Purification and characterization of fructokinase from developing tomato (Lycopersicon esculentum Mill.) fruits

A procedure is described which allows the purification of fructokinase (EC 2.7.1.4) from young tomato fruit. The procedure yielded a 400-fold purification and two isoenzymes designated fructokinase I and II (FKI and FKII) were separated by anion-exchange chromatography. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) the molecular mass was estimated to be 35 kDa. Gel filtration on Sepharose-12 indicated that for both fructokinases the functional form is a dimer. Two dimensional isoelectric focusing/SDS-PAGE combined with immunoblotting showed that FKI has two components with isoelectric points (pIs) of 6.42 and 6.55, while four components with pIs from 6.07 to 6.55 were detected for FKII. A mixture of both fructokinases showed that the components of FKI match the more alkaline components of FKII. The activity of both fructokinases increased with increasing pH to around 8.0 and equal activity was observed from 8.0 to 9.5. Both fructokinases were specific for fructose with K _m values for fructose of 0.131 and 0.201 mM for FKI and FKII, respectively. At high concentrations (> 0.5 mM), fructose was also a strong inhibitor with inhibition constants (K _i) of 1.82 and 1.39 mM for FKI and FKII, respectively. The preferred phosphate donor for both isoforms was ATP, and K _m values of 0.11 and 0.15 mM were observed for FKI and FKII. At low concentrations (0.05–0.2 mM), fructose exhibited noncompetitive inhibition with respect to ATP for both fructokinases. This inhibition pattern changed to uncompetitive when higher fructose concentrations (0.5–10 mM) were used. These data indicated that substrate addition is ordered, with ATP adding first. Inhibition by ADP was also affected by the fructose concentrations. At 0.5 mM fructose, FKI showed non-competitive inhibition by ADP with respect to ATP and this inhibition changed to uncompetitive when 3 mM fructose was used. The isoform FKII showed a competitive inhibition pattern for ADP at 0.5 mM fructose which also changed to uncompetitive when 3 mM fructose was used. The features of the regulation of both fructokinases suggest that this enzyme might have a relevant role in carbon metabolism during tomato fruit development.     

Characterization of DNA kinase from calf thymus

DNA kinase has been purified to homogeneity from calf thymus. The purified enzyme, with a specific activity of 16.7 units/mg protein at 25 degrees C, exhibited a sharp pH/activity curve with a pH optimum at 5.5 and low activity at alkaline pH. The molecular weight of the enzyme was estimated by dodecylsulfate/polyacrylamide gel electrophoresis to be 5.4 X 10(4). The enzyme has a sedimentation coefficient of 4.0 S. An apparent molecular weight of 5.6 X 10(4) and a Stokes' radius of 3.3 nm were estimated by gel-filtration on Sephadex G-100. The enzyme phosphorylates neither yeast RNA nor poly(A) instead of DNA. Compared with rat liver DNA kinase, calf thymus DNA kinase is relatively resistant to the inhibition by sulfate (Ki = 7 mM) and pyrophosphate (Ki = 5 mM). The enzyme activity is markedly stimulated by polyamines at the sub-optimal concentration of Mg2+ but not by monovalent cations.     

Characterization of native and yeast-expressed tomato fruit fructokinase enzymes.

Three fructokinase isozymes (FKI, FKII, FKIII) were separated from both immature and ripe tomato fruit pericarp. All three isozymes were specific for fructose with undetectable activity towards glucose or mannose. The three isozymes could be distinguished from one another with respect to response to fructose, Mg and nucleotide donor concentrations and this allowed the comparison of the fruit enzymes with the gene products of the two known cloned tomato fructokinase genes, LeFRK1 and LeFRK2. FKI was characterized by both substrate (fructose), as well as Mg, inhibition; FKII was inhibited by neither fructose nor Mg; and FKIII was inhibited by fructose but not by Mg. ATP was the preferred nucleotide donor for all three FKs and FKI showed inhibition by CTP and GTP above 1 mM. All three FKs showed competitive inhibition by ADP. During the maturation of the tomato fruit total FK activity decreased dramatically. There were decreases in activity of all three FKs, nevertheless, all were still observed in the ripe fruit. The two tomato LeFRK genes were expressed in yeast and the gene products were characterized with respect to the distinguishing characteristics of fructose, Mg and nucleotide inhibition. Our results indicate that FKI is the gene product of LeFRK2 and FKII is probably the gene product of LeFRK1.     

