Isolation and purification of cytokinin binding protein from tobacco leaves by affinity column chromatography

Cytokinin binding protein from tobacco leaves was isolated and purified to a single protein by means of affinity chromatography on benzyladenine-linked Sepharose column combined with polyacrylamide gel electrophoresis. In vitro binding of this protein to [¹⁴C] benzyladenine was inhibited remarkably by cold benzyladenine and kinetin and slightly by adenine, but not adenosine. The molecular weight of the protein was determined to be about 4,000 daltons by gel filtration and SDS polyacrylamide gel electrophoresis.     

Purification and characterization of chitin-binding proteins from the hemolymph of sweet potato hornworm, Agrius convolvuli

Three chitin-binding proteins (CBPs: CBP9, CBP15, CBP66) were identified from the larval hemolymph of sweet potato hornworm, Agrius convolvuli.Two (CBP9 and CBP15) of them have been isolated and purified by gel filtration (Superdex HR 75), cation-exchange chromatography (Mono S), and reverse-phase chromatography (μRPC PC 2.1/3). In experiments to detect CBPs in hemolymph, we examined whether ionic strength and existence of bovine serum albumin in the incubation solution influenced binding affinity of CBPs to chitin. The N-terminal sequences of three CBPs were determined by the automated Edman degradation and showed the sequence homology in basic local alignment search tool search. CBP15 and CBP66 were quite similar to lysozymes and bovine serum albumins, respectively. In contrast, CBP9 is not similar to any other known protein, as judged from databank comparisons. Therefore, we concluded that CBP9 is a novel protein with binding capacity to chitin that is a component of the fungal cell wall. CBP9 has no antibacterial activity against Escherichia coli and Micrococcus luteus, and also showed negative response in hemagglutination assay. CBP9 is confirmed as a monomer with a molecular mass of 9.14 kDa by electron spray ionization and matrix-assisted laser desorption ionization mass spectrometry.     

Isolation and structural studies on a galactofuranosyl-containing glycopeptide fromAscobolus furfuraceus

A galactofuranosyl-containing glycopeptide has been isolated from mycelium ofAscobolus furfuraceus by extraction with water. The glycoconjugate was purified by DEAE-cellulose chromatography followed by gel filtration. A molecular weight of about 20 000 was determined by the latter method using standard dextrans. Neutral sugars accounted for 94.5% of the glycopeptide and were characterized as mannose, galactose, and glucose. Glucosamine was estimated colorimetrically (1.8%). The molar ratio of Man:Gal:Glc:GlcNH₂ was 68:32:16:2. A trace amount of total phosphorus (0.2%) was found. The predominant amino acids were threonine and serine. The peptide moiety was labeled with [¹⁴C]formaldehyde and the elution of radioactivity was coincident with sugar on gel filtration in the presence of sodium dodecyl sulfate. The peak of radioactivity was retarded on release of galactose by mild acid hydrolysis. These results confirm the sugar-peptide linkage.     

Synthesis of 1-(2,6,6-Trimethyl-4-hydroxy-1-cyclohexen-1-yl)-1,3-butanedione

Streptococcus dysgalactiae IID 678, belonging to group C of the streptococci, secreted a large amount of hyaluronidase (hyaluronate lyase, EC 4.2.2.1) into a culture medium containing hyaluronic acid. The purification procedures of hyaluronidase were 70% ammonium sulfate precipitation, ECTEOLA-cellulose chromatography, phospho-cellulose chromatography, and gel filtration on Sephacryl S-300. The hyaluronidase was purified approximately 27,000-fold from the culture filtrate. The purified enzyme was homogeneous by SDS-poIyacrylamide gel electrophoresis. The enzyme degradated only hyaluronic acid and chondroitin to zl 4,5-unsaturated disaccharides and did not act on other glycosaminoglycans containing sulfate groups, while the degradation rate of chondroitin was about 1/60 of that of hyaluronic acid. The optimum pH was wide, from pH 5.8 to pH 6.6, and the optimum temperature was 37°C. Fe^{2 +}, Cu^{2 +}, Pb^{2 +}, and Hg^{2 +} ions inhibited the activity strongly and Zn²⁺ inhibited it by half. The molecular weight of the enzyme was estimated to be 125,000 by gel filtration and 117,000 by SDS-polyacrylamide gel electrophoresis. The enzyme was different immunochemically from the hyaluronidase from Streptococcus pyogenes belonging to group A.     

