倍,酶活力回收43.4%,比活274.9U/mg。鸭血清胆碱酯酶性质研究表明:此酶是糖蛋白和酸性蛋白水解酶,等电点 4.2 左右,最适 pH7.5 左右;对底物碘化硫代丁酰胆碱的 K_m=9.8×10^-5mol/L;SDS-PAGE 电泳和聚丙烯酰胺梯度电泳表明,鸭血清胆碱酯酶以相同亚基组成的不同聚合体形式存在,亚基分子量 78000,具有完整的酶活性.不同聚合体带电状态相同.     

Hyaluronidase and esterase activities of the venom of the poisonous brown recluse spider

Venom of Loxosceles reclusa free from impurities was expressed from venom glands collected by microdissection. Polyacrylamide gel electrophoresis of the venom at pH 8.3 demonstrated 7 or 8 major plus 3 or 4 minor components. Upon electrophoresis at pH 4.9 two major components plus 3 or 4 minor components were noted. Monophoretic hyaluronidase prepared by Sephadex gel filtration and electrophoresis at pH 8.3 exhibited optimum activity from pH 5.0 to 6.6. Sodium dodecyl sulfate gel electrophoresis of purified hyaluronidase revealed two components with estimated molecular weights of 33,000 and 63,000. The purified hyaluronidase exhibited activity against chondroitin sulfate, types A, B, and C at approximately 20–30% of that upon hyaluronic acid. The enzyme was inhibited 10–20% by the heavy metal ions, Fe⁺³ and Cu⁺². Rabbit antivenom inhibited the spreading effect of whole venom in vivo and completely inhibited hyaluronidase in vitro.Incorporation of [¹⁴C]leucine into the spider venom led to the separation of hyaluronidase from the dermonecrotic activity of the venom.The venom demonstrated activity against carbobenzoxy-l-tyrosine-p-nitrophenyl ester and β-naphthylacetate which was inhibited approximately 65% by 2.5 × 10^?3m levels of EDTA and EGTA but not by 2.5 × 10^?4mo-phenanthroline. The esterase activity resisted concentrations of p-chloromercuribenzoate which totally inactivated papain. The venom appeared devoid of collagenase, dipeptidase, acetylcholinesterase, phosphodiesterase, ribonuclease A, and deoxyribonuclease.     

The effect of growth temperature on esterase patterns in psychrotrophic Bacillus species and other Gram-positive genera

The growth temperature of Bacillus stearothermophilus was previously reported to affect the esterase band pattern obtained after gel electrophoresis. In this study gel electrophoresis of esterases was done on a group of Antarctic bacterial strains cultivated at different temperatures to investigate whether band shift due to a change in growth temperature was a general phenomenon or limited only to select groups. Most strains studied were in the Bacillus genus. Standard strains of known Bacillus species and other Gram-positive genera were examined for comparative purposes. A change in the esterase band pattern was observed as a result of variation in growth temperature. Two major esterase band were dominant in this diverse group of species.     

Purification and properties of carbonic anhydrase from Chlamydomonas reinhardii

Carbonic anhydrase (CA) was purified from the unicellular green alga Chlamydomonas reinhardii, and the purity of the preparation was established by gradient gel electrophoresis. The purified enzyme exhibited a MW of 165 000 and contained 6 atoms of Zn. The subunit MW, as determined by dodecyl sulfate electrophoresis, was 27 000. These results are consistent with a quarternary structure which is hexameric, each monomer containing 1 g atom of Zn. Like spinach CA, and in contrast to other oligomeric plant CAs, a sulfhydryl reducing agent is not needed to stabilize the enzyme. CO₂-hydrase activity was inhibited by both acetazolamide (I₅₀ = 7.8 × 10^?9M) and sulfanilamide (I₅₀ = 1.3 × 10^?5M), as well as by certain inorganic anions. The purified enzyme showed relatively weak esterase activity with p-nitrophenyl acetate but was an extremely effective esterase with 2-hydroxy-5-nitro-α-toluenesulfonic acid sultone as the substrate. Both esterase activities could be completely inhibited by adding acetazolamide. In its gross structural characteristics, the C. reinhardii enzyme resembles the CAs from higher plants. However, in its esterase activity and the inhibition by sulfonamides it is markedly different from plant CAs and bears more resemblance to erythrocyte CAs.     

