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)

To analyze direct effects of steroids on the rates of synthesis (and/or degradation) of newly synthesized proteins of the rat heart, we have used high resolution two-dimensional gel electrophoresis and autoradiography. A collective steroid domain of nineteen proteins, comprising fifteen with an increased rate of synthesis and four with a decreased rate of synthesis, was consistently seen in cultures of cardiac muscle and non-muscle cells from neonatal rats following 24 h incubation with 10(-7) dexamethasone. Similarly, incubation with 10(-7) M sex steroids, mineralocorticoids, and other glucocorticoids including the highly selective compound RU26988, established the glucocorticoid-specificity of the response. Different subsets of this glucocorticoid domain were seen for collagenase- or trypsin-dispersed primary cultures of cardiac muscle and non-muscle cells or for passaged cultures of cardiac non-muscle cells. Six polypeptides were consistently induced in all cardiac cultures, regardless of cell morphology. Two polypeptides were consistently induced only in those cultures containing cardiac non-muscle cells, whereas protein l, of identical Mr(approximately 52K) and pI (approximately 5.3) to desmin, was induced only in cultures of spontaneously contractile cardiac muscle cells. The glucocorticoid domain proteins described herein represent direct steroid effects on cardiac cells and are therefore candidate mediators of physiological glucocorticoid effects on, for example, differentiation and contractility.
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PMID:Glucocorticoid effects on newly synthesized proteins in muscle and non-muscle cells cultured from neonatal rat hearts. 651 49

The light chain fraction was separated from myosin extracted from chicken cardiac muscle. Two light chain components, L-1 and L-2 in the fraction were isolated by chromatography on a column of DEAE-cellulose (DE-52) in the presence of4 M urea. After performic acid oxidation, the L-1 chain was digested with trypsin and the resulting peptides were isolated. The amino acid sequences of the peptides were established. The ordering of these tryptic peptides in the L-1 chain was deduced from the amino acid compositions and the partial sequences of peptic peptides from S-carboxymethylated L-1 chain. Comparing the whole sequence of the L-1 chain thus established with that of alkali light chain of rabbit skeletal muscle myosin, 67 amino acid substitutions and two insertions were recognized.
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PMID:Amino-acid sequence of the L-1 light chain of chicken cardiac-muscle myosin. 677 48

In order to define further the nature of the apparent close interaction between mitochondrial creatine phosphokinase and oxidative phosphorylation (Erickson-Viitanen, S., Viitanen, P., Geiger, P. J., Yang, W. C. T., and Bessman, S. P. (1982) J. Biol. Chem. 257, 14395-14404), rabbit heart and rat skeletal muscle mitochondria prepared by gentle mechanical homogenization were compared with preparations isolated after tryptic digestion of tissues, a method which has been reported to yield superior mitochondria (Reichert, M., Schaller, H., Kung, W., and Gerber, G. (1978) Acta Biol. Med. Germ. 37, 1167-1176). The ability of de novo synthesized and exported mitochondrial ATP to interact with creatine phosphokinase prior to total mixing of the ATP pool, which we consider to be evidence of compartmentation, could not be demonstrated with mitochondria prepared via the trypsin procedure. Mitochondria from rabbit cardiac muscle treated with digitonin synthesized ATP and creatine phosphate, but failed to show apparent compartmentation of creatine phosphokinase. Km values for ATP were compared for four conditions: 1) respiring, digitonin-treated rabbit heart mitochondria, 2) atractyloside-inhibited, digitonin-treated rabbit heart mitochondria supplied with a pyruvate kinase-phosphoenolpyruvate regenerating system, 3) respiring rabbit heart mitochondria, 4) atractyloside-inhibited rabbit heart mitochondria supplied with an ATP regenerating system. The observed Km values for ATP for conditions 1, 2, and 3 were similar but lower than that for condition 4. These findings suggest that an outer membrane diffusion barrier influences or controls mitochondrial creatine phosphokinase compartmentation.
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PMID:Compartmentation of mitochondrial creatine phosphokinase. II. The importance of the outer mitochondrial membrane for mitochondrial compartmentation. 714 18

Two isoforms of the ryanodine receptor (termed alpha and beta) are coexpressed in avian fast twitch skeletal muscle, whereas a single isoform is expressed in avian cardiac muscle. We have investigated the relationship between these three proteins, comparing several different properties. First, the three receptor isoform subunits have different mobilities on SDS-polyacrylamide gels. Second, monoclonal antibodies against the chicken skeletal muscle receptor isoforms recognize shared and unique epitopes in each receptor protein, indicating there is not a simple antigenic relationship between the isoforms. Third, the three receptor isoforms exhibit different susceptibilities to proteolysis by trypsin, and limited tryptic digestion yields a different peptide map for each isoform. Fourth, in native sarcoplasmic reticulum membranes, the chicken muscle receptor isoforms are phosphorylated to different extents by the multifunctional calcium/calmodulin-dependent protein kinase II (beta > cardiac > alpha). Fifth, the sites phosphorylated by the calcium/calmodulin-dependent protein kinase in the chicken cardiac and skeletal receptor isoforms are not equivalent. A polyclonal serum, produced against a synthetic peptide containing the site phosphorylated by this kinase in the mammalian cardiac muscle receptor, by immunoprecipitation showed markedly different avidities for the receptor isoforms, and recognized only the cardiac receptor isoform on Western blots. Sixth, the chicken ryanodine receptor isoforms differ in the extent to which they bind azido[125I]calmodulin (alpha > beta > cardiac). These results indicate that three distinct ryanodine receptor proteins are expressed in chicken striated muscles.
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PMID:Three ryanodine receptor isoforms exist in avian striated muscles. 768 27

