Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Kinetic measurements indicate that the energy-independent transhydrogenation of 3-acetylpyridine-NAD+ by NADPH in membranes of Escherichia coli follows a rapid equilibrium random bireactant mechanism. Each substrate, although reacting preferentially with its own binding site, is able to interact with the binding site of the other substrate to cause inhibition of enzyme activity. 5'-AMP (and ADP) and 2'-AMP interact with the NAD+- and NADP+-binding sites, respectively. Phenylglyoxal and 2,3-butanedione in borate buffer inhibit transhydrogenase activity presumably by reacting with arginyl residues. Protection against inhibition by 2,3-butanedione is afforded by NADP+, NAD+, and high concentrations of NADPH and NADH. Low concentrations of NADPH and NADH increase the rate of inhibition by 2,3-butanedione. Similar effects are observed for the inactivation of the transhydrogenase by tryptic digestion in the presence of these coenzymes. It is concluded that there are at least two conformations of the active site of the transhydrogenase which differ in the extent to which arginyl residues are accessible to exogenous agents such as trypsin and 2,3-butanedione. One conformation is induced by low concentrations of NADH and NADPH. Under these conditions the coenzymes could be reacting at the active site or at an allosteric site. The stimulation of transhydrogenase activity by low concentrations of the NADH is consistent with the latter possibility.
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PMID:Steady-state kinetics and the inactivation by 2,3-butanedione of the energy-independent transhydrogenase of Escherichia coli cell membranes. 38 87

Previous studies have shown that platelet membranes bind radiolabeled ADP and have nucleoside diphosphokinase activity which transforms added ADP to ATP. In order to further characterize these reactions, the ADP-binding and nucleoside diphosphokinase activity of purified platelet membranes were solubilized by freeze-thaw injury followed by extraction with isotonic buffered saline. Up to 80% of membrane ADP-binding activity was solubilized along with 20% of the total membrane protein, a 4-fold purification. A Millipore filter binding assay was developed to detect the soluble binding protein using [3H]ADP as radioligand. Binding of [3H]ADP was rapid, reversible, saturable, and was destroyed by heat, trypsin digestion, and 1 mM N-ethylmaleimide. By Scatchard analysis, there was a single class of binding sites with a Kd of 3.8 x 10(-7) M. Unlabeled nucleotides competed with [3H]ADP with the following potency series: ATP = ADP greater than AMP greater than adenosine. The solubilized nucleoside diphosphokinase activity could be separated from ADP-binding activity by ultracentrifugation on 5 to 20% sucrose density gradients containing 0.6 M KCl suggesting that the activities reside on separate molecules. Hydrodynamic parameters were calculated for the binding protein by gel filtration and ultracentrifugation. The s20,w was 4.1, Stoke's radius 35 x 10(-8)cm, axial ratio (f/fo) 1.09, and the Mr = 61,000. The studies suggest that this platelet ADP-binding protein may act as the receptor for initiating ADP-induced aggregation and release.
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PMID:Solubilization and characterization of a platelet membrane ADP-binding protein. 43 64

Endothelial cells in culture can modulate platelet aggregation and vascular tone, in part by producing prostacyclin (PGI2), a powerful vasodilator and inhibitor of platelet aggregation, but also by their ecto-ADPase activity, which initiates the conversion of pro-aggregating ADP to adenosine, a potent vasodilator and platelet inhibitor. We have now demonstrated that cultured aortic endothelial cells exposed to trypsin, thrombin or other stimuli can liberate a high proportion of their adenine nucleotides without substantial loss of lactate dehydrogenase. ADP rapidly accumulates extracellularly, reaching biologically active concentrations before there is further breakdown to adenosine. Whether this selective release of nucleotides is a response to damage, or whether it represents a specific secretory mechanism remains to be resolved. Cultured aortic smooth muscle cells can secrete adenine nucleotides in a similar manner, but extracellular conversion to adenosine occurs much faster.
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PMID:Vascular endothelial and smooth muscle cells in culture selectively release adenine nucleotides. 48 3

