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)

The binding characteristics of histogranin (HN), an endogenous peptide first recognized for its antagonism of N-methyl-D-aspartate (NMDA) responses, were determined in membrane preparations of rat brain. [125I][Ser1]HN, a stable bioactive analog of HN, bound specifically and reversibly to a homogenous population of high-affinity sites with a Kd of 25 nM and a Bmax of 410 fmol/mg protein. The binding of [125I][Ser1]HN increased linearly with membrane protein concentration and was destroyed upon membrane pretreatment with trypsin. The binding displayed rapid association and dissociation kinetics and was blocked by peptides possessing close homology with HN in the following order: [Ser1]HN-(1-15) > HN > [Ser1]HN-(1-14) > HN-(2-15) > [Ser1]-HN-(1-10) > HN-(6-10). Unrelated peptides such as substance P, beta-endorphin, neuropeptide Y, [Met5]enkephalin, [Leu5]enkephalin, dynorphin A(1-13) and neuromedin C were inactive in competition binding assays against [125I]Ser1]HN. Ligands of the binding domains of the NMDA receptor, such as (+)3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid, (+) 5-methyl-10,11-dihydro 5H-dibenzo[a, d]cyclohepten-5,10-imine hydrogen maleate, 1-N-(2-thienyl)cyclohexylpiperidine, glycine and glutamate were also ineffective in competing for [125I][Ser1]HN binding sites. Interestingly, specific ligands for the polyamine site on the NMDA receptor, as well as the cations Mg++ and Zn++ inhibited [125I][Ser1]HN binding. The polyamine antagonist diethylenetriamine produced a noncompetitive inhibition with an IC50 (175 nM) comparable to that of HN (75 nM). The cations Zn++ and Mg++ displaced [125I][Ser1]HN binding with IC50 values of 18 and 240 microM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characterization of [125I][Ser1]histogranin binding sites in rat brain. 822 61

In a recent study [Wang & Beattie (1992) Biochemistry 31, 8445-8459], we reported that dicyclohexylcarbodiimide (DCCD) was bound to either aspartate-155 or glutamate-166 localized in an amphiphilic, non-membrane-spanning, helix of cytochrome. Moreover, DCCD inhibits proton translocation in a cytochrome bf complex reconstituted into proteoliposomes without significant inhibition of electron transfer, suggesting that the helix containing aspartate-155 and glutamate-166 may play a role in proton movements. In order to explore the environment of this amphiphilic helix, we employed a fluorescent derivative of DCCD, N-cyclohexyl-N'-[4-(dimethylamino)naphthyl]carbodiimide (NCD-4). After incubation of NCD-4 with a cytochrome bf complex isolated from spinach chloroplasts, a fluorescent compound was formed with a 331-nm excitation peak and 440-nm emission peak. NCD-4 was selectively bound to cytochrome b6 and inhibited proton translocation with only a minimal inhibitory effect on electron transfer in the cytochrome bf complex reconstituted into proteoliposomes. Exhaustive digestion of the NCD-4-labeled cytochrome b6 with trypsin resulted in the formation of a single 6-kDa fluorescent peptide with similar properties to the peptide labeled with radioactive DCCD. The fluorescence of NCD-4 bound to the cytochrome bf complex reconstituted into proteoliposomes was quenched by CAT-16, an amphiphilic spin label that intercalates at the membrane surface, as well as by nitroxide derivatives of stearic acid in the order 5-doxylstearic acid > 7-doxylstearic acid > 12-doxylstearic acid. At higher concentrations, the hydrophilic membrane-impermeant quenchers, CAT-1 and D-569, also quenched the fluorescence of NCD-4.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Topographical organization of cytochrome b6 in the thylakoid membrane of spinach chloroplasts determined by fluorescence studies with N-cyclohexyl-N'-[4-(dimethylamino)naphthyl]carbodiimide. 837 66

We present a model for the three-dimensional structure of the glutamate-specific endopeptidase from Streptomyces griseus based on the crystal structures of other bacterial proteases of the trypsin family. For the first time a structural model is described which attempts to explain the basis of P1 glutamate specificity in serine proteases. Several important changes to the S1 pocket with respect to other members of the family of different specificity are described. Of particular interest is the presence of a histidine at position 213 and the substitution of Arg-138 by lysine. Other biochemical evidence concerning substrate preferences can be rationalized on the basis of the model.
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PMID:A structural model for the glutamate-specific endopeptidase from Streptomyces griseus that explains substrate specificity. 850 58

