Purification and properties of two ribonucleases and a nuclease from barley seeds

Three enzymes possessing RNAase activity were isolated from barley seeds. These enzymes were further purified by ammonium sulphate precipitation DEAE-cellulose chromatography, gel filtration on Sephadex G-75 and DEAE-Sephadex A-50 chromatography. These enzymes have been characterized and classified as: 1. Plant RNAase I (EC 3.1.27.1). It has a pH optimum at 5.7 and molecular weight of 19 000. 2. Plant RNAase II (EC 3.1.27.1). It has a pH optimum at 6.35 and molecular weight of 19 000. 3. Plant nuclease I (EC 3.1.30.2). It has a pH optimum at 6.8 and molecular weight of 37 000. Two RNAases were purified to homogeneity by means of affinity chromatography on poly(G)-Sepharose 4B, as shown by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.     

Purification and characterization of acid proteinase from Neosartorya fischeri var. spinosa IBT 4872

An acid proteinase from Neosartorya fischeri var. spinosa IBT 4872 was purified 38-fold with a yield of 11% by ultrafiltration, ammonium sulphate fractionation, Sephadex-G200 gel filtration, DEAE-Sephadex anion exchange chromatography, and hydroxyapatite chromatography. The enzyme was most active at pH 3·0 and 50 °C and had a molecular weight of 45 kDa, as determined by SDS-PAGE. It was stable over a pH range of 3·0 to 6·0 and exhibited thermal stability up to 50 °C. The Km value for haemoglobin was 0·44% (w/v). The activity was inhibited by pepstatin, suggesting that the enzyme is an aspartic proteinase.     

PURIFICATION AND CHARACTERISTICS OF RNase INHIBITOR FROM PIG CEREBRAL CORTEX

Abstract— An RNase inhibitor has been purified from pig cerebral cortex by DEAE-cellulose and hydroxylapatite chromatography and Sephadex G-100 gel filtration. The purified RNase inhibitor could be resolved into a major band (about 80–85 per cent of total protein) and several minor components by polyacrylamide gel electrophoresis.
The ultraviolet absorption curve of the purified RNase inhibitor indicated a typical protein spectrum. The inhibitor was inactivated by digestion with trypsin or prozyme, and by heating at 70ºC for 5 min. The inhibitor was also inactivated by an SH reagent such as p -chloromercuribenzoate. The inhibitor did not affect RNase T₁. It has been suggested that the inhibitor is an acidic protein and also a SH-protein. The molecular weight of the RNase inhibitor was estimated to be about 60,000.     

Amylase from Lactobacillus cellobiosus D-39 isolated from vegetable wastes: Purification and characterization

An organism producing α-amylase and identified as Lactobacillus cellobiosus D-39 was recently isolated from vegetable wastes. Its amylase was purified by (NH₄)₂SO₄ precipitation and DEAE cellulose column chromatography and was obtained in crystalline form. It was fairly stable at a broadly neutral pH range and maximally active at 50°C. The molecular weight was 22 500 daltons as determined by SDS gel electrophoresis.     

Emergence and disappearance of an immune molecule,an antimicrobial lectin,in basal metazoa. A tachylectin-related protein in the sponge Suberites domuncula

Sponges (phylum Porifera) represent the evolutionarily oldest metazoans that comprise already a complex immune system and are related to the crown taxa of the protostomians and the deuterostomians. Here, we demonstrate the existence of a tachylectin-related protein in the demosponge Suberites domuncula, termed Suberites lectin. The MAPK pathway was activated in response to lipopolysaccharide treatment of the three-dimensional cell aggregates, the primmorphs; this process was abolished by the monosaccharide D-GlcNAc. The cDNA encoding the S. domuncula lectin was identified and cloned; it comprises 238 amino acids (26 kDa) in the open reading frame. The deduced protein has one potential transmembrane region, three characteristic Cys residues, and six internal tandem repeats; it shares the highest sequence similarity with lectins from the horseshoe crab Tachypleus trunculus. The steady-state level of expression of the Suberites lectin rises in primmorphs in response to lipopolysaccharide, an effect that was prevented by co-incubation with D-GlcNAc. The natural sponge lectin was purified by affinity chromatography; it has a size of 27 kDa and displays antibacterial activity against the Gram-negative bacteria Escherichia coli and the Gram-positive bacteria Staphylococcus aureus. The putative protein, deduced from the cloned gene, is identical/similar to the purified natural protein, as demonstrated by immunological cross-reactivity with specific antibodies. We conclude that the S. domuncula lectin acts as an antibacterial molecule involved in immune defense against bacterial invaders.     

