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1.
  • 1.1. Phospholipase A2 was isolated from Agkistrodon bilineatus venom by Sephadex G-75 and CM-Cellulose column chromatographies.
  • 2.2. The purified phospholipase A2-I gave a single band on disc polyacrylamide gel electrophoresis, isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis.
  • 3.3. The enzyme preparation had a molecular weight of 14,000, isoelectric point of pH 8.77 and possessed 123 amino acid residues.
  • 4.4. The purified phospholipase A2 possessed lethal, indirect hemolytic and anticoagulant activities.
  • 5.5. The enzyme hydrolyzed the phospholipids phosphatidyl choline (PC), phosphatidyl ethanolamine (PE), phosphatidyl inositol (PI) and phosphatidyl serine (PS).
  • 6.6. The concentration of mouse diaphragm was inhibited and the contraction of guinea pig left atrium was increased by phospholipase A2-I.
  • 7.7. Phospholipase A2 activity of this preparation was inhibited by ethylenediamine tetraacetic acid, p-bromo phenacyl bromide, n-bromo succinimide or dithiothreitol, but not by diisopropyl fluorophosphate or benzamidine.
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2.
  • 1.1. The major phospholipase A2 (PLA-DE4) of the venom of Trimeresurus purpureomaculatus (shore pit viper) has been purified to electrophoretic homogeneity.
  • 2.2. The isoelectric point of the purified enzyme was determined to be 4.20, and the mol. wt was 31,700 as estimated by Sephadex G-75 gel filtration chromatography; and 14.000 as estimated by SDS-polyacrylamide gel electrophoresis.
  • 3.3. The purified enzyme hydrolyzed phosphatidylcholine (PC) faster than phosphatidylethanolamine (PE), whereas phosphatidylserine (PS) was not hydrolyzed at all (PC > PE > PS = 0). However, in reaction system consisted of mixtures of PC and PS, phosphatidylserine was effectively hydrolyzed by the enzyme.
  • 4.4. The phospholipase A2 exhibited edema-forming activity but not hemolytic, hemorrhagic or anticoagulant activities. It was not lethal to mice at a dosage of 10 μg/g by i.v. route.
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3.
  • 1.1. A thermostable orthophosphoric monoester phosphohydrolase (EC 3.1.3.1) from Thermus sp strain Rt41A has been purified 400-fold to give a specific activity of 25 U/mg at 60°C in IM diethanolamine (pH 11.1).
  • 2.2. The enzyme has a Mr of 160,000 and is trimeric.
  • 3.3. The half-life of the enzyme is 5 min at 85°C.
  • 4.4. The enzyme has a wide specificity for a number of phosphate monoesters.
  • 5.5. The Hm of the enzyme is pH dependent, so the pH optimum of the enzyme is affected by the substrate concentration.
  • 6.6. The enzyme is inhibited 50% by 20 mM Ca2+ or Mg2+.
  • 7.7. The Ki for phosphate, EDTA-di sodium salt and arsenate (in 1 M diethanolamine, pH 11.1) is approx 1.2, 1.6 and 4mM respectively.
  • 8.8. Urea (200 mM) is not inhibitory.
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4.
  • 1.1. An alkaline p-nitrophenylphosphate phosphatase has been purified 440-fold from extracts of Hatobacterium halobium.
  • 2.2. The enzyme has an apparent molecular weight of 24,000.
  • 3.3. A Km value for p-nitrophenylphosphate of 1.12mM has been found under optimal conditions.
  • 4.4. The enzyme is selectively activated and stabilized by Mn2+.
  • 5.5. It requires high salt concentrations for stability and maximum activity.
  • 6.6. It displays an unusual restricted substrate specificity of 25 phosphate esters tested, only phosphotyrosine and casein were hydrolysed besides p-nitrophenylphosphate.
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5.
  • 1.1. The copepod Acartia clausi exhibited two laminarinases (exo- and endo-acting forms) purified by gel chromatography followed by affinity chromatography. Specific antibodies have been raised against the purified exolaminarinase antigen.
  • 2.2. A single band of protein appeared on a polyacrylamide disc gel electrophoresis; its mol. wt is 21,000.
  • 3.3. Biochemical properties of the purified enzyme showed a maximum activity at pH 5.2 and a temperature of 40°C with laminarin as substrate. The thermal stability of the enzyme and the effect of various cations on its activity were examined. The enzyme hydrolyses specifically the β(1–3) linked polysaccharides and had no activity against the α(1–4) or β(1–4) disaccharides or polysaccharides.
  • 4.4. The kinetic parameters Vm and Km vary with the temperature; the affinity constant (Ka) was maximum between 25–30°C. The Arrhenius plot defined two values of energy of activation: 7980 cal/mole and 17,506 cal/mole.
  • 5.5. From the purification scheme the exoacting form appears to be largely dominant over the endoacting form.
