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)

Incorporation of beta-alanine or gamma-aminobutyric acid in position P2 of ACHPA or Leu psi [CHOHCH2]Val-based tetrapeptides gave highly active renin inhibitors (compounds V, VI, and XVII) with high specificity for renin and a remarkable stability against chymotrypsin. Replacement of the amide bond between P2 and P3 by isosteres (ketomethylenes, hydroxyethylenes, and the corresponding thio-insertion analogues) led to compounds (VIII-XIII, XVIII, and XIX) with renin inhibitory activity in the nanomolar range. Oral activity was achieved by incorporation of polar functionalities at the N-terminus of beta-alanine-containing tetrapeptides. One of these compounds (XXVIII) was chosen for further studies. This inhibitor demonstrated excellent efficacy and a long duration of action after intravenous and oral administration to cynomolgus monkeys.
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PMID:Substrate analogue renin inhibitors containing replacements of histidine in P2 or isosteres of the amide bond between P3 and P2 sites. 195 45

The nature of the non-adrenergic non-cholinergic (NANC) relaxation was studied in the proximal (duodenum) and distal (ileum) regions of the rat small intestine. In rat duodenum ATP (1 microM-1 mM) produced a concentration-dependent transient relaxation. In ileal segments it produced a slight inhibitory effect at low concentrations (1-10 microM) and a powerful concentration-dependent contractile effect at concentrations equal to or higher than 100 microM. Relaxation similar to that elicited by ATP can be induced in rat duodenum with the nicotinic stimulant dimethylphenylpiperazinium (DMPP, 0.1 mM) and with gamma-aminobutyric acid (GABA, 1 mM). DMPP had a similar inhibitory effect on distal ileum while GABA barely affected spontaneous activity in this preparation. TTX (0.5 microM)-sensitive relaxation can be elicited in both duodenal and ileal tissues by field stimulation at 0.1 Hz. In the rat duodenum this nerve-mediated relaxation was sensitive to ATP desensitization, nucleotide pyrophosphatase (0.25 U/ml) but resistant to the proteolytic enzyme alpha-chymotrypsin (2 U/ml). On the other hand the field stimulation (0.1 Hz)-induced relaxation in the distal ileum was unaffected by ATP desensitization (by using both low or high concentration of ATP) and by incubation of the preparation with the two enzymes. These findings provide pharmacological evidence that low frequency field stimulation activates at least two different inhibitory NANC systems in the rat small intestine. Adenosine-5'-triphosphate (ATP) appears to be involved as a major transmitter in the duodenal but not in the ileal NANC inhibitory mechanism(s).
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PMID:Pharmacological evidence that at least two different non-adrenergic non-cholinergic inhibitory systems are present in the rat small intestine. 287 76

The nature of the inhbitory non-adrenergic non-cholinergic (NANC) neurotransmitter responsible for neurogenic relaxation of rat duodenum was studied with in vitro techniques. Adenosine 5'-triphosphate (ATP)(1 mM), gamma-aminobutyric acid (GABA, 1 mM), dimethylphenylpiperazinium (DMPP, 0.1 mM) and field stimulation (60 V, 2 ms, 0.1 Hz) produced transient relaxation followed by rebound contraction. In contrast vasoactive intestinal polypeptide (VIP) (0.3 microM) and noradrenaline (1 microM) induced relaxation which set in more slowly and lasted longer. Tetrodotoxin (0.85 microM) abolished field stimulation-induced relaxation but not ATP-, VIP- or noradrenaline-induced relaxation. Nucleotide pyrophosphatase (0.25 U/ml), but not the proteolytic enzyme alpha-chymotrypsin (2 U/ml), selectively antagonized NANC relaxation. The rank order of potency of various adenine derivatives for inducing relaxation was adenosine-5'-triphosphate greater than adenosine-5'-diphosphate much greater than adenosine greater than adenosine-5'-monophosphate. ATP-induced relaxation was selectively antagonized by the putative P2 purinoceptor antagonist reactive blue 2, but unaffected by the selective P1 purinoceptor antagonist 8-phenyltheophylline. The duration of ATP- as well as beta-gamma-methylene adenosine-5'-triphosphate (a stable analogue of ATP)-induced relaxation was similar and was unaffected by indomethacin 10 microM (which abolished the rebound contraction). In those preparations whose contractile tone was increased by using a high-K+ medium the ability of ATP to elicit relaxation was markedly reduced, while GABA- and DMPP-induced relaxation was abolished. On the other hand, ATP-, GABA- and DMPP-induced relaxation of the tonic component of 5-hydroxytryptamine (5-HT)(0.1 mM)-induced contraction was similar to that observed in control conditions. These findings add further weight to the proposal that endogenous ATP is involved in determining NANC relaxation of rat duodenum.
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PMID:Further evidence for involvement of adenosine-5'-triphosphate in non-adrenergic non-cholinergic relaxation of the isolated rat duodenum. 299 70

