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)

We have identified four discrete proteolytic fragments of von Willebrand factor (vWF) that define two collagen-binding domains. Two of the fragments tested, T 96 kDa and T 55 kDa, were generated by digestion with trypsin, and two, Fragments I and III, with Staphylococcus aureus V8 protease. The larger Fragment III, a disulfide-linked homodimer, extends between residues 1 and 1365 of the 2050-residue vWF subunit and comprises the sequence of all the others. T 96 kDa, also a disulfide-linked homodimer, extends between residues 449 and 728. T 55 kDa and Fragment I, both single-chain polypeptides, have a partial sequence overlap corresponding to residues 911-1114, and together extend from residue 730 to 1365. The ability of the fragments to interfere with the vWF-collagen interaction was evaluated by measuring inhibition of 125I-labeled vWF binding to fibrillar bovine collagen types I and III. All the four fragments tested inhibited binding. Native conformation was essential for expression of this function; denaturation with guanidine hydrochloride and reduction of disulfide bonds resulted in marked reduction or complete loss, respectively, of the inhibitory activity at all the concentrations tested. Two monoclonal antibodies were prepared, one directed against T 96 kDa and the other against Fragment I. Both antibodies partially inhibited vWF binding to collagen, and their inhibitory effect was enhanced when they were used together. 125I-Labeled Fragment I bound to collagen in a saturable manner, and the binding was completely blocked by both T 96 kDa and T 55 kDa. Thus, we have identified at least two distinct functional domains of vWF that concurrently mediate the vWF-collagen interaction. The two domains appear to share a common recognition site on collagen.
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PMID:Isolation and characterization of two domains of human von Willebrand factor that interact with fibrillar collagen types I and III. 349 19

The nonenzymatic glycosylation (glycation) of Cu-Zn-superoxide dismutase led to gradual inactivation of the enzyme (Arai, K. Iizuka, S., Tada, Y., Oikawa, K., and Taniguchi, N. (1987) Biochim. Biophys. Acta 924, 292-296). The purified superoxide dismutase from human erythrocytes comprises both glycated and nonglycated forms. The nonglycated Cu-Zn-superoxide dismutase was isolated by boronate affinity chromatography. Incubation of the nonglycated superoxide dismutase with D-[6-3H]glucose in vitro resulted in the gradual accumulation of radioactivity in the enzyme protein, and Schiff base adducts were trapped by NaBH4. The sites of glycation of the superoxide dismutase were identified by amino acid analysis after reverse-phase high performance liquid chromatography of the trypsin-treated peptides. Lysine residues, i.e. Lys3, Lys9, Lys30, Lys36, Lys122, and Lys128, were found to be glycated. Three of the glycated sites lie in Lys-Gly, two in Lys-Ala, and one in Lys-Val. The inactivation of the superoxide dismutase on the glycation is due mainly to the glycation of Lys122 and Lys128, which are supposed to be located in an active site liganding loop. The remaining five sites, such as Lys-Glu, Lys-Asp, Lys-His, and Lys-Thr are relatively inactive as to the formation of either a Schiff base or an Amadori adduct.
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PMID:Glycation and inactivation of human Cu-Zn-superoxide dismutase. Identification of the in vitro glycated sites. 368 Feb 84

Several years ago this laboratory presented evidence that SBP is a dimer composed of two subunits having similar molecular weights. The question of whether or not these subunits are identical and therefore products of a single gene remained unanswered. We now report that the two polypeptide chains are identical and that SBP is a homodimer. The experimental approach was to reduce and [14C]alkylate cystine residues in human SBP, digest the product with trypsin or cyanogen bromide and determine the number of unique amino acid sequences around each [14C]carboxymethylcysteine residue. Only four unique sequences were found when all the radioactive peptides were analyzed. Since there are eight half-cystine residues per dimer, the results support a homodimeric structure.
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PMID:Molecular organization of the sex steroid-binding protein (SBP) of human plasma. 370 28

Light-mediated conformational changes in highly purified 124-kDa phytochrome preparations from etiolated oat seedlings have been identified by steric exclusion high performance liquid chromatography and limited proteolytic studies. Steric exclusion high performance liquid chromatography studies of oat and rye phytochromes show photoreversible changes in retention times, with the red absorbing form of phytochrome (Pr form) eluting later than the far red absorbing form of phytochrome produced by saturating red light illumination of Pr (Pfr form) in a variety of different mobile phase buffers. Molecular mass calibration with globular protein standards in Tris-glycol buffers provides estimates of 318-349 and 363-366 kDa for the molecular sizes of the Pr and Pfr forms, respectively. These analyses support earlier studies that phytochrome is a nonglobular homodimer of 124-kDa subunits in vitro. Limited proteolytic dissection of phytochrome in nondenaturing buffers with seven different endoproteases provides evidence for two "operational" domains within the 124-kDa subunit with molecular mass values of 69-72 and 52-55 kDa. The larger 69-72-kDa domain contains the site for the chromophore attachment as shown by gel electrophoresis derived enzyme-linked immunosorbent assay utilizing site-directed rabbit antiserum to a synthetic undecapeptide which is homologous with the chromophore binding site on oat phytochrome. This chromophore domain exhibits a compact structure, resistant to further proteolysis except near its N terminus. By contrast, the 52-55-kDa nonchromophore domain contains multiple sites for further proteolytic cleavage as revealed by rapid cleavage to smaller polypeptide fragments. Detailed kinetic analyses of the limited proteolytic cleavage of phytochrome with four endoproteases, subtilisin BPN', thermolysin, trypsin, and clostripain, has mapped specific regions within the 124-kDa subunit that participate in light-induced conformational changes. These include a 4-10-kDa region near the N terminus of the chromophore binding domain and at least two regions within the nonchromophore domain. A comprehensive peptide map of the oat phytochrome subunit is presented, which incorporates the results of these proteolytic studies with the recent, yet unpublished sequence analyses of Avena phytochrome cDNA clones which show the N-terminal localization of the chromophore binding site (Hershey, H. P., Colbert, J. T., Lissemore, J. L., Barker, R. F., and Quail, P. H. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 2332-2336).
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PMID:Structure function studies on phytochrome. Identification of light-induced conformational changes in 124-kDa Avena phytochrome in vitro. 388 93

