Production of multiple δ-endotoxins by Bacillus thuringiensis: δ-endotoxins produced by strains of the subspecies galleriae and wuhanensis

1994 ◽  
Vol 40 (12) ◽  
pp. 1026-1034 ◽  
Author(s):  
Galina G. Chestukhina ◽  
Lyubov I. Kostina ◽  
Igor A. Zalunin ◽  
Lyudmila P. Revina ◽  
Alla L. Mikhailova ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Limited Proteolysis ◽  
Protein Composition ◽  
Amino Acid Sequences ◽  
Sequence Determination ◽  
Crystal Proteins ◽  
Sds Page ◽  
Functional Consequences ◽  
Terminal Amino ◽  
Fast Performance Liquid Chromatography

A method was developed to assess the number of δ-endotoxins contained in Bacillus thuringiensis entomocidal crystals. It utilized proteolytic conversion of 130-kDa protoxin into 60- to 65-kDa "true" toxin via limited proteolysis with trypsin and separation of stable N-terminal domains by fast-performance liquid chromatography. Immunodiffusion experiments and N-terminal sequence determination (applied to the major component isolated by SDS-PAGE) completed the analysis of the crystal protein composition. The application of this approach to crystals produced by cells of B. thuringiensis subsp. galleriae and wuhanensis allowed us to identify at least seven and eight different δ-endotoxins, respectively. Among those δ-endotoxins assigned to previously described families, CryIA, CryID, Cry IF, and CryIG were found, as well as crystal proteins, which possess N-terminal amino acid sequences very different from those of all known δ-endotoxins. Possible functional consequences of δ-endotoxin multiplicity are discussed.Key words: Bacillus thuringiensis, δ-endotoxins, multiplicity of crystal proteins, separation of true toxins.

Characterization of Glycoprotein Fraction from Carp Pituitaries Isolated Using Concanavalin A as the Affinity Ligand

1998 ◽  
Vol 63 (3) ◽  
pp. 434-440 ◽  
Author(s):  
Irena Hulová ◽  
Jana Barthová ◽  
Helena Ryšlavá ◽  
Václav Kašička
Keyword(s):  
Concanavalin A ◽  
Reversed Phase ◽  
Amino Acid Sequences ◽  
Gel Chromatography ◽  
Affinity Ligand ◽  
Sds Page ◽  
Terminal Amino ◽  
Α And Β Subunits

Glycoproteins that have affinity to Concanavalin A were isolated from the acetone-dried pituitaries of common carp (Cyprinus carpio L.). Two fractions of glycoproteins were separated using gel chromatography on Superdex 75HR. The fraction with lower molecular weight (30 000) corresponding to the carp gonadotropin cGtH II was composed of two subunits as determined using SDS-PAGE. This protein fraction was further divided into four components using reversed-phase HPLC. Two fractions were pure α and β subunits of cGtH II as follows from immunodetection and from determination of N-terminal amino acid sequences. The other two were a mixture of α and β subunits as was also revealed by N-terminal analysis. Capillary electrophoresis was also used for characterization of isolated glycoproteins.

scholarly journals Autoantibodies against triosephosphate isomerase. A possible clue to pathogenesis of hemolytic anemia in infectious mononucleosis.

1990 ◽  
Vol 171 (2) ◽  
pp. 565-570 ◽  
Author(s):  
K Ritter ◽  
H Brestrich ◽  
B Nellen ◽  
H Kratzin ◽  
H Eiffert ◽  
...  
Keyword(s):  
Infectious Mononucleosis ◽  
Triosephosphate Isomerase ◽  
Clinical Symptoms ◽  
Glycolytic Enzyme ◽  
Amino Acid Sequences ◽  
Cellular Proteins ◽  
51Cr Release ◽  
Terminal Amino Acid ◽  
Sds Page ◽  
Terminal Amino

In sera from patients with acute EBV, infection and the clinical symptoms of infectious mononucleosis antibodies of the Ig class M were found that are directed against two cellular proteins. The molecular mass of these proteins was determined to be 29 (p29) and 26 kD (p26), respectively, in SDS-PAGE. P29 was identified as part of the glycolytic enzyme triosephosphate isomerase (TPI) by comparison of the NH2-terminal amino acid sequences. A purified antibody against TPI induces a 51Cr release from human erythrocytes. Possibly, anti-TPI causes hemolysis, which is an infrequent but serious symptom of infectious mononucleosis.

