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

This study examines the interaction of titin and myosin. In order to analyze the domains of myosin contributing to the binding for titin, we conducted a solid phase binding assay. Different portions of myosin (heavy chains, light chains and myosin fragments) were coated on the microtiter wells and reacted with biotinylated titin. Then the binding of biotinylated titin to these polypeptides was detected by using the avidinbiotin-peroxidase method. The results demonstrated that light meromyosin and subfragment 1 were the major domains of myosin interacting with titin. Titin fragments obtained by trypsin digestion were allowed to react with myosin in an affinity column, and the bound fragments were isolated by an acidic elution. Immunoblot analysis of myosin-bound titin fragments revealed that an A-band domain of titin was responsible for the binding of myosin. In addition, biotinylated titin labelled the outer A-bands and Z-bands in intact myofibrils, thus confirming the in situ binding of titin to myosin.
 ...
PMID:Studies on the interaction between titin and myosin. 150 52

Limited proteolysis of titin with trypsin yielded a number of polypeptides which were electrophoresed and transferred to a nitrocellulose membrane. Proteolytic removal of the C-terminal residues on the nitrocellulose-bound polypeptides was achieved by using carboxypeptidase Y. The species of the polypeptides left after the digestion was quantified by immunoblotting with two distinct monoclonal anti-titin antibodies A2 and A12 of which the epitopes were located at 0.74 micron and 0.69 micron away from the center of an A-band, respectively. Two polypeptides (266 kd and 84 kd) reactive to both antibodies were identified in the control group. Fifteen minutes after the digestion, the immunoreactivities of A2 on 266 kd and 84 kd polypeptides were disappeared, while those of A12 on these polypeptides were not affected. The results indicate that the C-terminal end of titin is located near the Z-line region and the N-terminal end at the M-line region in the sarcomere.
 ...
PMID:Location of the C-terminus of titin at the Z-line region in the sarcomere. 170 43

Muscle needs an elastic framework to maintain its mechanical stability. Removal of thin filaments in rabbit skeletal muscle with plasma gelsolin has revealed the essential features of elastic filaments. The selective removal of thin filaments was confirmed by staining with phalloidin-rhodamine for fluorescence microscopy, examination of arrowhead formation with myosin subfragment 1 by electron microscopy, and analysis by SDS-PAGE. Thin section electron microscopy revealed the elastic fine filaments (approximately 4 nm in diameter) connecting thick filaments and the Z line. After removal of thin filaments, both rigor stiffness and active tension generation were lost, but the resting tension remained. These observations indicate that the thin filament-free fibers maintain a framework composed of the serial connections of thick filaments, the elastic filaments, and the Z line, which gives passive elasticity to the contractile system of skeletal muscle. The resting tension that remained in the thin filament-free fibers was decreased by mild trypsin treatment. The only protein component that was digested in parallel with the decrease in the resting tension and the disappearance of the elastic filaments was alpha-connectin (also called titin 1), which was transformed from the alpha to the beta form (from titin 1 to 2, respectively). Thus, we conclude that the main protein component of the elastic filaments is alpha-connectin (titin 1).
 ...
PMID:Elastic filaments in skeletal muscle revealed by selective removal of thin filaments with plasma gelsolin. 215 47

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.
 ...
PMID:Passive tension in cardiac muscle: contribution of collagen, titin, microtubules, and intermediate filaments. 775 23

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.
 ...
PMID:Titin develops restoring force in rat cardiac myocytes. 878 95

