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)

Actin-activated Mg2+-ATPase activity of myosin II from Acanthamoeba castellanii is regulated by phosphorylation of three serine residues located at the carboxyl-terminal end of each of the two 185,000-Da heavy chains; the phosphorylated molecule has full Ca2+-ATPase activity but no actin-activated Mg2+-ATPase activity. Under controlled conditions, chymotrypsin removes a small peptide containing all three phosphorylation sites from the ends of the myosin II heavy chains producing a molecule with heavy chains of 175,000 Da and undigested light chains. The length of the myosin II tail decreased from 89 to 76 nm. Chymotrypsin-cleaved myosin II has complete Ca2+-ATPase activity but no actin-activated Mg2+-ATPase activity under standard assay conditions and binds to F-actin as well as undigested myosin II in the absence, but not in the presence, of MgATP. In the presence of MgCl2, undigested myosin II forms biopolar filaments but chymotrypsin-cleaved myosin II forms only parallel (monopolar) dimers, as assessed by analytical ultra-centrifugation and rotary shadow electron microscopy. We conclude that the short segment very near the end of the myosin II tail that contains the three phosphorylatable serines is necessary for the formation of biopolar filaments and, probably as a consequence of filament formation, for the high-affinity binding of myosin II to F-actin in the presence of ATP and the actin-activated Mg2+-ATPase activity of native myosin II. This supports our previous conclusion that actin-activated Mg2+-ATPase of native myosin II is expressed only when the enzyme is in bipolar filaments with the proper conformation as determined by the state of phosphorylation of the heavy chains.
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PMID:Filament formation and actin-activated ATPase activity are abolished by proteolytic removal of a small peptide from the tip of the tail of the heavy chain of Acanthamoeba myosin II. 315 41

Dictyostelium myosin was associated into dimers and small oligomers at very low ionic strength, filamentous at intermediate ionic strength, and monomeric in solution conditions of high ionic strength. These different associations were probed by fragmenting myosin with chymotrypsin, trypsin, or V-8 protease. All three proteases digested monomeric myosin giving rise to multiple fragments with a wide range of molecular weights. Filamentous myosin was not digested by the V-8 protease, was preferentially cleaved at a single site in the middle of the heavy chain by chymotrypsin, and was cleaved at several sites by trypsin. If the reaction was carried out in very low ionic strength, however, two of these proteases generated stable fragments of high molecular weight. Electron microscopic analysis of these stable fragments showed that tails were shorter than in intact myosin, indicating that the cleavage sites were in the rod portion of the molecule. Under the same conditions of enzymatic digestion, myosin that had been radio labeled in vivo with 32P was analyzed by SDS-PAGE and autoradiography. By comparing the state of phosphorylation and the size of the stable fragments, it was determined that the heavy chain phosphorylation site was located between 55 and 70 kD from the tip of the myosin tail, near a region where the tail displayed sharp bends.
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PMID:Proteolytic fragmentation of Dictyostelium myosin and localization of the in vivo heavy chain phosphorylation site. 320 94

We have produced and characterised five monoclonal antibodies against myosin isolated from chicken intestinal epithelial brush border cells. The binding sites of the antibodies on the rod portion of brush border myosin were localised using rotary shadowing/electron microscopy of myosin-antibody complexes. Two antibodies were shown to bind to the tip of the myosin tail, two antibodies to sites about two thirds down the length of the rod, and one antibody about one third down the length of the rod. Brush border myosin was digested with papain, trypsin and alpha-chymotrypsin, and they myosin fragments obtained were analysed by western blots with the monoclonal antibodies and polyclonal antiserum, and by gel overlay with 125I-labelled light chains. Using this approach we were able to identify and map the protease cleavage sites and thus characterise the proteolytic substructure of brush border myosin. Solid-phase assays, western blots and immunofluorescence were used to study the cross-reactivity of these monoclonal antibodies against a variety of myosins from different species and cell types, to assess the immunological relatedness between brush border myosin and homologous molecules present in different tissues and species. Finally, we used a competitive solid-phase assay to measure the 'relative affinities' of the antibodies towards the three possible conformational states of brush border myosin, i.e. filament, extended monomer and folded monomer.
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PMID:Studies on the structure and conformation of brush border myosin using monoclonal antibodies. 329 86

