Purification and characterization of a hemolysin of Vibrio mimicus that relates to the thermostable direct hemolysin of Vibrio parahaemolyticus

A hemolysin (designated Vm-rTDH) from Vibrio mimicus (AQ0915-E13) was purified by ammonium sulfate fractionation and successive column chromatography with DEAE-Sephadex A-25, hydroxyapatite, Mono Q, Superose 12 and Phenyl-Superose. The Mr of the subunit was estimated to be about 22,000 by sodium dodecyl sulfate-slab gel electrophoresis. The isoelectric point of Vm-rTDH was approximately pH 4.9. The hemolytic activity of Vm-rTDH was stable upon heating at 100 degrees C for 10 min, similar to that of the thermostable direct hemolysin (Vp-TDH) of V. parahaemolyticus. Vm-rTDH also showed lytic activities similar to those of Vp-TDH. Immunological cross-reactivity between Vp-TDH and Vm-rTDH was demonstrated by the Ouchterlony double-diffusion test. Thus we conclude that V. mimicus produces a newly discovered type of hemolysin (Vm-rTDH) which is similar to Vp-TDH.     

Homogeneity of a hemolysin(Vh-rTDH) produced by clinical and environmental isolates of Vibrio hollisae

The characteristics of Vh-rTDH, a hemolysin similar to Vp-TDH of Vibrio parahaemolyticus produced by clinical and environmental isolates of Vibrio hollisae, were comparatively studied. All 7 strains of V. hollisae tested were found to produce indistinguishable Vh-rTDH when they were examined by heat-stability test, Western blotting analysis and conventional polyacrylamide slab gel electrophoresis.     

Comparison of hemolysins of Vibrio cholerae non-O1 and Vibrio hollisae with thermostable direct hemolysin of Vibrio parahaemolyticus

Hemolysin (Vh-rTDH) produced by Vibrio hollisae and hemolysin (NAG-rTDH) produced by Vibrio cholerae non-O1 were characterized and compared with hemolysin (Vp-TDH) produced by Vibrio parahaemolyticus. These three hemolysins are each composed of two subunits and have similar, but not identical, molecular weights. The amino acid compositions of Vp-TDH and NAG-rTDH are similar, but are different from that of Vh-rTDH. The three hemolysins showed similar lethal toxicities to mice. The effects of temperature on hemolysis and the time dependencies of hemolysis by the three hemolysins were similar. The three were concluded to be immunologically related, but not identical, and to have common and also unique antigenic determinants.     

Comparative amino acid sequence analysis of hemolysins produced by Vibrio hollisae and Vibrio parahaemolyticus.

Vibrio hollisae produces a hemolysin (Vh-rTDH) that is related to the thermostable direct hemolysin of Vibrio parahaemolyticus (Vp-TDH). Although both hemolysins are essentially similar biologically and immunologically, they differ markedly in heat stability; Vp-TDH is heat stable, whereas Vh-rTDH is heat labile. To elucidate the relationships between their characteristics and molecular structures, we analyzed the amino acid sequence of Vh-rTDH and compared it with that of Vp-TDH. Vh-rTDH consisted of 165 residues, of which 23 residues, spread over the peptide chain, differed from those of Vp-TDH.     

Properties of a hemolysin related to the thermostable direct hemolysin produced by a Kanagawa phenomenon negative, clinical isolate of Vibrio parahaemolyticus

A hemolytic toxin (Vp-TRH) produced by a Kanagawa phenomenon negative, clinical isolate of Vibrio parahaemolyticus was further characterized. The purified Vp-TRH showed various biological activities, such as fluid accumulation in rabbit ileal loops, increase of rabbit skin vascular permeability, and cardiotoxicity on cultured myocardial cells, all of which are essentially similar to the activities found with thermostable direct hemolysin (Vp-TDH), a pathogenic toxin produced by Kanagawa phenomenon positive V. parahaemolyticus. Immunological similarities of Vp-TRH not only to Vp-TDH but also to hemolytic toxins produced by Vibrio hollisae and Vibrio cholerae non-O1, both of which are also enteropathogens closely related to V. parahaemolyticus, were demonstrated. The amino acid composition and sequence of N-terminal amino acids of Vp-TRH were determined. These results suggest that Vp-TRH has biological and immunological characters similar to Vp-TDH, although they are distinct molecules.     

