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:3.4.21.1 (chymotrypsin)
10,938 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

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

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

Glycoprotein (GP) IIb-IIIa serves as the platelet fibrinogen receptor. Studies of the tertiary structure of GPIIIa have shown that the protein has a large loop structure of at least 325 amino acids in length. To further characterize this loop structure, intact platelets were digested with alpha-chymotrypsin. Digestion products were examined using the anti-GPIIIa monoclonal antibodies (MoAbs) AP3, D3GP3, and C5GP3, as well as the human alloantibody, anti-PLA1. AP3 recognized GPIIIa digestion products of 109, 95, and 68 Kd. D3GP3 and C5GP3 recognized an additional band of 51 Kd. Time course digestions demonstrated that the 51-Kd fragment was generated by proteolysis of the 68-Kd peptide. Sequence analysis of the reduced 51-Kd peptide showed that this fragment began at amino acid 422. The nonreduced 51-Kd peptide was reactive with antibodies directed against the first 13 amino acids of GPIIIa, demonstrating the presence of a covalently attached N-terminal peptide. These data suggest that: (1) the minimum length of the loop structure is at least 384 amino acids; (2) the AP3 epitope is formed at least in part by a determinant contained within residues 348 to 421; and (3) the D3GP3 and C5GP3 epitopes are contained within amino acids 422 to 692 of GPIIIa, a region that may be flexible and involved in conformational changes that occur after ligand binding.
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PMID:Further characterization of the loop structure of platelet glycoprotein IIIa: partial mapping of functionally significant glycoprotein IIIa epitopes. 172 Jun 99

During proteolytic digestion of myosin to prepare HMM or HMM-S-1 subfragments, myosin light chains are affected variously according to experimental conditions. In the presence of Ca2+ at low ionic strength trypsin rapidly degrades the DTNB light chain to a 18 K peptide. This new DTNB light chain is compared to a DTNB (17K) light chain obtained by chymotryptic digestion under similar conditions as shown here and in parallel studies. (Weeds and Pope (1977), J. Mol. Biol, 111, 129--157). Whereas the chymotryptic DTNB (17K) has lost its phosphorylation site (Ser-15), tryptic DTNB (18K) has lost only a strongly basic N-terminal peptide. A transitory (ca 14K) fragment is formed when digestion occurs in the presence of EDTA. A-1 light chain (20.7K) is cut to form a 20K species when myosin (of (CT)-HMM obtained ina high ionic strength medium) is digested with trypsin whether Me2+ is present or not. The new formed species has also lost its strongly basic N-terminal peptide and assumes a primary structure closer to that of A-2. Chymotrypsin was shown to have no effect on the A-1 light chain under the present conditions, whereas A-2 is not affected by chymotrypsin or trypsin under any of the conditions described in the present study.
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PMID:Fate of the light chains in the course of proteolytic digestion of rabbit fast skeletal myosin. 676 1

Myoglobin isolated from red muscle of the gummy shark M. antarcticus was purified by gel filtration and ion-exchange chromatography on carboxymethyl cellulose in 8 M urea-thiol buffer. Amino acid analysis and sequence determination showed 148 amino acid residues. The amino terminal residue is acetylated as shown by nuclear magnetic resonance and mass spectrographic analysis of an N-terminal peptide. There is a deletion of four residues at the amino terminal end as well as one residue in the CD interhelical area relative to other myoglobins. These overall differences were also found previously in myoglobin of Heterodontus portusjacksoni. The complete amino acid sequence has been determined following digestion with trypsin, chymotrypsin, thermolysin, staphylococcal protease and cyanogen bromide. Sequences of purified peptides were determined by the dansyl-Edman procedure. The amino acid sequence showed approximately 88 differences from mammalian, monotreme, bird and tuna myoglobins, slightly more than previously reported for H. portusjacksoni usually considered a more primitive animal. There were 24 residues common to both shark myoglobins that were different from those present in other myoglobins. The sequence has been compared to the myoglobin of yellowfin tuna and other myoglobins.
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PMID:Myoglobins of cartilaginous fishes. II. Isolation and amino acid sequence of myoglobin of the shark Mustelus antarcticus. 743 64

