The cellulase of Fusarium solani. Resolution of the enzyme complex

1. Culture filtrates from Fusarium solani were fractionated by ion-exchange chromatography on DEAE-Sephadex, followed by gel chromatography on Sephadex G-100, into a C(1) component, a C(x) component (CM-cellulase) and a beta-glucosidase (cellobiase) component. 2. The individual components showed little capacity for the solubilization of cotton fibre (cellulase activity), but when recombined in their original proportions 81% of the original cellulase activity was recovered. 3. The C(1) components of F. solani and Trichoderma koningii were similar in their pH optima, heat stabilities over the pH range 5-8 and elution volumes on Sephadex G-100. 4. The C(1) component of F. solani synergized with the C(x) component of T. koningii and conversely. 5. The C(1) and the beta-glucosidase components of F. solani were devoid of the swelling-factor (S-factor) activity associated with the C(x) component.     

Purification of lysophospholipase of Vibrio parahaemolyticus and its properties.

Lysophospholipase [EC 3.1.1.5] was solubilized from the cells of Vibrio parahaemolyticus with Triton X-100 and purified by the following procedure; precipitation with ammonium sulfate, acid treatment and ion exchange column chromatography using DEAE-cellulose, DEAE-Sephadex A-50, and CM-cellulose, successively. The purified preparation was shown to be homogeneous by polyacrylamide gel disk electrophoresis. The isoelectric point of the enzyme was found to be around pH 3.64 by isoelectric focusing electrophoresis, and its molecular weight was estimated to be 89,000 at pH 7.6 by gel filtration on Sephadex G-200. The minimal molecular weight (15,000) was found at pH 3 by gel filtration on Sephadex G-100 and also by SDS-polyacrylamide disk electrophoresis. The enzyme hydrolyzed 1-acyl-GPC, 1-acyl-GPE, 2-acyl-GPE, and lysocardiolipin but did not attack monoacylglycerol, triacylglycerol, or phosphatidylcholine at all. The enzyme activity required no bivalent cations, and was unaffected by reagents specific to SH-groups, although it was inhibited by Hg2+. The enzyme activity was completely inhibited by preincubation with diisopropylfluorophosphate. The enzyme lost its activity on preincubation with either 1% SDS or 8 M urea at 37 degrees C for 30 min, but the activity lost with urea was recovered by dialysis against distilled water.     

Purification and properties of cytochrome c-556 from Agrobacterium tumefaciens B2a.

Cytochrome c-556 from Agrobacterium mefaciens B2a was isolated in a pure, homoneous state. The best purification procedure volved ammonium sulphate fractionation, delting on Sephadex G-25, column chromatographic fractionation on DEAE- and CM-cellulose, and gel filtration on Sephadex G-75 superfine. Substitution of the CM-cellulose step by isoelectric focusing was successful. The purity of the final preparation is warranted by the purity index value, the electrophoretic patterns in the absence and presence of sodium dodecylsulphate, the sedimentation profile and the N-terminal amino acid analysis (alanine). The absorption spectrum of reduced cytochrome c-556 has maxima at 318, 419, 526 and 555.5 nm. The molar extinction coefficient for the alpha-band is 20 200M-1cm-1. The isoelectric point, determined both by preparative and analytical isoelectric focusing, is 5.55 +/- 0.10. The molecular weight of cytochrome c-556 was determined by gel filtration as 12000 and by dodecylsulphate gel electrophoresis as 11 500.     

