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1.
Polygalacturonase from Rhizopus stolonifer, an Elicitor of Casbene Synthetase Activity in Castor Bean (Ricinus communis L.) Seedlings 总被引:25,自引:15,他引:10
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Apparently homogeneous polygalacturonase-elicitor purified from the filtrates of Rhizopus stolonifer cultures stimulates germinating castor bean seedlings to produce greatly increased levels of casbene synthetase activity. The purification procedure involved gel-filtration chromatography on Sephadex G-25 and G-75 columns followed by cation-exchange chromatography on a Sephadex CM C-50 column. Homogeneity of the purified preparation was indicated by the results of cationic polyacrylamide disc gel electrophoresis and isoelectric focusing (pI = 8.0). The identity of the casbene elicitor activity and polygalacturonase were indicated by the coincidence of the two activities at all stages of purification, the coincidence of both activities with the single protein-staining band detected on a cationic polyacrylamide disc gel and an isoelectric focusing gel, and the identical behavior of both activities on an agarose gel affinity column. The purified polygalacturonase-elicitor is a glycoprotein with approximately 20% carbohydrate content and an estimated molecular weight of 32,000 by polyacrylamide disc gel electrophoresis. 相似文献
2.
Acyl hydrolase activities have been purified from the leaves of Phaseolus multiflorus. The purification procedure involved heat treatment, DEAE-cellulose chromatography, Sephadex G-100 filtration and hexyl agarose chromatography. The elution pattern from hexyl agarose columns together with substrate competition experiments indicated the presence of two hydrolase enzymes. The first could hydrolyse oleoylglycerol and phosphatidylcholine while the second would deacylate glycosylglycerides and oleoylglycerol. Overall purification of both enzymes was ca 70-fold and the MW of the glycosylglyceride-hydrolysing enzyme was in the range 70–78000. 相似文献
3.
Invertase inhibitor from potatoes: purification, characterization, and reactivity with plant invertases 总被引:8,自引:6,他引:2
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Pressey R 《Plant physiology》1967,42(12):1780-1786
Invertase inhibitor was extracted from potato tubers and purified nearly 1000-fold. The purification procedure involved precipitation at pH 4.0, fractionation with ammonium sulfate, adsorption on alumina Cγ gel, and gel filtration on Sephadex G-100 and DEAE-Sephadex A-50. The product obtained was homogeneous to electrophoresis on polyacrylamide gel. Exclusion chromatography on Sephadex G-100 indicated a molecular weight of about 17,000. The inhibitor did not inhibit yeast, Neurospora, and several plant invertases. It completely inhibited potato tuber invertase and a number of other plant invertases. Some plant invertases were partially inhibited. 相似文献
4.
John C. Cox 《Applied microbiology》1975,29(4):464-468
Diptheria toxin of high purity was prepared in batch cultures of 1 to 40 liters by procedures capable of processing 200-liter batches without modification. The procedure incorporates preliminary purification of the growth medium and, after deep fermentation or surface culture of Corynebacterium diphtheriae, both concentration and partial purification of the toxin by membrane ultrafiltration. Final purification is achieved by Sephadex G-100 gel filtration. Purities of 2,000 to 2,500 flocculation units per mg of protein nitrogen (260 to 410 flocculation units per unit of absorbance at 280 nm) were routinely obtained with only a 10% loss of toxin. The toxin appeared pure on immunoelectrophoresis and ultracentrifugation, and only minor amounts of lower-molecular-weight impurities were revealed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Significant advantages of the procedure are its rapidity and reproducibility and the fact that all stages are performed at 4 C in neutral isotonic buffer. 相似文献
5.
