Enzymatic and nonenzymatic synthesis of glutathione conjugates: application to the understanding of a parasite's defense system and alternative to the discovery of potent glutathione S-transferase inhibitors

A primary pathway for metabolism of electrophilic compounds in Schistosoma japonicum involves glutathione S-transferase (SjGST)-catalyzed formation of glutathione (GSH) conjugates. As part of a program aimed at gaining a better understanding of the defense system of parasites, a series of aromatic halides (1-8), aliphatic halides (9, 10), epoxides (11-20), alpha,beta-unsaturated esters (21, 22), and alpha,beta-unsaturated amides (23, 24) were prepared, and their participation in glutathione conjugate formation was evaluated. Products from enzymatic and nonenzymatic reactions of these substances with glutathione were characterized and quantified by using reverse-phase high-performance liquid chromatography (HPLC), NMR, and fast atom bombardment mass spectrometry (FAB-MS) analysis. Mechanisms for formation of specific mono(glutathionyl) or bis(glutathionyl) conjugates are proposed. Although the results of this effort indicate that SjGST does not catalyze addition or substitution reactions of 1, 3, 4, 7-9, 11-13, 15-17, 19-21, and 24, they demonstrate that 2, 5, 6, 14, 18, and 23 undergo efficient enzyme-catalyzed conjugation reactions. The kcat values for SjGST with 23 and 18 are about 886-fold and 14-fold, respectively, larger than that for 5. This observation suggests that 23 is a good substrate in comparison to other electrophiles. Furthermore, the initially formed conjugation product, 23a, is also a substrate for SjGST in a process that forms the bis(glutathionyl) conjugate 23b. Products arising by enzymatic and nonenzymatic pathways are generated under the conditions of SjGST-activated GSH conjugation. Interestingly, production of nonenzymatic GSH conjugates with electrophilic substrates often overwhelms the activity of the enzyme. The nonenzymatic GSH conjugates, 9a-11a, 16a, 21a, and 22a, are inhibitors of SjGST with respective IC50 values of 1.95, 75.5, 0.96, 19.0, 152, and 0.36 microM, and they display moderate inhibitory activities against human GSTA2. Direct evidence has been gained for substrate inhibition by 10 toward SjGST and GSTA2 that is more potent than that of its GSH conjugate 10a. The significance of this work is found in the development of a convenient NMR-based technique that can be used to characterize glutathione conjugates derived from small molecule libraries as part of efforts aimed at uncovering specific potent SjGST and GSTA2 inhibitors. This method has potential in applications to the identification of novel inhibitors of other GST targets that are of chemotherapeutic interest.     

Binding properties of ferrocene-glutathione conjugates as inhibitors and sensors for glutathione S-transferases

The binding properties of two electroactive glutathione-ferrocene conjugates that consist in glutathione attached to one or both of the cyclopentadienyl rings of ferrocene (GSFc and GSFcSG), to Schistosoma japonica glutathione S-transferase (SjGST) were studied by spectroscopy fluorescence, isothermal titration calorimetry (ITC) and differential pulse voltammetry (DPV). Such ferrocene conjugates resulted to be competitive inhibitors of glutathione S-transferase with an increased binding affinity relative to the natural substrate glutathione (GSH). We found that the conjugate having two glutathione units (GSFcSG) exhibits an affinity for SjGST approximately two orders of magnitude higher than GSH. Furthermore, it shows negative cooperativity with the affinity for the second binding site two orders of magnitude lower than that for the first one. We propose that the reason for such negative cooperativity is steric since, i) the obtained thermodynamic parameters do not indicate profound conformational changes upon GSFcSG binding and ii) docking studies have shown that, when bound, part of the first bound ligand invades the second site due to its large size. In addition, voltammetric measurements show a strong decrease of the peak current upon binding of ferrocene-glutathione conjugates to SjGST and provide very similar K values than those obtained by ITC. Moreover, the sensing ability, expressed by the sensitivity parameter shows that GSFcSG is much more sensitive than GSFc, for the detection of SjGST.     

A calorimetric study of the binding of S-alkylglutathiones to glutathione S-transferase.

