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1.
Alkaline Serine Protease Is an Exotoxin of Vibrio alginolyticus in Kuruma Prawn,Penaeus japonicus 总被引:1,自引:0,他引:1
An extracellular lethal toxin produced by Vibrio
alginolyticus strain Swy originally isolated from diseased kuruma prawn
(Penaeus japonicus) was partially purified by Fast Protein Liquid
Chromatography with hydrophobic interaction (Phenyl Sepharose High
Performance) chromatography and gel filtration columns. The toxin is an
alkaline serine protease, inhibited by phenyl-methylsulfonyl fluoride (PMSF),
and showed maximal activity at pH 10, having a molecular weight of about 33
kDa estimated by SDS-PAGE and gel filtration chromatography. In addition, the
toxin was also completely inhibited by FeCl2 but partially
inhibited by CaCl2, CuCl2, CoCl2,
MnCl2, and ZnCl2, and not inhibited by ethylenediamine
tetraacetic acid (EDTA), ethylene glycol-bis(β-amino-ethyl ether)
N,N,N′,N′-tetraacetic acid (EGTA), iodoacetamide, pepstatin A, sodium dodecyl
sulfate (SDS), and N-tosyl-l-phenyl-alanine chloromethyl ketone (TPCK). Both
the crude extracellular products (ECP) and the partially purified toxin are
lethal for kuruma prawn at LD50 values of 0.30 and 0.27 μg
protein/g body weight, respectively. The addition of PMSF completely
inhibited the lethal toxicity of both the ECP and the partially purified
toxin, indicating that this serine protease is a lethal factor produced by
the bacterium. The 33-kDa protease is, therefore, suggested to be a new toxic
protease produced by V. alginolyticus strain Swy.
Received: 12 April 1996 / Accepted: 31 July 1996 相似文献
2.
SUMMARY.
- 1 A soluble cell fraction lethal to Daphnia magna was isolated by the disruption of net phytoplankton from eutrophic Aculeo Lake. The phytoplankton of this lake is dominated by Microcystis sp.
- 2 The soluble fraction was also lethal to common native zooplankton, including the rotifers Keratella sp., Trichocerca similis, the copepod Boeckella sp., and the cladoceran Chydorus sphaericus. In addition, a partially purified soluble component of the lethal fraction caused the death of the fish Gambusia affinis.
- 3 The specific toxicity of the soluble fraction from phytoptankton varied four- to six-fold seasonally with peaks during periods of warm temperature.
- 4 Our results suggest that releases of a toxic peptide from Microcystis sp. may be the cause of massive fish kills at the study site, Aculeo Lake.
3.
The sea anemone Actineria villosa expresses a lethal protein toxin. We isolated a novel 120-kDa protein, Avt120, from partially purified toxin and found it
to possess extremely strong lethal activity. The 3,453-bp Avt120 gene translates to a 995-amino acid protein. The 50% lethal
dose (LD50) of purified Avt120 in mice was 85.17 ng. Among several tested cell lines, Colo205 cells were most sensitive to Avt120: 50%
of them were damaged by 38.4 ng/mL Avt120. Avt120 exerted ATP degradation activity (10 μmol ATP h−1 mg−1), which was strongly inhibited by ganglioside GM1 to decrease the cytotoxicity of Avt120. 相似文献
4.
Takayasu Tsuchida Kazuo Nakamuka Yoshitsugu Fujii Hajime Takahashi 《Bioscience, biotechnology, and biochemistry》2013,77(11):1355-1361
Formation of pectolytic enzyme system in Erwinia aroideae was stimulated to a remarkable extent when the cells were incubated in a pectin medium containing carrot extracts. The active principle in the carrot extract preparation was resistant to acid hydrolysis, digestion by Pronase, RNase, DNase or α-amylase, and to ninhydrin and charcoal treatments. The factor lost, however, its stimulating activity upon alkaline hydrolysis or periodate oxidation. The factor was partially purified by the combination of gel filtration with Sephadex G-10 and ninhydrin and charcoal treatments. The molecular weight of the partially purified factor was presumed to be around 400 by the gel filtration. 相似文献
5.
