Ascidian phenoloxidase: its release from hemocytes, isolation, characterization and physiological roles

Hemocytes of the solitary ascidian Halocynthia roretzi released phenoloxidase in response to sheep red blood cells and yeast cells but not to latex beads. Phenoloxidase was also released from the hemocytes by treatments with zymosan and lipopolysaccharides but not with β1–3 glucan. EDTA scarcely inhibited the activity of phenoloxidase but inhibited the release of the enzyme. Phenoloxidase was purified from H. roretzi hemocytes by SP-Sephadex chromatography and Sephadex G-100 gel filtration. The molecular weight of the purified enzyme was estimated to be 62 000. Phenoloxidase activity was strongly inhibited by diethyldithiocarbamate, phenylthiourea and reducing agents. H. roretzi phenoloxidase was characterized as a metalloenzyme that required copper ions for the expression of full activity. The phenoloxidase showed antibacterial activity in the presence of -(3,4-dihydroxy)-phenylalanine and H. roretzi plasma. Thus, it can be concluded that phenoloxidase released from H. roretzi hemocytes functions as a humoral factor in the defense system of H. roretzi.     

Purification and characterization of prophenoloxidase from the haemolymph of Locusta migratoria

Prophenoloxidase (proPO) was purified from plasma of the locust, Locusta migratoria. This was achieved in three steps (gel filtration on 5300, anion exchange on QMA Memsep, and affinity chromatography on blue Trisacryl) without the use of anticoagulant buffer or inhibitors. The native protein had an apparent molecular mass of 250 kDa as determined by gel filtration and was likely composed of three non-covalently associated subunits of 81 kDa. Its amino acid composition was found to be very similar to that of Bombyx mori proPO. Purified locust proPO could be converted into phenoloxidase (PO) by α-chymotrypsin. Using L-dopa as substrate, K_in and V_max were determined to be 1.5 mM and 5 μM/s, respectively. © 1996 Wiley-Liss, Inc.     

Isolation of latent phenoloxidase from prepupae of the housefly,Musca domestica

Latent phenoloxidase was purified from prepupae of the housefly, Musca domestica vicina Maquart. The purification procedures included DEAE-cellulose column chromatography, sucrose density gradient centrifugation adn second sucrose density gradient centrifugation. The final preparations appear to be homogeneous based on results obtained from polyacrylamide gel electrophoresis in the presence of EDTA. Electrophoresis in the absence of EDTA caused spontaneous activation of latent phenoloxidase. While latent phenoloxidase was fairly stable over the range of temperatures between 0 and 40°C, it was quite sensitive to changes in pH, being stable only around pH 6.0. The molecular weight of latent phenoloxidase was estimated to be 178,000, as determined by gel filtration and sucrose density gradient centrifugation. Furthermore, phenoloxidase formed by the activation of latent phenoloxidase indicated a higher molecular weight (340,000) than that of latent phenoloxidase. Thus, it appears that the mechanism of the activation of latent phenoloxidase involves the association and disassociation system.     

Types of Amylases in Rice Grains

The subunit structure of phenoloxidase from the larvae of housefly, Musca domestica vicina Maquart, was investigated by urea treatment, SDS-polyacrylamide gel electrophoresis and electron microscopy. Phenoloxidase was dissociated into a single type of subunit (MW 6.5 × 10⁴) by 5 m urea treatment. A similar subunit (MW 6.1 × 10⁴) was also detected by SDS-polyacrylamide gel electrophoresis. Furthermore, four additional protein bands with molecular weights of 4.8 × 10⁴, 3.5 × 10⁴, 2.4 × 10⁴ and 1.2 × 10⁴ were detected by SDS-polyacrylamide gel electrophoresis by extensive incubation of phenoloxidase with SDS. A cylindrical molecular structure was deduced from the electron microscopic analysis. The outside and inside diameter and the height of phenoloxidase molecule were evaluated to be 100, 50 and 70 Å, respectively.     

Assimilation of Ammonia by Gluconobacter suboxydans through Glutamine Synthetase/Glutamate Synthase Pathway

A gram negative bacterium isolated from soil was found to produce an endo-α-N-acetylgalactosaminidase in culture. The microorganism was identified as Alcaligenes sp. from various bacteriological characteristics. The enzyme was purified from the culture fluid by fractionation with ammonium sulfate, and column chromatographies on DEAE-Sephadex A-50 and hydroxylapatite. The molecular weight of the enzyme was estimated to be 160,000 by gel filtration and SDS-polyacrylamide gel electrophoresis. The optimum pH was found to be 4.5 and the stable pH range was 4.5 ~ 6.5. The Km values were 3.7 mm and 3.2 mm with asialofetuin and asialo κ-casein glycopeptide as substrates, respectively. The enzyme released the disaccharide, Galβ1→3GalNAc, from glycoproteins possessing serine or threonine O-glycosidic linkages. The enzyme production was highly induced by porcine gastric mucin added to the medium.     