The binding of three competitive glutathione analogue inhibitors (S-alkylglutathione derivatives) to glutathione S-transferase from Schistosoma japonicum, SjGST, has been investigated by isothermal titration microcalorimetry at pH 6.5 over a temperature range of 15--30 degrees C. Calorimetric measurements in various buffer systems with different ionization heats suggest that no protons are exchanged during the binding of S-alkylglutathione derivatives. Thus, at pH 6.5, the protons released during the binding of substrate may be from its thiol group. Calorimetric analyses show that S-methyl-, S-butyl-, and S-octylglutathione bind to two equal and independent sites in the dimer of SjGST. The affinity of these inhibitors to SjGST is greater as the number of methylene groups in the hydrocarbon side chain increases. In all cases studied, Delta G(0) remains invariant as a function of temperature, while Delta H(b) and Delta S(0) both decrease as the temperature increases. The binding of three S-alkylglutathione derivatives to the enzyme is enthalpically favourable at all temperatures studied. The temperature dependence of the enthalpy change yields negative heat capacity changes, which become less negative as the length of the side chain increases.     

Production and characterization of glutathione-S-transferase fused with a poly-histidine tag

Schistosoma japonicum glutathione-S-transferase (SjGST) was genetically engineered with a poly-histidine tag at the C-terminus and highly expressed in Escherichia coli. Both SjGST and the tagged protein, SjGST/His, were purified with glutathione Sepharose 4B gels and subsequently studied for their activities, antibody-binding abilities, and metal affinities. The production level of active SjGST/His was higher than that of SjGST. Both proteins had similar specific catalytic activities and binding abilities with anti-SjGST antibody, while the antibody against poly-histidine recognized only SjGST/His. Proteolytic degradation was occasionally observed in aged dialyzed SjGST/His preparation. Under a native condition, the Co(2+)-chelated TANOL gel (Co-TANOL) had a better binding specificity to the tagged protein than did the Ni(2+)-chelated nitriloacetic acid (Ni-NTA) agarose gel. However, the binding capacity of the Ni-NTA gel for SjGST/His was 2-fold higher than that of the Co-TANOL one. To increase the native binding specificity of the Ni-NTA gel, 20 mM imidazole had to be added to the washing solution. In a denatured state, both gels could only capture SjGST/His, and the binding capacity of the Ni-NTA gel was nearly 2-fold higher than that of the Co-TANOL gel. The binding association constants of both gels with SjGST/His did not differ greatly under either condition. The study demonstrated that the C-terminal addition of the poly-histidine tag to SjGST increased the metal affinity of the enzyme to the Co-TANOL gel under both native and denaturing conditions and to the Ni-NTA gel under denaturing conditions, whereas the enzymatic activity and antibody-binding ability were not affected.     

Crystallographic and thermodynamic analysis of the binding of S-octylglutathione to the Tyr 7 to Phe mutant of glutathione S-transferase from Schistosoma japonicum

Glutathione S-transferases are a family of multifunctional enzymes involved in the metabolism of drugs and xenobiotics. Two tyrosine residues, Tyr 7 and Tyr 111, in the active site of the enzyme play an important role in the binding and catalysis of substrate ligands. The crystal structures of Schistosoma japonicum glutathione S-transferase tyrosine 7 to phenylalanine mutant [SjGST(Y7F)] in complex with the substrate glutathione (GSH) and the competitive inhibitor S-octylglutathione (S-octyl-GSH) have been obtained. These new structural data combined with fluorescence spectroscopy and thermodynamic data, obtained by means of isothermal titration calorimetry, allow for detailed characterization of the ligand-binding process. The binding of S-octyl-GSH to SjGST(Y7F) is enthalpically and entropically driven at temperatures below 30 degrees C. The stoichiometry of the binding is one molecule of S-octyl-GSH per mutant dimer, whereas shorter alkyl derivatives bind with a stoichiometry of two molecules per mutant dimer. The SjGST(Y7F).GSH structure showed no major structural differences compared to the wild-type enzyme. In contrast, the structure of SjGST(Y7F).S-octyl-GSH showed asymmetric binding of S-octyl-GSH. This lack of symmetry is reflected in the lower symmetry space group of the SjGST(Y7F).S-octyl-GSH crystals (P6(3)) compared to that of the SjGST(Y7F).GSH crystals (P6(3)22). Moreover, the binding of S-octyl-GSH to the A subunit is accompanied by conformational changes that may be responsible for the lack of binding to the B subunit.     

