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)

Previous studies have shown that the non-alpha-helical, amino-terminal head region of vimentin is essential for the formation and stability of vimentin intermediate filaments (IFs). In order to specify its target site on companion protein subunits, it was cut off from vimentin at amino acid position 96 with lysine-specific endoproteinase and allowed to react with intact vimentin and other IF proteins. In solution of high salt concentration (500 mM KCl), the isolated polypeptide (vim NT) showed a high affinity for all cytoplasmic IF proteins tested, but not for nuclear lamins. Employing limited digestion of the IF proteins with different proteinases, the binding site was shown to reside in their alpha-helical rod domains. Other polypeptides possessing alpha-helical regions with the potential to form coiled-coil structures like tropomyosin and myosin subfragment 2 did not react with vim NT. The binding to IF proteins was strongly inhibited by phosphorylation of vim NT and totally abolished in the presence of 200 mM arginine hydrochloride, whereas the same concentration of lysine hydrochloride was ineffective. Limited chymotryptic digestion of vim NT produced polypeptides that were unable to react with the alpha-helical region of vimentin at high salt concentration. Consistent with these observations, vim NT strongly inhibited filament formation in vitro from protofilamentous vimentin. A 14-mer oligopeptide comprising the amino acids 3 to 16 of the amino terminus also inhibited filament formation, though to a lesser extent. Conversely, vim NT and, with a lower efficiency, the 14-mer oligopeptide also severely affected the structure of preformed vimentin filaments by unraveling them. Phosphorylated vim NT was considerably less active in this respect. Further digestion of the rod domain of vimentin with chymotrypsin yielded 17.4 and 21 kDa polypeptides, which were tentatively characterized as originating from the carboxy- and amino-terminal half of the rod domain, respectively. Both formed salt-stable complexes with vim NT, the smaller polypeptide with a higher efficiency than the larger one. These results suggest that the staggered, antiparallel arrangement of the two coiled-coils in the protofilaments of IF proteins is, at least in part, determined by the twofold, symmetrical association of the amino-terminal head regions of one coiled-coil rope structure with the carboxy-terminal halves of the alpha-helical rod domains of the other coiled-coil and that similar interactions occur during filament assembly and in the intact filament.
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PMID:Salt-stable interaction of the amino-terminal head region of vimentin with the alpha-helical rod domain of cytoplasmic intermediate filament proteins and its relevance to protofilament structure and filament formation and stability. 162 50

The 70-kDa neurofilament protein subunit (NF-L) is phosphorylated in vivo on at least three sites (L1 to L3) (Sihag, R. K. and Nixon, R. A. (1989) J. Biol. Chem. 264, 457-464). The turnover of phosphate groups on NF-L during axonal transport was determined after the neurofilaments in retinal ganglion cells were phosphorylated in vivo by injecting mice intravitreally with [32P]orthophosphate. Two-dimensional phosphopeptide maps of NF-L from optic axons of mice 10 to 90 h after injection showed that radiolabel decreased faster from peptides L2 and L3 than from L1 as neurofilaments were transported. To identify phosphorylation sites on peptide L2, axonal cytoskeletons were phosphorylated by protein kinase A in the presence of heparin. After the isolated NF-L subunits were digested with alpha-chymotrypsin, 32P-peptides were separated by high performance liquid chromatography on a reverse-phase C8 column. Two-dimensional peptide mapping showed that the alpha-chymotrypsin 32P-peptide accepting most of the phosphates from protein kinase A migrated identically with the in vivo-labeled phosphopeptide L2. The sequence of this peptide (S-V-R-R-S-Y) analyzed by automated Edman degradation corresponded to amino acid residues 51-56 of the NF-L sequence. A synthetic 13-mer (S-L-S-V-R-R-S-Y-S-S-S-S-G) corresponding to amino acid residues 49-61 of NF-L was also phosphorylated by protein kinase A. alpha-Chymotryptic digestion of the 13-mer generated a peptide which contained most of the phosphates and co-migrated with the phosphopeptide L2 on two-dimensional phosphopeptide maps. Edman degradation of the phosphorylated 13-mer identified serine residue 55 which is located within a consensus phosphorylation sequence for protein kinase A as the major site of phosphorylation. Since protein kinase A-mediated phosphorylation influences intermediate filament assembly/disassembly in vitro, we propose that the phosphopeptide L2 region is a neurofilament-assembly domain and that the cycle of phosphorylation and dephosphorylation of Ser-55 on NF-L, which occurs relatively early after subunit synthesis in vivo, regulaaes a step in neurofilament assembly or initial interactions during axonal transport.
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PMID:Identification of Ser-55 as a major protein kinase A phosphorylation site on the 70-kDa subunit of neurofilaments. Early turnover during axonal transport. 171 55