A laccase-like phenoloxidase is correlated with lignin biosynthesis in Zinnia elegans stem tissues

Stem tissues from different internodes of 4–6 week-old Zinnia elegans cv. Envy plants were sectioned and stained with chromogenic substrates previously used in studies of laccases (p-diphenol:O₂ oxidoreductases) isolated from tree tissues. The pattern of color development found when stem sections were stained in the presence and absence of H₂O₂ suggested that p-diphenol:O₂ oxidoreductase activity was tightly correlated spatially and temporally with the lignification of secondary cell walls in developing primary xylem. The correlation between this laccase-like phenoloxidase activity and lignification appeared tighter than that between lignification and peroxidases stained using the same substrates. Zymogram analysis of the phenoloxidase activities catalyzed by enzymes that were not boiled prior to separation by SDS—PAGE suggested that a single enzyme was predominantly responsible for the laccase-like phenoloxidase activity in Zinnia stems. Some of this enzyme was released from cell wall residue by washing with high ionic strength buffer; however, substantial amounts of the enzyme could only be recovered after treatment of the residue with cell wall-degrading enzymes. This phenoloxidase appears to share significant characteristics with the coniferyl alcohol oxidase isolated from developing secondary xylem in pines, which suggests that such enzymes may be widespread in vascular plants.     

Purification and Characterization of an Endopolygalacturonase Produced by Sclerotinia Sclerotiorum

An endopolygalacturonase (endo-PG), was purified from the culture medium of a local isolate of Sclerotinia sclerotiorum with ammonium sulphate precipitation, cation exchange chromatography and gel filtration. The purified endo-PG had a molecular mass of approximately 18 kDa estimated by gel filtration. The isoelectric point was determined by isoelectric focusing to be approximately 8, suggesting that PG II possesses a net positive charge at physiological pHs. The pH optimum for the enzyme was at pH 4.5. The endo-PG showed essentially the same affinity for pectin and polygalacturonic acid as substrates. This revised version was published online in July 2006 with corrections to the Cover Date.     

A Novel Synthesis of (±)-Munduserone via Acetylenic Intermediates

A riboflavin α-glucoside-synthesizing enzyme from the acetone powder of pig liver was purified by a procedure including fractionation with ammonium sulfate, heat treatment, fractionation with acetone, gel filtration on a Sephadex G-150 column, calcium phosphate gel treatment, and isoelectric focusing. A final enzyme preparation was homogeneous on polyacrylamide disc gel electrophoresis and in the ultracentrifuge. The enzyme had a sedimentation coefficient of 9.90 S and an isoelectric point of pH 3.7. The enzyme had a pH optimum at 6.0 with maltose as substrate. The enzyme catalyzed the hydrolysis of diverse kinds of α-glucosidic substrates, and the transfer of α-glucosyl residue from these substrates to riboflavin. The Km value for maltose was 1.20×10^?3m. The enzyme hydrolyzed phenyl α-maltoside to glucose and phenyl α-glucoside. Amylose was almost completely hydrolyzed to glucose by the enzyme. Maltotriose was obtained as the main transfer product after the treatment of maltose with the enzyme. The enzyme also catalyzed the transfer of α-glucosyl residue from maltose to pyridoxine, esculin, rutin, and adenosine. It was recognized that a single enzyme catalyzed not only the hydrolysis of maltose and α-glucosidic substrates but also the transfer of the α-glucosyl residue of these substrates to suitable acceptors.     

Purification and Properties of Acid Carboxypeptidase IV from Aspergillus oryzae

Acid carboxypeptidase IV from Aspergillus oryzae was purified from the rivanol precipitable fraction by column chromatography on DEAE-cellulose, DEAE-Sephadex A–50, hydroxylapatite and P-cellulose and gel filtration through Sephadex G–100. The optimum pH is at pH 3.0 for carbobenzoxy-l-glutamyl-l-tyrosine. The enzyme activity was inhibited by sulfhydryl reagents and diisopropylphosphorofluoridate, but was not inhibited by metal chelating agents. The molecular weight of the enzyme was estimated to be about 43,000 by gel filtration method.     

Purification and Some Properties of β-Xylosidase from Malbranchea pulchella var. sulfurea No. 48

A β-xylosidase of a thermophilic fungus, Malbranchea pulchella var. sulfurea No. 48, was purified 99-fold from the culture filtrate after ammonium sulfate fractionation, DEAE-cellulose column chromatography, column electrophoresis and gel filtration on Sephadex G–200. The purified enzyme was found to be homogeneous upon ultracentrifugal analysis, disc electrophoresis and gel filtration. The molecular weight of the enzyme was estimated to be 26,000 by gel filtration, and the sedimentation coefficient was calculated to be 2.78S. at 280 nm in phosphate buffer (pH 6.7) was 13.2. The optimum pH was found to be in the range of 6.2~6.8, and the optimum temperature was 50°C.     

