Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

A computer graphic molecular display system has been used to construct a three-dimensional model of the B chain of bovine thrombin. The model is derived from the bovine alpha-chymotrypsin structure as determined by X-ray crystallographic studies. The amino acid sequence of bovine thrombin has been substituted for that of alpha-chymotrypsin, preserving the beta-barrel structure and maximizing homology of the amino acid sequence of the two proteins. With the exception of an area in the vicinity of the specificity binding pocket, most of the changes observed in thrombin occur on the surface of the molecule. The most notable changes observed in the model are the increases on the surface of positively charged (arginine and lysine) and negatively charged (glutamate and aspartate) residues. A glutamate replaces methionine 192 near the entrance to the specificity binding pocket. The nature of this site was further altered by the substitution of an aspartate for serine 189 and an alanine for serine 190. The structure of the resulting specificity binding pocket is consistent with that of serine proteases, which have trypsin-like substrate specificity. The computer graphics molecular display system has been used to insert models of synthetic thrombin inhibitors into the active site of the thrombin B chain model. With the model, it has been possible to correlate the interaction of thrombin with the observed binding constants of two inhibitors of trypsin-like serine proteases, p-amidinophenylmethylsulfonylfluoride (Ki = 1.27 x 10(-6) M) and m-[m-(trifluoromethyl)phenoxypropoxy]benzamidine (KD = 2.9 x 10(-6) M).
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PMID:A computer-generated three-dimensional model of the B chain of bovine alpha-thrombin. 694 67

Aspartase (L-aspartate ammonia-lyase, EC 4.3.1.1) of Escherichia coli, already of full activity, is 3-5-fold activated by a limited proteolysis with trypsin (Mizuta, K. and Tokushige, M. (1976) Biochim. Biophys. Acta 452, 253-261). Structural bases for the activation were investigated. The NH2-termini of the native enzyme and of the trypsin-activated enzyme were found to be equally serine, as analyzed by the dansylation method. However, the COOH-terminal glutamate of the native enzyme was altered to arginine upon activation, as revealed by treatments with carboxypeptidases Y, A and B. The released peptides were obtained by molecular sieve membrane filtration following trypsin activation of the enzyme. The peptides were separated by high voltage paper electrophoresis, and the amino acid composition and the terminal residues were determined. The results showed that one or a few related peptides consisting of 7-17 residues were released from the COOH-terminal upon activation. The circular dichroism spectrum of the enzyme suggested that the helical content of the activated enzyme was about 5% less than that of the native enzyme, an indication that the trypsin-activated enzyme has a somewhat looser conformation than the native enzyme. Determination of the fluorescence decay time of the enzyme protein indicated that the tryptophan residue became more exposed to outer environment than that of the native enzyme upon trypsin-activation.
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PMID:Studies on aspartase. VI. Trypsin-mediated activation releasing carboxy-terminal peptides. 701 87

Schizolobium parahyba seed chymotrypsin inhibitor (SPC) is a protein with M(r) of 20,000 and four half-cystine residues and no free thiol group. SPC is stable at temperatures up to 75 degrees at pH 7 but gradually loses activity when kept at 95 degrees for 1 hr and total inactivation occurs after 5 hr. Amino acid analysis shows a high content of glycine, aspartate, glutamate and alanine residues. A pI of 4.52 predicted from the amino acid content agrees with experimental results. A stable binary complex with M(r) of 45,000, Ki = 5.85 x 10(-8) M and molar ratio of 1:1 is formed between SPC and chymotrypsin. The determined single N-terminal sequence of SPC shows homology with Kunitz type soybean trypsin inhibitors.
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PMID:Purification and partial characterization of a Schizolobium parahyba chymotrypsin inhibitor. 757 50