The thiol-specific photoactivatable reagent 4-(2-iodoacetamido)benzophenone (BPIA) can be selectively incorporated into the SH-1 of myosin subfragment 1 (S1), and upon photolysis an intramolecular cross-link is formed between SH-1 and the N-terminal 25-kDa region of S1. If a Mg2+-nucleotide is present during photolysis, cross-links can be formed either with the 25-kDa or with the central 50-kDa region [Lu, R. C., Moo, L., & Wong, A. G. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 6392-6396]. Heavy chains with these two types of intramolecular cross-links and un-cross-linked heavy chain have different mobility on sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gels and therefore can be purified electrophoretically. Each type of heavy chain was cleaved with Staphylococcus aureus protease, chymotrypsin, or lysyl endopeptidase. The cleavage points were determined on the basis of the molecular weights of weights of peptides containing the N-terminus, which was identified with the use of an antibody. Locations of the cross-links were deduced by comparing the peptide maps of cross-linked and un-cross-linked heavy chains. The results indicate that the segment located about 12-16 kDa from the N-terminus of the heavy chain can be cross-linked to SH-1 via BPIA independently of the presence of a nucleotide, whereas the segment located 57-60 kDa from the N-terminus can be cross-linked to SH-1 only in the presence of a Mg2+-nucleotide. With use of the avidin-biotin system, it has been shown that SH-1 is located 13 nm from the head/rod junction [Sutoh, K., Yamamoto, K., & Wakabayashi, T. (1984) J. Mol. Biol. 178, 323-339]. Since BPIA spans less than 1 nm, our results show that two regions, separated by approximately 400 amino acid residues and located in the 25- and 50-kDa domains of S1, respectively, are also part of the head structure about 12-14 nm from the head/rod junction.
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PMID:Identification of two segments, separated by approximately 45 kilodaltons, of the myosin subfragment 1 heavy chain that can be cross-linked to the SH-1 thiol. 331 Nov 49

The heavy chain fragments generated by restricted proteolysis of the smooth chicken gizzard myosin subfragment-1 (S-1) with trypsin, Staphylococcus aureus V8 protease, and chymotrypsin were isolated and submitted to partial amino acid sequencing. The comparison between the smooth and striated muscle myosin sequences permitted the unambiguous structural characterization of the two protease-vulnerable segments joining the three putative domain-like regions of the smooth head heavy chain. The smooth carboxyl-terminal connector is a serine-rich region located around positions 632-640 of the rabbit skeletal sequence and would represent the "A" site that is conformationally sensitive to the myosin 10 S-6 transition and to its interaction with actin (Ikebe, M., and Hartshorne, D. J. (1986) Biochemistry 25, 6177-6185). A third site which undergoes a nucleotide-dependent chymotryptic cleavage which inactivates the Mg2+-ATPase (Okamoto, Y., and Sekine, T. (1981) J. Biochem. (Tokyo) 90, 833-842, 843-849) was identified at Trp-31/Ser-32. It is vicinal to Lys-34 that is monomethylated in the skeletal heavy chain but not at all in the smooth sequence. However, the two trimethyl lysine residues present in the skeletal sequence are conserved in the same regions of the smooth S-1 and may play a general functional role in myosin. The smooth central 50-kDa segment could be selectively destroyed by a mild tryptic digestion in the absence of any unfolding agent, with a concomitant inhibition of the ATPase activities. This feature is in line with the proposed domain structure of the S-1 heavy chain and also suggests a relationship between the specific biochemical properties of the smooth S-1 and the particular conformation of its 50-kDa region.
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PMID:Comparative structure of the protease-sensitive regions of the subfragment-1 heavy chain from smooth and skeletal myosins. 331 20

