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 Vicia angustifolia proteinase inhibitor was incubated with p-toluenesulfonyl-L-phenylalanine chloromethyl ketone-trypsin (EC 3.4.21.4) and a main product was isolated. The purified product was different to the first trypsin-modified V. angustifolia inhibitor. The C-terminal residues of the new derivative were arginine, which was also the C-terminal of the cleaved antitryptic site; lysine was a newly exposed C-terminal. These results suggest that the new derivative lacks the C-terminal portion of the native inhibitor, which has asparagine at its C-terminus. The liberated C-terminal peptide had the following amino acid sequence: H-Glu-Glu-Val-Ile-Lys-Asn-OH. The derivative lacking the C-terminal hexapeptide still possesses inhibitory activities against trypsin and alpha-chymotrypsin (EC 3.4.21.1), however, its antichymotryptic activity was inactivated by incubation with chymotrypsin at pH 8.0.
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PMID:Isolation and activities of the trypsin-modified Vicia angustifolia proteinase inhibitor lacking carboxyl-terminal hexapeptide. 3 67

Trypsin is a prototype of a large group of enzymes belonging to serine proteinases. The X-ray crystal-structure analyses of its proenzyme trypsinogen, of the active trypsin and of their complexes formed with the pancreatic trypsin inhibitor (PTI) have considerably enhanced our understanding of the mechanisms of activitation, action and inhibition. The trypsinogen is an incompletely folded molecule. Its substrate-binding site becomes only completely fixed upon the enzymatic cleavage of an N-terminal peptide. The contact regions of trypsin and PTI are almost complementary. The complex formed is a (stable) intermediate in the normal tryptic substrate-cleavage reaction.
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PMID:[Activation, activity and inhibition of bovine trypsin]. 38 46

Conformational studies on an isolated integral membrane protein are reported. Lipoprotein of Escherichia coli outer membrane was released from murein by treatment with either lysozyme or trypsin. The isolated lysozyme-released lipoprotein (lipoprotein I) contained 2 or 3 muropeptides covalently linked at the C-terminal end, while the trypsin-released lipoprotein (lipoprotein II) was free of muropeptides and lacked the C-terminal peptide Tyr-Arg-Lys. Circular dichroism spectra of the two preparations were essentially identical, and they show an alpha-helix content of about 80%. According to calculations based on the Chou-Fasman rules for proteins of known sequence, lipoprotein is 64% alpha-helix and 15% beta-structure. Infrared spectroscopy qualitatively supports these values. The conformation was stable in the pH range of 5 - 12. Danaturation of lipoprotein by heat, 8 M urea, or sodium dodecylsulphate was a fully reversible, cooperative process. The thermal denaturation of lipoprotein occurs in two steps with transition points at 79.4 degrees C for lipoprotein I and at 85.1 degrees C for lipoprotein II. Lioprotein markedly changes conformation at dodecylsulphate concentrations where micelle formation sets in. The unusual behaviour of the lipoprotein convormation in sodium dodecylsulphate is discussed in relation to the lipoprotein conformation and aggregation within the membrane.
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PMID:Conformational studies on murein-lipoprotein from the outer membrane of Escherichia coli. 79 57

Performic-acid-oxidized isoinhibitor K of snails (Helix pomatia) was subjected to arginine-directed tryptic proteolysis. Six peptide fragments including one overlap peptide from limited cleavage of the Arg-3-Pro-4 bond were purified to homogeneity. Four arginine peptides and the C-terminal peptide were sequenced by automatic Edman degradation using a special peptide program. The phenylthiohydantoins were all identified by chemical ionization mass spectrometry, except four cysteic acid residues that were identified on an amino acid analyzer after acid hydrolysis. Quantitative evaluation of the phenylthiohydantoins by chemical ionization mass spectrometry using total molecular-ion beam integration greatly facilitated sequencing. The mass spectrum of the dipeptide less than Glu-Gly revealed that the N-terminus was blocked by pyroglutamic acid. The complete amino acid sequence of isoinhibitor K was determined. An almost 50% homology between the sequences of the snail inhibitor and the bovine trypsin-kallikrein inhibitor (Kunitz) became obvious. A comparison of all homologous sequences of this particular class of proteins known to date is presented.
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PMID:The amino-acid sequence of isoinhibitor K form snails (Helix pomatia). A sequence determination by automated Edman degradation and mass-spectral identification of the phenylthiohydantoins. 118 46

