玉米酯酶同工酶和过氧化物酶同工酶的分子量研究

本试验利用聚丙烯酰胺凝胶梯度电泳分步染色法直接对玉米苗期酯酶同工酶和过氧化物酶同工酶各酶带的分子量进行了比较测定。酯酶同工酶 E_1、E_2、E_3~F、E_3~S、a、b、c 各酶带的分子量分别为<20000,35200、33000、38500、29900、28500、34000道尔顿过氧化物酶同工酶 PX_4~F和 PX_4~S酶带的分子量分别为131000和149000道尔顿。根据酶带在均匀胶和梯度胶中的位置变化对各酶带的生化性质作了初步分析,发现 E_3~F和 E_3~S、PX_4~F 和 PX_4~S 在迁移率上的差异主要是分子量的差异。本文为同工酶的分子量测定提供了一个简便的方法。     

Studies on the Molecular Weights of Esterase and Peroxidase Isozymes of Maize

The molecular weights of esterase and peroxidase isozymes of maize seedlings were directly determined by improved polyacrylamide gradient gel electrophoresis. The different isozyme bands developed in polyacrylamide slab gel electrophoresis (uniform gel) were identified in polyacrylamide gradient gel electrophoresis by means of isozyme variants. The molecular weights of esterase isozymes E1, E2, E3F, E3S, a, b, c, named according to isozyme patterns in uniform gel, are <20000, 35200, 33000, 38500, 29900, 28500, 34000 doltons respectively. The molecular weights of peroxidase isozymes PX4F and PX4S are 131000 and 149000 doltons respectively. According to the band location in uniform gel and in gradient gel, some biochemical properties of the isozyme bands and relationships between the isozyme bands were analyzed. The possible errors in the determination of smaller molecular weight isozymes are discussed.     

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.     

The esterase profile of a lipase from Candida cylindracea

A commercial preparation of a lipase produced by Candida cylindracea catalysed the hydrolysis of both long- and short-chain esters of p-nitrophenol. Six major bands of hydrolytic activity to alpha-naphthyl acetate were detected on polyacrylamide gel electrophoresis and two on isoelectric focusing. The esterase activity fractionated into two major peaks of activity on ion-exchange chromatography and into several peaks of activity on hydrophobic interaction chromatography. These esterase activities showed different substrate specificities to p-nitrophenyl esters, tributyrin and cetyl palmitate.     

Pregnancy-associated esterase in sera of baboons

Baboon serum samples were resolved by starch gel electrophoresis and polyacrylamide gradient gel electrophoresis and stained with naphthol substrates for esterase activity. An esterase that hydrolyzed alpha-naphthyl butyrate in preference to alpha-naphthyl acetate was found in very high activities in some individuals but not others. It migrated just cathodal of the albumin band in starch gels. In polyacrylamide gradient gels, it co-migrated with albumin and had an apparent molecular weight of approximately 65,000 daltons. Electrophoretic analysis by gel electrophoresis of random serum samples from male and female baboons indicated that this esterase was present only in the sera of pregnant baboons. Further investigation of serial samples collected from carefully monitored baboons confirmed that the amount of activity of this esterase was correlated with stage of pregnancy. Therefore, it was named pregnancy esterase (PE). PE was detectable by gel electrophoresis and chromogenic staining techniques as early as day 30 of pregnancy; its activity gradually increased with progressive pregnancy and reached maximum activity near full term (182 days). Soon after parturition, the activity of PE decreased rapidly and was not detected in maternal sera by day 14 postpartum. No evidence of PE was detected in sera of pregnant humans.     

Hydrolysis of plant cuticle by plant pathogens. Purification, amino acid composition, and molecular weight of two isozymes of cutinase and a nonspecific esterase from Fusarium solani f. pisi.