A protein has been isolated from the small intestine and bile duct which inhibits the binding of [3H]diazepam to specific benzodiazepine binding sites on synaptosomal membranes. When ion-exchange chromatography and gel filtration chromatography are used, this protein has been purified to apparent homogeneity. "Nepenthin" has been chosen as a name for this protein, which has an approximate molecular weight of 16 000, as determined by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography. Purified nepenthin is a competitive inhibitor of [3H]diazepam binding with a Ki = 4.6 X 10(-8) M. It does not inhibit the binding of specific ligands to the enkephalin, beta-adrenergic, gamma-aminobutyrate, or dopamine binding sites in the CNS. Neither gamma-aminobutyric acid nor glycine alters the inhibition of [3H]diazepam binding by this protein. Nepenthin can be extensively treated with proteases (trypsin, chymotrypsin, and Pronase), and inhibition of diazepam binding remains stable, indicating that a lower molecular weight fragment retains activity. Antibodies raised against this purified effector have been used in in situ double antibody labeling studies with rat brain slices. These studies indicate that cells containing an immunologically similar material are present in the deep cortical region of the forebrain.
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PMID:Endogenous effector of the benzodiazepine binding site: purification and characterization. 626 85

The irreversible incorporation upon ultraviolet illumination of the glycine receptor antagonist, [3H]strychnine, into synaptic membrane fractions of rat spinal cord has been investigated. The specificity of this photoaffinity-labelling reaction for the glycine receptor was demonstrated by the following results: (a) the Kd value (9.7 nM) of the glycine-displaceable irreversible incorporation of [3H]strychnine was similar to the previously reported Kd of [3H]strychnine binding to the glycine receptor; (b) pre-illumination of the membranes with unlabelled strychnine led to a corresponding reduction in the number, but not the affinity, of reversible glycine-displaceable [3H]strychnine binding sites; (c) the ultraviolet light-induced incorporation into the membranes of [3H]strychnine was inhibited by different glycine receptor agonists; other neurotransmitter substances had little or no effect. Also, [3H]strychnine alone was shown to be stable upon illumination with ultraviolet light; this suggests that photocrosslinking of [3H]strychnine may require energy transfer from specific groups of its high-affinity receptor binding site. Upon sodium dodecyl sulphate/polyacrylamide gel electrophoresis a single labelled polypeptide with a relative molecular mass of 48000 was revealed from spinal cord membranes photoaffinity-labelled with [3H]strychnine. Spinal cord membranes photoaffinity-labelled with the gamma-aminobutyric acid receptor ligand [3H]flunitrazepam, however, gave a single polypeptide with a relative molecular mass of 5- 0000. Treatment of membranes, labelled with [3H]strychnine, by endoglycosidase H did not alter the relative molecular mass of the 48000-Mr labelled polypeptide. Trypsin treatment, on the other hand, successively produced major fragments of relative molecular masses of 42000 and 37000. Also, even after extensive treatment with trypsin or chymotrypsin, greater than or equal to 90% of the radioactivity incorporated into the labelled membranes remained membrane-associated. It is concluded that the strychnine binding site of the glycine receptor is located on a protease-inaccessible, i.e. probably hydrophobic domain of the 48000-Mr subunit.
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PMID:Photoaffinity-labelling of the glycine receptor of rat spinal cord. 630 11

