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1.
Journal of Muscle Research and Cell Motility - Double mutation D208Q:K450L was introduced in the beta isoform of human cardiac myosin to remove the salt bridge D208:K450 connecting loop 1 and the... 相似文献
2.
Summary X-ray diffraction patterns from frog sartorius muscle were recorded during steady shortening with various loads. The intensity of the third meridional reflection from the thick filament decreased on shortening to an extent proportional to the drop in tension. The intensity correlated more closely with the tension than with the shortening velocity. The Bragg spacing of the third meridional reflection decreased in proportion to the decrease in tension. The intensity decrease of the actin layer lines at 1/5.1 and 1/5.9 nm–1 was roughly proportional to the decrease in the load, indicating that the number of cross-bridges decreases similarly. The intensity of the (1,1) equatorial reflection showed a significant decrease only with low loads. Assuming that a steady structural state is attained during steady shortening, the results are consistent with the cross-bridge model in which the number of myosin cross-bridges decreases during shortening. 相似文献
3.
W. Glenn L. Kerrick James D. Potter Phyllis E. Hoar 《Journal of muscle research and cell motility》1991,12(1):53-60
Summary The effect of Ca2+ activation on the apparent rate constant governing the dissociation of force generating myosin cross-bridges was studied in skinned rabbit adductor magnus fibres (fast-twitch) at 21±1 °C. Simultaneous measurements of Ca2+-activated isometric force and ATPase activity were conducted in parallel with simultaneous measurements of DANZ-labelled troponin C (TnCDANZ) fluorescence and isometric force in fibres whose endogenous troponin C had been partially replaced with TnCDANZ. The Ca2+ activation of isometric force occurred at approximately two times higher Ca2+ concentration than did actomyosin ATPase activity at 2.0 mM MgATP. Since increases in both TnCDANZ fluorescence and ATPase activity occurred over approximately the same Ca2+ concentration range at substantially lower concentrations of Ca2+ than did force, this data suggests that the TnCDANZ fluorescence is associated with the Ca2+ activation of myosin crossbridge turnover (ATPase) rather than force. According to the model of Huxley (1957) and assuming the hydrolysis of one molecule of ATP per cycle of the crossbridge, the apparent rate constantg
app for the dissociation of force generating myosin crossbridges is proportional to the actomyosin ATPase/isometric force ratio. This measure ofg
app shows approximately a fivefold decrease during Ca2+ activation of isometric force. This change ing
app is responsible for separation of the Ca2+ sensitivity of the normalized ATPase activity and isometric force curves. If the MgATP concentration is reduced to 0.5 mM, the change ing
app is reduced and consequently the difference in Ca2+ sensitivity between normalized steady state ATPase and force is also reduced.The abbreviations used are TnC
troponin C
- DANZ
5-dimethylaminonapthalene-2-sulphonyl aziridine
- TnCDANZ
DANZ-labelled TnC
-
a
the number of half sarcomeres
-
A
the cross-sectional area of the fibre
- AP5A
P1,P5-di(adenosine-5)pentaphosphate
- EGTA
ethyleneglycolbis-(betaaminoethyl ether)-N,N,N,N-tetraacetic acid
-
F
force a muscle fibre develops
- fapp
apparent rate of formation of force generating myosin crossbridges
- Fav
the average force per myosin head
-
Fs
steady-state fraction of cycling myosin crossbridges in the force generating state
-
g
app
apparent rate of dissociation of force generating myosin crossbridges
- L1/2s
the length of a half sarcomere
- LDH
lactate dehydrogenase
- [M]
is the concentration of myosin per fibre volume
- NAD
nicotinamide adenine dinucleotide
- NADH
reduced form of NAD
- pCa
-log10 of the free Ca2+ concentration
- PEP
phosphenol pyruvate
- PK
pyruvate kinase 相似文献
4.