Purification and properties of a highly enantioselective l-menthyl acetate hydrolase from Burkholderia cepacia

A highly enantioselective l-menthyl acetate esterase was purified to homogeneity from Burkholderia cepacia ATCC 25416, with a recovery of 4.8% and a fold purification of 22.7. The molecular weight of the esterase was found to be 37 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The N-terminal amino acid sequence was “MGARTDA”, and there was no homology in contrast to other Burkholderia sp. esterases. This enzyme preferentially hydrolyzed short-chain fatty acid esters of menthol with high stereospecificity and high hydrolytic activity, while long-chain l-menthyl esters were poor substrates. Considered its substrate specificity and N-terminal sequence, this esterase was concluded as a new enzyme belonging to the carboxylesterase group (EC 3.1.1.1) of esterase family. The optimum temperature and pH for enzyme activity using racemic menthyl acetate as substrate were 30 °C and 7.0, respectively. The esterase was more stable in the pH range of 7.0–9.0 and temperature range of 30–40 °C. Hydrolytic activity was enhanced by Ca²⁺, K⁺ and Mg²⁺, but completely inhibited by Hg²⁺, Cu²⁺, ionic detergents and phenylmethylsulfonyl fluoride (PMSF) at 0.01 M concentration.     

Serum esterase genetics in rabbits. IV. The prealbumin and β-globulin systems

Discontinuous starch gel electrophoresis revealed a fourth allele of rabbit prealbumin serum esterase at locus Est-2. This allele is designated Est-2 ^f and appears to be silent. In addition to the prealbumin serum esterases, another serum esterase system was studied in rabbits. This system is localized in the β-globulin region. Genetic analysis indicated that one locus with two codominant alleles controls the variation in this region. Linkage of this system with Est-1 and Est-2 of the prealbumin serum esterases was demonstrated. Comparison of the arrangement of these esterase loci on linkage group VI with the esterase loci on chromosome 8 of the mouse gives additional support for the theory of evolutionary conservation of chromosomal segments coding for mammalian esterases.     

Esterase D polymorphism in a French-Canadian population

Summary Red blood cell esterase D (ESD) polymorphism was studied in a French-Canadian population from Quebec city, Canada, by means of high voltage electrophoresis on agarose gel followed, in heterozygotes for ESD1, by IEF to reveal the possible allele ESD^*5. Frequencies of the ESD alleles in 904 unrelated individuals were ESD^*1: 0.888, EDS^*2: 0.095 and ESD^*5: 0.017. The segregation pattern observed in 275 families confirmed a Mendelian inheritance of three autosomal alleles.     

Studies on the characterization of the sodium-potassium transport adenosine triphosphatase. Purification and properties of the enzyme from the electric organ of Electrophorus electricus

An unspecific carboxylesterase was purified 180-fold from acid-precipitated human liver microsomes. The final preparation was homogeneous on disc electrophoresis and polyacrylamide gel electrophoresis in the presence of 6.25 M urea at pH 3.2. A single symmetrical peak was also found on gel filtration and on velocity sedimentation in the analytical ultracentrifuge, whereas slight heterogeneity was observed on isoelectric focusing.The amino acid composition of the purified enzyme is presented. From the results the partial specific volume (0.745 ml × g^?1) and the minimal molecular weight (60,000) could be calculated. Fingerprint maps of tryptic peptides from the carboxymethylated enzyme are shown.The molecular weight as determined by gel filtration, disc electrophoresis, and analytical ultracentrifugation is in the range of 181,000–186,000. For the molecular weight of the subunits a value of 61,500 has been obtained by sodium dodecylsulfate polyacrylamide gel electrophoresis. The equivalent weight of the enzyme has been estimated to be 62,500 from stoichiometry of its reaction with diethyl-p-nitrophenyl-phosphate. Partial cross-linking of the subunits with dimethyl suberimidate and subsequent sodium dodecylsulfate polyacrylamide gel electrophoresis yielded three bands with molecular weights of 60,000, 120,000, and 180,000.From these results it is concluded that human liver esterase is a trimeric protein. It is composed of three subunits of equal size, and there is one active site per subunit.     

Gene diversity in Indian populations

Summary The phenotypes of glyoxalase I (GLO) were determined in a random population from Hessen (Germany) by high-voltage agarose gel electrophoresis. The gene frequencies in 1150 unrelated individuals were 0.4391 for GLO¹ and 0.5609 for GLO². Rare phenotypes were not observed. The segregation of phenotypes in 50 families and 32 mother-child combinations supports the assumed autosomal codominant inheritance. The possibility of a simultaneous typing for GLO, esterase D (EsD), and carbonic anhydrase₂ (CA₂) on one gel is discussed.     

The occurrence of an adult reproductive diapause in the univoltine life cycle of the beech leaf mining weevil,Rhynchaenus fagi L.