Membrane vesicles from rat brain have been subjected to trypsin treatment in the absence and presence of substrates of the (Na+ + K+)-coupled L-glutamate transporter GLT-1. The fragments of this transporter have been detected upon immunoblotting employing several antibodies raised against sequences from this transporter. At the amino terminus, initially a fragment of an apparent molecular mass of 30 kDa is generated. This fragment is subsequently cleaved to one of 16 kDa. The generation of these bands is greatly inhibited in the presence of lithium. Moreover, lithium abolishes the positive cooperative activation of the transporter by sodium. The generation of the 30- and 16-kDa fragments is accelerated in the presence of L-glutamate and other transportable analogues, provided sodium is present as well. The 30-kDa fragment also contains an epitope from the loop connecting the putative membrane-spanning alpha-helices 3 and 4. This epitope, in contrast with the amino-terminal one, is destroyed with time. The carboxyl-terminal epitope is predominantly located on a 43-kDa fragment which is slowly converted to one of 35 kDa. This conversion is not inhibited by lithium. It is, however, stimulated by L-glutamate and other transportable analogues, but only in sodium-containing media. Potassium also stimulates this conversion regardless of the presence of L-glutamate. The stimulation of generation of amino- and carboxyl-terminal fragments by L-glutamate is not mimicked by the nontransportable analogue dihydrokainate. However, the analogue blocks the stimulation exerted by L-glutamate. In addition to new experimental information on the transporters topology, our observations provide novel information on the function of the GLT-1 transporter. Although lithium by itself does not sustain transport, it may occupy one of the sodium sites and be transported. Furthermore, the transporter-glutamate complex appears to exist in at least two states. After the initial binding (suggested to be important for the decay of synaptic glutamate), it undergoes a conformational change which represents, or is tightly associated with, the transport step.
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PMID:Conformational changes monitored on the glutamate transporter GLT-1 indicate the existence of two neurotransmitter-bound states. 762 23

To eliminate highly antigenic substances, bovine pericardium was washed in 5% sodium chloride (NaCl) for 24 hours, followed by incubation in trypsin for 40 minutes. To achieve adequate fixation, NaCl-trypsin-treated pericardium was preserved in glutaraldehyde (GA) solution with gradually increasing concentrations from 0.1% to 0.25%. To inactivate the free aldehyde groups and residual GA on the surface of the implant, NaCl-trypsin-GA-treated pericardial samples were posttreated separately with 1% lysine, 8% monosodium glutamate, and 4% chitosan. Fresh (untreated) and 0.1%, 0.2%, and 0.625% GA-treated and NaCl-trypsin-GA-treated pericardial specimens were prepared for comparative study. All samples were implanted subdermally in rats for 2, 4, 8, and 12 weeks for calcification studies. Morphologic and chemical analyses showed mild calcification in fresh pericardia (Ca, 10.5 +/- 1.25 micrograms/mg, von Kossa +) and in glutamate-posttreated pericardia (Ca, 11.5 +/- 3.45 micrograms/mg, von Kossa +). Calcium was practically undetectable in chitosan-posttreated implants (Ca, 1.1 +/- 0.27 micrograms/mg, von Kossa 0), whereas severe calcification was noticed in the rest of the samples (mean Ca greater than 200.0 micrograms/mg, von Kossa ) at 12 weeks. This study suggests that posttreatment with an amino compound such as chitosan would prevent the calcification of GA-treated bioprostheses at an early implantation stage, but elimination of antigenic factors and adequate GA fixation would prevent tissue degeneration, thus enabling the prosthesis to function over a long period.
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PMID:Prevention of calcification of heart valve bioprostheses: an experimental study in rat. 764 84

