Crystals of phospholipase A2 inhibitor. The non-toxic component of vipoxin from the venom of Bulgarian viper (Vipera ammodytes).

Phospholipase A2 inhibitor, the non-toxic, acidic component of vipoxin from the venom of Bulgarian viper (Vipera ammodytes), has been crystallized. The tetragonal crystals obtained, exhibit the symmetry of space group P4(1)22 (or 4(3)22) with unit cell dimensions a = b = 59.9 A; c = 141.1 A; alpha = beta = gamma = 90 degrees. For two molecules per asymmetric unit, this would give Vm = 2.3 A3/Da, indicating normal packing. The crystals diffract to 2.5 A and a native data set to 3.2 A resolution has been collected.

Crystallization and preliminary X-ray analysis of vipoxin, a complex between a toxic phospholipase A2 and its natural polypeptide inhibitor.

The toxin vipoxin, which is a complex between a basic toxic phospholipase A2 and an acidic non-toxic protein inhibitor, is found in the venom of the Bulgarian viper (Vipera ammodytes ammodytes), the most toxic snake in Europe. The two polypeptide chains each consist of 122 residues and are highly homologous (62%). The vipoxin complex is the first reported example of a high degree of structural homology between an enzyme and its natural inhibitor. The present crystals diffract in the X-ray beam to 1.8 A resolution. The space group is P2(1)2(1)2(1). The cell dimensions are a = 45.80 A, b = 55.36 A and c = 107.69 A. Native data to a resolution of 2.8 A have been recorded.

Crystal structure of vipoxin at 2.0 A: an example of regulation of a toxic function generated by molecular evolution.

Vipoxin is the main toxic component in the venom of the Bulgarian snake Vipera ammodytes meridionalis, the most toxic snake in Europe. Vipoxin is a complex between a toxic phospholipase A2 (PLA2) and a non-toxic protein inhibitor. The structure is of genetic interest due to the high degree of sequence homology (62%) between the two functionally different components. The structure shows that the formation of the complex in vipoxin is significantly different to that seen in many known structures of phospholipases and contradicts the assumptions made in earlier studies. The modulation of PLA2 activity is of great pharmacological interest, and the present structure will be a model for structure-based drug design.

Spectroscopic investigation of calcium binding sites in the neurotoxin vipoxin and its components-relation with the X-ray structure.

Vipoxin is a neurotoxin from the venom of Vipera ammodytes meridionalis, the most toxic snake in Europe. It is a unique complex of a toxic phospholipase A2 (PLA2) and a non-toxic PLA2-like protein inhibitor (Inh) which probably evolved from the enzyme and reduces its activity and toxicity. The enzymatic activity of Vipoxin is Ca2+-dependent and the interaction of this metal ion with the neurotoxic complex and its separated components was investigated using the fluorescent probe ANS. Vipoxin binds two calcium ions, one per each subunit. The X-ray model of the Ca2+-free neurotoxin shows that the potential metal-binding sites require minor structural changes to bind calcium. The dissociation constants K(2+)Ca of the calcium complexes of Vipoxin and its components, PLA2 and Inh, were determined to be 16, 10 and 9 mM, respectively. The affinity for calcium of Vipoxin is reduced in comparison to those of PLA2 and Inh. The X-ray model shows that the potential Ca2+-binding sites in the two components are partially 'shielded' in the complex. The affinity of the neurotoxin to Sr2+ and Ba2+ is lower and the respective K(2+)Ca are 20 and 30 mM. The saturation of Ca2+-binding sites increased the melting point Tm of Vipoxin by 11 degrees C and the activation energy for the thermal deactivation of the excited tryptophans Ea by 11 kJ mol(-1) x Ca2+ is important not only for the enzymatic activity of Vipoxin but also for its thermostability.

Spectroscopic investigation of phenolic groups ionization in the vipoxin neurotoxic phospholipase A2: comparison with the X-ray structure in the region of the tyrosyl residues.

The neurotoxin vipoxin is the major lethal component of the venom of Vipera ammodites meridionalis, the most toxic snake in Europe. It is a complex between a toxic phospholipase A2 (PLA2) and a non-toxic protein inhibitor (Inh). Tyrosyl residues are involved in the catalytic site (Tyr 52 and 73) and in the substrate binding (Tyr 22). Spectroscopic studies demonstrated differences in the ionization behavior of the various phenolic hydroxyl groups in the toxic PLA2. The tyrosyl side chains of the enzyme can be classified into three groups: (a) three phenolic hydroxyls are accessible to the solvent and titrate normally, with a pKeff = 10.45; (b) three residues are partially 'buried' and participate in hydrogen bonds with neighboring functional groups. They titrate anomalously with a pKeff = 12.17; (c) two tyrosines with a pKeff = 13.23 are deeply 'buried' in the hydrophobic interior of PLA2. They became accessible to the titrating agent only after alkaline denaturation of the protein molecule. The spectroscopic data are related to the X-ray structure of the vipoxin PLA2. The refined model was investigated in the region of the tyrosyl side chains. The accessible surface area of each tyrosyl residue and each phenolic hydroxyl group was calculated. A good correlation between the spectrophotometric and the crystallographic data was observed. The ionization behavior of the phenolic groups is explained by peculiarities of the protein three-dimensional structure and the participation of tyrosines in the catalytic site hydrogen bond network. Attempts are made to assign the calculated pKeff values to individual residues. The high degree of 'exposure' on the protein surface of Tyr 22 and 75 is probably important for their function as parts of the substrate binding and pharmacological sites.