Yagi N Okuyama H Toyota H Araki J Shimizu J Iribe G Nakamura K Mohri S Tsujioka K Suga H Kajiya F 《Pflügers Archiv : European journal of physiology》2004,448(2):153-160
We examined the sarcomere length-dependence of the spacing of the hexagonal lattice of the myofilaments and the mass transfer of myosin cross-bridges during contraction of right ventricular papillary muscle of the rat. The lattice spacing and mass transfer were measured by using X-ray diffraction, and the sarcomere length was monitored by laser diffraction at the same time. Although the lattice spacing and the sarcomere length were inversely related, their relationship was not exactly isovolumic. The cell volume decreased by about 15% when the sarcomere length was shortened from 2.3 µm to 1.8 µm. Twitch tension increased with sarcomere length (the Frank–Starling law). At the peak tension, the ratio of the intensity of the (1,0) equatorial reflection to that of the (1,1) reflection was smaller when the tension was greater, showing that the larger tension at a longer sarcomere length accompanies a larger amount of mass transfer of cross-bridges from the thick to the thin filament. The result suggests that the Frank–Starling law is due to an increase in the number of myosin heads attached to actin, not in the average force produced by each head. 相似文献
5.
Kuster DW Bawazeer AC Zaremba R Goebel M Boontje NM van der Velden J 《Journal of muscle research and cell motility》2012,33(1):43-52
Perturbations in sarcomeric function may in part underlie systolic and diastolic dysfunction of the failing heart. Sarcomeric dysfunction has been ascribed to changes in phosphorylation status of sarcomeric proteins caused by an altered balance between intracellular kinases and phosphatases during the development of cardiac disease. In the present review we discuss changes in phosphorylation of the thick filament protein myosin binding protein C (cMyBP-C) reported in failing myocardium, with emphasis on phosphorylation changes observed in familial hypertrophic cardiomyopathy caused by mutations in MYBPC3. Moreover, we will discuss assays which allow to distinguish between functional consequences of mutant sarcomeric proteins and (mal)adaptive changes in sarcomeric protein phosphorylation. 相似文献
6.
7.
Expression of the slow myosin heavy chain (MyHC) 2 gene defines slow versus fast avian skeletal muscle fiber types. Fetal, or secondary, skeletal muscle fibers express slow MyHC isoform genes in developmentally regulated patterns within the embryo, and this patterning is at least partly dependent on innervation in vivo. We have previously shown that slow MyHC 2 gene expression in vitro is regulated by a combination of innervation and cell lineage. This pattern of gene expression was indistinguishable from the pattern observed in vivo in that it was restricted to innervated muscle fibers of slow muscle origin. We show here that slow MyHC 2 gene expression in the slow muscle fiber lineage is regulated by protein kinase C (PKC) activity. Inhibition of PKC activity induced slow MyHC 2 gene expression, and the capacity to express the slow MyHC 2 gene was restricted to muscle fibers of slow muscle (medial adductor) origin. Fast muscle fibers derived from the pectoralis major did not express significant levels of slow MyHC 2 with or without inhibitors of PKC activity. This differential expression pattern coincided with different inherent PKC activities in fast versus slow muscle fiber types. Furthermore, over-expression of an unregulated PKCalpha mutant suppressed slow MyHC 2 gene expression in muscle fibers of the slow lineage. Lastly, denervation of skeletal muscles caused an increase in PKC activity, particularly in the slow medial adductor muscle. This increase in PKC activity was associated with lack of slow MyHC 2 gene expression in vivo. These results provide a mechanistic link between innervation, an intracellular signaling pathway mediated by PKC, and expression of a muscle fiber type-specific contractile protein gene. Dev Dyn 1999;216:177-189. 相似文献
8.