Crystal structure of non-fused glutathione S-transferase from Schistosoma japonicum in complex with glutathione

Schistosoma japonicum glutathione S-transferase (SjGST) is a common fusion tag in recombinant protein production, and its 3-dimensional structure has been studied in the context of drug design. We have determined the crystal structure of non-fused SjGST complexed with glutathione, and compare it to complexes between glutathione and SjGST fusion proteins.     

Combined IL-12 Plasmid and Recombinant SjGST Enhance the Protective and Anti-pathology Effect of SjGST DNA Vaccine Against Schistosoma japonicum

Schistosomiasis is listed as one of most important tropical diseases and more than 200 million people are estimated to be infected. Development of a vaccine is thought to be the most effective way to control this disease. Recombinant 26-kDa glutathione S-transferase (rSjGST) has previously been reported to achieve a worm reduction rate of 42–44%. To improve the efficiency of the vaccine against Schistosoma japonicum, we immunized mice with a combination of pcDNA vector-encoded 26-kDa SjGST (pcDNA/SjGST), IL-12 expressing-plasmid (pIL-12), and rSjGST. Co-vaccination with pcDNA/SjGST, pIL-12, and rSjGST led to a reduction in worm burden, hepatic egg burden, and the size of liver tissue granulomas than that in the untreated infection controls. In addition, we detected high levels of specific IgG, IgG1, and IgG2a against the rSjGST antigen in infected mice vaccinated with this combination of pcDNA/SjGST, pIL-12, and rSjGST. Moreover, high expression levels of Th2 cytokines, including IL-4 and IL-10, were also detected in this group, without diminished levels of IL-12, INF-γ, and TNF-α cytokines that are related to parasite killing. In conclusion, we have developed a new vaccination regimen against S. japonicum infection and shown that co-immunization with pcDNA/SjGST vaccine, pIL-12, and rSjGST has significant anti-parasite, anti-hepatic egg and anti-pathology effects in mice. The efficacy of this vaccination method should be further validated in large animals such as water buffalo. This method may help to reduce the transmission of zoonotic schistosomiasis japonica.     

日本血吸虫(中国大陆株)谷胱甘肽转移酶编码区基因的体外扩增、克隆及在原核细胞的高效表达

为表达、获取日本血吸早谷胱甘肽转移酶（ＳｊＧＳＴ）基因工程重组蛋白,以日本血吸虫（中国大陆株）ｃＤＮＡ为模板,设计、合成特定寡核苷酸引物,ＲＴ－ＰＣＲ法扩增ＧＳＴ编码基因序列,将扩增产物连接ｐＧＥＭ－Ｔ克隆载体,再亚克隆到真、原核表达质粒ｐＢＫ－ＣＭＶ中,转染大肠杆菌ＸＬ１－ｂｌｕｅ,经ＩＰＴＧ诱导后用ＳＤＳ－ＰＡＧＥ分析表达效果。结果 ＲＴ－ＰＣＲ法特异性扩增出日本血吸虫ＧＳＴ编码区基因片段,其     

Thermodynamic analysis of the binding of glutathione to glutathione S-transferase over a range of temperatures.

