Initial selectivity of the antiplatelet covalent action of biogenic chloramines on platelet-rich plasma

To describe in full the peculiarities of the antiplatelet action of covalent inhibitors on platelet-rich plasma, we have proposed to take into account the initial selectivity that determines the elevated efficacy of inactivation of platelet molecular target (receptor). The quantitative index of initial selectivity is the ratio of rate constant of inactivation of the platelet molecular target to the rate constant of the chemical reaction of an inhibitor with reactive atomic groups in plasma proteins. For the important case of the domination of the inhibitor expenditure in the reaction with plasma proteins, a formula was derived which depicts the dependence of the share of inactivated targets on the concentration of the inhibitor introduced and reactive atomic groups contained in plasma. In the case of chloramine derivatives of amino acids, evidence was obtained indicating that the degree of inhibition of platelet aggregation measured by the turbidimetric method is equal to the square of the share of inactivated receptors. The index of initial selectivity can be evaluated by measuring the degree of inhibition of platelet aggregation and the operating concentration of the inhibitor. According to experimental evidence, the effects of a number of chloramine derivatives of amino acids (biochloramines) on aggregation of platelets stimulated by ADP show selectivity at the molecular target level, so that the index of initial selectivity is greater than 1. The mechanism of the selective action of the biochloramines having significant molecular masses (150-200 Da) probably consists in the inactivation of the molecular target via chemical modification of several reactive atomic groups in its different sites. One may suppose that the biochloramines with lower molecular masses (150-100 Da) exhibit a high anti-aggregatory capacity owing to another mechanism of initial selectivity, which involves the modification of highly sensitive sulfur-containing atomic groups.     

Theory and experimental method for determining individual kinetic constants of fast-acting, irreversible proteinase inhibitors

A theory and experimental method are presented to characterize the kinetics of fast-acting, irreversible proteinase inhibitors. The theory is based upon formal analysis of the case of an irreversible inhibitor competing with a substrate for the active-site of a proteinase. From this theory, an experimental method is described by which the individual microscopic kinetic constants for the interaction of the inhibitor with the proteinase can be determined. These are, for a two-step inhibition reaction sequence, the equilibrium dissociation constant and the first-order rate constant for inhibition, and, for a one-step inhibition reaction sequence, the second-order rate constant for inhibition. The theory and experimental method were validated by an analysis of the inhibition of trypsin by the two-step synthetic inhibitor p-nitrophenyl p-guanidinobenzoate and the one-step protein inhibitor bovine pancreatic trypsin inhibitor. The substrate used in these experiments is a new, fluorogenic substrate for trypsin-like serine proteinases (Cbz-Ile-Pro-Arg-NH)2-Rhodamine, the synthesis and properties of which are described.     

Presence of a renin inhibitor in the plasma of intact dogs]

The authors elaborated a method of determination of the plasma renin inhibitor based on the statement that with successive dilution of the plasma the velocity of the renin + substrate reaction in the presence of an inhibitor fell more slowly than the extent of dilution, and, on the contrary, in the plasma without any inhibitor the rate of the reaction decreased more than the value of the plasma dilution. This statement follows from the equations of the reaction suggested by Dixon and Webb (1966). With the aid of this method the presence of the renin inhibitor in the plasma was found in 8 of 19 intact dogs examined.     

Analysis of lymphocyte adhesion and the phenomenon of immunologic adhesion inhibition

Investigations were performed in rabbits to elucidate the cell physiological parameters of the inhibitor reaction of adhesion. For this purpose, an improved method of measuring the cell adhesion was used. The well-known fact that culture cells will reveal a much stronger reaction could be confirmed. This may be due to the high increase of T-lymphocytes because of findings concerning cell differentiation with acid alpha-naphthylacetate esterase (ANAE), whereas no corresponding results could be found for the particular role of macrophages. It could be proved that the inhibitor reaction already began 20 minutes after the antigen contact. On the basis of these facts beta-lymphotoxin might be assumed to play a part in the inhibitor reaction. A direct relationship between oxygen consumption and the inhibitor reaction of adhesion could not be proved. The respiratory depression frequently observed might be due to the cytotoxic effect of excreted lymphotoxins. As the inhibitor reaction of adhesion could be identified even after 1-1 1/2 years, it can be regarded as a longterm immunophenomenon. The findings with metabolic blockers underline that the primary adhesion of lymphocytes is supported by the oxidative metabolism and not by glycolysis which is generally characteristic of granulocytes.     

