Characterization of Enterobacter cloacae and E. sakazakii by electrophoretic polymorphism of acid phosphatase, esterases, and glutamate, lactate and malate dehydrogenases

Acid phosphatase, esterases, and glutamate, lactate and malate dehydrogenases of 34 strains of Enterobacter cloacae and 22 strains of Enterobacter sakazakii were analysed by horizontal polyacrylamide agarose gel electrophoresis and by isoelectrofocusing in thin-layer polyacrylamide gel. The two species could be separated on the basis of distinct electrophoretic patterns of all enzymes analysed. Glutamate dehydrogenase and acid phosphatase were detected exclusively in E. cloacae, whereas esterase bands were more intensively stained in E. sakazakii. For each species, two zymotypes could be distinguished, on the basis of electrophoretic mobilities of malate dehydrogenase and banding patterns of esterase for E. cloacae, and by both isoelectric point and electrophoretic mobilities of an esterase and of lactate and malate dehydrogenases for E. sakazakii. The high degree of enzyme polymorphism within the two species permitted precise identification of strains. The variations in electrophoretic patterns might therefore provide useful epidemiological markers.     

Esterase and Malate Dehydrogenase Phenotypes in Portuguese Populations of Meloidogyne Species

Nonspecific esterases and malate dehydrogenases of 1-5 females from 40 root-knot nematode populations from Portugal were analyzed by electrophoresis in 0.4-mm-thick polyacrylamide gels. Fourteen major bands of esterase activity were detected, corresponding to 10 distinct phenotypes, Meloidogyne javanica and M. hapla had distinct species-specific phenotypes. Two phenotypes occurred in M. arenaria. The most variability was found among M. incognita populations. Of the remaining two phenotypes, one was associated with M. hispanica and the other belonged to a new species. Three malate dehydrogenase phenotypes were discerned on the basis of particular combinations of the eight main bands of activity found. As previously found, esterases were more useful than malate dehydrogenases in identification of the major Meloidogyne species. The host plant had no effect on the nematode esterase or malate dehydrogenase phenotypes.     

Biochemical characterization of insecticide resistance in the German cockroach, Blattella germanica, from Malaysia

The possible insecticide resistance mechanisms of four Malaysian field-collected strains of the German cockroach, Blattella germanica (Linnaeus) (Dictyoptera: Blattellidae), were characterized with biochemical assays and native polyacrylamide gel electrophoresis (PAGE). Elevated esterase activity (at low to moderate frequency) and altered acetylcholinesterase (low frequency) were detected in all field strains, while elevated glutathione S-transferase levels were present in only two strains. Seven esterase bands were separated by native PAGE; a greater intensity occurred in three bands in the resistant strains compared to the susceptible strain. Inhibition studies using specific inhibitors on polyacrylamide gels suggested that the slowest of these three esterases is a cholinesterase, while the other two are carboxylesterases with a preference for beta- over alpha-naphthyl acetate.     

Distinctive electrophoretic and isoelectric focusing patterns of esterases from Yersinia enterocolitica and Yersinia pseudotuberculosis

Esterases of 53 strains of Yersinia enterocolitica sensu stricto, including five previously defined biotypes, and 30 strains of Yersinia pseudotuberculosis were analysed by horizontal polyacrylamide-agarose gel electrophoresis and by isoelectrofocusing in thin-layer polyacrylamide gel. Esterase bands were defined by their range of activity towards several synthetic substrates, their resistance to heat and to di-isopropyl fluorophosphate. The two species were characterized by distinct electrophoretic patterns of their esterases. The apparent molecular weights of the heat-resistant esterase of Y. enterocolitica and of the major heat-resistant esterase of Y. pseudotuberculosis, as determined by polyacrylamide gradient gel electrophoresis, were estimated to be 52 000 and 250 000, respectively. On the basis of electrophoretic mobilities and isoelectric points of esterases produced by strains of Y. enterocolitica, five principal zymotypes were observed: two for strains of biotype 1, two for strains of biotypes 2 and 3, respectively, and only one for strains of both biotypes 4 and 5. The zymotypes of strains of biotypes 2, 3, 4 and 5 appeared to be more closely related to one another than to zymotypes of strains of biotype 1. Variations in number or mobility of bands observed within each biotype of Y. enterocolitica and within some serotypes of Y. pseudotuberculosis could represent an additional marker for epidemiological analysis.     

