[Effects of biogenic phosphates on protease-induced protein cleavage and functioning of plasminogen activators].

We showed, using the method of lysis of fibrin plates and five substrate proteins in a thin layer of agar gel, that inorganic orthophosphate (0.001-0.06 M) enhances by 50-250% the activatory functions of streptokinase, urokinase, and tissue plasminogen activator and, in general, by 1.2-12.0 times enhances protein lysis by trypsin, alpha-chymotrypsin, subtilisin, papain, bacterial metalloprotease, and even pepsin at a concentration < 4 mM. At higher concentrations, phosphate sharply inhibited pepsin activity and inhibited by 40-50% gelatin lysis by papain and gelatin (at a peak concentration) and casein lysis by metalloprotease. Inorganic pyrophosphate ions at concentrations of 10(-8)-10(-1) M enhanced the cleavage of a number of proteins by serine proteases and, at concentrations of 10(-5) -10(-3) M, the activities of pepsin, plasminogen tissue activator, and streptokinase by 100 and 40%, respectively. The pyrophosphate concentrations of > 10(-3) and >10(-4) M inhibited pepsin- and metalloprotease-induced lysis of virtually all proteins. ATP increased casein lysis by serine proteases, metalloprotease, and pepsin by 20-60% at concentration of 10(-3) M and by 30-260% at 10(-2) M concentration. At concentrations of 10-2 M, it inhibited the cleavage of some proteins by trypsin, chymotrypsin, papain, and metalloprotease by 20-100%, and, at concentrations of 10(-3) M, lysis of albumin with pepsin and other proteins (except for fibrinogen) by metalloprotease. A GTP concentration of 10(-7)-10(-2) M increased protein degradation by serine proteases, papain, and gelatin lysis by pepsin by 20-90%, whereas albumin lysis was inhibited by 40-70%. The presence of 10(-6)-10(-5) M GTP led to a slightly increased degradation of hemoglobin and casein by bacterial metalloprotease, while 10(-3) M GTP induced a drop in the activity of the metalloprotease by 20-50%. ADP could enhance gelatin lysis by trypsin, casein lysis by pepsin and papain, and inhibited metalloprotease activity by 20-100% (at 10(-3) M). Peculiarities of the effects of AMP and GD(M)P on gelatin lysis were found.

[Synthesis of modified fragments of fibrinogen and their effect on the activity of proteolytic enzymes].

New analogues of the Gly-Pro-Arg and Arg-Gly-Asp fragments of fibrinogen were synthesized: Gly-Pro-Arg-Pro (I), Gly-Pro-Arg-Pro-Met-OMe (II), Gly-Pro-Arg-Pro-Phe (III), Gly-Pro-Arg-Pro-Asp (IV), Gly-Pro-Arg-Pro-Glu (V), and Arg-Asn-Trp-Asp (VI). Their effect on the activity of proteases of various types was studied with the method of lysis of fibrin plates. All the peptides were found to inhibit plasmin activity (by 60-85%) and the gamma-subunit of nerve growth factor (by 55-93%). Tetrapeptide (VI) proved to be an effective inhibitor of tissue activator of plasminogen and the gamma-subunit of nerve growth factor (by 96 and 93%, respectively). The peptides exerted practically no effect on the activity of urokinase and moderately inhibited the activity of streptokinase [(III), (IV), and (VI)], papain [(I), (II), (IV), and (VI)], subtilisin [(V) and (VI)], alpha-chymotrypsin [(III), (V), and VI)], and Bacillus subtilis metalloprotease (VI). They inhibit trypsin [except for (I) and (III)] when applied on fibrin plates at a concentration of 1 x 10(-2) M, while, at a concentration of 1 x 10(-3) M, (I) and (II) induced an increase in proteolytic activity by 35 and 47%, respectively.

Some unusual manifestations of proteolysis.

In the article the results of the long-term researches revealing the essence of the following three phenomena are generalized. 1. Participation of active oxygen species (especially, superoxide radical) in activation of zymogens--plasminogen, trypsinogen, chymotrypsinogen, pepsinogen and in realization of the catalytic (proteolytic) function of a number of proteinases. The essence of the hypothesis of oxygen-dependent reactions of proteolysis is stated. As shown by the example of mouse brain homogenate fractions, the plasminogen-activating ability of the fractions can also be realized via this way. From the positions of these views the experimental facts obtained about the influence of streptokinase and plasminogen on vital activity of nervous tissue cells are analysed. 2. Suppression of proteolytic reactions by ATP: plasminogen-activating ability of streptokinase, gamma- and betasubunits of the nerve growth factor, proteolytic activity of Arthrobothrys longa proteinases, destroyed cells of Corynebacterium diphtheriae PW-8. In some cases a significant effect was reached at concentration of ATP < or = 0.001 M. The effect depends on protein substrates used. 3. The increase of fibrinolytic activity of the mitochondrial fraction of the mouse brain and liver, proteolytic activity of human lymphoblasts of transplanted lines in the presence of inorganic orthophosphate. Judging by the results of inhibitory analysis, it is not caused by the resynthesis of ATP in the system and has an independent character--"phosphate effect" in proteolysis. 4. The results of our researches of formation of stable equimolar complexes of streptokinase or plasminogen with enzymes of carbohydrate-energetic metabolism are briefly analysed. The results of researches of functional properties of the molecules of diphtheria toxin, the nerve growth factor and it subunits are summarized. A number of fundamental and applied consequences of these phenomena are considered.