Characterisation of 20-kDa lectin-spermagglutinin from Arum maculatum that prevents Chlamydia pneumoniae infection of L-929 fibroblast cells

A novel lectin from the root of Arum maculatum was isolated by saline extraction and purified by cold ethanol precipitation and subsequent fractionation on Superose 6 column. The lectin named A. maculatum agglutinin is a non-glycosylated protein with 20-kDa molecular mass agglutinating human ejaculated spermatozoa, but not human erythrocytes. The agglutination was blocked in the presence of N-acetylneuraminic acid indicating that the lectin is sialoglycoprotein specific. Chlamydia pneumoniae strain AR-39 showed considerable potential to grow in murine L-929 fibroblast cells. Pretreatment of the cell monolayers with purified lectin reduced the entry and intracellular replication of C. pneumoniae. These results suggest that the isolated lectin prevents attachment by binding to a C. pneumoniae specific sialoglycoprotein receptor expressed on the surface of L-929 fibroblast cells.     

Purification of a High Molecular Weight Kininogen from Rat Plasma

A high molecular weight kininogen has been isolated from rat plasma and purified. At each preparative step the kininogen concentration and purity were monitored by assay on the perfused isolated rat uterus in terms of bradykinin equivalents formed per mg protein following incubation of the plasma fractions with rodent acid protease for 24 hours at 37 and pH 4.0. Kinin formation by crystalline trypsin and human pancreatic kallikrein also was compared. Citrated rat plasma first was precipitated with 43% ammonium sulfate. The kininogen fractions then were subjected to a series of gel filtration ion exchange chromatographic columns that included G-200 Sephadex, G-200: G-100 Sephadex interconnected columns, DEAE-A50 Sephadex, and hydroxylapatite. The kininogen fractions finally were subjected to preparative polyacrylamide gel electrophoresis, resulting in a final purification of 92.9-fold compared to the initial rat plasma. A single major kininogen protein band and a minor band of protein impurity were obtained on disc gel electrophoresis. Only the pancreatic kallikrein did not form kinin from this purified kininogen. The apparent molecular weight was estimated by SDS polyacrylamide gel technique to be 110,000.     

BRAIN PEPTIDASES: CONVERSION AND INACTIVATION OF KININ HORMONES

Abstract— Two enzymes that selectively hydrolyse kinins at pH 7.5 were obtained in partially purified form from the supernatant fraction of homogenates of previously frozen rabbit brain by gel filtration on Sephadex G-100. The enzymes were detected and their activity estimated by bioassay with the isolated guinea pig ileum The products of the enzymic reactions were identified by high voltage electrophoresis at pH 3.5 and by the determination with the amino acid analyser of the amino acids released from the kinins.
One enzyme, kinin-converting enzyme, catalyses the hydrolysis of kinin-10 (Lysbradykinin) and kinin-11 (Met-Lys-bradykinin) into kinin-9 (bradykinin). It also hydrolyses the aminoacyl-8-naphthylamides of methionine, lysine, arginine and leucine. The conversion of kinin-10 to kinin-9 was inhibited by puromycin (K_i 3.5 × 10⁻⁵ M) These properties are similar to those of brain arylamidases described in the literature.
Kininase, the second enzyme, inactives kinins 9, 10 and 11 by peptide-bond hydrolysis. Similar rates of release of arginine and phenylalanine were observed for the three kinins, suggesting that kininase acts at the carboxy-terminus of these peptides.
Our results suggest that brain contains proteases which apparently selectively metabolize polypeptide hormones that exert definite pharmacological effects on the central and peripheral nervous systems.     

