Instantaneous Inhibition Research Articles

Inhibition and potentiation of platelet function by lysolecithin

The effects of lysolecithin (LPC) on aggregation, serotonin release, shape, and lysis of rabbit, pig, or human platelets in platelet-rich plasma (PRP) or Tyrode albumin solution were examined during prolonged incubation. LPC added to citrated or heparinized PRP from humans or rabbits at a final concentration above 100 muM caused instantaneous inhibition of platelet aggregation induced by adenosine diphosphate (ADP), epinephrine (human PRP only), collagen, or thrombin. The inhibitory effect of LPC was found to be partially reversible over a period of 60–90 min. LPC at final concentrations above 30 muM also caused inhibition of ADP-, collagen-, and thrombin-induced aggregation and collagen- and thrombin-induced release of serotonin in suspensions of rabbit, pig, or human platelets. With washed platelets, the inhibitory effect not only rapidly disappeared but was followed by transient potentiation of aggregation and serotonin release. This potentiating effect of LPC was most pronounced when thrombin was used as stimulus. Both inhibition and potentiation were observed at concentrations of LPC that did not cause a significant change in platelet shape or loss from platelets of lactic dehydrogenase. Inhibition and potentiation were also observed when platelets were added to suspending medium containing LPC, although considerably higher concentrations of LPC were required under these conditions. Potentiation was not observed when LPC was added to citrated or heparinized rabbit or human PRP or to washed rabbit platelets suspended in a medium containing 4% bovine serum albumin. It seemed likely that some or all of the observed effects of LPC on platelet function were due to structural modification of the platelet membrane insufficient to result in gross membrane damage or platelet lysis. In addition, the results of experiments using 14C-LPC seemed to indicate that the observed potentiating effect of LPC on platelet function may be related to its rapid uptake and metabolism by the platelets.

Isolation and characterization of alpha2-plasmin inhibitor from human plasma. A novel proteinase inhibitor which inhibits activator-induced clot lysis.

A procedure is presented for purifying a novel proteinase inhibitor in human plasma whose apparent unique biological property is to inhibit efficiently the lysis of fibrin clots induced by plasminogen activator. The final product is homogeneous as judged by disc gel electrophoresis, and immunoelectrophoresis. Its molecular weight estimated by sodium dodecyl sulfate gel electrophoresis or sedimentation equilibrium is 67,000 and 63,000, respectively. The inhibitor is a glycoprotein consisting polypeptide chain containing 11.7% carbohyrate. It migrates in the alpha2-globulin region in immunoelectrophoresis. The inhibitor is chemically and immunologically different from all the other known inhibitors in plasma. Inhibition of plasmin by the inhibitor is almost instantaneous even at 0 degrees, in contrast to the slow inhibition of urokinase (plasminogen activator in urine). Plasminogen activation by urokinase-induced clot lysis is inhibited by the inhibitor mainly through a mechanism of instantaneous inhibition of plasmin formed and not through the inhibition of urokinase. The inhibitor also inhibits trypsin. Consequently, it is suggested that this newly identified inhibitor is named alpha2-plasmin inhibitor or alpha2-proteinase inhibitor. A specific antibody directed against the inhibitor neutralizes virtually all inhibitory activity of plasma to activator-induced clot lysis. Immunochemical quantitation of the inhibitor was specific antiserum to the inhibitor and the purified inhibitor as a standard indicates that the concentration of the inhibitory in the serum of a healthy man is in or near the range of 5 to 7 mg/100 ml, which is the lowest concentration among the concentration of the proteinase inhibitors in plasma. The inhibitor and plasmin, trypsin, or urokinase form a complex which cannot be dissociated with denaturing and reducing agents. The formation of the enzyme-inhibitor complex occurs on a 1:1 molar basis and is associated with the cleavage of a unique peptide bone, which is most clearly demonstrated in the interaction of the inhibitor and beta-trypsin. In the complex formation between the inhibitor and plasmin, the inhibitor is cross-linked with the light chain which contains the active site of plasmin. It is suggested that, in a fashion analogous to complex formation between alpha1-antitrypsin and trypsin, the cross-links are formed between the active site serine of the enzyme and the newly formed COOH-terminal residue of the inhibitor, with cleavage of a peptide bond.