Six potential hydrophobic sites of the Tus-TerB complex were analyzed using bromodeoxy-uridine and iododeoxyuridine as isosteric analogues of thymine. Analogues were incorporated at individual sites, and dissociation rates were measured in 150 mM potassium glutamate, pH 8.0, using a nitrocellulose filter assay. These halogenated analogues serve as a probe of the environment in which they reside. Our measurement revealed at least two types of responses. Three sites showed increases in stability with the introduction of the bromo and iodo derivatives. The enhanced stability is proposed to result from polar or charged molecules in the vicinity of the halogenated analogues through dipole-dipole, dipole-ion, or dipole-induced dipole interactions. The other three sites exhibited the opposite trend, being destabilized by the introduction of these analogues. The destabilizing effects are attributed to a hydrophobic environment which cannot accommodate polar molecules. The photoreactivity of these analogues was utilized to specifically cross-link the Tus protein and TerB DNA. Using the substitution of bromodeoxyuridine at position 8 in the TerB DNA, Tus protein was covalently attached to the DNA, and by trypsin digestion a fragment of Tus was isolated. Sequencing of the peptide fragment revealed a segment that matched the amino acid composition from 122-139 of the Tus protein.
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PMID:Identification of a Tus protein segment that photo-cross-links with TerB DNA and elucidation of the role of certain thymine methyl groups in the Tus-TerB complex using halogenated uracil analogues. 895 91

We studied the binding of cationic liposomes, including didodecyl N-(alpha-(trimethylammonio)acetyl)-D-glutamate chloride (TMAG), to a mouse macrophage-like cell line RAW264.7 to clarify which molecules contribute to the binding of TMAG liposomes to the cell surface. Several types of TMAG liposomes encapsulating [3H]inulin, intra-aqueous markers of liposomes, were prepared and their binding characteristics were compared with those of neutral and negatively charged liposomes. The binding of TMAG liposomes to cells was superior to those of neutral and negatively charged liposomes and increased with increasing TMAG content. Scatchard plots for the binding of TMAG liposomes to the cells were approximately linear, indicating a single class of binding sites. Pretreatment of the cell surface with heparinase, heparitiase, chondroitinase ABC, or neuraminidase did not reduce the binding of TMAG liposomes. These results suggested that neuraminic acid and glycosaminoglycan on the cell surface have little contribution to TMAG liposome binding. Pretreatment of the cells with trypsin reduced the binding of TMAG liposomes in a concentration-dependent manner but did not detach the cells from the culture plates. In addition, alpha-chymotrypsin pretreatment had no effect even up to 5 micrograms/mL. Post-treatment with trypsin enhanced the release of TMAG liposomes from the cell surface in a concentration-dependent manner. These results demonstrated that TMAG liposomes bind to trypsin-sensitive proteins on the cell surface.
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PMID:Contribution of trypsin-sensitive proteins to binding of cationic liposomes to the mouse macrophage-like cell line RAW264.7. 923 17

When incubated with their substrates, human phosphomannomutase and L-3-phosphoserine phosphatase are known to form phosphoenzymes with chemical characteristics of an acyl-phosphate. The phosphorylated residue in phosphomannomutase has now been identified by mass spectrometry after reduction of the phosphoenzyme with tritiated borohydride and trypsin digestion. It is the first aspartate in a conserved DVDGT motif. Replacement of either aspartate of this motif by asparagine or glutamate resulted in complete inactivation of the enzyme. The same mutations performed in the DXDST motif of L-3-phosphoserine phosphatase also resulted in complete inactivation of the enzyme, except for the replacement of the second aspartate by glutamate, which reduced the activity by only about 40%. This suggests that the first aspartate of the motif is also the phosphorylated residue in L-3-phosphoserine phosphatase. Data banks contained seven other phosphomutases or phosphatases sharing a similar, totally conserved DXDX(T/V) motif at their amino terminus. One of these (beta-phosphoglucomutase) is shown to form a phosphoenzyme with the characteristics of an acyl-phosphate. In conclusion, phosphomannomutase and L-3-phosphoserine phosphatase belong to a new phosphotransferase family with an amino-terminal DXDX(T/V) motif that serves as an intermediate phosphoryl acceptor.
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PMID:A new class of phosphotransferases phosphorylated on an aspartate residue in an amino-terminal DXDX(T/V) motif. 960 9

The heme prosthetic group from the bovine milk enzyme lactoperoxidase (LPO), termed heme l, is isolated through an approach that combines proteolytic hydrolysis and reverse-phase high performance liquid chromatographic separation of the resulting digest. Application of different proteases yields either a peptide-bound heme (with trypsin and chymotrypsin) or a peptide-free heme (with proteinase K). Both heme l and heme l-peptide species were investigated by paramagnetic 1H NMR spectroscopy, electrospray mass spectrometry, and peptide sequence analysis. Paramagnetic 1H NMR experiments on the low spin bis(cyano)-Fe(III)heme l complex conclusively define the heme l structure as a 1,5-bis(hydroxymethyl) derivative of heme b. The electrospray mass spectrum of heme l confirms the two-site hydroxyl functionalization on this heme. Paramagnetic 1H NMR spectra of the high spin bis(dimethyl sulfoxide)-Fe(III) complexes of the isolated heme species provide information regarding peptide content. Sequence analyses of peptides released from two heme l-peptide species by base hydrolysis suggest that heme-protein ester linkages in lactoperoxidase occur between the two hydroxyl groups of heme l and the carboxylic side chains of glutamate 275 and aspartate 125. These results confirm the earlier reported structural proposal (Rae, T. D., and Goff, H. M. (1996) J. Am. Chem. Soc. 118, 2103-2104).
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PMID:The heme prosthetic group of lactoperoxidase. Structural characteristics of heme l and heme l-peptides. 977 11