Proline iminopeptidase from Bacillus coagulans: purification and enzymatic properties

Proline iminopeptidase [EC 3.4.11.5] was purified about 2,700-fold from cell-free extract of Bacillus coagulans by a series of column chromatographies on DEAE-Toyopearl, PCMB-T-Sepharose, and hydroxyapatite, and gel filtration on Sephadex G-150. The purified enzyme was homogeneous as judged by disc gel electrophoresis. The enzyme was most active at pH 7.3 with Pro-beta-naphthylamide (Pro-2-NNap) as the substrate, and hydrolyzed Pro-X (X = amino acid including proline, peptide, amide, and arylamide) bonds when the proline residue was at the amino terminus. Pro-D-amino acid bonds were also susceptible to the enzyme. The enzyme was completely inhibited by p-chloromercuribenzoate (PCMB) and partially by proline but not by metal chelators, diisopropylphosphorofluoridate (DFP), or phenylmethanesulfonyl fluoride (PMSF). The enzyme inactivated with PCMB was reactivated by incubation with 2-mercaptoethanol. These results and the chromatographic profile on PCMB-T-Sepharose suggest that the enzyme is a sulfhydryl enzyme. The isoelectric point of the enzyme was 4.0, and the molecular weight of the enzyme was estimated to be 40,000 by gel filtration on Sephadex G-100 and 35,000 by sodium dodecyl sulfate (SDS) gel electrophoresis, indicating that the enzyme exists as a monomer.     

The Structures of Myocotrienins I. and II,a Novel Class of Ansamycin Antibiotic

Endopolygalacturonase I [EC 3.2.1.15], the major component of endopolygalacturonases causing silver-leaf symptoms, was purified from culture liquids of Stereum purpureum by column chromatographies on CM-52 and Sephadex G-100. The purified enzyme was homogeneous on Polyacrylamide gel electrophoresis and ultracentrifugation. The sedimentation coefficient (S_20,W) was determined to be 3.21 S, and the molecular weight was estimated to be 40,000 by gel filtration, 41,000 by SDS-polyacrylamide gel electrophoresis and 44,000 by sedimentation equilibrium. The enzyme had an isoelectric point of pH 8.5. The optimal pH of the enzyme was 3.5 for trigalacturonic acid, 4.0 for tetragalacturonic acid, and 4.5 for pectic acid. The enzyme was stable in the range of pH 4.0 to 9.0 and up to 70%C for 30 min. The amount of the enzyme which was required to induce silver-leaf symptoms on apple trees was 20 μg/tree.     

创新霉素产生菌吲哚丙酮酸甲基转移酶的研究

在创新霉素产生菌济南游动披线菌的无细胞提取物中检测到吲哚丙酮酸甲基转移酶活性,并进行了分离提取。该酶能利用S-腺苷-L-甲硫氨酸对吲哚丙酮酸进行甲基化,它可能作用于创新霉素中间体的甲基化。经过硫酸铵分部盐析和DEA F一纤维素柱层析,得到了纯化60倍的甲基转移酶,比活0．66mu／ms。酶的最适底物是吲哚丙酮酸,最适pH7．5,对于底物s一腺苷一L一甲硫氨酸和吲哚丙酮酸的米氏常数(KⅢ)分别是4×10-,mol／L和1．8×10-7moI／L。用Sephadcx G-150凝胶过滤测得分子量是55000士5000道尔顿。     

Prolidase from bovine intestine: purification and characterization

Prolidase [iminodipeptidase, EC 3.4.13.9] was highly purified from the cytosol fraction of bovine small intestine by a series of column chromatographies on DEAE-Toyopearl, Sephadex G-150, PCMB-T-Sepharose and hydroxyapatite. The purified enzyme appeared homogeneous as judged by disc gel electrophoresis. The enzyme was most active at pH 7.2 with Gly-Pro as substrate. It was stable between pH 5.5 and 8.5 for 30 min at 30 degrees C and retained half of the activity after 15 min at 40 degrees C. It was completely inactivated by p-chloromercuribenzoate (PCMB) but not inhibited by diisopropylphosphorofluoridate (DFP), phenylmethane sulfonylfluoride (PMSF) and metal chelators. Its amino acid composition was determined. Its molecular weight was estimated to be 116,000 by gel filtration on Sephadex G-150 and 56,000 by sodium dodecyl sulfate (SDS) gel electrophoresis, suggesting that it is a dimer. It hydrolyzed dipeptides represented as X-Pro (X = amino acid).     

