Characterization of Potent Fibrinolytic Enzymes in Earthworm,Lumbricus rubellus

Stable and potent fibrinolytic enzymes (six homogeneous proteins) were purified to homogeneity from extracts of the lyophilized powder of an earthworm, Lumbricus rubellus. The molecular weight of each enzyme estimated by SDS–polyacrylamide gel electrophoresis was different from those by gel filtration chromatography in the six purified proteins. The exact molecular weight of each enzyme (F-III-2, F-III-1, F-II, F-I-2, F-I-l, and F-I-0) measured by ionspray MS analysis was 29, 662, 29, 667, 24, 664, 24, 220, 24, 196, and 23,013, respectively. The isoelectric point (pI) of each enzyme was 3.40, 3.60, 4.20, 4.00, 4.30, and 4.85, respectively. The enzymes were single polypeptide chains. They had a very strong fibrinolytic activity and the maximum reactivity for chromogenic substrates from pH 9-11. The enzymes, acidic proteins that had abundant asparagine and aspartic acid, and low lysine in their amino acid composition, did not contain component sugars. The enzymes were stable at from pH 1-11 and up to 60°C. Studies on substrate specificity and inhibition indicated that these enzymes were alkaline trypsin-like serine proteases. N-Terminal amino acid sequences of the enzymes had local similarities to those of trypsin-like enzymes such as elastase and coagulation factor IX. From the results of amino acid sequence, amino acid composition analyses and immunological analyses, it was suggested that these six enzyme proteins were derived as isozyme(s) from at least four different genes.     

Purification and properties of alkaline phosphatase from the mucosa of rat small intestine

Alkaline phosphatase [orthophosphoric monoester phosphohydrolase, EC 3.1.3.1] was purified from the mucosa of rat small intestine by butanol extraction, ethanol fractionation, gel filtration, with controlled-pore glass-10 and DEAE-cellulose column chromatography. On the gel filtration, the enzyme activity was separated into three peaks; A in the void volume, B and C at lower molecular weight positions. Enzyme A was purified to homogeneity. The activity of enzymes A, B, and C was detected even on sodium dodecyl sulfate-polyacrylamide gel electrophoresis at the position of the protein of enzyme A, which had a molecular weight of 110,000 daltons. Enzymatic properties such as pH optimum, Km value for the substrate, heat inactivation and inhibition by amino acids were the same in all three enzymes. Based on these findings, together with the elution positions on gel filtration, enzyme A was regarded as an aggregate, and enzymes B and C as dimer and monomer molecules, respectively.     

Oxidation of fatty alcohol in the cotyledons of jojoba seedlings.

An externally accessible polypeptide has been purified from hepatoma tissue culture cells. The purification involves four steps: deoxycholate extraction of whole cells, isoelectric focusing of deoxycholate-insoluble material in the presence of 8 m urea and Triton X-100, hydroxylapatite chromatography in the presence of sodium dodecyl sulfate, and preparative acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The final preparation is homogeneous, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by isoelectric focusing in polyacrylamide. The polypeptide has an apparent molecular weight of 55,000 and is labeled following in situ lactoperoxidase-catalyzed iodination of the hepatoma tissue culture cells. The polypeptide can also be labeled by growing cells in the presence of labeled amino acids, but is not labeled by growth in labeled sugars. The purified protein does not react with the periodate-Schiff reagent. Hence, it does not appear to be a glycoprotein that contains mannose, fucose, glucosamine, or sialic acids.     

Structural Proteins of Simian Virus 40

Sodium dodecyl sulfate acrylamide gel electrophoresis of the solubilized proteins from purified simian virus 40 (SV40) virions revealed two major and two minor structural polypeptide components. The major components which comprise over 75% of the total virion were shown to be the capsid proteins by immunological and isoelectric focusing fractionation analysis. These two polypeptides have estimated molecular weights of 45,000 daltons as determined by gel electrophoresis. One of the two minor components was identified as the nucleocapsid protein and has an approximate molecular weight of 16,000. The other unidentified minor component has an average molecular weight of 29,000.     

ISOLATION OF AN ACID-SOLUBLE PROTEIN WITH LOW MOLECULAR WEIGHT FROM MONKEY BRAIN

Abstract— The isolation of a perchloric acid-soluble low molecular weight protein from brain of Macaca irus is reported. Sodium dodecyl sulphate polyacrylamide gel electrophoresis and gel isoelectric focusing indicate that the protein is free of impurities. The molecular weight, as determined by gel filtration and sodium dodecyl sulphate gel electrophoresis, is shown to be 10,400 and 9900, respectively. This is in agreement with the value of 10,700 obtained from amino acid analysis. The protein contains 27 per cent acid amino acids and 15 per cent basic amino acids. However, the relatively high amide content gives the protein a neutral nature as shown by isoelectric point determination using gel isoelectric focusing.     