The occurrence of an adult reproductive diapause in the univoltine life cycle of the beech leaf mining weevil,Rhynchaenus fagi L.

Summary

Juvenile hormone (JH) and α-naphthyl acetate (α-NA) esterase activity was measured on a daily basis during embryogenesis of the house cricket, Acheta domesticus. In eggs dissected from the lateral oviducts and embryos through blastokinesis, there were elevated levels of nonspecific JH esterase activity. The JH esterase activity could not be resolved from the α-NA esterase activity by gel filtration chromatography and the metabolism of both substrates was inhibited equally by 0,0-diisopropyl phosphorofluoridate (DFP). From blastokinesis through egg hatch, the JH esterase activity was maintained at relatively low levels and was resolved from the α-NA esterase activity by gel filtration. The α-NA esterase activity was inhibited by DFP while the JH esterase activity was relatively unaffected. Low JH titers in eggs must be maintained through blastokinesis for normal development. Elevated JH esterase activity in eggs during this period appears to have a functional role in the metabolism of maternal JH in the egg.     

Purification and Some Properties of Dipeptidyl Carboxypeptidase from Bacillus pumilus

An intracellular protease from a bacterium, Bacillus pumilus HL721, was purified about 5000-fold by Chromatography with a Q-Sepharose Fast Flow column, TSK-gel HA-1000 glass column, and TSK-gel G3000SWXL column using Bz-Gly-Ala-Pro as a substrate. The enzyme was the most active at pH around 7.5 and stable from 4.5 and 8.0. The enzyme activity was inhibited by Cu²⁺, EDTA, N-ethylmaleimide, o-phenanthroline, and p-chloromercuribenzoic acid. The molecular weight of the enzyme was 155,000 by gel filtration. The enzyme removed dipeptide from the carboxyl end of some peptides used as substrates. From these results the enzyme seems to be a dipeptidyl carboxypeptidase.     

Purification and Some Properties of α-Galactosidase from Tubers of Stachys affinis

An α-galactosidase from tubers of S. affinis was purified about 130 fold by ammonium sulfate fractionation, chromatography on DEAE-cellulose and gel filtration on Sephadex G-75. The purified enzyme showed a single protein band on disc gel electrophoresis. The molecular weight of the enzyme was determined to be approximately 42,000 by gel filtration and 44,000 by SDS disc gel electrophoresis. The optimum reaction pH was 5.2. The enzyme hydrolyzed raffinose more rapidly than planteose. The activation energy of raffinose and planteose by the enzyme was estimated to be 7.89 and 11.4 kcal/mol, respectively. The enzyme activity was inhibited by various galactosides and structural analogs of d-galactose. Besides hydrolytic activity, the enzyme also catalyzed the transfer reaction of d-galactosyl residue from raffinose to methanol.     

Guanosine Deaminase and Guanine Deaminase from Tea Leaves

Guanosine deaminase and guanine deaminase were partially purified from tea leaves. The optimum activity of guanosine deaminase was observed at pH 7.5 and that of guanine deaminase was at pH 7.0–7.5 and 8.5. Guanosine deaminase was an unstable enzyme. The activities of these deaminases were significantly inhibited by heavy metals. Molecular weights of guanosine deaminase and guanine deaminase as measured by gel filtration were about 18,000 and 54,000, respectively. The K_m for the respective substrates, guanosine and guanine, were 9.5 μm and 41.7 μm. Guanosine deaminase was considered to catalyze the deamination of 2′-deoxyguanosine besides guanosine. It is suggested that guanosine deaminase as well as guanine deaminase in tea leaves not only acts on the catabolic pathway, but also is involved in the biosynthesis of caffeine from guanosine or guanine nucleotides.     

Characterization of Endo β-1,3-Glucanase IV from Flavobacterium dormitator var. glucanolyticae FA-5

Endo β-1,3-glucanase IV (E.C. 3.2.1.6, endo-1,3(4)-β-d-glucanase) from Flav. dormitator var. glucanolyticae FA-5 was shown to be a glycoprotein by gel filtration and sodium dodecyl sulfate gel electrophoresis. The carbohydrate moiety was composed of 17 hexose units. The enzyme had an apparent molecular weight of 3.3 x 10⁴, determined by gel filtration, sodium dodecyl sulfate gel electrophoresis and ultracentrifugation. The enzyme showed maximum reactivity at pH 6.0 and 6.5 for living yeast cells and laminaran, respectively. The enzyme predominantly released laminaripen-taose from a variety of linear β-1,3-glucans and showed transglucanosylation activity. The amino acid composition of the enzyme and some of its physicochemical and enzymatic properties are described.     