Purification and properties of a chitinase from Penicillium oxalicum autolysates

A chitinase (EC. 3.2.1.14) from autolysed culture filtrate of Penicillium oxalicum was purified by precipitation with ammonium sulphate, gel filtration and ion exchange chromatographies. The purified enzyme showed a single protein band in SDS gel electrophoresis. The enzyme is an acidic protein with a pI of 4.5 and has a molecular weight of 54 900 as estimated from SDS gel electrophoresis and 21 500 from gel filtration. The optimum pH and temperature were 5.0 and 35°C, respectively. The enzyme was stable at temperatures up to 45°C and in a pH range between 4.0 and 6.0. The K_m was 2.5 mg ml^-1 for colloidal chitin, Hg²⁺ and Ag⁺ were effective inhibitors. The viscosimetric study carried out using carboxymethyl chitin as substrate revealed the endotype action of this enzyme.     

Rhodanese from Cercopithecus aethiops (vervet monkey) liver. I. Purification and some physical characteristics

Rhodanese (thiosulphate sulphurtransferase , EC 2.8.1.1.) from Cercopithecus aethiops (vervet monkey) liver has been isolated and purified by means of extraction, ammoniumsulphate and pH fractionation, anion-exchange chromatography, Sephacryl S-300 gel chromatography and cation-exchange chromatography. A yield of about 10% pure enzyme with a specific activity of 242 U/mg protein corresponding to a purification factor of 523 was obtained. The enzyme was physically characterized and its homogeneity determined by electrophoretic studies and gel chromatography. The rhodanese enzyme has a molecular weight of 37,000 daltons, a D020 ,w value of 7.6 X 10(-7) cm2 sec-1, a Stokes radius (molecular size) of 2.75 X 10(-7) cm and a frictional ratio of 1.071.     

Purification and characterization of an extracellular amylase from Lactobacillus plantarum strain A6

Extracellular amylase from Lactobacillus plantarum A6 was purified by fractionated precipitation with ammonium sulphate and by anion exchange chromatography. The homogeneity of the purified fraction was tested by polyacrylamide gel electrophoresis and showed multiple amylase forms. A major form had an estimated molecular weight of 50 kDa. It was identified as an α-amylase, with an optimum pH of 5.5, an optimum temperature of 65°C and K _m value of 2.38 g l^-1 with soluble starch substrate. The enzyme was inhibited by N -bromosuccinimide, iodine and acetic acid. The enzyme activation energy was 30.9 kJ mol^-1.     

THE DIAPAUSE FACTOR FROM THE SILKWORM, BOMBYX MORI L.: PURIFICATION AND INACTIVATION EXPERIMENTS

The diapause factor, which is responsible for the induction of èmbryonic dia pause in the silkworm ( Bombyx mori L.), has been partially purified from the extract of adult heads by means of protein purification procedures, including the use of gel filtration of Sephadex, column chromatography on Dowex 1, isoelectric focusing and phenol extraction.
Two species of the diapause factor could be recognized in respect to their molecular weight. They were separated by Sephadex G-25 and their molecular weights were estimated to be about 2,000 and 5,000 from the gel filtration results. The smaller species was purified about 90-fold in specific activity, and its isoelectric point was determined by isoelectric focusing to be at about pH 4.5.
The biological activity of the partially purified principle could be abolished by incubation with several proteolytic enzymes (trypsin, α-chymotrypsin and pronase), or by treatment with amino acid-modifying reagents such as tyrosinase, N-bromosuccinimide or 2-hydroxy-5-nitrobenzyl bromide, but was not affected by incubation with neuraminidase, cyanogen bromide or photooxidation in the presence of methylene blue.     