Purification and characterization of an amidase from an acrylamide-degrading Rhodococcus sp.

A constitutively expressed aliphatic amidase from a Rhodococcus sp. catalyzing acrylamide deamination was purified to electrophoretic homogeneity. The molecular weight of the native enzyme was estimated to be 360,000. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified preparation yielded a homogeneous protein band having an apparent molecular weight of about 44,500. The amidase had pH and temperature optima of 8.5 and 40 degrees C, respectively, and its isoelectric point was pH 4.0. The amidase had apparent K(m) values of 1.2, 2.6, 3.0, 2.7, and 5.0 mM for acrylamide, acetamide, butyramide, propionamide, and isobutyramide, respectively. Inductively coupled plasma-atomic emission spectometry analysis indicated that the enzyme contains 8 mol of iron per mol of the native enzyme. No labile sulfide was detected. The amidase activity was enhanced by, but not dependent on Fe(2+), Ba(2+), and Cr(2+). However, the enzyme activity was partially inhibited by Mg(2+) and totally inhibited in the presence of Ni(2+), Hg(2+), Cu(2+), Co(2+), specific iron chelators, and thiol blocking reagents. The NH2-terminal sequence of the first 18 amino acids displayed 88% homology to the aliphatic amidase of Brevibacterium sp. strain R312.     

Purification, properties, and immunological characterization of folate-binding proteins from human leukemia cells

A new matrix for affinity chromatography using pteroylglutamic acid coupled to an epoxy-activated matrix via hexanediamine resulted in negligible ligand leakage and permitted the purification of soluble and membrane-associated folate-binding proteins from human leukemia cells contained in a human spleen. Two species of membrane-associated folate-binding proteins were purified from the solubilized membrane fraction of the tissue using 2 M guanidine-HCl to elute the proteins from the affinity matrix. The higher molecular weight binding protein had an Mr of approximately 310,000 and the smaller species had an Mr of approximately 28,000 by gel filtration. By SDS-polyacrylamide gel electrophoresis the smaller species of membrane-associated protein had a molecular weight of 35,500, but the molecular weight of the larger membrane-associated species could not be determined by this method because of the high concentration of residual Triton X-100 in the sample which interfered with the silver staining of the gel. Two folate-binding proteins, which by SDS-polyacrylamide gel electrophoresis had molecular weights of 34,500 and 32,000, were purified from the 44,000 X g supernatant fraction of the tissue homogenate by acid elution from the affinity matrix. Despite the different cell components from which the soluble and membrane-associated folate-binding proteins were purified, the amino acid compositions were similar, especially with respect to the apolar amino acids. All these forms of folate-binding proteins had higher affinity for oxidized than for reduced folates, and very low affinity for 5-formyltetrahydrofolate and methotrexate. Although these proteins cross-react with one antiserum raised previously to a folate-binding protein from other human leukemia cells, they do not cross-react with the folate-binding proteins purified from two other sources of human leukemia cells, from human placenta, or from the human KB cell line.     

Characterization of Candida albicans dihydrofolate reductase

Dihydrofolate reductase from Candida albicans was purified 31,000-fold and characterized. In addition, the C. albicans dihydrofolate reductase gene was cloned into a plasmid vector and expressed in Escherichia coli, and the enzyme was purified from this source. Both preparations showed a single protein-staining band with a molecular weight of about 25,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzymes were stable and had an isoelectric point of pH 7.1 on gel isoelectric focusing. Kinetic characterization showed that the enzymes from each source had similar turnover numbers (about 11,000 min-1) and Km values for NADPH and dihydrofolate of 3-4 microM. Like other eukaryotic dihydrofolate reductases, the C. albicans enzyme exhibited weak binding affinity for the antibacterial agent trimethoprim (Ki = 4 microM), but further characterization showed that the inhibitor binding profile of the yeast and mammalian enzymes differed. Methotrexate was a tight binding inhibitor of human but not C. albicans dihydrofolate reductase; the latter had a relatively high methotrexate Ki of 150 pM. The yeast and vertebrate enzymes also differed in their interactions with KCl and urea. These two agents activate vertebrate dihydrofolate reductases but inhibited the C. albicans enzyme. The sequence of the first 36 amino-terminal amino acids of the yeast enzyme was also determined. This portion of the C. albicans enzyme was more similar to human than to E. coli dihydrofolate reductases (50% and 30% identity, respectively). Some key amino acid residues in the C. albicans sequence, such as E-30 (human enzyme numbering), were "vertebrate-like" whereas others, such as I-31, were not. These results indicate that there are physical and kinetic differences between the eukaryotic mammalian and yeast enzymes.     

