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 number of biomolecules were coupled covalently by nucleophilic displacement to agarose preparations substituted with tosyl groups. In one series of experiments N6-(6-aminohexyl)-adenosine 5'-monophosphate and N6-(6-aminohexyl)adenosine 2',5'-bisphosphate were bound by their terminal amino groups to the polysaccharide support. It could be shown that from a mixture of lactate and 6-phosphogluconate dehydrogenase the immobilized monophosphate showed bio-affinity only for NAD+-dependent lactate dehydrogenase, whereas the immobilized bisphosphate showed affinity only for the NADP+-dependent 6-phosphogluconate dehydrogenase. Furthermore, the immobilized monophosphate (5 mumol/g wet gel) was applied for the single-step purification of lactate dehydrogenase from crude beef heart extract. To demonstrate the immobilization of proteins, soybean trypsin inhibitor (75 mg/g dry support) was immobilized to tosylated agarose, tested as affinity chromatography material and shown to bind 60 mg trypsin/g dry gel. Horseradish peroxidase and horse liver alcohol dehydrogenase were used as model enzymes. Although no optimization had been attempted, the former (approximately 70 mg/g dry support) had a coupling yield of approximately 18% with a specific activity (relative to soluble enzyme) of approximately 10%, whereas approximately 60% of alcohol dehydrogenase was coupled (approximately 100 mg/g dry support) with a specific activity of approximately 25%.
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PMID:p-Toluenesulfonyl chloride as an activating agent of agarose for the preparation of immobilized affinity ligands and proteins. 746 Sep 29

Transhydrogenase catalyses the reversible transfer of reducing equivalents between NAD(H) and NADP(H) to the translocation of protons across a membrane. Uniquely in Rhodospirillum rubrum, the NAD(H)-binding subunit (called Ths) exists as a separate subunit which can be reversibly dissociated from the membrane-located subunits. We have expressed the gene for R. rubrum Ths in Escherichia coli to yield large quantities of protein. Low concentrations of either trypsin or endoproteinase Lys-C lead to cleavage of purified Ths specifically at Lys227-Thr228 and Lys237-Glu238. Observations on the one-dimensional 1H-NMR spectra of Ths before and after proteolysis indicate that the segment which straddles the cleavage sites forms a mobile loop protruding from the surface of the protein. Alanine dehydrogenase, which is very similar in sequence to the NAD(H)-binding subunit of transhydrogenase, lacks this segment. Limited proteolytic cleavage has little effect on some of the structural characteristics of Ths (its dimeric nature, its ability to bind to the membrane-located subunits of transhydrogenase, and the short-wavelength fluorescence emission of a unique Trp residue) but does decrease the NADH-binding affinity, and does lower the catalytic activity of the reconstituted complex. The presence of NADH protects against trypsin or Lys-C cleavage, and leads to broadening, and in some cases, shifting, of NMR spectral signals associated with amino acid residues in the surface loop. This indicates that the loop becomes less mobile after nucleotide binding. Observation by NMR during a titration of Ths with NAD+ provides evidence of a two-step nucleotide binding reaction. By introducing an appropriate stop codon into the gene coding for the polypeptide of E. coli transhydrogenase cloned into an expression vector, we have prepared the NAD(H)-binding domain equivalent to Ths. The E. coli protein is sensitive to proteolysis by either trypsin or Lys-C in the mobile loop. Judging by the effect of NADH on its NMR spectrum and on the fluorescence of its Trp residues, the protein is capable of binding the nucleotide though it is unable to dock with the membrane-located subunits of transhydrogenase from R. rubrum.
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PMID:Conformational dynamics of a mobile loop in the NAD(H)-binding subunit of proton-translocating transhydrogenases from Rhodospirillum rubrum and Escherichia coli. 755 67