A thermophilic extracellular -amylase from Bacillus licheniformis

A strain of Bacillus licheniformis isolated from soil produced an extracellular α-amylase(s) with unusual characteristics. The enzyme was purified 126-fold by starch adsorption, DEAE-cellulose treatment, and CM-cellulose column chromatography. Four active protein bands were detected by disc electrophoresis in poly-acrylamide gel although the enzyme behaved as a single peak during both ultracen-trifugation and chromatography using CM-cellulose and Sephadex G-100. The enzyme showed a very broad pH-activity curve and had substantial activity in the alkaline range. The optimal temperature was 76 °C at pH 9.O. The enzyme was stable between pH 6 and 11 at 25 °C, and below 60 °C at pH 8.0. Using Sephadex G-100 gel filtration, a molecular weight of 22,500 was estimated for the enzyme. The action pattern on amylose and amylopectin is unique in that the predominant product during all stages of hydrolysis is maltopentaose.     

The cellulase of Trichoderma koningii. Purification and properties of some endoglucanase components with special reference to their action on cellulose when acting alone and in synergism with the cellobiohydrolase.

1. Four principal endoglucanase components of Trichoderma koningii cellulase were separated and purified by gel filtration on Sephadex G-75, ion-exchange chromatography on DEAE- and sulphoethyl-Sephadex and isoelectric focusing. 2. All four endoglucanases hydrolysed CM-cellulose, H3PO4-swollen cellulose, cellotetraose and cellopentaose, but differed in the rate and mode of attack. 3. Attack on cotton fibre by the endoglucanases was minimal, but resulted in changes that were manifested by an increased capacity for the uptake of alkali, and a decrease in tensile strength. 4. All four endoglucanases acted synergistically with the exoglucanase [cellobiohydrolase; Wood & McCrae (1972) Biochem. J. 128, 1183-1192] of T. koningii during the early stages of the breakdown of cotton fibre, but only two could produce extensive solubilization of cotton cellulose when acting in admixture with the exoglucanase component. 5. The mode of action of the enzymes is discussed in relation to these synergistic effects. It is suggested that the results are compatible with the interpretation that the 'crystalline' areas of cotton cellulose are hydrolysed only by those endoglucanases capable of forming of forming an enzyme-enzyme complex with the cellobiohydrolase on the surface of the cellulose chains.     

Isolation and some properties of a nonspecific extracellular ribonuclease of Aspergillus clavatus]

RNase has been isolated from the homogenate of the Aspergillus clavatus mycelium by gel filtration through Sephadex G-75, chromatography on CM-cellulose and DEAE-cellulose. By gel filtration and electrophoresis in polyacrylamide gel the preparation has been shown to be homogeneous. The enzyme is acid protein with the isoelectric point at pH 4.4 and molecular weight of 27,000. RNase has pH optimum at 6.0--6.2 and temperature optimum 60 degrees for RNA action. The enzyme splits RNA completely in the absence of metal ions. Ions Zn2+, Cu+2, Ag+1 and Ni+2 at a concentration of 10(-4) M are strong inhibitors of RNase activity.     

Beta-endorphin-like and adrenocorticotropin-like materials in turtle heart and intestine

1. Turtle heart and intestine acetone powders were extracted with an acetone-water-HCl mixture. An acid acetone powder resulted by adding a copious volume of acetone to the extract. The powder was subjected to gel filtration on Sephadex G-25 and ion exchange chromatography on CM-cellulose. 2. In turtle heart, corticotropin-like bioactivity was distributed among chromatographic fractions (derived from material unretarded on Sephadex G-25) unadsorbed and adsorbed on CM-cellulose. The highest opiate receptor binding activity and beta-endorphin-like immunoreactivity were adsorbed on CM-cellulose. 3. In turtle intestine, corticotropin-like bioactivity was absent. Opiate receptor binding activity was present in fractions unretarded as well as in fractions retarded on Sephadex G-25, indicating a molecular weight of greater and smaller than 5000 respectively. 4. The highest opiate receptor binding activity and beta-endorphin-like immunoreactivity were found in a fraction adsorbed on CM-cellulose.     