Purification and characterization of an extracellular beta-1,4-mannanase from a marine bacterium, Vibrio sp. strain MA-138.

A beta-mannanase (EC 3.2.1.78) from Vibrio sp. strain MA-138 was purified by ammonium sulfate precipitation and several chromatographic procedures including gel filtration, adsorption, and ion-exchange chromatographies. The final ion-exchange chromatography Mono Q yielded one major active fraction and three minor active fractions. The major active fraction was purified to homogeneity on the basis of native polyacrylamide gel electrophoresis (PAGE). This purified enzyme was identified as a glycoprotein by periodic acid-Schiff staining and a monomeric protein with a molecular mass of 49 kDa by sodium dodecyl sulfate-PAGE. The pI of the enzyme was 3.8. The purified enzyme exhibited maximal activity at pH 6.5 and 40 degrees C and hydrolyzed at random the internal beta-1,4-mannosidic linkages in beta-mannan to give various sizes of oligosaccharides. The first 20 N-terminal amino acid sequence of the purified enzyme showed high homology with the N-terminal region of beta-mannanase from Streptomyces lividans 66.     

Characterization of a new thermostable direct haemolysin produced by a Kanagawa-phenomenon-negative clinical isolate of Vibrio parahaemolyticus

The production of two haemolysins, thermostable direct haemolysin (Vp-TDH) and a Vp-TDH-related haemolysin (Vp-TRH), by clinical isolates of Vibrio parahaemolyticus has previously been reported. Here we describe a third type of haemolysin (named Vp-TDH/I), which is produced by a clinical isolate (strain TH012) that is Kanagawa phenomenon negative. Vp-TDH/I was purified by a series of column chromatographies on DEAE-Sephadex A25, hydroxyapatite, Sepharose 4B and Mono Q. By physicochemical, biological and immunological analyses, Vp-TDH/I was demonstrated to be similar, but not identical, to Vp-TDH and Vp-TRH. The gene encoding Vp-TDH/I was cloned and the deduced amino acid sequence of Vp-TDH/I confirmed that Vp-TDH/I has a sequence different from those of previously known Vp-TDH and Vp-TRH. Not only purified Vp-TDH/I but also live cells of the Vp-TDH/I-producing strain induced fluid accumulation in ligated rabbit intestine. We conclude that this clinical isolate produces a new type of Vp-TDH-related haemolysin, which may be involved in the pathogenesis of this organism.     

Production by non-O1 Vibrio cholerae of hemolysin related to thermostable direct hemolysin of Vibrio parahaemolyticus

Abstract The production by non-O1 Vibrio cholerae of a hemolysin immunologically related to the thermostable direct hemolysin (TDH) of Vibrio parahaemolyticus was demonstrated by enzyme-linked immunosorbent assay and a double gel diffusion test. Although results by the double gel diffusion test suggested the immunological identities of TDH and the TDH-related hemolysin of non-O1 V. cholerae , conventional polyacrylamide gel disc electrophoresis and immunoelectrophoresis suggested some differences between the two, at least with respect to charge. The TDH-related hemolysin of non-O1 V. cholerae was also shown to differ from the hemolysin of non-O1 V. cholerae reported previously.     

Purification and Characterization of Vibrio vulnificus Protease

A protease was purified from a strain of Vibrio vulnificus isolated from the blood of a septicemic human. The vibrio was cultured in bacto peptone-yeast extract medium, and the protease was purified by a purification procedure including ultrafiltration of the culture supernatant with an Amicon YM 5 membrane, diethylaminoethyl-Sephacel column chromatography, Sephacryl S-200 column chromatography and fast protein liquid chromatography on Mono Q column. The protease preparation revealed homogeneity on polyacrylamide gel electrophoresis and about 30,000-fold purification was achieved, with a yield of about 30%. The isoelectric point of the purified V. vulnificus protease was about 5.80 and its molecular weight was ca. 45,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The optimum pH of the protease activity was 8.0. The V. vulnificus protease was inhibited by a metalloprotease inhibitor and zinc ion and/or ferrous ion were essential for its enzyme activity. No cysteine residue was detected in the V. vulnificus protease. The protease had caseinolytic, elastolytic and collagenolytic activities.     

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.     

Phosphodiesterase-phosphomonoesterases from Fusarium moniliforme. VI. A modified method of purification and identification of isozymes.

