Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.1 (chymotrypsin)
 10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Normal human platelets aggregated by thrombin undergo the release reaction and are not readily deaggregated by the combination of inhibitors hirudin, chymotrypsin, and prostaglandin E1 (PGE1). In contrast, thrombin-induced aggregates of platelets from patients with delta-storage pool deficiency (delta-SPD), which lack releasable nucleotides, are readily deaggregated by the same combination of inhibitors. The ease with which delta-SPD platelets are deaggregated is caused by the lack of stabilizing effects of released ADP, since: (1) exogenous adenosine diphosphate (ADP) (10 mumol/L), but not serotonin (2 mumol/L), abolishes the ability of these inhibitors to deaggregate delta-SPD platelets; (2) thrombin-induced aggregates of platelets from a patient (V.R.) (whose platelets have a severe, selective impairment of sensitivity to ADP, but normal amounts of releasable nucleotides) can be readily deaggregated, and addition of ADP does not stabilize the platelet aggregates; (3) apyrase or creatine phosphate (CP)/creatine phosphokinase (CPK), added before thrombin, make control platelets more easily deaggregated by hirudin, chymotrypsin, and PGE1, and do not change the deaggregation response of delta-SPD platelets and of V.R.'s platelets. Thrombin-induced aggregation and release of beta-thromboglobulin in control, delta-SPD, and in V.R.'s platelets was similar and not inhibited by apyrase or CP/CPK. The stabilizing effect of ADP on platelet aggregates is specific, since epinephrine in the presence of apyrase to remove traces of released ADP does not stabilize the aggregates of control, delta-SPD, or of V.R.'s platelets. Because epinephrine increases fibrinogen binding to thrombin-stimulated platelets to a greater extent than ADP, but does not stabilize the aggregates, it is unlikely that the additional fibrinogen binding sites induced by ADP have a major role in inhibiting deaggregation by the combination of inhibitors.
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PMID:Released adenosine diphosphate stabilizes thrombin-induced human platelet aggregates. 213 16

Human platelets that have undergone the release reaction do not deaggregate readily. We examined conditions under which washed human platelets can be deaggregated after they have undergone an extensive release reaction induced by thrombin (1 or 5 U/ml). To make fibrinogen receptors unavailable, either CP/CPK (or apyrase) was used to remove released ADP, or PGE1 was used to increase cAMP. Chymotrypsin was used to digest proteins that might link platelets, and heparin to interact with released proteins and interfere with their binding to platelets and to each other. Individually, none of these caused deaggregation; heparin did not inhibit the effect of thrombin because no antithrombin III was present. Platelets exposed to thrombin (1 U/ml) which was neutralized at 90 sec by hirudin, could be deaggregated by combinations of CP/CPK (or apyrase) and chymotrypsin, or PGE1 and chymotrypsin. When a higher concentration of thrombin was used (5 U/ml) these combinations caused platelets to deaggregate only when heparin was added before thrombin induced the release reaction. Thus, when extensive release occurs three mechanisms may come into play to link human platelets: one that requires the fibrinogen receptor; a heparin-sensitive reaction that may involve the binding of released proteins; and a linkage that can be disrupted only by proteolysis, providing the other two mechanisms are also inhibited.
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PMID:Deaggregation of human platelets aggregated by thrombin. 298 9