Extracellular tyrosinase from Streptomyces sp. KY-453: purification and some enzymatic properties

A strain of Streptomyces isolated from soil was found to produce a large amount of tyrosinase (monophenol, dihydroxy-L-phenylalanine: oxygen oxidoreductase: EC 1.14.18.1) extracellularly. The enzyme was purified from the culture filtrate about 550-fold by a series of column chromatographies on Duolite A-2 and CM-cellulose and gel filtration on Sephadex G-100. The purified enzyme appeared homogeneous as judged by disc gel electrophoresis. The enzyme catalyzed the hydroxylation of monophenols and the oxidation of diphenols and was most active at pH 6.8 with dihydroxy-L-phenylalanine (L-DOPA) as the substrate. It was inhibited by kojic acid, diethyldithiocarbamate, and inhibitors obtained from micro-organisms. The isoelectric point of the enzyme was 9.9, and the molecular weight was estimated to be 36,000 by gel filtration on Sephadex G-100 and 29,000 by sodium dodecyl sulfate (SDS) gel electrophoresis, which suggests that the enzyme is a monomer. Metal analysis by atomic absorption spectroscopy indicated that the enzyme contains nearly 1 gram atom of copper per mol.     

Isolation and characteristics of a nerve growth factor from the venom of the Central Asiatic viper Echis multisquamatus

The nerve growth factor (NGF) was isolated from the Echis multisquamatus venom by ultrafiltration on PM-10 filter, chromatography on TSK-55 gel, ion-exchange chromatography on CM-cellulose and gel filtration on Sephadex G-75. The protein exhibited a marked nerve growth activity within the concentration range of 10-15 ng/ml in cultures of chicken embryo spinal ganglia. The molecular mass of NGF is equal to 33,000-37,000 Da according to Sephadex G-75 gel filtration data; however, according to SDS electrophoresis data its Mr is 13,000 Da. Isoelectrofocusing data suggest that the pI of the isolated factor lies in the region of 7.0-7.2; sugar content is 1-2%.     

A protease-like permeability factor in the guinea pig skin 1. Patial purification and characterization

A permeability factor was extracted in a latent from from guinea pig skin and separated by ammonium fraction into the pseudoglobulin fraction (30–50% saturation). The activation of the latent form of t he permeability factor seemed to be caused in the desalting step by gel filtration with Sephadex G-50. The factor was partially purified by streptomycin treatment and column chromatography using hydroxyapatite, diethylaminoethyl cellulose and Sephadex G-75, in this order. Gel filtration showed that its molecular weight was approx. 35 000. Its permeability activity was heat stable at 61δC for 60 min at neutral pH, resistant at pH 5–10 and at ionic strengts from deionized water to 1 M NaCl at 4°C. Its activity was transient and suppressed by guneia pig serum, but insensitive to an anti-histamic agent (triprolidine). Furthermore, its permeability activity was inhibited by diisopropylfluorophosphate, soybean trypin inhibitor and leupeptin, and completely adsorbed by soybean trypsin inhibitor affinity column. These findings suggested that the permeability factor was a ser ine-type protease.     

Substances with alpha-melanotropin-like immunoreactivity in bovine brains.

1. Bovine cerebral hemispheres were extracted with an acidic medium (acetone-water-hydrochloric acid mixture, 40:5:1 by volume, pH 1.8). The precipitate which formed upon addition of a copious volume of cold acetone to the extract was designated acid acetone powder (AAP). 2. The AAP was then subjected to ion exchange chromatography on carboxymethyl (CM)-cellulose, gel filtration on Sephadex G100 and Sephadex G25, second ion exchange chromatography on CM-cellulose and high performance liquid chromatography. The absorbance of all fractions was measured at 280 nm and their alpha-melanotropin-(alpha-MSH)-like immunoreactivity was monitored with radioimmunoassay. 3. It was found that alpha-MSH-like immunoreactivity and bioactivity (lipolytic activity) was due to low molecular weight materials as evidenced by their retardation on Sephadex G-100 and Sephadex G-25. The immunoreactivity was distributed among fractions adsorbed and fractions unadsorbed on CM-cellulose and also among high performance liquid chromatographic fractions signifying the presence of multiple alpha-MSH-like molecules.     

