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)

Experiments have explored the possible relationships between the flagellar surface motility of chlamydomonas, visualized as translocation of polystyrene beads by paralyzed (pf) mutants (Bloodgood, 1977, J. Cell Biol. 15:983-989), and the capacity of gametic flagella to participate in the mating reaction. While vegetative and gametic flagella bind beads with equal efficiencies and are capable of transporting them along entire flagellar lengths, beads on vegetative flagella are primarily associated with the proximal half of the flagella whereas those of gametic flagella exhibit no such preference. This difference may relate to the "tipping" response of gametes during sexual flagellar agglutination (Goodenough and Jurivich, 1978, J. Cell Biol. 79:680-693). Colchicine, vinblastine, chymotrypsin, cytochalasins B and D, and anti-beta-tubulin antiserum are all able to inhibit the binding of beads to the flagellar suface. Trysin digestion and an antiserum directed against whole chlamydomonas flagella have no effect on the ability of flagella to bind beads, but the beads remain immobile. These results suggest that at least two flagellar activities participate in surface motility: (a) bead binding, which may involve a tubulin-like component at the flagellar surface; and (b) bead translocation, which may depend on a second component (e.g. an ATPase) of the flagellar surface. Surface motility is shown to be distinct from gametic adhesiveness per se, but it may participate in concentrating dispersed agglutinins, in driving them toward the flagellar tips, and/or in generating a signal-to-fuse from the flagellar tips to the cell body. Directly supporting these concepts is the observation that bound beads remain immobilized at the flagellar tips during the "tip-locking" stage of pf x pf matings, and the observation that bound ligands such as antibody fail to be tipped by trypsinized flagella.
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PMID:Experimental dissection of flagellar surface motility in Chlamydomonas. 740 Feb 20

Tubulin has been found to be synthesized on both membrane-bound and free polyribosomes prepared from brain. Cell-free studies indicate that tubulin made on rough microsomes is incorporated into the endoplasmic reticulum membrane as it is synthesized. This tubulin remains associated with the membrane after sedimentation and washing. The tubulin is not removed from the membrane after stripping ribosomes from the membranes in KCl-puromycin, followed by repeated washing by either sedimentation or flotation in 0.05 M-KCl. The membrane tubulin is partially susceptible to proteolysis by trypsin and chymotrypsin: beta-tubulin is more accessible to the proteases than in alpha-tubulin. Nonionic detergents extract mostly beta-tubulin from the microsomal membrane. Newly synthesized tubulin which has been extracted from microsomal membranes in 0.5% Nonidet P-40, coassembles and disassembles with carrier microtubule protein. The insertion of newly synthesized tubulin into endoplasmic reticulum membrane may be the first step in the incorporation of tubulin into the plasma membrane.
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PMID:Association of newly synthesized tubulin with brain microsomal membranes. 745 9

Colchicine, the classic antimitotic poison, disrupts cell division by preventing proper assembly of microtubules in the mitotic spindle. Colchicine is known to act by binding to tubulin, the heterodimeric subunit of microtubules. How this binding to tubulin changes the structure of the protein and results in polymerization poisoning has not been characterized. The structural locus of spectroscopically detected conformational changes induced by colchicine is unknown. We report here that colchicine induces the unfolding of a small region in the carboxyl-terminal region of beta-tubulin, around Arg-390. This unfolding is detected by proteolysis with trypsin and chymotrypsin. Chymotrypsin cleaves this region after Phe-389, and trypsin cleaves after Lys-392. The unfolded region appears to be the carboxyl end of an amphipathic helix in the absence of colchicine, and we propose that this unfolding prevents contacts necessary for assembly. Our results suggest that beta-tubulin is exposed on the growing end of the microtubule, which provides a mechanism for coupling GTP hydrolysis to polymerization.
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PMID:Molecular mechanism of colchicine action: induced local unfolding of beta-tubulin. 825 91

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

Cryptophycin 1 is a new cytotoxic antimicrotubule agent with excellent antitumor activity. The methods of Sackett (Biochemistry, 34, 7010-7019, 1995), utilizing the selective and specific proteolysis of alpha- and beta-tubulin by trypsin and chymotrypsin, was used to identify the cryptophycin 1 binding site on tubulin. Occupancy of the colchicine or vinca binding sites causes changes in the structure of tubulin that can be detected by proteolysis with trypsin and chymotrypsin. The addition of cryptophycin 1 to tubulin causes changes in both the tryptic and chymotryptic cleavage of tubulin consistent with occupation of the vinca binding site and distinct from occupation of the colchicine binding site. The effects of cryptophycin 1 on the tryptic digests are identical to the effects seen with vinblastine and differ saliently from the effects of maytansine and rhizoxin, other agents known to bind to the vinca site. The data suggest that the binding site of cryptophycin 1 may overlap the vinca binding site on tubulin.
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PMID:Cryptophycin 1 binds to tubulin at a site distinct from the colchicine binding site and at a site that may overlap the vinca binding site. 891 66