Trypanosoma brucei ornithine decarboxylase was reconstituted by coexpression of two polypeptides corresponding to residues 1-305 and residues 306-425 in Escherichia coli. The two peptides were coexpressed, at wild-type levels, from a single transcriptional unit that was separated by a 15-nucleotide untranslated region containing a ribosome binding site. The fragmented enzyme was purified and analyzed. The N- and C-terminal peptides are tightly associated into a fully active tetramer which has the same molecular weight as the native dimer. The kinetic constants (Km and kcat) measured for the decarboxylation of ornithine are identical to those obtained for the wild-type enzyme. These results suggest that the enzyme is organized into two structural domains, with a domain boundary in the region of amino acid 305. In contrast, the individual N- and C-terminal peptides are expressed primarily as inclusion bodies. Small quantities of soluble N-terminal peptide could be purified. This truncated protein is capable of inhibiting the wild-type enzyme, suggesting that it is folded into a native-like structure. Limited proteolysis with trypsin or chymotrypsin identifies a likely surface loop at amino acids 160-170, present in both the mouse and T. brucei enzyme, which positions one or more functionally important active site residues (e.g., Lys169). Kinetic analysis of a chimeric enzyme composed of T. brucei and mouse ornithine decarboxylase suggests that the substrate carboxylate binding determinant is located between residues 1 and 170.
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PMID:Domain organization and a protease-sensitive loop in eukaryotic ornithine decarboxylase. 757 30

The pregnancy and tumor marker human chorionic gonadotropin (hCG) belongs to the family of the glycoprotein hormones. Information on epitope forming sequences of hCG and its subunits hCG alpha and hcg beta has significant impact on the examination of intra- and extracellular metabolism and the standardization of diagnostic assay systems. Variants of hCG appear in biological fluids with variable modifications on different parts of the molecule. These changes may influence the binding patterns of monoclonal antibodies (MCA), thereby causing erroneous results in hCG immunoassays. The aim of the present work was to investigate the influence of peptide bond cleavages and the loss of certain segments of the molecule, which were induced by proteases on the expression of the seven hCG alpha-(alpha 1-alpha 7), nine hCG beta- (beta 1-beta 9) and four hCG beta-core-fragment-epitopes (beta 10-beta 13), previously identified by us [1-10]. To this end, we digested hCG alpha and hCG beta with chymotrypsin. Hormone fragments were separated by high performance liquid chromatography (HPLC) and subsequently immunochemically examined by direct binding radioimmunoassay (DB-RIA), competitive RIA and immunoenzymometric assays (IEMA). Fractions containing hCG-like immunoreactivity were sequenced by Edman and carboxypeptidase-Y degradation. It appeared that: (I) Amino acids (AA) alpha 41-47 and the peptide bonds between AA alpha 40/41, alpha 47/48 and alpha 29/30 do not influence the expression of the 7 alpha-epitopes, (II) The absence of the hCG beta N-terminus plays a crucial role for the formation of epitopes beta 10 and beta 13. (III) Neither the presence nor the absence of the C-terminal peptide of hCG beta (hCG beta CTP, AA beta 114-145) has any importance for the expression of epitopes beta 1-beta 7 and beta 10-beta 13 (IV).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:[Fragments of human chorionic gonadotropin (hCG): diagnosis of pregnancy and tumors]. 787 85

Wheat serpin genes have been identified by Southern blot hybridization with three distinct barley protein Z probes. Immunoblot analysis with a monoclonal antibody towards barley protein Z confirmed expression of related M(r) approximately 40 kDa proteins in wheat grain. The wheat serpins were extracted under reducing conditions and separated from beta-amylase and other seed proteins by thiophilic adsorption and anion-exchange chromatography. One molecular form possessing chymotrypsin inhibitory activity was isolated in a reactive site cleaved form on a chymotrypsin affinity column. N-terminal amino acid sequences of a CNBr fragment and of the C-terminal peptide from the cleaved inhibitor (M(r) 4574 +/- 4 Da) verified homology with barley protein Z and mammalian serpins. The native inhibitory serpin was demonstrated to form an SDS-stable complex with alpha-chymotrypsin.
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PMID:Serpins from wheat grain. 816 22

We have previously shown that rat brain tubulin, a heterodimer consisting of an alpha and beta monomer, can be covalently labeled with [3H]colchicine by near UV irradiation. Most of the label appears in beta-tubulin. We show here that beta-tubulin can be separated and purified from SDS preparative gels and analyzed by proteolysis. Chymotrypsin yielded a labeled approximately 4-kDa band that contained two peptides. Tryptic digestion also yielded an approximately 4-kDa band containing two peptides. Sequence analysis revealed a peptide of residues 1-36 and 213-242 for chymotrypsin and a peptide of residues 1-46 and 214-241 for trypsin. To identify which peptide carried the label, limited hydrolysis of beta-tubulin was done with trypsin; this procedure yielded a labeled 16-kDa N-terminal peptide and a 35-kDa C-terminal peptide, as identified by antibodies. Isolation of these peptides and extensive digestion with trypsin yielded two labeled peptides corresponding to residues 1-46 from the 16-kDa N-terminal fragment and residues 214-241 from the 35-kDa C-terminal fragment. These results show that at least two regions in beta-tubulin are specifically involved in colchicine binding and that the span of the colchicine molecule, < or = 11 A, bridges these two regions in the native beta monomer.
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PMID:Localization of the colchicine-binding site of tubulin. 826 96


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