The extracellular fluid of the plant pathogen, Fusarium solani f. pisi, grown on the plant cuticular polymer, cutin, was shown to contain cutinase and p-nitrophenyl palmitate hydrolase activities (R.E. Purdy and P.E. Kolattukudy (1973), Arch. Biochem. Biophys. 159, 61). From this extracellular fluid two isozymes of cutinase and a nonspecific esterase (p-nitrophenyl palmitate hydrolase) were isolated using Sephedex G-100 gel filtration, QAE-Sephadex chromatography, and SE-Sephedex chromatography. Phenolics contained in the extracellular fluid were found to be associated with the cutinase but not with the nonspecific esterase, and the phenolic materials were removed from cutinase at the QAE-Sephedex step. A 34-fold purification of the nonspecific esterase and a 6.5-fold purification of cutinase were achieved by the procedure described. The two isozymes of cutinase (I and II) and the nonspecific esterase were homogeneous as judged by polyacrylamide disc gel electrophoresis and sedimentation equilibrium centrifugation. Molecular weights of cutinase I, cutinase II, and the nonspecific esterase were determined by Sephedex G-100 gel filtration, sedimentation equilibrium centrifugation, amino acid composition, and sodium dodecyl sulfate polyacrylamide disc gel electrophoresis. The values obtained with these techniques agreed with each other and were about 22,000 for both cutinases and 52,000 for the nonspecific esterase. The dodecyl sulfate gel electrophoresis indicated that a small portion of cutinase II contained proteolylic clips, near the middle of the polypeptide chain, and that the nonspecific esterase might also have undergone some proteolylic modification. The amino acid composition of cutinase I was similar to that of cutinase II except for the presence of a larger number of tryptophan residues in the latter, while the amino acid composition of the nonspecific esterase showed more differences from that of either cutinase.     

A new variant of the Es-1 locus in the rat

A new prealbumin plasma esterase was demonstrated by the use of miniaturized polyacrylamide slab gel electrophoresis. Genetic analysis indicated that the new variant is controlled by the Es-1 locus and this gene was designated Es-1c. Among 11 rat strains only one strain, WJ, possessed the gene. Two random-bred stocks, Jcl:Wistar and Jcl:SD, also maintained it in their populations. Miniaturized polyacrylamide slab gel electrophoresis showed that the ES-1 band consisted of two close bands with the cathodal one staining darker.     

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.     

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.     

东亚飞蝗山西两地理种群酯酶特性的比较研究

对东亚飞蝗山西临猗和永济2个地理种群的酯酶特性进行了比较研究.非变性聚丙烯酰胺凝胶电泳图谱显示:以a-乙酸萘酯为底物染色,2个东亚飞蝗种群谱带差别不明显.但是,酯酶动力学研究结果表明:以a-乙酸萘酯和α-丁酸萘酯为底物时,永济种群的酯酶活性分别是临猗种群的1.81倍和1.20倍.永济种群酯酶活性的增高可能与非变性聚丙烯酰胺凝胶电泳图谱显示出较临猗种群多出的酶带有关.体外酯酶抑制动力学研究表明:永济和临猗2种群所含酯酶大都为B型酯酶,其含量分别为84.94%和91.47%.永济种群对对氧磷的耐受性要高于临猗种群,我们推测可能与2种群马拉硫磷使用背景不同有关.     

东亚飞蝗山西两地理种群酯酶特性的比较研究

对东亚飞蝗山西临猗和永济2个地理种群的酯酶特性进行了比较研究。非变性聚丙烯酰胺凝胶电 泳图谱显示：以α-乙酸萘酯为底物染色,2个东亚飞蝗种群谱带差别不明显。但是,酯酶动力 学研究结果表明：以α-乙酸萘酯和α-丁酸萘酯为底物时,永济种群的酯酶活性分别是临猗 种群的1.81倍和1.20倍。永济种群酯酶活性的增高可能与非变性聚丙烯酰胺凝胶电泳图谱显 示出较临猗种群多出的酶带有关。体外酯酶抑制动力学研究表明：永济和临猗2种群所含酯酶大 都为B型酯酶,其含量分别为84.94%和91.47%。永济种群对对氧磷的耐受性要高于临猗种群 ,我们推测可能与2种群马拉硫磷使用背景不同有关。     

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.     

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.     

