Induction of macrophage interleukin-1 production by Listeria monocytogenes hemolysin.

Listeriolysin O produced by a hemolytic strain of Listeria monocytogenes was purified from the ammonium sulfate precipitate of a culture supernatant through the steps of ion-exchange chromatography and gel filtration. The purified hemolysin finally gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight of 58,000. When peritoneal exudate macrophages were stimulated with purified hemolysin, we found a high level of IL-1 activity as determined by thymocyte costimulator assay in the culture supernatant. Cell-associated and intracellular IL-1 activity was also detected. The activity in the supernatant or membrane was blocked by polyclonal antibody to murine IL-1 alpha. Moreover, IL-1-specific mRNA expression could be detected in the macrophages stimulated with listeriolysin O by Northern blot analysis. Possible contamination by LPS of the listeriolysin O preparation did not seem to contribute to the induction of macrophage IL-1 production.     

Purification and some properties of cytotoxin produced by Clostridium difficile

The cytotoxin produced by Clostridium difficile was highly purified by using ammonium sulfate fractionation and successive column chromatographies of DEAE-Sephadex A-25, hydroxyapatite, Bio-Gel A-0.5m, Phenyl-Sepharose CL-4B, and Mono Q. The purified cytotoxin gave a single band on conventional and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of dithiothreitol. Its molecular weight was estimated to be 260,000 and 50,000 by gel filtration and sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis in the presence of dithiothreitol, respectively. Thus it was supposed that the toxin consists of 5 subunits having molecular weight of approximately 50,000. It had an isoelectric point of 6.6. The toxin was heat-labile (60 C for 10 min) and inactivated by treatment with trypsin and pronase, or at pH below 4 or over 10. The minimum cytotoxic dose of the cytotoxin against Chinese hamster ovary cells was 3 ng. It was also demonstrated that the toxin is antigenically different from enterotoxin of C. difficile.     

Liver microsomal expoxide hydrase. Solubilization, purification, and characterization.

Epoxide hydrase was solubilized from liver microsomes of phenobarbital-treated rats by treatment with cholate and purified to apparent homogeneity by ammonium sulfate fractionation and column chromatography in the presence of the nonionic detergent Emulgen 911 on DEAE-cellulose and hydroxylapatite. The purified enzyme preparation had a single major band with a molecular weight of 53,000 to 54,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Other studies indicated that in the absence of sodium dodecyl sulfate, purified epoxide hydrase exists as high molecular weight aggregates. The preparation was essentially free of heme and flavin, but still contained small amounts of lipids and Emulgen 911.     

Purification and properties of streptococcal nicotinamide adenine dinucleotide glycohydrolase.

Highly purified streptococcal nicotinamide adenine dinucleotide glycohydrolase (NADase) was obtained by utilizing disodium tetrathionate to protect the enzyme by blocking the sulfhydryl groups of streptococcal proteinase. This was followed by two-step ion-exchange chromatography. The pure enzyme, demonstrated as a single band on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, had a specific activity of 11,200 NADase units per mg of protein and was devoid of hemolytic activity. NADase had a molecular weight of about 55,000 as determined by gel filtration, by summation of amino acid residues, and by sodium dodecyl sulfate/gel electrophoresis. The purified enzyme had optimal activity at pH 7.3 and at 40 C and a calculated Km of 5.1 times 10- minus 4 mM. It was inhibited by alpha-iodoacetamide.     

Partial characterization of oat and rye phytochrome

Purified oat and rye phytochrome were examined by analytical gel chromatography, polyacrylamide gel electrophoresis, N-terminal, and amino acid analysis. Purified oat phytochrome had a partition coefficient on Sephadex G-200 (sigma(200)) of 0.350 with an estimated molecular weight of 62,000; sodium dodecyl sulfate polyacrylamide electrophoresis gave an equivalent weight estimate. Purified rye phytochrome had a sigma(200) value of 0.085 with an estimated molecular weight of 375,000; sodium dodecyl sulfate electrophoresis gave a weight estimate of 120,000, indicating a multimer structure for the nondenatured protein. Comparative sodium dodecyl sulfate electrophoresis with purified phycocyanin and allophycocyanin gave a molecular weight estimate of 15,000 for allophycocyanin, and two constituent classes of subunits for phycocyanin with molecular weights of 17,000 and 15,000. Amino acid analysis of oat phytochrome confirmed a previous report; amino acid analysis of rye phytochrome differs markedly from a previous report. Oat phytochome has four detectable N-terminal residues (glutamic acid, serine, lysine, and leucine, or isoleucine); rye phytochrome has two detectable groups (aspartic and glutamic acids). Model experiments subjecting purified rye phytochrome to proteinolysis generate a product with the characteristic spectral and weight properties of oat phytochrome, as it has been described in the literature. It is concluded that the structural characteristics of purified rye phytochrome are likely those of the native protein.     