The functional changes, associated with the sequential transformation of L'4 into L4 pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) were studied. L'4 enzyme from human erythrocytes shows strong hysteretic behaviour: the initial rate of the enzyme preincubated with an unsaturating concentration of phosphoenolpyruvate is much higher than of the enzyme preincubated with ADP, at the same phosphoenolpyruvate concentration, although the "final activity" (the activity of the linear part of the reaction progress curve) was the same in both cases. This phenomenon was observed both in the presence and absence of fructose 1,6-diphosphate. High concentrations of both Mg2+free and MgATP2- diminish the difference in initial rate, between the ADP and phosphoenolpyruvate preincubated enzymes: Mg2+free by stabilizing the phosphoenolpyruvate-induced form; ATPMg2- by stabilizing the ADP-induced form. The magnitude of the difference in initial rates of the ADP-or phosphoenolpyruvate-preincubated enzyme is a function of both substrates. L4 pyruvate kinase (either from human liver or trypsin treated L'4 enzyme) does not, or to a very slight extent, show such behaviour. L'2L2 pyruvate kinase shows behaviour intermediate between L'4 and L4 enzymes. A model is proposed to describe the kinetic behaviour of L'4 and L4 enzymes.
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PMID:Functional changes associated with the sequential transformation of L'4 into L4 pyruvate kinase. 49 28

1. The isolation of the ADP/ATP translocator from beef heart mitochondria as the bongkrekateprotein complex is described, using hydroxyapatite chromatography and gel filtration in Triton X-100 solution. 2. The inhibitor is bound to the protein prior to solubilization with detergent for protection against denaturation. Only the intact bongkrekate-protein passes easily through the hydroxyapatite column. Bongkrekate shileds the protein in contrast to carboxyatractylate only partially against proteinases present in the crude extract. 3. The isolated bongkrekate protein shows the same molecular weights in dodecylsulfate and Triton X-100, the same amino acid composition and the same isoelectric point as the earlier isolated carboxyatractylate-protein complex. It differs by its higher sensitivity against trypsin and thermolysin. 4. The identity of both proteins is demonstrated by interconversion of the bongkrekate-protein into the carboxyatractylate-protein. The process requires the catalysis by ADP or ATP, the natural substrates of the protein. 5. The formation of the extractable [3H]bongkrekate-protein complex in mitochondria requires the presence of ADP or ATP. 6. These data, the immunological studies presented earlier, and the differences in the reactivity of -SH groups of the isolated bongkrekate and carboxyatractylate complexes (to be published) indicate that both proteins represent different conformational states of the translocator protein (m-state and c-state).
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PMID:Isolation of the ADP/ATP translocator from beef heart mitochondria as the bongkrekate-protein complex. 64 34

The possible participation of proteases in human platelet aggregation was explored using various protease inhibitors and substrates. Protease inhibitors used included naturally occurring inhibitors of serine proteases and synthetic inhibitors that modify the active site of protease. Substrates used were synthetic substrates for the trypsin type as well as for the chymotrypsin type of protease. All these inhibitors and substrates inhibited platelet aggregation and serotonin release induced by ADP, collagen, epinephrine, or thrombin. In ADP- and epinephrine-induced platelet aggregation the second phase of aggregation was most efficiently inhibited. The inhibitors suppressed the formation of malondialdehyde during platelet aggregation. Release by aggregating agents of arachidonate and its metabolites from indomethacin-treated platelets as well as nontreated platelets was also inhibited. The inhibitors apperar to interact with stimulated platelets but not with unstimulated platelets. These observations suggest that the interaction of an aggregating agent with its platelet receptor activates a unique precursor serine protease that in turn activates platelet phospholipase to liberate arachidonic acid (the precursor of the potent platelet aggregating agent thromboxane A2) from platelet phospholipids.
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PMID:Inhibition of platelet aggregation by protease inhibitors. Possible involvement of proteases in platelet aggregation. 65 19

Trypsin was studied as an aggregating and release-inducing agent with gel-filtered platelets (GFP) and was compared with ADP, epinephrine and collagen. GFP aggregated irreversibly with final concentrations of 0.5-4 microgram/ml trypsin, 1.6-3.2 micrometer ADP, 2.5-5 micrometer epinephrine and 40 microlite 1/ml soluble collagen. Addition of human fibrinogen to the Tyrode-suspending buffer was required for ADP and epinephrine, but was not necessary for trypsin or collagen. Release of (14C)5HT was obtained with trypsin and collagen using the same concentrations as used in aggregation. GFP stored at room temperature for 48 h were still responsive to trypsin and collagen, whereas aggregability and (14C)5HT release induced by ADP and epinephrine were already impaired 5 h after collection of blood. CP-CPK, an ADP-removing reagent, blocked aggregation and release induced by low trypsin concentrations, suggesting that ADP plays an intermediate role in the mechanism by which trypsin activates platelets. Trypsin appears to be a valuable reagent for studying platelet physiology, particulary following storage.
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PMID:The effect of trypsin and storage on aggregation and release of human gel-filtered platelets. 65 86