The purification and characterization of homologous high molecular weight storage proteins from grain of wheat,rye and barley

Summary Homologous high molecular weight storage prolamins were purified from grain of wheat, rye and barley using combinations of gel filtration, ion-exchange chromatography and preparative isoelectric focusing. Sodium dodecylsulphate polyacrylamide gel electrophoresis showed that the components were single bands with apparent mol.wts. of above 100,000. Molecular weights determined by sedimentation equilibrium ultracentrifugation were considerably lower; 54,700, 67,600 and 69,600 for the components from barley, rye and wheat respectively. Amino acid analysis showed the presence of 13.6 to 16.5 mol% glycine, 29.6 to 34.0 mol% glutamate + glutamine, 11.4 to 13.7 mol% proline and a total of 4.0 to 5.7 mol% basic amino acids. Automated N-terminal amino acid sequencing of the component from wheat showed the presence of cysteine residues at positions 5 and 10, and this is discussed in relation to the possible role of these proteins in the visco-elastic gluten network.     

Kallikrein-like enzyme from Crotalus ruber ruber (red rattlesnake) venom

1. A kallikrein-like enzyme was isolated and characterized from the venom of Crotalus ruber ruber (red rattlesnake). 2. The kallikrein-like enzyme was shown to be homogeneous as demonstrated by a single band on acrylamide gel electrophoresis, isoelectric focusing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunodiffusion and reverse-phase (RP) HPLC. 3. The enzyme has a molecular weight of 31,000 and isoelectric point of 4.6. It consists of 271 total amino acid residues, 24% of which are acidic amino acids. 4. Specific esterolytic activities of the kallikrein-like enzyme on N-tosyl-L-arginine methylester (TAME) and N-benzoyl-L-arginine ethylester (BAEE) are 109.5 and 23.6 mumol/min/mg, respectively. 5. The enzyme differs from trypsin as the soybean trypsin inhibitor does not inhibit the enzyme's action. Diisopropylfluorophosphate (DFP) inhibits the enzyme, suggesting that the serine hydroxyl group is important for enzyme activity. 6. The enzyme is not lethal at 15 micrograms/g in mice and has no hemorrhagic activity, yet the injection of the purified enzyme intradermally, produced capillary permeability-increasing activity as shown by the use of Evans blue dye, and immediate drop in blood pressure. It also contracted the rat uterus.     

Cloning and sequence analysis of the heat-stable acrylamidase from a newly isolated thermophilic bacterium, Geobacillus thermoglucosidasius AUT-01

A thermophilic bacterium capable of degrading acrylamide, AUT-01, was isolated from soil collected from a hot spring area in Montana, USA. The thermophilic strain grew with 0.2 % glucose as the sole carbon source and 1.4 mM acrylamide as the sole nitrogen source. The isolate AUT-01 was identified as Geobacillus thermoglucosidasius based on 16S rDNA sequence. An enzyme from the strain capable of transforming acrylamide to acrylic acid was purified by a series of chromatographic columns. The molecular weight of the enzyme was estimated to be 38 kDa by SDS-PAGE. The enzyme activity had pH and temperature optima of 6.2 and 70 ºC, respectively. The influence of different metals and amino acids on the ability of the purified protein to transform acrylamide to acrylic acid was evaluated. The gene from G. thermoglucosidasius encoding the acrylamidase was cloned, sequenced, and compared to aliphatic amidases from other bacterial strains. The G. thermoglucosidasius gene, amiE, encoded a 38 kDa, monomeric, heat-stable amidase that catalysed the cleavage of carbon–nitrogen bonds in acrylamide. Comparison of the amino acid sequence to other bacterial amidases revealed 99 and 82 % similarity to the amino acid sequences of Bacillus stearothermophilus and Pseudomonas aeruginosa, respectively.     