Wang D Luo Y Cooke R Grammer J Pate E Yount RG 《Journal of muscle research and cell motility》1999,20(8):743-753
A spin-labeled photoaffinity ATP analogue 3(2)-O-{4-[4-oxo-(4-amido-2,2,6,6-tetramethyl-piperidino-1-oxyl)]-benzoyl}benzoyl adenosine 5-triphosphate (SL-Bz2ATP) was synthesized and used to photolabel myosin in muscle fibers. Previous work has shown that 3(2)-O-(4-benzoyl)benzoyl adenosine 5-triphosphate (Bz2ATP) photolabeled Ser-324 of the 50 kDa tryptic fragment of skeletal S1 heavy chain. In this work, [-32P]SL-Bz2ATP was hydrolyzed and trapped as the diphosphate analogue with Co2+ and orthovanadate at the active site of myosin in rabbit psoas muscle fibers. After UV irradiation, the myosin heavy chain was the only protein band found to be significantly photolabeled as assayed by gel electrophoresis and radioactivity counting. The labeling was localized after brief trypsin digestion by SDS-PAGE to be on the 50 kDa tryptic fragment of the S1 heavy chain. Ca. 35% of the myosin in fibers was covalently photolabeled. The fibers photolabeled with SL-Bz2ATP had the same active tension and maximum shortening velocity as the control fibers. The resulting spin label on myosin was too mobile to report the orientation of the heads in fibers. Nonetheless, this is the first work to show the feasibility of utilizing active site binding and photoaffinity labeling to place covalent spectroscopic probes at the myosin active site in fibers with high specificity and yield without affecting mechanical function. 相似文献
9.
Temperature dependence of the force-generating process in single fibres from frog skeletal muscle 总被引:11,自引:7,他引:4
G. Piazzesi M. Reconditi N. Koubassova V. Decostre M. Linari L. Lucii V. Lombardi 《The Journal of physiology》2003,549(1):93-106
Generation of force and shortening in striated muscle is due to the cyclic interactions of the globular portion (the head) of the myosin molecule, extending from the thick filament, with the actin filament. The work produced in each interaction is due to a conformational change (the working stroke) driven by the hydrolysis of ATP on the catalytic site of the myosin head. However, the precise mechanism and the size of the force and length step generated in one interaction are still under question. Here we reinvestigate the endothermic nature of the force-generating process by precisely determining, in tetanised intact frog muscle fibres under sarcomere length control, the effect of temperature on both isometric force and force response to length changes. We show that raising the temperature: (1) increases the force and the strain of the myosin heads attached in the isometric contraction by the same amount (∼70 %, from 2 to 17 °C); (2) increases the rate of quick force recovery following small length steps (range between −3 and 2 nm (half-sarcomere)−1 ) with a Q 10 (between 2 and 12 °C) of 1.9 (releases) and 2.3 (stretches); (3) does not affect the maximum extent of filament sliding accounted for by the working stroke in the attached heads (10 nm (half-sarcomere)−1 ). These results indicate that in isometric conditions the structural change leading to force generation in the attached myosin heads can be modulated by temperature at the expense of the structural change responsible for the working stroke that drives filament sliding. The energy stored in the elasticity of the attached myosin heads at the plateau of the isometric tetanus increases with temperature, but even at high temperature this energy is only a fraction of the mechanical energy released by attached heads during filament sliding. 相似文献
10.
Shigeru Takemori Maki Yamaguchi Naoto Yagi 《Journal of muscle research and cell motility》1995,16(6):571-577
Summary Using a technique to obtain a detailed X-ray diffraction pattern from a single skinned frog muscle fibre, we studied the effects of ADP on the structure and arrangement of myosin heads. An imaging plate and a cooled-CCD X-ray detector were used to record the diffraction patterns. Addition of 1 mm ADP to a rigor fibre increased the intensity of the third-order meridional reflection of the myosin repeat by 50–85%. The intensity of the sixth-order meridional reflection also increased. After removing the ADP, these intensities decreased but did not return to the level before the ADP was added. No significant changes were observed in the intensities of the equatorial reflections and the actin layer-lines. These results suggest that, upon ADP binding, the conformation of a myosin head changes without detaching from actin. The structural change may involve a relative motion between domains of the myosin head by the closure of the cleft to which an ADP molecule binds. 相似文献
11.
Preston LC Lipscomb S Robinson P Mogensen J McKenna WJ Watkins H Ashley CC Redwood CS 《Pflügers Archiv : European journal of physiology》2007,453(6):771-776
We recently reported a dilated cardiomyopathy (DCM) causing mutation in a novel disease gene, TNNC1, which encodes cardiac troponin C (TnC). We have determined how this mutation, Gly159Asp, affects contractile regulation
when incorporated into muscle fibres. Endogenous troponin in rabbit skinned psoas fibres was partially replaced by recombinant
human cardiac troponin containing either wild-type or Gly159Asp TnC. We measured both the force–pCa relationship of these
fibres and the activation rate using the caged-Ca2+ compound nitrophenyl-EGTA. Gly159Asp TnC had no significant effect on either the Ca2+ sensitivity or cooperativity of force generation when compared to wild type. However, the mutation caused a highly significant
(ca. 50%) decrease in the rate of activation. This study shows that whilst not affecting the force–pCa relationship, the mutation
Gly159Asp causes a significant decrease in the rate of force production and a change in the relationship between the rate
of force production and generated force. In vivo, this mutation may cause both a slowing of force generation and reduction
in total systolic force. This represents a novel mechanism by which a cardiomyopathy-causing mutation can affect contractility. 相似文献
12.