The respiratory chain of Azotobacter vinelandii. III. The effect of cyanide in the presence of substrates

1. 1. Cyanide (100 μM) causes a rapid disappearance of the band (648 nm) of oxidized cytochrome d in particles of Azotobacter vinelandii oxidizing NADH. The rate of disappearance of the band can be related to the rate of inhibition of the oxygen consumption. 2. 2. The kinetics of the disappearance of the 648-nm band of cytochrome d with excess cyanide in the presence of substrates deviate from first-order, indicating that at least two conformations of the enzyme are involved. 3. 3. The rate of binding of cyanide to cytohrrome d increases the larger the rate of turnover of the oxidase. From this it is concluded that cyanide binds preferentially to the enzymically active oxidized conformation of cytochrome d. 4. 4. The instantaneous inhibition of the oxidation of ascorbate plus N, N, N′, N′-tetramethyl- p-phenylenediamine (TMPD) by cyanide is related neither to the binding of cyanide to cytochrome d as determined spectrophotometrically, nor to the rate of inhibition of the oxidation of NADH. This indicates that different oxidases are involved in the oxidation of NADH and of ascorbate plus N, N, N′, N′-tetramethyl- p-phenylenediamine (TMPD), in line with the conclusion of Jones and Redfearn (1967) Biochim. Biophys. Acta 143, 340–353. 5. 5. From experiments in which the change in redox states of cytochrome b 1 and c-551 were related to the rate of binding of cyanide to cytochrome d, it is concluded that the cytochromes b 1 and d are located in the main pathway to oxygen, whereas cytochrome c-551 functions in the branch via cytochrome o. 6. 6. After prolonged incubation of particles with cyanide in the presence of NADH a residual activity (5%) was found in which a b-type cytochrome is involved. This shows the existence of a third but minor pathway to oxygen.

The absence of zinc in the mitochondrial and supernatant forms of malate dehydrogenase

The malate dehydrogenase enzyme system from porcine heart ( l-malate:NAD) oxidoreductase, EC 1.1.1.37) has previously been shown to be a multiple enzyme system 1. This discussion is limited to a consideration of the two basic systems; i.e. enzyme isolated from the cytoplasm or supernatant form and enzyme of a mitochondrial origin. No consideration is made at this time of the microheterogeneity observed in each major type. An efficient method for the purification of the supernatant form of the enzyme in tandem with a previously reported procedure for the mitochondrial enzyme is described. The possibility that either or both enzymes are zinc metalloenzymes has been investigated. Although instantaneous inhibition by known metal-chelating reagents was observed, atomic absorption analysis of samples of highly purified enzyme of both types failed to indicate the presence of a stoichiometric amount of zinc. Inhibition of both enzymes has been observed in the presence of a non-chelating analogue of 1,10-phenanthroline, (1,7-phenanthroline). This information coupled with the analytical data indicating the absence of intrinsic metal ions in either enzyme system has suggested that the inhibition by metal-chelating agents observed by these authors and others was the result of hydrophobic interaction of the chelating agents at or near the enzymatic active center, and not due to the specific chelating properties of the reagents employed.

Regulation of Glyceraldehyde 3-Phosphate Dehydrogenase by Phosphocreatine and Adenosine Triphosphate: IV. FACTORS AFFECTING IN VIVO CONTROL OF ENZYMATIC ACTIVITY