以靛酚乙酸酯为底物的酯酶同工酶显色方法的研究

建立一种以靛酚乙酸酯为底物的酯酶同工酶的显色新方法。酯酶样品的聚丙烯酰胺凝胶电泳（PAGE）凝胶用磷酸缓冲液漂洗约10min后,浸入含有0.002%靛酚乙酸酯的溶液显色5～10min,可显出清晰的蓝色酯酶带。先将酯酶凝胶板浸于有机磷农药溶液中,然后再用靛酚乙酸酯显色液显色,比较同工酶谱,从同工酶带由深蓝色变为浅蓝色的颜色变化,可以看出对有机磷农药敏感的同工酶所受到的抑制程度。     

Female specific proteins in Triatoma infestans haemolymph

The presence of female specific proteins in triatoma infestans haemolymph, as well as the relationship between the female specific proteins and egg proteins, were analysed. At the same time, the presence of specific female proteins in different instars was studied. Cellulose acetate electrophoresis, polyacrylamide gel electrophoresis, and immunochemical methods were used.No differences between immature female and male haemolymph were established. Female haemolymph obtained from insects with ovary development revealed quantitative differences, with cellulose acetate, with respect to the control males. With polyacrylamide gel electrophoresis, one component that is not detected in control males was detected in mature female haemolymph. With immunochemical assays, at least two antigenic components that were not observed in male haemolymph were detected.Egg extract showed, with cellulose acetate, two bands with a mobility similar to that of the proteins increased in the haemolymph of the mature female; with polyacrylamide gel, two major bands with a mobility similar to that of the specific female haemolymph protein were detected. Egg extract contains at least two components demonstrated by double-diffusion assays and three components by immunoelectrophoresis, with immunological identity to specific mature female haemolymph proteins.The extract obtained from recently hatched insects revealed two components with immunological identity to specific female proteins. Haemolymph from first, second, third, fourth and fifth instars do not appear to contain any femalespecific haemolymph protein.     

吡虫啉对不同发育阶段蜘蛛酯酶同工酶活性的影响

本文采用聚丙烯酰胺凝胶电泳技术研究了吡虫啉对不同发育阶段星豹蛛酯酶同工酶活性的影响.结果表明,星豹蛛酯酶同工酶酶带可分为三个区域,其中EST-3区的酶带颜色最深,酶活性最强;EST-1区和EST-2区酶带颜色较浅,酶活性较弱.星豹蛛不同发育阶段酶带数由多到少的顺序为:雌成蛛＞卵袋＞雄成蛛＞若蛛,酶活性由强到弱的顺序为:...     

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

The electrophoretic mobilities of non-specific esterases in vertical polyacrylamide slab gels were determined for 184 strains of staphylococci, representing a total of 18 proposed species and subspecies. Markedly uniform esterase patterns were seen within species demonstrating a high degree of human host specificity, while those species demonstrating a wide host range were polytypic and often showed considerable polymorphism. The unique banding patterns found in several species indicate that this technique may serve as a valuable aid to existing taxonomic schemes. Starch gel electrophoresis of representative strains usually produced sharper esterase bands than were found with polyacrylamide electrophoresis. However, the additional molecular-sieving effect produced by the polyacrylamide gels differentiated esterases to a greater extent.     

Purification and characterization of the tween-hydrolyzing esterase of Mycobacterium smegmatis.