The pyridine nucleotide transhydrogenase of Escherichia coli catalyzes the reversible transfer of hydride ion equivalents between NAD+ and NADP+ coupled to translocation of protons across the cytoplasmic membrane. Recently, transhydrogenation of 3-acetylpyridine adenine dinucleotide (AcPyAD+), an analog of NAD+, by NADH has been described using a solubilized preparation of E. coli transhydrogenase [Hutton, M., Day, J.M., Bizouarn, T., and Jackson, J.B. (1994) Eur. J. Biochem. 219, 1041-1051]. This reaction depended on the presence of NADP(H). We show that (a) this reaction did not require NADP(H) at pH 6 in contrast to pH 8; (b) the reaction occurred at pH 8 in the absence of NADP(H) in the mutant beta H91K and in a mutant in which six amino acids of the carboxy-terminus of the alpha subunit had been deleted; (c) the mutant transhydrogenases contained bound NADP+ and were in a conformation in which the beta subunit was digestible by trypsin; (d) the conformation of the beta subunit of the wild-type enzyme was made susceptible to trypsin digestion by NADP(H) or by placing the enzyme at pH 6 in the absence of NADP(H). It is concluded that reduction of AcPyAD+ by NADH does not involve NADPH as an intermediate and that the role of NADP(H) in this reaction at pH 8 is to cause the transhydrogenase to adopt a conformation favouring transhydrogenation between NADH and AcPyAD+.
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PMID:The mechanism of hydride transfer between NADH and 3-acetylpyridine adenine dinucleotide by the pyridine nucleotide transhydrogenase of Escherichia coli. 757 17

Limited hydrolysis of EF-2 with trypsin in mild conditions leads to cleavage at the N-terminal part of the protein, at the region of phosphorylation, at the Arg54 and Arg65 residues. The trypsinolysis product, fragment T1', containing Thr56 and Thr58, which are phosphorylated in EF-2, is also phosphorylated by EF-2-kinase at the same residues. In the phosphorylated EF-2, digestion by trypsin takes place only at Arg65, resulting in a reduction of the rate of hydrolysis in comparison with the native EF-2. Digestion of EF-2 with elastase results in the formation of two fragments E1 and E2 (60 and 40 kDa, respectively). Fragment E1 represents the N-terminal part of EF-2. It is resistant to the further action of elastase, is not cleaved by trypsin, and loses its capability for phosphorylation. Fragment E2, the C-end part of the molecule, is not resistant to the further action of elastase and retains its capability for ADP-ribosylation with the A fragment of diptheria toxin and NAD+. Electrophoretic analysis of EF-2 and its proteolytic fragments according to O'Farrell showed that the modification, resulting in the presence of two initial forms of EF-2, is located between the amino acid residues 66 and 506 of the polypeptide chain. In conclusion a possibility of studying the formation of partial functional activities within the framework of individual structure-functional domains using a set of N-terminal fragments of various length is discussed.
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PMID:[Structure-activity domain of elongation factor EF-2. Analysis of fragments of limited EF-2 hydrolysis, obtained using trypsin and elastase]. 782 14

Photoaffinity labeling of ovine prolactin with the NAD+ photoaffinity analog [alpha-32P]nicotinamide-2-azidoadenine dinucleotide has been used to identify an NADH/NADPH binding site. Specificity of nucleotide interaction was demonstrated by saturation and protection of labeling at physiologically relevant concentrations. Saturation of photoinsertion was observed at approximately 100 microM probe with an apparent Kd of approximately 25 microM. Protection of photoinsertion was observed with NAD+ and NADH. The photoinsertion was decreased by 75% and greater than 95%, respectively, upon addition of 200 microM of the above-mentioned compounds. The protection obtained with NADP+ and NADPH was of the same order, respectively. The adenine ring binding domain of NADH/NADPH binding site was identified by trypsin and chymotrypsin digestion of the photolabeled prolactin and purification of the photolabeled peptide by boronate affinity chromatography and immobilized Fe3+ affinity chromatography. The peptide was identified to be Ala22-Tyr28. These studies demonstrate that prolactin contains an NADH/NADPH binding site which may be significant in the mechanism of action of this hormone.
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PMID:Identification and characterization of a nucleotide binding site of ovine prolactin with 2-azido-NAD. 832 98