Summary

Juvenile hormone (JH) and α-naphthyl acetate (α-NA) esterase activity was measured on a daily basis during embryogenesis of the house cricket, Acheta domesticus. In eggs dissected from the lateral oviducts and embryos through blastokinesis, there were elevated levels of nonspecific JH esterase activity. The JH esterase activity could not be resolved from the α-NA esterase activity by gel filtration chromatography and the metabolism of both substrates was inhibited equally by 0,0-diisopropyl phosphorofluoridate (DFP). From blastokinesis through egg hatch, the JH esterase activity was maintained at relatively low levels and was resolved from the α-NA esterase activity by gel filtration. The α-NA esterase activity was inhibited by DFP while the JH esterase activity was relatively unaffected. Low JH titers in eggs must be maintained through blastokinesis for normal development. Elevated JH esterase activity in eggs during this period appears to have a functional role in the metabolism of maternal JH in the egg.     

Isozyme Specificity to Temperature

TEMPERATURE treatment of styles leads to self-compatibility in otherwise self-incompatible species of Oenothera^2,3, Lilium⁴ and Trifolium^5,6. I have studied the temperature treatment of esterase in styles of several species and hybrids of Lilium by using gel electrophoresis.     

Growth of Pseudomonas chlororaphis on apolyester–polyurethane and the purification andcharacterization of a polyurethanase–esterase enzyme

A Pseudomonas chlororaphis was found to degrade and utilize apolyester polyurethane as a sole carbon and energy source. Polyurethane utilization by P.chlororaphis followed simple Michaelis–Menten kinetics. The K_s and μ_max values were 0.802 mg·ml⁻¹ and 1.316 doublings·h⁻¹, respectively. The enzymes from P. chlororaphis responsible for polyurethanedegradation were found to be extracellular. Analysis of the polyurethane degrading proteins, usingnon-denaturing polyacrylamide gel electrophoresis, revealed three active protein bands with R_f values of 0.25, 0.417 and 0.917. A polyurethane degrading enzyme was purifiedand displayed esterase activity. This enzyme was inhibited by phenylmethylsulfonyl fluoride andhad a molecular weight of 27,000 daltons.     

Genetic control of two esterases of rat plasma (Rattus norvegicus)

Three electrophoretic variants of plasma esterase in the albumin zone, presumably carboxylesterase, have been demonstrated in 250 rats representing a laboratory population of Wistar rats. Electrophoretic variants of the enzyme are believed to be controlled by two codominant alleles at the autosomal locus referred to as Es-2. The variant of carboxylesterase represented by a fast-migrating single band on starch gel electrophoresis is determined by the gene named Es-2 ^a, whereas the slow-migrating variant, represented by two bands, is under control of the allelic gene Es-2 ^b. Animals with Es-2 ^a/Es-2 ^b genotype have three bands of carboxylesterase in the albumin zone. Genetically determined polymorphism of plasma esterase, presumably carboxylesterase, in the prealbumin zone was shown in both laboratory and wild populations of rats. Breeding tests suggest that the gene referred to as Es-1 ^a, responsible for the presence of carboxylesterase in the prealbumin zone, is inherited dominantly, whereas animals homozygous for the allele Es-1 ^b locked this esterase fraction.     

几种杀虫药剂敏感蚊类酯酶多态性的研究

通过蚊虫酯酶蛋白的淀粉凝胶电泳分析和基因组DNA的限制性酶切片段长度多态性(RFLPs)比较, 对尖音库蚊Culex pipiens、三带喙库蚊Culex tritaeniorhynchus和中华按蚊Anopheles sinensis有机磷杀虫药剂敏感种群的酯酶蛋白和结构基因的多态性进行分析。发现在蛋白质水平上,三带喙库蚊敏感种群(n=54)在酯酶α和β位点分别存在2个和3个等位基因,在DNA水平上有2.9%的个体具有与酯酶β1¹基因1.3 kb Cdna片段同源的1.3 kb单拷贝带存在。发现中华按蚊敏感种群 (n= 50)中具有低活性的非特异性酯酶存在,在蛋白质水平上,酯酶α和β位点各有一个等位基因;在DNA水平上,通过对单个蚊虫基因组DNA的研究未发现有与酯酶β1¹基因同源的酯酶编码基因的存在。对尖音库蚊北京敏感种群(n= 64)的研究发现,在酯酶α和β位点都存在5个等位基因,在DNA水平上,使用一个限制性内切酶(EcoRI),15只蚊虫的样本在酯酶β位点发现了5个等位基因,说明在尖音库蚊北京敏感种群的酯酶β基因周围存在着较大的中性多态性,在有机磷杀虫剂的选择下,这些中性多态性可能会成为基因扩增的潜在因素。     

Isozyme variability in species of the genusDrosophila. V. ejaculatory bulb esterases inDrosophila phylogeny

The presence or absence of certain esterase types in ejaculatory bulbs of 93 Drosophilaspecies was determined by appropriate staining after starch gel electrophoresis. One entire subgenus (Sophophora)and several primitive species were found lacking in one type of esterase. This esterase was present in numerous species of the large Drosophilasubgenus, but presence and absence did not follow closely the lines of taxonomic subdivisions in the subgenus. Correlation of presence-absence of the bulb esterase and previous results in interspecific hybridization tests indicated a greater chance for a successful interspecific cross when males possessed the bulb esterase.     

