Purification and properties of glutamine synthetase from Douglas fir roots

Glutamine synthetase (GS. EC 6.3.1.2) was purified to apparent electrophoretic homogeneity from roots of Pseudotsuga menziesii (Mirb) Franco by a three-step procedure involving diethylaminoethyl (DEAE)-Trisacryl chromatography, affinity chromatography on Matrex Gel Red A. and preparative polyacrylamide gel electrophoresis. The enzyme was purified 40-fold with a 16% recovery. The native enzyme had a molecular mass of 460 ± 5 kDa as estimated by gel filtration, interpolation of the Ferguson plots and non-denaturing gradient-PAGE. It was composed of two different subunits of 54 and 64 kDa. Affinity constants for glutamate (Glu), glutamine (Gln), ATP and ADP were 2.6, 10.5, 0.5 and 0.083 m M . respectively. The enzyme exhibited a negative cooperativity for ammonium (Hill number of 0.7) with two K_m values which were 11 and 75 μ M in the presence of ammonium concentrations lower and higher than 1.3 m M , respectively. Glycine and ADP appeared as potential inhibitors of the GS activity. The optimum pH values were 7.2 and 7.6 for the transferase and the biosynthetic assays, respectively. The enzyme lost 30% of its activity within 25 days of storage at 4°C. The optimum temperatures of activity were 40°C and 45°C for the transferase and bio-synthetic activities, respectively.     

Purification and Properties of L-Arginine Decarboxylase of Evernia prunastri

An L-arginine decarboxylase was isolated from Evernia prunastrithallus. The enzyme was purified about 117-fold and showed apH optimum of 7.1 and a temperature optimum at 26°C. Itsmolecular weight was estimated as 300,000. The Evernia argininedecarboxylase was significantly inhibited by L-ornithine, ureaand putrescine. The K_m for L-arginine was about 12.5 mM. (Received March 9, 1981; Accepted June 26, 1981)     

Shikimate dehydrogenase from pepper (Capsicum annuum) seedlings. Purification and properties

Shikimate dehydrogenase (SKDH, EC 1.1.1.25) was extracted from seedlings of pepper ( Capsicum annuum L.) and purified 347-fold. The purification procedure included precipitation with ammonium sulphate and chromatography in columns of Reactive Red-agarose, Q-Sepharose and Sephadex G-100. Pepper SKDH isozymes are separable only using PAGE. The purified enzyme has a relative molecular mass of 67 000 as estimated by gel filtration. The optimum pH of enzyme activity is 10.5 and the optimum temperature is 50°C, but the enzyme is quickly inactivated at temperatures higher than 40°C. The purified enzyme exhibited typical Michaelis-Menten kinetics and K_m values are 0.087 m M for shikimic acid and 0.017 m M for NADP. The mechanism of reaction is sequential considering NADP as a cosubstrate. Ions such as Ca²⁺, Mg²⁺ and Mn²⁺ activate the enzyme, but Zn²⁺ and Cu²⁺ are strong inhibitors. Some phenolic compounds such as guaiacol, protocatechuic acid and 2,4-D are competitive inhibitors of pepper SKDH, showing K_i values of 0.38 m M , 0.27 m M and 0.16 m M , respectively.     

Partial purification and characterization of stomatal phosphoenolpyruvate carboxylase from Vicia faba

