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)

A panel of monoclonal antibodies specific to alpha- or beta-tubulin subunits was used to study the location of tubulin molecules in microtubules. Limited proteolysis of tubulin with trypsin and chymotrypsin followed by immunoblotting demonstrated that the antibodies discriminated between structural domains of tubulin subunits. Antibodies against N-terminal domains were tested for their ability to interfere with the formation of microtubules in vitro. Although the antibodies exhibited similar association constants when tested on immobilized tubulin, they differed in their inhibitory effect on microtubule assembly. The sedimentation assay using microtubules prepared from purified tubulin showed an almost undetectable binding of the antibodies with the strongest inhibitory power to the microtubules. Immunofluorescence staining of unfixed detergent-extracted cells revealed that antibodies to determinants on C-terminal domains labelled microtubules, but these were not decorated with antibodies against N-terminal domains. The same results were obtained after a microinjection of antibodies into living cells. The data indicate that while parts of C-terminal domains of both subunits are exposed on the exterior of microtubules, considerable regions of the N-terminal domains are not. The surface regions of N-terminal domains appear to be involved in the formation of microtubules.
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PMID:Tubulin orientation in microtubules probed with domain-specific antibodies. 172 30

By chemically modifying carboxyl groups we have investigated the role of the highly acidic COOH-terminal domains of alpha- and beta-tubulin in regulating microtubule assembly. Using a carbodiimide-promoted amidation reaction, as many as 25 carboxyl groups were modified by the addition of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and an amine nucleophile, [14C] glycine ethyl ester or [3H]methylamine, to assembled microtubules. Modification occurred primarily in the carboxyl-terminal region as demonstrated by limited proteolysis of modified tubulin by trypsin, chymotrypsin, subtilisin, and carboxypeptidase Y. Modified tubulin polymerized into microtubules with a critical concentration that was 15% of that for unmodified tubulin. Assembly of modified tubulin and microtubules formed from modified tubulin were less sensitive to Ca2+ and high ionic strength. Ca2+ binding studies under low ionic strength conditions indicated that modified tubulin does not contain the high affinity Ca2+ binding site. While assembly of unmodified tubulin was stimulated by Mg2+ up to 10 mM, assembly of the modified protein was inhibited by concentrations greater than 1 mM. When 24 residues were modified, polymerization was no longer stimulated by microtubule-associated proteins (MAPs) or polylysine and incorporation of high molecular weight MAPs into the polymers was reduced by about 70% compared to unmodified tubulin. These studies demonstrate that chemical modification of carboxyl groups in tubulin, most of which are localized in the COOH-terminal region, leads to an enhanced ability to polymerize and a decrease in interaction with MAPs and other positively charged species.
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PMID:Assembly properties of tubulin after carboxyl group modification. 198 23

We synthesized five peptides homologous to the potentially antigenic positions alpha(214-226), alpha(430-443), alpha(415-443), beta(241-256), and beta(412-431) of the porcine brain tubulin sequences. These peptides were successfully employed to raise tubulin-cross-reactive antibodies. The antibodies are specific of the regions of tubulin spanned by the peptides. They react specifically with the tubulin bands in immunoblots and with microtubules in immunofluorescence assays of cytoskeletons. The peptides of the C-terminal regions have also been employed to localize determinants recognized by two available monoclonal antibodies to tubulin in the positions alpha(415-430) and beta(412-431), respectively. In a first application of the anti-peptide antibodies, we have mapped the fragments of limited proteolysis of purified calf brain tubulin by trypsin, chymotrypsin, papain, thermolysin, subtilisin, and protease V8 from Staphylococcus aureus. Thirty-seven peptides have been identified, of which 32 have been unequivocally aligned into the tubulin sequences on the basis of their antigenic reactivity. There are three major, well-defined zones of preferential cleavage by the proteases: the C-termini and two internal zones in each chain. C-Terminal cleavages of both chains by subtilisin do not remove the antigenic reactivity of the zones alpha(415-430) and beta(412-431). C-Terminal cleavages by protease V8 are preferential of beta-tubulin. All six proteases tested cleave alpha- and/or beta-tubulin at one or both of the internal zones. These zones are located roughly at one-third and two-thirds of the chain length in both subunits. Therefore, a model of the tubulin monomers is proposed which consists of three major, proteolytically defined, compact regions (N-terminal, middle, and C-terminal thirds) and the cleavable zones. This model is discussed with the tubulin structural information presently available.
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PMID:Tubulin structure probed with antibodies to synthetic peptides. Mapping of three major types of limited proteolysis fragments. 245 62