The effect of metals on isolated pea pyruvate decarboxylase

Pyruvate decarboxylase (PDC) was prepared from four-day-old pea seedlings by a procedure which involves extraction of plant material with a phosphate buffer, fractionation of the extract with ammonium sulphate, desalting by dialysis or gel filtration on Sephadex G-25 column, chromatography on DEAE cellulose and filtration on Sepharose 4B. The PDC preparation activity 10 000 U g^-1 protein was about 600 fold higher than that of the sodium phosphate buffer extract. According to the enzyme behaviour during the gel filtration on different carriers the molecular mass of pea PDS was estimated at about 10⁶. Magnesium ions and thiamine pyrophosphate were found to be coenzymes of PDC. Cofactors can be removed by 48 h dialysis followed by chromatography on DEAE cellulose. Apoenzym is activated optimally with the concentration of cofactors of 0.002 M. Magnesium ions can be replaced in their activation function by ions of Fe²⁺, Ni²⁺, Co²⁺, Zn²⁺ and Mn²⁺. Another ions,i.e. Ba²⁺, Ca²⁺, Cd²⁺, Hg²⁺ and Cu²⁺ are inactive. Assumption about the relation between the ion diameter and degree of activation is formulated.     

Purification and properties of a high specific activity protein kinase from wheat germ

A protein kinase was extensively purified to near-homogeneity from wheat germ by a procedure involving affinity chromatography on casein-Sepharose 4B, gel filtration, and repeated chromatography on carboxymethyl-Sepharose CL-6B. The protein kinase preparations have the highest specific activities (up to 656 nanomoles phosphate incorporated per minute per milligram of protein) yet reported for plant protein kinases. The major polypeptides in purified preparations were revealed as two barely-resolved bands (molecular weight 31,000) on polyacrylamide gel electrophoresis in subunit-dissociating conditions. The molecular size of the protein kinase as determined from gel filtration is 30,000. The protein kinase catalyzes the phosphorylation of casein, phosvitin, and the wheat germ cyclic AMP-binding protein cABPII but not of bovine serum albumin and histones nor of the wheat germ cytokinin-binding protein CBP. The protein kinase has a pH optimum of 7.9 and a K_m value for ATP of 10 micromolar. The protein kinase differs from wheat germ CBP kinase in molecular weight, differential sensitivity to inhibitors, and in substrate specificity.     

Resolution and properties of a protein kinase catalyzing the phosphorylation of a wheat germ cytokinin-binding protein

The major cytokinin binding protein of wheat germ (CBP) was extensively purified employing chromatography on Cibacron F3GA-Sepharose CL6B and concanavalin A-agarose as key purification steps. The major polypeptides present in the purified CBP preparations have molecular weights of 60,000 ± 4,000, 42,000 ± 3,000, and 37,000 ± 3,000, respectively. A protein kinase that catalyzes the phosphorylation of CBP (CBP kinase) was extensively purified from wheat germ by affinity chromatography on casein-Sepharose 4B and CBP-Sepharose 4B. The purification procedure resolves CBP kinase from an abundant casein kinase that does not phosphorylate CBP. CBP kinase catalyzes the phosphorylation of casein, phosvitin, CBP, and the wheat germ cyclic AMP-binding protein cABPII. CBP kinase phosphorylates the major 60,000 dalton subunit of CBP as well as 16,000 to 18,000 dalton polypeptides present in CBP preparations. CBP fractions with differing activities as substrates for CBP kinase were partly resolved by gel filtration and by chromatography on DEAE-Sephacel.     