At least three distinct ryanodine receptor genes appear to be expressed in mammalian brain. We have used biochemical and immunological methods to characterize the major form of ryanodine binding protein purified from brain. [3H]Ryanodine binding to the purified brain receptor is stimulated by Ca2+, ATP, KCl, and phosphorylation and is inhibited by calmodulin, Mg2+, and ruthenium red. Immunoblot and immunoprecipitation analysis using a panel of monoclonal and polyclonal antibodies against skeletal and cardiac muscle ryanodine receptors, and two novel polyclonal antibodies against the brain ryanodine receptor, reveals that the major form of ryanodine receptor expressed in brain is immunologically similar to the cardiac ryanodine receptor, but is distinct from the skeletal muscle receptor. Digestion of cardiac and brain ryanodine receptors with trypsin or alpha-chymotrypsin generates similar proteolytic patterns as detected by immunoblot analysis or by autoradiography after labeling with a hydrophobic probe, suggesting that the two proteins are similar in both their large cytoplasmic and hydrophobic transmembrane domains. Taken together, these data indicate that the cardiac ryanodine receptor/Ca2+ release channel is the major form of ryanodine receptor expressed in brain, and that it likely functions in releasing Ca2+ from caffeine-sensitive intracellular Ca2+ stores in neurons by a mechanism of regulated Ca(2+)-induced Ca2+ release.
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PMID:Characterization of the major brain form of the ryanodine receptor/Ca2+ release channel. 769 41

The passive tension-sarcomere length relation of rat cardiac muscle was investigated by studying passive (or not activated) single myocytes and trabeculae. The contribution of collagen, titin, microtubules, and intermediate filaments to tension and stiffness was investigated by measuring (1) the effects of KCl/KI extraction on both trabeculae and single myocytes, (2) the effect of trypsin digestion on single myocytes, and (3) the effect of colchicine on single myocytes. It was found that over the working range of sarcomeres in the heart (lengths approximately 1.9-2.2 microns), collagen and titin are the most important contributors to passive tension with titin dominating at the shorter end of the working range and collagen at longer lengths. Microtubules made a modest contribution to passive tension in some cells, but on average their contribution was not significant. Finally, intermediate filaments contributed about 10% to passive tension of trabeculae at sarcomere lengths from approximately 1.9 to 2.1 microns, and their contribution dropped to only a few percent at longer lengths. At physiological sarcomere lengths of the heart, cardiac titin developed much higher tensions (> 20-fold) than did skeletal muscle titin at comparable lengths. This might be related to the finding that cardiac titin has a molecular mass of 2.5 MDa, 0.3-0.5 MDa smaller than titin of mammalian skeletal muscle, which is predicted to result in a much shorter extensible titin segment in the I-band of cardiac muscle. Passive stress plotted versus the strain of the extensible titin segment showed that the stress-strain relationships are similar in cardiac and skeletal muscle. The difference in passive stress between cardiac and skeletal muscle at the sarcomere level predominantly resulted from much higher strains of the I-segment of cardiac titin at a given sarcomere length. By expressing a smaller titin isoform, without changing the properties of the molecule itself, cardiac muscle is able to develop significant levels of passive tension at physiological sarcomere lengths.
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PMID:Passive tension in cardiac muscle: contribution of collagen, titin, microtubules, and intermediate filaments. 775 23

Calsequestrin is an intermediate affinity, high capacity Ca(2+)-binding protein found in the lumen of the sarcoplasmic reticulum of both skeletal and cardiac muscle cells. Previous sequence analysis suggested that calsequestrin may contain a hydrophobic binding site for the drug trifluoperazine, a site shared by the calmodulin family and shown to play a role in calmodulin/calmodulin receptor interaction. Previous studies showed that, upon Ca2+ binding, calsequestrin undergoes a conformational change, burying the trifluoperazine-binding site, folding into a more compact structure that is trypsin-resistant, and increasing the negative ellipticity of the circular dichroism spectrum. In this study, the structural and functional roles of the trifluoperazine-binding site in the Ca(2+)-induced conformational change of calsequestrin are further studied using the calmodulin antagonists trifluoperazine and melittin. If trifluoperazine or melittin is added to calsequestrin prior to Ca2+ addition, then Ca(2+)-induced folding is inhibited as determined by the changes in circular dichroism spectra and protein sensitivity to trypsin digestion. If, however, Ca2+ is added prior to trifluoperazine or melittin, calsequestrin remains resistant to trypsin digestion, just as if the calmodulin antagonists are not present, suggesting that the conformational change is not affected. Aggregates of calsequestrin that exhibit high Ca2+ binding capacity have previously been shown to occur at high Ca2+ and calsequestrin concentrations. By preventing a prerequisite folding step, trifluoperazine or melittin also prevents the Ca(2+)-induced aggregation of calsequestrin, thus decreasing the maximal Ca2+ binding by calsequestrin. These data suggest that the trifluoperazine-binding site is critically involved in the Ca(2+)-induced intramolecular folding step required for the intermolecular interactions leading to high capacity Ca(2+)-binding by calsequestrin.
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PMID:Ca(2+)-induced folding and aggregation of skeletal muscle sarcoplasmic reticulum calsequestrin. The involvement of the trifluoperazine-binding site. 822 22