Column Chromatography and Cell Culture Assay of Pseudomonas aeruginosa Toxin Z Preparations 总被引:1,自引:0,他引:1
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Toxic material produced by Pseudomonas aeruginosa in cell culture was concentrated and partially purified. This toxic material, designated toxin Z, was produced during the growth of strain PA Z or PA 103 in HEp-2 monolayer cultures using Eagle minimal essential medium with 10% serum. Toxin Z, concentrated fourfold by Lyphogel or ultrafiltration, was used to produce antiserum in rabbits and also was fractionated by column chromatography, Twentyfold purification of toxin Z was obtained on a Sephadex G-200 column. Toxic column fractions were confirmed to have toxin Z by neutralization with specific antiserum. During concentration, purification, and neutralization procedures, the toxin was assayed exclusively by the cytopathic effect it produced in cell culture. 相似文献
6.
Bacteriolytic enzymes from Staphylococcus aureus. Purification of an endo-β-N-acetylglucosaminidase 总被引:1,自引:1,他引:0
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On cultivation of Staphylococcus aureus in a complex liquid medium, bacteriolytic activity is found extracellularly. The maximal amount was found at the end of the exponential growth phase in batch culture, but in continuous culture run under similar conditions the yield was doubled. Isoelectric focusing of dialysed crude culture supernatants showed that the bacteriolytic activity of all four strains studied (M18, 524, Wood 46 and Duncan) was heterogeneous. The most alkaline peak of activity (isoelectric point 9.5±0.1) was assayed against Micrococcus lysodeikticus turbidimetrically. This bacteriolytic activity was purified more than 70-fold after continuous dialysis by adsorption on CM-Sephadex, precipitation with ethanol, heat purification, isoelectric focusing and Sephadex G-100 chromatography. The purified enzyme (isoelectric point 9.6±0.1) was found to give a single band on polyacrylamide-gel and cellulose acetate electrophoresis and was devoid of all 14 staphylococcal enzymes and toxins assayed for. The molecular weight is 70000±5000 as estimated by Sephadex G-100 and G-200 chromatography. The marked instability of the partially and highly purified enzyme was investigated. The mode of action and some properties of this enzyme are given in the following papers (Wadström & Hisatsune, 1970; Wadström, 1970). These results indicate that this extracellular enzyme which is produced by several strains of S. aureus is not a `lysozyme' (endo-β-N-acetylmuramidase) as previously suggested, but an endo-β-N-acetylglucosaminidase. 相似文献
7.
Allosteric interactions in glycerol dehydratase. Purification of enzyme and effects of positive and negative cooperativity for glycerol 总被引:2,自引:0,他引:2
An improved method for the purification of the apoenzyme (AB complex) of glycerol dehydratase from Aerobacter aerogenes is presented. One hundredfold purification Was achieved. This purification was possible due to stabilization of the AB complex by glycerol. Using chromatography on Sephadex G-200, the highest degree of association of AB complex was found in glycine buffer in the presence of glycerol.Potassium ions, in contrast to glycerol, seem to weaken the forces which bind subunits A and B together. This may be the action of potassium necessary for the performance of glycerol dehydratase activity, since potassium is required for holoenzyme activity.From kinetic studies it appears that the enzyme exhibits homotrophic effects with regard to glycerol binding sites, which can he extended from positive (in the presence of glycine buffer) to negative (in the presence of ethanolamine buffer) cooperativity. The high cooperativity between glycerol binding sites on the enzyme, in glycine buffer, can be abolished by the addition of phosphate. By decreasing the value of the Hill coefficient and increasing the V of the enzymatic reaction, phosphate seems to act as an allosteric activator. 相似文献
8.
Purification and Characterization of Sucrose Synthetase from the Shoot of Bamboo Leleba oldhami 总被引:6,自引:5,他引:1
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Su JC 《Plant physiology》1977,60(1):17-21
A 108-fold purification of the sucrose synthetase from the extract of the shoot of bamboo Lelaba oldhami was achieved by ammonium sulfate fractionation, calcium phosphate gel adsorption, and chromatographic separations on Sephadex G-100 and diethylaminoethyl-cellulose columns. Some properties of this enzyme, namely thermal and pH stabilities, stabilization by aqueous glycerol, pH optimum, substrate specificities, effects of metallic ions, effects of sulfhydryl reagents, molecular weight, sedimentation constants, isoelectric point, and substrate saturation kinetics had been investigated. 相似文献
9.
