Toxin-binding proteins from midgut epithelium membranes of Anopheles stephensi larvae.

Proteins of 65 and 57 kD were isolated from the apical membranes of midgut epithelium of Anopheles stephensi larvae by affinity chromatography. These proteins can specifically bind endotoxin Cry11A and activate toxin Cry4B (Cry4B-tox) under conditions of ligand blotting, and both Cry proteins compete for this binding. At least in the case of Cry4B-tox, the binding with 65 and 57 kD proteins is reversible. The ability of the products of limited proteolysis of Cry11A and Cry4B to bind the 65 and 57 kD proteins correlates with their toxicity to A. stephensi larva. The N-terminal amino acid sequence of the 57 kD protein is unique and absent in the NCBI GenBank. The proteins of 65 and 57 kD share most of the properties studied with Aedes aegypti toxin-binding proteins. It is possible that they altogether represent a novel class (or classes) of delta-endotoxin receptors.

Reconstruction of Bacillus thuringiensis ssp. israelensis Cry11A endotoxin from fragments corresponding to its N- and C-moieties restores its original biological activity.

Subtilisin hydrolyzes Cry11A endotoxin (of 70 kD) produced by Bacillus thuringiensis ssp. israelensis to fragments of 33- and 36-kD, which correspond to N- and C-terminal halves of the endotoxin molecule. Thermitase (a serine protease from Thermoactinomyces vulgaris) and insect gut proteases from Diptera and Lepidoptera exhibit the same hydrolytic effect on Cry11A. Hydrolyzates maintain high toxicity with respect to larvae of Aedes aegypti, Anopheles stephensi, and Culex pipiens. The 33- and 36-kD Cry11A endotoxin components purified by ion-exchange chromatography from the subtilisin hydrolyzate were inactive; however, equimolar mixture of these proteins exhibited almost the same activity as the initial hydrolyzate.

[Cytopathological effect of Bacillus thuringiensis israelensis endotoxins on the intestines of Aedes aegypti mosquito larvae]. / Tsitopatologicheskoe vliianie éndotoksinov Bacillus thuringiensis israelensis na kishechnik lichinok komarov Aedes aegypti.

The dynamics of pathological changes in the intestine of Aedes aegypti larvae under the influence of toxins Cry11A and Cry4B produced by Bacillus thuringiensis israelensis was studied by means of electron microscope. Most significant ultrastructure changes in the intestine of the second instar larvae were observed in the midgut. The cytoplasm of cells disintegrated, and elongated lacunae appeared. The number of microvilli decreased, or they disappeared in the result of destruction. The peritrophic membrane displaced to the lumen of midgut. Any changes in epithelial cells and cuticle in time of foregut and hindgut were not observed in a comparison to control. The toxin Cry4B caused the most effective destruction of the midgut epithelium.

Interaction of 65- and 62-kD proteins from the apical membranes of the Aedes aegypti larvae midgut epithelium with Cry4B and Cry11A endotoxins of Bacillus thuringiensis.

A protein with the molecular weight of 65 kD is the only component of Aedes aegypti larvae BBM capable to specifically bind mosquitocidal toxins Cry4B and Cry11A of Bacillus thuringiensis. This protein lacks the leucine aminopeptidase activity which is characteristic for the toxin-binding proteins from the membranes of caterpillars. Cry-toxins inactive against A. aegypti larvae either fail to bind to the 65-kD protein and to a putative product of its proteolysis with the molecular weight of 62 kD (Cry1Ab), or bind but do not compete for this binding with mosquitocidal proteins (Cry9A). The proteolytic splitting out of the first five alpha-helices in the Cry4B toxin molecule does not affect its binding to the 65- and 62-kD proteins, but an additional removal of 20-30 amino acids from the C-terminal of the molecule sharply spoils this binding. Monosaccharide residues are not involved in the binding of the 65- and 62-kD proteins with Cry4B, Cry11A, and Cry9A.

Two types of entomocidal crystals of Bacillus thuringiensis ssp. finitimus have the same set of unique delta-endotoxins.

The strain B-1166 differs from the other strains of Bacillus thuringiensis ssp. finitimus because it has two crystal types with different localization in the sporulating cell, i.e., inside and outside of exosporium membrane. Two dissociants of the strain were obtained containing only one of the crystal types. The initial strain produces at least three various delta-endotoxins (Fin2, Fin3, and Fin5) differing from all other known entomocidal proteins; Fin2 and Fin3 are similar to each other but differ from Fin5. Both crystal types contain the same endotoxins (Fin2, Fin3, and Fin5). In the B-1166 strain the site of crystal deposition is not determined by their protein composition.

Membrane proteins of Aedes aegypti larvae bind toxins Cry4B and Cry11A of Bacillus thuringiensis ssp. israelensis.