The intrinsic cellular mechanisms by which length regulates myocardial contraction, the basis of the Frank-Starling relation, are uncertain. The aim of this work was to test the hypothesis that passive force, possibly via titin, participates in the modulation of Ca2+ sensitivity of cardiac contractile proteins induced by stretch. Titin degradation by a mild trypsin digestion modulated passive force induced by increasing from 1.9 to 2.3 microm sarcomere length in skinned rat cardiac cells. Force-pCa curves were established at these two sarcomere lengths after various durations of trypsin application that induced different passive force levels. They allowed us to evaluate myofilament Ca2+ sensitivity by the pCa of half-maximal activation (pCa50). In control conditions, stretching cells from 1.9 to 2.3 microm induced a leftward shift of pCa50 (DeltapCa50) of 0.39+/-0.03 pCa units (mean+/-SEM, n=8 cells), reflecting an increase in Ca2+ sensitivity of the contractile machinery. Passive force measured every 2 min decreased exponentially after the beginning of the trypsin application (t1/2 approximately 12 min). The first 30% decrease of passive force did not affect the stretch-induced variation in Ca2+ sensitivity. Then, with further decrease in passive force, DeltapCa50 decreased. At the lowest passive force investigated 20% of initial passive force, DeltapCa50 decreased by approximately 55%. These effects were not accompanied by a significant modification of either maximal activated force at pCa 4.5 solution or pCa50 at 1.9 microm sarcomere length. This indicates that there was no major functional alteration of the contractile machinery during the protocol as also suggested by contractile and regulatory protein electrophoresis on 2.5-12% gradient and 15% SDS-PAGE gels. Thus, besides modulation induced by the reduced lattice spacing during enhanced heart refilling, Ca2+ sensitivity of the cardiac contractile machinery may be controlled at least partially by internal passive load, which is known to be largely attributable to titin.
 ...
PMID:Length modulation of active force in rat cardiac myocytes: is titin the sensor? 1037 96

One of the most salient physiological characteristics of cardiac muscle is that a dilated heart pumps more vigorously, a phenomenon known as the Frank-Starling relationship (see Allen and Kentish, 1985). At least two cellular mechanisms participate in this phenomenon: the reduction of the interfilament lattice spacing which favors the formation of cross-bridges (Wang and Fuchs, 1995) and the increased affinity of troponin C (TnC) for calcium (Ca2+) (Babu et al., 1988). In the latter case, it has been established that TnC itself is not the length sensor (Moss et al., 1991). The intracellular structure(s) able to sense changes in cell length has always been challenged and is still not known. We previously observed on intact isolated cardiac cells that active tension is more closely related to passive tension than to sarcomere length per se (Cazorla et al., 1997). This might have some physiological implications in the working heart since we found that sub-epicardial cells are more supple than sub-endocardial cells. In the present work on skinned cells, we studied the relationship between different levels of passive tension (modulated by a mild trypsin digestion) and the shift in pCa50 of tension-pCa relations induced by a stretch of cells from 1.9 to 2.3 microns sarcomere length. A significant correlation was obtained between passive tension and the stretch-induced shift in pCa50, or stretch-sensitivity of the active force. These observations led us to assume that titin might play a role in sensing cell length to modulate the contractile activity. Besides, it is known that myocardial infarcted cells are less sensitive to stretch. We propose that, in such a rat model, alterations of titin might participate in heart failure.
 ...
PMID:Is titin the length sensor in cardiac muscle? Physiological and physiopathological perspectives. 1098 82

Alpha-connectin/titin-1 exists as an elastic filament that links a thick filament with the Z-disk, keeping thick filaments centered within the sarcomere during force generation. We have shown that the connectin filament has an affinity for calcium ions and its binding site(s) is restricted to the beta-connectin/titin-2 portion. We now report the localization and the characterization of calcium-binding sites on beta-connectin. Purified beta-connectin was digested by trypsin into 1700- and 400-kDa fragments. which were then subjected to fluorescence calcium-binding assays. The 400-kDa fragment possesses calcium-binding activity; the binding constant was 1.0 x 10(7) M(-1) and the molar ratio of bound calcium ions to the 400-kDa fragment reached a maximum of 12 at a free calcium ion concentration of approximately 1.0 microM. Antibodies against the 400-kDa fragment formed a sharp dense stripe at the boundary of the A and the I bands, indicating that the calcium-binding domain constitutes the N-terminal region of beta-connectin, that is, the elastic portion of connectin filaments. Furthermore, we estimated the N-terminal location of beta-connectin of various origins (n = 26). Myofibrils were treated with a solution containing 0.1 mM CaCl2 and 70 microM leupeptin to split connectin filaments into beta-connectin and a subfragment, and chain weights of these polypeptides were estimated according to their mobility in 2% polyacrylamide slab gels. The subfragment exhibited a similar chain weight of 1200+/-33 kDa (mean+/-SD), while alpha- and beta-connectins were variable in size according to their origin. These results suggest that the apparent length of the 1200-kDa subfragment portion is almost constant in all instances, about 0.34 microm at the slack condition, therefore that the C-terminus of the 1200-kDa subfragment, that is, the N-terminus of the calcium-binding domain, is at the N2 line region of parent filaments in situ. Because the secondary structure of the 400-kDa fragment was changed by the binding of calcium ions, connectin filaments could be expected to alter their elasticity during the contraction-relaxation cycle of skeletal muscle.
 ...
PMID:Calcium binding to an elastic portion of connectin/titin filaments. 1151 38