Rabbit liver cytosolic serine hydroxymethyltransferase exists in several subforms which have different isoelectric points. Incubation of the purified enzyme with chymotrypsin cleaves the enzyme at Trp14. The released amino-terminal 14-mer peptide was shown to exist in three forms of equal concentration. The peptides differ in structure only at the asparaginyl residue at position 5. In addition to asparagine at this position we found both aspartyl and isoaspartyl residues. The deamidation of Asn5 does not appear to occur during the purification of the enzyme. The in vitro rate of deamidation of Asn5 in the enzyme is more than 5-fold slower than the rate of deamidation of this residue in the free 14-mer peptide. The isoaspartyl residue at position 5 serves as a substrate for protein carboxyl methyltransferase both in the free 14-mer peptide and the native enzyme. The enzyme which has had the amino-terminal 14 residues removed by digestion with chymotrypsin still exists in several forms with different isoelectric points. Reaction of peptides from this enzyme with carboxyl methyltransferase suggests that there is at least one more asparaginyl residue in this enzyme other than Asn5 which has undergone deamidation with the formation of isoaspartyl bonds.
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PMID:Evidence for the in vivo deamidation and isomerization of an asparaginyl residue in cytosolic serine hydroxymethyltransferase. 231 67

Two human tumor cell lines were analyzed for the production of human antileucoprotease (ALP). One of them, a human squamous lung carcinoma cell line (HS-24) synthesized, as confirmed by Western blot analysis, high amounts of ALP in serum-free medium. The supernatant inhibited elastase, chymotrypsin and trypsin. Northern blot analysis with an 18-mer radiolabelled oligonucleotide, derived from an ALP specific cDNA clone, revealed a specific mRNA of about 700-800 nucleotides in HS-24 tumor cells. In contrast, a secondary human lung tumor cell line (SB-3), derived from the adrenal cortex, did not synthesize ALP when assayed under identical conditions. The supernatant inhibited only trypsin and chymotrypsin.
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PMID:Secretion of antileucoprotease from a human lung tumor cell line. 367 87

The urea-induced dissociation and subsequent conformational transitions of the nucleotide-bound form of GroEL were studied by light scattering, 4,4'-bis(1-anilino-8- naphthalenesulfonic acid) binding, and intrinsic tyrosine fluorescence. Magnesium ion alone (10 mM) stabilizes GroEL and leads to coordination of the structural transitions monitored by the different parameters. The midpoint of the light-scattering transition that monitored dissociation of the 14-mer with bound magnesium was raised to approximately 3 M, which is considerably higher than the ligand-free form of the protein, which exhibits a transition with a midpoint at approximately 2 M urea. Binding of ADP results in destabilization of the GroEL oligomeric structure, and complete dissociation of the 14-mer in the presence of 5 mM ADP occurs at about 2 M urea with the midpoint of the transition at approximately 1 M urea. The same destabilization by ADP and stabilization by Mg2+ were seen when the conformation was followed by the intrinsic fluorescence. Complexation with the nonhydrolyzable ATP analog, 5'-adenylimidodiphosphate gave an apparent stability of the quaternary structure that was between that observed with Mg2+ and that with ADP. The ADP-bound form of the protein demonstrated increased hydrophobic exposure at lower urea concentrations than the uncomplexed GroEL. In addition, the GroEL-ADP complex is more accessible for proteolytic digestion by chymotrypsin than the uncomplexed protein, consistent with a more open, flexible form of the protein. The implication of the conformational changes to the mechanism of the GroEL function is discussed.
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PMID:The chaperonin GroEL is destabilized by binding of ADP. 749 69