Purification and characterization of human plasma lecithin:cholesterol acyltransferase.

A highly purified (approximately 12 000-fold) homogeneous preparation of human plasma lecithin:cholesterol acyltransferase (LCAT) with 16% yield was obtained by a combination of density ultracentrifugation, high density lipoprotein affinity column chromatography, hydroxylapatite chromatography, and finally chromatography on anti-apolipoprotein D immunoglobulin-Sepharose columns to remove apolipoprotein D. This enzyme preparation was homogeneous by the following criteria: a single band by polyacrylamide gel electrophoresis in 8 M urea; a single band on sodium dodecyl sulfate gel electrophoresis with an apparent molecular weight of 68 000 +/- 1600; a single protein peak with a molecular weight of 70 000 on a calibrated Sephadex G-100 column. Its amino acid composition was different from human serum albumin and all other apoproteins isolated from lipoprotein fractions.     

Isolation and characterization of bovine factor VII.

Factor VII (proconvertin) has been purified approximately 5 x 10(5)-fold from bovine plasma with an overall yield of 30%. The isolation procedure involves barium sulfate adsorption and elution, DEAE-Sephadex batchwise adsorption and elution, benzamidine-agarose column chromatography, heparin-agarose column chromatography, and preparative polyacrylamide gel disc electrophoresis. The final product was homogeneous when examined by gel electrophoresis in the presence of sodium dodecyl sulfate. A minimal molecular weight of 45,500 was determined by sedimentation equilibrium. The molecular weight estimated by sodium dodecyl sulfate gel electrophoresis was 54,000. Factor VII is composed of a single polypeptide chain possessing an amino-terminal sequence of Ala-Asn-Gly-Phe-Leu-. The amino acid and carbohydrate compositions of factor VII are also reported.     

Purification and properties of oxytocinase (cystine amino-peptidase) from monkey placenta.

Oxytocinase (cystyl-aminopeptidase) [EC 3.4.11.3] was isolated from monkey placenta in a purified form by a six-step prodedure comprising extraction from monkey placenta homogenate, ammonium sulfate fractionation, repeated chromatography on hydroxylapatite, chromatography on a column of DEAE-cellulose and gel filtration on a column of Sephadex G-200. The purified enzyme showed a single band on polyacrylamide disc electrophoresis. Oxytocin was inactivated by this enzyme preparation. The enzyme hydrolyzed several aminoacyl-beta-naphthylamides. A terminal amino group was required for enzyme activity. The molecular weight of the purified enzyme was estimated to be 87,000 by gel filtration and 83,000 by sodium dodecyl sulfate gel electrophoresis. Other properties of the enzyme, the effects of metal ions and various chemical reagents on the enzyme activity, the pH optimum, and Km values for a number of aminoacyl-beta-naphthylamides were also examined.     

Isolation of a 32P-labeled polypeptide of low molecular weight from phosphorylated human erythrocyte membranes.

The incorporation of 32P into well washed human erythrocyte membranes was studied in a medium containing [gamma-32P]ATP, Mg2+, and EGTA. Following phosphorylation, the membranes were completely solubilized in 1% sodium dodecyl sulfate and subjected to gel electrophoresis in dodecyl sulfate polyacrylamide. A large incorporation of radioactivity was observed in a band which migrated faster than component 7 (nomenclature of T. L. Steck, (1972), J. Mol. Biol. 66, 295) but slower than the bromophenol blue tracking dye, and did not stain with Coomassie Blue. Isolation of this band by preparative gel electrophoresis revealed that 41% of the radioactivity was associated with a 32P-labeled polypeptide. This polypeptide was further purified by gel chromatography on Sephadex LH-20 in chloroform-methanol-HCl, and Bio-Gel A 1.5m in dodecyl sulfate. Its amino acid composition is characterized by a high content of acidic residues. The calculated minimal molecular weight is 15084. Based upon the recovery of amino acids, the polypeptide fraction comprises at least 1.8% by weight of the total erythrocyte membrane proteins. An apparent molecular weight of 15000 was estimated by gel chromatography in dodecyl sulfate, while a range of 14000-16000 was estimated by electrophoresis in dodecyl sulfate polyacrylamide. The state of phosphorylation of this peptide may reflect a physiological function in the intact red cell.     

Purification of homogeneous glutamine-dependent carbamyl phosphate synthetase from ascites hepatoma cells as a complex with aspartate transcarbamylase and dihydroorotase.