Transfer of phosphatidylcholine between different membranes in Tetrahymena as studied by spin labeling.

A permeability factor was extracted in a latent form from guinea pig skin and separated by ammonium sulfate fractionation into the pseudoglobulin fraction (30--50% saturation). The activation of the latent form of the permeability factor seemed to be caused in the desalting step by gel filtration with Sephadex G-50. The factor was partially purified by streptomycin treatment and column chromatography using hydroxyapatite, diethylaminoethyl cellulose and Sephadex G-75, in this order. Gel filtration showed that its molecular weight was approx. 35000. Its permeability activity was heat stable at 61 degrees C for 60 min at neutral pH, resistant at pH 5--10 and at ionic strengths from deionized water to 1 M NaCl at 4 degrees C. Its activity was transient and suppressed by guinea pig serum, but insensitive to an anti-histamic agent (triprolidine). Furthermore, its permeability activity was inhibited by diisopropylfluorophosphate, soybean trypsin inhibitor and leupeptin, and completely adsorbed by soybean trypsin inhibitor affinity column. These findings suggested that the permeability factor was a serine-type protease.     

Beta-endorphin-like and adrenocorticotropin-like materials in heart tissues of the rat, gerbil, hamster and guinea pig

1. Heart tissues of several rodent species including the rat, gerbil (Meriones unguiculatus), hamster (Mesocricetus auratus) and guinea pig (Cavia porcellus) were extracted with an acetone-water-HCl mixture. An acid acetone powder was obtained by adding a copious volume of acetone to the extract. 2. Rat heart acid acetone powder was subjected to ion exchange chromatography on CM-cellulose. Gerbil heart acid acetone powder was subjected to salt fractionation, gel filtration on Sephadex G-10 and then ion exchange chromatography on CM-cellulose. Hamster and guinea pig heart acid acetone powders were subjected to gel filtration on Sephadex G-25. 3. The fractions were assayed for the ability to stimulate corticosterone production in isolated rat adrenal decapsular (zona fasciculata, zona reticularis and medulla) cells, to displace D-ala2-D-leu5-(tyrosyl-3,5-3H) enkephalin from binding to rat brain membranes, and to inhibit 125I-human beta-endorphin from binding to its antibodies. 4. The widespread occurrence of beta-endorphin-like immunoreactivity among the rat heart CM-cellulose fractions may reflect different species of beta-endorphin. The fraction with the highest beta-endorphin-like immunoreactivity and opiate receptor binding activity was strongly adsorbed on CM-cellulose. 5. In hamster and guinea pig hearts, beta-endorphin-like immunoreactivity and opiate receptor binding activity were distributed among high molecular weight and low molecular weight fractions. 6. In gerbil hearts, opiate receptor binding activity was present in fractions unretarded on Sephadex G-10 (i.e. with a molecular weight greater than 700) as well as in the retarded fractions (i.e. with a molecular weight smaller than 700).(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of two human erythrocyte arylamidases preferentially hydrolysing N-terminal arginine or lysine residues.