Spectroscopic properties and stability of the neurotoxic complex. Vipoxin and its components.

The neurotoxin Vipoxin from the venom of Vipera ammodytes meridionalis is a complex between a toxic basic phospholipase A2 (PLA2) and a non-toxic acidic protein inhibitor (Inh). Tryptophan fluorescence parameters are determined for the complex and for its components. Iodide, caesium and acrylamide are not efficient quenchers of the Vipoxin indole emission. Increased accessibilities of tryptophans to ionic and neutral quenchers are found after the dissociation of the complex. Trp 20 and Trp 31 became more 'exposed' in the separated individuals proteins. The indole rings of the complex are located in a positively charged environment. Inspection of the Vipoxin X-ray model showed that the three tryptophyl side chains are located in the interface region between the enzyme and the inhibitor and are completely 'exposed' in the separated components of the complex. In Vipoxin an efficient 'interchain' energy transfer between tyrosyl and tryptophyl residues from different polypeptide chains occurs. Static quenching with acrylamide is also detected in PLA2 and Inh. The free energy changes deltaG D for the unfolding reactions of Vipoxin, PLA2 and Inh are determined in circular dichroism spectroscopy. The complex formation between the toxic PLA2 and the inhibitor increases deltaG HD2O to 23.5 kJ mol-1.

[The antibacterial activity of some substituted cysteine sulphonamides and peptides containing cysteine sulphonamide]. / Antibakterielle Aktivität einiger substituierter Cysteinsulfonamide und cysteinsulfonamidhaltiger Peptide.

The authors investigated the antibacterial activity of some cysteine sulphonamides substituted in the sulphonamide group as well as that of some peptides containing cysteine sulphonamide. They found that the antibacterial activity of these compounds was in part considerably stronger than that of cysteine sulphonamide.

[Synthesis and in vivo antitumor effect of N,N-di(2-chlorethyl) hydrazides of natural alpha-aminocarboxylic acids]. / Synthese und geschwulsthemmende Wirkung in vivo von N,N-Di(2-chlorethyl)hydraziden natürlicher alpha-Aminocarbonsäuren.

N,N-Di(2-chlorethyl)hydrazides of the following alpha-amino-carboxylic acids were synthesized: glycine, valine, norvaline, lysine, phenylalanine, tyrosine, cystine, homocystine, aspartic and glutamic acid as well as the N,N-diethylhydrazides of glycine, phenylalanine and cystine. The N,N-di-(2-chlorethyl)hydrazides have a pronounced effect on solid tumours (tumour growth inhibition by 30-100%), whereas their inhibition activity with ascite tumours is negligible. N,N-diethylhydrazides show analogous but less expressed biological effect.

[Synthesis and antitumor action of N,N-di(2-chlorethyl)-hydrazines of alpha-aminocarboxylic acid antimetabolites]. / Synthese und geschwulsthemmende Wirkung von N,N-Di(2-chlorethyl)-hydrazinen der alpha-Aminocarbonsäureantimetabolite.

The N,N-di(2-chlorethyl)hydrazides of the following alpha-aminocarboxylic acid antimetabolites: methioninsulphoxide, ethionine, 2-, 3- and 4-fluorophenylalanine, 4-nitrophenylalanine and 2,2-dimethyl-thiazolidine-4-carboxylic acid were synthesized. Preliminary studies of the activity on experimental tumour models were carried out. It was shown that these compounds have a high antitumour effect (80-100%) on sarcoma Yoshida and carcinosarcoma Walker.

[Combined ultrastructural and immunocytochemical studies of pancreatic endocrine tumors]. / Kompleksnye ul'trastrukturnye i immunotsitokhimicheskie issledovaniia éndokrinnykh opukholei podzheludochnoi zhelezy.

9 surgically removed endocrine tumours of pancreas of female patients aged from 19 to 71 years were studied. The material was stained with hematoxylin and eosin, by Grimelius and Fontana--Masson; PAP method with polyclonal antibodies to insulin, glucagon and somatostatin was also used. Immunoreactivity was weaker in tumours with clinically pronounced hormonal activity as compared to the normal endocrine cells in the Langerhans islets. This is confirmed ultrastructurally: tumour cells contain comparatively low number of neuroendocrine granules. Functionally inactive tumours show the opposite immunohistochemical results. The multipotential properties of cells forming pancreatic endocrine tumours are discussed.