Some properties of partially purified phytase from Aspergillus niger.

Phytase was purified from Aspergillus niger culture fluid by molecular sieve filtration on Sephadex G-200, followed by thermal inactivation of acid phosphatase and CM-cellulose chromatography. The 12-fold purified enzyme had two pH optima at 2.7 and 5.5 and was characterized by high thermal stability in alkaline environment and broad substrate specificity. The Michaelis constant of phytase relative to myo-inositol hexaphosphate sodium salt is 4.8 X 10(-4) M and activation energy 9,217 cal/mole. The molecular weight of the enzyme is estimated at 200,000.     

Isolation and characterization of pteroylpolyglutamate hydrolase from rat intestinal mucosa

Pteroylpolyglutamate hydrolase was isolated from rat intestinal mucosa and purified with the aid of affinity chromatography. The affinity ligand was poly-gamma-glutamic acid (Mr approximately 12,000) derived from Bacillus subtilis. The specific enzymatic activity was increased 2,000-fold over the 100,000 X g supernatant of the mucosal homogenate with a yield of 20%. Sephadex G-200 gel filtration yielded an estimated molecular mass of 80,000 daltons. The isoelectric point was pH 8.2. The pH optimum in acetate buffer containing 1 mM zinc was 4.5. The KM values for pteroylheptaglutamate and pteroyltriglutamate were 0.21 and 0.67 microM, respectively. Polyanionic compounds, poly-gamma-glutamic acid, dextran sulfate, and heparin were noncompetitive inhibitors. Studies of the time course of hydrolysis of synthetic [3H]pteroylheptaglutamate by three separate techniques demonstrated the appearance of [3H]pteroylmonoglutamate, synchronous with substrate cleavage. Intermediate pteroyloligoglutamates were not detected. An endopeptidase-like mode of hydrolysis was further established by identification of a hexaglutamyl peptide as the other reaction product.     

Purification and properties of the Pichia guilliermondii acid nucleotide pyrophosphatase hydrolyzing flavin adeninine dinucleotide

Acid nucleotide pyrophosphatase was isolated from the cell-free extracts of Pichia guilliermondii Wickerham ATCC 9058. The enzyme was 25-fold purified by saturation with ammonium sulphate, gel-filtration on Sephadex G-150 column and ion-exchange chromatography on DEAE-Sephadex A-50 column. The pH optimum was 5.9, temperature optimum--45 degrees C. The enzyme catalyzed the hydrolysis of FAD, NAD+ and NADH, displaying the highest activity with NAD+. The Km, values for FAD, NAD+ and NADH were 1.3 x 10(-5) and 2.9 x 10(-4) M, respectively. The hydrolysis of FAD was inhibited by AMP, ATP, GTP, NAD+ and NADP+. The K1 for AMP was 6.6 x 10(-5) M, for ATP--2.0 X 10(-5) M, for GTP--2.3 X 10(-6) M, for NAD+--1.7 X 10(-4) M. The molecular weight of the enzyme was 136 000 as estimated by gel-filtration on Sephadex G-150 and 142 000 as estimated by thin-layer gel-filtration chromatography on Sephadex G-200 (superfine). Protein-bound FAD of glucose oxidase was not hydrolyzed by acid nucleotide pyrophosphatase. The enzyme was stable at 2 degrees C in 0.05 M tris-maleate buffer, pH 6.2. Alkaline nucleotide pyrophosphatase hydrolyzing FAD was also detected in the cells of P. guilliermondii.     