John O. Hui Alfredo G. Tomasselli Ilene M. Reardon June M. Lull David P. Brunner Che-Shen C. Tomich Robert L. Heinrikson 《The protein journal》1993,12(3):323-327
The protease encoded by the human immunodeficiency virus type 1 (HIV-1) was engineered inEscherichia coli as a construct in which the natural 99-residue polypeptide was preceded by an NH2-terminal methionine initiator. Inclusion bodies harboring the recombinant HIV-I protease were dissolved in 50% acetic acid and the solution was subjected to gel filtration on a column of Sephadex G-75. The protein, eluted in the second of two peaks, migrated in SDS-PAGE as a single sharp band ofM r ≈ 10,000. The purified HIV-1 protease was refolded into an active enzyme by diluting a solution of the protein in 50% acetic acid with 25 volumes of buffer atpH 5.5. This method of purification, which has also been applied to the purification of HIV-2 protease, provides a single-step procedure to produce 100 mg quantities of fully active enzyme. 相似文献
10.
sn-Glycerol-1-Phosphate-Forming Activities in Archaea: Separation of Archaeal Phospholipid Biosynthesis and Glycerol Catabolism by Glycerophosphate Enantiomers 总被引:1,自引:0,他引:1
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Masateru Nishihara Tomoko Yamazaki Tairo Oshima Yosuke Koga 《Journal of bacteriology》1999,181(4):1330-1333
In Methanobacterium thermoautotrophicum, sn-glycerol-1-phosphate (G-1-P) dehydrogenase is responsible for the formation of the Archaea-specific backbone of phospholipids, G-1-P, from dihydroxyacetonephosphate (DHAP). The possible G-1-P-forming activities were surveyed in cell-free extracts of six species of Archaea. All the archaeal cell-free homogenates tested revealed the ability to form G-1-P from DHAP. In addition, activities of G-3-P-forming glycerol kinase and G-3-P dehydrogenase were also detected in four heterotrophic archaea, while glycerol kinase activity was not detected in two autotrophic methanogens. These results show that G-1-P is produced from DHAP by G-1-P dehydrogenase in a wide variety of archaea while exogenous glycerol is catabolized via G-3-P. 相似文献
11.
A simple and rapid purification procedure is described for the routine preparation of large quantities of purified chlorophyllase (chlorophyll chlorophyllido-hydrolase, EC 3.1.1.14) from Chlorella protothecoides. The enzyme with specific activity of 960 nmol chlorophyll a hydrolyzed (mg protein)?1 min?1 was prepared by treating the homogenate with n-butanol, ammonium sulfate fractionations and gel filtration through Sephadex G-200 and Sepharose CL-6B, with a yield of 53% of activity based on the butanol extract. The enzyme preparation showed apparent homogeneity as judged by polyacrylamide gel electrophoresis. The procedures take only 4 days and can be operated routinely without column repacking. 相似文献
12.
A long chain acyl-CoA reductase of Clostridium butyricum has been partially purified from the 100,000g supernatant fraction of cell extracts. The enzyme reduces acyl-CoA derivatives to aldehydes in the presence of NADH. It is stable in dithiothreitol-containing buffers at 4 °C, heat-labile, and sensitive to sulfhydryl reagents. It is active with palmitoyl-CoA, stearoyl-CoA, oleoyl-CoA, and myristoyl-CoA. Its apparent molecular weight on Sephadex G-100 column chromatography is 50,000. In crude extracts and at low purification, an NADPH-dependent reduction of palmitaldehyde to cetyl alcohol was also observed. An acyl-CoA hydrolase was also observed in crude extracts. 相似文献
13.
