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)

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

The incubation of chloroplast fructose-1,6-bisphosphatase with both dithiothreitol and protein denaturants made sulfhydryl groups available for reaction with [1-14C]iodoacetamide (10-12 mol iodoacetamide incorporated/mol enzyme). Digestion of S-carboxyamidomethylated enzyme with trypsin and polyacrylamide gel electrophoresis, in the presence of sodium dodecylsulfate, yielded two 14C-labeled fragments whose apparent molecular mass were 10 kDa and 16 kDa. In the absence of either dithiothreitol or protein denaturants the incorporation of iodoacetamide to the enzyme was lower than 4 mol. When chloroplast fructose-1,6-bisphosphatase was initially incubated with dithiothreitol (2.5 mM) and (a) high concentrations of both fructose 1,6-bisphosphate (4 mM) and Ca2+ (0.3 mM) or (b) low concentrations of both fructose 1,6-bisphosphate (0.8 mM) and Ca2+ (0.05 mM) in the presence of either 2-propanol (15%, by vol.), trichloroacetate (0.15 M) or chloroplast thioredoxin-f (0.5 microM) and subsequently subjected to proteolysis and electrophoresis, S-carboxyamidomethylated tryptic fragments had similar molecular masses. Thus, conditions that stimulated the specific activity of chloroplast fructose-1,6-bisphosphatase caused conformational changes which favoured both the reduction of disulfide bridges and the exposure of sulfhydryl groups. In this aspect, thioredoxin exerted structural and kinetic effects similar to compounds not involved in redox reactions (organic solvents, chaotropic anions). These results indicated that the modification of hydrophobic (intramolecular) interactions in chloroplast fructose-1,6-bisphosphatase constituted the underlying mechanism in light-activation by the ferredoxin-thioredoxin system.
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PMID:Concerted action of cosolvents, chaotropic anions and thioredoxin on chloroplast fructose-1,6-bisphosphatase. Reactivity to iodoacetamide. 255 90

Chloroplast fructose-1,6-bisphosphatase is an essential enzyme in the photosynthetic pathway of carbon dioxide fixation into sugars and the properties of this enzyme are clearly distinct from cytosolic gluconeogenic fructose-1,6-bisphosphatase. Light-dependent activation via a ferredoxin/thioredoxin system and insensitivity to inhibition by AMP are unique characteristics of the chloroplast enzyme. In the present study, purified spinach chloroplast fructose-1,6-bisphosphatase was reduced, S-carboxymethylated with iodoacetic acid, and cleaved with either cyanogen bromide or trypsin. The resulting peptides were purified by reversed-phase high performance liquid chromatography. Automated Edman degradation of some of the purified peptides showed amino acid sequences highly homologous to residues 72-86, 180-199, and 277-319 of pig kidney fructose-1,6-bisphosphatase. These findings suggest a common evolutionary origin for mammalian gluconeogenic and chloroplast fructose-1,6-bisphosphatase, enzymes catalyzing the same reaction but having different functions and modes of regulation.
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PMID:Amino acid sequence similarity between spinach chloroplast and mammalian gluconeogenic fructose-1,6-bisphosphatase. 300 49

The amino acid sequence of the thioredoxin isolated from rabbit bone marrow was determined chiefly by high performance tandem mass spectrometry and fast atom bombardment mass spectrometry combined with manual Edman degradation. The sequences of peptides generated by digestion with trypsin alone or in combination with Staphylococcus aureus protease V8 or thermolysin were determined from their collision-induced dissociation 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 S. aureus protease V8 and alpha-chymotrypsin. The resulting sequence of 104 residues is as follows: Val-Lys-Gln-Ile-Glu-Ser-Lys-Ser-Ala-Phe-Gln- Glu-Val-Leu-Asp-Ser-Ala-Gly-Asp-Lys-Leu-Val-Val- Val-Asp-Phe-Ser-Ala-Thr-Trp-Cys-Gly-Pro-Cys-Lys- Met-Ile-Lys-Pro-Phe-Phe-His-Ala-Leu-Ser-Glu-Lys- Phe-Asn-Asn-Val-Val-Phe-Ile-Glu-Val-Asp-Val-Asp- Asp-Cys-Lys-Asp-Ile-Ala-Ala-Glu-Cys-Glu-Val-Lys- Cys-Met-Pro-Thr-Phe-Gln-Phe-Phe-Lys-Lys- Gly-Gln-Lys-Val-Gly-Glu-Phe-Ser-Gly-Ala-Asn-Lys- Glu-Lys-Leu-Glu-Ala-Thr-Ile-Asn-Glu-Leu-Leu.
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PMID:Amino acid sequence of thioredoxin isolated from rabbit bone marrow determined by tandem mass spectrometry. 316 11

