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)

Substructure of the myosin rod and its correlation to the filament formation were investigated by using fish myosin rod. It was found that fish rod contains a unique chymotrypsin susceptible site, 40 kDa from the COOH-terminus or 20 kDa downstream from the subfragment-2/light meromyosin junction (S-2/LMM junction). Cleavage at this new site produced subfragment-2 possessing 95 kDa subunit (95k S-2) and light meromyosin possessing 40 kDa subunit (40k LMM). The latter is the shortest unit ever reported to exhibit filament formation. Moreover, the 40k LMM was able to form filaments independently of the presence of Mg2+, while filament formation of rod and ordinary LMM (70k LMM) was promoted by Mg2+ addition. These results indicated that the Mg2+ binding sites are present within the NH2-terminal 20 kDa region of the 70k LMM. We concluded that the COOH-terminal 40 kDa portion of rod is responsible for the self-assembly ability of myosin, while the NH2-terminal 20 kDa region of the 70k LMM is the regulatory domain for thick filament formation through Mg(2+)-binding.
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PMID:Filament-forming domain of carp dorsal myosin rod. 845 73

Myosin-I refers to a class of proteins with a molecular weight of approximately 110-kDa, which have characteristics of conventional myosin but are unable to form filaments. Previous studies have implicated myosin-I in motile cellular processes including cell migration and phagocytosis. Although the first example of myosin-I in higher eukaryotes was the intestinal 110K-calmodulin complex, which forms in microvilli the lateral links connecting the core bundle of actin filaments to the membrane, myosin-I has now been shown to be a component of rat kidney and to be present in bovine adrenal gland and brain. We have now purified and characterized two polypeptides from rat liver which have several characteristics of the intestinal 110K-calmodulin complex. Both liver polypeptides are solubilized with ATP and co-elute on gel filtration with calmodulin. The polypeptides, of 110-kDa and 130-kDa, bind calmodulin in 1 mM EGTA. Both polypeptides bind to F-actin in an ATP reversible fashion, and crosslink actin filaments. The purified polypeptides possess an actin-activated Mg(2+)-ATPase activity typical of brush border myosin-I. A polyclonal antiserum directed against the chicken intestinal 110-kDa polypeptide recognizes both rat liver polypeptides, whereas another serum recognizes the 130-kDa but not the 110-kDa rat liver polypeptide. Controlled proteolysis of the purified polypeptides with alpha-chymotrypsin indicates that the two polypeptides are distinct but related. Immunofluorescence microscopy on isolated hepatocytes shows distribution of myosin-I to be vesicular, distributed throughout the cytoplasm, but more concentrated near the nucleus. These data contribute new evidence by several functional criteria that multiple myosin-I molecules are present in higher organisms and may coexist in a single cell type.
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PMID:Myosin-I in mammalian liver. 846 25

A complex of 110-kDa heavy chain and calmodulin was isolated from porcine aorta media smooth muscle and identified as myosin I. The isolated myosin I consisted of equimolar amounts of 110-kDa heavy chain and calmodulin. The addition of exogenous calmodulin to the complex revealed that a maximum of two molecules of calmodulin could be bound to the heavy chain. Isolated complex bound to F-actin in an ATP-dependent manner and its Mg(2+)-ATPase activity was activated by F-actin. In addition, it bound to phospholipid, which is a characteristic property of myosin I. Calcium ions induced a structural change, which was revealed by a difference in the cleavage pattern and for rate of cleavage by alpha-chymotrypsin. This behavior was similar to that reported for brush border myosin I [L.M. Collins and A. Bretscher (1988) J. Cell. Biol. 106, 367-373]. Calcium-dependent structural change of a complex of 110-kDa heavy chain and calmodulin was found from its solubility change at various NaCl concentrations in the presence of ATP. A complex of 116-kDa heavy chain and calmodulin, possibly another type of myosin I, was also isolated. A polyclonal antibody against the complex of 110-kDa heavy chain and calmodulin did not recognize the 116-kDa heavy chain. This result suggests that at least two types of myosin Is may exist at the protein level in porcine aorta media smooth muscle.
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PMID:Isolation and identification of myosin I from porcine aorta media smooth muscle. 898 54

The partial amino acid sequence of subfragment-1 of adult chicken atrial myosin was determined by direct protein sequencing. Subfragment-1 was prepared by limited digestion of adult chicken atrial myosin with alpha-chymotrypsin. Ten peptides were then obtained by cleaving this subfragment with cyanogen bromide. The amino acid composition and amino acid sequence of the obtained peptides were subsequently determined. By sequence comparison with the corresponding region of adult chicken ventricular myosin, three peptides, with differing sequences that corresponded to the same position in subfragment-1, were detected. This indicates that at least three isoforms of atrial myosin exist in adult chicken atrial muscle. One of the three peptides was identical to ventricular subfragment-1 while the remaining two peptides were markedly different. Furthermore, four of these ten peptides were completely different from ventricular subfragment-1. These four peptides were presumed to be fragments of atrial-specific myosin heavy chain protein. Results suggest the expression of at least two species of atrial-specific myosin heavy chain in the atrial muscle of adult chickens.
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PMID:Identification of atrial-specific myosin heavy chain in cardiac muscle of adult chickens. 908 35