Scoparone inhibits ultraviolet radiation-induced lipid peroxidation

Antioxidant capabilities of scoparone, the component of Artemisia scoparia and other medicinal plants, against lipid peroxidation induced by ultraviolet radiation or Fenton reaction have been analyzed. Lipid peroxidation was monitored by measuring the absorption spectra of the conjugated dienes and quantified by the Klein oxidation index. Obtained results imply that scoparone is a very efficient inhibitor of ultraviolet radiation-induced lipid peroxidation and damage.     

Reversible cross-linking of alpha 2-plasmin inhibitor to fibrinogen by fibrin-stabilizing factor

alpha 2-Plasmin inhibitor, a primary inhibitor of fibrinolysis, is cross-linked to fibrin by plasma transglutaminase (glutaminyl-peptide:amine gamma-glutamyltransferase, EC 2.3.2.13, activated fibrin-stabilizing factor) when blood coagulation takes place. alpha 2-Plasmin inhibitor was found also to be cross-linked to fibrinogen by plasma transglutaminase. The inhibitor was corss-linked exclusively to the A alpha-chain of fibrinogen, and the cross-linking reaction proceeded very rapidly. The reaction was almost completed before the formation of the gamma-chain dimers of fibrinogen which precedes cross-linking polymerization of the A alpha-chain of fibrinogen. The maximum level of inhibitor cross-linking achieved was approx. 30% of the inhibitor present at the start of the reaction. The level of cross-linking of the inhibitor was not changed when the cross-linking reaction was preceded by dimerization of fibrinogen. The cross-linking reaction was found to be a reversible one, since the cross-linked complex of the inhibitor and fibrinogen was partly dissociated to each of its components when the complex was incubated with plasma transglutaminase. These results suggest that the self-limiting nature of the cross-linking reaction between alpha 2-plasmin inhibitor and fibrin(ogen) is due to the reaction equilibrium favoring dissociation of the complex, and not due to the development of structural hindrance in polymerizing fibrin(ogen).     

pK changes of ionizable reporter groups as an index of conformational changes in proteins. A study of fluorescein-labelled ribonuclease A.

A chemical derivative of bovine pancreatic ribonuclease A (RNase A) has been prepared by reaction with fluorescein-isothiocyanate at pH 6. This derivative has a fluorescein group covalently attached to the alpha-amino group of the protein. The enzymic properties of the modified protein are similar to those of RNase A. It is shown that the pK of the fluorescein group can be used as an index of protein conformation to monitor structural changes in the protein. In this work, the binding of a specific inhibitor (cytidine 2'-monophosphate) to RNase A, the isomerization process occurring in RNase A around pH 6, and the thermal unfolding of RNase A, were studied by mean of the pK changes of the fluorescein group. The results obtained by this method are fully consistent with those obtained by other methods. It is proposed that using ionizable reporter groups and their changes in pK to monitor conformational changes in proteins may be a sensitive tool both in equilibrium and kinetic studies.     

One-step enzyme immunochromatographic assay for theophylline

The convenience of the previously described enzyme immunochromatography method for visually quantifying theophylline in whole blood has been improved with the development of a one-step protocol. The capillary migration and color generation in the two-step enzyme immunochromatographic assay have been combined into a single step. Ascorbic acid is used as a signal inhibitor to delay enzymatic color product formation until the inhibitor itself is consumed. The concept of internal delay reaction is presented and the mechanism of ascorbate's action as an inhibitor to temporarily delay color generation is described. The internal delay reaction has been applied to a practical one-step quantitative visual enzyme immunochromatographic assay for theophylline in whole blood.     