The non-specific esterases of mouse lung

The non-specific esterases of the lung of the house mouse, M. musculus, were examined by polyacrylamide electrophoresis and by isoelectric focusing. At least 13 different esterases were distinguished and identified, mainly by their catalytic properties, susceptibility to inhibition, developmental patterns and phenotypic variation amongst different strains. A list of diagnostic features of the 13 esterases was presented. None of the esterases was lung-specific. However, the pattern of esterases found in the adult lung was characteristic of that organ. It was pointed out that this pattern is associated with the high degree of tissue differentiation in the adult lung. At least 8 esterases were found which belong to the isozyme system of carboxylesterase EC 3.1.1.1, under the control of genes located on chromosome 8. These esterases accounted for about 90% of the esterase activity in the lung.     

Production, Purification, and Properties of Extracellular Carboxyl Esterases from Bacillus subtilis NRRL 365

Bacillus subtilis NRRL 365 produced high extracellular carboxyl esterase activity in submerged culture media containing wheat bran, corn steep liquor, and salts. Supplementation of this medium with glucose reduced esterase activity to 37% of that in the unsupplemented control. Esterase activity was purified by ammonium sulfate fractionation, DEAE-Sephadex A-50 ion-exchange chromatography with sodium chloride gradient elution, and preparative polyacrylamide gel electrophoresis. The resultant purified components, esterases I and II, manifested single bands following silver staining of polyacrylamide gel electrophoresis gels and had final specific activities of 80 and 520 U/mg, respectively. Molecular weights for components I and II were 36,000 and 105,000 to 110,000, respectively. Esterases I and II both had a pH optimum of 8.0, with relative activities of 10 and 85%, respectively, at pH 9.0. K_ms with p-nitrophenylacetate were 0.91 mM for esterase I and 0.67 mM for esterase II. In general, patterns of enzyme inhibition were similar for both components. Differences were observed in the relative activities of esterases I and II towards p-nitrophenyl esters of acetate, propionate, and butyrate; Activity ratios for components I and II were 100:94:48 and 100:36:23, respectively. The purified components did not hydrolyze long-chain triglycerides and did not manifest proteolytic activity.     

六种疫霉菌体可溶性蛋白质和酯酶的聚丙烯酰胺凝胶平板电泳研究

本文对槐生疫霉(Phytophthora robinicola)、掘氏疫霉(P.drechsleri)、樟疫霉(P.cinnamomi)、烟草疫霉(P.nicotianae)、棕榈疫霉(P.palmivora)及辣椒疫霉(P.capsici)等6种疫霉81株菌菌体可溶性蛋白质和酯酶进行了聚丙烯酰胺凝胶平板电泳研究。结果指出,种间蛋白质图谱差异明显,种内菌株间基本一致。就酯酶图谱来说,种内菌株间存在一定差异,但种间差异更为显著。因此,本试验进一步支持槐生疫霉新种的建立。同时表明,菌体可溶性蛋白质电泳图谱在疫霉种的鉴定和分类上具有重要意义,而酯酶电泳图谱在一定程度上也有助于疫霉种的研究。     

Lipoprotein substrates of lipoprotein lipase and hepatic triacylglycerol lipase from human post-heparin plasma.

The number and the substrate specificities of glutathione thiol esterases of human red blood cells have been investigated by gel electrophoresis and isoelectric focusing and staining methods devised for the location of these enzymes on gels. Several glutathione thiol esterase forms, both unspecific (with respect to the S-acyl group of the substrate) and specific were found. Electrophoresis on both polyacrylamide and agarose gels resolved three enzyme components with apparently similar substrate specificity. Isoelectric focusing in liquid column separated two unspecific thiol esterase components with S-lactoylglutathione (pI = 8.4) and S-propionylglutathione (pI = 8.1) as the best substrates, respectively, and two specific enzymes, S-formylglutathione hydrolase (pI = 5.2) and S-succinylglutathione hydrolase (pI = 9.0). Isoelectric focusing on polyacrylamide gel resolved nine unspecific glutathione thiol esterase bands (between pH values 7.0 and 8.4). Partially purified glyoxalase II (S-2-hydroxyacylglutathione hydrolase, EC 3.1.2.6) from erythrocytes or liver still gave three components on electrophoresis and several activity bands on gel electrofocusing. These results indicate that human red cells contain at least four separate glutathione thiol esterases. Glyoxalase II, one of these enzymes, apparently occurs in multiple forms. These were neither influenced by preptreatment of the samples with neuraminidase or thiols nor were interconvertible during the fractionations.     