The method of high-voltage paper electrophoresis may be applied not only for peptide separation, but also in modifications and combinations with other methods, so, by means of aminoethylation and maleylation it is possible to broaden or narrow the range of trypsin action. This, in its turn, makes it possible to isolate preparatively lysin- and arginine-containing peptides, oxidation with performic acid enables the thyol-containing fragments to be isolated and application of carboxypeptidase A-C-terminal peptide of protein. When studying the primary structure of proteins the method has already found its widest application but with an increase in the number of methods of protein specific modification its potentiabilities will be even wider.
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PMID:[High-voltage electrophoresis and its application in combination with other methods for protein structure studies]. 121 56

A method was developed for direct microsequencing of N alpha-acetylated proteins electroblotted onto polyvinylidene difluoride membranes from polyacrylamide gels. N alpha-Acetylated proteins (greater than 32 pmol), including horse heart cytochrome c, five mutants of yeast cytochrome c, and bovine erythrocyte superoxide dismutase, were separated by SDS-PAGE and electroblotted onto polyvinylidene difluoride membranes. The portions of the membrane carrying the bands were cut out and treated with 0.5% polyvinylpyrrolidone in acetic acid solution at 37 degrees C for 30 min. The protein was digested on the membrane with 5-10 micrograms of trypsin at 37 degrees C for 24 h. During tryptic digestion, the resultant peptides were released from the membrane and the N-terminal peptide was efficiently deblocked with 50 mU of acylamino acid-releasing enzyme at 37 degrees C for 12 h. Picomole levels of the deblocked proteins could be sequenced directly by use of a gas-phase protein sequencer.
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PMID:Deblocking and subsequent microsequence analysis of N alpha-blocked proteins electroblotted onto PVDF membrane. 132 65

The channel-forming protein aerolysin is secreted as a protoxin which can be activated by proteolytic removal of a C-terminal peptide. The activation and subsequent oligomerization of aerolysin were studied using a variety of spectroscopic techniques. Mass spectrometric determination of the molecular weights of proaerolysin and aerolysin permitted identification of the sites at which the protoxin is processed by trypsin and chymotrypsin. The results of far- and near-UV circular dichroism measurements indicated that processing with trypsin does not lead to major changes in secondary or tertiary structure of the protein. An increase in tryptophan fluorescence intensity and a small red shift in the maximum emission wavelength of tryptophans could be observed, suggesting that there is a change in the environment of some of the tryptophans. There was also a dramatic increase in the binding of the hydrophobic fluorescent probe 1-anilino-8-naphthalenesulfonate during activation, leading us to conclude that a hydrophobic region in the protein is exposed by trypsin treatment. Using measurements of light scattering, various parameters influencing oligomerisation of trypsin-activated aerolysin were determined. Oligomerization rates were found to increase with the concentration of aerolysin, whereas they decreased with increasing ionic strength.
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PMID:Spectroscopic study of the activation and oligomerization of the channel-forming toxin aerolysin: identification of the site of proteolytic activation. 138 79

A pentapeptide which potently inhibits primary IgE antibody formation, Asp-Ser-Asp-Gly-Lys (DSDGK), has been efficiently produced with the aid of the dihydrofolate reductase (DHFR) handle [M. Iwakura, et al. (1992) J. Biochem. 111, 37-45]. The genes coding fused proteins comprising DHFR and multimeric forms of DSDGK, namely, DHFR-(DSDGK)3, DHFR-(DSDGK)14, and DHFR-(DSDGK)28, were constructed and expressed in Escherichia coli. The C-terminal peptides attached to DHFR did not affect the expression or the function of the DHFR handle, even when the length of the C-terminal peptide was as long as 160 amino acid residues. The fused proteins were easily purified by methotrexate affinity chromatography, one of the major advantages of the DHFR handle. The fused proteins were digested with trypsin and the monomeric peptide, DSDGK, was purified by HPLC. The yields of the peptide were estimated to be 11, 43, and 99 mg per 1 gram of the total cell proteins from E. coli cells producing DHFR-(DSDGK)3, DHFR-(DSDGK)14, and DHFR-(DSDGK)28, respectively.
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PMID:Efficient production of a small peptide by expression as a multimeric form fused with the dihydrofolate reductase affinity handle. 147 25