When relaxed after contraction, isolated cardiac myocytes quickly relengthen back to their slack length. The molecular basis of the force that underlies passive relengthening, known as restoring force, is not well understood. In a previous study of titin's elasticity in cardiac myocytes, we proposed that titin/connectin develops restoring force, in addition to passive force. This study tested whether titin indeed contributes to the restoring force in cardiac myocytes. Skinned rat cardiac myocytes in suspension were shortened by approximately 20%, using Ca(2+)-independent shortening, followed by relaxation. Cells were observed to relengthen until they reached their original slack sarcomere length. However, the ability to relengthen was abolished after cells had been treated for 12 minutes with trypsin (0.25 microgram/mL, 20 degrees C). Gel electrophoresis showed that this treatment had degraded titin without clearly affecting other proteins, and immunoelectron microscopy revealed that the elastic segment of titin in the I band was missing from the sarcomere. Restoring force was also directly measured, before and after trypsin treatment. Restoring force of control cells was -61 +/- 20 micrograms (per cell) at a sarcomere length of 1.70 microns. Comparison of our results with those of activated trabeculae indicated that a large fraction of restoring force of cardiac muscle originates from within the myocyte. Restoring force of myocytes was found to be depressed after titin had been degraded with trypsin. We conclude that cardiac, titin indeed develops restoring force in shortened cardiac myocytes, in addition to passive force in stretched cells, and that titin functions as a bidirectional spring. Our work suggests that at the level of the whole heart, part of the actomyosin-based active force that is developed during systole is harnessed by titin, allowing for elastic diastolic recoil and aiding in ventricular filling.
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PMID:Titin develops restoring force in rat cardiac myocytes. 878 95

Increased ATP-sensitive potassium (KATP) channel activity in cardiac muscle during hypertension and myocardial hypertrophy may be induced by the release of endogenous proteases, altering inhibitory binding sites for intracellular ATP. To test this hypothesis, we studied the effects of trypsin (1.5 mg/ml) on channel sensitivity to ATP in myocytes from control (WKY) and spontaneously hypertensive rats (SHR). Trypsin increased channel activity in 63% of membrane patches from WKY rats, but in only 29% from SHR. Pre-treatment with trypsin decreased sensitivity to the inhibitory effects of ATP in both groups. These results support the possibility that KATP channel modification during chronic metabolic stress is caused by intracellular proteolysis, which decreases sensitivity to [ATP]i.
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PMID:Trypsin alters ATP sensitivity of KATP channels in control and hypertrophied myocytes. 896 Aug 72

Both regular physical exercise and low levels of H(2)O(2) administration result in increased resistance to oxidative stress. We measured the accumulation of reactive carbonyl derivatives and the activities of proteasome complex and DT-diaphorase in cardiac muscle of trained and untrained rats after chronic i.p. administration of 1 ml t-butyl H(2)O(2) (1 mmol/kg for 3 weeks every second day). Twenty-four rats were randomly assigned to a control group administered with saline, control administered with H(2)O(2), and exercised administered either saline or H(2)O(2). The activity of DT-diaphorase significantly increased in H(2)O(2) administered and exercised groups, indicating that an increase in H(2)O(2) levels stimulate the activity of this enzyme. The cardiac muscle of H(2)O(2) administered nonexercised animals accumulated significantly more carbonyl than control group (P < 0.05). The exercise and H(2)O(2) administration resulted in less oxidatively modified protein than found in nonexercised groups (P < 0.05). The peptide-like activity of proteasome complex was induced by the treatment of H(2)O(2) and exercise and exercise potentiate the effect of H(2)O(2). On the other hand, the chymotrypsin-like and trypsin-like activities were stimulated only by physical training and H(2)O(2) administration. The data suggest that chronic administration of H(2)O(2) after exercise training decreases the accumulation of carbonyl groups below the steady-state level and induces the activity of proteasome and DT-diaphorase. Hence, the stimulating effect of physical exercise on free radical generation is an important phenomenon of the exercise-induced adaptation process since it increases resistance to oxidative stress. Regular exercise training is a valuable physiological means of preconditioning the myocardium to prolonged oxidative stress.
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PMID:Exercise preconditioning against hydrogen peroxide-induced oxidative damage in proteins of rat myocardium. 1077 9


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