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1.
Purification and molecular properties of rabbit liver esterase ES-1A   总被引:1,自引:0,他引:1  
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.  相似文献   

2.
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.  相似文献   

3.
The molecular masses of three rat-plasma carboxylesterases (ES-1, ES-2, and ES-14) were estimated by transverse-gradient polyacrylamide gel electrophoresis and subsequent application of Ferguson-plot-based calculation methods. Two electrophoretic buffer systems were used and the data subjected to either weighted or unweighted regression analysis. The Tris-boric acid buffer system produced significantly higher retardation coefficients than the Tris-glycine system. Molecular mass estimates were significantly higher with the Tris-glycine buffer system. Unweighted instead of weighted analysis produced significantly higher molecular mass estimates. Molecular mass estimates also depended on the calculation method, that is, the choice of calibration relationship with molecular size as a function of retardation coefficient. Three commonly used calibration relationships were compared. On the basis of their accuracy, both the weighted log[retardation coefficient] versus log[molecular mass] plot and the square root of retardation coefficient versus molecular radius were found suitable, provided that the Tris-boric acid buffer was used for electrophoresis. Using the former calibration relationship, the molecular masses of rat-plasma ES-1, ES-2, and ES-14 were 55.5, 61.1, and 65.3 kDa, respectively.  相似文献   

4.
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.  相似文献   

5.
ES-62 is an immunomodulatory phosphorylcholine (PC)-containing glycoprotein secreted by the rodent filarial nematode Acanthocheilonema viteae. Previously, the use of knockout mice has revealed the effects of ES-62 on macrophages and dendritic cells to be dependent on TLR4. However, it is possible that ES-62 may interact with additional proteins on the surfaces of target cells and hence that cells may vary with respect to receptor usage. In this study, we identified by molecular weight, proteins that interact with ES-62 and found differences amongst the immune system cells studied. Thus, whereas lymphocytes appear to have two major interacting proteins of ~135 and ~82 kDa, U937 monocytes only contain an ES-62-binding protein of the latter molecular weight. Binding to the proteins on B cells and U937 cells was blocked by PC, suggesting a critical role for this ES-62 moiety in facilitating interaction. Finally, ES-62 binding is followed by internalization in both macrophages and B cells but only in the former was absence of TLR4 found to block internalization. These findings are consistent with differences in receptor usage by ES-62 amongst different cell-types.  相似文献   

6.
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.  相似文献   

7.
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.  相似文献   

8.
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.  相似文献   

9.
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.  相似文献   

10.
Filarial nematodes, parasites of vertebrates, including humans, secrete immunomodulatory molecules into the host environment. We have previously demonstrated that one such molecule, the phosphorylcholine-containing glycoprotein ES-62, acts to bias the immune response toward an anti-inflammatory/Th2 phenotype that is conducive to both worm survival and host health. For example, although ES-62 initially induces macrophages to produce low levels of IL-12 and TNF-alpha, exposure to the parasite product ultimately renders the cells unable to produce these cytokines in response to classic stimulators such as LPS/IFN-gamma. We have investigated the possibility that a TLR is involved in the recognition of ES-62 by target cells, because phosphorylcholine, a common pathogen-associated molecular pattern, appears to be responsible for many of the immunomodulatory properties of ES-62. We now demonstrate that ES-62-mediated, low level IL-12 and TNF-alpha production by macrophages and dendritic cells is abrogated in MyD88 and TLR4, but not TLR2, knockout, mice implicating TLR4 in the recognition of ES-62 by these cells and MyD88 in the transduction of the resulting intracellular signals. We also show that ES-62 inhibits IL-12 induction by TLR ligands other than LPS, bacterial lipopeptide (TLR2) and CpG (TLR9), via this TLR4-dependent pathway. Surprisingly, macrophages and dendritic cells from LPS-unresponsive, TLR4-mutant C3H/HeJ mice respond normally to ES-62. This is the first report to demonstrate that modulation of cytokine responses by a pathogen product can be abrogated in cells derived from TLR4 knockout, but not C3H/HeJ mice, suggesting the existence of a novel mechanism of TLR4-mediated immunomodulation.  相似文献   