1-Aminocyclopropane-1-carboxylate (ACC) N-malonyltransferase converts ACC, an immediate precursor of ethylene, to the presumably inactive product malonyl-ACC (MACC). This enzyme plays a role in ethylene production by reducing the level of free ACC in plant tissue. In this study, ACC N-malonyltransferase was purified 3660-fold from etiolated mung bean (Vigna radiata) hypocotyls, with a 6% overall recovery. The final specific activity was about 83,000 nmol of MACC formed mg−1 protein h−1. The five-step purification protocol consisted of polyethylene glycol fractionation, Cibacron blue 3GA-agarose chromatography using salt gradient elution, Sephadex G-100 gel filtration, MonoQ anion-exchange chromatography, and Cibacron blue 3GA-agarose chromatography using malonyl-CoA plus ACC for elution. The molecular mass of the native enzyme determined by Sephadex G-100 chromatography was 50 ± 3 kD. Protein from the final purification step showed one major band at 55 kD after sodium dodecyl sulfate polyacrylamide gel electrophoresis, indicating that ACC N-malonyltransferase is a monomer. The mung bean ACC N-malonyltransferase has a pH optimum of 8.0, an apparent Km of 0.5 mm for ACC and 0.2 mm for malonyl-coenzyme A, and an Arrhenius activation energy of 70.29 kJ mol−1 degree−1. 相似文献
14.
Kensuke Kameda Lidia K. Suzuki Yoh Imai 《Biochimica et Biophysica Acta (BBA)/General Subjects》1985,840(1):29-36
Acyl-CoA synthetase was further purified fromEscherichia coli in good yield and fold purification by affinity chromatography on CoA-Sepharose 4B. The molecular weight of the active form of the purified enzyme was estimated as 45 000 by Sephadex G-100 and 47 000 by Sephadex G-200. Sedimentation equilibrium ultracentrifugation analysis revealed a molecular weight of 50 000. The sedimentation coefficient was calculated as 4.4. S. An absorption maximum at 276 nm was observed in the ultraviolet light absorption spectrum. The molar extinction coefficient was 9.2 · 104. Kinetic constants were determined fortrans fatty acids. All ions tested, including chaotropic and lyotropic ions, stimulated or inhibited acyl-CoA synthetase activity depending on their concentrations in the assay system. In a series of chaotropes, the lower concentration required to maximally activate acyl-CoA synthetase in increasing order of potency of chaotropic ions. The inhibitory effect of chaotrope on the enzyme activity was reversible. These data suggest that salts have a common mode of action and influence acyl-CoA synthetase activity primarily through their effect on the solution structure. 相似文献
15.
Glyoxalase I and glyoxalase II (EC. 4.4.1.5 and EC 3.1.2.6) were separated by gel filtration on Sephadex G-75 and G-100. This simple procedure permitted also the partial purification of glyoxalase II. The purification coefficient in a single run from supernatant from beef liver was about 1 : 30 compared with 1 : 15 after the fifth step of purification with classical methods. 相似文献
16.
Hajime Fugo Hitoshi Saito Hiromichi Nagasawa Akinori Suzuki 《Journal of insect physiology》1985,31(4):293-298
The ultimate timing of hatching in the silkworm, Bombyx mori, is controlled by a circadian oscillator. The presence of eclosion hormone in developing embryos of the silkworm is demonstrated. Eclosion hormone activity first becomes detectable in embryos which have developed almost to the stage of the differentiation of the neuroendocrine system. Hormonal activity increases sharply to a maximum level 1 day before hatching and falls by about a half in the newly hatched larvae. Eclosion hormone was partially purified from the pharate first-instar larvae and approx, a 2100-fold purification was achieved. The molecular weight of the embryo eclosion hormone is estimated to be 7000 ~ 9000 Daltons by gel-filtration on Sephadex G-50 (superfine). The role of eclosion hormone on hatching behaviour of the silkworm, Bombyx mori, is discussed. 相似文献
17.