[A complex approach to the diagnosis of HIV-1 infection: the use of the polymerase chain reaction as an alternative confirmatory test]. / Kompleksnyi podkhod k diagnostike VICh-1-infektsii: ispol'zovanie polimeraznoi tsepnoi reaktsii v kachestve al'ternativnogo podtverzhdaiushchego testa.

The results of the three-year observation of female patient St. in whose blood antibodies to HIV disappeared in the course of time and the results of the laboratory study of the blood sera of children born of HIV-infected mothers are presented. In these patients correct diagnosis could be made only with the use of a complex of virological and molecular-biological methods.

Effect of active oxygen species scavengers on fibrinolytic activity of some proteinases.

Scavengers of different active oxygen species affect fibrin plate lysis, catalysed by various proteinases, only at relatively high concentrations (> 10(-2) M). Singlet oxygen scavengers change proteinase activity insignificantly except for strong inhibition of pepsin and papain by sodium azide, but pepsin-by histidine, and fibrinolytic urokinase activity-by all used O2 delta 1 scavengers. Of all used scavengers of OH-radical only ethanol caused significant changes in the proteinases under study, except for alpha-chymotrypsin. The most strong inhibitory effect on proteinase activity was demonstrated by scavengers of superoxide radical. Thus, nitrotetrazolium blue strongly inhibited the activity of plasmin, urokinase (fibrinolytic activity), papain and pepsin. Catalase changed proteinase activity insignificantly, though it leads to total inhibition of pepsin activity at final 4.5 x 10(-4) M concentration. These facts and our previous findings on generating of active oxygen species by proteinases give us grounds to suppose that minor active oxygen species, endogenous for the "proteinase-substrate" system, can participate in the catalytic function of some proteinases.

[Recombinant monoclonal antibodies to the Lassa virus: the formation of reactive paratopes]. / Rekombinantnye monoklonal'nye antitela k virusu Lassa: formirovanie reaktivnykh paratopov.

Eighteen hybrid lines secreting recombinant monoclonal antibodies to Lassa virus were produced by fusion of mouse splenocytes with antibody-secreting X-63 myeloma cells. Interrelations between the structure and reactivity of the antibodies were studied by different serological and immunochemical methods. Monoclonal antibodies were divided into different groups according to their serological properties and macromolecular structure. A comparative analysis of the structure and reactivity of the recombinant monoclonal antibodies showed that the light and heavy Ig-specific chains could form the reactive antibodies when the chains were present in different paratopes of Ig molecules.

Activation of human plasminogen by streptokinase: new aspects of fibrinolysis regulation.

It is established fact, that SK-initiated fibrinolysis is decelerated, when oxygen is removed from solutions; that SK possesses superoxide dismutase-like activity and that its activating function is sharply inhibited by superoxide radical scavengers. The point in discussion is the possibility of oxygen-dependent Pg activation, which is not related to functions of serine proteinase-like activators. The realization of oxygen-radical mechanisms is supposed to depend on activation of zymogens of other serine proteinases and proteinase reactions effect.

[Effect of adenine nucleotides on the activator function of streptokinase]. / Vliianie adenilovykh nukleotidov na aktivatornuiu funktsiiu streptokinazy.

The addition of ATP or 3,5-AMP (but not UTP, GTP, CTP, AMP, 2,3-AMP, ADP, inorganic pyrophosphate) at a final concentration of 10(-1) M into streptokinase solution, pH 7.0 or 9.5, causes a dramatic inhibition of streptokinase-induced fibrinolysis. The specificity of ATP effect is fully lost at pH 3.0, when all nucleotides completely inhibit the activating function of streptokinase. Ribose-5-phosphate causes a similar effect at pH 3.0. The character of nucleotide action on the activating function of streptokinase considerably differs from their influence on proteolytic reactions.

[Effect of organic solvents on streptokinase-initiated fibrinolysis]. / Vliianie organicheskikh rastvoritelei na initsiiruemyi streptokinazoi fibrinoliz.

Proton-developing (ethanol, I-propanol, I-butanol, I-pentanol) and aproton solvents (dioxane, dimethylsulfoxide, dimethylformamide) at concentrations 1.5-50% activated the streptokinase-initiated fibrinolysis. Methanol and acetone were not effective. The rate of fibrinolysis, studied in presence of glycerol and phenol (used at 0.1-9.3% concentrations), depended on concentrations of these reagents in samples. Efficiency of aliphatic alcohols was increased with elongation of hydrocarbonic chain. The activating effect of ethanol was not removed by epsilon-aminocapronic acid 10(-2) M.