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)

A small redox-active protein has been purified to homogeneity from cell-free extracts of the strictly anaerobic thermophilic methanogen, Methanobacterium thermoautotrophicum (strain Marburg). The purification consisted of streptomycin sulfate and acid treatments and three chromatographic steps using Sephadex G-75, Mono Q HR 10/10, and Superose 12 HR 10/30 columns. When these procedures were carried out under strictly anaerobic conditions, approximately 3 mg of this protein could be isolated from 45 g of wet cell paste. Like the thioredoxins and glutaredoxins, it is a small acidic protein (pI = 4.2) consisting of 83 amino acids (M(r) = 9136). In the presence of dithiothreitol or dihydrolipoate, the protein serves as a hydrogen donor for the ribonucleotide reductase from Escherichia coli, and it catalyzes the reduction of insulin. However, it does not interact with the thioredoxin reductases from E. coli or Corynebacterium nephridii and does not function as a hydrogen donor for the ribonucleotide reductase of C. nephridii. The amino acid sequences determined by automated Edman degradation of the 14C-carboxymethylated protein and of peptides derived from trypsin and chymotrypsin digestions show a redox-active site -Cys-Pro-Tyr-Cys-, typical of the glutaredoxins. Its amino acid sequence shows moderate identity with the known glutaredoxins (E. coli, yeast, rabbit bone marrow, calf thymus, and pig liver) when the proteins are aligned at the active site. The secondary structure of the glutaredoxin-like protein predicted by the Chou-Fasman procedure shows that it is similar to the known glutaredoxins. However, surprisingly, the protein does not function as a glutathione-disulfide oxidoreductase in the presence of glutathione and glutathione reductase. This glutaredoxin-like protein may be a component of a ribonucleotide-reducing system distinct from the previously described systems utilizing thioredoxin or glutaredoxin.
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PMID:The purification, characterization, and primary structure of a small redox protein from Methanobacterium thermoautotrophicum, an archaebacterium. 158 36

Polyclonal antibodies against pig liver thioltransferase were raised in a New Zealand rabbit. These antibodies completely neutralized the thioltransferase activity of the homogeneous enzyme and that in the crude cytosolic homogenate at an equivalent titer. The antibodies also cross-reacted equally with calf thymus glutaredoxin and calf liver thioltransferase, but not with Escherichia coli thioredoxin, suggesting that thioltransferase and glutaredoxin from the same species are identical. Immunoblotting analysis of the cytosolic proteins from 14 different pig tissues revealed that most pig tissues contain a 12-kDa protein which reacts with these antibodies. This protein is found in greater abundance in stomach, small intestine, liver, skeletal muscle, kidney, heart, lung, and cerebral cortex, whereas retina, cerebellum, spleen, pancreas, and thymus have low levels of the protein. No reactive protein was detected in the lens. The tissue distribution of the protein was also determined by assay of the enzyme activity and was generally in good agreement with that obtained from the immunoblotting survey. Pig liver thioltransferase was cleaved by trypsin, chymotrypsin, Staphylococcus aureus V8 protease, and cyanogen bromide. The selected peptides purified by reversed phase high performance liquid chromatography or ion exchange fast protein liquid chromatography were subjected to reaction with the polyclonal antibodies against pig liver thioltransferase. Four antigenically reactive fragments were detected by dot-blotting analysis. These peptides are located in the first 30-amino acid residues from the NH2 terminus and the sequence from amino acid residues 39-67, indicating that the active site of the enzyme, Cys22 and Cys25, is located on one of the antigenic determinant domains.
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PMID:Immunological characterization of thioltransferase from pig liver. 245 32

A glutaredoxin was purified from rabbit bone marrow, and its amino acid sequence was determined by high performance tandem mass spectrometry. The sequences of peptides generated by digestion with trypsin alone or in combination with thermolysin were determined from their collision-induced dissociation (CID) mass spectra. Alignment of these sequences and additional sequence information were obtained from the collision-induced dissociation mass spectra of peptides obtained from digestion of the intact protein with Staphylococcus aureus V8 protease and alpha-chymotrypsin. The resulting sequence of 106 amino acids is as follows: Ac-Ala-Gln-Glu-Phe-Val-Asn-Ser-Lys-Ile-Gln-Pro-Gly-Lys-Val-Val-Val-Phe- Ile-Lys-Pro-Thr-Cys-Pro-Tyr-Cys-Arg-Lys-Thr-Gln-Glu-Ile-Leu-Ser-Glu-Leu- Pro-Phe - Lys-Gln-Gly-Leu-Leu-Glu-Phe- Val-Asp-Ile-Thr-Ala-Thr-Ser-Asp-Met-Ser-Glu-Ile- Gln-Asp-Tyr-Leu-Gln-Gln-Leu-Thr-Gly-Ala-Arg- Thr-Val-Pro-Arg-Val-Phe-Leu-Gly-Lys-Asp-Cys-Ile- Gly-Gly-Cys-Ser-Asp-Leu-Ile-Ala-Met-Gln-Glu-Lys- Gly-Glu-Leu-Leu-Ala-Arg-Leu-Lys-Glu-Met-Gly- Ala-Leu-Arg-Gln. This glutaredoxin strongly resembles the corresponding calf and pig proteins (known as glutaredoxin and thioltransferase, respectively) with respect to its primary structure and enzymatic activity as a GSH:disulfide thioltransferase, an activity also found for the glutaredoxin from Escherichia coli. However, rabbit glutaredoxin was not active as a hydrogen donor for the reduction of ribonucleotides in the presence of the ribonucleotide reductases from rabbit bone marrow, Lactobacillus leichmannii, and Corynebacterium nephridii.
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PMID:Glutaredoxin from rabbit bone marrow. Purification, characterization, and amino acid sequence determined by tandem mass spectrometry. 268 77