The method of Hill's coefficient calculation for the region of inhibition of an enzyme by excess of the substrate. Inhibition of phosphofructokinase by excess of ATP]

The new method for Hill's coefficient (nH) calculation in the region of substrate concentrations where the latter acts as an inhibitor has been developed. The method does not need preliminary determination of maximum value of enzyme reaction rate (V) for ascending branch of the plot of enzyme reaction rate versus substrate concentration and allows to avoid over-estimation of value of nH when the magnitude of optimal reaction rate is less than value of V. The literature data for inhibition of phosphofructokinase by excess of ATP are used for illustration of applicability of the suggested method of Hill's coefficient calculation.     

Soluble high molecular weight derivatives of trypsin pancreatic inhibitor. Isolation and properties of dextran-bound pancreatic inhibitor]

A method of isolating preparations of pancreatic inhibitor of trypsin, bound with soluble polysaccharide carriers, is worked out. It is demonstrated that the reaction of a pancreatic inhibitor and cyanuric chloride-activated dextran proceeds for OH groups of tyrosine residues and for-epsilon-NH2 groups of lysine residues. A method is offered of the protection of amino groups with citraconic anhydride for the complete retaining of the inhibitory activity during attachment to dextran. Thermic denaturation of pancreatic inhibitor preparations at pH 4.7 and 97 degrees C is studied. It is found that the modification by 2-amino-4.6-dichloro-s-triazine stabilizes the protein molecule, while the interaction with the matrix of soluble dextran does not carry any contribution to thermostability of the pancreatic inhibitor.     

Determination of UDP-glucuronosyltransferase UGT2B7 activity in human liver microsomes by ultra-performance liquid chromatography with MS detection

A rapid and specific ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS) method was developed for the qualitative and quantitative determination of UGT2B7 activity using 3'-azido-3'-deoxythymidine (AZT) as probe substrate in human liver microsomes (HLMs). The method was validated for the determination of AZT glucuronidation (AZTG) with respect to specificity, linearity, detection limit, recovery, stability, precision and accuracy. The chromatographic separation was achieved on a UPLC BEH C18 column (50 mm x 2.1mm i.d., 1.7 microm), with phase of acetonitrile-water (ratio 6:94). Selective ion reaction (SIR) monitor was specific for AZT, AZTG and I.S. The method was linear over the concentration range 0.5-500 microM for AZTG in spiked HLMs. Good precision and accuracy were obtained for concentrations over the standard curve range. AZTG was stable at 4 degrees C for at least 72 h in spiked liver microsomes samples. The method was successfully used to determine the kinetics of UGT activities toward AZT in HLMs. In addition, the method could determine the effects of fluconazole, a known UGT2B7 selective inhibitor, on AZTG in HLMs. Therefore, this method is suitable for in vitro studies using AZTG formation as an index reaction for UGT2B7 activity.     

The reaction of ribonuclease inhibitor with modified ribonucleases

1. The effect of chemical modification of ribonuclease on its reaction with ribonuclease inhibitor has been studied. 2. Removal of free amino groups from the enzyme with nitrous acid or by acetylation did not affect the reaction. Some changes altered the stoicheiometry of the reaction and ribonuclease S was found to be inhibited linearly by increasing amounts of ribonuclease inhibitor, in contrast with ribonuclease A, which is inhibited in a non-linear way. One derivative of ribonuclease containing dimethylaminonaphthalenesulphonyl groups actually reacted with ribonuclease inhibitor to a greater extent (and linearly) than did the unaltered enzyme. 3. The positively charged histidine at the active site and the active enzyme did not appear to be necessary for the reaction since 1-carboxymethylhistidine-119-ribonuclease reacted with ribonuclease inhibitor to almost the same extent as the native enzyme. In general, any significant change in the conformation of ribonuclease was accompanied by a loss in its ability to combine with inhibitor. The presence of 8m-urea also prevented reaction of ribonuclease with inhibitor. 4. Some characteristics of the reaction of ribonuclease inhibitor, ribonuclease and deaminated ribonuclease with RNA and deaminated RNA were investigated.     

The localization and properties of membrane adenosine triphosphatases in the guinea-pig placenta.