Considerable evidence indicates that the glycoprotein (GP) IIb/IIIa complex on human platelets functions as a receptor for fibrinogen, but little is known about the mechanism of receptor "exposure." To investigate this mechanism, our previously described murine monoclonal antibody (10E5) and a new monoclonal antibody (7E3), both of which block the binding of fibrinogen to platelets and bind to GPIIb and/or GPIIIa, were radiolabeled and their rates of binding to native and ADP-activated platelets were studied. At low concentrations, 125I-10E5 bound nearly equally rapidly to both native and activated platelets, whereas 125I-7E3 bound slowly to native platelets and much more rapidly to activated platelets. This increased rate of 7E3 binding is unlikely to be due to an increase in the number of GPIIb/IIIa sites on the surface of activated platelets because: (a) the rate of 10E5 binding was unchanged; (b) the total number of surface GPIIb/IIIa sites increased by only 2-10% with activation as judged by equilibrium binding of near-saturating concentrations of 10E5 and 7E3, and (c) there was less than 1% release of platelet factor 4 with activation, indicating minimal fusion of alpha-granule membranes (a potential source of GPIIb/IIIa) with the plasma membrane. Other activators (epinephrine, thrombin, and ionophore A 23187) also increased the rate of 7E3 binding, as did digestion of platelets with chymotrypsin. Aspirin did not affect the rate of binding of 7E3, whereas apyrase, prostaglandin E1, and dibucaine all inhibited the enhancement of the 7E3-binding rate produced by ADP. These data provide evidence for an activation-dependent change in the conformation and/or microenvironment of the GPIIb/IIIa complex, and offer a method of studying the receptor exposure mechanism that does not rely on the binding of fibrinogen itself.
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PMID:A new murine monoclonal antibody reports an activation-dependent change in the conformation and/or microenvironment of the platelet glycoprotein IIb/IIIa complex. 299 35

By means of CM-Sephadex C-50 column chromatography and gel filtration on Sephadex G-75 and G-50 columns, a potent platelet aggregation inhibitor was purified and characterized. It was a glycoprotein with a molecular weight of 31,000. It was devoid of phospholipase A, ADPase, esterase and fibrino(geno)lytic activities. It inhibited dose-dependently the aggregation of washed platelets induced by collagen, thrombin, sodium arachidonate, platelet activating factor and ionophore A23187 with a similar IC50 (5-10 micrograms/ml). It was also active in platelet-rich plasma, with an IC50 of 10-15 micrograms/ml. The venom inhibitor reduced the elasticity of whole blood clot and inhibited the thrombin-induced clot retraction of platelet-rich plasma. These activities were related to its inhibitory activity on platelet aggregation rather than blood coagulation. The venom inhibitor had various effects on [14C]serotonin release stimulated by aggregation agonists. It had no effect on thromboxane B2 formation of platelets stimulated by sodium arachidonate, collagen and ionophore A23187. The presence of this venom inhibitor prior to the initiation of aggregation was a prerequisite for the maintenance of its maximal activity. It showed a similar inhibitory effect on collagen or thrombin-induced aggregation even when it was added after the platelets had undergone the shape change. High fibrinogen levels partially antagonized its activity. The venom inhibitor completely inhibited the fibrinogen-induced aggregation of alpha-chymotrypsin-treated platelets. It is concluded that this venom inhibitor interferes with the interaction of fibrinogen with fibrinogen receptors, leading to inhibition of aggregation.
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PMID:Characterization of a potent platelet aggregation inhibitor from Agkistrodon rhodostoma snake venom. 362 Apr 99

Close platelet-to-platelet contact induced by weak agonists in a medium with a low concentration of Ca2+ leads to thromboxane A2 (TXA2) formation, release of granule contents, and secondary aggregation. These responses do not occur in a medium containing Ca2+ in the physiological range (1 to 2 mmol/L). Experiments were done to determine whether feedback amplification is required to generate amounts of TXA2 that are sufficient to cause secondary aggregation and the reactions associated with it, or whether close platelet-to-platelet contact alone is sufficient to generate enough TXA2 to produce these responses. Platelets were washed and resuspended in a modified Tyrode solution to which no calcium salt was added that contained 0.35% albumin and apyrase. This medium contains 20 mumol/L Ca2+ and 1 mmol/L Mg2+. Platelets were aggregated with adenosine diphosphate (ADP) in the presence of fibrinogen, agglutinated with polylysine, or after pretreatment with chymotrypsin, aggregated with fibrinogen. In the low-Ca2+ medium, all these agonists caused platelets to adhere to each other, followed by secondary aggregation with TXA2 formation and release of granule contents. When Ca2+ (1 to 2 mmol/L), aspirin, or the thromboxane receptor blocker BM 13.177 was present, the secondary responses did not occur; dazoxiben decreased thromboxane formation, but did not prevent secondary aggregation or release. Aspirin-treated platelets were less responsive to ADP, U46619, or TXA2 in the low-Ca2+ medium, which indicated that the secondary responses of untreated platelets were not caused by a generalized increase in sensitivity. The reactions that result from close platelet-to-platelet contact in a low-Ca2+ medium can be caused by a wide variety of weak agonists; the secondary aggregation response and release of granule contents are dependent on TXA2 formation and on feedback amplification by TXA2 or the prostaglandin endoperoxides. The secondary responses caused by weak agonists in citrated platelet-rich plasma (which has a concentration of Ca2+ similar to the low-Ca2+ medium used in the present studies) do not occur at the concentration of Ca2+ in circulating blood and thus may have little biologic relevance.
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PMID:Thromboxane A2 causes feedback amplification involving extensive thromboxane A2 formation on close contact of human platelets in media with a low concentration of ionized calcium. 362 Jun 98

