Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Interactions between troponin C (TnC) and troponin I (TnI) play an important role in the Ca2(+)-dependent regulation of vertebrate striated muscle contraction. Previous attempts to elucidate the molecular details of TnC-TnI interactions, mainly involving chemically modified proteins or fragments thereof, have led to the widely accepted idea that the "inhibitory region" (residues 96-116) of TnI binds to an alpha-helical segment of TnC comprising residues 89-100 in the nonregulatory, COOH-terminal domain. In an attempt to identify other possible physiologically important interactions between these proteins, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) was used to produce zero-length cross-links in the complex of rabbit skeletal muscle TnC and TnI. TnC was activated with EDC and N-hydroxysuccinimide (NHS) and then mixed with an equimolar amount of TnI [Grabarek, Z., & Gergely, J. (1988) Biophys. J. 53, 392a]. The resulting cross-linked TnCXI was cleaved with cyanogen bromide, trypsin, and Staphylococcus aureus V8 protease (SAP). Cross-linked peptides were purified by reverse-phase HPLC and characterized by sequence analysis. The results indicated that residues from the regulatory Ca2(+)-binding site II in the NH2-terminal domain of TnC (residues 46-78) formed cross-links with TnI segments spanning residues 92-167. The most highly cross-linked residues in TnI were Lys-105 and Lys-107, located in the inhibitory region. These results yield the first evidence for an interaction between the N-terminal domain of TnC and the inhibitory region of TnI.
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PMID:Characterization of zero-length cross-links between rabbit skeletal muscle troponin C and troponin I: evidence for direct interaction between the inhibitory region of troponin I and the NH2-terminal, regulatory domain of troponin C. 210 19

Fluorescence energy transfer measurements were carried out between landmarks on wheat germ calmodulin to measure the interdomain distance. Tb3+ ions bound at the four Ca2+-binding sites were used as energy donors, and an organic chromophore, [4-(dimethylamino)-phenyl-4'-azophenyl]maleimide, attached to the single cysteine residue at position 27, was used as the acceptor. At pH's near neutrality all bound Tb3+ ions emit luminescence with shortened lifetimes as a result of energy transfer to the acceptor; at pH 5, however, part of the metal emission becomes unquenched. When the protein is subjected to limited digestion with trypsin in the presence of Ca2+, resulting in the formation of two fragments, each corresponding to half of the molecule, the decay of Tb3+ emission is no longer pH sensitive. These results suggest that, like rabbit skeletal troponin C [Wang, C.-L. A., Zhan, Q., Tao, T., & Gergely, J. (1987) J. Biol. Chem. 257, 8372-8375], wheat germ calmodulin exists in a relatively compact conformation at neutral pH's, but becomes more elongated at pH 5.
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PMID:pH-dependent conformational changes of wheat germ calmodulin. 250 82

Tritiated calmodulin (T-CM) was bound to the EGTA-treated particulate fraction of cardiac muscle in a calcium-dependent manner with half-maximal binding occurring between 0.8 to 1.2 microM calcium. Binding exhibited high specificity at an optimum pH of 7.4-7.6. An excess of parvalbumin and other globular proteins did not displace T-CM. The Kd for the interaction was 2.5 +/- 0.83 microM. Binding was trypsin-sensitive, inhibited by high ionic strength and was heat inactivated at a midpoint of 48 - 50 degrees C. Competitive displacement of T-CM occurred with unlabeled troponin C and calmodulin over the same concentration range. The first-order rate constant of T-CM dissociation was 3.27 min-1. Calcium-dependent binding of T-CM was inhibited equally by both mepacrine and trifluoperazine with 50 percent inhibition occurring at 70 microM.
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PMID:Interaction of calmodulin with the particulate fraction of cardiac muscle. 312 61

Rabbit muscle troponin C was selectively modified at Cys-98 by 1,3-difluoro-4,6-dinitrobenzene. The second function of the bifunctional reagent was triggered at alkaline pH in the presence and absence of Ca2+. The crosslinked troponin C was hydrolyzed by trypsin and the peptides containing a dinitrobenzene moiety were isolated. When troponin C was crosslinked in the presence of Ca2+, the single dinitrobenzene-containing peptide was Gly-89-Arg-100, in which Cys-98 was crosslinked with Lys-90. When crosslinking was performed in the absence of Ca2+, beside the above peptide two additional peptides containing dinitrobenzene were found. One of these peptides is made up of two fragments, Ser-91-Arg-100 and Asn-105-Arg-120, crosslinked between Cys-98 and Tyr-109. The second peptide, Ala-121-Lys-140, contains modified Lys-136, presumably crosslinked with His-135. The data indicate that the distances between the alpha-carbon of Cys-98 and those of Lys-90, Tyr-109, Lys-136 and probably the alpha-carbon distance His-125-Lys-136, do not exceed 14 A. Comparison with the X-ray structure of troponin C (Herzberg, O. and James, M.N.G. (1985) Nature 313, 653-659) indicates that some of the above distances increase on Ca2+-binding.
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PMID:Ca2+-induced structural change in the Ca2+/Mg2+ domain of troponin C detected by crosslinking. 394 41