Two arylamidases (I and II) were purified from human erythrocytes by a procedure that comprised removal of haemoglobin from disrupted cells with CM-Sephadex D-50, followed by treatment of the haemoglobin-free preparation subsequently with DEAE-cellulose, gel-permeation chromatography on Sephadex G-200, gradient solubilization on Celite, isoelectric focusing in a pH gradient from 4 to 6, gel-permeation chromatography on Sephadex G-100 (superfine), and finally affinity chromatography on Sepharose 4B covalently coupled to L-arginine. In preparative-scale purifications, enzymes I and II were separated at the second gel-permeation chromatography. Enzyme II was obtained as a homogeneous protein, as shown by several criteria. Enzyme I hydrolysed, with decreasing rates, the L-amino acid 2-naphtylamides of lysine, arginine, alanine, methionine, phenylalanine and leucine, and the reactions were slightly inhibited by 0.2 M-NaCl. Enzyme II hydrolysed most rapidly the corresponding derivatives of arginine, leucine, valine, methionine, proline and alanine, in that order, and the hydrolyses were strongly dependent on Cl-. The hydrolysis of these substrates proceeded rapidly at physiological Cl- concentration (0.15 M). The molecular weights (by gel filtration) of enzymes I and II were 85 000 and 52 500 respectively. The pH optimum was approx. 7.2 for both enzymes. The isoelectric point of enzyme II was approx. 4.8. Enzyme I was activated by Co2+, which did not affect enzyme II to any noticeable extent. The kinetics of reactions catalysed by enzyme I were characterized by strong substrate inhibition, but enzyme II was not inhibited by high substrate concentrations. The Cl- activated enzyme II also showed endopeptidase activity in hydrolysing bradykinin.     

Isolation and Some Properties of a beta-d-Xylosidase from Clostridium acetobutylicum ATCC 824

A beta-d-xylosidase from C. acetobutylicum ATCC 824 was purified by column chromatography on CM-Sepharose, hydroxylapatite, Phenyl Sepharose, and Sephadex G-200. The enzyme had an apparent molecular weight of 224,000 as estimated by gel filtration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the enzyme consisted of two subunits of 85,000 and one subunit of 63,000 daltons. It exhibited optimal activity at pH 6.0 to 6.5 and 45 degrees C. the enzyme had an isoelectric point of 5.85. It hydrolyzed p-nitrophenylxyloside readily with a K(m) of 3.7 mM. The enzyme hydrolyzed xylo-oligosaccharides with chain lengths of 2 to 6 units by cleaving a single xylose from the chain end. It showed little or no activity against xylan, carboxymethyl cellulose, and other p-nitrophenylglycosides.     

Cellulolytic enzyme system of Trichoderma koningii. Separation of components attacking native cotton

1. Cell-free culture filtrates from Trichoderma koningii were concentrated by precipitation with ammonium sulphate between the limits of 20% and 80% saturation. 2. Removal of a low-molecular-weight carboxymethylcellulase (CM-cellulase) component by chromatography on Sephadex G-75 had no effect on the ability of the enzyme complex to solubilize cotton. 3. Further chromatography on DEAE-Sephadex separated a component (C(1)) from the C(x) (CM-cellulase) and beta-glucosidase activities. Separately these components had little ability to produce soluble sugars from cotton, but when recombined in their original proportions this capacity was almost completely recovered. 4. The C(x) component was further fractionated on SE-Sephadex into a fraction containing only CM-cellulase and a fraction showing CM-cellulase and beta-glucosidase activities: the latter two components could be separated by heat treatment. 5. The C(1) component had no swelling factor (S-factor) activity (Marsh, Merola & Simpson, 1953; Reese & Gilligan, 1954) on its own, but it had a synergistic effect on the S-factor activity associated with the CM-cellulase and beta-glucosidase components.     

Purification and characterization of a human urine ribonuclease (RNAase 1) showing genetic polymorphism