1. The action of beryllium on the following enzymes has been examined: alkaline phosphatase (Escherichia coli and kidney), acid phosphatase, phosphoprotein phosphatase, apyrase (potato), adenosine triphosphatase (liver nuclei, liver mitochondria, brain microsomes), glucose 6-phosphatase, polysaccharide phosphorylases a and b, phosphoglucomutase, hexokinase, phosphoglyceromutase, ribonuclease, A-esterase (rabbit serum), cholinesterase (horse serum), chymotrypsin. Alkaline phosphatase and phosphoglucomutase are inhibited by 1mum-beryllium sulphate whereas the other enzymes are largely unaffected by 1mm-beryllium sulphate. 2. Possible mechanisms for the inhibition of phosphoglucomutase and alkaline phosphatase are discussed.
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PMID:The inhibition of enzymes by beryllium. 428 87

Two highly purified neutral proteases from human leukocytes i.e. elastase-like protease (ELP) and chymotrypsin-like protease (CLP) do not destroy human platelets since no difference was found in 51Cr liberation from control and enzyme-treated platelets. As with pancreatic chymotrypsin (alpha-CT) ELP does not induce the release of 3H-serotonin while CLP provokes 3H-serotonin secretion, in an enzyme concentration and time dependent fashion. The rate and degree of 3H-serotonin release by CLP is similar to that produced by thrombin. Incubation of platelets at 37 degrees C for 30 min with alpha-CT or ELP renders them resistant to thrombin-releasing activity. Thrombin did not liberate any additional label from platelets which lost over 60% of serotonin during the preceding incubation with CLP. alpha-CT and ELP do not aggregate platelets either in the presence or absence of apyrase. CLP does aggregate platelets suspended in Tyrode buffer without apyrase but not in the presence of apyrase (100 mg/l). The action of alpha-CT, ELP and CLP on washed platelets induces a progressive prolongation of lag phase and a decrease in changes of light transmission during aggregation by thrombin. Similarly to alpha-CT-treated platelets, those subjected to CLP action aggregate in the presence of human fibrinogen. It is concluded that: (1) neutral proteases possibly contribute to development of defects in platelet function in pathological states associated with liberation of leukocyte content into the circulation, (2) CLP similarly to alpha-CT, exposes fibrinogen receptors but in contrast to alpha-CT, CLP aggregates platelets and stimulates serotonin secretion.
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PMID:Effect of neutral proteases from blood leukocytes on human platelets. 632 Apr 86