The oligomeric form (14-mer) of the chaperonin protein, Cpn60 (GroEL) from Eschericia coli, displays restricted hydrophobic surfaces and binds tightly one to two molecules of the fluorescent hydrophobic reporter, 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid (bisANS). The 14-mer is resistant to proteolysis by chymotrypsin, and none of the three sulfhydryl groups/monomer react with 6-iodoacetamidofluorescein. When monomers of Cpn60 that are folded and competent to participate in protein folding are formed by low concentrations of urea (< 2.5 M), the hydrophobic exposure increases to accommodate approximately 14 molecules of bisANS/14-mer, the binding affinity for bisANS decreases, and 1 sulfhydryl group/monomer reacts with 6-iodoacetamidofluorescein. These monomers display limited proteolysis by chymotrypsin at several points within a hydrophobic sequence centered around residue 250 to produce a relatively stable N-terminal fragment (approximately = to 26 kDa) and a partially overlapping C-terminal fragment (approximately = to 44 kDa). The exposure of hydrophobic surfaces is facilitated by ATPMg. Ions increase hydrophobic exposure more effectively than urea without dissociation of Cpn60. For example, subdenaturing concentrations of guanidinium chloride (< or = 0.75 M) or the stabilizing salt, guanidinium sulfate, as well as NaCl or KCl are effective. The trivalent cation, spermidine, induces maximum exposure at 5 mM. The results suggest that hydrophobic surfaces can be involved in stabilizing the oligomer and/or in binding proteins to be folded, and they are consistent with suggestions that amphiphilic structures, presenting hydrophobic surfaces within a charged context, would be particularly effective in binding to Cpn60.
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PMID:Hydrophobic surfaces that are hidden in chaperonin Cpn60 can be exposed by formation of assembly-competent monomers or by ionic perturbation of the oligomer. 782 81

beta protein, a key component of Red-pathway of phage lambda is necessary for its growth and general genetic recombination in recombination-deficient mutants of Escherichia coli. To facilitate studies on structure-function relationships, we overexpressed beta protein and purified it to homogeneity. A chemical cross-linking reagent, glutaraldehyde, was used to stabilize the physical association of beta protein in solution. A 67-kDa band, corresponding to homodimer, was identified after separation by SDS-polyacrylamide gel electrophoresis. Stoichiometric measurements indicated a site-size of 1 monomer of beta protein/5 nucleotide residues. Electrophoretic gel mobility shift assays suggested that beta protein formed stable nucleoprotein complexes with 36-mer, but not with 27- or 17-mer DNA. Interestingly, the interaction of beta protein with DNA and the stability of nucleoprotein complexes was dependent on the presence of MgCl2, and the binding was abolished by 250 mM NaCl. The Kd of beta protein binding to 36-mer DNA was on the order of 1.8 x 10(-6) M. Photochemical cross-linking of native beta protein or its fragments, generated by chymotrypsin, to 36-mer DNA was performed to identify its DNA-binding domain. Characterization of the cross-linked peptide disclosed that amino acids required for DNA-binding specificity resided within a 20-kDa peptide at the N-terminal end. These findings provide a basis for further understanding of the structure and function of beta protein.
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PMID:Characterization of the DNA-binding domain of beta protein, a component of phage lambda red-pathway, by UV catalyzed cross-linking. 898 71

The roles of the G-protein-linked thrombin receptor and platelet glycoprotein Ib (GPIb) as alpha-thrombin-binding sites on platelets remain controversial. alpha-Thrombin has been proposed to bind to both GPIb and the hirudin-like domain of the G-protein-linked receptor (from which it cleaves the NH2-terminal extracellular domain to release a 41-mer peptide (TR-(1-41), where TR is alpha-thrombin receptor)) to initiate platelet activation. Using affinity-purified rabbit anti-human TR-(1-41) IgG and immunoblotting, we demonstrated TR-(1-41) release from platelets suspended in Tyrode's buffer containing 2 mM CaCl2 and incubated with >/=0.5 nM alpha-thrombin for 10-60 s at 37 degrees C. As quantified by enzyme-linked immunosorbent assay, 0.32-0.59 nM TR-(1-41) was released from washed platelets (5 x 10(11) platelets/liter) after their incubation with 10 nM alpha-thrombin for 10 s. Parallel binding of alpha-thrombin to and activation of the platelets were confirmed by flow cytometry. A monoclonal antibody against the hirudin-like domain of the G-protein-linked receptor abrogated alpha-thrombin binding to platelets, cleavage of TR-(1-41), and platelet activation by </=1.0 nM (but not 10 nM) alpha-thrombin. Proteolysis of platelet GPIb with Serratia marcescens protease or O-sialoglycoprotein endopeptidase had no effect on alpha-thrombin binding to platelets or their subsequent activation. In contrast, chymotrypsin, which cleaves both GPIb and the G-protein-linked receptor, abrogated alpha-thrombin binding to platelets, TR-(1-41) release, and platelet activation. Furthermore, monoclonal antibodies directed against the reported alpha-thrombin-binding site on GPIb inhibited neither alpha-thrombin binding to nor activation of the platelets. Thus, alpha-thrombin binds to and cleaves the G-protein-linked receptor when it activates platelets, and GPIb does not appear to serve as an important binding site when alpha-thrombin activates platelets.
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PMID:Binding of thrombin to the G-protein-linked receptor, and not to glycoprotein Ib, precedes thrombin-mediated platelet activation. 899 92