Purification and characterization of the extracellular alpha-amylase activity of the yeast Schwanniomyces alluvius

The extracellular alpha-amylase activity of the yeast Schwanniomyces alluvius has been purified by anion-exchange chromatography on DEAE-cellulose and gel-filtration chromatography on Sephadex G-100. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and N-terminal amino acid analysis of the purified sample indicated that the enzyme preparation was homogeneous. The enzyme is a glycoprotein having a molecular mass of 52 kilodaltons (kDa) estimated by SDS-PAGE and 39 kDa by gel filtration on Sephadex G-100. Chromatofocusing shows that it is an acidic protein. It is resistant to trypsin but sensitive to proteinase K. Its activity is inhibited by the divalent cation chelators EDTA and EGTA and it is insensitive to sulfhydryl-blocking agents. Exogenous divalent cations are inhibitory as are high concentrations of monovalent salts. The enzyme has a pH optimum between 3.75 and 5.5 and displays maximum stability in the pH range of 4.0-7.0. Under the conditions tested, the activity is maximal between 45 and 50 degrees C and is very thermolabile. Analysis of its amino acid composition supports its acidic nature.     

Purification and biologic properties of fibroblast somatomedin

Cultured human fibroblasts produce a peptide growth factor that cross-reacts with antisera to human somatomedin-C (Sm-C). To determine the identity of this species and compare its molecular properties to pure Sm-C, 2 liters of conditioned medium derived from human fibroblast monolayers were concentrated (X10) by ultrafiltration. The concentrated conditioned medium was purified further by CM-Sephadex ion-exchange chromatography. Following elution in 1.0 M NaCl, pH 8.0, the active material was purified by gel filtration on Sephadex G-150. The active fractions which eluted at Kd 0.45 (Mr estimated at 32,000) were further purified by isoelectric focusing. Two peaks of activity electrofocused at pI 5.4 and 7.2, respectively. The pI 5.4 peak contained only binding protein activity. The active fractions from the neutral pool were further purified by reverse-phase high pressure liquid chromatography on a C-18 Bondapak with a linear gradient of acetonitrile (10-60%). The active single peak which eluted at 55% acetonitrile gave a single band when analyzed by polyacrylamide gel electrophoresis. This material stimulated [3H]thymidine incorporation into human fibroblast DNA with approximately 3.2 times the potency of pure Sm-C but was equipotent in stimulating BALB/c 3T3 fibroblasts. It was degraded by fibroblast cultures at a slower rate compared to Sm-C, although it had a similar affinity for Sm-C-binding protein. We conclude that human fibroblasts produce two peptides that react with anti-Sm-C antibody but are chemically distinct from Sm-C. The greater response to fibroblast somatomedin may be due to its affinity for somatomedin-binding protein and slower degradation. These findings may have implications for understanding the regulation of human fibroblast replication.     

trans-2, cis-4-Heptadienoic Acid,One of Metabolites of Sporobolomyces odorus

Water-soluble phospholipase B was purified to homogeneity from Torulaspora delbrueckii cell washings. The washings were concentrated by ultrafiltration, and then a fraction with phospholipase B activity was precipitated with ammonium sulfate, and purified by sequential column chromatographies on Octyl-Sepharose CL-4B, DEAE-Sephacel, and Sepharose 6B. The molecular weight of the enzyme was estimated to be 170,000~200,000 by SDS-polyacrylamide gel electrophoresis and by gel filtration with a Sephadex G-200 column. The isoelectric point of the enzyme was 4.0. The purified enzyme had two pH optima at pH 2.5 and pH 7.5. The activity at acidic pH was greatly stimulated by the divalent metal ions tested, but the activity at alkaline pH was stimulated mainly by Ca²⁺ and Fe²⁺. The purified enzyme had both lysophospholipase activity and phospholipase B activity in a ratio of 37:1 at acidic pH and 73:1 at alkaline pH. The amino acid composition of the enzyme was characterized by high contents of Asp, Ser, Leu, and Gly.     