A protein capable of inhibiting trypsin and other pancreatic proteases has been purified to homogeneity from Escherichia coli by conventional procedures and affinity chromatography. It is stable for at least 30 min at 100 degrees C and pH 1.0, but it is inactivated by digestion with pepsin. The inhibitor has an apparent molecular weight of 38,000 as determined by gel filtration and must be a homodimer since it contains a single 18,000-dalton subunit upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The inhibitor has an isoelectric point of 6.1. One dimeric molecule of the inhibitor can bind two trypsin molecules to form a mixed tetrameric complex, in which trypsin molecules are completely inhibited. The inhibitor is not digested by the trypsin. When N-benzoyl-DL-arginine-p-nitroanilide was used as a trypsin substrate, half-maximal inhibition was observed at 22 nM. This protein also inhibits chymotrypsin, pancreatic elastase, rat mast cell chymase, and human serosal urokinase, but it does not inhibit human pulmonary tryptase, kallikrein, papain, pepsin, Staphylococcus aureus V8 protease, subtilisin, and thermolysin. Surprisingly, it did not inhibit any of the eight soluble endoproteases recently isolated from E. coli (i.e. proteases Do, Re, Mi, Fa, So, La, Ci, and Pi) nor the chymotrypsin-like (protease I) and trypsin-like (protease II) esterases in E. coli. The inhibitor is localized to the periplasmic space and its level did not change with different growth media or stages of cell growth. The physiological function of this E. coli trypsin inhibitor is unknown. We suggest that E. coli trypsin inhibitor be named "Ecotin."
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PMID:Purification from Escherichia coli of a periplasmic protein that is a potent inhibitor of pancreatic proteases. 641 24

The complete amino-acid sequence of the copper-zinc superoxide dismutase of the Photobacterium leiognathi was determined. The fragmentation strategy employed included cyanogen bromide cleavage at its methionine residues and the only tryptophan residue. The S-carboxymethylated chain was further cleaved by means of trypsin, in order to obtain overlapping fragments. For sequence determination automated solid or liquid-phase techniques of Edman degradation were used. C-Terminal amino acids of the entire chain were determined after treatment with carboxypeptidase A. Comparison of the primary structure of this bacterial Cu-Zn superoxide dismutase with the established amino-acid sequences of the other eukaryotic Cu-Zn superoxide dismutases revealed clear homologies. Correspondingly, the Cu-Zn-binding amino-acid residues of the active centre were localized: His45, His47, His70, His79, His125 and Asp91. The two cysteine residues in position 52 and 147 were homologous to the cysteine residues, modelling the essential intrachain disulfide bridge of the corresponding bovine enzyme. As only 25-30% of aligned sequence positions were found to be identical, the enzyme of P. leiognathi shows only a remote phylogenetic relationship towards eukaryotic Cu-Zn superoxide dismutases. When compared to the established phylogenetic tree of the cytochrome c family, this indicates a separate evolution of Cu-Zn superoxide dismutase in Photobacterium. Therefore, a natural gene transfer from the eukaryotic host (ponyfish) to the prokaryotic photobacterium, which Martin and Fridovich postulated 1981 (J. Biol. Chem. 256, 6080-6089) on the basis of amino-acid compositions, can be excluded.
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PMID:The primary structure of Cu-Zn superoxide dismutase from Photobacterium leiognathi: evidence for a separate evolution of Cu-Zn superoxide dismutase in bacteria. 688 93