The growth factor for malo-lactic fermentation bacteria was partially purified from tomato juice, and some properties of the factor were examined. The factor was comparatively a low molecule, thermo-stable and insoluble in nonpolar organic solvents. The purified factor fraction showed positive reaction with Molisch-, tetrazolium- and Nessler-reagents, suggesting the existence of sugar and nitrogen as the chemical components. The treatment with cellobiase or cellulase-II resulted in the inactivation of the factor and simultaneously released the sugar constituent composed of glucose from the factor. These results indicated that the glucose residue might exist as the β-1,4-glucosidic linkage in the growth factor. 相似文献
6.
《Bioscience, biotechnology, and biochemistry》2013,77(6):1142-1143
The transglycosylation reaction of partially purified β-glucosidase from sesame seeds with cellobiose is described. Sesame β –glucosidase was partially purified by ammonium sulfate fractionation and gel filtration. The molecular weight of the enzyme was 200,000 by gel filtration. Sesame β-glucosidase showed strong transfer activity to synthesize the trisaccharide from cellobiose. The optimum pH and temperature of the transglycosylation reaction were pH 4.0 and 60°C. 相似文献
7.
One or more proteinaceous factors with insecticidal activities in the locust pathogen Serratia marcescens HR-3 culture filtrates were found to cause the death of grassland locusts. A novel insecticidal protein was purified to homogeneity.
It was a monomer of 61 kDa. The purified protein showed a strong insecticidal effect with a median lethal dosage of 12.1 μg
locust−1 and contained a high level of protease activity (101 U ml−1). Insecticidal activity was significantly decreased when the protein was pretreated with ethylene diamine tetraacetic acid
and 1-10-phenanthroline, and it was restored when the treated protein was incubated with Zn2+. The N-terminal amino acid sequence of insecticidal protein showed sequence similarity with metalloprotease from S. marcescens SM6 and Serratia spp. E15. Our results suggested that the factor primarily responsible for insecticidal activity toward locusts was a zinc-dependent
61-kDa metalloprotease. 相似文献
8.
The lethal factor of Bacillus anthracis is a major factor for lethality of anthrax infection by this bacterium. With the aid of the protective antigen, lethal factor gains excess to the cell cytosol where it manifests toxicity as a metalloprotease. For better understanding of its specificity, we have determined its residue preferences of 19 amino acids in six subsites (from P3 to P3′) as relative kcat/Km values (specificity constants). These results showed that lethal factor has a broad specificity with preference toward hydrophobic residues, but not charged or branched residues. The most preferred residues in these six subsites are, from P1 to P3′, Trp, Leu, Met, Tyr, Pro, and Leu. The result of residue preference was used to design new substrates with superior hydrolytic characteristics and inhibitors with high potency. For better use of the new findings for inhibitor design, we have modeled the most preferred residues in the active site of lethal factor. The observed interactions provide new insights to future inhibitor designs. 相似文献
9.
Vitamin B12-dependent methionine synthetase (N5-methyItetrahydrofolate-homocysteine Bi2-methyltransferase; EC 2.1.1.13) was partially purified from two different types of photo-synthetic bacteria, Chromatium D and Rhodospirillum rubrum.Chromatium D, which does not produce vitamin B12, possessed apomethionine synthetase when grown in the absence of the vitamin. Partially purified apoenzyme was converted to holoenzyme efficiently with CH3B12 or OHB12. Holo-methionine synthetase was purified 244 fold with 56.4 % recovery from Chromatium D cells grown with vitamin B12 added. The partially purified enzyme required reductants but was only partially dependent on S-adenosylmethionine.On the other hand, Rsp. rubrum methionine synthetase which was always present as holoenzyme, in contrast with that of Chromatium D, was purified 40 fold with 2.8% recovery. The obtained preparation required S-adenosylmethionine and reductants for the enzyme activity. The optimal pH of Chromatium D enzyme and of Rsp. rubrum enzyme was in the range of 7.5~7.8 and 6.5~6.75, respectively. 相似文献
10.
Kazutsugu Matsumoto Hidehiko Kitajima Tadashi Nakata 《Journal of Molecular Catalysis .B, Enzymatic》1995,1(1)
Enantioselectivity-promoting factor enhances enantioselectivity of protonation in lipase AP-catalyzed asymmetric hydrolysis of enol esters. The factor was partially purified by chromatography using Phenyl-TOYOPEARL 650M and Sephacryl S-200HR. The hydrolysis of 2-benzyl-l-cyclohexenyl acetate by PLE in the presence of the purified factor produced (R)-2-benzylcyclo-hexanone in 92% ee, while the reaction without the factor gave the racemate. 相似文献
11.
