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1.
In skeletal muscle cells, myofibrillar proteins are highly organized into sarcomeres in which thick filaments interdigitate with thin filaments to generate contractile force. The size of thick filaments, which consist mainly of myosin molecules, is strictly controlled. However, little is known about the mechanisms by which myosin molecules assemble into thick filaments. Here, we assessed the ability of each domain of myosin heavy chain (Myh) to form thick filaments. We showed that exogenously expressed subfragment 2 (S2) + light meromyosin (LMM) of Myh was efficiently incorporated into thick filaments in muscle cells, although neither solely expressed S2 nor LMM targeted to thick filaments properly. In nonmuscle COS7 cells, S2+LMM formed more enlarged filaments/speckles than LMM. These results suggest that Myh filament formation is induced by S2 accompanying LMM. We further examined the effects of Myh C‐terminus on thick filament assembly. C‐terminal deletion mutants were incorporated not into entire thick filaments but rather into restricted regions of thick filaments. Our findings suggest that the elongation of myosin filaments to form thick filaments is regulated by S2 as well as C‐terminus of LMM.  相似文献   

2.
Slow-tonic myofiber to fast-twitch myofiber conversion was induced in chickens by feeding thyroxine. Incorporation of newly synthesized myosin heavy chain (MHC) into myofibers and myofibrils was followed by immunofluorescence with antibodies specific for fast-twitch MHC and slow-tonic MHC. Presence of more than one type of myosin heavy chain was detected in thyroxine-induced transitional myofibers of chicken pectoralis. Myofibers undergoing a transition were histochemically identical to immunologically cross-reacting, transitional myofibers of normal anterior latissimus dorsi. Newly synthesized MHC appeared to be incorporated uniformly across the cross sectional area of transitional myofibers and incorporated homogeneously into each sarcomere of transitional myofibrils. These observations are consistent with a theory of continuous protein exchange between myofibrillar protein and a non-myofibrillar protein pool, such that every sarcomere of a myofibril, and every myofibril of a myofiber, would be turned over simultaneously.  相似文献   

3.
Image analysis procedures for immunofluorescence microscopy were developed to measure muscle thin filament lengths of beef, rabbit, and chicken myofibrils. Strips of beef cutaneous trunci, rectus abdominis, psoas, and masseter; chicken pectoralis; and rabbit psoas muscles were excised 5 to 30 min postmortem. Fluorescein phalloidin and rhodamine myosin subfragment-1 (S1) were used to probe the myofibril structure. Digital images were recorded with a cooled charge-coupled device controlled with IPLab Spectrum software (Signal Analytics Corp.) on a Macintosh operating system. The camera was attached to an inverted microscope, using both the phase-contrast and fluorescence illumination modes. Unfixed myofibrils incubated with fluorescein phalloidin showed fluorescence primarily at the Z-line and the tips of the thin filaments in the overlap region. Images were processed using IPLab and the National Institutes of Health's Image software. A region of interest was selected and scaled by a factor of 18.18, which enlarged the image from 11 pixels/microm to approximately 200 pixels/microm. An X-Y plot was exported to Spectrum 1.1 (Academic Software Development Group), where the signal was processed with a second derivative routine, so a cursor function could be used to measure length. Fixation before phalloidin incubation resulted in greatest intensity at the Z lines but a more-uniform staining over the remainder of the thin filament zone. High-resolution image capture and processing showed that thin filament lengths were significantly different (P < 0.01) among beef, rabbit, and chicken, with lengths of 1.28 to 1.32 microm, 1.16 microm, and 1.05 microm, respectively. Measurements using the S1 signal confirmed the phalloidin results. Fluorescent probes may be useful to study sarcomere structure and help explain species and muscle differences in meat texture.  相似文献   

4.
In striated muscles, approximately 300 myosin molecules form a single thick filament in myofibrils. Each myosin is continuously displaced by another myosin to maintain the thick filament structure. Our previous study using a fluorescence recovery after photobleaching (FRAP) technique showed that the myosin replacement rate is decreased by inhibition of protein synthesis, but myosin is still exchangeable. This result prompted us to examine whether myosin in the cytoplasm is involved in myosin replacement in myofibrils. To address this, FRAP was measured in green fluorescent protein (GFP)‐tagged myosin heavy chain 3 (Myh3) expressing myotubes that were treated with streptolysin‐O (SLO), which forms pores specifically in the plasma membrane to induce leakage of cytoplasmic proteins. Our biochemical data demonstrated that the cytoplasmic myosin content was reduced in SLO‐permeabilized semi‐intact myotubes. Furthermore, FRAP experiments showed a sluggish substitution rate of GFP‐Myh3 in SLO‐permeabilized myotubes. Taken together, these results demonstrate that the myosin substitution rate is significantly reduced by a decreased amount of myosin in the cytoplasm and that cytoplasmic myosin contributes to myosin replacement in myofibrils.  相似文献   