A panel of 11 monoclonal antibodies specific to alpha- or beta-tubulin subunits was used to study the location of tubulin molecules in cytoplasmic microtubules. Specificity of antibodies was confirmed by immunoblotting and immunofluorescence experiments on fixed cells. The limited proteolysis of tubulin with trypsin and chymotrypsin followed by immunoblotting demonstrated that the antibodies discriminated between structural domains of both subunits. Epitope mapping of isolated alpha-tubulin revealed that a set of antibodies against the N-terminal domain of the alpha-subunit (TU-01, TU-02, TU-03, TU-09, 6-11B-1) recognized at least four different antigenic determinants. Immunofluorescence staining of unfixed detergent-extracted cells showed that antibodies to determinants on C-terminal domains labelled microtubules, but these were not decorated with antibodies to N-terminal domains. The same results were obtained after microinjection of antibodies into living cells. The unchanged distribution of microtubules in injected cells was confirmed by double-label immunofluorescence with polyclonal antibodies. The data indicate that while parts of C-terminal domains of both subunits are exposed on the exterior of the microtubules, considerable regions of the N-terminal domains are either not exposed on the surface of cytoplasmic microtubules, or are masked by interacting proteins.
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PMID:Differences in the exposure of C- and N-terminal tubulin domains in cytoplasmic microtubules detected with domain-specific monoclonal antibodies. 248 Mar 56

The alpha and beta subunits of the tubulin dimer each possess a distal C-terminal subtilisin cleavage site which, when cleaved, releases an acidic, small peptide. In addition, each possesses an internal site, cleaved by trypsin in alpha and chymotrypsin in beta, which connects the amino and carboxyl structural domains. A model of the dimer is presented which suggests that the beta C-terminal subtilisin site may be more accessible in the monomer than in the dimer. Kinetics of cleavage at this site on the dimer yield straight-line plots of log (undigested fraction) versus time, from which pseudo-first-order rate constants are obtained. Temperature effects on the rate constant are due to changes in the activity of subtilisin, not to temperature-induced unfolding around this site. The rate constant is proportional to the subtilisin/tubulin ratio, whether this is varied by changing the concentration of subtilisin or of tubulin. However, if the rate constant increases due to decreasing tubulin concentration, the extrapolated zero time intercept decreases. The decrease in zero time intercept is interpreted as being due to the appearance of a rapidly digested fraction upon dilution of tubulin. The increase observed in this fast fraction with dilution of tubulin is fully reversible upon reconcentration. It is suggested that this fast fraction represents monomeric beta-tubulin and the concentration dependence of this fast fraction indicates a dissociation constant of about 1.5 X 10(-7) M.
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PMID:Tubulin dimer dissociation and proteolytic accessibility. 265 76

A characterization is reported of the major cytoskeletal protein, called IEF (isoelectric focusing)-51K, of marginal band microtubule coils from human blood platelets (Kenney, D. M. and Linck, R. W. (1985) J. Cell Sci. 78, 1-22). IEF-51K is a unique biochemical species which is distinguishable from platelet and mammalian neuronal alpha-tubulin and beta-tubulin by 1) its faster mobility on discontinuous sodium dodecyl sulfate electrophoresis corresponding to an apparent Mr 51,000; 2) its more alkaline relative isoelectric point at pH 5.7 compared with that of alpha- and beta-tubulin at pH 5.3 and 5.5, respectively; 3) lack of coincidence in peptide maps prepared with chymotrypsin or Staphylococcus aureus V8 protease; and 4) lack of immunochemical cross-reactivity of polyclonal anti-IEF-51K with alpha- and beta-tubulin and of monoclonal anti-alpha-tubulin and anti-beta-tubulin with IEF-51K. In contrast to its chemical uniqueness, IEF-51K is tubulin-like in some of its properties. IEF-51K is localized in the marginal band of intact platelets by immunofluorescence; it undergoes cycles of microtubule disassembly/reassembly both in vitro and in vivo. Furthermore, IEF-51K was not extracted from isolated Taxol-stabilized marginal band microtubules by elevated NaCl concentrations (to 0.45 M), conditions that do not disrupt the polymeric structure of alpha- and beta-tubulin. These results indicate that IEF-51K together with alpha-tubulin and beta-tubulin are the major structural polypeptides of platelet marginal band microtubules. The unusual subunit composition of the platelet marginal band microtubule may be related to specialization(s) of microtubule structure and function in the marginal band coil of platelets.
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PMID:A novel microtubule protein in the marginal band of human blood platelets. 312 30

Limited proteolysis and chemical cross-linking techniques have been used to study the interaction between alpha- and beta-tubulin subunits. Trypsin digestion of tubulin dimer resulted in the cleavage of the alpha-subunit into two fragments, whereas chymotrypsin cleaved the beta-subunit into two distinct fragments. All of these fragments have been mapped on the tubulin subunits by further proteolysis with formic acid. Cross-linking of trypsin- and chymotrypsin-cleaved subunits has been performed with two different cross-linker agents of different cross-linking distance. The addition of formaldehyde resulted in the cross-linking of the alpha-tubulin N-terminal fragment with beta-tubulin C-terminal domain. The same result was obtained when methyl 4-mercaptobutyrimidate was used.
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PMID:The interaction between subunits in the tubulin dimer. 390 10