Estimation of the position and effect of a lethal factor locus on a molecular marker linkage map 总被引:3,自引:0,他引:3
R. Cheng A. Saito Y. Takano Y. Ukai 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1996,93(4):494-502
In the mapping of DNA markers the distortion of segregation of marker genotypes is often observed, which may be caused by a lethal factor acting in filial generations derived from distant crosses. A method is presented for estimating the recombination values between a lethal factor locus and neighboring molecular markers, and the relative viability or fertilization ability of gametes or zygotes affected by the lethal factor in an F2 population using the maximum likelihood method and the expectation conditional maximization (ECM) algorithm. Three selection models of gamete or zygote were considered, and the most likely one was determined by goodness of fit of the observed frequency of the phenotypes to the expected ones under the models. The method was applied to segregation data of molecular markers of an F2 population consisting of 144 individuals derived from a cross between an Indica and a Japonica rice variety. The presence of a lethal factor locus (L) located on chromosome III that caused partial gametic selection in both the male and female sides was suggested. The locus L was tightly linked to RFLP marker number 23 of the RFLP linkage map of Saito et al. (1991a), and the fertilization chance of a male or female gamete possessing the lethal factor was, on average, 41.5% of that of the normal gamete. 相似文献
12.
Jordan Konisky Billie S. Cowell Mary J. Gilchrist 《Journal of cellular biochemistry》1973,1(3):208-219
The specific binding of 125 Iodine labelled colicin Ia and Ib to Escherichia coli cell envelopes and partially purified cell walls is demonstrated. Neither partially purified cytoplasmic membranes isolated from a wild type sensitive strain nor envelopes or cell walls prepared from an E. coli mutant known to be defective in the colicin I receptor could bind the colicins. Competition studies suggest that colicins Ia and Ib have a common bacterial receptor which resides in the bacterial cell wall. 相似文献
13.
Bacillus anthracis lethal factor (LF) is a 90-kDa zinc metalloprotease that plays an important role in the virulence of the organism. LF has previously been purified from Escherichia coli and Bacillus anthracis. The yields and purities of these preparations were inadequate for crystal structure determination. In this study, the genes encoding wild-type LF and a mutated, inactive LF (LF-E687C) were placed in an E. coli–Bacillus shuttle vector so that LF was produced with the protective antigen (PA) signal peptide at its N-terminus. The resulting vectors, pSJ115 and pSJ121, express wild-type and mutated LF fusion proteins, respectively. Expression of the LF genes is under the control of the PA promoter and, during secretion, the PA signal peptide is cleaved to release the 90-kDa LF proteins. The wild-type and mutated LF proteins were purified from the culture medium using three chromatographic steps (Phenyl–Sepharose, Q–Sepharose, and hydroxyapatite). The purified proteins were greater than 95% pure and yields (20–30 mg/L) were higher than those obtained in other expression systems (1–5 mg/L). These proteins have been crystallized and are being used to solve the crystal structure of LF. Their potential use in anthrax vaccines is also discussed. 相似文献
14.
The ability of partially purified phospholipase C preparations (Bacillus cereus) to disaggregate elongation factor 1 from rabbit reticulocytes is due to the presence of carboxypeptidase A in the preparations. A similar factor has been purified from the growth medium of B. cereus. In contrast, the disaggregation of elongation factor 1 observed with extracts of Artemia salina nauplii appears to be due to a protease. These results show that different types of proteolytic modification of elongation factor 1 can result in disaggregation of the factor. 相似文献
15.
Endogenous extractable factors associated with auxin action in plant tissues were investigated, especially their effects
on elongation of 1-mm coleoptile segments of maize (Zea mays L.), in the presence of saturating 10 μM indole-3-acetic acid (IAA). The relative growth response, to auxin alone, was much
smaller in segments shorter than 2–3 mm compared to 10-mm segments. Fusicoccin-induced elongation, however, was less affected
by shortening the segments. A reduced auxin response may result from the depletion through cut surfaces of a substance required
for IAA-mediated growth. Sucrose, phenolics like flavonoids, and vitamins were ruled out as the causal factors. A partially
purified methanol extract of maize coleoptiles supported long-term, auxin-controlled elongation. The active material was also
found among substances bleeding from scrubbed maize coleoptiles. The active factor from maize was further purified by HPLC
and characterised by the UV spectrum and its pH shift. This factor was identified as 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one
(DIMBOA) by mass spectroscopy. Activity tests confirmed that pure DIMBOA from other sources sustained auxin-induced elongation
of short maize coleoptile segments. However, DIMBOA only partially restored the activity lost from short segments. This indicates
that an additional factor, other than DIMBOA, is required. Extracts from Avena or Cucurbita did not contain the factor DIMBOA; it was active on maize elongation, but not on Avena coleoptiles or Cucurbita hypocotyls. This narrow specificity and the lack of DIMBOA action in short-term tests with maize indicate that DIMBOA is
not the general auxin cofactor but may specifically “spare” the co-auxin in maize.