The binding properties of a glutathione S-transferase (EC 2.5.1.18) from Schistosoma japonicum to substrate glutathione (GSH) has been investigated by intrinsic fluorescence and isothermal titration calorimetry (ITC) at pH 6.5 over a temperature range of 15-30 degrees C. Calorimetric measurements in various buffer systems with different ionization heats suggest that protons are released during the binding of GSH at pH 6.5. We have also studied the effect of pH on the thermodynamics of GSH-GST interaction. The behaviour shown at different pHs indicates that at least three groups must participate in the exchange of protons. Fluorimetric and calorimetric measurements indicate that GSH binds to two sites in the dimer of 26-kDa glutathione S-transferase from Schistosoma japonicum (SjGST). On the other hand, noncooperativity for substrate binding to SjGST was detected over a temperature range of 15-30 degrees C. Among thermodynamic parameters, whereas DeltaG degrees remains practically invariant as a function of temperature, DeltaH and DeltaS degrees both decrease with an increase in temperature. While the binding is enthalpically favorable at all temperatures studied, at temperatures below 25 degrees C, DeltaG degrees is also favoured by entropic contributions. As the temperature increases, the entropic contributions progressively decrease, attaining a value of zero at 24.3 degrees C, and then becoming unfavorable. During this transition, the enthalpic contributions become progressively favorable, resulting in an enthalpy-entropy compensation. The temperature dependence of the enthalpy change yields the heat capacity change (DeltaCp degrees ) of -0.238 +/- 0.04 kcal per K per mol of GSH bound.     

Metal binding ability of glutathione transferases conserved between two animal species, the vanadium-rich ascidian Ascidia sydneiensis samea and the schistosome Schistosoma japonicum

Glutathione transferases (GSTs) are multifunctional enzymes found in many organisms. We recently identified vanadium-binding GSTs, designated AsGSTs, from the vanadium-rich ascidian, Ascidia sydneiensis samea. In this study, the metal-selectivity of AsGST-I was investigated. Immobilized metal ion affinity chromatography (IMAC) analysis revealed that AsGST-I binds to V(IV), Fe(III), and Cu(II) with high affinity in the following order Cu(II)>V(IV)>Fe(III), and to Co(II), Ni(II), and Zn(II) with low affinity. The GST activity of AsGST-I was inhibited dose-dependently by not V(IV) but Cu(II). A competition experiment demonstrated that the binding of V(IV) to AsGST-I was not inhibited by Cu(II). These results suggest that AsGST-I has high V(IV)-selectivity, which can confer the specific vanadium accumulation of ascidians. Because there are few reports on the metal-binding ability of GSTs, we performed the same analysis on SjGST (GST from the schistosome, Schistosoma japonicum). SjGST also demonstrated metal-binding ability although the binding pattern differed from that of AsGST-I. The GST activity of SjGST was inhibited by Cu(II) only, as that of AsGST-I. Our results indicate a possibility that metal-binding abilities of GSTs are conserved among organisms, at least animals, which is suggestive of a new role for these enzymes in metal homeostasis or detoxification.     

两种血吸虫病DNA疫苗的候选抗原基因研究

目的：以日本血吸虫基因SjFABP和SjGST原核表达产物检测二价DNA疫苗pVIVO2-SjFABP-SjGST在体内诱发的特异性抗体。方法：克隆日本血吸虫抗原基因SjFABP和sjGST,构建重组原核表达载体pET30a-SjFABP、pET30a-SjGST及真核表达载体pVIVO2-SjFABP-SjGST;将pET30a-SjFABP和pET30a-sjGST进行原核表达,并将表达产物用镍亲和柱分离纯化;采用Western印迹对日本血吸虫DNA疫苗pVIVO2-SjFABP-SjGST免疫4周后的BALB／c小鼠血清进行特异性抗体检测。结果：克隆了日本血吸虫抗原基因SjFABP（399bp）和町GST（657bp）,并构建了pET30a-SjFABP、pET30a-SjGST及pVIVO2-SjFABP-SjGST重组质粒;经Western印迹检测,pET30a-SjFABP及pET30a-SjGST原核表达的抗原蛋白均能够与经日本血吸虫二价DNA疫苗pVIVO2-SjFABP-SjGST免疫的小鼠的血清产生特异性免疫反应。结论：日本血吸虫町尉即和町GST基因的原核表达系统成功建立;原核表达的抗原蛋白具有免疫原性;以原核表达产物可检测日本血吸虫DNA疫苗pVIVO2-SiFABP-SiGST在体内诱发的特异性抗体。     