Purification and characterization of a novel extracellular esterase from pathogenic Streptomyces scabies that is inducible by zinc.

Native polyacrylamide gels of extracellular proteins produced by several Streptomyces isolates grown with suberin were assayed in situ for esterase activity. Two pathogenic isolates of Streptomyces scabies from different geographical regions were found to produce a similar esterase activity that was not produced by nonpathogenic strains. After treatment with EDTA, suberin no longer induced esterase production. Expression was restored when EDTA-treated suberin was supplemented with zinc. The optimal concentration of zinc required for esterase production was 2 microM. This esterase was purified from one of the pathogenic isolates and characterized. The enzyme was 38,000 daltons when determined by gel filtration on Sephadex G-100 and 36,000 daltons when determined by denaturing polyacrylamide gel electrophoresis. The esterase showed maximal activity in sodium phosphate buffer above pH 8.0, was stable to temperatures of up to 60 degrees C, and had an apparent Km of 125 microM p-nitrophenyl butyrate.     

六种疫霉菌体可溶性蛋白质和酯酶的聚丙烯酰胺凝胶平板电泳研究*

本文对槐生疫霉(Phytophthora robinicola)、掘氏疫霉(P.drechsleri)、樟疫霉(P.cinnamomi)、烟草疫霉(P.nicotianae)、棕榈疫霉(P.palmivora)及辣椒疫霉(P.capsici)等6种疫霉81株菌菌体可溶性蛋白质和酯酶进行了聚丙烯酰胺凝胶平板电泳研究。结果指出,种间蛋白质图谱差异明显,种内菌株间基本一致。就酯酶图谱来说,种内菌株间存在一定差异,但种间差异更为显著。因此,本试验进一步支持槐生疫霉新种的建立。同时表明,菌体可溶性蛋白质电泳图谱在疫霉种的鉴定和分类上具有重要意义,而酯酶电泳图谱在一定程度上也有助于疫霉种的研究。     

Alterations in esterases are associated with malathion resistance in Habrobracon hebetor (Hymenoptera: Braconidae)

Biochemical mechanisms of malathion resistance were investigated in a malathion-resistant strain of the parasitoid Habrobracon hebetor Say collected from a farm storage in Kansas. General esterase activities were significantly lower in the resistant strain compared with those in a susceptible strain. However, no significant differences were found in activities of malathion specific carboxylesterase (MCE), glutathione S-transferase and cytochrome P450 dependent O-demethylase activities, cytochrome P450 contents, and sensitivity of acetylcholinesterase to inhibition by malaoxon between the 2 strains. Because MCE was not elevated in the resistant strain, the weak malathion resistance in H. hebetor may result from a different mechanism compared with that hypothesized for some insect species in which reduced general esterase activity is accompanied by an elevated MCE. Decreased esterase activity in the resistant strain suggested that null alleles of some esterases were associated with the resistance. Indeed, E1 and E2, major esterases in the susceptible strain, were not present in the resistant strain on polyacrylamide gels that were stained for esterase activity using the model substrate 1-naphthyl acetate. In contrast, the activity of esterase E3 on the gels was much higher in the resistant strain as compared with that of the susceptible strain. These findings indicate that malathion resistance in H. hebetor is associated with both an increased activity of the esterase E3 and null alleles of the esterases E1 and E2.     