Sequences located in the N-terminal region of the high M(r) 2-oxoglutarate dehydrogenase (E1) enzyme of the mammalian 2-oxoglutarate dehydrogenase multienzyme complex (OGDC) exhibit significant similarity with corresponding sequences from the lipoyl domains of the dihydrolipoamide acetyltransferase (E2) and protein X components of eukaryotic pyruvate dehydrogenase complexes (PDCs). Two additional features of this region of E1 resemble lipoyl domains: (i) it is readily released by trypsin, generating a small N-terminal peptide with an apparent M(r) value of 10,000 and a large stable 100,000 M(r) fragment (E1') and (ii) it is highly immunogenic, inducing the bulk of the antibody response to intact E1. This 'lipoyl-like' domain lacks a functional lipoamide group. Selective but extensive degradation of E1 with proteinase Arg C or specific conversion of E1 to E1' with trypsin both cause loss of overall OGDC function although the E1' fragment retains full catalytic activity. Removal of this small N-terminal peptide promotes the dissociation of dihydrolipoamide dehydrogenase (E3) from the E2 core assembly and also affects the stability of E1 interaction. Thus, structural roles which are mediated by a specific gene product, protein X in PDC and possibly also the E2 subunit, are performed by similar structural elements located on the E1 enzyme of the OGDC.
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PMID:Sequences directing dihydrolipoamide dehydrogenase (E3) binding are located on the 2-oxoglutarate dehydrogenase (E1) component of the mammalian 2-oxoglutarate dehydrogenase multienzyme complex. 150 15

We have investigated the transmembrane topology of the bovine heart mitochondrial porin by means of proteases and antibodies raised against the amino-terminal region of the protein. The antisera against the human N-terminus reacted with porin in Western blots of NaDodSO4-solubilized bovine heart mitochondria and with the membrane-bound porin in enzyme-linked immunosorbent assay (ELISA). The immunoreaction with mitochondria coated on microtiter wells showed that the amino-terminal region of the protein is not embedded in the lipid bilayer but is exposed to the cytosol. Back-titration of unreacted anti-N-terminal antibodies after their incubation with intact mitochondria demonstrated that the porin N-terminus is also exposed in "noncoated" mitochondria. No difference in antisera reactivity was observed between intact and broken mitochondria. Intact and broken mitochondria were subjected to proteolysis by specific proteases. The membrane-bound bovine heart porin was strongly resistant to proteolysis, but a few specific cleavage sites were observed. Staphylococcus aureus V8 protease gave a large 24K N-terminal peptide, trypsin produced a 12K N-terminal and an 18K C-terminal peptide, and chymotrypsin gave two peptides of Mr 19.5K and 12.5K, which were both recognized by the antiserum against the human N-terminus. Carboxypeptidase A was ineffective in cleaving the membrane-bound porin in both intact and broken mitochondria. Thus, the carboxy-terminal part of the protein is probably not exposed to the water phase. The cleavage patterns of membrane-bound porin, obtained with S. aureus V8 protease, trypsin, and chymotrypsin, showed no difference between intact and broken mitochondria, thus indicating that all porin molecules have the same orientation in the membrane. The computer analysis of the sequence of human B-lymphocyte porin suggested that 16 beta-strands can span the phospholipid bilayer. This result, together with the overall information presented, allowed us to draw a possible scheme of the transmembrane arrangement of mammalian mitochondrial porin.
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PMID:Peptide-specific antibodies and proteases as probes of the transmembrane topology of the bovine heart mitochondrial porin. 171 14


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