The paper deals with the role of metals in the catalytic action of Asp. oryzae aminopeptidase. Cobalt ions are more specific activators than Mn2+ and Mn2+ and evoke its maximal activity. Sinergic activation of Co2+ in combination with Mn2+ and Mg2+ was not found in contrast to some aminopeptidases of animal origin. Activation of the enzyme with cobalt chloride depends on temperature. By the 10th minute of incubation the activation reaches its maximum: 650 and 900% at 20 and 40 degrees C, respectively. EDTA (10(-2) M) inactivates completely aminopeptidase for 3h; this process is intensified in the presence of leucine (10(-3) M). Aminopeptidase is also inactivated by chelating agents, such as o-phenantroline and 2,2'-dipyridyl. When the temperature rises from 20 to 40 degrees C the intensification of these reagents effect is insignificant. Cobalt ions reduce and activate to some extent the enzyme after inhibition with EDTA.

2,2'-Dipyridyl↗

PubMedDate not supplied

[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.

Amino Acid Sequence↗

PubMedDate not supplied

[Binding of Kunitz-Northrop inhibitor on some sorbents].

This work is devoted to the problem of sorption and desorption of Kunitz--Northrop inhibitor on different sorbents. By passing through Dowex 1.10 column 0.05 M glycine-NaOH buffer, pH 10, two fractions with 100% inhibitor activity were identified, while different admixtures and inert proteins remained resin-bound. In trypsin-Spheron 300, trypsin-agarose and anhydrochymotrypsin-Spheron columns the contamination of sorbents-bound inhibitor complex was eliminated by washing out with 0.1 M NaCl, pH 8.0. The resin-bound inhibitor was released at pH 1. The specific activity of the preparation obtained was shown to increase in 200-240-fold, but in the case of anhydrochymotrypsin-Spheron 300 the inhibitors activity was detected both at pH 8.0 and pH 1.7. In this case the increase in specific activity was only 2 and 68-fold, respectively. The most effective inhibitor-peptide desalting was defined at application of dialysis membranes "Spectra/Por" (MWCo: 3000-5000 USA). While applying PAAG electrophoresis the standard SDS-system were shown to be ineffective. Therefore some modifications of this method were used. Being compared with molecular weight of Contrycal and other known peptides this preparation revealed the presence of protein contamination. Within 1-9 mg the peptides demonstrated a linear dependence in trypsin inhibition. The weight and molar ration of inhibitor: trypsin was found to be 1:1 and 3:1, respectively. It was calculated that IUE of the inhibitor had inhibited 0.73 mg of trypsin, IUE of Contrycal--0.15 of the enzyme, that was 4.6 fold less effective than the separated peptide.

Animals↗

PubMedDate not supplied

[Human thrombin: enzymatic properties, stability and standardization of preparation].

The work deals with estimation of thrombin preparation having such features as: sedimentation activity 3000-3200 NIH un. per 1 mg of protein and 97% of active centres. The enzyme isolated has been estimated according to the amidolytic activity on synthetic substrates S-2160 and BAPNA being equal 5200 and 185 milli un/mg of protein, respectively. According to the electrophoresis in PAAG in the presence of Ds-Na the preparation is homogenous, its molecular mass is 36000. The fibrinogen sedimentation time dependence on the isolated thrombin concentration has been estimated as well as the comparative analysis with the thrombin of the firm "Sigma" with the previously calibrated activity using the international standartion (coded P4) has been conducted. The absence of proportionality between the substrate sedimentation time and the preparation concentration has been determined. It has been revealed, that if the experimental findings are presented in the units 1/t against the thrombin units NIH the right lines are received within the limits used. The defreezing and secondary freezing of the preparation preserved under -20 degrees C have been showed as rendering an essential effect on thrombin activity. In order of the enzyme stabilizing at preserving the thrombin isolated has been concentrated applying the amycon membranes (MWCo: 30,000). While applying the thrombin water-saline solution in the conditions selected the preparation has showed itself practically stable during a year without utilizing any admixtures. The essential effect on thrombin has been found from the side of 1% glycin, 0.5% PEG, 1% saccharose and so on. The thrombin isolated high functional homogeneity, its stability permit to recommend the preparation as an operative standard.

Binding Sites↗

PubMedDate not supplied

[Hydrophobic interactions of serine proteases with low molecular weight compounds: partial effector displacement from the activated enzyme by the substrate].

1,2,3-tris-tri-beta-phenylethylaminopropane (a compound which can simultaneously interact with S1- and S2'-enzyme sites and has complicated grouping in the region analogous to "leaving group" of a substrate) has been studied for the effect on chymotriptic hydrolysis. Anomalously high inhibitory effect of some cumbersome organic compounds is explained on the base of the obtained data.

Binding Sites↗

PubMedDate not supplied

[Interrelation between thrombin structure and its stability].

Data concerning peculiarities of fermentative nature and structure of thrombin in water-salt solution have been generalized; regularities of stabilizing effect made on thrombin by various polyols and other substances have been analyzed. It has been shown that formation of thrombin optimum macrostructure is one of the methods of its stabilization. Presence of different dissolving additives changes this enzymes hydration and this affects its stability and activity. There exist some systems to stabilize thrombin solutions. The systems consist of various salts, low-molecular and high-molecular polyols, surfactants, protein chain, composition buffer, etc. It has been shown that optimal concentrations of polyols, buffer salts and surfactants, as well as protein interaction increase considerably thrombin stability, preserving secondary structure even under its low concentration in the solution.