The distribution and properties of cytochemically demonstrable phosphatases in the near-term guinea-pig placenta were examined using a strontium capture technique for sodium- and potassium-dependent adenosine triphosphatase (Na+, K+-ATPase) and a lead capture technique for magnesium-dependent adenosine triphosphatase (Mg2+-ATPase). Localizations with the strontium technique in the presence of an alkaline phosphatase inhibitor were mainly on the syncytiotrophoblast plasma membranes; the reaction was potassium-dependent and ouabain-sensitive. Reaction product using the lead capture method was found on both trophoblast and endothelial cell plasma membranes and was independent of magnesium and insensitive to p-hydroxymercuribenzoate (POHMB), an inhibitor of membrane ATPases. However, a very large proportion of this reaction could be blocked by an alkaline phosphatase inhibitor. It is concluded that the strontium capture technique gave a reliable localization for Na+, K+-ATPase. However, the lead capture method mainly demonstrated alkaline phosphatase, and does not offer a useful approach to specific ATPase studies in this particular system.     

Estimation of kinetic parameters for substrate and inhibitor in a reaction with an enzyme sample containing different types of inhibitor

The method of kinetic analysis is developed to obtain the maximum velocity (V_m), the Michaelis constant (K_m) and the parameters characterizing the inhibitors in an impure enzyme reaction, contaminated with one of four types of inhibitor (competitive, noncompetitive, uncompetitive and mixed-type). Although the reaction rate decreases with the increasing concentration of the enzyme sample containing an inhibitor, the double-reciprocal plot of the rate against the sample concentration becomes linear. The slopes of these linear plots at several different concentrations of substrate provide K_m and the specific enzyme activity, which is proportional to V_m, in the sample. These linear straight lines intersect in a point, of which the coordinates give the unique parameters for the inhibitor. To prove the validity of this kinetic method, the model experiments were carried out with acetylcholinesterase and its inhibitors, phenyltrimethylammonium and trimethylammonium. The present method was applied to the measurement of the specific activity of galactosylceramide galactosidase in the mouse cerebral homogenate. In addition, a kinetic method is indicated for the inhibition of an enzymatic reaction by a contaminant which binds the substrate to reduce the fraction available to the enzyme.     

Kinetic evidence for a two-step mechanism for the binding of chymotrypsin to alpha 1-proteinase inhibitor.

We have used the proflavin displacement method and a stopped-flow apparatus to measure the rate constant for the binding of 2 microM-chymotrypsin to 20-125 microM-alpha 1-proteinase inhibitor. The observed pseudo-first-order constant showed a hyperbolic dependence on alpha 1-proteinase inhibitor concentration, suggesting a reaction mechanism in which a fast pre-equilibrium (K = 0.19 mM) is followed by a first-order formation of the final complex (k = 252 s-1).     

Kinetics of plasmin inhibition in the presence of a synthetic tripeptide substrate. The reaction with pancreatic trypsin inhibitor and two forms of alpha 2-plasmin inhibitor.

The progressive inhibition of plasmin by pancreatic trypsin inhibitor and by alpha 2-plasmin inhibitor in the presence of D-valyl-L-leucyl-L-lysine 4-nitroanilide was investigated. The kinetics with plasmin were compared with those with miniplasmin. The kinetic properties of two functionally different forms of alpha 2-plasmin inhibitor described by Clemmensen [(1979) in The Physiological Inhibitors of Coagulation and Fibrinolysis (Collen. D., Wiman, B & Verstraete, M., eds.), pp 131-136, Elsevier, Amsterdam] were characterized. The two forms differ in their plasminogen-binding capability, and this difference can account for a difference in secondary site interaction suggested from the kinetics. The binding of inhibitor to miniplasmin is a simple pseudo-first-order reaction with both pancreatic trypsin inhibitor and the two alpha 2-plasmin inhibitor forms. Such simple kinetics are also observed for the reaction between plasmin and the non-plasminogen-binding form of alpha 2-plasmin inhibitor. More complicated kinetics are obtained for the reaction between plasmin and the alpha 2-plasmin inhibitor form that binds to plasminogen. With both forms of the alpha 2-plasmin inhibitor, a complex stable to acetic acid/urea and gel electrophoresis is present and fully developed 15 s after initiation of the reaction with plasmin.     