Native tubulin alpha beta dimers and microtubules have been subjected to limited proteolysis with trypsin, chymotrypsin, elastase, clostripain, proteinase lysine-C, thermolysin, protease V8, papain, subtilisin, proteinase K, proteinase aspartic-N, and bromelain. Eighty nicking points have been mapped onto the alpha- and beta-tubulin sequences with the aid of site-directed antibodies, of which 18 sites have been exactly determined by N-terminal sequencing, and the probable position of 6 others deduced from protease specificities. Proteolytic sites cluster into five characteristic zones, including the C termini of both chains. Residues accessible to proteases in the tubulin dimer include alpha-tubulin Lys40-Thr41-Ile42, Glu168-Phe169-Ser170, Ser178-Thr179-Ala180-Val181, Lys280-Ala281, Glu290-Ile291, Ala294-Cys295, Arg339-Ser340 (plus probably Lys60-His61 and Glu183-Pro184) and beta-tubulin Gly93-Gln94, Lys174-Val175, Gly277-Ser278, Tyr281-Arg282-Ala283, Cys354-Asp355 (plus probably Arg121-Lys122, Phe167-Ser168, Tyr183-Asn184, and Glu426-Asp427 or Ala430-Asp431). While the majority of these sites remain accessible at the outer surface of taxol-induced microtubules, alpha-tubulin Lys280-Ala281, Arg339-Ser340 and beta-tubulin Tyr281-Arg282-Ala283 (and probably Arg121-Lys122) become protected from limited proteolysis, suggesting that they are close to or at intermolecular contacts in the assembled structure. The protease nicking points constitute sets of surface constraints for any three-dimensional model structures of tubulin and microtubules. The dimer tryptic site at alpha-tubulin 339-340 jumps approximately 12-22 residues upstream (probably to Lys326-Asp327 or Lys311-Tyr312) in taxol microtubules, suggesting a tertiary structural change. The cleavage of the approximately 10 C-terminal residues of alpha-tubulin by protease V8, papain, and subtilisin is inhibited in taxol microtubules compared to tubulin dimers, while the approximately 20 C-terminal residues of beta-tubulin are similarly accessible to protease V8, subtilisin, proteinase K, proteinase AspN, and bromelain and show enhanced papain cleavage. This is consistent with models in which the alpha-tubulin C-terminal zone is near the interdimer contact zone along the protofilaments, whereas the C terminus of beta is near the interface between both subunits.
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PMID:Mapping surface sequences of the tubulin dimer and taxol-induced microtubules with limited proteolysis. 891 4

In the axonemes of Tetrahymena cilia, beta-tubulin was digested more rapidly by chymotrypsin than alpha-tubulin. On the other hand, in the solubilized state, both tubulins were digested by the protease at almost the same rate. Among alpha-tubulin isotypes within the axonemes, alpha 5-tubulin was more rapidly digested by chymotrypsin than other alpha-tubulin isotypes. The addition of ATP and vanadate (Vi) to the axonemes significantly protected the alpha 5-isotype from chymotryptic proteolysis. These tendencies could not be observed in tubulins solubilized from axonemes. Based on the case of the alpha 5-tubulin isotype, it is possible to negate the idea that all tubulin isotypes are distributed homogeneously and play similar functional roles within the axonemes.
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PMID:Proteolysis of tubulin isotypes within Tetrahymena axonemes. 907 48

Dendritic cells from the mesenteric lymph nodes (MLN) contain dense esterase-positive inclusions that may originate in effete intestinal epithelial cells and reach MLN without degradation. The MLN esterases have the electrophoretic mobilities of both intestinal and mononuclear cells. Cryptosporidium parvum (CP)-infected mice have CP Ag-positive cells in MLN and also increased numbers of dense esterase-positive cells, but the CP Ag-positive cells do not stain for esterase. To characterize the handling of epithelial cell products by dendritic cells, we analyzed mRNAs in the MLN of control and CP-infected recombination-activating gene(-/-)DO11.10 mice by oligoarrays. mRNAs for 115 proteins were increased in MLN after CP infection, of which the principal increases in trypsin and chymotrypsin approximated to 250-fold. Colipase, reg-1, C-reactive protein-ductin, and amyloid were also up-regulated >10-fold and all returned to baseline by 28 days after infection. mRNAs for the same proteins were detected in intestinal epithelial cells of infected mice by oligoarrays and RT-PCR after infection. mRNA for CP beta-tubulin was detectable in intestinal epithelial cells between 5 and 18 days after infection but was not detected in the MLN throughout the observation period. It appears that host response to CP infection includes expression of mRNA for some pancreatic enzymes by intestinal epithelial cells and their subsequent transport to the MLN. The esterase and trypsin, and mRNAs for chymotrypsin, colipase, and others that may derive from uninfected epithelial cells, appear to be transported to the MLN intact, while mRNA for CP beta-tubulin that is derived from infected cells is degraded.
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PMID:Intact intestinal mRNAs and intestinal epithelial cell esterase, but not Cryptosporidium parvum, reach mesenteric lymph nodes of infected mice. 1167 48


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