Animals↗

PubMedDate not supplied

[Interaction of human alpha-thrombin with organic ligands of ionic nature].

Investigations results of human thrombin interaction with organic ligands of ion nature containing nonpolar groups are presented. It is shown that electrostatic interaction is the basic one under enzyme binding, while hydrophobic binding is only additional function in the reaction enzyme-ligand, this fact is confirmed by the absence of interaction between thrombin and rivanol which has a positive charge side by side with cumbrous hydrophobic group. New data are presented about the ligand specificity of binding sites of thrombin active centre. The importance of relative arrangement of hydrophobic ligand groups for interaction with enzyme is shown. It is supposed that thrombin binding with organic ligands occurs owing anionic site of beta-domain of active thrombin centre with the major aminoacids arginine and lysine (Lys 68, Arg 78, Arg 77, Arg 66 etc.). It is shown that the compounds containing negative group SO3 and have some cunbours hydrophobic groups interact more intensively with the enzyme. Thus, rosseline--with symmetrical hydrophobic nucleus (four benzene rings)--is the most efficient ligand for the binding with thrombin. The obtained investigation results evidence for bacteriostatical and stabilizing effect of low-molecular asobenzene ligands on rather labile thrombin molecules.

Binding Sites↗

PubMedDate not supplied

[Interrelation between thrombin structure and its stability].

Temperature inactivation of human thrombin has been studied when finding out the mechanism of this enzyme stabilization by amino acids. Effect of a number of amino acids on thrombin in the conditions (pH) of the highest activity of proteinase has been investigated. It is established that most amino acids are characterized to more or less extent by the protective action, when hampering the temperature inactivation of the enzyme. The correspondence was mainly found between the stabilizing effect of amino acids and thrombin specificity. Thrombin is stabilized by L-arginine and DL-lysine more intensively than by other amino acids. A stabilizing effect of L-glutamic acid was shown in contrast to the action of the latter on trypsin that was obviously connected with the original structure of the active centre of thrombin, that is the availability of anionic binding centre which includes Lys68, Arg72, Arg77. High thrombin stabilization by such amino acids as phenylalanine, DL-serine, DL-methonine was an exception. It was established that amino acids stabilize thrombin with formation of a compound with the reactive centre of its molecule, like the compounds enzyme-substrate. The macrostructure stability probably depends, to a considerable extent, on the state of the enzyme reactive centre: thrombin molecules, which contain a free reactive centre, are more labile than those which reactive centre is bound to the reagent of more or less specific character. The inhibition of the autolysis process may be another manifestation of thrombin stabilization by amino acids.

Amino Acids↗

PubMedDate not supplied

[Hydrophobic interactions of serine proteases with low molecular compounds: role of the S'2-site in substrate activation and interaction with serpines].

An attempt is made to simulate the P1-P'2 site of the reactive centre of protein inhibitors of serine proteases (serpines). On the basis of data from literature structure requirements are formulated and compound 1,5 bis-dibenzyl-aminopentane is synthesized. It may simultaneously interact with S1- and S'2-sites of chymotrypsin and contains no bonds adequate to the hydrolytic centre of proteinase. The compound is studied for its effect on hydrolysis of low-molecular substrates and proteins by chymotrypsin. Results obtained are discussed as well as the possible role of the S'2-binding site in the substrate activation of serine proteinases and their interaction with serpines.

Binding Sites↗

PubMedDate not supplied

[Enzyme activity and lipids of a preparation from the pyloric ceca of salmon].

Proteolytic activity and lipid composition of enzymic preparations of water extract from the pyloric caeca of salmon fishes have been studied. Phospholipids and sterols are tightly bound with proteins. The participation of lipids in the proteolytic activity is discussed.

Amino Acids↗

PubMedDate not supplied

[Comparative study of the properties of serine proteases of lower and higher vertebrates].

Results of the comparative study of trypsin- and chymotrypsin-like serine proteases from pyloric caeca of salmon fishes and trypsin and chymotrypsin of bulls are presented in the paper. The hydrolytic activity of salmon proteases with respect to methyl ethers of N-benzoyl-L-leucine is 2.4 times higher than that of bull chymotrypsin, but with respect to methyl esters of N-benzoyl-L-tyrosine and N-benzoyl-L-arginine the activity of salmon proteases is 6.5 and 80 times lower than that of bull chymotrypsin and trypsin. Salmon proteases in contrast to bull trypsin and chymotrypsin hydrolyze but slightly N-glutaryl-L-phenylalanine para-nitroanilide. It shown that fish proteases are not absolutely specific to synthetic substrates, which is a result of their less pronounced (than in case of bull trypsin and chymotrypsin) differences in structures of binding centres. The study of the salmon protease interaction with some immobilized ligands has confirmed the higher affinity of enzymes to reagents with two space-separated aromatic rings in their composition. It is supposed that salmon proteases interact with such reagents through two sites: hydrophobic "pockets" and probably additional binding site of the active centre. The salmon protease preparation demonstrates higher resistance to inactivating action of formaldehyde within the range of concentrations 2-16% than bull chymotrypsin does.

Animals↗