Purification and Characterization of γ-Glutamylmethylamide Synthetase from Methylophaga sp. AA-30

γ-Glutamylmethylamide synthetase [L-glutamate: methylamine ligase (ADP-forming), EC 6.3.4.12] was purified about 70-fold from a cell-free extract of Methylophaga sp. AA-30 by ammonium sulfate fractionation, Octyl-Sepharose column chromatography, and Sephacryl S-300 gel filtration. Only a single protein band was detected after SDS-polyacrylamide gel electrophoresis of the purified preparation; the band was at a position corresponding to a molecular weight of 56,000. The molecular weight of the enzyme was calculated to be 440,000 by Superose 6HR gel filtration, so we suggest that the enzyme is an octomer of identical subunits. The enzyme had maximum activity at pH 7.5 and 40°C. It could use ethylamine and propylamine instead of methylamine as the substrate, but it could not use D-glutamate or L-glutamine instead of L-glutamate.     

Purification and Properties of Leucine Aminopeptidase I–III from Aspergillus oryzae

An aminopeptidase from Aspergillus oryzae 460 was purified from the rivanol precipitable fraction. The partially purified enzyme was not homogeneous in disc electrophoresis, although symmetric profiles were obtained for enzyme protein and activity in Sephadex gel filtration. Its optimum pH is at pH 8.5 for l-leucyl-β-naphthylamide. The enzyme activity was inhibited by metal chelating agents and S-S dissociating agents, but not inhibited by SH reagents. The molecular weight of the enzyme was estimated to be about 26,500 by gel filtration. The enzyme was named leucine aminopeptidase I of Asp. oryzae 460, since it preferentially hydrolyzed oligopeptides that possess leucine as the amino terminal amino acid.     

Occurrence of Endogenous Pollen Growth Inhibitors, 4-Hydroxybenzoic Acid and 4-(β-D-Glucopyranosyloxy)benzoic Acid,in the Pollen of Pinus densiflora†

A Pseudomonas strain capable of using pyrazinamide as the sole source of nitrogen was isolated from soil. An aromatic amidase from the bacterium was purified 400-fold to homogeneous on polyacrylamide gel electrophoresis. The enzyme had a molecular weight of 43,000 by gel filtration on Sephadex G-150 and consisted of two identical subunits. The isoelectric point was at 4.45. Among the compounds tested, pyrazinamide (relative activity, 100%), nicotinamide (60%), and 5-methylpyrazinamide (3.4%) were hydrolyzed at considerable rates. Benzamide, picolinamide, and isonicotinamide were not substrates. Apparent Km of the enzyme for pyrazinamide and nicotinamide were 5.6 × 10 ^?5 m and below 5 × 10^?6 m, respectively. The enzyme was not able to hydrolyze aliphatic amides. The enzyme was most active between pH 6.5 and 10 and 75°C, and was stable between pH 5.5 and 8.5 and below 45°C.     

Purification and Properties of Alkaline Proteinase from Aspergillus oryzae

Alkaline proteinase was purified from culture extract of a strain of Aspergillus oryzae. The process consists of the Amberlite IRC-50 adsorption, column chromatography on DEAE-cellulose and CM-cellulose and Sephadex G-100 gel filtration. The molecular weight of the enzyme was estimated to be about 23,000 by a gel filtration method. Alkaline proteinase showed neither carboxypeptidase activity nor aminopeptidase activity, but degraded 10101010 poly-l,α-glutamic acid, poly-l-lysine, 10101010 and 10101010. The enzyme was completely inhibited by diisopropylphos-phorofluoridate (10^?2 m) or potato inhibitor (250 μg/ml).     

Study on a proteolytic enzyme from Trypanosoma congolense

Summary A protease has been purified from Trypanosoma congolense bloodstream forms by osmotic disruption, freeze-thawing of the cells, followed by chromatography using Thiopropyl-Sepharose and gel filtration.The enzyme is a thiolprotease. A combination of SDS-polyacrylamide gel electrophoresis and contact print zymograms using casein as substrate showed a single proteolytic band with a molecular weight of 31 000. The isoelectric point of the enzyme as ascertained by isoelectric focusing extended from pH 4.4 to 5.5 with a maximum at pH 5.0. The protease cleaved various heat denatured substrates such as casein, hemoglobin, albumin and ovalbumin. The highest enzyme activity was observed at pH 5.5 and pH 6.0 using casein and hemoglobin as substrates respectively. The max. temperature was found to be 50 °C. The enzyme is inactivated by mercurial compounds, iodoacetamide, iodoacetate, chloromethylketones and leupeptin and is activated by dithioerythritol.