Received: 27 June 2000 / Accepted: 16 October 2000 相似文献
16.
Leonard Machlis 《American journal of botany》1962,49(2):171-177
Machlis , Leonard . (U. California, Berkeley.) The nutrition of certain species of the green alga Oedogonium. Amer. Jour. Bot. 49 (2) : 171–177. Illus. 1962.—Evidence is presented showing that O. cardiacum and O. geniculatum require vitamin B12 and an unknown growth factor released from soil into water during autoclaving. The unknown growth factor is shown not to be a number of things, to be retained on anion exchange columns, and to be partially purified by fractionation on the polydextran, Sephadex 50. It appears to be an organic substance with acidic properties. Isolates of Oedogonium presently in unialgal and axenic culture are listed and the methods used to obtain the axenic cultures and to grow them for nutritional studies are described. 相似文献
17.
J.F. Cloix M. Bachelet A. Ulmann J.L. Funck-Brentano 《Biochemical and biophysical research communications》1978,83(4):1456-1463
The 25-hydroxycholecalciferol-binding protein has been partially purified (purification factor: 37) from rat duodenal cytosol, using chromatographic procedures on gel and ionic exchange resin. This partially purified protein bound 25-hydroxycholecalciferol with high affinity (KD = 5.7 × 10?9M) and low binding capacity (23 × 10?12 mole/mg of protein. Using a rabbit antiserum obtained against such partially purified protein, we demonstrated that 25-hydroxycholecalciferol cytosolic binder and 25-hydroxycholecalciferol plasmatic binding share common antigenic sites. 相似文献
18.
Entomopathogenic nematodes are used for insect control. Herein, an extracellular protease was partially purified from a culture
supernatant ofXenorhabdus nematophilus, a symbiotic bacterium of an entomopathogenic nematode,Steinernema glaseri, using precipitation with 80% v/v isopropyl alcohol followed by gel permeation chromatography with a packed Sephacryl S-300
HR media. The partially purified protease exhibited maximal activity at pH 7 in the presence of 1 mM CaCl2. The protease was identified as a metallo-protease based on the inhibition of its activity by the metal chelating agent,
EDTA. 相似文献
19.
V. Cabiaux A. Phalipon R. Wattiez P. Falmagne J. M. Ruysschaert M. Kaczorek 《Molecular microbiology》1988,2(3):339-346
The toxB gene of Corynebacterium diphtheriae bacteriophage β encoding the B fragment of diphtheria toxin was cloned into an inducible expression vector. When expressed In Escherichia coli, fragment B was not proteolysed and was indistinguishable, by immunological criteria, from wild-type C. diphthsriae derived fragment B. Soluble fragment B was partially purified from the cytoplasm by saline precipitation steps and was shown to compete with the wild-type diphtheria toxin for binding to receptors of sensitive eukaryotic cells. A complete diphtheria toxin was reconstituted by formation of the disulphide bridge between purified fragment A and recombinant fragment B, which migrates at the expected Mr on Western blots and which was able to block protein synthesis by ADP-ribosylation of elongation factor–2, thereby indicating that the recombinant fragment B had retained its biological activity. 相似文献
20.
Endogenous gibberellins from floating plants and in vitro induced turions of Wolffiella floridana (Lemnaceae) were extracted and partially purified. Gibberellin-like activity was detected in two zones of the chromatogram corresponding to Rf 0-0.1 and Rf 0.4-0.5 by dwarf pea bioassay. The active compounds in the two zones have been referred to in this paper as factor I and factor II respectively. There were quantitative differences in the gibberellin-like substances of floating plants and turions. The floating plants contained more of factor I but less of factor II as compared to the turions. The chromatographic behavior of factor I was similar to either gibberellin A1 and A3 while the identity of factor II was uncertain. 相似文献