Esterases in Folsomia candida (Collembola: Isotomidae). Characterization of enzymes among parthenogenic strains

Esterase enzymes from four strains of Folsomia candida were investigated using polyacrylamide gel electrophoresis. Up to 12 bands of enzymatic activity were present in each strain. Esterase bands were classified as choline esterases or as one of two groups of carboxyl esterases, based on mobility, on substrate specificity and on activity remaining after inhibition by class-specific chemicals. One strain-specific choline esterase was discovered which resisted the effects of many organophosphate inhibitors. Organophosphate inhibitor concentrations had to be 10 to 100 times greater to reduce the staining activity of this resistant choline esterase to the level of comparable esterases in other strains.     

Characterization of Yersinia enterocolitica, Y. intermedia, Y. aldovae, Y. frederiksenii, Y. kristensenii and Y. pseudotuberculosis by electrophoretic polymorphism of acid phosphatase, esterases, and glutamate and malate dehydrogenases

Acid phosphatase, esterases, and glutamate and malate dehydrogenases of 192 strains of Yersinia enterocolitica, Y. intermedia, Y. aldovae, Y. frederiksenii, Y. kristensenii and Y. pseudotuberculosis were analysed by horizontal polyacrylamide agarose gel electrophoresis and by isoelectrofocusing in thin-layer polyacrylamide gels. The six species were clearly separated from each other by their distinct enzyme electrophoretic polymorphism. For Y. enterocolitica, the strains of biotype 5 were differentiated from the other biotypes by the mobility of glutamate dehydrogenase. For Y. frederiksenii, six zymotypes were delineated by pI and by the mobility of the enzymes. Variation in number or mobility of esterases within each species could represent a marker for epidemiological and ecological analyses. A linear relationship was obtained between the mean genetic diversity coefficient of enzymes and the mean percentage DNA-DNA relatedness of Y. intermedia, Y. aldovae, Y. enterocolitica and Y. frederiksenii.     

Kinetics of inhibition of platelet calpain II by human kininogens.

Organophosphate-resistant and -susceptible strains of Culex quinquefasciatus (mosquito) have been compared on the basis of their esterase activities. The homozygous resistant strain (Dar) shows two highly active esterases after starch-gel electrophoresis, of Rm 0.2 and 0.4, which are absent from susceptible strains (Apo, Mon), and which previous selection studies have shown to be inseparable from organophosphate resistance. After SDS/polyacrylamide-gel electrophoresis and silver staining of total C. quinquefasciatus proteins, a 62 kDa band is observed in strain Dar at high concentrations, and in susceptible strains in trace amounts. After Western blotting, this 62 kDa protein is recognized by antisera raised against the two esterases eluted from starch gels. After chromatofocusing of Dar proteins, the 62 kDa protein is seen to be associated with esterase activity, and of a similar pI to that observed for esterases after isoelectric focusing. Post-translational modification is not required for recognition of the 62 kDa putative esterase, since the protein is immunoprecipitated by the anti-esterase serum from products of translation of Dar mRNA in vitro.     

Diagnostic assays based on esterase-mediated resistance mechanisms in western corn rootworms (Coleoptera: Chrysomelidae)

Resistance to methyl-parathion among Nebraska western corn rootworm, Diabrotica virgifera virgifera LeConte, populations is associated with increased hydrolytic metabolism of an organophosphate insecticide substrate. An electrophoretic method to identify resistant individuals based on the staining intensity of esterase isozymes on nondenaturing polyacrylamide gels was developed. Three groups of esterases (I, II, and III) were visible on the gels, but only group II esterase isozymes were intensified in resistant populations. A total of 26 and 31 field populations of western corn rootworms from Nebraska (in 1998 and 1999, respectively) were assessed with nondenaturing polyacrylamide gel electrophoresis (PAGE) assays and diagnostic concentration bioassays. Significant correlations were observed between the two diagnostic assays. Group II esterase isozymes provide a reliable biochemical marker for detection of methyl-parathion resistance in individual western corn rootworms and a tool for monitoring the frequency of resistant individuals in field populations.     