N-Pivaloyl-leucyl-gamma-aminobutyric acid (PLG) is a synthetic dipeptide with a partition coefficient of 1.67 in an ethyl acetate/water system that partially inhibits the synaptosomal uptake and activates the release of [U-14C]gamma-aminobutyric acid [( U-14C]GABA). The displacement of GABA from crude synaptic membranes by PLG occurs with an IC50 of 10(-5) M. The compound has the capacity to cross the blood-brain barrier and increase central GABA levels. Its ED50 on cardiazol -induced convulsions is 60-65 mg/kg. PLG is resistant to hydrolysis by chymotrypsin and partially inhibits the proteolytic activity of trypsin.
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PMID:An N-protected gamma-aminobutyric acid dipeptide with anticonvulsant action. 654 64

Knowledge on complete sequences is pivotal to identify splice variants, generate specific antibodies and predict conformation. A simple analytical approach to obtain 100% sequence coverage, however, is currently not available. Recombinant gamma-aminobutyric acid A receptor subunits were from insect SF9 cells that were co-transfected with rat alpha1 and His-tag beta 3. The complex of these two subunits was run on blue-native PAGE, followed by multidimensional electrophoretic steps. Spots resolved at the third electrophoretic step were in-gel digested with trypsin, chymotrypsin and Asp-N. In-gel modification of lysines by acetylation was carried out to increase sequence coverage. Subsequently, peptides were analyzed by nano-ESI-LC-MS/MS using both, collision-induced dissociation and electron transfer dissociation principles. When results from trypsin, chymotrypsin and Asp-N digestion were combined, a single peptide [424 KKTHLRRRSSQL 435] was still not identified. In-gel lysine acetylation leads to unambiguous identification of this peptide by the use of MASCOT v2.2. The use of the Modiro software along with MASCOT, however, was able to provide 100% sequence coverage even without the use of in-gel lysine acetylation. It was observed that the use of trypsin, chymotrypsin and Asp-N with bioinformatic handling by the MASCOT and Modiro software is sufficient to obtain complete sequencing of a highly hydrophobic membrane protein.
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PMID:Complete sequencing of GABAA receptor subunit beta 3 by a rapid technique following in-gel digestion of the protein. 1958 15

The analysis of highly hydrophobic proteins is still an analytical challenge. Using a recombinant gamma-aminobutyric acid A (GABAA)-receptor subunit as a model protein, we developed a gel-based proteomic approach for high MS/MS-peptide sequence coverage identification. Protein samples were separated by multi-dimensional gel electrophoresis and the three protein spots representing the GABAA-receptor subunit alpha-1 from the last electrophoretic step were used for in-gel digestion with trypsin, chymotrypsin and subtilisin, followed by subsequent mass-spectrometric identification by nano-ESI-LC-MS/MS Qstar XL (quadrupole time-of-flight (qQTOF)) and linear ion trap (LIT) LTQ XL identification. This protocol allows the unambiguous identification of the GABAA-receptor alpha-1 subunit protein with 100% sequence coverage, thus covering all four hydrophobic transmembrane domains. This protocol differs from other methods in the selection of enzymes, digestion conditions and use of the two mass spectrometry principles. The protocol takes approximately 10 d to complete and may represent a step forward in the complex analysis of other membrane or hydrophobic proteins.
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PMID:Gel-based mass spectrometric analysis of a strongly hydrophobic GABAA-receptor subunit containing four transmembrane domains. 1970 90