A human brain E-type ATPase (HB6 ecto-apyrase) was subjected to site-directed mutagenesis to assess the functional significance of two highly conserved tryptophan residues (Trp 187 and Trp 459), the only two tryptophans conserved in nearly all E-type ATPases. Mutation of tryptophan 187 to alanine yielded a poorly expressed ecto-apyrase completely devoid of nucleotidase activity. Immunolocalization of the W187A mutant in mammalian COS cells showed a cellular distribution clearly different from that of the wild-type enzyme, with the majority of the immunoreactivity concentrated in the interior of the cell. Unlike the wild-type enzyme, this mutant did not bind the nucleotide analogue Cibacron Blue and was sensitive to proteolytic digestion by chymotrypsin. These results suggest alteration of the tertiary structure, causing the enzyme to be improperly folded and retained within the cell. In contrast, mutation of tryptophan 459 to alanine resulted in an ecto-apyrase with enhanced NTPase activity, but diminished NDPase activity. Immunolocalization of this active mutant ecto-apyrase revealed a cellular pattern similar to that of the wild-type enzyme, distributed along the cell periphery and in cell processes. Coupling this active W459A mutation to a previously described mutation (D219E) resulted in an enzyme which preferentially hydrolyzes nucleoside triphosphates over diphosphates. The D219E/W459A double mutant had an ATPase:ADPase ratio of 11:1 and a UTPase:UDPase ratio of 148:1. In addition, the double mutant is substantially less sensitive to inhibition by azide, a more potent inhibitor of ecto-apyrases than ecto-ATPases. Thus, mutation of only two amino acids of an E-type ATPase essentially converts an ecto-apyrase to an ecto-NTPase.
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PMID:Mutagenesis of two conserved tryptophan residues of the E-type ATPases: inactivation and conversion of an ecto-apyrase to an ecto-NTPase. 1023 36

Anopheles stephensi is the main urban mosquito vector of malaria in the Indian subcontinent, and belongs to the same subgenus as Anopheles gambiae, the main malaria vector in Africa. Recently the genome and proteome sets of An. gambiae have been described, as well as several protein sequences expressed in its salivary glands, some of which had their expression confirmed by amino terminal sequencing. In this paper, we randomly sequenced a full-length cDNA library of An. stephensi and performed Edman degradation of polyvinylidene difluoride (PVDF)-transferred protein bands from salivary homogenates. Twelve of 13 proteins found by aminoterminal degradation were found among the cDNA clusters of the library. Thirty-three full-length novel cDNA sequences are reported, including a novel secreted galectin; the homologue of anophelin, a thrombin inhibitor; a novel trypsin/chymotrypsin inhibitor; an apyrase; a lipase; and several new members of the D7 protein family. Most of the novel proteins have no known function. Comparison of the putatively secreted and putatively housekeeping proteins of An. stephensi with An. gambiae proteins indicated that the salivary gland proteins are at a faster evolutionary pace. The possible role of these proteins in blood and sugar feeding by the mosquito is discussed. The electronic tables and supplemental material are available at http://www.ncbi.nlm.nih.gov/projects/Mosquito/A_stephensi_sialome/ .
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PMID:Exploring the salivary gland transcriptome and proteome of the Anopheles stephensi mosquito. 1282 99

One approach to genetic control of transmission of the parasites that cause human malaria is based on expressing effector genes in mosquitoes that disable the pathogens. Endogenous mosquito promoter and other cis-acting DNA sequences are needed to direct the optimal tissue-, stage- and sex-specific expression of the effector molecules. The mRNA accumulation profiles of eight different genes expressed specifically in the midgut, salivary glands or fat body tissues of the malaria vector, Anopheles gambiae, were characterized as a measure of their suitability to direct the expression of effector molecules designed to disable specific stages of the parasites. RT-PCR techniques were used to determine the abundance of the gene products and their duration following multiple blood meals. Transcription from the midgut-expressed carboxypeptidase-encoding gene, AgCP, follows a cyclical, blood-inducible expression pattern with maximum accumulation every 3 h post blood meal. Other midgut-expressed genes encoding a trypsin and chymotrypsin, Antryp2 and Anchym1, respectively, and the fat body-expressed genes, Vg1 and Cathepsin, also show a blood-inducible pattern of expression with maximum accumulation 24 h after every blood meal. Expression of the Lipophorin gene in the fat body and apyrase and D7-related genes (AgApy and D7r2) in the salivary glands is constitutive and not significantly affected by blood meals. Promoters of the midgut- and fat body-expressed genes may lead to maximum accumulation of antiparasite effector molecule transcripts after multiple blood meals. The multiple feeding behaviour of An. gambiae thus can be an advantage to express high levels of antiparasite effector molecules to counteract the parasites throughout most of adult development.
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PMID:The accumulation of specific mRNAs following multiple blood meals in Anopheles gambiae. 1566 79


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