11.
12.
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.  相似文献   

13.
Beta-Glucuronidase has been purified from mouse kidneys previously induced by gonadotrophin to a specific enzyme activity 15 times higher than the non-induced kidney. The purification procedure includes ultrasonication to solubilize the enzyme, acid and ammonium sulfate precipitations, gel filtration in Sephadex G-200, DEAE-ion exchange chromatography, and isoelectric focusing. The resulting product has a specific activity of 284,000 Fishman units/mg of protein, representing a 1,090-fold purification and is 17,000-fold higher than the level in the non-induced kidney. The purified beta-glucuronidase is apparently homogeneous by criteria of gel filtration, sodium dodecyl sulfate gel electrophoresis, and immunodiffusion. Characterization of the purified enzyme showed that it is identical with the lysosomal isoenzymic from electrophoretically, has subunit molecular weight of 74,000 (estimated by sodium dodecyl sulfate gel electrophoresis) and oligomer molecular weight of 300,000. The purified enzyme is stable at high temperature (up to 55 degrees) and at wide range of pH (from 4 to 11). It has a pH optimum for its activity at 4.7 and a Km of 1.18 times 10- minus 4 M. The purification and characterization of this enzyme from mouse kidney will have significance in the understanding of the molecular nature of the isoenzymes of beta-glucuronidase and will be useful in future studies on the mechanism of intracellular transport and distribution of this hydrolase.  相似文献   

14.
Genetic variation of a new codominantly inherited esterase, designated ES-17, has been discovered in the house mouse using isoelectric focusing in polyacrylamide gels. The ES-17 A phenotype (three bands; isoelectric points, betweenpH 5.55 andpH 5.90) was found in C57 BL/10Sn. LP/J possessed the Es-17B phenotype (three bands; isoelectric points,pH 5.05–5.55). ES-17 was present in all tissues examined, except for hemolysate and serum, and was most clearly expressed in the small intestine. Because of its reaction toward various substrates and inhibitors, ES-17 has tentatively been classified as acetyl esterase (EC 3.1.1.6). ES-17 was shown to be controlled by the structural locusEs-17, located on chromosome 9. From test-cross data, a gene order ofEs-17-8.7±2.5 map units-Mpi-1-10.2±2.7 map units-Mod-1 was established. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 46). This is communication No. 35 of a research program devoted to the cellular distribution and genetics of nonspecific esterases.  相似文献   

15.
1. Phosphatase II is a form of phosphoprotein phosphatase originally found in rat liver extract; it has a molecular weight of 160 000 by gel filtration and is highly active towards phosphorylase alpha. This phosphatase has been purified 1800-fold by using DEAE-cellulos (DE-52), aminohexyl--Sepharose-4B, protamine--Sepharose-4B and Sephadex G-200 chromatography. Throughout the purification steps, the original molecular weight and substrate specificity of phosphatase II were almost perfectly preserved. 2. The product of the final purification step migrated predominantly as a single protein band on non-denaturing gel electrophoresis. Sodium dodecyl sulfate gel electorphoresis revealed that the enzyme contains two types of subunit, alpha and beta, with molecular weights of 35 000 and 69 000, respectively. When treated with 0.2 M 2-mercaptoethanol at -20 degrees C, phosphatase II was dissociated to release the catalytically active alpha subunit. The beta subunit may be catalytically inactive but interacts with the alpha subunit so that phosphatase II becomes much less susceptible than the alpha subunit to inactivation by ATP or pyrophosphate.  相似文献   

16.
ES-62, a protein secreted by filarial nematodes, parasites of vertebrates including humans, has an unusual posttranslational covalent addition of phosphorylcholine to an N-type glycan. Studies on ES-62 from the rodent parasite Acanthocheilonema viteae ascribe it a dominant role in ensuring parasite survival by modulating the host immune system. Understanding this immunomodulation at the molecular level awaits full elucidation but distinct components of ES-62 may participate: the protein contributes aminopeptidase-like activity whereas the phosphorylcholine is thought to act as a signal transducer. We have used biophysical and bioinformatics-based structure prediction methods to define a low-resolution model of ES-62. Sedimentation equilibrium showed that ES-62 is a tightly bound tetramer. The sedimentation coefficient is consistent with this oligomer and the overall molecular shape revealed by small angle x-ray scattering. A 19 A model for ES-62 was restored from the small-angle x-ray scattering data using the program DAMMIN which uses simulated annealing to find a configuration of densely packed scattering elements consistent with the experimental scattering curve. Analysis of the primary sequence with the position-specific iterated basic local alignment search tool, PSI-BLAST, identified six closely homologous proteins, five of which are peptidases, consistent with observed aminopeptidase activity in ES-62. Differences between the secondary structure content of ES-62 predicted using the consensus output from the secondary structure prediction server JPRED and measured using circular dichroism are discussed in relation to multimeric glycosylated proteins. This study represents the first attempt to understand the multifunctional properties of this important parasite-derived molecule by studying its structure.  相似文献   