scholarly journals β-Galactosidase II in Ripening Tomatoes

HortScience ◽  
1995 ◽  
Vol 30 (4) ◽  
pp. 817A-817
Author(s):  
Russell Pressey ◽  
C.M. Sean Carrington
Keyword(s):  
Amino Acid ◽  
Cell Walls ◽  
Amino Acid Sequences ◽  
Cell Wall Polysaccharides ◽  
Terminal Amino Acid ◽  
Molecular Weights ◽  
Sds Page ◽  
Original Procedure ◽  
Terminal Amino ◽  
Terminal Amino Acid Sequence

Tomatoes contain several isozymes of β-galactosidase, but only one, β-galactosidase II, can hydrolyze the β-1,4-galactans in tomato cell walls. β-galactosidase II has now been highly purified by modification of the original procedure. The molecular weight of this isozyme is ≈62 kDa according to gel infiltration, but SDS-PAGE of the purified enzyme separated three components with molecular weights of 29, 42, and 82 kDa. The 82-kDa peptide may be the intact enzyme and the smallest peptides are subunits as proposed for other β-galactosidases. The N-terminal amino acid sequence of β-galactosidase II showed high homology with amino acid sequences reported for other plant β-galactosidases. A new assay for β-galactosidase II in tomato extracts has been developed using FPLC. This isozyme was not detected in mature-green tomatoes but appeared at about the breaker stage and increased during ripening. The increase in b-galactosidase II was accompanied by a decrease in galactose content of cell wall polysaccharides, suggesting that this enzyme may be involved in the loss of galactose during tomato ripening.

scholarly journals Identification of one- and two-chain forms of trypsinogen 1 produced by a human gastric adenocarcinoma cell line

1994 ◽  
Vol 303 (1) ◽  
pp. 187-190 ◽  
Author(s):  
N Koshikawa ◽  
H Yasumitsu ◽  
Y Nagashima ◽  
M Umeda ◽  
K Miyazaki
Keyword(s):  
Gastric Adenocarcinoma ◽  
Amino Acid Sequences ◽  
Molecular Forms ◽  
Adenocarcinoma Cell ◽  
Sds Page ◽  
Human Gastric Adenocarcinoma ◽  
Proteolytic Activities ◽  
Terminal Amino ◽  
The One ◽  
Chain Form

It has previously been reported that two kinds of human gastric adenocarcinoma cell lines (STKM-1 and MKN28) secrete a trypsin-like enzyme. In this study, four molecular forms of the enzyme (26, 25, 24 and 23 kDa on non-reducing SDS/PAGE) were purified from the serum-free conditioned medium of STKM-1 cells. Analysis of N-terminal amino acid sequences showed that the 26 kDa protein was a two-chain form of trypsinogen 1 which had been produced by proteolytic cleavage of the Arg107-Val108 bond of trypsinogen 1, and the 24 kDa protein was the one-chain form of trypsinogen 1. The 25 and 23 kDa proteins were the activated forms of the two-chain and one-chain trypsinogen 1 respectively. Isoelectric focusing gave pI values of 6.3 and 6.6 for the 26 kDa two-chain form and the 24 kDa one-chain form of trypsinogen 1 respectively. Comparison of the proteolytic activities indicated that the one-chain trypsin 1 had amidolytic activity about four times higher than that of the two-chain enzyme.

A Novel Fibrinogen-clotting Enzyme, TL-BJ, from the Venom of the Snake Bothrops jararaca: Purification and Characterization

2000 ◽  
Vol 83 (03) ◽  
pp. 438-444 ◽  
Author(s):  
Claudio Sampaio ◽  
Reinhard Mentele ◽  
Antonio Camargo ◽  
Edwin Fink ◽  
Solange Serrano
Keyword(s):  
Amino Acid Sequences ◽  
Amidolytic Activity ◽  
Purification And Characterization ◽  
Terminal Amino Acid ◽  
Bothrops Jararaca ◽  
Sds Page ◽  
Competitive Inhibitors ◽  
Molecular Masses ◽  
Terminal Amino ◽  
Serine Endopeptidases

SummaryThree chromatographically distinct forms of a novel fibrinogenclotting serine endopeptidase, TL-BJ 1, 2 and 3, were purified from the venom of Bothrops jararaca by a combination of ammonium sulfate precipitation and chromatographic steps. The three forms of TL-BJ have similar amidolytic and plasma coagulating activities. TL-BJ 1, TL-BJ 2 and TL-BJ 3 cause the specific clotting of fibrinogen with release of fibrinopeptide A, the specific activities are 16.8 NIH U/mg (TL-BJ 1), 16.7 NIH U/mg (TL-BJ 2) and 20.8 NIH U/mg (TL-BJ 3). The most sensitive chromogenic substrates for measuring the amidolytic activity of TL-BJ 3 were D-Pro-Phe-Arg-pNA, D-Phe-pipecolyl-ArgpNA and Z-D-Arg-Gly-Arg-pNA. The amidolytic and coagulant activities of TL-BJ were inhibited by phenylmethylsulfonyl fluoride but not by hirudin. Benzamidine derivatives, which are competitive inhibitors of trypsin-like serine endopeptidases, also inhibited the amidolytic activity of TL-BJ. In SDS/PAGE the main bands of TL-BJ 1, 2 and 3 showed molecular masses of 30 kDa, 31 kDa and 32 kDa. Upon incubation with N-glycosidase F only TL-BJ 3 remained unchanged, whereas TL-BJ 1 and TL-BJ 2 showed products with molecular masses around 23 kDa. Thus, TL-BJ 3 does not seem to be N-glycosylated. The N-terminal amino acid sequences of TL-BJ 2 and TL-BJ 3 are identical while TL-BJ 1 has five substitutions.