Fusarium phosphodiesterase-phosphomonesterase was purified 1,630-fold with 19% yield from dried powder of the culture medium by a modified method consisting of seven steps. The purified preparation was shown to be devoid of inactive protein by disc electrophoresis. The preparation was homogeneous with respect to size as demonstrated by gel filtration, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and ultracentrifugation. The molecular weights determined by gel filtration on Sephadex G-200 and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 106,000 and 100,000, respectively. The sedimentation coefficient at infinite dilution was 5.71 S. Isoelectric focusing of the purified preparation showed the presence of at least four isozymes with isoelectric points of 6.6, 6.3, 6.2, and 5.9.     

Purification of undegraded ceruloplasmin from outdated human plasma

A method for the rapid isolation of homogeneous undegraded ceruloplasmin from outdated human plasma is reported. The procedure consists of a precipitation step with polyethylene glycol 4000, batchwise adsorption and elution from QAE-Sephadex, and gradient elution from DEAE-Sepharose CL-6B. Ceruloplasmin was purified 1740-fold and the yield from outdated plasma was 67%. The purified ceruloplasmin was found to be homogeneous on anionic polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulfate-PAGE, isoelectric focusing, and low-speed equilibrium centrifugation. The isoelectric point as determined by isoelectric focusing was 4.4. The purified enzyme was sensitive to storage; when a sample was resubmitted to PAGE after 4 months of storage at 4 degrees C, two bands were obtained and the fast-moving band showed no oxidase activity. The molecular weight estimated by gel electrophoresis and sedimentation equilibrium centrifugation was 130,000.     

Purification and characterization of nitrate reductase from nodule cytosol of soybean plants

Nodulated soybean ( Glycine max [L.] Merr.) plants were grown in a nitrogen-free liquid culture medium prepared with distilled water. The cytosol fraction from root nodules showed a significant level of NADH-dependent nitrate reductase activity, even when the root did not show activity. This nitrate reductase was purified by column chromatography and native polyacrylamide gel electrophoresis (PAGE). The purified protein showed a main band at 100 kDa on sodium dodecyl sulfate (SDS)-PAGE. The K _m value for nitrate was 0.16 m M , and the highest activity was obtained at around pH 7.5. These characteristics are very similar to the inducible type of nitrate reductase, previously purified from soybean leaves. The developmental change in activity of this enzyme corresponded to that in nitrogenase activity.     

Production of monoclonal antibody against a hemolysin (Vp-TRH) produced by Vibrio parahaemolyticus

The antigenicity of a hemolysin (Vp-TRH: Vp-TDH related hemolysin) produced by Kanagawa phenomenon-negative clinical isolates of Vibrio parahaemolyticus was studied using monoclonal antibodies (MAbs). A total of 12 hybridoma clones which produced MAbs against Vp-TRH were established. All MAbs contained the Kappa light chain and were IgG type. These MAbs were divided into a minimum of 5 different specificity groups, including antibodies specific to Vp-TRH and common to both Vp-TRH and Vp-TDH, a possible pathogenic toxin of Kanagawa phenomenon-positive V. parahaemolyticus. These results clearly show the immunological similarity and dissimilarity (specificity) of Vp-TRH and Vp-TDH.     

A mutant hemolysin with lower biological activity produced by a mutant Vibrio parahaemolyticus

Abstract A mutant toxin (m-TDH) of thermostable direct hemolysin (Vp-TDH) of Vibrio parahaemolyticus w was isolated from the culture of a strain of this organism mutagenized with N -methyl- N '-nitro- N -nitrosoguanidine. Although the m-TDH had a molecular structure similar to the native Vp-TDH, the m-TDH retained only about 7% residual hemolytic activity of the native toxin. Furthermore, other biological activities of m-TDH, such as lethality in mice and enterotoxicity in rabbit ileal loops, were also weakened. The m-TDH was immunologically indistinguishable from the native Vp-TDH. These results suggest that the m-TDH is only slightly different in structure from the native Vp-TDH. Also, the mutagenized site in m-TDH, which is not immunogenic, seems to be involved in expressing not only hemolytic activity but also lethal and enterotoxic activity.     