17.
The molecular size of bovine brain mitochondrial monoamine oxidase (MAO) was investigated Mitochondria were solubilized with an anionic detergent. Emarl 20C, and fractionated by ammonium sulfate. Ammonium sulfate-fractionated MAO was subjected to detergent-containing gel chromatography and detergent-containing gel electrophoresis. MAO activity appeared as single symmetrical peak in gel chromatography in the presence of 1% Emarl 20C, and the molecular weight was estimated to be 44,000. Polymerization of MAO was observed when gel chromatography was performed in lower (0.1%, 0%) concentrations of Emarl 20C. Activity staining of MAO after electrophoresis on a gel containing 0.1% Emar 20C was successful. The molecular weight of MAO estimated from the mobility of this stained band was 89,000. It is suggested that the molecular weight of MAO is 44,000 and that it recombines in low concentrations of the detergent to form complex particles with molecular weights of 89,000 or more.  相似文献   

18.
Esterase-27A (ES-27A) was characterized in strain A/WySnA by a cascade of seven bands seen after disc electrophoresis of serum and subsequent staining for esterase. ES-27A catalyses the hydrolysis of thiocholine butyrate and is strongly inhibited by 100 microM tetraisopropyl pyrophosphamide (isoOMPA). Hence, the enzyme was concluded to be a cholinesterase EC 3.1.1.8. A heat-labile form termed ES-27B was represented by strain AKR/Han. From a three-point cross (AKR/Han, A/Wy) and a five-point cross (AKR/Han, SEG/1), the gene order on chromosome 3 was concluded to be centromere-Car-2-Es-26-Es-27-Amy-1-Adh-1.  相似文献   

19.
Parasitic nematodes manufacture various carbohydrate-linked phosphorylcholine (PCh)-containing molecules, including ES-62, a protein with an N-linked glycan terminally substituted with PCh. The PCh component is biologically important because it is required for immunomodulatory effects. We showed that most ES-62 was bound to a single protein, C-reactive protein (CRP), in normal human serum, displaying a calcium-dependent, high-avidity interaction and ability to form large complexes. Unexpectedly, CRP binding to ES-62 failed to efficiently activate complement as far as the C3 convertase stage in comparison with PCh-BSA and PCh-containing Streptococcus pneumoniae cell wall polysaccharide. C1q capture assays demonstrated an ES-62-CRP-C1q interaction in serum. The three ligands all activated C1 and generated C4b to similar extents. However, a C2a active site was not generated following ES-62 binding to CRP, demonstrating that C2 cleavage was far less efficient for ES-62-containing complexes. We proposed that failure of C2 cleavage was due to the flexible nature of carbohydrate-bound PCh and that reduced proximity of the C1 complex was the reason that C2 was poorly cleaved. This was confirmed using synthetic analogues that were similar to ES-62 only in respect of having a flexible PCh. Furthermore, ES-62 was shown to deplete early complement components, such as the rate-limiting C4, following CRP interaction and thereby inhibit classical pathway activation. Thus, flexible PCh-glycan represents a novel mechanism for subversion of complement activation. These data illustrate the importance of the rate-limiting C4/C2 stage of complement activation and reveal a new addition to the repertoire of ES-62 immunomodulatory mechanisms with possible therapeutic applications.  相似文献   

20.
Polysomes or mRNA prepared from cultured AtT-20/D16v mouse pituitary adenocarcinoma cells direct the efficient incorporation of amino acid into newly synthesized material in the presence of wheat germ translational factors. A significant franction of the total cell-free product is specifically immunoprecipitable with corticotropin antibody purified by immune affinity chromatography. Analysis of the cell-free synthesized immunoreactive products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals that two high molecular weight corticotropin species (Mr congruent to 32,500 and 28,000) are synthesized in an approximate 2:1 ratio. Neither product contains carbohydrate based upon concanavalin A chromatography or exposure to polysaccharidases. The smaller molecular weight product does not appear to arise from proteolytic processing since both species are synthesized in approximately the same ratio in cell-free reaction mixtures directed by either polysomes or mRNA. These results suggest that AtT-20/D16v cells contain two distinct mRNA poluations specifying the synthesis of two different high molecular weight forms of mouse corticotropin.  相似文献   

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