The substructure of the tubulin molecule was studied by limited proteolysis and high affinity polyclonal antibodies specific for alpha or beta-tubulin. Brief enzymatic cleavage separates the tubulin monomer into two domains of unequal size. Trypsin splits alpha-tubulin into components with Mr values of 36 X 10(3) and 14 X 10(3), chymotrypsin splits beta-tubulin into 31 X 10(3) Mr and 20 X 10(3) Mr fragments. The cleavage occurs at Arg339 (alpha) and Tyr281 (beta), as determined by sequencing several N-terminal residues of the small domains, i.e. the small domains are the C-terminal parts of the molecules, the large ones are the N-terminal parts. There is a second cleavage site of chymotrypsin within Mr 10(3) to 2 X 10(3) of the C terminus of beta-tubulin. The fragments can be separated only under denaturing conditions. They copolymerize into microtubules and incomplete microtubule walls joined by a wall junction, forming S-shapes and hooks in cross-section. The antibodies were raised against electrophoretically purified tubulin monomers. Those produced with alpha-tubulin are directed predominantly against the large domains; they are either specific for alpha-tubulin or cross-react with the large domain of beta-tubulin. Conversely, antibodies raised against beta-tubulin are directed predominantly against the small domains (beta-specific and beta-cross-reacting fractions). Thus the antibodies discriminate not only between the tubulin chains but also between the domains generated by the proteases. The complementary antigenicity correlates well with the stability of the domains. Potential sites of antigenic determinants are located within the polypeptide chains by comparing theoretical predictions with the pattern of immunoblots. Two epitopes of the alpha-cross-reacting antibodies have been located approximately. One is very close to the C terminus (within about 20 residues), the other is close to the N terminus (within about Mr 8 X 10(3) ). The epitope of the beta-cross-reacting antibody is also located within Mr 12 X 10(3) of the C terminus. The antibodies prevent microtubule assembly and cause disassembly of preformed microtubules. A variety of breakdown products are observed by electron microscopy. They include fibres of about 10 nm width, sheets with undefined substructure, thick tapered fibrous bundles and wispy filaments.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Tubulin domains probed by limited proteolysis and subunit-specific antibodies. 405 49

Three alpha-tubulins and two beta-tubulins have been resolved by two-dimensional gel electrophoresis of whole cell lysates of Physarum myxamoebae or plasmodia. Criteria used to identify the tubulins included migration on two-dimensional gels with myxamoebal tubulins purified by self-assembly into microtubules in vitro, peptide mapping with Staphylococcus V8 protease and with chymotrypsin, immunoprecipitation with a monoclonal antibody specific for beta-tubulin, and, finally, hybrid selection of specific mRNA by cloned tubulin DNA sequences, followed by translation in vitro. Differential expression of the Physarum tubulins was observed. The alpha 1- and beta 1-tubulins were detected in both myxamoebae and plasmodia; alpha 2 and beta 2 were detected only in plasmodia, alpha 3 was detected only in the myxamoebal phase, and may be specific to the flagellate. Observation of more tubulin species in plasmodia than in myxamoebae was remarkable; the only microtubules detected in plasmodia are those of the mitotoic spindle, whereas myxamoebae display cytoplasmic, centriolar, flagellar, and mitotic-spindle microtubules. In vitro translation of myxamoebal and plasmodial RNAs indicated that there are distinct mRNAs, and therefore probably separate genes, for the alpha 1-, alpha 2-, beta 1-, and beta 2-tubulins. Thus, the different patterns of tubulin expression in myxamoebae and plasmodia reflect differential expression of tubulin genes.
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PMID:Cell type-dependent expression of tubulins in Physarum. 619 70

beta-Tubulin subunits isolated from chicken brain tissue and erythrocytes are distinguishable as unique biochemical species by electrophoretic and peptide mapping procedures. 1) The subunits of beta-tubulin exhibit major differences in electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels that vary according to the pH and ionic strength of the gel. 2) The isoelectric points of urea-denatured beta subunits from brain tissue and erythrocytes are pH 5.1 and 5.4, respectively, whereas those of both alpha subunits are approximately pH 5.2.3) Two-dimensional peptide maps prepared with alpha-chymotrypsin or V8 protease show that alpha-tubulin peptides are indistinguishable, whereas beta-tubulin peptides are very different. Only one-third of the 15 major tyrosine-containing beta-tubulin peptides prepared with alpha-chymotrypsin are common to both beta-tubulin species. The data indicate that the beta-tubulin subunits of brain tissue and erythrocytes are biochemically distinct and may be different gene products. The presence of tubulin variants in brain tissue and erythrocytes may indicate special requirements for microtubule assembly and function in different cell types.
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PMID:Brain and erythrocyte microtubules from chicken contain different beta-tubulin polypeptides. 634 31


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