Clark Richard J. Nyitrai Miklós Webb Martin R. Geeves Michael A. 《Journal of muscle research and cell motility》2003,24(4-6):317-323
The detailed kinetic analysis of novel myosin motors is often limited by the quantity of stable protein available for study.
We show here that the use of coumarin based fluorescent ADP analogues allows the assay of ADP affinities and dissociation
rate constants in a flash photolysis apparatus using μg quantities of the rabbit muscle myosin S1. We go on to use the analogues
to characterise two other rat muscle myosin S1 and the motor domain of Dictyostelium cytoplasmic myosin II. The results show that the fluorescence change for the binding of a coumarin based ADP analogue to
a myosin motor domain is variable in sign as well as amplitude for the different proteins. The analysis also provided estimates
of the affinities of caged-ATP for S1 which were ≤10 μM for muscle S1s and >200 μM for the non-muscle myosin.
This revised version was published online in August 2006 with corrections to the Cover Date. 相似文献
13.
Measurement of the dissociation rate constant of antigen/antibody complexes in solution by enzyme-linked immunosorbent assay 总被引:1,自引:0,他引:1
Marie-Pierre Larvor Lisa Djavadi-Ohaniance Barry Nall Michel E. Goldberg 《Journal of immunological methods》1994,170(2):167-175
A reliable, convenient ELISA based method has been developed for measuring the dissociation rate constants of antigen/antibody complexes in solution. Its rationale is as follows: a solution containing the preformed antigen/antibody complex is diluted well below the equilibrium dissociation constant to initiate the dissociation and, at various times after the dilution, the amount of dissociated antibody contained in an aliquot is determined by a classical ELISA, using a brief incubation of the solution in antigen coated wells. To test the validity of this method, the dissociation rate constants for several antigen/antibody complexes were compared with those obtained by classical fluorescence based methods. The good agreement between both sets of data validates the ELISA procedure. The present method offers several advantages. It uses only minute amounts of sample which need not be purified; it requires no radioactive or fluorescent labelling of the antibody or antigen, and it can, in principle, be applied toa ny type of complex between macromolecules if an ELISA test can be set up to detect quantitatively one of the macromolecules. 相似文献
14.
Manish K. Gupta Jeffrey Robbins 《Pflügers Archiv : European journal of physiology》2014,466(2):231-236
Cardiac myosin binding protein C (cMyBP-C) is an integral sarcomeric protein that associates with the thick, thin, and titin filament systems in the contractile apparatus. Three different isoforms of MyBP-C exist in mammalian muscle: slow skeletal (MyBPC1), fast skeletal (MyBP-C2, with several variants), and cardiac (cMyBP-C). Genetic screening studies show that mutations in MYBPC3 occur frequently and are responsible for as many as 30–35 % of identified cases of familial hypertrophic cardiomyopathy. The function of cMyBP-C is stringently regulated by its post-translational modification. In particular, the addition of phosphate groups occurs with high frequency on certain serine residues that are located in the cardiac-specific regulatory M domain. Phosphorylation of this domain has been extensively studied in vitro and in vivo. Phosphorylation of the M domain can regulate the manner in which actin and myosin interact, affecting the cross bridge cycle and ultimately, cardiac hemodynamics. 相似文献
15.
16.