The complete amino acid sequence of pig liver thioltransferase has been determined. The homogeneous protein was cleaved by trypsin, chymotrypsin, Staphylococcus aureus V8 protease, and cyanogen bromide. The resulting peptides were purified by reversed-phase high performance liquid chromatography and ion-exchange fast protein liquid chromatography. Sequencing of the fragments was achieved with either automated Edman degradation or fast atom bombardment-mass spectrometry. Pig liver thioltransferase is a single polypeptide with 105 amino acid residues and an acetylated glutamine N terminus. The protein has 2 cysteine pairs with sequences of -Cys-Pro-Phe-Cys- and -Cys-Ile-Gly-Gly-Cys-, the first pair of which (Cys22 and Cys25) is located at the potential active site of the enzyme. The sequence of pig liver thioltransferase displays close homology (82%) with calf thymus glutaredoxin, suggesting that they belong to the same evolutionary family.
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PMID:The primary structure of pig liver thioltransferase. 357 Dec 78

An immunosorbent method using antiglutaredoxin-Sepharose was developed for purification of glutaredoxin in high yield from a mutant strain of Escherichia coli K 12 lacking thioredoxin reductase (C 10-17). The primary structure of the protein was determined by analyses of [14C]carboxymethylated glutaredoxin and its proteolytic fragments obtained by digestions with trypsin, clostripain, chymotrypsin and staphylococcal Glu-specific extracellular protease. The single active-center disulfide has the structure-Cys-Pro-Tyr-Cys-, with the half-cystine residues located at positions 11 and 14 in the polypeptide chain. In total the protein was deduced to have 85 residues corresponding to a molecular weight of 9674 for the reduced form of glutaredoxin, making it one of the smallest known enzymes (a glutathione-disulfide transhydrogenase). The half-cystines are identically spaced and similarly positioned in the N-terminal part of the protein when compared with a corresponding functionally active disulfide/dithiol in thioredoxins. Glutaredoxin is also distantly homologous with thioredoxins from phage T4 and E. coli, but extensive differences, even around the redox-active disulfide, distinguish glutaredoxin from the thioredoxins. Allowing for deletions in the glutaredoxin sequence (or insertions in the T4 thioredoxin sequence) at four places, there are identical residues at 25 positions of the 77 compared (= 32% identity). The results establish that glutaredoxin belongs to the same superfamily of small redox proteins as the thioredoxins. The structures are, however, subject to large changes, only four positions have residues identical among all presently analyzed forms. The fluorescence of reduced and oxidized glutaredoxin demonstrates an increase in the quantum yield of the tyrosine emission upon reduction with dithiothreitol. Differences in the spectra support the presence of tyrosine adjacent to the redox-active disulfide bridge. They also confirm that glutaredoxin lacks the disulfide-adjacent tryptophan residues of E. coli thioredoxin. There are known to be great differences between the bacterial E. coli and phage T4 forms of thioredoxin. The glutaredoxin structure is most similar to the phage type, both with respect to size of the polypeptide chain and to actual sequence. From the structural results and the previously known functional similarities it appears possible that the phage thioredoxin may have evolved from an early glutaredoxin gene. The mixed properties are compatible with this conclusion, the superfamily assignment, and the differences in biological activity.
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PMID:The primary structure of Escherichia coli glutaredoxin. Distant homology with thioredoxins in a superfamily of small proteins with a redox-active cystine disulfide/cysteine dithiol. 635 62

The primary structure of calf thymus glutaredoxin was determined by analysis of the [14C]carboxymethylated protein and the proteolytic fragments obtained by treatments with trypsin, chymotrypsin, CNBr and staphylococcal Glu-specific extracellular protease. The active center has the structure Cys-Pro-Tyr-Cys, with the redox-active cysteines/half-cystines located at positions 22 and 25 in the polypeptide chain. This active center is identical in amino acid sequence and similar in position to that of Escherichia coli glutaredoxin, suggesting this structure to be typical for glutaredoxins and distinguishing them from the distantly related thioredoxins. However, the two glutaredoxins also exhibit considerable differences. Calf thymus glutaredoxin is extended at both ends and has 31% overall residue identities with the corresponding E. coli protein. In contrast to the bacterial glutaredoxin, the calf thymus protein contains two additional half-cystines/cysteine residues at positions 74 and 78, which may be of regulatory significance.
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PMID:The primary structure of calf thymus glutaredoxin. Homology with the corresponding Escherichia coli protein but elongation at both ends and with an additional half-cystine/cysteine pair. 638 71