The shortening velocity of skeletal muscle fibres is determined principally by actomyosin cross-bridges. However, these contractile elements are in parallel with elastic elements, whose main structural basis is thought to be the titin filaments. If titin is stretched, it may contribute to sarcomere shortening simply because it can recoil 'passively'. The titin-based contribution to shortening velocity (V(p)) was quantified in single rabbit psoas myofibrils. Non-activated specimens were rapidly released from different initial sarcomere lengths (SLs) by various step amplitudes sufficient to buckle the myofibrils; V(p) was calculated from the release amplitude and the time to slack reuptake. V(p) increased progressively (upper limit of detection, approximately 60 microm s(-1) sarcomere(-1)) between 2.0 and 3.0 microm SL, albeit more steeply than passive tension. At very low passive tension levels already (< 1-2 mN mm(-2)), V(p) could greatly exceed the unloaded shortening velocity measured in fully Ca(2+)-activated skinned rabbit psoas fibres. Degradation of titin in relaxed myofibrils by low doses of trypsin (5 min) drastically decreased V(p). In intact myofibrils, average V(p) was faster, the smaller the release step applied. Also, V(p) was much higher at 30 degrees C than at 15 degrees C (Q(10): 2.0, 3.04 or 6.15, for release steps of 150, 250 or 450 nm sarcomere(-1), respectively). Viscous forces opposing the shortening are likely to be involved in determining these effects. The results support the idea that the contractile system imposes a braking force onto the passive recoil of elastic structures. However, elastic recoil may aid active shortening during phases of high elastic energy utilization, i.e. immediately after the onset of contraction under low or zero load or during prolonged shortening from greater physiological SLs.
 ...
PMID:Titin-based contribution to shortening velocity of rabbit skeletal myofibrils. 1192 78

Titin, a giant protein spanning half the sarcomere, is responsible for passive and restoring forces in cardiac myofilaments during sarcomere elongation and compression, respectively. In addition, titin has been implicated in the length-dependent activation that occurs in the stretched sarcomere, during the transition from diastole to systole. The purpose of this study was to investigate the role of titin in the length-dependent deactivation that occurs during early diastole, when the myocyte is shortened below slack length. We developed a novel in vitro assay to assess myocyte restoring force (RF) by measuring the velocity of recoil in Triton-permeabilized, unloaded rat cardiomyocytes after rigor-induced sarcomere length (SL) contractions. We compared rigor-induced SL shortening to that following calcium-induced (pCa) contractions. The RF-SL relationship was linearly correlated, and the SL-pCa curve displayed a characteristic sigmoidal curve. The role of titin was defined by treating myocytes with a low concentration of trypsin, which we show selectively degrades titin using mass spectroscopic analysis. Trypsin treatment reduced myocyte RF as shown by a decrease in the slope of the RF-SL relationship, and this was accompanied by a downward and leftward shift of the SL-pCa curve, indicative of sensitization of the myofilaments to calcium. In addition, trypsin digestion did not alter the relationship between SL and interfilament spacing (assessed by cell width) after calcium activation. These data suggest that as the sarcomere shortens below slack length, titin-based restoring forces act to desensitize the myofilaments. Furthermore, in contrast to length-dependent activation at long SLs, length-dependent deactivation does not depend on interfilament spacing. This study demonstrates for the first time the importance of titin-based restoring force in length-dependent deactivation during the early phase of diastole.
 ...
PMID:Titin determines the Frank-Starling relation in early diastole. 1256 38


1 2 Next >>