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6.
  • 1.1. Aminopeptidase N was selectively released from larval midgut of silkworm, Bombyx mori, by phosphatidylinositol-specific phospholipase C, and purified to a homogeneous state by ion exchange, gel filtration. Con A-Sepharose and 4-aminobenzyl phosphonic acid-agarose column chromatographies.
  • 2.2. The purified aminopeptidase N preparation showed 190.8 U/mg of specific activity. Its molecular weight was estimated to be around 100 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
  • 3.3. Purified aminopeptidase N molecule preferentially hydrolyzed Leu-, Ala- and Met-p-nitroanilide as substrates. Especially, Leu-p-nitroanilide proved to be the best substrate for aminopeptidase N from larval midgut of silkworm.
  • 4.4. By treatment with phosphatidylinositol-specific phospholipase C, two other hydrolases, alkaline phosphatase and alkaline phosphodiesterase I, were also solubilized from silkworm midgut.
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7.
  • 1.1. The acid phosphatase (AcPase, EC 3.1.3.2) IV from rat testicular tissue was purified to apparent homogeneity.
  • 2.2. The enzyme displays a native molecular weight of 70 kDa determined on gel permeation chromatography on a Sephadex G-100 column and 68 kDa using linear 5–20% sucrose density gradient centrifugation. The subunit molecular weight on SDS-PAGE analysis is 67 kDa, suggesting that the enzyme is a monomeric protein.
  • 3.3. The enzyme does not bind to Concanavaline A-Sepharose 4B column, indicating that it is not a glycoprotein.
  • 4.4. The rat testis AcPase IV is a metal activated enzyme in which Mg2+ is the metal activating agent with a Ka, = 0.88 × 10−3 M. The Michaelis constant for p-nitrophenylphosphate, in the presence of saturating concentrations of Mg2+ ions, is 0.23 × 10−3 M.
  • 5.5. The enzyme preferentially hydrolizes p-nitrophenylphosphate, phenylphosphate and ATP.
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8.
  • 1.1. An ld-dipeptidase (EC 3.4.13.-) that hydrolyzes the unrelated dipeptides l-Ala-d-Glu (sp. act. 0.85 μmol·min−1·mg−1) and l-Lys-d-Ala (sp. act. 11 μmol · min−1·mg−1) has been purified 250-fold from the sporulation medium of Bacillus sphaericus with a 4% recovery of lytic activity.
  • 2.2. Throughout the purification steps, followed with both substrates, the enzyme peaks of activities were congruent and the ratios of activities were constant. Both activities were activated 50-fold by cobalt. Polyacrylamide gel electrophoresis of the final preparation showed the two enzyme activities to be coincident. The data are consistent with those activities being due to a single enzyme.
  • 3.3. Sodium dodecylsulfate polyacrylamide gel electrophoresis of the purified enzyme showed a single protein band (Mr 38,000).
  • 4.4. This dipeptidase hydrolyzes some other ld-dipeptides with a free amino and carboxyl group. Although dipeptides having a di-amino acid as the amino terminus are the best of the substrates tested, the hydrolysis occurs also when neutral amino acids are N-terminal. The activity is higher with neutral C-terminal residues such as Gly or d-Ala than with a di-acid residue such as d-Glu.
  • 5.5. This enzyme may have a function in peptidoglycan metabolism.
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9.
  • 1.1. The purified enzyme hydrolyzes the linear l-lysinamide and the cycle amide of l-lysine—l-α-amino-ϵ-caprolactam.
  • 2.2. The apparent relative molecular mass is 180,000. The enzyme consists of four subunits and the molecular mass of a single subunit was found to be 47,000.
  • 3.3. The coefficient of molecular sedimentation equals 8.3 S, the isoelectric point was determined to be pH 4.3
  • 4.4. The enzyme is not a glycoprotein. p-Mercuribenzoate binds 10 SH-groups of the native enzyme molecule and 20 SH-groups in the presence of 0.7% SDS.
  • 5.5. pH- optimum for the hydrolysis of l-lysine amides was observed to be 7.5–7.7. The enzyme is strictly dependent on Mn2+ and Mg2+.
  • 6.6. The kinetic parameters for the hydrolysis of l-lysinamide where Km = 3.8 mM and kcat = 3000 sec−1 For the hydrolysis of cyclic L-lysinamide Km = 4.8 mM and kcat = 2600 sec.
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10.
  • 1.1. A lipoxygenase activity was purified from Thermoactinomyces vulgaris and some of its properties were characterized.
  • 2.2. The enzyme showed a temperature activity range of 40–55°C with still significant activity over 60°C.
  • 3.3. The pH of activity on linoleic acid had a broad range with an optimum at pH 6.0 and a weaker one at pH 11.0.