Elongation factor 2 (eEF-2) can interact not only with guanylic nucleotides but also with adenylic ones, as was shown by intrinsic fluorescence quenching studies [Sontag, B., Reboud, A.M., Divita, G., Di Pietro, A., Guillot, D. & Reboud, J.P. (1993) Biochemistry 32, 1976-1980]. Here we studied sites of these interactions by using photoactivable 8-azido-[gamma-32P]GTP and 8-azido-[gamma-32P]ATP. Photoincorporation of the radioactive GTP derivative into eEF-2 was prevented by the previous addition of GTP and GDP. The addition of adenylic nucleotides (ATP, ADP) and some adenylic derivatives [NAD+, NADH,poly(A)] decreased the photoincorporation by only 40% at most. However, photoincorporation of the radioactive ATP derivative was prevented by the previous addition not only of adenylic compounds [ATP, ADP, NAD+, NADH, poly(A)] but also of GTP and GDP. Photoincorporation of radioactive nucleotide derivatives was not decreased by the addition of other nucleotidic compounds [UTP, poly(U), ITP, NADP+, NADPH]. ATP and GTP acted as non-competitive inhibitors of the photoincorporation of 8-azido-[gamma-32P]GTP and 8-azido-[gamma-32P]ATP, respectively. eEF-2 photolabeled with these radioactive nucleotide derivatives was submitted to trypsin digestion under different conditions and the labeled peptidic fragments identified after HPLC purification and gel electrophoresis by N-terminal sequencing. An octapeptide, Y264FDPANGK271, was the only peptide photolabeled with 8-azido-[gamma-32P]GTP whereas a N-terminal fragment of about 7 kDa was the only one photolabeled with 8-azido-[gamma-32P]ATP. The different results support the hypothesis that guanylic and adenylic nucleotides do not interact with the same site of eEF-2.
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PMID:Photoaffinity labeling of elongation factor-2 with 8-azido derivatives of GTP and ATP. 861 59

Photoaffinity labeling with [alpha-32P]-8-azidoadenosine 5'-diphosphate (8N3ADP) and [beta-32P]-2-azidoadenosine 5'-diphosphate (2N3ADP) was used to identify overlapping tryptic and chymotryptic generated peptides within the adenine binding domain of the regulatory ADP site of bovine liver glutamate dehydrogenase (GDH). In the absence of UV irradiation, 8N3ADP was able to activate the reverse reaction catalyzed by GDH as well as ADP. Photoinsertion of both [alpha 32P] 8N3ADP and [beta 32P]2N3ADP was reduced best by ADP in comparison to other nucleotides. Photolabeling of GDH with [alpha 32P]8N3-ADP appeared to be biphasic, with saturation occurring near 80 and 130 microM, whereas [beta 32P]2N3ADP showed saturation near 50 microM. When 60 microM [alpha 32P]8N3ADP (below the first saturation value) was used to identify peptides within the ADP binding domain, peptides corresponding to residues G156-K200 and E175-K200 (tryptic) and I158-Y183 (chymotryptic) were photolabeled. However, when 160 microM [alpha 32P]8N3ADP (above the second saturation value) was used, the peptide D403-R418 was also photolabeled. Digestion with both trypsin and chymotrypsin resulted in isolation of peptides E175-Y183 and A184-I192. [beta 32P]2N3ADP at 90 microM also photolabeled tryptic peptides G156-K200 and C270-K289. C270-K289 was shown earlier to be within the NAD+ binding site [Kim, H., and Haley, B. (1991) Bioconjugate Chem. 2, 142-147]. These results are consistent with the residues E175-[192 being within the adenine binding domain of the ADP regulatory site.
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PMID:Identification of adenine binding domain peptides of the ADP regulatory site within glutamate dehydrogenase. 881 52