The complete primary structure of thioredoxin from Rhodobacter sphaeroides Y has been determined by analysis of peptides after cleavage with cyanogen bromide, chymotrypsin and trypsin. Peptides were separated by HPLC and analyzed by liquid-phase and gas-phase sequencer degradations. The protein consists of 105 residues (Mr = 11,180); its amino acid sequence shows a clear homology to the five known thioredoxins from plant or bacterial sources, with 40-56% residue identity when the proteins are aligned at the active-site disulfide. Not only the active-site regions are conserved, but also residues which belong to the hydrophobic surface suggested to be important for binding of procaryote thioredoxins in redox interactions with other proteins (residues 75-76; 91-93 in Escherichia coli). A three-dimensional model of Rb. sphaeroides thioredoxin has been derived from the E. coli crystallographic structure with computer graphics. This model indicates that the overall structures as well as the active sites are closely similar; however, the residue substitutions allow both proteins to adopt different local folding as shown in the hydrophobic core.
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PMID:Amino acid sequence determination and three-dimensional modelling of thioredoxin from the photosynthetic bacterium Rhodobacter sphaeroides Y. 328 Mar 8

The amino acid sequence of the thioredoxin isolated from the photosynthetic green sulfur bacterium Chlorobium thiosulfatophilum was determined chiefly by fast atom bombardment mass spectrometry combined with Edman degradation and tandem mass spectrometry. For this purpose, the protein was digested with trypsin, alpha-chymotrypsin, thermolysin, and Staphylococcus aureus protease or combinations thereof. Chemical cleavage with cyanogen bromide was also used alone or in combination with trypsin. The resulting sequence of 108 amino acids is as follows: Ala-Gly- Lys-Tyr-Phe-Glu-Ala-Thr-Asp-Lys-Asn-Phe-Gln- Thr-Glu-Xle-Xle-Asp-Ser-Asp-Lys-(Ala-Val)-Xle- Val-Asp-Phe-Trp-Ala-Ser-Trp-Cys-Gly-(Pro-Cys)- Met-Met-Xle-Gly-Pro-Val-Xle-Glu-Gln-Xle-Ala-Asp- Asp-Tyr-Glu-Gly-Lys-Ala-Xle-Xle-Ala-Lys-Xle-Asn- Val-Asp-Glu-Asn-Pro-Asn-Xle-Ala-Gly-Gln-Tyr-Gly- Xle-Arg-Ser-Xle-Pro-Thr-Met-Xle-Xle-Xle-Ly s- (Gly-Gly-Lys)-Val-Val-Asp-Gln-Met-Val-Gly-Ala- Xle-Pro-Lys-Asn-Met-Xle-Ala-Lys-Lys-Xle-Asp-Glu-His-Il e-Gly (where Xle represents leucine or isoleucine; sequences in parentheses are based on homology considerations). It exhibits less than 53% homology with Escherichia coli thioredoxin.
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PMID:Mass spectrometrically derived amino acid sequence of thioredoxin from Chlorobium, an evolutionarily prominent photosynthetic bacterium. 329 35

The primary structure of thioredoxin, a redox protein isolated from Chromatium vinosum, was determined by high-performance tandem mass spectrometry, which permitted sequencing of the 14 peptides (ranging in length from 2 to 18 amino acids) generated by digestion with trypsin and of several peptides produced by Staphylococcus aureus protease. The mass spectrometrically determined molecular weights of the peptides from the latter digest were used to properly align the tryptic peptides, which could also be accomplished on the basis of the considerable homology with Escherichia coli thioredoxin. Finally, the molecular weight of the Chromatium thioredoxin was determined by mass spectrometry and found to be 11,748.0, in good agreement with 11,750.2 calculated for the proposed sequence. Although it was difficult to establish by mass spectrometry, five leucines and three isoleucines could be identified, leaving only eight undifferentiated.
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PMID:The primary structure of thioredoxin from Chromatium vinosum determined by high-performance tandem mass spectrometry. 356 66