Structural Proteins of Adenovirus-Associated Viruses

The structural proteins of adenovirus-associated virus (AAV) types 1, 2, and 3 were analyzed by acrylamide gel electrophoresis. In each case, one major protein (C) and two minor proteins (A and B) were identified. Component C had an estimated molecular weight of 62,000 daltons, and the molecular weights of components A and B were found to be 87,000 and 73,000 daltons, respectively. Coelectrophoresis of adenovirus and AAV proteins revealed an overlap only between the adenovirus fiber-penton component and the AAV C polypeptide. Among AAV serotypes, homologous components were electrophoretically identical, except that the C component of AAV-2 was of slightly lower molecular weight than the C components of AAV-1 and AAV-3. The relative incorporation of (14)C-arginine and (14)C-mixed amino acids into the three polypeptides of AAV-2 was similar, indicating an absence of an arginine-rich component. In addition, AAV-2 was found to have a substantially lower arginine content than helper adenoviruses.     

Keratin Degradation by Fervidobacterium pennavorans, a Novel Thermophilic Anaerobic Species of the Order Thermotogales

From a hot spring of the Azores islands a novel thermophilic bacterium belonging to the Thermotogales order was isolated. This strain, which grows optimally at 70(deg)C and pH 6.5, is the first known extreme thermophile that is able to degrade native feathers at high temperatures. The enzyme system converts feather meal to amino acids and peptides. On the basis of physiological, morphological, and 16S rDNA studies the new isolate was found to be a member of the Thermotogales order and was identified as Fervidobacterium pennavorans. The strain was highly related to Fervidobacterium islandicum and Fervidobacterium pullulanolyticum. The cell-bound keratinolytic enzyme system was purified 32-fold by detergent treatment with CHAPS (3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was characterized as a serine protease with a molecular mass of 130 kDa and an isoelectric point of 3.8. Optimal activity was measured at 80(deg)C and pH 10.0. Furthermore, 19 anaerobic thermophilic archaea and bacteria belonging to the orders Thermococcales, Thermoproteales, Thermotogales, and Clostridiales (growth temperatures between 60 and 105(deg)C) were tested for their abilities to grow on feathers and produce heat-stable keratinolytic enzymes. None of the tested extremophilic microorganisms was able to attack the substrate in a native form.     

The purification and characterization of multiple forms of mouse submaxillary gland renin

Five forms of renin, A₀, A, C, D and E, from mouse submaxillary gland were purified by a two-step procedure including chromatography on the immunoaffinity column and CM-cellulose column. Four renin fractions, A₀, A, C and E were purified to homogeneity by the criteria of polyacrylamide gel electrophoresis, analytical isoelectric focusing and Ouchterlony double immunodiffusion. All these forms of renin have molecular weights of 40 000 as determined by gel filtration on Sephadex G-100 column. No high molecular weight renin could be demonstrated. Individual renin fractions showed similar angiotensin I formation activity, 52–158 ng angiotensin I/ng protein per h. No other protease activity could be detected with hemoglobin or casein as substrate. These purified proteins showed a discrete pattern of migration under polyacrylamide gel electrophoresis. Under denaturing condition in SDS-gel electrophoresis, all but fraction D showed a protein band with a molecular weight of 30 000. Fraction D showed a major component with molecular weight of 33 000. The isoelectric points of these renin forms varied from 5.46 to 5.76. They all reacted with antibody raised against renin A and showed similar pressor response activity with 20 ng quantities of the purified proteins. The closely related characteristics of these five forms of renin were further demonstrated by their similarity in peptide mapping patterns after limited digestion with Staphylococcus aureus V8 protease. The data suggest that these proteins are homologous proteins.     

Purification and properties of a proteinaceous metallo-proteinase inhibitor from Streptomyces nigrescens TK-23.

A novel metallo-proteinase inhibitor which is capable of inhibiting the activities of metallo-proteinases such as the thermolysin, was isolated from the culture filtrates of Streptomyces nigrescens TK-23. The inhibitor was purified batch-wise from the culture filtrate by Amberlite IRC-50 and column chromatographies on CM-Sephadex C-50 and Sephadex G-50. The purified inhibitor showed a single band on 15% polyacrylamide gel electrophoresis at pH 4.3, and at pH 7.5 on SDS-gels. The inhibitor retained 80% of its original activity after treatment of 100 degrees C for 5 min between pH and 7. The molecular weight was estimated to be 12 000 by gel filtration and SDS-polyacrylamide gel electrophoresis, and calcuated as 11 950 from its amino acid composition. The isoelectric point was pH 10.3. The inhibitor showed a high content of hydrophobic amino acids, did not contain tryptophan, and had two disulfide bridges. It also showed specific inhibitory activity for metallo-proteinases but not for serine-, thio- and carboxyl-proteinases.     