Endo-β-Glucanase from Acetobacter xylinum: Purification and Characterization

A cellulose-producing acetic acid bacterium, Acetobacter xylinum KU-1, abundantly produces an extracellular endo-β-glucanase (EC 3.2.1.4) in the culture broth. The enzyme was purified to homogeneity by DEAE- and CM- Toyopearl 650M ion-exchange chromatography, Butyl-Toyopearl 650M hydrophobic chromatography, and Toyopearl HW-50 gel filtration. The purified enzyme showed the maximum activity at pH 5 and 50°C: it was stable up to 50°C at pH 5, activated by Co²⁺, and competitively inhibited by Hg²⁺; the apparent K _i was 7 μM. The molecular weight of the enzyme was determined to be about 39,000 by sodium dodesyl sulfate/polyacrylamide gel electrophoresis, and about 41,000 by Toyopearl HW-50 gel filtration; the enzyme is monomeric. The enzyme hydrolyzed carboxymethylcellulose with an apparent K _m of 30 mg/ml and V _max of 1.2 μM/min. It hydrolyzed cellohexaose to cellobiose, cellotriose and cellotetraose, and also cellopentaose to cellobiose and cellotriose, but did not act on cellobiose, cellotriose, or cellotetraose. Received: 3 October 1996 / Accepted: 5 November 1996     

Purification of Cytosine Deaminase from Pseudomonas aureofaciens

Cytosine deaminase from Pseudomonas aureofaciens was purified about 480-fold by means of ammonium sulfate fractionation, ethyl alcohol fractionation, chromatography on columns of DEAE-Sephadex A–50 and hydroxylapatite and by gel filtration on a Sephadex G–200 column. The enzyme was homogeneous by the criteria of sedimentation and electrophoretic analysis. The molecular weight of the purified enzyme was estimated to be 630,000 by gel filtration and consisted of twelve to sixteen identical subunits having a molecular weight of about 45,000. The enzyme catalyzed the deamination of cytosine and 5-methylcytosine.     

Multiple Shoot Formation from Almond Seeds and an Excised Single Shoot

An amylase which produces maltotriose from starch as the main product was found in the culture filtrate of a strain of Bacillus subtilis newly isolated from soil. The enzyme was purified to almost complete homogeneity, as judged by disc electrophoresis in polyacrylamide gel, by means of ammonium sulfate fractionation, DEAE-Sepharose column chromatography and Sephadex gel filtration.

The optimum pH and temperature of the enzyme were around 6~7 and 50°C, respectively. Metal ions such as Hg²⁺, Cu²⁺, Zn²⁺, Ni²⁺ and Fe²⁺ strongly inhibitied the enzyme activity. The molecular weight was found to be about 25,000 by gel filtration. The yields of maltotriose from short-chain amylose (DP 17), amylopectin, soluble starch and glycogen were about 69, 56, 56 and 40%, respectively.     

Composition and properties of the sexual agglutinins of the flagellated green alga Chlamydomonas eugametos