Proteins of molecular weight 65 and 62 kD and having affinity for toxins Cry4B and Cry11A produced by Bacillus thuringiensis ssp. israelensis have been isolated from brush border membranes of Aedes aegypti larvae using affinity chromatography. Using a ligand blotting technique, we show that the binding of these proteins to the biotinylated toxins is reversible and that the two toxins compete for binding to the two proteins. These proteins are likely to be Cry4B and Cry11A toxin receptors in gut epithelial cells of Aedes aegypti larvae.

Two novel delta-endotoxin gene families cry26 and cry28 from Bacillus thuringiensis ssp. finitimus.

Genes cry26Aal and cry28Aal were cloned from Bacillus thuringiensis ssp. finitimus strain B-1166 VKPM. This strain forms insecticidal crystal bodies either outside or inside the exosporium. The deduced amino acid sequence of the cry26Aal gene product included seven residues determined to be an N-terminal part of a chymotrypsin-treated delta-endotoxin isolated from the same strain. Earlier this protein was detected in both free and spore-associated types of crystals [Revina et al., Biokhimia (1999) in press]. Neither BtI nor BtII promoter sequences were found upstream of the open reading frames in both genes. Southern hybridization has shown that the surroundings of both genes at least 3 kb upstream and downstream of the open reading frames are unique. We suggest that the protein Cry26Aal in both types of crystal bodies is synthesized under the control of one and the same genomic locus.

Limited proteolysis of Bacillus thuringiensis CryIG and CryIVB delta-endotoxins leads to formation of active fragments that do not coincide with the structural domains.

Bacillus thuringiensis "true" toxins consist of three domains: the N-terminal, alpha-helical domain followed by two beta-structural domains. Their limited proteolysis does not proceed at the domain boundaries, but is directed to the loops within the domains. There are at least two patterns of the limited proteolysis of "true" toxins. The first pattern, observed for CryIA and CryIVD delta-endotoxins, results in the proteolysis of the loops connecting beta-strands of the second domain. The second pattern, detected for CryIG and CryIVB proteins, consists in the cleavage of the loop connecting the fifth and the sixth alpha-helices of the first domain. The choice between the routes depends on the size, sequence, and dynamics of the loop that define its accessibility to a proteinase. Bioassay of CryIG and CryIVB delta-endotoxin fragments indicates that only two alpha-helices, the sixth and the seventh within the first domain, followed by the two beta-structural domains are sufficient for the insecticidal activity.

Macluralisin--a serine proteinase from fruits of Maclura pomifera (Raf.) Schneid.

A serine proteinase was isolated from fruits of Maclura pomifera (Raf.) Schneid. by affinity chromatography on bacitracin-containing sorbents and gel-filtration. The enzyme, named macluralisin, is a glycoprotein with a molecular mass of 65 kDa; its protein moiety corresponds to a molecular mass of 50 kDa. The substrate specificity of macluralisin towards synthetic peptides and insulin B-chain is similar to that of cucumisin, a subtilisin-like proteinase from melon fruit. The enzyme is completely inhibited by diisopropylfluorophosphate. Its amino-acid composition resembles that of a serine proteinase isolated from the Cucurbitaceae. The N-terminal sequence has 33% of its residues identical to those of the sequence of fungal subtilisin-like proteinase K. Hence, Maclura pomifera serine proteinase belongs to the subtilisin family, which seems to be broadly distributed in the plant kingdom.

[Primary structure of the intracellular serine proteinase from Bacillus amyloliquefaciens. III. Amino acid sequence of peptides obtained by hydrolysis with a Glu,Asp-specific proteinase. Reconstruction of the entire amino acid sequence of the proteinase]. / Pervichnaia struktura vnutrikletochnoi serinovoi proteinazy Bacillus amyloliquefaciens. III. Aminokislotnaia posledovatel'nost' peptidov, poluchennykh gidrolizom Glu,Asp-spetsifichnoi proteinazoi. Rekonstruktsiia polnoi aminokislotnoi posledovatel'nosti proteinazy.

Glu,Asp-specified protease hydrolysate of intracellular serine proteinase (ISP) was separated by ion-exchange chromatography on a sulphocationite resin followed by HPLC to yield 30 individual peptides. Their sequences, spanning to 243 amino acid residues, were determined by the manual Edman procedure. Four overlapping fragments were reconstructed by comparing their sequences with those of tryptic and chymotryptic peptides. To arrange these fragments in the proteinase polypeptide chain and to reconstruct the enzyme's total sequence, additional peptides were isolated from the tryptic hydrolysate and analysed. Primary structure of ISP, corresponding to 297 amino acid residues, was reconstructed. Its comparison with related serine proteinases revealed the following levels of homology: with Bacillus subtilis intracellular serine proteinase, 88%; with secretory subtilisin BPN' produced by B. amyloliquefaciens, 46%.