Purification and partial characterization of manganese peroxidase from immobilized Phanerochaete chrysosporium

Solid-state culture of the white-rot fungus Phanerochaete chrysosporium BKMF-1767 (ATCC 24725) has been carried out, using an inert support, polystyrene foam. Suitable medium and culture conditions have been chosen to favor the secretion of manganese peroxidase (MnP). The enzyme was isolated and purified from immobilized P. chrysosporium and partially characterized. Partial protein precipitation in crude enzyme was affected using ammonium sulphate, polyethylene glycol, methanol, and ethanol methods. Fractionation of MnP was performed by DEAE-Sepharose ion exchange chromatography followed by Ultragel AcA 54 gel filtration chromatography. This purification attained 23.08% activity yield with a purification factor of 5.8. According to data on gel filtration chromatography and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), the molecular weight of the enzyme was 45 000±1000 Da. The optimum pH and temperature of purified MnP were 4.5 and 30 °C, respectively. This enzyme was stable in the pH range 4.5–6.0, at 25 °C and also up to 35 °C at pH 4.5 for 1 h incubation period. MnP activity was inhibited by 2 mM NaN₃, ascorbic acid, β-mercaptoethanol and dithreitol. The K_m values of MnP for hydrogen peroxide and 2.6-dimetoxyphenol were 71.4 and 28.57 μM at pH 4.5, respectively. The effects of possible inhibitors and activators of enzyme activity were investigated.     

Production, purification and characterization of an α-amylase produced by Saccharomycopsis fibuligera

Saccharomycopsis fibuligera ST 2 produced high levels of extracellular amylase during the stationary phase of growth. Glucose or other low molecular weight metabolizable sugars did not repress the synthesis of the amylase, indicating the lack of catabolite repression in this organism. Of the nitrogen sources examined, yeast extract and corn steep liquor stimulated the highest yield of amylase. Ammonium sulphate inhibited α-amylase synthesis. The enzyme was purified 118-fold from the culture supernatant fluid by isopropanol precipitation and DEAE-Sephadex A50 chromatography. The purified enzyme was characterized as an α-amylase. The α-amylase had the following properties: molecular weight, 40900 ± 500; optimum temperature, 60°C; activation energy, 1600 cal/mol; optimum pH, 4·8–6·0; range of pH stability, pH 4·0–9·4; K_m (50°C, pH 5·5) for soluble starch, 0·572 mg/ml; final products of starch hydrolysis—glucose, maltose, maltotriose and maltotetraose.     

Calf-spleen nicotinamide--adenine dinucleotide glycohydrolase. Solubilization purification and properties of the enzyme.

NAD glycohydrolase of calf spleen was solubilized with pancreatic lipase and purified approximatively 800-fold to a specific activity of 7 units/mg of protein by successive DEAE-cellulose and carboxymethyl-cellulose chromatography. The purified enzyme has a molecular weight of 24,000 and is characterized by a double band on disc gel electrophoresis. Some kinetic properties of the NAD-glycohydrolase-catalyzed hydrolsis of NAD have been examined using a titrimetric assay for enzyme activity. The reaction is subject to inhibition be excess of substrate, which disappears at high ionic strength and low pH. At a pH below 5 the kinetic displays an apparent activation by substrate. The effects of pH (4.5-9.0) on the kinetic parameters do not reveal an essential ionizable group in the catalytic process.     

Origin of the integrin-mediated signal transduction. Functional studies with cell cultures from the sponge Suberites domuncula.

Sponges (phylum Porifera) represent the phylogenetically oldest metazoan animals. Recently, from the marine sponge Geodia cydonium a first cDNA encoding a putative integrin receptor molecule was isolated. In the present study basic functional experiments have been conducted to test the hypothesis that in sponges integrin polypeptides also function as adhesion molecules and as outside-in signaling molecules. The sponge Suberites domuncula has been used for the experiments because from this sponge only has a cell culture been established. Here we report that aggregation factor (AF)-mediated cell-cell adhesion is blocked by the RGDS peptide which is known to interact with beta integrin. Both RGDS and AF were found to stimulate DNA synthesis within 24 h. The beta subunit of the integrin receptor was cloned from S. domuncula; the estimated 91-kDa molecule comprises the characteristic signatures. Evolutionary conservation of the beta integrin was assessed by comparison with corresponding beta integrin subunits from evolutionary higher metazoan taxa. Addition of RGDS or of AF to isolated cells of S. domuncula causes a rapid (within 1-2 min) increase in the intracellular Ca2+ concentration which is further augmented in the presence of Ca2+. Furthermore, incubation of the cells with RGDS or AF causes an activation of the GTP-binding protein Ras. In addition it is shown that after a prolonged incubation of the cells with RGDS and AF the expression of the genes coding for Ras and for calmodulin is upregulated. These results suggest that the integrin receptor functions in the sponge system not only as adhesion molecule but also as a molecule involved in outside-in signaling.     