Optical ellipsometry studies of single, skinned muscle fibers conducted on the diffraction orders have yielded spectra that are sensitive to the state of the fiber. The linearly polarized light field vector becomes elliptically polarized as it passes through the fiber and may be collected at the diffraction orders. Fibers that have been subjected to extraction of myosin (0.6 M KCl) retain a weak diffraction pattern and exhibit a substantially decreased depolarization of incident linearly polarized light. A significant decrease in polarization is seen in skinned fibers that are subject to an increase in pH from 7.0 to 8.0. This increase in pH results in a decrease of approximately 30% in the depolarization angle of single fibers. The major decrease in depolarization angle that we observe at pH 8.0 is consistent with the notion that as cross-bridges move out from the shaft of the thick filament, their ability to cause depolarization of the incident linearly polarized light decreases. This interpretation is also consistent with the work of Ueno and Harrington where the decrease in the ability to cross-link S-1 and S-2 to the thick filament at pH 8.2 suggests cross-bridge movement away from the thick filament. A large decrease in birefringence, seen after treatment of skinned fibers with alpha-chymotrypsin, appears to be related to the breakdown of myosin into rod, S-1, heavy meromyosin, and light meromyosin.
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PMID:Optical depolarization changes in single, skinned muscle fibers. Evidence for cross-bridge involvement. 348 81

The heavy chains and the 19-kDa and 20-kDa light chains of bovine brain myosin can by phosphorylated. To localise the site of heavy-chain phosphorylation, the myosin was initially subjected to digestion with chymotrypsin and papain under a variety of conditions and the fragments thus produced were identified. Irrespective of the ionic strength, i.e. whether the myosin was monomeric or filamentous, chymotryptic digestion produced two major fragments of 68 kDa and 140 kDa; the 140-kDa fragment was further digested by papain to yield a 120-kDa and a 23-kDa fragment. These fragments were characterised by (a) a gel overlay technique using 125I-labelled light chains, which showed that the 140-kDa and 23-kDa polypeptides contain the light-chain-binding sites; (b) using myosin photoaffinity labelled at the active site with [3H]UTP, which showed that the 68-kDa fragment contained the catalytic site, and (c) electron microscopy, using rotary shadowing and negative-staining techniques, which demonstrated that after chymotryptic digestion the myosin head remains attached to the tail whereas on papain digestion isolated heads and tails were observed. Thus the 120-kDa polypeptide derived from the 140-kDa fragment is the tail of the myosin, and the 68-kDa fragment containing the catalytic site and the 23-kDa fragment, with the light-chain-binding sites, form the head (S1) portion of the myosin. When [32P]-phosphorylated brain myosin was digested with chymotrypsin and papain it was shown that the heavy-chain phosphorylation site is located in a 5-kDa peptide at the C-terminal end of the heavy chain, i.e. the end of the myosin tail. Using hydrodynamic and electron microscopic techniques, no significant effect of either light-chain or heavy-chain phosphorylation on the stability of brain myosin filaments was observed, even in the presence of MgATP. Brain myosin filaments appear to be more stable than those of other non-muscle myosins. Light-chain phosphorylation did, however, have an effect on the conformation of brain myosin, for example in the presence of MgATP non-phosphorylated myosin molecules were induced to fold into a very compact folded state.
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PMID:Proteolytic fragmentation of brain myosin and localisation of the heavy-chain phosphorylation site. 348 10