Maria E. Gallego Claudio F. Heredia 《Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression》1982,696(1):57-65
This paper describes the purification and properties of an enzyme present in Artemia larvae which hydrolyzes aminoacyl-tRNA by splitting the ester bond between the amino acid and the tRNA chain. The hydrolase has a molecular weight of 55 000 as estimated by gel filtration in Sephadex G-150, is maximally active in the presence of a divalent cation (Mg2+, Mn2+) and has a pH maximum at around neutrality. The enzyme has a wide substrate specificity, hydrolyzing with practically the same efficiency aminoacyl-tRNAs with the amino group free or substituted. This property distinguishes this enzyme from the widely distributed peptidyl-tRNA hydrolase and other more specific aminoacyl-tRNA hydrolases. The expression of the hydrolase during Artemia larval development is blocked by inhibitors of protein synthesis. 相似文献
18.
De Novo Synthesis of Isocitritase in Peanut (Arachis hypogaea L.) Cotyledons 总被引:17,自引:14,他引:3
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Germination of peanut seed is accompanied by a rapid increase in isocitritase (isocitrate lyase, EC 4.1.3.1) during the first 4 days. The presence of cycloheximide (50 μg/ml) during water imbibition inhibited the increase in isocitritase activity. Actinomycin D conversely did not inhibit isocitritase activity until the second day of imbibition while RNA synthesis was inhibited. Germination of peanut seed in 14C-reconstituted amino acids followed by fractionation of a 20 to 35% ammonium sulfate preparation on a Sephadex G-200 column (57-fold purification) showed that the active enzymic fraction coincided with a large peak of radioactivity. Germination of peanut seed in 45% D2O followed by enzyme purification and CsCl equilibrium centrifugation revealed that all the enzyme from D2O seed had a higher density than normal isocitritase. These data indicate that isocitritase in peanut seed is synthesized de novo. 相似文献
19.
《Comparative biochemistry and physiology. C: Comparative pharmacology》1978,59(1):147-151
1. The authors studied certain characteristics of the acetylcholinesterase present in Allolobophora caliginosa; a good purification of the enzyme was achieved by homogenization, ultracentrifugation and then by Sephadex G-200 and DEAE-cellulose chromatographies.2. Three enzymatic forms, probably monomeric, dimeric and tetrameric were isolated. The monomeric one is quantitatively prevailing and shows a higher specific activity; it seems to be composed of of two subunits with the same molecular weight.3. The various active fractions are inhibited by eserine and hydrolyse acetylthiocholine more rapidly than butyrylthiocholine; besides, this latter does not cause substrate inhibition.4. The enzyme is classifiable as acetylcholine hydrolase (E.C. 3.1.1.7). 相似文献
20.
《Biochimica et Biophysica Acta (BBA) - Enzymology》1976,422(1):231-240
Glucosaminephosphate synthase (glucosaminephosphate isomerase (glutamine-forming), EC 5.3.1.19) prepared from rat liver by extraction in the presence of glucose 6-phosphate (Glc-6-P) followed by precipitation with (NH4)2SO4 is susceptible to digestion by trypsin. This enzyme, designated form A, can be converted to tryptic-insusceptible form B upon incubation with Glc-6-P or fructose 6-phosphate (Fru-6-P) at 37°C. The two forms also differ in the degree of activation by dithiothreitol, the degree of inhibition by methylglyoxal and the behavior on DEAE-Sephadex and Sephadex G-200 column chromatography.During purification with DEAE-Sephadex followed by hydroxyapatite, form B is converted to form A if Fru-6-P is absent and form A to form B if Fru-6-P is present. The two forms are therefore interconvertible. Under the conditions of purification, form B is more stable than form A, since the purity and yield of the final product are greater with form B than with form A.These findings suggest that the two forms of glucosaminephosphate synthase differ conformationally and that the equilibrium position depends on the concentration of Fru-6-P. Glc-6-P is effective only when it gives rise to Fru-6-P by mediation of glucose-phosphate isomerase. 相似文献