  • 4.4. On arachidonic acid the pattern was narrow bell-shaped with an optimum at pH 6.5.
  • 5.5. The purified lipoxygenase from Th. vulgaris showed an apparent Km of 1 mM and Vmax of 0.84 μmol diene/min/mg protein.
  • 6.6. It was inhibited by the oxidation products, 9-HPOD and 13-HPOD.
  • 7.7. A 160,000 Da molecular weight of the enzyme was determined by molecular filtration. Methionine, tyrosine, tryptophan and cysteine are apparently involved in its activity.
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11.
  • 1.1. Biliverdin reductase from the liver of eel, Anguilla japonica was characterized and purified with a novel enzymatic staining method on polyacrylamide electrophoretic gel.
  • 2.2. This enzyme could use both NADPH and NADH as coenzyme. The Km of NADPH was 5.2 μM, while that of NADH was 5.50 μM.
  • 3.3. The optimum reaction pH for using HADPH as coenzyme was 5.3. That for NADH was 6.1. The optimum reaction temperature is 37°C.
  • 4.4. When NADPH was used as coenzyme, the Km of biliverdin was 0.6 μM. When NADH was used as coenzyme, the Km of biliverdin was 7.0 μM.
  • 5.5. The activity of the enzyme was inhibited by the concentration of biliverdin. Also, the potency of the enzyme was much less than that of the analogous enzyme isolated from mammals.
  • 6.6. This is a fairly stable enzyme with a mol. wt around 67,000. Its estimated pI was pH 3.5–4.0.
  • 7.7. This is the first time biliverdin reductase has been isolated and characterized from a vertebrate other than mammals. The property of it is quite different from that of mammals.
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12.
  • 1.1. NADH-dependent isocitrate dehydrogenase has been purified 110-fold from the crude extract of the flight muscle mitochondria of Aldrichina grahami.
  • 2.2. The purification procedure involved Triton X-100 treatment of isolated mitochondria, column chromatography on DEAE-cellulose, Affi-gel blue, and P-cellulose.
  • 3.3. The purified enzyme was homogeneous by criteria of the polyacrylamide gel electrophoresis.
  • 4.4. The enzyme of the blowfly contains more acidic amino acids and less hydrophobic amino acids than that of pig heart.
  • 5.5. The molecular weight was determined to be 330,000 daltons. The subunit construction differs from ghat of mammalian isocitrate dehydrogenase.
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13.
  • 1.1. Glucose-6-phosphate dehydrogenase (G6PDH EC 1.1.1.49) from mouse liver has been purified 1100-fold by extraction, ion-exchange chromatography on DE-52, absorption chromatography on Bio-Gel HTP and gel filtration through sepharose 6 HR 10/30. The purified enzyme showed a single band in silver stained SDS-PAGE.
  • 2.2. The native and subunit molecular weight were 117 and 31 kDa respectively.
  • 3.3. The kinetic studies and the patterns obtained from the inhibition by-products suggest that the enzyme follows an ordered sequential kinetic mechanism.
  • 4.4. The reduced Km values for the substrates favour the operativity of the enzyme. The “fine control” of the enzymatic activity was exerted by the NADPH, whose Ki is several fold lower than the in vivo concentration.
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14.
  • 1.1. Alkaline phosphatase (EC 3.1.3.1) from the dinoflagellate Peridinium cinctum, the Lake Kinneret bloom alga, has been partially purified by gel filtration.
  • 2.2. The enzyme could be easily extracted using a distilled water/chloroform mixture suggesting that the alkaline phosphatase of Peridinium is particularly labile.
  • 3.3. The molecular weight of the enzyme was estimated as 158,000 ± 5000. The enzyme showed a broad pH optimum (in the range pH 8.0–8.5), had a Km of 0.45 mM for p-nitrophenylphosphate as substrate and was stable to repeated freeze/thawing cycles.
  • 4.4. The enzyme was strongly activated by Mg2+ whereas Zn2+ (and to a lesser extent Cd2+) was an effective inhibitor of the enzyme. Cu2+ activated the enzyme at low concentrations, although at higher concentrations inhibited the enzyme. This effect of metals on the Peridinium alkaline phosphatase could be environmentally important since underwater hot springs, containing high concentrations of copper, enter the lake.
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15.
  • 1.1. Pseudomonas aeruginosa phospholipase C from culture supernatants of bacteria grown in high-Pi basal salt medium with choline, as the sole carbon and nitrogen source, was purified by precipitation with 70% saturation ammonium sulfate in the presence of celite.
  • 2.2. The PLC activity was eluted of this mixture by the use of a reverse gradient of 70-0% ammonium sulfate.
  • 3.3. The peak containing the PLC activity revealed a single protein after SDS-PAGE.
  • 4.4. The method could also be applied to purify PLC produced in a low-Pi complex medium. The resultant preparation was not homogeneous.