The main kinetic parameters for purified phosphorylase kinase from chicken skeletal muscle were determined at pH 8.2: Vm = 18 micromol/min/mg; apparent Km values for ATP and phosphorylase b from rabbit muscle were 0.20 and 0.02 mM, respectively. The activity ratio at pH 6.8/8.2 was 0.1-0.4 for different preparations of phosphorylase kinase. Similar to the rabbit enzyme, chicken phosphorylase kinase had an absolute requirement for Ca2+ as demonstrated by complete inhibition in the presence of EGTA. Half-maximal activation occurred at [Ca2+] = 0.4 microM at pH 7.0. In the presence of Ca2+, the chicken enzyme from white and red muscles was activated 2-4-fold by saturating concentrations of calmodulin and troponin C. The C0.5 value for calmodulin and troponin C at pH 6.8 was 2 and 100 nM, respectively. Similar to rabbit phosphorylase kinase, the chicken enzyme was stimulated about 3-6-fold by glycogen at pH 6.8 and 8.2 with half-maximal stimulation occurring at about 0.15% glycogen. Protamine caused 60% inhibition of chicken phosphorylase kinase at 0.8 mg/ml. ADP (3 mM) at 0.05 mM ATP caused 85% inhibition with Ki = 0.2 mM. Unlike rabbit phosphorylase kinase, no phosphorylation of the chicken enzyme occurred in the presence of the catalytic subunit of cAMP-dependent protein kinase. Incubation with trypsin caused 2-fold activation of the chicken enzyme.
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PMID:[Regulatory properties of phosphorylase from chicken skeletal muscle]. 407 75

We report here that a precipitating antibody prepared against Tetrahymena pyriformis calmodulin recognizes calcium-dependent determinants in the native protein. The ability of the antibody to precipitate 35S-labeled Tetrahymena calmodulin in direct radioimmunoassays was enhanced at least 3-fold in the presence of calcium. Competitive radioimmunoassay using homogeneous preparation of endogenously 35S-labeled Tetrahymena calmodulin and protein A-Sepharose-purified immunoglobulin G demonstrated that this antibody preparation is specific for protozoan calmodulin. Homogeneous vertebrate, invertebrate, and plant calmodulins, as well as rabbit skeletal muscle troponin C, did not show significant competition with the 35S-labeled Tetrahymena protein at concentrations 100-fold greater than that at which the homologous unlabeled Tetrahymena calmodulin produced 50% competition. A cyanogen bromide digest of Tetrahymena calmodulin also showed partial competition with the intact 35S-labeled protein, but only in the presence of calcium. The major antigenic determinants were localized to the carboxyl-terminal half of the molecule by immunoassay of limited trypsin fragments of Tetrahymena calmodulin. The antibody bound native calmodulin complexed to bovine brain phosphodiesterase (EC 3.1.4.17) but failed to recognize the Tetrahymena calmodulin carboxyl-terminal fragment (76-147) when complexed to the enzyme.
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PMID:Polyclonal antibody that recognizes calcium-dependent determinants in Tetrahymena calmodulin. 608 17

A major protein constituent of a rat islet cell tumour that exhibited Ca2+-dependent changes in electrophoretic mobility has been purified to homogeneity and compared in its physicochemical and biological properties with bovine brain and rat brain calmodulin (synonymous with phosphodiesterase activator protein, calcium-dependent regulator, troponin C-like protein and modulator protein). The protein, like these calmodulins, contained trimethyl-lysine, exhibited a blocked N-terminus and had an identical amino-acid composition and molecular weight on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Peptide "maps' prepared after digestion of the three proteins with trypsin, papain or Staphylococcus V-8 proteinase were virtually superimposable. Ca2+ altered the electrophoretic mobilities the enhanced the native protein fluorescence in an equivalent manner with all three proteins. Equilibrium dialysis experiments demonstrated in each case the binding of 4g-atoms of calcium/mol of protein; the binding sites were equivalent and showed Kd 0.8 microM. Tumour and brain proteins were equipotent as Ca2+-dependent activators of partially purified rat brain cyclic nucleotide phosphodiesterase, and in this action were inhibited in an identical manner by trifluoperazine. The proteins also exhibited the common property of Ca2+-dependent binding to troponin I, histone H2B and myelin basic protein. The estimated tumour content of calmodulin was 450 mg/kg fresh wt., a value similar to that reported in islets of Langerhans. These results further document the validity of the islet cell tumour as an experimental model of Ca2+-mediated molecular events associated with insulin secretion. They also suggest that brain calmodulin may be substituted for endogenous calmodulin in experimental investigations into the mechanism of insulin secretion.
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PMID:Isolation and characterization of calmodulin from an insulin-secreting tumour. 627 21