Photoaffinity labeling with [2'-32P]2N3NADP+ and [32P]2N3NAD+ was used to identify two overlapping tryptic and chymotryptic generated peptides within the adenine binding domain of NADP(+)-dependent isocitrate dehydrogenase (IDH). Photolysis was required for insertion of radiolabel, and prior photolysis of photoprobes before addition of IDH prevented insertion. Photoincorportion of 2N3NAD+ inhibited the enzymatic activity of IDH. Photolabeling of IDH with both [32P]2N3NAD+ and [2'-32P]2N3-NADP+ showed saturation effects with apparent Kds of 20 and 14 microM (+/-12%), respectively. The efficiency of photoincorporation at saturation of binding sites was determined to be about 50%. Also, photolabeling was observed with [32P]8N3ATP and [32P]2N3ATP but with saturation effects observed at lower affinity. With all radiolabeled probes reduction of photoinsertion was effected best by the addition of NADP+ followed by NAD+ and then ATP, indicating that photoinsertion with all the probes was within the NADP+ binding site. Isolation of [32P]2N3NAD+ and [2'-32P]2N3NADP+ photolabeled peptides by use of immobilized boronate and immobilized Al3+ chromatography, respectively, followed by HPLC purification resulted in the identification of overlapping peptides corresponding to Ile244-Arg249 and Leu121-Arg133 (tryptic fragments) and Lys243-His248 and Leu121-His135 (chymotryptic fragments). Trp125 and Trp245 were identified as the sites of photoinsertion based on these residues not being detectable on sequencing, the lack of chymotryptic cleavage at these residues, and the decreased rate of trypsin digestion at nearby Lys243 and Lys127. Sequence analysis of [32P]8N3ATP and [32P]2N3ATP photolabeled peptides gave essentially the same peptide regions being photolabeled but at much lower efficiency, indicating that the effects of ATP on IDH activity are dependent on competition for the same site.
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PMID:Identification of adenine binding domain peptides of the NADP+ active site within porcine heart NADP(+)-dependent isocitrate dehydrogenase. 888 29

Photoaffinity labelling of the human poly(ADP-ribose) polymerase (PARP) catalytic domain (40 kDa) with the NAD+ photoaffinity analogue 2-azido-[alpha-32P]NAD+ has been used to identify NAD+-binding residues. In the presence of UV, photo-insertion of the analogue was observed with a stoichiometry of 0.73 mol of 2-azido-[alpha-32P]NAD+ per mol of catalytic domain. Competition experiments indicated that 3-aminobenzamide strongly protected the insertion site. Residues binding the adenine ring of NAD+ were identified by trypsin digestion and boronate affinity chromatography in combination with reverse-phase HPLC. Two major NAD+-binding residues, Trp1014 of peptide Thr1011-Trp1014 and Lys893 of peptide Ile979-Lys893, were identified. The site-directed mutagenesis of these two residues revealed that Lys893, but not Trp1014, is critical for activity. The close positioning of Lys893 near the adenine ring of NAD+ has been confirmed by the recently solved crystallographic structure of the chicken PARP catalytic domain [Ruf, Menissier-de Murcia, de Murcia and Schulz (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 7481-7485].
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PMID:Photoaffinity labelling of human poly(ADP-ribose) polymerase catalytic domain. 906 65

Rat liver d-3-phosphoglycerate dehydrogenase was purified to homogeneity and digested with trypsin, and the sequences of two peptides were determined. This sequence information was used to screen a rat hepatoma cDNA library. Among 11 positive clones, two covered the whole coding sequence. The deduced amino acid sequence (533 residues; Mr 56493) shared closer similarity with Bacillus subtilis 3-phosphoglycerate dehydrogenase than with the enzymes from Escherichia coli, Haemophilus influenzae and Saccharomyces cerevisiae. In all cases the similarity was most apparent in the substrate- and NAD+-binding domains, and low or insignificant in the C-terminal domain. A corresponding 2.1 kb mRNA was present in rat tissues including kidney, brain and testis, whatever the dietary status, and also in livers of animals fed a protein-free, carbohydrate-rich diet, but not in livers of control rats, suggesting transcriptional regulation. The full-length rat 3-phosphoglycerate dehydrogenase was expressed in E. coli and purified. The recombinant enzyme and the protein purified from liver displayed hyperbolic kinetics with respect to 3-phosphoglycerate, NAD+ and NADH, but substrate inhibition by 3-phosphohydroxypyruvate was observed; this inhibition was antagonized by salts. Similar properties were observed with a truncated form of 3-phosphoglycerate dehydrogenase lacking the C-terminal domain, indicating that the latter is not implicated in substrate inhibition or in salt effects. By contrast with the bacterial enzyme, rat 3-phosphoglycerate dehydrogenase did not catalyse the reduction of 2-oxoglutarate, indicating that this enzyme is not involved in human D- or L-hydroxyglutaric aciduria.
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PMID:Cloning, sequencing and expression of rat liver 3-phosphoglycerate dehydrogenase. 916 25


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