Fluoro ketone inhibitors of hydrolytic enzymes

The use of fluoro ketones as inhibitors of hydrolytic enzymes has been investigated. The acetylcholine analogues 6,6-dimethyl-1,1,1-trifluoro-2-heptanone and 3,3-difluoro-6,6-dimethyl-2-heptanone are inhibitors of acetylcholinesterase with Ki values of 16 X 10(-9) M and 1.6 X 10(-9) M, respectively. These fluoro ketones are 10(4)-10(5) times better as inhibitors than the corresponding methyl ketone. Since nucleophiles readily add to fluoro ketones, it is likely that these compounds inhibit acetylcholinesterase by formation of a stable hemiketal with the active-site serine residue. Fluoro ketone substrate analogues are also inhibitors of zinc metallo- and aspartylproteases. 2-Benzyl-4-oxo-5,5,5-trifluoropentanoic acid is an inhibitor of carboxypeptidase A (Ki = 2 X 10(-7) M). Trifluoromethyl ketone dipeptide analogues are good inhibitors of angiotensin converting enzyme. An analogue of pepstatin that contains a difluorostatone residue in place of statine has been prepared and found to be an extremely potent inhibitor of pepsin (Ki = 6 X 10(-11) M). The hydrated ketones are probably the inhibitory species since they are structural mimics of the tetrahedral intermediate that forms during the hydrolysis of peptide substrates.     

The efficacy and selectivity of tumor cell killing by Akt inhibitors are substantially increased by chloroquine

This study was to evaluate the enhancement value of chloroquine (CQ) in cancer cell killing when used in combination with Akt inhibitors. The results showed that the combination of CQ and Akt inhibitors is much more effective than either one alone. Importantly, the CQ-mediated chemosensitization of cell killing effects by Akt inhibitors is cancer specific. In particular, when combined with 10 microM CQ, 1,3-dihydro-1-(1-((4-(6-phenyl-1H-imidazo[4,5-g]quinoxalin-7-yl)phenyl)methyl)-4-piperidinyl)-2H-benzimidazol-2-one (an Akt1 and 2 inhibitor; compound 8) killed cancer cells 10-120 times more effectively than normal cells. Thus, CQ is a very effective and cancer-specific chemosensitizer when used in combination with Akt inhibitors.     

The binding of HIV-1 protease inhibitors to human serum proteins

The non-specific binding of a drug to plasma proteins is an important determinant of its biological efficacy since it modulates the availability of the drug to its intended target. In the case of HIV-1 protease inhibitors, binding to human serum albumin (HSA) and alpha(1)-acid glycoprotein (AAG) appears to be an important modulator of drug bioavailability. From a thermodynamic point of view, the issue of drug availability to the desired target can be formulated as a multiple equilibrium problem in which a ligand is able to bind to different proteins or other macromolecules with different binding affinities. Previously, we have measured the binding thermodynamics of HIV-1 protease inhibitors to their target. In this article, the binding energetics of four inhibitors currently in clinical use (saquinavir, indinavir, ritonavir and nelfinavir) and a second-generation inhibitor (KNI-764) to human HSA and AAG has been studied by isothermal titration calorimetry. All inhibitors exhibited a significant affinity for AAG (K(a) approximately 0.5-10 x 10(5) M(-1)) and a relatively low affinity for HSA (K(a) approximately 5-15 x 10(3) M(-1)). It is shown that under conditions that simulate in vivo concentrations of serum proteins, the inhibitor concentrations required to achieve 95% protease inhibition can be up to 10 times higher than those required in the absence of serum proteins. The effect is compounded in patients infected with drug resistant HIV-1 strains that exhibit a lower affinity for protease inhibitors. In these cases the required inhibitor concentrations can be up to 2000 times higher and beyond the solubility limits of the inhibitors.     

Effects of PDE4 inhibitors on lipopolysaccharide-induced priming of superoxide anion production from human mononuclear cells.