Abstract Phosphocreatine inhibited glyceraldehyde 3-phosphate dehydrogenase crystallized from rabbit muscle or yeast. Creatine at the same concentration showed no inhibition. The inhibition was competitive with respect to glyceraldehyde 3-phosphate. At the approximate physiological concentrations of substrate (0.01 mm) and phosphocreatine (20 mm), the enzyme was 65 % inhibited. Phosphocreatine is not competitive with respect to NAD or orthophosphate. The inhibition by phosphocreatine was maximal at pH 7.4 and decreased about 17 % with a pH fall to 6.8 or a rise to 8.5. Incubation of the muscle enzyme with phosphocreatine produced a time-dependent inactivation which was prevented by the addition of glyceraldehyde 3-phosphate. This time-dependent inactivation and the instantaneous inhibition were additive. Magnesium partially relieved the inhibitory effects of phosphocreatine. The properties of the inhibitory effects of phosphocreatine and ATP are compared. Since the phosphocreatine level in muscle may decrease very rapidly during muscle contraction, the inhibition on triose phosphate dehydrogenase may be lessened substantially, and ATP resynthesis will be promoted. Further physiological consequences of phosphocreatine and ATP inhibition in the resting and contracting muscle are considered.

Interaction between Adenosine Triphosphate and Glyceraldehyde 3-Phosphate Dehydrogenase: III. MECHANISM OF ACTION AND METABOLIC CONTROL OF THE ENZYME UNDER SIMULATED IN VIVO CONDITIONS

Abstract The inhibitory effect of ATP on glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12) has been investigated under conditions simulating the intracellular milieu. The inhibition is strongly pH-dependent. With the physiological concentrations of ATP, NAD, and orthophosphate, the dehydrogenase reaction is 87 % inhibited at pH 6.8, 67 % at pH 7.4, and only 20 % at pH 8.1. ADP, adenosine 5'-monophosphate, and cyclic adenosine 3':5'-monophosphate are almost equally inhibitory but have less physiological significance since the intracellular levels are much lower than ATP. The ATP inhibition is mixed type inhibition in which ATP is nearly competitive with respect to either NAD or Pi. ATP (6 mm) increases Km for NAD from 0.06 to 0.3 mm and decreases Vmax from 91 to 65 µmoles of NADH per min per mg of protein. Similarly the Km for Pi is changed from 1.2 to 6.1 mm whereas Vmax is decreased from 89 to 61. Experiments using AMP or pyrophosphate as the component parts of ATP have also been carried out. AMP reacts competitively with respect to NAD but not Pi, whereas PPi is nearly competitive with Pi but not NAD. These data suggest that ATP simultaneously obstructs both NAD and Pi binding in the active center. ATP is noncompetitive with regard to glyceraldehyde 3-phosphate. Mg2+ or Mn2+ reduces the inhibitory effect of ATP by complexing with the nucleotide. ATP also effects a time-dependent inactivation, which is additive with the instantaneous inhibition mentioned above. This time-dependent inhibition is counteracted by NAD but not Pi. The physiological implication of ATP inhibition on the dehydrogenase is discussed in the connection with muscle physiology. ATP inhibition may in part account for the high levels of glyceraldehyde 3-phosphate dehydrogenase in the muscle, protect against excessive acidity due to lactic production, limit maximal glycolytic flux, and modify the turnover of the enzyme.

Activation of human plasminogen by an insoluble derivative of urokinase. Structural changes of plasminogen in the course of activation to plasmin and demonstration of a possible intermediate compound.

Activation of plasminogen was performed by perfusing a plasminogen solution through a small column loaded with insolubilized urokinase. The effluent was immediately collected in an excess of plasmin inhibitor (p‐nitrophenyl‐p‐guanidino‐benzoate). This provides a practically instantaneous inhibition of the plasmin formed and autocatalytic degradation is thus reduced to a minimum. Different degrees of activation are obtained by changing the perfusion rate.Dodecylsulfate‐polyacrylamide electrophoresis, amino acid and NH2‐terminal amino acid analyses show that the activation of human plasminogen involves cleavage of at least two peptide bonds, the first event being the release of a peptide of 64 amino acid residues from the NH2‐terminal end. An intermediate inactive product with a molecular weight of about 86000 and methionine as NH2‐terminal amino acid is formed. Cleavage at the second site results in formation of a molecule with two chains connected by disulfide bridges. One chain which has a molecular weight of about 63000 is very sensitive to proteolytic degradation in its NH2‐terminal part. However, there is evidence that methionine is the true NH2‐terminal amino acid of this fragment. The other chain has a molecular weight of about 25000 and has valine as NH2‐terminal amino acid.