A ribonuclease (RNAase) was isolated and purified from the urine of a 45-year-old man by column chromatographies on DEAE-Sepharose CL-6B, cellulose phosphate and CM-cellulose followed by gel filtrations on Bio-Gel P-100 and Sephadex G-75, and finally to a homogeneous state by SDS-polyacrylamide gel electrophoresis. The enzyme was designated RNAase 1. It was possible to detect RNAase 1 isozymes in urine and serum without difficulty using isoelectric focusing electrophoresis followed by immunoblotting with a rabbit antibody specific to RNAase 1. The existence of genetic polymorphism of RNAase 1 was detected in human serum utilizing this technique (Yasuda, T. et al. (1988) Am. J. Hum. Genet., in press). RNAase 1 in serum and urine seemed to exist in multiple forms with regard to molecular weight and pI value. Genetically polymorphic RNAase 1 was a glycoprotein, containing three mannose, one fucose, four glucosamine and no sialic acid residues per molecule, with a molecular weight of 16,000 and 17,500 determined by gel filtration and SDS-polyacrylamide gel electrophoresis, respectively. The enzyme was most active at pH 7.0 on yeast RNA substrate and inhibited remarkably by Cu2+, Hg2+ and Zn2+. It also showed definite substrate preference for poly(C) and poly(U), but much less activity against poly(A) and poly(G). Thus, the enzyme is a pyrimidine-specific RNAase.     

Purification and some properties of rabbit liver cytosol thioltransferase

Thioltransferase was purified 650-fold from rabbit liver by procedures including acid treatment, heat treatment, gel filtration on Sephadex G-50, column chromatography on DEAE-cellulose, isoelectric focusing (pH 3.5-10) and gel filtration on Sephadex G-75. The final enzyme preparation was almost homogeneous in polyacrylamide gel electrophoretic analysis. Only one active peak with an apparent molecular weight (Mr) of 13,000 was detected by gel filtration on Sephadex G-50 and only a single protein band with a molecular weight of 12,400 was detected by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Isoelectric focusing revealed only one enzyme species, having an isoelectric point (pI) of 5.3. The enzyme has an optimum pH about 3.0 with S-sulfocysteine and GSH as substrates. The purified enzyme utilized some disulfides including S-sulfocysteine, alpha-chymotrypsin, trypsin, bovine serum albumin, and insulin as substrates in the presence of GSH. The enzyme does not act as a protein : disulfide isomerase (the activity of which can be measured in terms of reactivation of randomly reoxidized soybean Kunitz trypsin inhibitor). The enzyme activity was inhibited by chloramphenicol, but not by bacitracin. The inhibition by chloramphenicol was non-competitive (apparent K1 of 0.5 mM). Thioltransferase activity was found in the cytosol of various rabbit tissues.     

Purification of hyaluronidase from human placenta.

Hyaluronidase [EC 3.2.1.35] was isolated from human placenta and purified by ammonium sulfate fractionation, DEAE-cellulose column chromatography and gel filtration on Sephadex G-150. Its isoelectric point was at pH 5.2 and the molecular weight was 7 X 10(4) based on Sephadex G-200 gel filtration data. This enzyme was very stable at temperatures below 30 degree, but was almost completely inactivated at 60degree within 30 min. Its optimum pH was 3.9, a characteristic property of a lysosomal hyaluronidase. The Michaelis constant was 1.18 x 10(-1) mg per ml with purified hyaluronate. This enzyme depolymerized hyaluronate, chondroitin, chondroitin 4-sulfate and 6-sulfate, and the end product formed from hyaluronate was tetrasaccharide. Its biological diffusing activity was statistically significant on intracutaneous injection of 1.86 mU of the hyaluronidase into the back skine of a rabbit.     

Toxic Metabolites of an Unidentified Filamentous Fungus Isolated from Zinnia Leaves

Two ribonucleases (RNases) designated RNase I and RNase II were found in Euphausia superba and isolated by (NH₄)₂SO₄ fractionation, 2 cycles of CM-cellulose chromatography and gel filtration on Sephadex G-100. This procedure resulted in a 2,116-fold purification of RNase I and a 130-fold purification of RNase II. The molecular weight of both purified enzymes was estimated by gel filtration to be 31,500. The isoelectric points were 6.0 (RNase I) and 7.0 (RNase II). Each enzyme hydrolyzed poly A-U, poly U but did not degrade poly G, poly C and DNA. Both enzymes were classified as endonuclease from the hydrolysis product of yeast RNA and poly A. The enzymes were located mainly in the cardiac and pyloric portion of the stomach.