Diversity of plant cell wall esterases in thermophilic and thermotolerant fungi

Fourteen thermophilic and thermotolerant fungal strains isolated from composting soils produced plant cell wall-acting esterases in a medium containing corn cobs and oat spelt xylan. The concentrated and dialyzed protein extracts of these fungi were fractionated using isoelectric-focusing, gels sliced and eluted protein in each slice was assayed for esterase activity against p-nitrophenyl acetate. A total of 84 esterases detected on the basis of pI were found to show distinct preferential substrate specificities towards p-nitrophenyl acetate, p-nitrophenyl ferulate and p-nitrophenyl butyrate, and were putatively classified as acetyl esterases and esterases types I and II. None of the esterases were active against p-nitrophenyl myristate. In addition, these esterases were characterized as acid, neutral or alkaline active.     

The quantitation of rat serum esterases by densitometry of acrylamide gels stained for enzyme activity.

Normal rat sera were electrophoresed on polyacrylamide gels. The gels were then stained for (a) esterase activity and (b) protein content. Subsequent analysis on a densitometer permitted quantitative estimation of both enzyme activity and protein concentration. (The specific activity (per mg of protein) of individual serum esterases were then calculated.) Two previously unreported esterases (Bands A and D), were identified and partially characterized. When carefully prepared, non-activated plasma was used, no differences in enzyme activity were found compared to serum.     

Enzyme electrophoretograms in the analysis of taxon relatedness of Micrococcus cryophilus, Branhamella catarrhalis and atypical Neisserias.

Extracts were prepared from Micrococcus cryophilus, several strains of Branhamella catarrhalis and Neisseria spp. Esterases, NADP-dependent isocitrate dehydrogenase and malate dehydrogenase activities were assayed after electrophoresis of extracts of polyacrylamide gels. Except for Neisseria perflava and N. sicca which resolved activity bands for the acetate-esterase only, the remaining bacteria exhibited species-specific esterase patterns also for the propionate and butyrate substrates. The multiple esterase patterns from B. catarrhalis ATCC25238 were qualitatively and quantitatively different from those of B. catarrhalis ATCC23246. This finding and other evidence supports a taxonomic shift of the latter to a species level of that genus. The atypical neisserias N. caviae and N. ovis appeared to exhibit an intrageneric specificity in their esterase patterns with those from B. catarrhalis but not to the other Neisseria spp. tested. The malate dehydrogenase patterns from the atypical neisserias and B. catarrhalis ATCC23246 were qualitatively similar; however, the patterns of isocitrate dehydrogenase activity were variable for these species. Micrococcus cryophilus was distinct in its esterase and dehydrogenase bands, strongly suggesting its taxon unrelatedness to the genus Branhammella or the atypical neisserias. Of the enzymes assayed, esterase proved to be the most reliable for taxonomic identifications.     

BIOCHEMICAL CHANGES DURING SEXUAL DEVELOPMENT IN MARCHANTIA POLYMORPHA: I. ESTERASES

Extracts of uninduced thalli, induced thalli, stalks, antheridiophore and archegoniophore disks of Marchantia polymorpha were subjected to starch-gel zone electrophoresis. Developed gels were treated with appropriate reaction mixtures to detect sites of activity for 12 enzyme systems; only phosphatases, esterases, and peroxidases were observed. Although common sites of phosphatase, peroxidase, and esterase activity were detected in all tested extracts, additional sites of peroxidase and esterase activity were found in extracts from antheridiophore disks. The antheridia provided the additional esterases as determined by the electrophoresis of antheridial extracts and by a histochemical test for esterases in sections of antheridiophores.     

Comparative zone electrophoresis of catalase of Staphylococcus species isolated from mammalian skin.

Vertical polyacrylamide slab gel electrophoresis was conducted for the catalase enzymes of representative strains of 18 proposed species and subspecies of the genus Staphylococcus. The catalase bands which resulted were predominantly monomorphic within each of the species and differences in catalase mobilities were observed between many of the species. The electrophoretic mobilities of the catalases were supportive to the scheme of classification used. Many strains of certain species demonstrated multiple catalase bands which are suggestive of multimolecular forms of the enzyme. Horizontal starch gel electrophoresis of representative strains of S. capitis produced catalase bands with relative mobilities that were different from those obtained with polyacrylamide electrophoresis, presumably due to a difference in molecular sieving between the gels.