Tag Archives: Bacillus thuringiensis
Mapping of residues of fibrinogen cleaved by protease II of Bacillus thuringiensis var. israelensis IMV B-7465
E. M. Stohniy1, V. O. Chernyshenko1*, N. A. Nidialkova2, A. V. Rebriev1,
L. D. Varbanets2, V. E. Hadzhynova1, T. M. Chernyshenko1,
I. M. Kolesnikova1, E. V. Lugovskoy1
1Palladin Institute of Biochemistry, National Academy
of Sciences of Ukraine, Kyiv;
2Zabolotny Institute of Microbiology and Virology, National Academy
of Sciences of Ukraine, Kyiv;
*e-mail: bio.cherv@gmail.com
The limited proteolysis of macromolecules allows obtaining the fragments that preserve the structure and functional properties of the whole molecule and could be used in the study of proteins structure and function. Proteases targeted to fibrinogen and fibrin are of interest as the tool for obtaining of functionally active fragments of fibrin(ogen) and for the direct defibrination in vivo. That is why the aim of the present work was to study the proteolytic action of Protease II (PII) purified from Bacillus thuringiensis var. israelensis IMV B-7465 on fibrinogen.
Hydrolysis products of fibrinogen by PII were analysed by SDS-PAGE under reducing conditions with further immunoprobing using the mouse monoclonal 1-5A (anti-Aα509-610) and ІІ-5С (anti-Aα20-78) antibodies. It was shown that PII cleaved preferentially the Aα-chain of fibrinogen splitting off the peptide with apparent molecular weight of 10 kDa that corresponded the C-terminal part of Aα-chain of fibrinogen molecule.
MALDI-TOF analysis of hydrolysis of fibrinogen was performed using a Voyager-DE. Results analyzed by Data Explorer 4.0.0.0 allowed to detect the main peak occurring at mass/charge (M/Z) ratio of 11 441 Da. According to “Peptide Mass Calculator” this peptide corresponded to fragment Аα505-610 of fibrinogen molecule. The result showed that PII cleaves the peptide bond AαAsp-Thr-Ala504-Ser505.
Thus, PII can be used for the obtaining of unique fragments of fibrinogen molecule. As far as αC-domain contains numerous sites of fibrin intermolecular interactions we can consider PII as a prospective agent for their study and for the defibrination.
Purification and physico-chemical properties of Bacillus thuringiensis ІМВ В-7324 peptidase with elastolytic and fibrinolytic activity
О. V. Matselyukh, N. A. Nidialkova, L. D. Varbanets
Institute of Microbiology and Virology, National Academy of Sciences of Ukraine, Kyiv;
e-mail: oivanko@yahoo.com
The scheme of isolation and purification of Bacillus thuringiensis ІMV B-7324 peptidase has been developed. This scheme includes ammonium sulfate precipitation and chromatography on neutral and charged TSK-gels. It was found that the enzyme hydrolyzes elastin and fibrin. The molecular weight is 26 kDa. It was shown that the enzyme is an alkaline serine peptidase. The optimal pH of hydrolysis of elastin and fibrin were 9.0 and 10.0, respectively. The optimal temperature of elastin and fibrin hydrolysis are 40 and 50 °C, respectively. The high stability of the purified preparation in the studied range of pH and temperature was shown. The stabilizing effect of zinc at a concentration of 1 mM on the elastase activity, and the inhibitory effect of other divalent cations under study have been established. The investigated chloride and acetate anions reduced activity by 20%, while phosphate anions increased activity by 15–30%.
Complexes of cobalt (II, III) with derivatives of dithiocarbamic acid – effectors of peptidases of Bacillus thuringiensis and α-L-Rhamnozidase of Eupenicillium erubescens and Cryptococcus albidus
L. D. Varbanets1, E. V. Matseliukh1, I. I. Seifullina2,
N. V. Khitrich2, N. A. Nidialkova1, E. V. Gudzenko1
1D. K. Zabolotny Institute of Microbiology and Virology,
National Academy of Sciences of Ukraine, Kyiv;
2I. I. Mechnikov Оdеssa National University, Ukraine;
e-mail: varbanets@serv.imv.kiev.ua
The influence of cobalt (II, III) coordinative compounds with derivatives of dithiocarbamic acid on Bacillus thuringiensis IMV B-7324 peptidases with elastase and fibrinolytic activity and Eupenicillium erubescens and Cryptococcus albidus α-L-rhamnosidases have been studied. Tested coordinative compounds of cobalt (II, III) on the basis of their composition and structure are presented by 6 groups: 1) tetrachlorocobaltates (II) of 3,6-di(R,R′)-iminio-1,2,4,5-tetratiane – (RR′)2Ditt[CoCl4]; 2) tetrabromocobaltates (II) of 3,6-di(R,R′)-iminio-1,2,4,5-tetratiane – (RR′)2Ditt[CoBr4]; 3) isothiocyanates of tetra((R,R′)-dithiocarbamatoisothiocyanate)cobalt (II) – [Co(RR′Ditc)4](NCS)2]; 4) dithiocarbamates of cobalt (II) – [Co(S2CNRR′)2]; 5) dithiocarbamates of cobalt (III) – [Co(S2CNRR′)3]; 6) molecular complexes of dithiocarbamates of cobalt (III) with iodine – [Co(S2CNRR′)3]∙2I2. These groups (1-6) are combined by the presence of the same complexing agent (cobalt) and a fragment S2CNRR′ in their molecules. Investigated complexes differ by a charge of intrinsic coordination sphere: anionic (1-2), cationic (3) and neutral (4-6). The nature of substituents at nitrogen atoms varies in each group of complexes. It is stated that the studied coordination compounds render both activating and inhibiting effect on enzyme activity, depending on composition, structure, charge of complex, coordination number of complex former and also on the enzyme and strain producer. Maximum effect is achieved by activating of peptidases B. thuringiensis IMV B-7324 with elastase and fibrinolytic activity. So, in order to improve the catalytic properties of peptidase 1, depending on the type of exhibited activity, it is possible to recommend the following compounds: for elastase – coordinately nonsaturated complexes of cobalt (II) (1-4) containing short aliphatic or alicyclic substituents at atoms of nitrogen and increasing activity by 17-100% at an average; for fibrinolytic – neutral dithiocarbamates of cobalt (II, III) (4-5) (by 29-199%). For increasing the fibrinolytic activity of peptidase it is better to use dibenzyl- or ethylphenyldithiocarbamates of cobalt (III), which increase activity by 15-40% at an average. The same complexes, and also compound {(CH2)6}2Ditt[CoCl4] make an activating impact on α-L-rhamnosidase C. albidus (by 10-20%).