Stomatal phosphoenolpyruvate carboxylase (PEPCase EC 4.1.1.31), extracted from abaxial epidermal peels of Vicia faba L. cv. Frühe Weiβkeimige, was partially purified by ammoniumsulfate precipitation, and molecular sieve (Sepharosc S-400) and ion exchange (DEAE-Sepharose) chromatography. The partially purified enzyme, essentially free of a PEPCase isoform existing in mesophyll and epidermal cells, had a specific activity of 300 nkat mg-¹ protein at 25°C. Western immunoblot analysis revealed that the stomatal enzyme had two bands (M_: of 110000 and 112000), crossreacting with PEPCase antibodies raised against PEPCase from Ka-lanchoe daigremontiana . The native molecular mass of the enzyme (467000) points to a tetrameric subunit structure. The temperature optimum was found to be 35°C; cold treatments of PEPCase before assaying were accompanied by inactivation. The energy of activation was calculated to 51 kJ mol-¹. The kinetic behaviour of the enzyme at fixed MgCl₂ concentrations is characterized by a pH optimum between pH 8.0–8.2 with or without 1 m M malate or 5 m M glucose-6-phosphate (Glc-6-P), but a combination of both effectors resulted in a shift of the optimum to pH 7.6. The enzyme showed a pH sensitive inhibition by 1 m M malate and an activation by Glc-6-P. At low pH (6–7), Glc-6-P was able to compensate for the malate induced inhibition of the enzyme. Malate and Glc-6-P both affected K_m(PEP), drastically and influenced V_max at pH 7, but not at pH 8.3. The inhibition constant of malate was determined to be 1.2 m M at pH 7. From the Dixon plot, a competitive inhibition of malate was assumed under defined assay conditions.     

Purification and characterization of an endoamylase from tulip (Tulipa gesneriana) bulbs

Amylase activity extracted from tulip ( Tulipa gesneriana L. cv. Apeldoorn) bulbs that had been stored for 6 weeks at 4°C was resolved to 3 peaks by anion-exchange chromatography on diethylaminoethyl-Sephacel. These 3 amylases exhibited different relative mobilities during non-denaturing polyacrylamide gel electrophoresis (PAGE). The most abundant amylase form (amylase I) was purified to apparent homogeneity using hydrophobic interaction chromatography, gel filtration and chromatofocusing. The apparent molecular mass of the purified amylase was estimated to be 51 kDa by sodium dodecyl sulfate-PAGE and 45 kDa by gel filtration chromatography. The purified amylase was determined to be an endoamylase (EC 3.2.1.1) based on substrate specificity and end-product analysis. The enzyme had a pH optimum of 6.0 and a temperature optimum of 55°C. The apparent K_m value with soluble starch (potato) was 1.28 mg ml⁻¹. The presence of Ca²⁺ increased the activity and thermal stability of the enzyme. The presence of dithiothreitol enhanced the activity, while β -mercaptoethanol and reduced glutathione had no significant effect. When pre-incubated in the absence of the substrate, N-ethylmaleimide and 5,5'-dithiobis-(2-nitrobenzoic acid) partially inhibited the enzyme. α -cyclodextrins or β -cyclodextrins had no effect on enzyme activity up to 10 m M . In addition to CaCl₂, CoCl₂ slightly enhanced activity, while MgCl₂ and MnCl₂ had no significant effect at a concentration of 2 m M . ZnCl₂, CuSO₄, AgNO₃ and EDTA partially inhibited enzyme activity, while AgNO₃ and HgCl₂ completely inhibited it at 2.0 m M .     

Purification and characterization of a cathepsin L-like enzyme from the body wall of the sea cucumber Stichopus japonicus

Cathepsin L-like enzyme was purified from the body wall of the sea cucumber Stichopus japonicus by an integral method involving ammonium sulfate precipitation and a series of column chromatographies on DEAE Sepharose CL-6B, Sephadex G-75, and TSK-GEL. The molecular mass of the purified enzyme was estimated to be 63 kDa by SDS-PAGE. The enzyme cleaved N-carbobenzoxy-phenylalanine-arginine7-amido-4-methylcoumarin with K(m) (69.92 microM) and k(cat) (12.80/S) hardly hydrolyzed N-carbobenzoxy-arginine-arginine 7-amido-4-methylcoumarin and L-arginine 7-amido-4-methylcoumarin. The optimum pH and temperature for the purified enzyme were found to be 5.0 and 50 degrees C. It showed thermal stability below 40 degrees C. The activity was inhibited by sulfhydryl reagents and activated by reducing agents. These results suggest that the purified enzyme was a cathepsin L-like enzyme and that it existed in the form of its enzyme-inhibitor complex or precursor.     