The Binding of Asparagine,Glutamine and Homoserine to Human Erythrocytes Containing Hemoglobins S and CS and to Soluble Hemoglobins S and CS

Abstract

Tritium labeled asparagine binds to oxyhemoglobin S and to a mixture of hemoglobins C and S in the molar ratio of 3.38:1 and 8.2:1 respectively. From the dialysis equilibrium studies it appears that labeled asparagine does not bind to oxy- or deoxy- hemoglobin A nor to deoxyhemoglobin S. The constant for equilibrium association of asparagine for oxyhemoglobin S is 7.38 × 10⁷ M^?1 and for'oxyhemoglobin CS 4.8 × 10⁴ M^?1 at 23°C. Tritium labeled asparagine is bound to oxyhemoglobin S and CS sufficiently strongly to prevent dissociation under the conditions of gel electrophoresis at pH 9.50. The protein with and without bound asparagine, gluta-mine or homoserine, is indistinguishable in molecular net charge and size by the criteria of quantitative polyacrylamide gel electrophoresis (PAGE). Also there were no significant differences in mobility between hemoglobin S and hemoglobin C in the presence and absence of asparagine, glutamine and homoserine as detectable in agar coated cellulose acetate electrophoresis at pH 6.3. Erythrocytes containing hemoglobin S and CS, after incubation with tritium labeled asparagine and lysis under the conditions of gel electrophoresis at pH 9.5, release hemoglobin S and C with bound tritiated asparagine. No tritiated asparagine remains bound to the ghost.     

Approaches to the purification of the juvenile hormone esterase from the cabbage looper,Trichoplusia ni

Several approaches to the purification of juvenile hormone (JH) esterase from second-day last-instar larvae of Trichoplusia ni were taken, including: ammonium sulphate precipitation, polyethylene glycol precipitation, hydrophobic interaction chromatography, anion exchange chromatography, and gel filtration chromatography. The most successful procedure involved a combination of polyethylene glycol precipitation with anion exchange chromatography on DEAE Sephacel which yielded a 134-fold purification of juvenile hormone esterase. When this preparation was subjected to semi-preparative electrophoresis followed by isoelectric focusing on a polyacrylamide slab gel, a single band of apparently homogeneous enzyme was obtained. Juvenile hormone esterase activity was unstable after electrophoresis and isoelectric focusing. The stability of juvenile hormone esterase activity in a water solution is influenced by protein concentration and by agents protecting sulphydryl groups. The results of this study support the hypothesis that a single protein is responsible for the majority of the JH hydrolysis catalyzed by haemolymph from the larvae of T. ni used in this study.     

Studies on the degradation of HeLa non-histone proteins

The hydrolysis of HeLa non-histone nuclear proteins over 24 h has been monitored in dilute alkali at 4, 15 and 25°C using the standard ninhydrin estimation, dansylation and various electrophoresis techniques. Under conditions (up to 0.2 N NaOH, 4°C) that do not release a significant quantity of ninhydrin-positive material or new N-terminal end group considerable breakdown was observed by two-dimensional electrophoresis analysis. The number of stained spots decreased from approx. 140 to 25–30. No internal protease activity could be found.Labelling studies (¹⁴C-labelled amino acids) showed that much of the hydrolysed material was extracted from the gel during normal staining and destaining procedures. Peptides could be extracted from alkali-hydrolysed non-histone protein with acid/ethanol and could be further separated by thin-layer chromatography on silica gel G. Short-term labelling of HeLa cells (¹⁴C-labelled amino acids for up to 60 min) revealed that these peptides probably have a high rate of turnover. [¹⁴C] Glucosamine studies also indicated the presence of considerable carbohydrate material in the low molecular weight products of this alkaline hydrolysis. Various standard proteins and histones were unaffected by hydrolysis in up to 0.2 N NaOH (4°C, 24 h) as judged by gel electrophoresis.Seven different phosphate-splitting enzymes and an esterase had no effect on the non-histone protein electrophoresis patterns but a preparation of phospholipase C which had no protease activity towards eight standard proteins did produce considerable breakdown in HeLa non-histone proteins similar to that produced by 0.2 N NaOH at 4°C.