The mitochondrial porin or VDAC (Voltage-Dependent Anion Channel), the pore-forming structure responsible for the high permeability of the outer mitochondrial membrane, was found to be one of only three mitochondrial proteins bound by [14C]dicyclohexylcarbodiimide (DCCD) at low dosages (1.5 nmol/mg of mitochondrial porin) (De Pinto, V., Tommasino, M., Benz, R., and Palmieri, F. (1985) Biochim. Biophys. Acta 813, 230-242). Treatment of intact mitochondria with DCCD results in the inhibition of their ability to binding hexokinase (Nakashima, R. A., Mangan, P. S., Colombini, M., and Pedersen, P. L. (1986) Biochemistry 25, 1015-1021). In the present study, mitochondrial porin was purified from [14C]DCCD-labeled mitochondria. The purified labeled porin was treated with the cleavage reagent CNBr and with the endoproteases trypsin and V8 from Staphylococcus aureus and blotted to polyvinylidene difluoride membrane. The transferred peptides were detected with Coomassie Blue dye, excised, and sequenced. The sequences of several labeled and unlabeled peptides were obtained and then overlapped. The region containing the [14C]DCCD radioactivity was limited to 50 amino acid residues and completely sequenced. Covalently incorporated [14C]DCCD was exclusively released at the position corresponding to glutamate 72. The DCCD-reactive residue is located in the 4th of 16 predicted transmembrane amphipathic beta-strands. When the sequence surrounding the DCCD site was compared to those surrounding the DCCD-reactive residue of other membrane proteins, no homology was apparent.
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PMID:Location of the dicyclohexylcarbodiimide-reactive glutamate residue in the bovine heart mitochondrial porin. 768 55

Glutamate receptors are the most abundant excitatory neurotransmitter receptors in vertebrate brain. We have previously cloned cDNAs encoding two homologous kainate receptors (GFKAR alpha, 45 kDa, and GFKAR beta, 41 kDa) from goldfish brain and proposed a topology with three transmembrane domains (Wo, Z. G., and Oswald, R. E. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 7154-7158). These studies have been extended using an in vitro translation/translocation system in conjunction with site-specific antibodies and point and deletion mutations. We report here that the entire region between the previously proposed third and fourth transmembrane segments is translocated and likely to be extracellular in mature receptors. This was based on the following results. 1) The entire segment was protected from Proteinase K and trypsin digestion and could be immunoprecipitated by a site-specific antibody. 2) Functional sites for N-glycosylation are present in the C-terminal half of the segment, and 3) a mutation, constructed with an additional consensus site for N-glycosylation in the N-terminal half of the segment, was found to be glycosylated at that site. Given the fact that the N terminus of the protein is likely to be extracellular, this would place an even number of transmembrane segments between the extracellular N terminus and the glycosylated segment. In addition, results of N-glycosylation and proteolysis protection assays of GFKAR alpha mutations indicated that the previously proposed second transmembrane segment is not a true transmembrane domain. These results provide further evidence in support of a topology with three transmembrane domains that has important implications for the relationship of structure to function in ionotropic glutamate receptors.
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PMID:A topological analysis of goldfish kainate receptors predicts three transmembrane segments. 783 26

The xylanase A (endo-1,4-beta-D-xylan xylanhydrolase) of the basidiomycete Schizophyllum commune was treated with the powerful carboxylate-modifying reagent 1-(4-azonia-4,4-dimethyl-pentyl)-3-ethylcarbodiimide iodide (EAC) in the presence of substrate. This treatment was followed by complete inactivation of the enzyme with [14c]EAC after the removal of excess reagent and protecting ligand. The inactivated enzyme was digested with endoproteinase Arg-C or trypsin, and peptides were separated and purified using reverse-phase high-performance liquid chromatography. Following sub-digestion of individual radioactive peptides with staphylococcal V8 protease and endoproteinase Lys-C, amino acid composition analysis and sequencing analysis revealed that the [14C]EAC label was bound exclusively to Glu87. Comparison of the primary sequences of related xylanase with that of xylanase A revealed that Glu87 is a highly conserved residue. Based on this similarity and the mechanism of carbodiimide action, Glu87 is proposed to act as the nucleophile in the catalytic mechanism of xylanase A. The possible environment of the putative catalytic glutamate residue was explored using hydrophobic-cluster analysis and secondary-structure prediction based on the primary sequence of xylanase.
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PMID:Identification of a glutamate residue at the active site of xylanase A from Schizophyllum commune. 790 49