Purification and properties of a second fructan exohydrolase from the roots of Cichorium intybus

A 1-FEH II (1-fructan exohydrolase, EC 3.2.1.80) was purified from forced chicory roots ( Cichorium intybus L. var. foliosum cv. Flash) by a combination of ammonium sulfate precipitation, concanavalin A (Con A) affinity chromatography and anion and cation exchange chromatography. This protocol produced a 70-fold purification and a specific activity of 52 nkat mg⁻¹ protein. The apparent size of the enzyme was 60 kDa as estimated by gel filtration and 64 kDa on SDS-PAGE. Optimal activity was found between pH 5.0 and 5.5. The temperature optimum was around 35°C. No product other than fructose could be detected with inulin as the substrate. The purified enzyme exhibited hyperbolic saturation kinetics with an apparent K_m of 58 m M for 1-kestose (Kes) and 64 m M for 1,1-nystose (Nys). The purified 1-FEH II hydrolyzed the β (2↠1) linkages in inulin, Kes and Nys at rates at least 5 times faster than the β (2↠6) linkages in levan oligosaccharides and levanbiose. Fructose did not affect the 1-FEH II activity but sucrose (Suc) was a strong inhibitor of this 1-FEH II (K_i=5.9 m M ). The enzyme was partially inhibited by Na-EDTA and CaCl₂ (1 m M ).     

Serine hydroxymethyltransferase from spinach leaf mitochondria. Purification and characterization

A mitochondrial serine hydroxymethyltransferase (EC 2.1.2.1) has for the first time been purified close to homogeneity from a photosynthetically active tissue, spinach ( Spinacea oleracea L. cv Viking II) leaves. The specific activity of the enzyme was 7.8 μmol (mg protein)⁻¹ min⁻¹ using L-serine as substrate. The enzyme was stable for at least 8 weeks at 4°C in the presence of folate. The pH optimum was at pH 8.5 where the enzyme had a K_m for L-serine of 0.9 m M . Carboxymethoxylamine was a strong competitive inhibitor with a K₁ of 1.4 μM. An absorption spectrum taken of the enzyme in the presence of glycine and tetrahydrofolate showed a peak at 492 nm, probably originating from a substrate-enzyme complex. The molecular weight obtained by gel filtration was 209 kDa. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the purified enzyme showed that the apparent molecular weight of the subunit was 53 kDa, indicating four subunits.     

Partial purification and properties of a 36-kDa 1-aminocyclopropane-1-carboxylate N-malonyltransferase from mung bean

A 36-kDa 1-aminocyclopropane-1-carboxylate (ACC) N-malonyltransferase, which converts the ethylene precursor ACC into the conjugated derivative malonyl-ACC (MACC), has been isolated from etiolated mung bean ( Vigna radiata ) hypocotyls, and partially purified in a four-step procedure. The enzyme is stimulated about 7-fold by 100 m M K⁺ salts or 0.5 m M Co²⁺ salts, and is inhibited competitively by D-phenylalanine (K_i= 1.3 m M ) and non competitively by CoASH (0.3 m M ). Beside malonyl-CoA, it is capable to use succinyl-CoA as an acyl donor. The 36-kDa enzyme described here exhibits a lower optimum temperature (40°C) and a 7- or 3-fold lower apparent K_m for ACC (68 μ M ) and malonyl-CoA (74 μ M ), respectively, when compared with its 55 kDa isoform already isolated from the same plant material. This data support the idea that several isoforms of ACC N-malonyltransferase exist in plants. These isoforms may play a differential role in regulating the availability of ACC, and consequently the rate of ethylene production, as well as detoxifying cells from D-amino acids.     