Effect of weak acids on amino acid transport by Penicillium chrysogenum: evidence for a proton or charge gradient as the driving force.

A variety of weak acids at and below their pK(a) are potent inhibitors of transport in Penicillium chrysogenum. The effective compounds include sorbate, benzoate, and propionate (common antifungal agents), indoleacetate (a plant hormone), acetylsalicylate (aspirin), hexachlorophene, and a yellow pigment produced by the mycelia under nutrient-deficient conditions, as well as the classical uncouplers 2,4-dinitrophenol, p-nitrophenol, and azide. The results suggest that a proton gradient or charge gradient is involved in energizing membrane transport in P. chrysogenum. The unionized form of the weak acids could discharge the gradient by diffusing through the membrane and ionizing when they reach an interior compartment of higher pH. Experiments with 2,4-dinitrophenol and p-nitrophenol established that the ionized species are not absorbed by the mycelium to any great extent. The transport inhibitors also caused a decrease in cellular adenosine 5'-triphosphate (ATP) levels, but there was no constant correlation between inhibition of transport and suppression of cellular ATP. A decrease in aeration of the mycelial suspension had the same effect on transport and ATP levels as the addition of a weak organic acid. The effects on transport rates and ATP levels were reversible. The instantaneous inhibition of [(14)C]l-leucine transport by NH(4) (-) (and vice-versa) in nitrogen-starved mycelia at pH values of 7 or below can be explained by competition for a common energy-coupling system. The inhibition is not observed in carbon-starved mycelia in which the NH(4) (+) transport system is absent or inactive (but the general amino acid transport is fully active), or in iodoacetate-treated mycelia in which the NH(4) (+) transport system has been differentially inactivated. At pH values greater than 7.0, NH(3) and HPO(4) (2-) inhibit transport, presumably by discharging the membrane proton or charge gradient. Aniline counteracts the inhibitory effect of NH(3) and HPO(4) (2-) possibly by acting as a proton reservoir or buffer within the membrane.

Glycerylphosphorylcholine diesterase: Effects of metal-binding agents

Glycerylphosphorylcholine diesterase ( l-3-glycerylphosphorylcholine glycerophosphohydrolase, EC 3.1.4.2) has been studied with respect to metal ion involvement. The effects of a number of metal-binding agents have been investigated by preincubation and instantaneous inhibition techniques. A high degree of selectivity of the metal-binding agent has been observed, with EDTA showing the greatest amount of inhibition. Structural analogs of EDTA have however, been found to be as effective as EDTA. The inhibition produced by EDDA has been determined to be competitive inhibition with respect to substrate. Heat and urea potentiate the inhibition seen with EDTA. Zn 2+ ions have been found to alleviate the EDTA-produced instantaneous inhibition and were observed to produce noncompetitive inhibition with respect to GPC. Dialysis against 0.01 m EDTA, pH 7.1, and water has been found to completely inactivate the enzyme, whereas addition of several cations to the EDTA-dialyzed enzyme were observed to bring about restoration of activity. Of 26 cations tested, Zn 2+ and Sr 2+ reactivated the diesterase 100% and 109%, respectively. Zn 2+ ions, when titrated with the enzyme obtained after dialysis with EDTA and water were found to restore the activity in a sigmoidal manner. The binding constant for Zn 2+ was calculated to be approximately 3 × 10 −6 m. The substrate, glycerylphosphorylcholine, was observed to reverse the instantaneous inhibition produced by EDTA, possibly by protecting the metal from the EDTA. These observations suggest that GPC diesterase may be a metalloenzyme, and a mechanism is proposed which suggests the inclusion of two molecules of Zn 2+ per molecule of enzyme.