Photoaffinity labeling with [32P]nicotinamide 2-azidoadenosine dinucleotide (2N3NAD+) was used to identify the NAD+ binding site within two types of glutamate dehydrogenase isoproteins (GDH I and GDH II) isolated from bovine brain. In the absence of photolysis, 2N3NAD+ is a substrate for the GDH isoproteins. When the enzymes were covalently modified by photolysis in the presence of saturating amounts of photoprobe, about 50% inhibition of the GDH activities was observed. Photoinsertion of probe was increased by GTP or glutarate and decreased by NAD+ or ADP. With the combination of immobilized boronate affinity chromatography and reversed-phase HPLC, photolabel-containing peptides generated with trypsin were isolated. This identified a portion of the adenine ring binding domain of GDH isoproteins as the region containing the sequence, CIAVGXSDGSIWNPDGIDPK for both GDH isoproteins, corresponding to Cys270 through Lys289 of the amino acid sequence of well known bovine liver GDH. The X indicates a position for which no phenylthiohydantoin-derivative could be assigned. The missing residue, however, can be designated as a photolabeled glutamate since the sequences including the glutamate residue in question have a complete identity with those of the other GDH species known. Photolabeling of these peptides was prevented by the presence of NAD+ during photolysis. These results demonstrate selectivity of the photoprobe for the NAD+ binding site and suggest that the peptide identified using the photoprobe is located in the NAD+ binding domain of the brain GDH isoproteins. Both amino acid sequencing and compositional analysis identified Glu275 as the site of photoinsertion.
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PMID:Identification of an NAD+ binding site of brain glutamate dehydrogenase isoproteins by photoaffinity labeling. 981 15

Ionotropic glutamate receptors constitute an important family of ligand-gated ion channels for which there is little biochemical or structural data. Here we probe the domain structure and boundaries of the ligand binding domain of the AMPA-sensitive GluR2 receptor by limited proteolysis and deletion mutagenesis. To identify the proteolytic fragments, Maldi mass spectrometry and N-terminal amino acid sequencing were employed. Trypsin digestion of HS1S2 (Chen GQ, Gouaux E. 1997. Proc Natl Acad Sci USA 94:13431-13436) in the presence and absence of glutamate showed that the ligand stabilized the S1 and S2 fragments against complete digestion. Using limited proteolysis and multiple sequence alignments of glutamate receptors as guides, nine constructs were made, folded, and screened for ligand binding activity. From this screen, the S1S21 construct proved to be trypsin- and chymotrypsin-resistant, stable to storage at 4 degrees C, and amenable to three-dimensional crystal formation. The HS1S21 variant was readily prepared on a large scale, the His tag was easily removed by trypsin, and crystals were produced that diffracted to beyond 1.5 A resolution. These experiments, for the first time, pave the way to economical overproduction of the ligand binding domains of glutamate receptors and more accurately map the boundaries of the ligand binding domain.
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PMID:Probing the ligand binding domain of the GluR2 receptor by proteolysis and deletion mutagenesis defines domain boundaries and yields a crystallizable construct. 986 57

We have used localized mutagenesis of the biotin domain of the Escherichia coli biotin carboxyl carrier protein coupled with a genetic selection to identify regions of the domain having a role in interactions with the modifying enzyme, biotin protein ligase. We purified several singly substituted mutant biotin domains that showed reduced biotinylation in vivo and characterized these proteins in vitro. This approach has allowed us to distinguish putative biotin protein ligase interaction mutations from structurally defective proteins. Two mutant proteins with glutamate to lysine substitutions (at residues 119 or 147) behaved as authentic ligase interaction mutants. The E119K protein was virtually inactive as a substrate for biotin protein ligase, whereas the E147K protein could be biotinylated, albeit poorly. Neither substitution affected the overall structure of the domain, assayed by disulfide dimer formation and trypsin resistance. Substitutions of the highly conserved glycine residues at positions 133 and 143 or at a key hydrophobic core residue, Val-146, gave structurally unstable proteins.
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PMID:Molecular recognition in a post-translational modification of exceptional specificity. Mutants of the biotinylated domain of acetyl-CoA carboxylase defective in recognition by biotin protein ligase. 988 May 19


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