Clark RJ Nyitrai M Webb MR Geeves MA 《Journal of muscle research and cell motility》2003,24(4-6):315-321
The detailed kinetic analysis of novel myosin motors is often limited by the quantity of stable protein available for study. We show here that the use of coumarin based fluorescent ADP analogues allows the assay of ADP affinities and dissociation rate constants in a flash photolysis apparatus using microg quantities of the rabbit muscle myosin S1. We go on to use the analogues to characterise two other rat muscle myosin S1 and the motor domain of Dictyostelium cytoplasmic myosin II. The results show that the fluorescence change for the binding of a coumarin based ADP analogue to a myosin motor domain is variable in sign as well as amplitude for the different proteins. The analysis also provided estimates of the affinities of caged-ATP for S1 which were < or = 10 microM for muscle S1s and > 200 microM for the non-muscle myosin. 相似文献
17.
Human single masseter muscle fibers contain unique combinations of myosin and myosin binding protein C isoforms 总被引:1,自引:0,他引:1
Striated craniofacial and limb muscles differ in their embryological origin, regulatory program during myogenesis, and innervation.
In an attempt to explore the effects of these differences on the striated muscle phenotype in humans, the expression of myosin
and myosin-associated thick filament proteins were studied at the single fiber level both in the human jaw-closing masseter
muscle and in two limb muscles (biceps brachii and quadriceps femoris muscles). In the masseter, unique combinations of myosin
heavy chain (MyHC) and myosin binding protein C (MyBP-C) isoforms were observed at the single fiber level. Compared to the
limb muscles, the MyHC isoform expression was more complex in the masseter while the opposite was observed for MyBP-C. In
limb muscles, a coordinated expression of three MyHC and three MyBP-C isoforms were observed, i.e., single fibers contained
one or two MyHC isoforms, and up to three MyBP-C isoforms. Also, the relative content of the different MyBP-C isoforms correlated
with the MyHC isoform expression. In the masseter, on the other hand, up to five different MyHC isoforms could be observed
in the same fiber, but only one MyBP-C isoform was identified irrespective MyHC isoform expression. This MyBP-C isoform had
a migration rate similar to the slow MyBP-C isoform in limb muscle fibers. In conclusion, a unique myofibrillar protein isoform
expression was observed in the human masseter muscle fibers, suggesting significant differences in structural and functional
properties between muscle fibers from human masseter and limb muscles.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
18.
Yagi N Saeki Y Kiyota H Kurihara S 《Pflügers Archiv : European journal of physiology》2002,444(1-2):38-42
An X-ray diffraction experiment on ferret heart muscle was made to examine the relationship between tension and mass transfer from the thick to the thin filament associated with the interaction of cross-bridges with actin. A ferret papillary muscle was electrically stimulated for 8 s in the presence of 5 microM ryanodine to give a tetanus. At different extracellular Ca2+ concentrations (2-20 mM), a linear relationship was found between the tension and the mass transfer. It was concluded that a change in tension caused by altering the extracellular Ca2+ concentration is due to a change in the number of myosin heads bound to actin. This is in contrast to the results obtained on skinned preparations, which showed a markedly nonlinear relationship between the number of heads in the vicinity of the thin filaments and force. It was found that the tension per myosin head is similar in twitch and tetanus of cardiac muscle. Also, a similar fraction of myosin heads is recruited in tetanus of cardiac and skeletal muscles. 相似文献
19.
20.
The present study assessed the efficacy of pattern feedback for producing integrative and dissociative patterns of heart rate (HR) and respiration rate (RR). 60 uninformed subjects were assigned to six groups in which beat-by-beat feedback was contingent upon production of a specific pattern of increased (↑), decreased (↓), or unchanged (=) HR and RR during 11 feedback trials. Concomitant changes in respiratory volume and general activity (GA) were also recorded. Groups given feedback for HR and RR changes in the same directions (HR↑RR↑ and HR↓RR↓) and for changes in opposite directions (HR↑RR↓ and HR↓RR↑) were generally unable to produce the respective patterns, indicating that pattern feedback does not enable subjects to produce a wide range of HR-RR patterns. However, evidence of dissociation of HR and RR was obtained in the HR↑RR= and HR↓RR= groups in which HR changed significantly in the appropriate directions without significant changes in RR. These results are not consistent with the view that RR changes are necessary for HR control, although significant concomitant changes in respiratory volume and GA indicated that HR control was non-specific relative to these variables. 相似文献