Two myosin heavy chain isoforms expressed in smooth muscle, SM1 (204 kDa) and SM2 (200 kDa), are derived from alternate splicing that results in different amino acid sequences at their non-helical C-terminal tail regions. These isoforms are developmentally regulated and differentially expressed in various smooth muscle tissues. The functional role of myosin isoforms differing at the C-terminal tail has been investigated both in vitro and in vivo. Removal of the C-terminal tail of SM1 by chymotrypsin activates the ATPase of myosin at low Mg2+ but does not change the maximum activity. Addition of peptides, mimicking C-terminal tail regions specific to the SM1 and SM2 isoforms, to permeabilized taenia coli smooth muscle fibers inhibits maximum shortening velocity (Vm) and decreases Ca2+ sensitivity but has no effect on maximum force. The inhibition of Vm by the SM1-peptide was not reversed on washout, whereas Vm inhibition by the SM2-peptide is reversible. We demonstrated that the SM1 peptide specifically bound to myosin at the subfragment 2-light meromyosin (S2-LMM) junction using crosslinking and immunomicroscopy. Modification at this site could have a direct effect on crossbridge function. The relation between C-terminal myosin isoforms and contractile function in vivo was examined using estrogen administration to ovariectomized rats to increase the relative expression of the SM1 C-terminal isoform in uterine smooth muscle. This increase in SM1 was significantly correlated with an increase in Vm. In contrast, the high ATPase N-terminal isoform was decreased by administration of estrogen to ovariectomized rats. Thus, changes in C-terminal isoform distribution appear to affect contractile function in vivo. We propose a mechanism whereby the interactions between the C-terminal tail of one myosin molecule and the S2-LMM region of another in the thick filament can modulate contractility in an isoform specific manner. Further work is needed to unequivocally identify the function of smooth muscle myosin isoforms. However, our evidence suggests that the C-terminal heavy chain isoforms may be important modulators of smooth muscle contractility.
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PMID:C-terminal isoforms of the myosin heavy chain and smooth muscle function. 918 9

Kinase-related protein, also known as KRP or telokin, is an independently expressed protein product derived from a gene within the gene for myosin light chain kinase (MLCK). KRP binds to unphosphorylated smooth muscle myosin filaments and stabilizes them against ATP-induced depolymerization in vitro. KRP competes with MLCK for binding to myosin, suggesting that both proteins bind to myosin by the KRP domain (Shirinsky, V. P., Vorotnikov, A. V., Birukov, K. G., Nanaev, A. K., Collinge, M., Lukas, T. J., Sellers, J. R., and Watterson, D. M. (1993) J. Biol. Chem. 268, 16578-16583). In this study, we investigated which regions of myosin and KRP interact in vitro. Using cosedimentation assays, we determined that KRP binds to unphosphorylated myosin with a stoichiometry of 1 mol of KRP/1 mol of myosin and an affinity of 5.5 microM. KRP slows the rate of proteolytic cleavage of the head-tail junction of heavy meromyosin by papain and chymotrypsin, suggesting it is binding to this region of myosin. In addition, competition experiments, using soluble headless fragments of nonmuscle myosin, confirmed that KRP interacts with the regulatory light chain binding region of myosin. The regions important for KRP's binding to myosin were investigated using bacterially expressed KRP truncation mutants. We determined that the acid-rich sequence between Gly138 and Asp151 of KRP is required for high affinity myosin binding, and that the amino terminus and beta-barrel regions weakly interact with myosin. All KRP truncations, at concentrations comparable to their KD values, exhibited some stabilization of myosin filaments against ATP depolymerization in vitro, suggesting that KRP's ability to stabilize myosin filaments is commensurate with its myosin binding affinity. KRP weakened the Km but not the Vmax of phosphorylation of myosin by MLCK, demonstrating that bound KRP does not prevent MLCK from activating myosin.
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PMID:Sites of interaction between kinase-related protein and smooth muscle myosin. 931 55

The major myosin light chain phosphatase is composed of three subunits with apparent molecular masses of 130, 38 and 20 kDa, corresponding to the myosin-binding, catalytic and a regulatory subunit of unknown function, respectively. In this work, we have amplified the cDNA coding for each of the three subunits by the polymerase chain reaction, and expressed the 130 kDa subunit in insect cells using the baculovirus expression system. Limited chymotrypsin digestion show that the folding of the expressed protein is similar to that in the native holoenzyme. N-Terminal sequencing reveals that our recombinant protein is authentic. Mass spectrometry shows that the expressed protein is full length. The recombinant protein is capable of binding myosin based on the ELISA assay and myosin affinity chromatography. Finally, rotary shadowing electron microscopy reveals an elongated structure with three globular domains connected by flexible strands. These results pave the way for future biochemical, structural and site-directed mutagenesis studies on the myosin light chain phosphatase. We also found that the cDNA of the 20 kDa subunit may code for a smaller protein with a molecular mass of 18.5 kDa.
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PMID:Expression in insect cells and characterization of the 110 kDa anchoring subunit of myosin light chain phosphatase. 942 58