Purification and characterization of a trypsin-like proteinase from the midgut of the larva of the hornet, Vespa orientalis

A proteinase from the larval midgut of Vespa orientalis was purified by exchange chromatography on DEAE-Sephadex A-50 and gel filtration on Sephadex G-75. This purified enzyme was proved to be homogeneous by electrophoresis on a cellulose acetate membrane. The molecular weight was calculated to be 27,000 by gel filtration. Optimum pH for the hydrolysis of N-benzoyl-arginine-ethyl ester (BAEE) was 7·5 to 8·5, and optimum temperature with casein as a substrate was 60°C at pH 8·0 for 20 min. According to studies with synthetic inhibitors the hornet protease belongs to the ‘serine proteases’, being inhibited by phenylmethyl sulphonylfluoride (PMSF) and tosyl-lysyl chloromethane (TLCK). The hydrolysis of different amino acid ester bonds and the cleavage specificity on the B chain of oxidized insulin allow us to speak of a trypsin-like protease.     

Purification and properties of NADP-dependent glutamate dehydrogenase from Sphaerostilbe repens

NADP-dependent glutamate dehydrogenase (EC 1.4.1.4) extracted from Sphaerostilbe repens was purified to homogeneity by using ammonium sullate fractionation hydroxyapatite and DEAE-cellulose column chromatography and, finally, preparative polyacrylamide gel electrophoresis. The turnover number of the enzyme for the amination reaction was about 66000 mol substrate transformed min^-1 (molecule of GDH)^-1. Molecular weight of the native enzyme was estimated to be 280000 dalton by polyacrylamide gradient gel electrophoresis. The same technique in the presence of sodium dodecyl sulfatc gave a single protein band that corresponded to the subunit molecular weight of 48000 dalton. Thus, it is concluded that NADP-GDH is composed of six identical polypeptidic chains.
The pH optimums were 6.9 and 8.4 for the forward and reverse reactions respectively. The NADP-GDH lost practically none of its activity for ten days at 4°C and for 15 h at room temperature, but was inactivated by higher temperatures. Thiol compounds such as 2-mercaptoethanol and dithiolhrcitol protected the enzyme from rapid inactivation. The Michaelis constants for GDH were 0.64, 0.049. 0.043 and 5.5 m M for α-ketoglutaratc. NADPH, NADP and glutamate, respectively. The enzyme had a negative cooperativity for ammonium (Hill number of 0.66), and its K_m value increased from 2.6 to 21.2 m M when the ammonium concentration exceeded 16 m M . The deamination reaction was highly sensitive to inhibition by ammonium, while the amination reaction was only slightly inhibited by glutamate. These results, considered together with the K_m values, indicate that the NADP-GDH in Sphaerostilbe repens is primarily concerned with glutamate biosynthesis.     

Interaction of sugars, polysaccharides and cells with boronate-containing copolymers: from solution to polymer brushes