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

A method was developed to assess the number of delta-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 delta-endotoxins, respectively. Among those delta-endotoxins assigned to previously described families, CryIA, CryID, CryIF, and CryIG were found, as well as crystal proteins, which possess N-terminal amino acid sequences very different from those of all known delta-endotoxins. Possible functional consequences of delta-endotoxin multiplicity are discussed.

[Primary structure of an intracellular serine proteinase from Bacillus amyloliquefaciens. II. Amino acid sequence of peptides in a chymotrypsin hydrolysate]. / Pervichnaia struktura vnutrikletochnoi serinovoi proteinazy Bacillus amyloliquefaciens. II. Aminokislotnaia posledovatel'nost' peptidov khimotripticheskogo gidrolizata.

Chymotrypsin hydrolyzate of the intracellular serine protease was separated by ion-exchange chromatography on a sulphocationite resin followed by HPLC to yield fifty one individual peptides. Their sequences, corresponding in total to 381 amino acid residues, were determined by the manual Edman procedure.

Primary structure of carboxypeptidase T: delineation of functionally relevant features in Zn-carboxypeptidase family.

The primary structure of carboxypeptidase T--a Zn-dependent extracellular enzyme of Thermoactinomyces vulgaris--was determined from the cloned cpT gene nucleotide sequence and compared to Zn-carboxypeptidases from various organisms. The compilation and analysis of multiple alignment accompanied by consideration of available tertiary structure data have shown that in the overall spatial structure and active site arrangement CpT is similar to other enzymes constituting the Zn-carboxypeptidase family. Nine of 16 amino acid residues found to be strictly invariant are presumably located close to the active site. The preservation of His69, Glu72, Asn144, Arg145, His196, Tyr248, and Glu270 identified previously as essential catalytic site participants implicates basically the same catalytic mechanism in the Zn-carboxypeptidase family. It is proposed that Pro205 and Asp256 should play an important role in proper S1'-pocket spatial arrangement. The comparative analysis of amino acid variations in S1'-pocket enabled us to reveal structural determinants of the Zn-carboxypeptidase primary specificity. The relatively reduced size of the pocket and negative charge of Asp253 are supposed to contribute correspondingly to A- and B-type substrate preferences of carboxypeptidase T endowed with dual primary specificity.

[Serine proteinase from the archaebacterium Halobacterium mediterranei--an analog of eubacterium subtilisin]. / Serinovaia proteinaza arkhebakterii Halobacterium mediterranei--analog subtilizina éubakterii.

A homogeneous serine proteinase was isolated from cultural filtrates of the extreme halophilic bacteria Halobacterium mediterranei 1538 using affinity chromatography on bacitracin-Sepharose, ultrafiltration and gel filtration on Sephadex G-75, with a 48% yield and 260-fold purification. The enzyme was completely inactivated by specific inhibitors of serine proteinases, PMSF and DFP, as well as by Hg2+ and PCMB. The enzyme activity was strongly dependent of NaCl concentration, the enzyme being inactivated below 0.75 M NaCl. Inactivation of the enzyme was also seen in the presence of 2-7% organic solvents. The pH optimum for Glp-Ala-Ala-Leu-pNA hydrolysis is 8.0-8.5; Km is 0.14 mM, kcat is 36.9 s-1. The stability optimum lies at pH 5.5-8.0, temperature optimum is at 55 degrees C. The enzyme molecular weight is 41,000 Da; pI is 7.5. The substrate specificity of the enzyme is comparable to that of secretory subtilisins; the extent of protein substrate hydrolysis is similar to that of proteinase K. The N-terminal sequence of Halobacterium mediterranei serine proteinase, Asp-Thr-Ala-Asn-Asp-Pro-Lys-Tyr-Gly-Ser-Gln-Tyr-Ala-Pro-Gln-Lys-Val-Asn- Ala- Asp-, reveals a 50% homology with the aminoterminal sequence of Thermoactinomyces vulgaris serine proteinase. Hence, the serine proteinase secreted by halophilic bacteria may be considered as a structural and functional analog of eubacterial enzymes.

A serine proteinase of an archaebacterium, Halobacterium mediterranei. A homologue of eubacterial subtilisins.

A homogeneous serine proteinase secreted by the extreme halophilic bacterium Halobacterium mediterranei 1538 was isolated by affinity chromatography on bacitracin-Sepharose with a yield of 48% (260-fold purification). The enzyme reveals an optimum for pyroglutamyl-Ala-Ala-Leu p-nitroanilide hydrolysis at pH 8.0-8.5 (Km 0.14 mM; k(cat). 36.9 s-1). Its activity increases linearly with NaCl concentration over the range 2-5 M. The substrate specificity of the enzyme is comparable with that of secretory subtilisins, the extent of protein degradation approaching that attained with proteinase K. The enzyme has a molecular mass of 41 kDa and a pI of 7.5. The N-terminal sequence of H. mediterranei serine proteinase reveals a 50% identity with that of Thermoactinomyces vulgaris serine proteinases, indicating that the enzyme belongs to the subtilisin family. Hence the serine proteinase secreted by the halophilic bacterium should be considered as a functional analogue, and a structural homologue, of eubacterial serine proteinases (subtilisins).