An esterase hydrolyzing Tween 80 (polyoxyethylene sorbitan monooleate) was purified from sonicated cell lysates of Mycobacterium smegmatis ATCC 14468 by DEAE-cellulose, Sephadex G-150, phenyl Sepharose, and diethyl-(2-hydroxypropyl) aminoethyl column chromatography and by subsequent preparative polyacrylamide gel electrophoresis. The molecular weight was estimated to be 36,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 41,000 by gel filtration on a Sephadex G-150 column. The esterase contained a single polypeptide. The esterase was stable to heat treatment at 100 degrees C and to a wide range of pH. The temperature and pH optima for the hydrolysis of Tween 80 were 50 degrees C and 8.3, respectively. The esterase had a narrow substrate specificity; it exhibited a high activity only on compounds having both polyoxyethylene and fatty acyl moieties, such as Tweens. Monoacylglyceride was hydrolyzed more slowly by this esterase and this enzyme exhibited a nonspecific esterase activity on p-nitrophenyl acyl esters, especially those having short chain acyl moieties. The Km and Vmax were 19.2 mM and 1,670 mumol/min per mg of protein for Tween 20, 6.6 mM and 278 mumol/min per mg of protein for Tween 80, and 0.25 mM and 196 mumol/min per mg of protein for p-nitrophenyl acetate, respectively. Observations of the effects of various chemical modifications on the activity of the esterase indicated that tyrosine, histidine, arginine, and methionine (with tryptophan) residues may be active amino acids which play important roles in the expression of Tween 80-hydrolyzing activity of the enzyme.     

Purification and characterization of esterases D1 and D2 from human erythrocytes. Evidence that they are monomers

Esterase D1 and esterase D2, two common esterase D (EC 3.1.1.1) isozymes, were isolated and purified from human erythrocytes. Their substrate specificity, pH profile and Km values were essentially identical. Their molecular mass was the same at 34 kDa on sodium dodecyl sulfate/polyacrylamide electrophoresis and at 27 kDa on Sephadex G-100 gel filtration. Antisera to each of the esterase D1 and esterase D2 isozymes were successfully raised in chickens; each antiserum reacted identically with both isozymes. These findings indicate that the isozymes are close to each other in structure. The fact that the molecular mass of the esterase D1 and esterase D2 isozymes computed on sodium dodecyl sulfate/polyacrylamide electrophoresis was close to that obtained on Sephadex G-100 gel filtration in non-dissociating buffer indicates that the isozymes are not dimers bound by disulfide bonds or a noncovalent force. These facts together indicate that the esterase D isozymes are monomers, contrary to the prevailing view that they are dimers.     

Carbonate dehydratase (carbonic anhydrase) in a spider. Association with the hemolymph lipoprotein

Carbonate dehydratase was detected dissolved in the hemolymph of the tarantula, Eurypelma californicum. The enzyme was purified 31-fold by gel filtration, anion-exchange chromatography, a second gel filtration, and finally, preparative polyacrylamide gel electrophoresis. Zinc content increased during purification to up to 2.4 mol Zn/100 000 g of protein (= 1.58 mg Zn/g protein). In the polyacrylamide electrophoresis of tarantula hemolymph under non-denaturing conditions three major protein bands were observed: hemocyanin, a 16 S lipoprotein and the active band which migrated closely behind the 16 S lipoprotein. After treatment with sodium dodecyl sulfate both the carbonate dehydratase-active protein and the lipoprotein revealed bands corresponding to Mr = 95 000 and 110 000, respectively, but the enzymatically active protein revealed an additional third band with Mr = 40 000. The latter band is though to represent the 'true' carbonate dehydratase protein. Upon isoelectric focusing of material containing carbonate dehydratase activity and lipoprotein, bands were obtained at pH 5.45, 5.6 and 5.7. The band at pH 5.6 contained the peak of enzyme activity, and upon dodecyl sulfate-polyacrylamide gel electrophoresis showed the highest proportion of the 40-kDa polypeptide. It is concluded that tarantula carbonate dehydratase, instead of forming a high molecular mass aggregate, is associated with the 16 S lipoprotein, the latter serving as a carrier for the enzyme. The lipoprotein is probably also involved in other transport processes. It is present in great excess and may therefore occur in two forms, charged with carbonate dehydratase or uncharged. Tarantula carbonate dehydratase is inhibited by acetazolamide and by dansylamide, but not by a number of other known inhibitors, most notably not by 4-(aminomethyl)benzenesulfonamide. Treatment with 1M urea does not affect specific enzyme activity, while 2M urea inhibits by 50%. 2-Mercaptoethanol inhibits activity by 50% at 0.1M. Like other carbonate dehydratases, the tarantula enzyme shows esterase activity. The Km for 4-nitrophenyl acetate is 5mM.