Sexual interaction between gametes of opposite mating type (mt) of the unicellular green alga Chlamydomonas eugametos starts with agglutination of the cells via particular glycoproteins on the flagellar surface. Purification of these socalled agglutinins was achieved by a three-step procedure consisting of, successively, gel filtration, anion-exchange chromatography, and high-performance gel filtration. The amino-acid and sugar compositions of both agglutinins showed a high degree of similarity; the most prominent amino acids were hydroxyproline, serine and glycine, and the main sugars were arabinose and galactose. The carbohydrate portions represented about half of the molecular mass of both agglutinins. Using high-performance gel filtration, a calibration curve was constructed for high-molecular-mass compounds from which the Stokes' radius of the sexual agglutinins could be estimated. The mt ⁺ agglutinin had a Stokes' radius of 39 nm and a sedimentation coefficient of 9.3 S. From these data its molecular mass was estimated to be 1.2·10⁶. The corresponding data for the mt ^- agglutinin were 38 nm, 9.7 S and 1.3·10⁶, respectively. The biological activity of both agglutinins was destroyed by mild periodate treatment. Treatment with specific glycosidases had a differential effect on the biological activity of the agglutinins. These observations indicate that carbohydrate side-chains are needed for biological activity and perhaps are responsible for the specifity of the sexual agglutinins. A comparison of both agglutinins is given and their possible structure is discussed in relation to their amino-acid and sugar compositions.Abbreviations HP high performance - mt mating type - SDS sodium dodecyl sulfate     

Characterization of a nicotinic acetylcholine receptor from rabbit skeletal muscle and reconstitution in planar phospholipid bilayers.

The nicotinic acetylcholine receptor from rabbit skeletal muscle was isolated by affinity chromatography and characterized by ¹²⁵I- α -Bungarotoxin binding and gel filtration chromatography. Quantal conductance events were observed when this material was added to planar phospholipid bilayers. These changes were stimulated by carbamylcholine and antagonized by curare, Butx, dithiothreitol and concanavalin A. The receptor preparation was found to bind 0.2 nMoles ¹²⁵I- α-Bungarotoxin per mg protein and the molecular weight was estimated to be 390,000.     

Purification of malic enzyme from bovine heart mitochondria by affinity chromatography

A simple two-step method for the purification of malic enzyme from bovine heart mitochondria in high yield is described. It consists of successive affinity chromatography steps on immobilized C⁸-(aminohexyl)-NADP and N⁶-(aminohexyl)-ADP. The molecular weight estimated by gel filtration of the homogeneous enzyme is 250,000 and the subunit molecular weight by SDS-polyacrylamide gel electrophoresis is 59,000.     

Purification and Characterization of Aminopeptidase from Euphausia superba

Krill aminopeptidase was purified about, 1,100-fold from an extract of Euphausia superba with DEAE-cellulose column chromatography, Toyopearl HW55, and hydroxyapatite column chromatography. The final preparation was electrophoretically homogeneous. The molecular weight was determined to be 140,000 by gel filtration and SDS-polyacrylamide disc gel electrophoresis. The optimum pH and optimum temperature were 8.4 and 45°C respectively. Krill aminopeptidase was inhibited by EDTA, Hg^{+ +} and amastatin, and partially by bestatin, and was activated by Co ^{+ +}. Alanyl-p-nitroanilide was hydrolyzed faster than leucyl-p-nitroanilide. Alanyl peptides (di-, tri-, tetra- and hexa-alanyl peptide) were hydrolyzed very fast.

These results suggest that krill aminopeptidase is an alanine aminopeptidase which is activated by cobaltous ion.     

Purification and partial characterization of mosquito egg development neurosecretory hormone: Evidence for gonadotropic and steroidogenic effects

Egg development neurosecretory hormone (EDNH), a hormone that stimulates vitellogenesis in mosquitoes, was purified 10,000-fold from the mosquito Aedes aegypti. The purification procedure included chromatography on hydrophobic, ion exchange, and gel filtration columns, and preparative electrophoresis to give an almost homogeneous preparation. The hormone is a polypeptide monomer of molecular weight of 18,700 ± 500 as determined from SDS electrophoresis and gel filtration chromatography. Using lowpressure chromatography throughout the purification procedure, the hormone was recovered at a high yield (39%). The amount of EDNH in a mosquito is about 0.6-1.6 ng, corresponding to 32–85 fmol. Injection of purified EDNH into female mosquitoes resulted in the conversion of [¹⁴C]cholesterol into labeled ecdysone and 20-hydroxyecdysone which were separated and identified using thin-layer chromatography and high-performance liquid chromatography separation procedures. Egg development and vitellogenin synthesis were also induced when EDNH was injected into several mosquito species, indicating that the hormone is not species-specific. This report is the first to show that a purified preparation of EDNH has both steroidogenic and a gonadotropic effects on female mosquitoes.     