Immunochemical demonstration of the iron-sulfur protein in Complex III of mitochondrial electron-transfer chain

An iron-sulfur protein of Complex III was purified by phenyl-Sepharose column chromatography and DEAE-Sepharose column chromatography. The purified preparation was homogeneous as demonstrated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, and a specific antibody directed against this protein was raised in a rabbit. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by electrophoretic blotting and immunoperoxidase reaction indicated that Complex III possesses a single polypeptide which reacts with the antibody. It was also found that the iron-sulfur center-containing subunits identified so far in Complex I did not cross-react with the antibody, indicating that they are antigenically unrelated to the iron-sulfur protein of Complex III.     

20 beta-hydroxysteroid dehydrogenase of neonatal pig testis: purification and some properties

20 beta-Hydroxysteroid dehydrogenase was purified from a cytosol fraction of neonatal pig testes to homogeneity as demonstrated by polyacrylamide gel electrophoresis (PAGE) and by isoelectric focusing. The molecular weight was estimated to be 30,500 using PAGE with sodium dodecyl sulfate and the gel filtration method. Molecular estimations showed that the purified enzyme consisted of a single polypeptide chain. It catalyzed the reduction of 17 alpha-hydroxyprogesterone to 17 alpha,20 beta-dihydroxypregn-4-en-3-one with NADPH. Furthermore, the C21-steroids, such as progesterone, pregnenolone, 17 alpha-hydroxypregnenolone, deoxycorticosterone, and deoxycortisol were also reduced by the purified enzyme. Apparent Km values for 17 alpha-hydroxyprogesterone, progesterone, pregnenolone, and deoxycorticosterone were 9.4, 1.5, 4.0, and 8.6 microM, respectively. The enzyme did not show 20 alpha-hydroxysteroid dehydrogenase activity. The maximum rate of enzyme activity was observed at 45 degrees C and optimum pH was at pH 5.5. The enzyme activity was strongly inhibited by heavy metal ions such as Hg2+ and Cu2+.     

Purification and characterization of an acidic amino acid specific endopeptidase of Streptomyces griseus obtained from a commercial preparation (Pronase)

A protease was purified 163-fold from Pronase, a commercial product from culture filtrate of Streptomyces griseus, by a series of column chromatographies on CM-Toyopearl (Fractogel), Sephadex G-50, hydroxyapatite, and Z-Gly-D-Phe-AH-Sepharose 4B using Boc-Ala-Ala-Pro-Glu-pNA as a substrate. The final preparation was homogeneous by polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and gel isoelectric focusing. Studies on the substrate specificity with peptide p-nitroanilides revealed that this protease preferentially hydrolyzed peptide bonds on the carbonyl-terminal side of either glutamic acid or aspartic acid. It was most active at pH 8.8 for the hydrolysis of Boc-Ala-Ala-Pro-Glu-pNA. The molecular weight of the protease was estimated to be 20,000 by gel filtration on Sepharose 6B using 6 M guanidine hydrochloride as an eluent, and 22,000 by SDS-PAGE in the presence of 2-mercaptoethanol. The isoelectric point of the enzyme was 8.4. The enzyme was inactivated by diisopropyl phosphofluoridate (DFP) but not by p-chloromercuribenzoate (PCMB) or EDTA.     

Two-step purification and partial characterization of a variant of the Vibrio cholerae non-O1 hemolysin

A two-step purification method using ammonium sulfate precipitation and gel filtration was developed for the purification of a variant of the El Tor hemolysin/cytolysin from supernatant fluids of a Vibrio cholerae non-O1 human isolate (strain 2194c). The toxin displayed delayed elution from a Sephacryl gel filtration column, eluting at between two and three column volumes. The molecular mass and isoelectric point of the purified 2194c toxin were 60 kDa and 5. 3, respectively. The N-terminal amino acid sequence was ASPAPANSETNTLPHVAFYI. Purified toxin was cytolytic for Chinese hamster ovary cells and erythrocytes from several animal species.     

Identification and preliminary characterization of Vibrio cholerae outer membrane proteins.

Outer membrane proteins of Vibrio cholerae were purified by sucrose density centrifugation and Triton X-100 extraction at 10 mM Mg2+. The proteins were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. V. cholerae outer membrane proteins presented a unique pattern when compared with the patterns of other gram-negative rods. There were 8 to 10 major bands (Mr 94,000 to 27,000), with most of the protein located in band 5 (Mr approximately 45,000), which thus appears to be the major structural protein of the outer membrane. Lipid and carbohydrate were associated with band 6.