Inhibitors to factor IX contain all IgG subclasses except IgG3.

Five per cent of patients with haemophilia B develop inhibitors to factor IX. It is of interest to know the immunoglobulin subclass of these IgG antibodies. We have developed a sensitive method for the characterization of the subclass nature of inhibitors to factor IX. The technique is a crossed immunoelectrophoresis for the isolation of factor IX-inhibitor complexes followed by an enzyme-linked immunoassay using monoclonal antibodies to IgG subclasses for the subclass identification. We studied seven inhibitors with both low and high titres. One patient was studied at a very early stage of inhibitor development. All inhibitors gave a strong reaction with antibody to IgG4. Depending on the titre of the inhibitor, a reaction was also found with antibodies to IgG1 and IgG2. No inhibitor contained any detectable IgG3. IgG4 does not bind complement and it is therefore of importance that IgG4 is the main subclass both in high-titred and in low-titred inhibitors. The inhibitors are polyclonal antibodies, also at an early stage of inhibitor development.     

A capillary electrophoresis method for the characterization of ecto-nucleoside triphosphate diphosphohydrolases (NTPDases) and the analysis of inhibitors by in-capillary enzymatic microreaction

A capillary electrophoresis (CE) method for the characterization of recombinant NTPDases 1, 2, and 3, and for assaying NTPDase inhibitors has been developed performing the enzymatic reaction within the capillary. After hydrodynamic injection of plugs of substrate solution with or without inhibitor in reaction buffer, followed by a suspension of an enzyme-containing membrane preparation, and subsequent injection of another plug of substrate solution with or without inhibitor, the reaction took place close to the capillary inlet. After 5 min, the electrophoretic separation of the reaction products was initiated by applying a constant current of −60 μA. The method employing a polyacrylamide-coated capillary and reverse polarity mode provided baseline resolution of substrates and products within a short separation time of less than 7 min. A 50 mM phosphate buffer (pH 6.5) was used for the separations and the products were detected by their UV absorbance at 210 nm. The Michaelis–Menten constants (K_m) for the recombinant rat NTPDases 1, 2, and 3 obtained with this method were consistent with previously reported data. The inhibition studies revealed pronounced differences in the potency of reactive blue 2, pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS), suramin, and N⁶-diethyl-β,γ-dibromomethylene-ATP (ARL67156) towards the NTPDase isoforms. Notably, ARL67156 does not inhibit all NTPDases, having only a minor inhibitory effect on NTPDase2. Dipyridamole is not an inhibitor of the NTPDase isoforms investigated. The new method is fast and accurate, it requires only tiny amounts of material (nanoliter scale), no sample pretreatment and can be fully automated; thus it is clearly superior to the current standard methods.     

Half-time analysis of the kinetics of irreversible enzyme inhibition by an unstable site-specific reagent

The half-time method for the determination of Michaelis parameters from enzyme progress-curve data (Wharton, C.W. and Szawelski, R.J. (1982) Biochem. J. 203, 351-360) has been adapted for analysis of the kinetics of irreversible enzyme inhibition by an unstable site-specific inhibitor. The method is applicable to a model in which a product (R) of the decomposition of the site-specific reagent, retaining the chemical moiety responsible for inhibitor specificity, binds reversibly to the enzyme with dissociation constant Kr: (formula; see text). Half-time plots of simulated enzyme inactivation time-course data are shown to be unbiased, and excellent estimates of the apparent second-order rate constant for inactivation (k +2/Ki) and Kr can be obtained from a series of experiments with varying initial concentrations of inhibitor. Reliable estimates of k +2 and Ki individually are dependent upon the relative magnitudes of the kinetic parameters describing inactivation. The special case, Kr = Ki, is considered in some detail, and the integrated rate equation describing enzyme inactivation shown to be analogous to that for a simple bimolecular reaction between enzyme and an unstable irreversible inhibitor without the formation of a reversible enzyme-inhibitor complex. The half-time method can be directly extended to the kinetics of enzyme inactivation by an unstable mechanism-based (suicide) inhibitor, provided that the inhibitor is not also a substrate for the enzyme.