Glutamine-dependent carbamyl phosphate synthetase [EC 2.7.2.9] was purified 1,300-fold from rat ascites hepatoma cells (AH 13) as a multienzyme complex with aspartate transcarbamylase[EC 2.1.3.2] and dihydroorotase[EC 3.5.2.3], using dimethyl sulfoxide, glycerol, and dithiothreitol as stabilizers. The purified complex was essentially homogeneous on agarose-acrylamide composite gel electrophoresis and analytical ultracentrifugation. Its molecular weight was estimated to be about 870,000 by sedimentation equilibrium studies. After alkylation with iodoacetamide or reduction with 0.6% dithiothreitol at 100 degrees, the complex gave a single band on polyacrylamide gel electrophoresis in sodium dodecyl sulfate in a position corresponding to a molecular weight of 210,000. These results indicate that the complex consists of four subunits of similar size.     

Purification of the restriction endonuclease PalI.

The restriction endonuclease PalI was purified from Providencia alcalifaciens 1650-fold with a yield of 33%. The purified protein moved as a single band upon polyacrylamide gel electrophoresis. When this was carried out in the presence of sodium dodecyl sulfate, a molecular weight of 31,000 was obtained for PalI. Gel filtration through Sephacryl S200 gave molecular weights ranging from 44,000 to 53,000 when 58 to 1870 ng/ml enzyme were used, respectively. Other properties of the enzyme are described.     

Purification and Properties of Arginase from Soybean, Glycine max, Axes

Arginase (EC 3.5.3.1) was purified to homogeneity from cytosol of soybean, Glycine max, axes by chromatographic separations on Sephadex G-200, DEAE-sephacel, hydroxyapatite, and arginine-affinity columns. The molecular weight of the enzyme estimated by pore gradient gel electrophoresis was 240,000, while sodium dodecyl sulfate polyacrylamide gel electrophoresis gave a single band at the molecular weight of 60,000. The optimal pH for activity was 9.5 and the K_m value was 83 millimolar. The enzyme was stimulated by polyamines such as putrescine.     

Purification and characterization of alpha-toxin of Clostridium oedematiens type A

The alpha-toxin of Clostridium oedematiens type A was purified from culture filtrate by two steps of column chromatography and repeated gel filtration. The purified alpha-toxin proved homogeneous in polyacrylamide gel electrophoresis and agar gel double diffusion. The molecular weight of the alpha-toxin was estimated at 280,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and at 260,000 by gel filtration on a Sephadex G-200 column. The isoelectric point determined by isoelectric focusing polyacrylamide gel electrophoresis was 6.1. No dissociation of the purified alpha-toxin into subunits was demonstrated in sodium dodecyl sulfate polyacrylamide gel electrophoresis. The 50% lethal and edematizing doses per mg protein of the purified alpha-toxin were 5.9 X 10(4) and 5.9 X 10(5), respectively. The L +/50 doses per mg protein of the toxin was 4.6 X 10(3). The purified alpha-toxin, when injected intradermally into the rabbit skin, induced increased vascular permeability. The toxin contained little or no hemolytic or lecithinase activity. These results attest that the lethal, edematizing and vascular permeability-enhancing activities elicited by C. oedematiens type A culture reside on the same protein molecule.     

Purification and Partial Characterization of an Antimicrobial Peptide Produced by a Novel <Emphasis Type="Italic">Bacillus</Emphasis> sp. Isolated from the Amazon Basin

An antimicrobial peptide produced by a new Bacillus species isolated from the Amazon Basin was purified and characterized. The antimicrobial peptide was purified by ammonium sulfate precipitation, gel filtration, and ion exchange chromatography, and after the final purification step, one active fraction was obtained, designated BLS P34. Direct activity on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was observed. A single band on SDS-PAGE suggested that the peptide was purified to homogeneity and had a molecular mass of about 5 kDa. The molecular weight (MW) was accurately determined by mass spectroscopy as 1456 Da. The purified BLS P34 remained active over a wide temperature range and was susceptible to all proteases tested.     

A dimer of a single polypeptide chain catalyzes the terminal four reactions of the L-tryptophan pathway in Euglena gracilis.

In Euglena gracilis the terminal four enzyme activities of the tryptophan biosynthetic pathway were found to be associated with a protein with an estimated molecular weight of 325,000 +/- 20,000. The protein was purified approximately 2,000-fold with relatively proportional recoveries of all four enzyme activities. The purified material was homogeneous by the criteria of analytical disc gel electrophoresis and gel isoelectric focusing. Disc gel electrophoresis after denaturation with sodium dodecyl sulfate gave a single protein band with a molecular weight of 155,000 +/- 5,000. Disc gel electrophoresis in 8 M urea also gave rise to a single protein band. We interpret these results as evidence for a single species of subunit. The pathway in Euglena is the only one known to the present in which the terminal enzyme, tryptophan synthase, is not a separate molecular species.     