Characterization ofBacillus thuringiensissubsp.kurstakistrain S93 effective against the fall armywormSpodoptera frugiperda)

1999 ◽  
Vol 45 (6) ◽  
pp. 464-471 ◽  
Author(s):  
Joseilde O Silva-Werneck ◽  
Marlene T De-Souza ◽  
José MC de S. Dias ◽  
Bergmann M Ribeiro
Keyword(s):  
Amino Acid ◽  
Bacillus Thuringiensis ◽  
Spodoptera Frugiperda ◽  
Fall Armyworm ◽  
Amino Acid Sequences ◽  
Dna Library ◽  
Sds Page ◽  
Total Dna ◽  
Type Gene ◽  
High Degree

A Brazilian strain of Bacillus thuringiensis subsp. kurstaki, designated S93, was analyzed regarding its cry gene and protein contents and activity against the fall armyworm (Spodoptera frugiperda, Smith 1797). Bioassays using lyophilized powders of S93 or HD-1 and third instar larvae of S. frugiperda showed a 12.3-fold lower LC50for the S93 strain when compared with the standard HD-1 strain. The spore-crystal mixture, analyzed by SDS-PAGE, showed two major polypeptides of 130 and 65 kDa, corresponding to Cry1 and Cry2 toxins, respectively. Western blot analysis showed that these proteins were immunologically related to the Cry1A protein from B. thuringiensis subsp. kurstaki HD-73. The polymerase chain reaction technique (PCR) using total DNA from the S93 strain and specific primers showed the presence of cry1Aa, cry1Ab, and cry1Ac genes, and a cry1A-type gene was localized in a plasmid of about 44 MDa. A cry1Ab gene was isolated from a S93 plasmid DNA library and completely sequenced. Computer analysis showed that the gene sequence (GenBank acession number AF059670) is identical to cry1Ab1 and has 91.6 and 85.9% identity with cry1Aa1 and cry1Ac1 genes, respectively. The deduced amino-acid sequence showed a high degree of similarity with the amino-acid sequences of the Cry1Ab1 (100%), Cry1Aa1 (93.8%), and Cry1Ac1 (90.6%) proteins.Key words: Bacillus thuringiensis, Spodoptera frugiperda, biological control, crystal protein, cry genes.

Isolation, purification and characterization of chymosin from riverine buffalo (Bubalos bubalis)

2003 ◽  
Vol 70 (1) ◽  
pp. 37-43 ◽  
Author(s):  
Ashok K Mohanty ◽  
Utpal K Mukhopadhyay ◽  
Jai K Kaushik ◽  
Sunita Grover ◽  
Virender K Batish
Keyword(s):  
Amino Acid ◽  
Proteolytic Activity ◽  
Gel Filtration ◽  
Maximum Activity ◽  
Amino Acid Sequences ◽  
Milk Clotting ◽  
Sds Page ◽  
Terminal Amino ◽  
The Stability

Chymosin, an aspartyl proteinase, is used for curdling of milk and manufacture of cheese. We report the purification and the physicochemical properties of chymosin isolated from the abomasal tissue of buffalo calves. The enzyme preparation extracted from buffalo abomasal tissues could be purified 29–fold using anion exchange and gel filtration chromatography. The molecular weight of the purified enzyme was 35·6 kDa on SDS-PAGE. Partial N-terminal amino acid sequence of the first eight amino acid sequences of buffalo chymosin was identical to the first eight amino acid sequences of cattle chymosin. Buffalo chymosin exhibited a skewed bell-shaped stability profile as a function of temperature with maximum activity near 55 °C. Milk clotting activity decreased gradually as pH increased. The enzyme became completely inactive, however, above pH 7·0. The ratio of milk clotting to proteolytic activity was 3·03. When compared with cattle chymosin, there were subtle differences in the stability and relative proteolytic activity of buffalo chymosin.

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