The Acanthamoeba myosin-IA heavy chain gene encodes a 134-kDa protein with a catalytic domain, three potential light chain binding sites, and a tail with separately folded tail homology (TH) -1, -2, and -3 domains. TH-1 is highly resistant to trypsin digestion despite consisting of 15% lysine and arginine. TH-2/3 is resistant to alpha-chymotrypsin digestion. The peptide link between TH-1 and TH-2/3 is cleaved by trypsin, alpha-chymotrypsin, and endo-AspN but not V8 protease. The CD spectra of TH-2/3 indicate predominantly random structure, turns, and beta-strands but no alpha-helix. The hydrodynamic properties of TH-2/3 (Stokes' radius of 3.0 nm, sedimentation coefficient of 1.8 S, and molecular mass of 21.6 kDa) indicate that these domains are as long as the whole myosin-I tail in reconstructions of electron micrographs. Furthermore, separately expressed and purified TH-1 binds with high affinity to TH-2/3. Thus we propose that TH-1 and TH-2/3 are arranged side by side in the myosin-IA tail. Separate TH-1, TH-2, and TH-2/3 each binds muscle actin filaments with high affinity. Salt inhibits TH-2/3 binding to muscle actin but not amoeba actin filaments. TH-1 enhances binding of TH-2/3 to muscle actin filaments at physiological salt concentration, indicating that TH-1 and TH-2/3 cooperate in actin binding. An intrinsic fluorescence assay shows that TH-2/3 also binds with high affinity to the protein Acan125 similar to the SH3 domain of myosin-IC. Phylogenetic analysis of SH3 sequences suggests that myosin-I acquired SH3 domain after the divergence of the genes for myosin-I isoforms.
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PMID:Organization and ligand binding properties of the tail of Acanthamoeba myosin-IA. Identification of an actin-binding site in the basic (tail homology-1) domain. 1057 99

It is well established that the bioavailability of non-haem iron from foods is enhanced by the presence of meat. However, the nature of the promoter in meat has not yet been characterised. The present study was designed to compare the effects of the myofibrillar protein fractions on the bioavailability of non-haem iron in an attempt to identify the 'meat factor'. Rabbit skeletal muscle was fractionated and whole muscle, myofibrillar protein, myosin and actin were isolated. Myosin was subjected to selective proteolysis with chymotrypsin and the heavy meromyosin, light meromyosin, rod region and head region were prepared. Protein fractions (1 g) were incorporated into 100 g semi-synthetic liquid meal and the in vitro dialysability of iron was determined. Egg albumin was used as a reference protein. When compared with egg albumin, all protein fractions significantly enhanced iron dialysability, except for light meromyosin which was inhibitory. Myosin had a greater enhancing effect than actin and, within myosin, the enhancing effect was greatest for the heavy meromyosin fraction. The enhancement appeared to coincide with the known distribution of cysteine residues in the myofibrillar proteins. The presence of the sulphydryl blocking agent, N-ethylmaleimide (NEM), in meals containing myosin reduced iron dialysability in a dose-related manner, but NEM had only a small effect in meals containing actin. Meanwhile, incorporation of cysteine into meals containing actin increased iron dialysability. The present results suggest that the enhancement of non-haem iron dialysability by meat is associated with myosin, in particular, with the heavy meromyosin region. Peptide fractions rich in cysteine residues, probably constitutes the 'meat factor'.
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PMID:Effect of myofibrillar muscle proteins on the in vitro bioavailability of non-haem iron. 1061 59

Physicochemical changes and in vitro digestibility of chicken breast myosin oxidized with a nonenzymic free-radical-generating system (FeCl(3)/H(2)O(2)/ascorbate) were studied by SDS-PAGE, differential scanning calorimetry, and o-phthaldialdehyde assay. Oxidation caused fragmentation and polymerization of myosin. Myosin polymers were cross-linked mainly through disulfide bonds. Hydroxyl radicals destabilized myosin, lowering its denaturation temperature by up to 4 degrees C. Oxidized myosin also produced a new thermal transition in the 60-80 degrees C temperature range, which could be attributed to the formation of disulfide-stabilized polymers. The proteolytic susceptibility of myosin to pepsin, trypsin, and chymotrypsin was increased by oxidation. Under nonreducing conditions, however, oxidized myosin showed decreased digestibility. The results may help explain variations in the functionality and nutritional quality of muscle foods in meat processing in which oxidation is involved.
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PMID:Electrophoretic pattern, thermal denaturation, and in vitro digestibility of oxidized myosin. 1072 25


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