Purification and characterization of a coagulant protein from the venom of Russell's viper.

The coagulant protein from the venom of Russell's viper was purified by means of successive chromatography on Sephadex G-50, DEAE-cellulose and Sephadex G-200. The purified coagulant protein was homogeneous by polyacrylamide gel electrophoresis and ultracentrifugation. The molecular weight was estimated to be about 100 000 by ultracentrifuge analysis and 130 000 by gel filtration. The coagulant protein contains 11.1% carbohydrate which includes 5.1% hexose (galactose: mannose = 1:1), 5% hexosamine (glucosamine), and 1% neuraminic acid (N-acetylneuraminic acid and N-glycolyneuraminic acid). The isoelectric point is pH 6.3. The results of both sodium dodecyl sulfate electrophoresis and gel filtration in 6 M guanidium chloride suggest that it consists of four polypeptide chains. The coagulant protein functions as an enzyme in activating blood coagulation factor X in the presence of Ca2+. N-a-p-Toluenesulfonyl-L-arginine methyl ester hydrolyzing activity in the preparation definitely decreased during purification and it suggests that the clotting activity is not associated with the esterase activity. The clotting activity is inhibited by diisopropyl phosphorofluoridate and by phenylmethylsulfonyl fluoride, suggesting that the coagulant protein is a serine protease. The optimum pH is between pH 7.0 and pH 8.0. At neutral pH the coagulant protein is stable below 50 degrees C, but is rapidly inactivated above 55 degrees C.     

Purification and properties of pig liver kynureninase.

Kynureninase [L-kynurenine hydrolase, EC 3.7.1.3] was purified from pig liver by a procedure including DEAE-cellulose chromatography, hydroxyapatite chromatography, ammonium sulfate fractionation, DEAE-Bio Gel chromatography, Sephacryl S-200 gel filtration, kynurenine-Sepharose affinity chromatography, and Sephadex G-200 gel filtration. The enzyme was found to be homogeneous by the criterion of disc-gel electrophoresis. The enzyme has a molecular weight of about 100,000 and exhibits absorption maxima at 280 and 420 nm. The optimum pH and the isoelectric point of the enzyme are 8.5 and 5.0, respectively. The Michaelis constants were determined to be as follows: L-kynurenine, 7.7 X 10(-4) M; L-3-hydroxykynurenine, 1.3 X 10(-5) M; and pyridoxal 5'-phosphate, 1.8 X 10(-6) M. L-3-Hydroxykynurenine is hydrolyzed more rapidly than L-kynurenine; the liver enzyme can be regarded as a 3-hydroxy-kynureninase.     

Affinity purification and properties of cathepsin-E-like acid proteinase from rat spleen.

A unique acid proteinase different from cathepsin D was purified from rat spleen by a method involving precipitation at pH 3.5, affinity chromatography on pepstatin-Sepharose 4B and concanavalin A-Sepharose 4B, chromatography on Sephadex G-100 and DEAE-Sephacel, and isoelectric focusing. A purification of 4200-fold over the homogenate was achieved and the yield was 11%. The purified enzyme appeared to be homogeneous on electrophoresis in polyacrylamide gels. The isoelectric point of the enzyme was determined to be 4.1-4.4. The enzyme hydrolyzed hemoglobin with a pH optimum of about 3.1. The molecular weight of the enzyme was estimated to be about 90000 by gel filtration on Sephadex G-100. In sodium dodecylsulfate polyacrylamide gel electrophoresis, the purified enzyme showed a single protein band corresponding to a molecular weight of about 45000. The hydrolysis of bovine hemoglobin by the enzyme was much higher than that of serum albumin. Various synthetic and natural inhibitors of the enzyme were tested. The enzyme was inhbited by Zn2+, Fe3+, Pb2+, cyanide, p-chloromercuribenzoate, iodoacetic acid and pepstatin, whereas 2-mercaptoethanol, phenylmethyl-sulfonyl fluoride and leupeptin showed no effect.