Local melting within the subfragment-2 region of activated rabbit skeletal glycerinated muscle fibers has been investigated over the temperature range 5 to 37 degrees C, using an enzyme (chymotrypsin)-probe method. The cleavage rates were determined from the time-course of formation of digestion products by electrophoresis on sodium dodecyl sulfate-containing polyacrylamide gels. We found the cleavage sites to be localized in a restricted region Mr = 64,000 to 90,000/polypeptide chain, measured from the C terminus of the myosin rod (the subfragment-2 hinge domain). The cleavage rate constant for activated muscle fibers in the presence of an ATP-regenerating system was about 100 times larger at each temperature than that for rigor or for relaxed muscle fibers and showed a marked increase in magnitude with increasing temperature. Comparative plots of the apparent rate-constant for cleavage within the subfragment-2 hinge domain and the isometric force generated by active fibers versus MgATP concentration gave closely similar profiles suggesting a strong positive correlation. Thus, there appears to be a close coupling between the conformational transition within the subfragment-2 hinge domain and contractile force when the cross-bridges undergo cycling.
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PMID:Local melting in the subfragment-2 region of myosin in activated muscle and its correlation with contractile force. 349 Dec 13

We have used alpha-chymotrypsin as an enzyme-probe to detect local melting in the subfragment-2 region of the cross-bridges of rigor myofibrils and glycerinated psoas fibers. The kinetics of proteolysis and the sites of cleavage were determined at various temperatures over the range 5 to 40 degrees C by following the decay of the myosin heavy chain and the rates of appearance of light meromyosin fragments, using electrophoresis on sodium dodecyl sulfate-containing polyacrylamide gels. Cleavage occurs primarily at the 72,000 Mr and 64,000 Mr (per polypeptide chain from the C terminus of myosin) sites within the light meromyosin-heavy meromyosin hinge domain of the subfragment-2 region, under all experimental conditions. At pH 8.2 to 8.3 and at low divalent metal ion (0.1 mM), where the actin-bound cross-bridges are thought to be released from the thick filament surface, the intrinsic cleavage rate constant (k) increases markedly as the temperature is raised. This suggests substantial thermal destabilization of the released cross-bridge in the intact contractile apparatus. Addition of divalent metal ion (10 mM) lowers the cleavage rate and shifts the k versus temperature profile to higher temperatures. Normalized rate constants for chymotryptic cleavage within the subfragment-2 hinge region of released cross-bridges (pH 8.2, low divalent metal) of rigor fibers were markedly lower than activated fibers at all temperatures investigated (5 to 40 degrees C). Results show that conformational melting within the subfragment-2 hinge region is amplified on activation and is well above that observed when the actin-attached rigor bridge is passively released from the thick filament surface.
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PMID:Temperature-dependence of local melting in the myosin subfragment-2 region of the rigor cross-bridge. 353 14

Incubation of human platelets with unilamellar vesicles composed of dilauroylphosphatidylcholine (DLPC) induces shedding of small vesicular structures from the platelet plasma membrane. No significant cell lysis is observed during the process of shedding. Isolated spicules contain the major membrane glycoproteins, Ib, IIb, and IIIa, which are used to define the sidedness of the spicule membrane. These glycoproteins are completely susceptible to chymotrypsin treatment, whereas cytoskeletal proteins are inaccessible towards this enzyme. This demonstrates that the spicule membranes have a right-side-out orientation in as far as membrane proteins are concerned. Isolated spicules were 30-fold more active than platelets in stimulating prothrombin conversion to thrombin by the prothrombinase complex (factors Xa, Va and Ca2+). The increased prothrombinase activity reflects an increased amount of phosphatidylserine in the outer leaflet of the spicule membrane. Protein analysis of platelet spicules and native platelets reveals a number of differences, the most conspicuous of which is the virtual absence of myosin in the spicule preparations. It is proposed that a lack of myosin produces a different cytoskeletal organization in the spicules. This enables phosphatidylserine to become exposed at the outer surface of the spicule membrane.
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PMID:Loss of phospholipid asymmetry in dilauroylphosphatidylcholine induced plasma membrane vesicles from human platelets. 365 53


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