Immunochemistry of phytochrome

Purified oat and rye phytochrome were examined by analytical gel chromatography, polyacrylamide gel electrophoresis, N-terminal, and amino acid analysis. Purified oat phytochrome had a partition coefficient on Sephadex G-200 (σ₂₀₀) of 0.350 with an estimated molecular weight of 62,000; sodium dodecyl sulfate polyacrylamide electrophoresis gave an equivalent weight estimate. Purified rye phytochrome had a σ₂₀₀ value of 0.085 with an estimated molecular weight of 375,000; sodium dodecyl sulfate electrophoresis gave a weight estimate of 120,000, indicating a multimer structure for the nondenatured protein. Comparative sodium dodecyl sulfate electrophoresis with purified phycocyanin and allophycocyanin gave a molecular weight estimate of 15,000 for allophycocyanin, and two constituent classes of subunits for phycocyanin with molecular weights of 17,000 and 15,000. Amino acid analysis of oat phytochrome confirmed a previous report; amino acid analysis of rye phytochrome differs markedly from a previous report. Oat phytochome has four detectable N-terminal residues (glutamic acid, serine, lysine, and leucine, or isoleucine); rye phytochrome has two detectable groups (aspartic and glutamic acids). Model experiments subjecting purified rye phytochrome to proteinolysis generate a product with the characteristic spectral and weight properties of oat phytochrome, as it has been described in the literature. It is concluded that the structural characteristics of purified rye phytochrome are likely those of the native protein.     

Yeast killer toxin: purification and characterisation of the protein toxin from Saccharomyces cerevisiae.

Killer toxin from killer strains of Saccharomyces cerevisiae was isolated from concentrates of extracellular medium by precipitation in poly(ethylene glycol) and chromatography through glyceryl-controlled-pore glass. The toxin migrated as a single protein band on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. A molecular weight of 11470 was determined for the toxin protein from its electrophoretic mobility and amino acid composition. Gel filtration of the active toxin indicated that the 11,470-Mr monomer was the active unit. Electrophoretic comparison of extracellular concentrates from a killer strain and an isogenic non-killer showed the presence of the toxin protein only in the killer-derived material. The activity of the toxin was most stable between pH 4.2 and 4.6. At 30 degrees C toxin from a superkiller strain was more stable than that from a normal killer.     

Purification of the insulin receptor from human placental membranes

Insulin receptors were purified from human placental microsomal membranes by solubilisation with Triton X-100 followed by Sepharose 6B chromatography, phosphate gradient elution from hydroxyapatite and affinity chromatography on concanavalin A-Sepharose. 2000-fold purification was achieved with 63% overall recovery. The purified receptor gave a single band on 3.75% polyacrylamide (0.1% Triton X-100) gel electrophoresis. On sodium dodecyl sulphate-polyacrylamide gel electrophoresis there was a major band at 75,000 and a minor band at 80,000 daltons. The purified receptor rechromatographed on Sepharose 6B with an apparent molecular weight of 300,000.     

Purification and some properties of alkaline pullulanase from a strain of bacillus no. 202-1, an alkalophilic microorganism.

Pullulanase (pullulan 6-glucanohydrolase EC 3.2.1.41) was purified about 290-fold from the culture fluid of Bacillus No. 202-1 by DEAE-cellulose adsorption, acetone fractionation, (NH4) 2SO4 precipitation and DEAE--cellulose column chromatography followed by Sephadex G-200 molecular sieve chromatography. The enzyme gave a single band of protein by disc polyacrylamide gel electrophoresis. The molecular weight was estimated as 92 000 by sodium dodecyl sulfate gel electrophoresis. The isolectric point was lower than pH 2.5. The optimum pH for enzyme action was about 8.5-9.0. The action of the enzyme on amylopectin and glycogen resulted in increase in the iodine coloration of 85% and 70%, respectively. The enzyme completely hydrolyzed 1,6-alpha-glucosidic linkages in amylopectin, glycogen and pullulan.     

Proteinases of small intestine enterocytes of swine. Purification and properties of aspartyl proteinase similar to cathepsin D

A new aspartic proteinase was isolated from porcine intestine mucosa by affinity chromatography on pepstatin-Sepharose 4B and gel filtration on Sephadex G-100. The enzyme was purified 1600-fold and appeared homogeneous upon polyacrylamide gel electrophoresis. The proteinase has a Mr 60 000 +/- 4000 Da. During sodium dodecyl sulfate polyacrylamide gel electrophoresis the enzyme produced a single protein band (Mr 30 000 +/- 3000 Da). Isoelectric focusing revealed that the enzyme has several multiple forms (pI 6.9, 7.5, 8,0). The enzyme is a glycoprotein containing 5.9% of carbohydrates; the mannose to galactose ratio is 1:3. The amino acid composition of the enzyme was studied. The proteinase splits an oxidized insulin B-chain and synthetic substrates. The pH optimum is 3.2. The enzyme is immunologically identical to porcine spleen cathepsin D.