Isolation and Characterization of a Cytosolic Phospholipase A2 from Bovine Adrenal Medulla

Abstract: We have recently demonstrated that bovine adrenal medulla contains a soluble phospholipase A₂ (PLA₂), which is localized in the cytosol. In the present study, this PLA₂ was purified 1,097-fold using sequential concanavalin A, hydrophobic interaction, anion exchange, gel filtration, and an additional anion exchange chromatography. The enzyme is activated over the range of 20–1,000 µ M Ca²⁺ and has a pH optimum near 8.0. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protein has a molecular mass of 26 kDa and an isoelectric point of 4.6 as revealed by isoelectric focusing. The cytosolic PLA₂ is not inhibited by NaCl, and the enzymatic activity is stimulated at low concentrations of Triton X-100 (0.01%) and deoxycholate (1 m M ) but inhibited at higher concentrations (0.1% and 3 m M , respectively) of these detergents. Furthermore, heat treatment (57°C, 5 min) reduced the enzymatic activity by 80%, whereas glycerol (30%) increased the activity. p -Bromophenacylbromide, a frequently used irreversible inhibitor of type II PLA₂, has little effect until 100 µ M , and 2–10 m M dithiothreitol totally inactivated the enzyme. The purified PLA₂ displays a preference for phosphatidylcholine as a substrate but hydrolyzes phospholipid substrates with arachidonic acid or linoleic acid esterified at the sn -2 position to the same extent. It is concluded that the chromaffin cell cytosolic PLA₂, which was isolated and characterized in this study, represents a type of PLA₂ that has not been formerly reported in chromaffin cells. Additional research on the chromaffin cell cytosolic PLA₂ will help to reveal whether the enzyme is important for exocytosis.     

Purification and characterization of β-amylase from leaves of potato (Solanum tuberosum)

Amylolytic activity is widely distributed in plants. In potato leaves ( Solanum tuberosum L.) the abundant amylolytic activity was found to be β-amylase (EC 3.2.1.2, a-1,4-D-glucan maltohydrolase). β-Amylase from potato leaves was purified to homogeneity for study of enzyme characteristics. The purification steps included ammonium sulphate precipitation, anion exchange chromatography, affinity chromatography and gel filtration. The end product of α-1,4-glucan degradation was maltose. The protein is a 111-kDa homo-dimer with a subunit molecular mass of 56 kDa and a pl of 5.6. The pH-optimum is 6.5 using p -nitrophenylmaltopentaoside (PNPG5) as substrate. The optimal temperature for hydrolysis is at 40°C. The enzyme is unstable at temperatures above 40°C. The K_nt-value for PNPG5 is 0.73 m M and the activity is inhibited by cyclodextrins. At a concentration of 1 m M , β-cyclodextrin is a stronger inhibitor than α-cyclodextrin (68 and 20% inhibition, respectively). Branched glucans (e.g. starch and amylopectin) are superior substrates as compared to long, essentially unbranched glucans (e.g. amylose). This study of the catalytic properties of β-amylase from potato leaves indicates the importance of β-amylase as a starch degrading enzyme.     

Purification and characterization of a neutral endoxylanase from Aspergillus nidulans

Abstract A neutral endoxylanase from a culture filtrate of Aspergillus nidulans grown on oat spelt xylan was purified to apparent homogeneity. The purified enzyme showed a single band on SDS-PAGE with a molecular mass of 22,000 and had an isoelectric point of 6.4. The enzyme was a non-debranching endoxylanase highly specific for xylans and completely free from cellulolytic activity. The xylanase showed an optimum activity at pH 5.5 and 62°C and had a K m of 4.2 mg oat spelt xylan per ml and a V max of 710 μmol min⁻¹ (mg protein)⁻¹.     