  • 5.5. The molecular weight for both PLC preparations was about 70 kDa.
  • 6.6. Both PLC used phosphatydilcholine and sphingomyelin as substrates, displayed hemolytic activity an exhibited an apparent KM of 25 mM for p-nitrophenylphosphorylcholine.
  • 7.7. They were not inhibited by 1% sodium deoxycholate but were 30% inhibited by 1% Triton X-100.
  • 8.8. 2% sodium dodecylsulfate and 1% tetradecyltrimethylammonium bromide inhibited the PLC from the HPl-BSM plus choline but not the enzyme from the LPl-CM.
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16.
  • 1.1. The enzyme fructose-1,6-bisphosphatase was purified from the mantle of the sea mussel Mytilus galloprovincialis Lmk. The purified enzyme showed a single band in SDS-polyacrylamide gel electrophoresis. The mol. wt and subunit mol. wt of the enzyme were 105,000 and 27,000, respectively.
  • 2.2. Divalent cations are essential for the enzyme activity. In the absence of chelating agents, FBPase 1 exhibits hyperbolic kinetics with respect to Mn2+, Zn2+ and Mg2+. The Km for Mg2+ is lower than the physiological concentration of cation in the tissue, whereas its Km for Mn2+ and Zn2+ is greater than the respective in vivo concentrations.
  • 3.3. The joint action of Mg2+ and Zn2+ increases the affinity of the enzyme for the substrate Fru-1,6-P2, though Vmax is reduced.
  • 4.4. Na+ strongly inhibits the enzyme even at very low concentrations. K+ has no effect whatsoever.
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17.
  • 1.1. A new tetralysine endopeptidase from Escherichia coli AJ005 has been purified about 135-fold.
  • 2.2. The peptidase seems to be specific to tetralysine among lysine homopolymers.
  • 3.3. The optimal pH was about 7.5
  • 4.4. The activity was inhibited by KCN but not inhibited by soybean trypsin inhibitor.
  • 5.5. The apparent Km value was 2.5 × 1O−3 M for tetralysine.
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18.
  • 1.1. A NAD+-dependent glutamate dehydrogenase (EC 1.4.1.2.) was purified 126-fold from Halobacterium halobium.
  • 2.2. Activity and stability of the enzyme were affected by salt concentration. Maximum activity of the NADH-dependent reductive amination of 2-oxoglutarate occurs at 3.2 M NaCl and 0.8 M KCl, and the NAD+-dependent oxidative deamination of l-glutamate occurs at 0.9 M NaCl and 0.4 M KCl. The maximum activity is higher with Na+ than with K+ in the amination reaction while the reverse is true in the deamination reaction.
  • 3.3. The apparent Km values of the various substrates and coenzymes under optimal conditions were: 2-oxoglutarate, 20.2 mM; ammonium, 0.45 M; NADH, 0.07 mM; l-glutamate, 4.0 mM; NAD+, 0.30 mM.
  • 4.4. No effect of ADP or GTP on the enzyme activity was found. The purified enzyme was activated by some l-amino acids.
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19.
  • 1.1. A quick and simple procedure is described for purifying kallikrein from human whole saliva. The enzyme has been purified about 2700-fold with a yield of approx. 30%.
  • 2.2. The procedure is based on the immediate fractionation of saliva by ion exchange chromatography. This is followed by a combination of affinity and high performance liquid chromatography.
  • 3.3. The results indicate that another protein component binds to the enzyme at pH 8.0.
  • 4.4. The homogeneity of the enzyme has been demonstrated by gel electrophoresis in the absence as well as in the presence of sodium dodecylsulfate.
  • 5.5. A mol. wt of 40,100±1800 has been calculated from gel electrophores is experiments.
  • 6.6. Sedimentation equilibrium in an analytical ultracentrifuge gave a mol. wt of 39,700.
  • 7.7. The amino acid composition has been determined and it confirms that the enzyme has a low isoelectric point.
  • 8.8. The presence of tryptophan has been demonstrated by absorption and fluorescence spectroscopy.
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20.
  • 1.1. The activities of S-adenosylmethionine decarboxylase (EC 4.1.1.50) were measured in cell extracts of mantle, hepatopancreas and foot from Mytilus edulis.
  • 2.2. The apparent molecular weights of the enzymes estimated by gel filtration chromatography were 65,000 ± 10,000.
  • 3.3. The enzymes do not require bivalent cations for catalysis and show optimum pH between 7.0–8.0 in phosphate buffer.
  • 4.4. The hepatopancreas enzyme shows different behavior to the other two enzymes against temperature and its activity is strongly inhibited by NH4+.
  • 5.5. The apparent Kms for S-adenosylmethionine were found to be 300, 200 and 250 μM for the hepatopancreas, mantle and foot enzymes, respectively.
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