We have developed a method for the purification in micromole amounts of the trypsin-derived ADP-ribosyl peptide from diphtheria toxin-modified yeast elongation factor 2 (EF-2). EF-2 was partially purified (15 to 20% purity) by ammonium sulfate precipitation and DEAE-Sephadex chromatography. After [3H]ADP-ribosylation by [3H]nad+ and diphtheria toxin, EF-2 was digested with trypsin and a homogeneous [3H]ADP-ribosyl peptide was isolated by chromatography on DEAE-Sephadex and dihydroxyboryl-substituted cellulose. Based on the amount of ADP-ribose acceptor activity in the crude extract, the overall yield of the peptide was 35%. The yeast peptide contains an unusual amino acid (X) which is the site of ADP ribosylation and is apparently identical to the amino acid reported from rat liver EF-2 by Robinson et al. (Robinson, E. A., Hendriksen, O., and Maxwell, E.S. (1974) J. Biol. Chem. 249, 5088-5093). The sequence of the 15-residue yeast peptide was determined to be: Val-Asn-Ile-Leu-Asp-Val-Thr-Leu-His-Ala-Asp-Ala-Ile-X-Arg. The 11 COOH-terminal residues of this peptide and of the homologous 15-residue peptide reported by Maxwell and co-workers from rat liver EF-2 are identical.
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PMID:Isolation and properties of the trypsin-derived ADP-ribosyl peptide from diphtheria toxin-modified yeast elongation factor 2. 72 6

Concanavalin A aggregated gel-filtered platelets in 0.9% NaCl solution signifying cross-bridging by the lectin. Aggregation of these platelets by concanavalin A was temperature dependent; it did not occur at 0-4 degrees C unless the platelets were previously trypsinized. The level of aggregation of trypsinized platelets by concanavalin A at 0-4 degrees C was similar to that of untreated platelets at 37 degrees C. It is suggested that trypsin facilitates platelet aggregation by concanavalin A at 0-4 degrees C by causing a configurational change in membrane glycoproteins which orientates concanavalin A receptor sites into positions that favour lectin cross-bridging. Concanavalin A failed to aggregate platelets in plasma. Radioisotope studies showed that the amount of [3H]concanavalin A which combined with platelets in plasma was extremely low compared with gel-filtered platelets in saline. The aggregation of Ehrlich ascites cells by concanavalin A was considerably reduced when platelet-free plasma was added to the medium suggesting that it was due to the presence of concanavalin A-reactive components in the plasma. Concanavalin A inhibited the ADP-induced aggregation of platelets suspended in plasma or in a salts solution supplemented with calcium and fibrinogen, although the inhibitory effect was more conspicuous in the latter case. The results suggests that concanavalin A produces its inhibitory effect on ADP-induced platelet aggregation by interacting with membrane glycoproteins, and this further suggests their involvement in aggregation.
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PMID:On the susceptibility of human platelets to aggregation by concanavalin A and the effect of this lectin on their response to ADP. 97 36

1. The sulphydryl reagent 2-2'dithio bis-(5-nitropyridine) (DTNP) inhibited photophosphorylation when the chloroplasts were preincubated with the reagent in the light. A maximum inhibition of about 50% was obtained in the presence of pyocyanine and MgCl 2 at 0.3 mumol DTNP per mg chlorophyll and was completed in about 40 s of preillumination. 2. Dithioerythritol, ADP plus Pi (or arsenate) and uncouplers prevented the inhibition when present during the preillumination while phloridzin, Dio-9 and discarine B were ineffective. Low concentrations of ADP or ATP afforded partial protection but other nucleotides had no effect. 3. DTNP inhibited the coupled electron transport rate to the basal level and had no effect on the uncoupled electron transport. The stimulation of proton uptake and inhibition of electron transport by ATP was prevented by DTNP. 4. The trypsin-activated but not the light- and dithioerythritol-triggered ATPase was inhibited by light preincubation of chloroplasts with DTNP. 5. Reversal of DTNP inhibition of photophosphorylation was obtained by a second preillumination in the presence of thiol groups. 6. More DTNP reacted with chloroplasts in the light than in the dark. Two mol of thione were formed in the light per mol of DTNP disappeared. 7. The results suggested that DTNP inhibition is related to the oxidation by DTNP of chloroplast vicinal dithiols probably exposed by a light-induced conformational change.
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PMID:Sulphydryl groups in photosynthetic energy conservation. I. Light-dependent inhibition of photophosphorylation by the sulphydryl reagent 2-2'dithio bis-(5-nitropyridine). 125 60


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