Rabbit polyclonal antibody was raised to a chemically synthesized nonapeptide (Trp-Ala-Glu-Trp-Cys-Gly-Pro-Cys-Lys) corresponding to the active-site sequence of Escherichia coli thioredoxin. The antiserum efficiently inhibited thioredoxin activity in the standard thioredoxin reductase/NADPH coupled assay. This inhibition was blocked by preincubation of the antiserum with the nonapeptide. Tight association of the E. coli thioredoxin to the active-site antibody required SDS denaturation. These results suggest that thioredoxin reductase (NADPH: oxidized-thioredoxin oxidoreductase, EC 1.6.4.5) alters the conformation of thioredoxin sufficiently to permit binding to the antibody. The antiserum bound to plant and liver thioredoxins. Bovine pancreatic trypsin inhibitor, whose active site (Gly-Pro-Cys-Lys) is homologous to that of thioredoxin, also competes for the active-site antibody. This result led to experiments showing that thioredoxin can inhibit the digestion of cytochrome c by trypsin. The ability of thioredoxin to act as a trypsin inhibitor analogue provides a rationale for thioredoxin's resistance to digestion by trypsin.
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PMID:The catalytic active site of thioredoxin: conformation and homology with bovine pancreatic trypsin inhibitor. 370 71

Thioredoxin from the cyanobacterium Anabaena 7119 serves as electron donor to ribonucleotide reductase and as a protein disulfide reductase. This small, heat-stable protein was found to have structural and functional similarities to thioredoxins from both bacterial and mammalian sources. We here report the complete primary structure of Anabaena thioredoxin. The structure was determined by analysis of peptides obtained after cleavage with cyanogen bromide, Staphylococcus aureus protease, and trypsin. The protein consists of 106 residues with the following amino acid sequence: Ser-Ala-Ala-Ala-Gln-Val-Thr-Asp- Ser-Thr-Phe-Lys-Gln-Glu-Val-Leu-Asp-Ser-Asp-Val-Pro-Val-leu-Val-Asp-Phe- Trp-Ala-Pro-Trp-Cys-Gly-Pro-Cys-Arg-Met-Val-Ala-Pro-Val-Val-Asp-Glu- Ile-Ala-Gln-Gln-Tyr-Glu-Gly-Lys-Ile-Lys-Val-Val-Lys-Val-Asn-Thr-Asp- Glu-Asn-Pro-Gln-Val-Ala-Ser-Gln-Tyr-Gly-Ile-Arg-Ser-Ile-Pro-Thr-Leu- Met-Ile-Phe-Lys-Gly-Gly-Gln-Lys-Val-Asp-Met-Val-Val-Gly-Ala-Val-Pro- Lys-Thr-Thr-Leu-Ser-Gln-Thr-Leu-Glu-Lys-His-Leu. The sequence of Anabaena thioredoxin shows a definite homology to the protein from Escherichia coli, with 49% residue identities occurring in the proteins when aligned at the active site disulfide.
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PMID:The primary structure of thioredoxin from the filamentous cyanobacterium Anabaena sp. 7119. 392 69

Two enzyme systems capable of reducing disulphide bonds both in low-Mr compounds and in polypeptides and proteins exist. One consists of thioltransferase in combination with reduced glutathione and glutathione reductase, and the second consists of thioredoxin in combination with thioredoxin reductase. Their relative effectiveness in catalysing disulphide reduction of various substrates in rat liver cytosol was evaluated in the present study. The thioltransferase-dependent system was found to be more efficient in reducing small molecules. Insulin was most effectively reduced by the thioredoxin system. Bovine trypsin was a better substrate for thioltransferase, and partially proteolysed bovine serum albumin was equally good for the two systems. Thus, in the case of protein disulphide bonds, the nature of the particular substrate used determines which of the two reducing systems is the more important.
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PMID:Relative contributions of thioltransferase-and thioredoxin-dependent systems in reduction of low-molecular-mass and protein disulphides. 635 44


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