The coding region of copper/zinc-superoxide dismutase (Cu/Zn-SOD) cDNA from sweet potato, Ipomoea batatas (L.) Lam. cv. Tainong 57, was introduced into an expression vector, pET-20b(+). The Cu/Zn-SOD purified by His-tagged technique showed two active forms (dimer and monomer). The amount of proteins of dimer and monomer appeared to be equal, but the activity of dimeric form was seven times higher than that of monomeric form. The enzyme was dissociated into monomer by imidazole buffer above 1.0 M, acidic pH (below 3.0), or SDS (above 1%). The enzyme is quite stable. The enzyme activity is not affected at 85 degrees C for 20 min, in alkali pH 11.2, or in 0.1 M EDTA and also quite resistant to proteolytic attack. Dimer is more stable than monomer. The thermal inactivation rate constant kd calculated for the monomer at 85 degrees C was 0.029 min-1 and the half-life for inactivation was about 28 min. In contrast, there is no significant change of dimer activity after 40 min at 85 degrees C. The enzyme dimer and monomer retained 83% and 58% of original activity, respectively, after 3 h incubation with trypsin at 37 degrees C, while those retained 100% and 31% of original activity with chymotrypsin under the same condition. These results suggest subunit interaction might change the enzyme conformation and greatly improve the catalytic activity and stability of the enzyme. It is also possible that the intersubunit contacts stabilize a particular optimal conformation of the protein or the dimeric structure enhances catalytic activity by increasing the electrostatic steering of substrate into the active site.
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PMID:Subunit interaction enhances enzyme activity and stability of sweet potato cytosolic Cu/Zn-superoxide dismutase purified by a His-tagged recombinant protein method. 759 15

Rabbit esophageal epithelium, a parakeratinized stratified epithelium, synthesizes as one of its major differentiation products a keratin pair consisting of a basic K4 (59 kDa) and an acidic K13 (41 kDa) keratin. Although immunohistochemical staining data suggest that in esophageal epithelia of some other species these two keratins are suprabasally located, antigenic masking of the epitopes in the basal cells has not been ruled out. Using several well-characterized monoclonal antibodies including AE8, which specifically recognizes K13, coupled with biochemical analysis of keratins of basal and suprabasal cells isolated from confluent rabbit esophageal epithelial culture, we have obtained direct evidence that K4 and K13 keratins are largely absent in the undifferentiated basal cells, but are present in large amounts in suprabasal cells. We also show that in the cornified cell layers that are formed during the terminal stage of esophageal epithelial differentiation, K4 and K13 keratins become disulfide-crosslinked to form three different dimers. Two of them (110 kDa and 100 kDa) are heterodimers and consist of equimolar amounts of K4 and K13; they presumably represent isomers crosslinked via different cysteine residues. The third dimer (90 kDa) was found to be a homodimer of the acidic K13 keratin. Trypsinization experiment established that at least some of the disulfide crosslinks in the K4/K13 heterodimer must involve cysteine residues residing in the trypsin-resistant rod domains of keratins. Air-oxidation of in vitro reconstituted filaments reproduced the two heterodimers, which most likely involve the crosslinking between type I and type II keratins of different coiled coils. The formation of these disulfide-crosslinked keratin dimers, instead of higher molecular mass oligomers or polymers as occurring in the epidermis and hair, may contribute to the formation of cornified cells with a physical stability and rigidity that are optimal for esophageal function. Our data also suggest that interactions involved in the formation of homodimers, thought to be metastable and unimportant during the initial step of filament assembly (i.e. tetramer formation), may actually play an important role in stabilizing a higher order structure in mature keratin filaments.
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PMID:Suprabasal change and subsequent formation of disulfide-stabilized homo- and hetero-dimers of keratins during esophageal epithelial differentiation. 768 69

A Forssman antigen (GalNAc alpha 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1Cer)-binding lectin has been purified from velvet bean (Mucuna derringiana) seeds by a combination of affinity chromatography and reversed phase HPLC. This lectin agglutinates both native and trypsin-treated sheep erythrocytes as well as trypsinized rabbit erythrocytes, but neither native rabbit nor human erythrocytes, irrespective of blood group type. SDS-PAGE and gel filtration chromatography reveal the lectin to be a homodimer consisting of two 54 kDa subunits linked by non-covalent bonds. The results obtained by quantitative precipitation, haemagglutination inhibition and TLC overlay assays indicate that the Mucuna lectin specifically recognizes Forssman antigen and Forssman disaccharide (GalNAc alpha 1-3GalNAc)-related structures.
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PMID:Isolation and characterization of a Forssman antigen-binding lectin from velvet bean (Mucuna derringiana) seeds. 769 47

To study the function of SecA protein and the protein translocation system of Bacillus subtilis, wild-type and mutant SecA proteins were characterized in vivo and in vitro. SecA protein was abundant in a wild-type strain (168) and existed in a stable homodimer. In contrast to this, SecA341 (ts) protein having an amino acid replacement from proline to leucine at residue 431 was undetectable by immunoblotting in the cell lysate of a secA341 mutant (TB301) at the nonpermissive temperature, 42 degrees C. Pulse-chase studies using 35S-methionine showed that newly synthesized SecA protein was rapidly degraded in the mutant at 42 degrees C. Purified SecA341 protein was more sensitive to trypsin and subtilisin than purified wild-type SecA protein in the presence of ATP. These results indicate that the secA341 mutation causes the rapid degradation of mutant SecA protein and a concomitant protein translocation defect in the cell.
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PMID:The rapid degradation of mutant SecA protein in the Bacillus subtilis secA341 (ts) mutant causes a protein translocation defect in the cell. 776 10


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