AIMS: Phosphodiesterase 4 (PDE4) inhibitors have been described as potent anti-inflammatory compounds, involving an increase in intracellular levels of cyclic 3'',5''-adenosine monophosphate (AMP). The aim of this study was to compare the effects of selective PDE4 inhibitors, rolipram and RP 73-401 with the cell permeable analogue of cyclic AMP, dibutyryl-cyclic AMP (db-cAMP) and the anti-inflammatory cytokine interleukin-10 (IL-10) on superoxide anion production from peripheral blood mononuclear cells preincubated with lipopolysaccharide (LPS). MAJOR FINDINGS: We report that, after incubation of the cells with LPS, a large increase in superoxide anion production was observed. Rolipram or RP 73-401 (10(-8) to 10(-5) M) induced significant reductions of fMLP-induced superoxide anion production in cells incubated with or without LPS. The db-cAMP (10(-5) to 10(-3) M) also elicited dose-dependent inhibitions of the fMLP-induced superoxide anion production. In contrast, IL-10 (1 or 10 ng/ml) did not elicit a reduction in fMLP-induced superoxide anion production in both conditions. PRINCIPAL CONCLUSION: These results suggest that the inhibitory activity of PDE4 inhibitors on fMLP-induced production of superoxide anion production is mediated by db-cAMP rather than IL-10.     

Kinetics of slow, tight-binding inhibitors of angiotensin converting enzyme

Five phosphorus-containing inhibitors of angiotensin converting enzyme were found to exhibit slow, tight-binding kinetics by using furanacryloyl-L-phenylalanylglycylglycine as substrate at pH 7.50 and T = 25 degrees C. Two of the inhibitors, (O-ethylphospho)-Ala-Pro (2) and (O-isopropylphospho)-Ala-Pro (3), are found to follow at minimum a two-step mechanism of binding (mechanism B) to the enzyme. This mechanism consists of an initial fast formation of a weaker enzyme-inhibitor complex (Ki = 130 nM for 2 and 180 nM for 3) followed by a slow reversible isomerization to a tighter complex with measurable forward (K3) and reverse (k4) rate constants (k3 = 4.5 X 10(-2) s-1 for 2 and 5.4 X 10(-2) s-1 for 3; k4 = 9.2 X 10(-3) s-1 for 2 and 3.5 X 10(-3) s-1 for 3). For the remaining three inhibitors, phospho-Ala-Pro (1), (O-benzyl-phospho)-Ala-Pro (4), and (P-phenethylphosphono)-Ala-Pro (5), a one-step binding mechanism (mechanism A) is observed under the conditions of the experiment. The second-order rate constants k1 (M-1 s-1) for the binding of these inhibitors to converting enzyme are found to have values more than 3 orders of magnitude lower than the diffusion-controlled limit for a bimolecular reaction involving the enzyme, viz., 3.9 X 10(5) for 1, 2.2 X 10(5) for 4, and 4.8 X 10(5) for 5.(ABSTRACT TRUNCATED AT 250 WORDS)     

Design of P1' and P3' residues of trivalent thrombin inhibitors and their crystal structures