[Comparison of amino acid sequences of sturgeon triprotamines using protamines from Acipenser stellatus gonads as an example]. / Sravnenie aminokislotnykh posledovatel'nostei triprotaminov osetrovykh na primere protaminov iz gonad Acipenser stellatus.

The amino acid sequence of the triprotamine stelline C from mature sperm nuclei of Acipenser stellatus has been established by automated sequence analysis of the protein and from data provided by thermolysine peptides. The complete amino acid sequence of stelline C is: R-R-R-R-R-H-A-S-T-K-L-K-R-R-R-R-R-R-R-H-G-K-K-S-H-K. The comparison of the primary structure of stelline C with that of other triprotamines from Acipenser stellatus shows that they are similar except for the absence of N-terminal alanine in the stelline A molecule. The main structure difference between stelline C and other fish protamines is that the stelline C molecule begins with five arginine residues.

[Thiol-dependent serine proteinase from Streptomyces thermovulgaris]. / Tiolzavisimaia serinovaia proteinaza Streptomyces thermovulgaris.

The cultural filtrates of S. thermovulgaris contain a proteinase which is active towards the chromogenic subtilisin substrate, Z-Ala-Ala-Leu-pNa, and azocasein. Pure enzyme preparations were obtained by affinity chromatography on bacitracin-Sepharose with subsequent rechromatography on the same adsorbent. The proteinase was completely inactivated by PMSF and DFP, the specific inhibitors for serine proteinase, by thiol reagents (HgCl2, PCMB) and by the protein inhibitor from S. jantinus. The pH activity optimum for the enzyme is 7.8-8.2, temperature optimum is 55 degrees C. The enzyme is stable at pH 6-9, has a pI of 5.0 and a molecular mass of 32 kDa. When tested against the peptide substrate, the enzyme shows a specificity characteristic for subtilisins. The N-terminal sequence of the enzyme, Tyr-Thr-Pro-Asn-Asp-Pro-Tyr-Phe-Ser-Ser-Arg-Gln-Tyr-Gly, shows a 100% homology with that of terminase, a thiol-dependent serine proteinase. On the basis of the above considerations the enzyme may be related to the subfamily of thiol-dependent serine proteinases.

[Pepsinogen activation by microbial proteinases]. / Aktivatsiia pepsinogena proteinazami mikroorganizmov.

Serine proteinase and metalloproteinase of Asp. oryzae, extracellular metalloproteinase of L. pneumophila and chymotrypsin-like proteinase of S. rutgersensis can hydrolyze pepsinogen by converting it into pepsin (pH 5.0, 37 degrees C). The localization of the site of hydrolysis depends on the nature of the enzyme: serine proteinase from Asp. oryzae induces the synthesis of a mixture of 60% pepsin, 25% leucyl-pepsin and 15% alanyl-leucyl-pepsin; metalloproteinase of Asp. oryzae converts pepsinogen only into leucyl-pepsin, while metalloproteinase of L. pneumophila yields a mixture of 33% pepsin, 53% leucyl-pepsin and 14% alanyl-leucyl-pepsin. Thus, the region of the activating pepsinogen peptide--Ala 42P-Ile 1 bond--seems to the most probable site for hydrolysis by exogenous proteinases. This site contains a Leu 44P-Ile 1 bond which is subjected to intermolecular hydrolysis during autocatalytic activation of pepsinogen. The experimental results emphasize the importance of the intermolecular pathway of pepsinogen activation.

[Primary structure of intracellular serine proteinase from Bacillus amyloliquefaciens. I. Isolation of the enzyme and amino acid sequence of peptides of tryptic hydrolysate]. / Pervichnaia struktura vnutrikletochnoi serinovoi proteinazy Bacillus amyloliquefaciens. I. Vydelenie fermenta i opredelenie aminokislotnoi posledovatel'nosti peptidov tripticheskogo gidrolizata.

Method of isolation of intracellular serine protease was modified. Gramicidin S-sepharose CL-4B with a higher content of the ligand, synthesized through a modified procedure, was used as an affinity sorbent which simplified the purification and led to the pure enzyme with high specific activity and 90% yield. Trypsin hydrolyzate of the protease was separated by ion-exchange chromatography on a sulphocationite resin followed by paper chromatography and paper electrophoresis to yield twenty-five individual peptides. Their complete or partial sequences, corresponding in total to 146 amino acid residues, were determined by the manual Edman procedure.