Rapid and sensitive method for detection of Salmonella strains using a combination of polymerase chain reaction and reverse dot-blot hybridization

Abstract Cell-free extracts of Thiobacillus acidophilus catalysed the stoichiometric conversion of tetrathionate to thiosulphate, sulphur and two protons. The pH optimum of the enzyme activity was 3.0 and its temperature optimum 40°C. The enzyme was unstable at 30 and 40°C, at which its activity decreased to zero within 100 and 20 h, respectively. Enzyme activity was not affected by incubation for 1 week on ice or by freezing and thawing of the extract. The K _m for tetrathionate was 0.3 mM. Enzyme activity was stimulated by ammonium sulphate up to a concentration of 1M. The results indicate that trithionate hydrolase cannot account for the observed conversion of tetrathionate.     

New Strains of Bacillus licheniformis and Bacillus coagulans producing Thermostable α-Amylase Active at Alkaline pH

Two species of Bacillus producing thermostable α-amylase with activity optima at alkaline pH are reported here. These organisms were isolated from soil and have been designated as Bacillus licheniformis CUMC 305 and B. coagulans CUMC 512. The enzymes released by these two species were partially purified up to about 81- and 72-fold respectively of the initial activity. The enzyme from B. licheniformis showed a wide temperature-range of activity, with optimum at 91°C. At this temperature it remained stable for 1 h. It retained 40–50% activity at 110°C and showed only 60% of its activity at 30°C. The enzyme showed a broad pH range of activity (4–10) retaining substantial activity on the alkaline side. The optimum pH was 9·5. The enzyme of B. coagulans showed activity up to 90°C, with optimum at 85°C and had a wide pH range with optimum at 7·5–8·5. The hydrolysis pattern of the substrate starch by these enzymes indicated that glucose, maltose, maltotriose and maltotetraose are the principal products rather than higher oligosaccharides.     

ACC oxidase from Carica papaya: Isolation and characterization

Most of the studies done on 1‐aminocyclopropane 1‐carboxylic acid (ACC) oxidase were done in vivo. It is only recently that in vitro studies have been carried out successfully on the enzyme. Here we report on in vitro studies of the enzyme that was isolated from Carica papaya . The enzyme had a K_m of 37 µ M and was inhibited by n ‐propyl gallate (0.240 m M ), sodium dithionite (0.022 m M ), sodium metabisulphite (0.021 m M ) and cobalt sulphate (0.100 m M ). The activity of the enzyme increased with ripening, the enzyme was somewhat labile and activity was lost after 4 days at 14°C; activity was prolonged when the crude homogenate was kept at −15°C. Isolation and purification were achieved with ammonium sulphate precipitation followed by gel‐filtration (Sephadex G 100‐120) and ion‐exchange chromatography (DEAE‐Sephadex). Gel electrophoresis of the purified enzyme gave a single band which corresponded to a molecular mass of 27.5 kDa. The amino acid content of the enzyme showed a relatively high percentage of valine (10.4%). Enzyme activity was enhanced when dithiothreitol (3 m M ) and bicarbonate ion (30 m M ) were added to the assay medium. The production of ethylene from Carica papaya did not require pretreatment of the fruit with ethylene.     

Purification and characterization of strongly chitin‐binding chitinases from salicylic acid‐treated leek (Allium porrum)

Six chitinases (EC 3.2.1.14) were purified from salicylate‐treated leek ( Allium porrum L.). They all strongly bind to chitin and can roughly be divided into two groups. One group has blocked N‐termini, is completely inhibited by 1 m M AgNO₃, has a relatively narrow pH optimum, a temperature optimum of 40°C and cannot degrade the tetramer of chitin. The other group has unblocked N‐termini showing homology to the chitin‐binding lectin WGA and is therefore considered as class I chitinases. This group is only moderately inhibited by 1 m M AgNO₃ (30%), has a relatively broad pH optimum, has a higher temperature optimum (50 to 60°C) and can degrade the tetramer of chitin to dimers. Furthermore, all isoforms have molecular masses around 34 kDa as estimated by SDS‐PAGE. They have isoelectric points ranging from 4 to 8 and no detectable lysozyme activity. Two isoforms investigated in more detail differ in their antifungal potential.     