粘虫颗粒体病毒增效因子的分离纯化及其生化性质

粘虫颗粒体病毒经０．０２ｍｏｌ／ＬＮａＯＨ碱溶,先用ＳｅｐｈａｄｅｘＧ－２００凝胶过滤层析柱从病毒蛋白粗提中分离增效因子,然后选用ＤＥＡＥ－ＳｅｐｈａｒｏｓｅＣＬ－６Ｂ离子交换层析柱进一步纯化增效因子,得到少量电泳纯的增效因子蛋白样品。     

Cathepsin D of rat spleen. Affinity purification and properties of two types of cathepsin D.

Two types of cathepsin D were purified from rat spleen by a rapid procedure involving an acid precipitation of tissue extract, affinity chromatography with pepstatin--Sepharose 4B and concanavalin-A--Sepharose 4B, and chromatography on Sephadex G-100 and DEAE-Sephacel. The purified major enzyme (85% of the cathepsin D activity after DEAE-Sephacel chromatography), termed cathepsin D-I, represented about a 1000-fold purification over the homogenate and about a 20% recovery. The purified minor enzyme (15%), termed cathepsin D-II, represented about a 900-fold purification and about a 3% recovery. Both enzymes showed four (pI: 4.2, 4.9, 6.1 and 6.5) and three (pI: 4.6, 5.6 and 5.8) multiple forms after isoelectric focusing, respectively. The purified enzymes appeared homogeneous on electrophoresis in polyacrylamide gel and had a molecular weight of about 44000. In sodium dodecylsulfate/polyacrylamide gel electrophoresis both enzymes showed a single protein band corresponding to a molecular weight of 44000. The enzymes had similar amino acid compositions except for serine, proline and methionine. Cathepsin D-I contained 6.6% carbohydrate, consisting of mannose, glucose, galactose, fucose and glucosamine in a ratio of 8:2:1:1:5 with a trace of sialic acid. The properties of purified enzymes were also compared.     

粗毛栓菌胞外锰过氧化物酶的纯化及性质

培养于麦草粉上的白腐担子菌粗毛栓菌分泌胞外木质纤维素降解酶(纤维素酶、木聚糖酶、漆酶、锰过氧化物酶和木质素过氧化物酶)。经过超滤、盐析、离子交换层析、凝胶过滤和活性聚丙烯酰胺凝胶电泳等步骤,获得了初步纯化的锰过氧化物酶组分。利用变性聚丙烯酰胺凝胶电泳和等电点聚焦技术所测定的锰过氧化物酶的相对分子质量和等电点分别为35.7 ku和pI 2.8。研究结果表明,所纯化的锰过氧化物酶在407nm处具有最大光吸收峰,该酶最适作用pH值和温度分别为pH 5.3和35℃。     

Purification and Properties of β-Glucosidase from Humicola insolens YH-8

The thermophilic fungus, Humicola insolens YH-8 exhibited high β-glucosidase activity when grown in solid wheat bran medium. The β-glucosidase was purified from the culture extract by consecutive column chromatographies and found to be homogeneous on polyacrylamide gel disc electrophoresis. The molecular weight was estimated to be 250,000 by SDS-gel electrophoresis, and the isoelectric point was at pH 4.23. The enzyme had an optimum pH of 5.0, an optimum temperature of 50°C, and showed significant resistance to urea, dimethyl sulfoxide and ethyl alcohol.     

Highly acidic proteins from human brain: purification and properties of Glu-50 protein

Three extremely acidic proteins were isolated from human brain and purified to apparent homogeneity. One of them, Glu-50 protein, contained much glutamic acid (about 50% of the total amino acids). Its purification involved ammonium sulfate fractionation, DEAE-Sephadex A-50 chromatography, and gel filtration on Sephadex G-100 and G-75. Its molecular weight was determined to be 11,000 by SDS polyacrylamide gel electrophoresis and 34,000-36,000 by gel filtration on Sephadex G-75, suggesting that it consists of three identical polypeptide chains. Its isoelectric point was pH 3.9. Its N-terminal amino acid sequence was NH2-Asp-Glu-Pro-Pro-Asp-Glu and its C-terminal amino acid was Lys. It contained no detectable carbohydrate.