Interaction of mono- and disaccharides, polysaccharide particles and yeast cells with boronate-containing copolymers (BCC) of N-acryloyl-m-aminophenylboronic acid (NAAPBA) with N,N-dimethylacrylamide (DMAA) or N-isopropylacrylamide (NIPAM) was studied. The binding of saccharides to BCC of NIPAM resulted in a shift of its phase transition temperature (DeltaTP), which provided a quantitative measure for the complex formation. Among the sugars typical of non-reducing ends of glycoproteins the DeltaTP decreased in the order: N-acetylneuraminic acid > xylose approximately galactose > mannose approximately fucose > N-acetylglucosamine. Strong specific adsorption of the BCC on the cross-linked agarose gel Sepharose CL-6B (15-30 mg/ml gel at pH 9.2) was registered. The copolymers adsorption was due to boronate-sugar interactions and decreased with pH. Multivalent interaction of the BCC with the agarose gel has been proven by liquid column chromatography exhibiting a weak reversible adsorption of NAAPBA and almost irreversible adsorption of DMAA-NAAPBA copolymer from 0.1 M sodium phosphate buffer, pH 7.9. The two studied BCCs could be completely desorbed from the gel by 0.1 M fructose in aqueous buffered media with pH from 7.5 to 9.2. In turn, the agarose particles and yeast cells were found to adhere to siliceous supports end-grafted with boronate-BCC of N,N-dimethylacrylamide at pH > or = 7.5, due to the actions. Quantitative detachment of adhered particles or cells could be attained by addition of 20 mM or 100 mM fructose, respectively, in the pH range from 7.5 to 9.2. Affinity adhesion of micron-size carbohydrate particles to boronate-containing polymer brushes fixed on solid supports was considered as a model system suggesting a new approach to isolation and separation of living cells.     

Characterization of a Thermostable α-Amylase from Bacillus licheniformis NCIB 6346

With one exception (NCIB 9668), the extracellular amylases from 10 strains of Bacillus licheniformis were thermostable and retained more than 98% of their original activity after incubation at 85°C for 60 min. The enzyme from B. licheniformis NCIB 6346 was purified 30-fold by ion-exchange chromatography and was characterized. It had an endo-action on starch yielding maltopentaose as the major product, and was identified as an α-amylase. The purified enzyme had a molecular weight of 62 650, was stable between pH 7 and 10 and was maximally active at 70-90°C at pH 7.0. It closely resembled commercial thermostable α-amylases in its general properties and it is concluded that B. licheniformis provides a good source of these enzymes.     

Purification of human renin substrate.

Renin substrate, angiotensinogen, has been purified from human plasma by methods which permit the processing of large amounts of outdated bank blood. The purified protein is homogeneous by disc gel electrophoresis at pH 9.5. The specific activity of 18 nmol/mg corresponds to a molecular weight of 56,000, while a higher value, 90,000, is found by gel filtration. Chromatography of partially purified renin substrate on DEAE-cellulose in a descending pH gradient shows evidence for the existence of multiple forms. However, some of these forms appear to be lost after chromatography on hydroxylapatite.     

Partial purification and properties of a chromatin bound endonuclease from the marine sponge Geodia cydonium

A chromatin bound endonuclease (Mr:107,000) has been extracted and partially purified from the siliceous sponge Geodia cydonium. Disc gel electrophoresis showed that only one enzyme was present in the partially purified preparation which was able to degrade DNA and poly(A). The enzyme liberates oligonucleotides on incubation with poly(A), which are further degraded to yield the 5'-mononucleotide, which has a pI of 6.5 and a pH optimum of 7.5-8.0. Cations are not required for enzymic activity and EDTA does not inhibit the enzyme. Only iodosobenzoic acid was found to completely inhibit the enzyme. The enzyme hydrolysed poly(A), poly(U), poly(C), DNA, poly[d(A-T)], poly[d(G-C)], but not poly (dA) or poly(G).     

Degradative acetolactate synthase of Bacillus subtilis: purification and properties.

A degradative acetolactate synthase (acetolactate pyruvate-lyase [carboxylating], EC 4.1.3.18) from Bacillus subtilis has been partially purified and characterized. The synthesis of the enzyme was induced by growth of cells in minimal medium plus isobutyrate or acetate. The enzyme was partially purified by ammonium sulfate fractionation, gel filtration, and hydroxyapatite chromatography. The pH optimum of the purified enzyme was 7.0 in phosphate buffer. When assayed in phosphate buffer (pH 7.0), activity was stimulated by acetate and inhibited by sulfate. When assayed in acetate buffer (pH 5.8), activity was inhibited both by sulfate and phosphate. Michaelis-Menten kinetics was observed when the enzyme was assayed in phosphate buffer (pH 6.0 or 7.0), and inhibition by sulfate was competitive and activation by acetate was noncompetitive. When assayed in acetate buffer (pH 5.8), nonlinear Lineweaver-Burk plots were obtained; inhibition by phosphate appeared to be competitive and that by sulfate was of the mixed type. The approximate molecular weight of the purified enzyme was 250,000 as determined by gel filtration.