Amylase production by a Gram-positive bacterium isolated from fermenting tef (Eragrostis tef)

Bacillus sp. A-001, which produced large amounts of amylase, was isolated from fermenting tef ( Eragrostis tef ) on tryptone soya agar supplemented with 1% starch. The organism grew between pH 4.5 and 10.5 with an optimum at 7–7.5. Growth occurred between 20 and 55°C but the optimum was about 35–40°C. At optimum medium pH (7.5) and a temperature of 35°C the organism entered the stationary phase after about 72 h and amylase production was at its highest (9.6 units ml^-1) at this time. Enzyme activity was optimal at pH 5.5 and 40°C and showed good thermal stability; it required 110 min to lose 50% of its activity at 70°C. The enzyme hydrolysed native starch (flour from tef, corn and kocho) to various oligosaccharides, including maltotriose, maltose and glucose.     

Thermolabile ribonucleases from antarctic psychrotrophic bacteria: detection of the enzyme in various bacteria and purification from Pseudomonas fluorescens

Abstract Thirteen terrestrial psychrotrophic bacteria from Antarctica were screened for the presence of a thermolabile ribonuclease (RNAase-HL). The enzyme was detected in three isolates of Pseudomonas fluorescens and one isolate of Pseudomonas syringae . It was purified from one P. fluorescens isolate and the molecular mass of the enzyme as determined by SDS-PAGE was 16 kDa. RNAase-HL exhibited optimum activity around 40°C at pH 7.4. It could hydrolyse Escherichia coli RNA and the synthetic substrates poly(A), poly(C), poly(U) and poly(A-U). Unlike the crude RNAase from mesophilic P. fluorescens and pure bovine pancreatic RNAase A which were active even at 65°C, RNAase-HL was totally and irreversibly inactivated at 65°C.     

Characterisation of maltase from enteropathogenic Escherichia coli

Abstract Enteropathogenic strains of faecal Escherichia coli produced significantly ( P < 0.01) more maltase than the non-pathogenic strains of the organism. The enzyme was induced by maltose but repressed by glucose and fructose. The maltase was partially purified by ammonium sulphate precipitation, followed by dialysis and gel permeation chromatography. The partially purified maltase had an M _r of 144500 and an apparent K _m of approx. 7.6 mM for maltose. The enzyme was stimulated by Ca²⁺, inhibited by Cu²⁺, Hg²⁺, Uo²⁺, IAA and EDTA, and exhibited optimum activity at pH 6.5 at 30°C.     

Reaction of Muscimol with 4-Aminobutyrate Aminotransferase

Abstract: The reaction of muscimol as amino donor substrate for GABA transaminase (GABA-T) has been studied using enzyme purified from rabbit brain. Enzyme activity was assayed by measuring the glutamate produced using glutamate dehydrogenase. Kinetic parameters determined at 37°C were for GABA, K _m (app) = 1.92 ± 0.24 m M , specific activity = 7.33 ± 0.27 μmol/min/mg ( k _cat= 13.7s⁻¹), and for muscimol, K _m (app) = 1.27 ± 0.15 m M , specific activity = 0.101 ± 0.009 μmol/min/mg ( k _cat= 0.19s⁻¹). Addition of muscimol to the enzyme caused the spectral changes associated with conversion of the pyridoxaldimine form to the pyridoxamine form, and the first-order rate constant for the reaction showed a dependence on muscimol concentration that followed saturation kinetics, with a K = 1.1 ±0.18 m M and k _max= 0.065 ± 0.004 s⁻¹ (19°C). The rate of spectral change observed on addition of muscimol to ornithine transaminase was extremely slow—at least an order of magnitude slower than that seen with GABA-T.