D-Amino Acid Oxidase: III. Studies of Flavin Adenine Dinucleotide Binding

Abstract A recently described spectrophotometric assay for d-amino acid oxidase was used to study the inhibition of the enzyme by a series of adenine and flavin derivatives. AMP, ADP, and ADP-ribose inhibited the enzyme competitively with respect to FAD. No instantaneous inhibition was observed with flavin derivatives. These inhibition studies indicated that the adenosine moiety and the pyrophosphate group of the FAD molecule contributed to the effective binding of FAD to d-amino acid oxidase. The isoalloxazine moiety of FAD appears to be less tightly bound than the adenosine moiety. FMN and riboflavin were found to inactivate the d-amino acid oxidase apoenzyme by a time-dependent photochemical process. This photoinactivation of the enzyme in the presence of flavin derivatives is enhanced by the adenine compounds. The various forms of d-amino acid oxidase were studied by gel filtration. This technique indicated that the apoenzyme form and holoenzyme form each have a molecular weight of approximately 50,000. This value corresponds to the minimum molecular weight calculated per FAD-binding site. The benzoate complex at low protein concentrations was found to contain a 50,000 molecular weight species and a 100,000 molecular weight species. At higher concentrations, the benzoate complex was found to exist entirely as the dimer.

Action of urea on human alkaline phosphatases: with a description of some automated techniques for the study of enzyme kinetics

An automated method is described for the study of enzyme kinetics. It employs a controlled substrate concentration gradient so that reaction velocities can be studied at a large number of substrate concentrations. Urea has two effects on human alkaline phosphatases: a reversible instantaneous inhibition and an irreversible time-dependent inactivation. The reversible inhibition is of the noncompetitive type. Alkaline phosphatases from different tissues show different susceptibilities to irreversible inactivation by urea. The placental enzyme is the most resistant, while bone enzyme is the most sensitive of the alkaline phosphatases examined. The alkaline phosphatases in human sera resemble those of the presumptive tissues of origin in their susceptibility to the actions of urea.

Action of zinc-complexing agents on nicotinamide adenine dinucleotide-linked aldehyde dehydrogenases from yeast and liver

Metal-complexing agents (1,10-phenanthroline (OP), 8-hydroxyquinoline (OHQ), EDTA, and histidine) produce essential activation of yeast aldehyde dehydrogenase (E.C.1.2.1.5), like cysteine and BAL. The extent of enzyme activation varies according to the coenzyme structure. Diethyldithiocarbamate and cyanide are weaker activators, and glycine, glycylglycine, adenine, and cupferron do not stimulate the enzyme activity. Liver aldehyde dehydrogenase (E.C.1.2.1.3) and cysteine-activated yeast aldehyde dehydrogenase are inhibited by OP, OHQ, diethyldithiocarbamate and α,α′-dipyridyl. The instantaneous inhibition is reversible, competitive between chelator and coenzyme and mostly noncompetitive with respect to substrate. The kinetics of inhibition is consistent wih the presence of zinc as an intrinsic constituent of both enzymes. OP, BAL, and other ligands produce time and temperature-dependent progressive inhibition of yeast aldehyde dehydrogenase (but not of liver aldehyde dehydrogenase). This inhibition increases with the specific activity of the enzyme preparation, or the presence of adenosine phosphates, nicotinamide and acetylpyridine dinucleotides, or sodium or ammonium ions. Thiol compounds, other ligands (EDTA, cyanide, OHQ, etc.), pyridine aldehyde dinucleotides, pyrophosphate, zinc, potassium, and rubidium ions prevent or decrease OP irreversible inhibition. The effects of most of ligands as enzyme activators and as enzyme protectors against OP inhibition are closely related, and with cysteine, the relationship is quantitative. Highly purified preparations of yeast aldehyde dehydrogenases contain traces of heavy metals that, by forming inactive enzyme-mercaptides, have apparently a significant role for the enzyme instantaneous activation and for the progressive inhibition by chelators. OP and OHQ increase the sensitivity of yeast aldehyde dehydrogenase toward SH-reagents and decrease that of the liver enzyme.

The Role of Zinc in Alcohol Dehydrogenase: IV. THE KINETICS OF THE INSTANTANEOUS INHIBITION OF HORSE LIVER ALCOHOL DEHYDROGENASE BY 1,10-PHENANTHROLINE

The Role of Zinc in Alcohol Dehydrogenase: IV. THE KINETICS OF THE INSTANTANEOUS INHIBITION OF HORSE LIVER ALCOHOL DEHYDROGENASE BY 1,10-PHENANTHROLINE