Glycerinated muscle fiber from rabbit psoas muscle often lost Ca2+-dependent regulation of its contraction with long-term extraction in a 50% glycerol solution containing 5 mM EGTA at -20 degrees C, designated as Ca2+-insensitive muscle fiber (CaIS-fiber). About 30 or 40% of glycerinated muscle fibers were CaIS-fibers after 1 to 3 months in the glycerol solution. We investigated the cause of the loss of Ca2+-sensitivity of the glycerinated muscle fiber by tension mechanogram and SDS polyacrylamide gel electrophoresis. This natural CaIS-fiber showed a new band of 30K daltons peptide on SDS gels. On the other hand, Ca2+-sensitive fiber (CaS-fiber) changed to CaIS-fiber by trypsin digestion for 40 sec. The tryptic CaIS-fiber had no troponin C and 30K daltons peptide bands in the electrophoretograms. Incubating with 2 mM CaCl2 for 40 hr at 25 degrees C, CaS-fiber changed easily to CaIS-fiber which had 30K daltons peptide and faint troponin T and I bands, as in natural CaIS-fiber. All CaIS-fibers could recover their Ca2+-dependent regulation by incubating with native tropomyosin from rabbit skeletal muscle for 2 days at 4 degrees C. These results indicate that the loss of Ca2+-dependent regulation of glycerinated muscle fiber is due to degradation of regulatory protein system by endogenous Ca2+-activated proteolytic enzymes.
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PMID:Loss of Ca2+-dependent regulation in glycerinated skeletal muscle contraction. 635 84

The exposure of hydrophobic sites on calmodulin, skeletal muscle troponin C and their tryptic fragments was investigated using Phenyl-Sepharose chromatography. A strong binding of both proteins and their fragments corresponding to the NH2-terminal halves of polypeptide chain of respective proteins in the presence of calcium ions was observed. Only a weak interaction with Phenyl-Sepharose or its lack was observed under these conditions for fragments corresponding to the COOH-terminal halves of calmodulin and troponin C, respectively. The elution of the samples from Phenyl-Sepharose column with ethylene glycol gradient allowed to compare relative hydrophobicity of both proteins and their fragments. The results show that hydrophobic properties of calmodulin and troponin C are virtually preserved in their fragments obtained as a result of their cleavage by trypsin in half. They also indicated that the exposure of hydrophobic residues caused by the binding of calcium ions takes place mainly in the NH2-terminal halves of polypeptide chains of both proteins. A simple method of purification of tryptic fragments of both proteins based on the difference in the strength of their interactions with Phenyl-Sepharose is described.
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PMID:Localization of hydrophobic sites in calmodulin and skeletal muscle troponin C studied using tryptic fragments: a simple method of their preparation. 661 40

The rate of tryptic digestion of troponin C has been shown to be dependent on Ca2+ (Drabikowski et al., Biochim. Biophys. Acta 490, 216-224). We have characterized the tryptic peptides produced both in the presence and absence of Ca2+ using amino acid composition and end-group analyses. In the presence of Ca2+ trypsin cleaves TnC at Arg-8, Lys-84 and Lys-88, leading to the formation of two large peptides, one containing the two low-affinity sites (TR1C), the other, the two high-affinity Ca2+-binding sites (TR2C). In the absence of Ca2+ (1 mM EDTA), digestion proceeds much more rapidly and takes place first at Arg-100, followed by Arg-104, Arg-120, Lys-153, Arg-8 and others. The data suggest that the points of cleavage are determined by the Ca2+-dependent conformational states of TnC, particularly in the C-terminal half of the protein where the cation is known to induce secondary structure.
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PMID:Digestion of troponin C with trypsin in the presence and absence of Ca2+. Identification of cleavage points. 732 66


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