Binding of urokinase-type plasminogen activator (uPA) to its cellular receptor (uPAR) renders the cell surface a favored site for plasminogen activation. Recently, a 15-mer peptide antagonist of the uPA-uPAR interaction, with an IC50 value of 10 nM, was identified using phage display technology [Goodson, R. J., Doyle, M. V., Kaufman, S. E., and Rosenberg, S. (1994) Proc. Natl. Acad. Sci. 91, 7129-7133]. In the present study, the molecular aspects of the interaction between this peptide and uPAR have been investigated. We have characterized the real-time receptor binding kinetics for the antagonist using surface plasmon resonance and identified critical residues by alanine replacements. The minimal peptide antagonist thus derived (SLNFSQYLWS) was rendered photoactivatable by replacing residues important for uPAR binding with photochemically active derivatives of phenylalanine containing either (trifluoromethyl)diazirine or benzophenone. These peptides incorporated covalently into purified soluble uPAR upon photoactivation, and this was inhibited by preincubation with receptor binding derivatives of uPA. The intact three-domain structure of uPAR was essential for efficient photoaffinity labeling. Proteolytic domain mapping using chymotrypsin revealed a specific labeling of both uPAR domain I and domains II + III dependent on the position of the photoprobe in the antagonist. On the basis of these studies, we propose the existence of a composite ligand binding site in uPAR combined of residues located in distinct structural domains. According to this model, a close spatial proximity between uPAR domain I and either domains II or III in intact uPAR is required for the assembly of this composite binding site. Since the receptor binding properties of the peptide antagonist closely mimic those of uPA itself, these two ligands presumably share coincident binding site in uPAR.
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PMID:Photoaffinity labeling of the human receptor for urokinase-type plasminogen activator using a decapeptide antagonist. Evidence for a composite ligand-binding site and a short interdomain separation. 952 80

The nature of the nucleation-collapse mechanism in protein folding is probed using 27-mer and 36-mer lattice models. Three different forms for the interaction potentials are used. Three of the four 27-mer sequences have maximally compact and identical native state while the other has a non-compact native conformation. All the sequences fold thermodynamically and kinetically by a two-state process. Analysis of individual trajectories for each sequence using a self-organizing neural net algorithm shows that upon formation of a critical set of contacts the polypeptide chain rapidly reaches the native conformation which is consistent with a nucleation-collapse mechanism. The algorithm, which reduces the identification of the folding nucleus for each trajectory to one of pattern recognition, is used to show that there are multiple folding nuclei. There is a distribution of nucleation contacts in the transition states with some of them occurring with more probability (when averaged over the denatured ensemble) than others. We also show that there is a distribution in the size of the nuclei with the average number of residues in the folding nuclei being less than about one-third of the chain size. The fluctuations in the sizes of the nuclei are large, suggestive of a broad transition region. The folding nuclei, the structures of each are the corresponding transition states, have varying degree of overlap with the native conformation. The distribution of the radius of gyration of the transition states shows that these structures are an expanded form (by about 25% in the radius of gyration) of the native conformation. Local contacts are most dominant in the folding nuclei while a certain fraction of non-local contacts is necessary to stabilize the transition states. The search for the critical nuclei initially involves the formation of local contacts, while non-local contacts are formed later. The fractional values of PhiF for the two 27-mer mutants found by using the protein engineering protocol are consistent with the microscopic picture of partial formation of structures involving these residues in the transition state. These observations lead to a multiple folding nuclei (MFN) model for nucleation-collapse mechanism in protein folding. The major implication of the MFN model is that, even if the residues whose tertiary interactions are formed nearly completely in the transition state are mutated, it does not disrupt the nature of the nucleation-collapse mechanism. We analyze the experiments on chymotrypsin inhibitor 2 and alpha-spectrin SH3 domain and two circular permutants in light of the MFN model. It is shown that the PhiF-value analysis for these proteins gives considerable support to the MFN model. The theoretical and experimental studies give a coherent picture of the nucleation-collapse mechanism in which there is a distribution of folding nuclei with some more probable than others. The formation of any specific nucleus is not necessary for efficient two-state folding.
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PMID:Lattice models for proteins reveal multiple folding nuclei for nucleation-collapse mechanism. 973 20


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