Synthetic bivalent thrombin inhibitors comprise an active site blocking segment, a fibrinogen recognition exosite blocking segment, and a linker connecting these segments. Possible nonpolar interactions of the P1' and P3' residues of the linker with thrombin S1' and S3' subsites, respectively, were identified using the "Methyl Scan" method [Slon-Usakiewicz et al. (1997) Biochemistry 36, 13494-13502]. A series of inhibitors (4-tert-butylbenzenesulfonyl)-Arg-(D-pipecolic acid)-Xaa-Gly-Yaa-Gly-betaAla-Asp-Tyr-Glu-Pro-Ile-Pro-Glu-Glu-Ala- (be ta-cyclohexylalanine)-(D-Glu)-OH, in which nonpolar P1' residue Xaa or P3' residue Yaa was incorporated, were designed and improved the affinity to thrombin. Substitution of the P3' residue with D-phenylglycine or D-Phe improved the K(i) value to (9.5 +/- 0.6) x 10(-14) or 1.3 +/- 0.5 x 10(-13) M, respectively, compared to that of a reference inhibitor with Gly residues at Xaa and Yaa residues (K(i) = (2.4 +/- 0.5) x 10(-11) M). Similarly, substitution of the P1' residue with L-norleucine or L-beta-(2-thienyl)alanine lowered the K(i) values to (8.2 +/- 0.6) x 10(-14) or (5.1 +/- 0.4) x 10(-14) M, respectively. The linker Gly-Gly-Gly-betaAla of the inhibitors in the previous sentence was simplified with 12-aminododecanoic acid, resulting in further improvement of the K(i) values to (3.8 +/- 0.6) x 10(-14) or (1.7 +/- 0.4) x 10(-14) M, respectively. These K(i) values are equivalent to that of natural hirudin (2.2 x 10(-14) M), yet the size of the synthetic inhibitors (2 kD) is only one-third that of hirudin (7 kD). Two inhibitors, with L-norleucine or L-beta-(2-thienyl)alanine at the P1' residue and the improved linker of 12-aminododecanoic acid, were crystallized in complex with human alpha-thrombin. The crystal structures of these complexes were solved and refined to 2.1 A resolution. The Lys(60F) side chain of thrombin moved significantly and formed a large nonpolar S1' subsite to accommodate the bulky P1' residue.     

Purification and characterization of serine proteinase inhibitors form gourd (Lagenaria leucantha Rusby var. Gourda Makino) seeds.

Gourd seed inhibitors were purified in the following manner: gourd seeds were ground and extracted with 10 mM ammonium carbonate, pH 7.8. The extract was precipitated with 65-90% acetone and the acetone precipitates were gel filtered in a Cellulofine GCL-90-m column. Fractions of 3000 Da showing trypsin inhibitory activity were combined and purified further by ion exchange and reversed phase chromatographies. Three inhibitors, LLTI-I, II, and III were thus purified to homogeneity and the amino acid sequences of these inhibitors were: [sequence: see text] The exact sequences are unique but very similar to proteinase inhibitors belonging to the squash family. Based on the sequence, it is assumed that the peptide bond (Arg-Ile) found in the three inhibitors is the reactive site for trypsin. The Ki values estimated for complexes of LLTI-I, II, and III with bovine trypsin were 3.6 x 10(-10) M, 6.5 x 10(-11) M, and 3.0 x 10(-11) M, respectively.     

Polypeptide serine proteinase inhibitors isolated from venom from several reptilian species

The survey encompasses literature data on the polypeptide inhibitors of some reptiles serine proteinases and their separation from adder Viperidae and cobra Elapidae species. The evolutionary comparison of physico-chemical and biological properties of them are also given and discussed within this work. Considerable homology (about 50%) in amino acid composition of adder, bee, mammal and others of different phylogenetic origin is being emphasized and high homology in structure of their functionally important inhibitors sites is observed. In the most cases the investigated peptide inhibitors of adder and cobra were observed to have an extremely high antitryptic activity with Ki ranging from 7.6 x 10(-10) M to 3.5 x 10(-12) M. The majority of polypeptide inhibitors are suggested by Laskowsky et al to interact with the proteinases in a standard way. The biological reactivity of the above preparations is a result of arginine and lysine presence in the substrate-binding sites of P2' and P3' or P4' centres.     

Effect of inhibitors on the viability and protein and nucleic acid synthesis of Escherichia coli

The object of this work was to study how the synthesis of protein, RNA and DNA in Escherichia coli M17 and its viability were influenced by chloramphenicol (50 and 300 micrograms/ml) an inhibitor of protein biosynthesis, and sodium azide (200 and 2000 microM) and aminazine (50 micrograms/ml), inhibitors of respiration. The exposed were inhibitors with the bacteria for 60 min at room temperature and for 1-4 months at -10 degrees C. The inhibition of the E. coli viability by chloramphenicol was shown to be reversible. The respiration inhibitors stabilized its viability upon storage at -10 degrees C for one month. The inhibitors were found to produce a different effect on the synthesis of RNA and protein in E. coli. The rates of DNA synthesis hardly changed. No correlation was established between changes in the synthesis of protein and nucleic acids by E. coli after the action of the inhibitors and its viability.