Primary neuronal cultures from 8-day-old rat cerebellum were incubated in the presence of exogenously added 16 nM [gamma-32P]ATP. Phosphorylation of a 45-kDa endogenous protein was detected within 1 min and increased linearly for approximately 20 min. Unlike what was seen with [gamma-32P]ATP, in the presence of [32P]orthophosphate no visible phosphorylation of protein was detected after 10 min, but a different pattern of phosphorylation was obtained in 30 min. The phosphorylation of the 45-kDa protein was reduced by 80-90% in the presence of 1 microM unlabeled ATP, 5 U/ml of apyrase, or 0.01% trypsin but not 1 mM PO4(3-). Phosphorylation was inversely proportional to cell density and was unaffected by addition to the cells of 56 mM KCl or 100 microM glutamate for 3 min. The presence of exogenously added cellular protein extracts or pretreatment of the cells for up to 20 min in phosphorylation buffer also did not affect the observed phosphorylation of the 45-kDa protein. The phosphorylation was found to be insensitive to MgCl2 but inhibited in the presence of MnCl2 or NaF and in the absence of CaCl2. Analogues of ATP suppressed phosphorylation of the 45-kDa protein by 80-90%. A similar inhibition was obtained in the presence of ADP or AMP. In this study, we establish via several different means that the phosphorylation of the 45-kDa protein in primary neuronal granule cultures occurs extracellularly through an ectokinase activity, which is furthermore distinguishable from a series of other presently characterized ecto-protein enzymes and intracellular kinases.
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PMID:Identification of an ectokinase activity in cerebellar granule primary neuronal cultures. 796 20

The vitamin D receptor (VDR) from a variety of animal species is a hormone-modulated substrate for phosphorylation in vivo. In this report, we utilize an expression vector to produce recombinant human VDR (hVDR) in 1,25-dihydroxyvitamin D3-treated COS-1 cells. Immunoprecipitation of the phosphorylated hVDR followed by gel purification and phosphoamino acid analysis revealed modification exclusively on one or more serine residues, consistent with previous studies of the VDR in other species. To identify the region of phosphorylation, immunoprecipitated and gel-purified hVDR from COS-1 cells was first mixed with purified hVDR isolated to homogeneity from Saccharomyces cerevisiae and then digested with trypsin or V8 protease, and the peptides were resolved on HPLC. The single phosphate-containing peptides were recovered and subjected to amino acid sequence analysis, revealing the modification to reside in a region extending from residue 171 to residue 206 common to both the tryptic- and the V8 protease-derived peptides. Sequential cleavage of similar VDR mixtures using trypsin and then CNBr, alpha-chymotrypsin, or thermolysin demonstrated an amino-terminal boundary of the phosphorylated peptide at 202. Selective manual Edman degradation of phosphorylated peptides beginning at 171, 195, and 200 revealed phosphate release only at serine 205. This peptide contained an average of 8-fold less radioactive phosphate in the absence of prior treatment of the culture cells with 1,25(OH)2D3. Site-directed modification of VDR serine 205 to alanine, aspartate, or glutamate each led to fully functional proteins when assessed in a transactivation assay using several VDRE-linked natural promoters. Unexpectedly, evaluation of the serine 205 to alanine hVDR mutant revealed that this protein continued to be phosphorylated in a hormone-dependent manner on an alternative site. These studies show directly that hVDR serine residue 205, a consensus site for casein kinase II, is modified in vivo in response to hormone.
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PMID:1,25-dihydroxyvitamin D3 modulates phosphorylation of serine 205 in the human vitamin D receptor: site-directed mutagenesis of this residue promotes alternative phosphorylation. 815 47


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