Macromolecular binding of (5,6-3H) prostaglandin E1 in liver cytosol in vitro

[³H] Prostaglandin E₁ (PGE₁) is bound extensively to macromolecules in liver cytosol $\text{in vitro}$. A principal binding protein accounts for 80% of the binding. This macromolecule is saturated at about 10^?10 M PGE₁. The partially purified protein has a molecular weight of 50,000 by gel filtration and a pI of about 3.5 by isoelectrofocusing. Binding is primarily noncovalent and the dissociated ligand behaves similarly to the parental [³H] PGE₁ on thin layer chromatography. Possible significance of this interaction is discussed.     

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).     

Isolation of an agent affecting the development of an internal parasitoid

The development of an internal braconid parasitoid, Glyptapanteles militaris, is adversely affected when its host, Pseudaletia unipuncta, is infected with the Hawaiian strain of granulosis virus. A plasma-derived agent, isolated from virus-infected hosts, was shown to elicit developmental aberrations in the parasitoid similar to those observed in virus-infected hosts. This agent was isolated by ammonium sulfate precipitation, gel filtration, and anion exchange chromatography, and its molecular weight, established by gel filtration and SDS-polyacrylamide gel electrophoresis, was determined to be about 64,000. The dilution end point of the agent and some stability properties were also established.     

Purification and partial characterization of a cellular retinol-binding protein from human liver

A protein with binding specificity for retinol was purified from human liver. [³H]Retinol was added to liver extracts and the [³H]retinol-binding protein isolated by conventional chromatographic techniques including ion-exchange chromatography on DEAE-Sepharose, gel filtration on Sephadex G-75 and G-50 and preparative isoelectric focusing. The yield was 10–15% in different preparations and the degree of purification was about 3000-fold. The purified protein had a molecular weight of about 15 000 as estimated from both gel filtration and polyacrylamide gel electrophoresis in sodium dodecyl sulphate and was homogeneous in several electrophoretic systems. Isoelectric focusing of the purified protein gave a doublet band. Only one fluorescent band at pH 4.70 was seen if the protein solution was incubated with excess retinol prior to isoelectric focusing. The isolated protein did not react with antiserum to the retinol-binding protein of plasma. The amino acid composition and the amino terminal amino acid sequence for the first sixteen amino acids of the purified protein differed significantly from that of the plasma retinol-binding protein.     

Sex Pheromone of Rice Stem Borer; Purification and Chemical Properties

Bacillus vitellinus, a butirosin-producing organism, was shown to possess butirosin 3′-phosphotransferase catalyzing the phosphorylation of butirosin A into butirosin A 3′-phosphate.

The enzyme was purified about 1200-fold from the cell-free extract of the organism by ammonium sulfate fractionation, affinity chromatography on butirosin A-Sepharose 4B and two gel filtrations on Sephadex G–100.

The molecular weight of the enzyme was estimated to be about 30,000 by gel filtration. The pH optimum was between 6.7 and 8.8. Mg²⁺ was required for maximal activity and could be partially replaced by Co²⁺. ATP and GTP were effective phosphoryl donors. The enzyme catalyzed the phosphorylation of aminoglycoside antibiotics such as butirosin A, butirosin B, xylostasin, ribostamycin, neomycin, paromomycin, kanamycin A and kanamycin B. The Km values for butirosin A and ATP were 4.0 × 10^?6 m and 5.6 × 10^?5 m, respectively. The enzyme was strongly inhibited by p-chloromercuribenzoate, Ag⁺ and Hg²⁺, and was competitively inhibited by 3′-deoxybutirosin A.