5.
The object of the present study was to reveal the action of inosine‐5'‐monophosphate (IMP) toward myofibrils in postmortem muscles. IMP solubilized isolated actomyosin within a narrow range of KCl concentration, 0.19‐0.20 mol/L, because of the dissociation of actomyosin into actin and myosin, but it did not solubilize the proteins in myofibrils with 0.2 mol/L KCl. However, IMP could solubilize both proteins in myofibrils with 0.2 mol/L KCl in the presence of 1 m mol/L pyrophosphate or 1.0–3.3 m mol/L adenosine‐5'‐diphosphate (ADP). Thus, we presumed that pyrophosphate and ADP released thin filaments composed of actin, and thick filaments composed of myosin from restraints of myofibrils, and then both filaments were solubilized through the IMP‐induced dissociation of actomyosin. Thus, we concluded that IMP is a candidate agent to resolve rigor mortis because of its ability to break the association between thick and thin filaments.  相似文献   

6.
Myofibrillar proteins of vertebrate skeletal muscles are insoluble in solutions of ionic strength that approximate physiological conditions. We established a method to solubilize more than 80% of chicken breast muscle myofibrillar proteins in water for the use of meat as a source of food protein. SDS‐polyacrylamide gel electrophoretic patterns of water‐soluble myofibrillar proteins demonstrated that all identified myofibrillar proteins except connectin/titin were soluble in water. A part of α‐actinin was released from myofibrils by repeated washing with 2.5 mmol/L NaCl and 5 mmol/L L‐histidine solution, and subsequent destruction of connectin/titin in washed myofibrils by ultrasonication resulted in solubilization of a large fraction of chicken breast muscle myofibrillar proteins in water. Myofibrillar proteins of chicken leg, pork loin, beef shoulder loin, and lamb were also solubilized in water using this procedure.  相似文献   

7.
不同遗传型鸡肌纤维超微结构的研究   总被引：5，自引：0，他引：5  
电镜观察红宝鸡,苏北地方鸡及杂种鸡肌纤维超微结构表明,不同遗传型肌原纤维在直径、肌节长度、Ⅰ带、A带长度不仅存在差异,而且在肌原纤维间肌质、线粒体、肌糖元、脂肪滴含量亦存在差异。分析表明,鸡肌纤维的超徼结构与鸡生长速度,肉质有密切关系。  相似文献   

8.
The intermediate (10-nm) filament subunit proteins (desmin and vimentin) in samples obtained from embryonic, neonatal, and postnatal porcine skeletal muscle were examined by two-dimensional electrophoresis (isoelectric focusing/sodium dodecylsulfate polyacrylamide gel electrophoresis). The skeletal muscle samples were taken from pig embryos at 45, 73 and 102 d of gestation; from neonatal pigs and from postnatal pigs at 1, 6 and 30 mo of age. Three fractions (namely, whole homogenized muscle, purified myofibrils and myofibrillar-protein-extracted residues) were prepared from each skeletal muscle sample for analysis. Vimentin was the major (approximately 75% vimentin: 25% desmin) 10-nm filament protein present in skeletal muscle samples obtained from the 45-d-old pig embryos. The relative proportion of vimentin decreased progressively during embryogenesis. At birth, the vimentin comprised approximately 15%, and desmin, 85%, of the 10-nm filament protein. The proportional amount of vimentin continued to decline postnatally, with the 10-nm filament protein of samples from the 30-mo-old animals consisting of less than approximately 5% vimentin and over 95% desmin. These results show a developmental stage-dependent pattern in the expression of vimentin and desmin intermediate filament subunit proteins in mammalian skeletal muscle. In the adult mammal, desmin is the significant 10-nm filament protein present.  相似文献   

9.
Denaturation of actin and myosin in myofibrils induced by heating at 50°C was investigated to reveal the mechanism of irreversible liberation of actin from myofibrils on heating at lower temperatures than conventional cooking. Denaturation of these proteins was determined by Mg2+‐ATPase (adenosine triphosphatase) and Ca2+‐ATPase activities. When minced meat was heated for 20 min, actin was liberated accompanying denaturation of 80% of actin and 50% of myosin. Heating of the myofibrillar fraction (MFF) isolated from meat homogenate induced much slower denaturation of actin than myosin. When MFF was heated with sarcoplasmic fractions, denaturation of actin was facilitated, suggesting that sarcoplasmic fractions contain factors to facilitate actin denaturation. Inosine‐5′‐monophosphate, a component of sarcoplasmic fractions, was shown to have no effect on actin and myosin denaturation. These results suggest that heating meat at 50°C dissociates binding (‘Bond A’) between actin and myosin participating in ATPase activities, resulting in denaturation of both proteins under influence of sarcoplasmic components. Although denaturation of actin and myosin disrupted Bond A, actin was not liberated simultaneously, suggesting the presence of another bond (‘Bond B’, more heat‐stable than Bond A) between both proteins and necessity of disruption of Bond B for actin release from myofibrils.  相似文献   

10.
A study was conducted to examine the effects that physiological levels of m-calpain (calpain requiring millimolar concentrations of Ca2+) extract and a lysosomal extract have on myofibrillar proteins in vitro, and the effects that zinc has on inhibiting proteolysis by these extracts. During a 22-h incubation period, the lysosomal extract degraded myosin heavy chain, alpha-actinin, desmin, troponin-I, and myosin light chains 1 and 2. The effectiveness of the lysosomal extract to degrade myofibrillar proteins was significantly affected by the presence or absence of EDTA. Zinc, which is a potent inhibitor of cysteine proteinases, prevented most, but not all, of the lysosomal extract-induced myofibrillar protein degradation. Incubation of myofibrils with m-calpain resulted in the hydrolysis of troponin-T, desmin, and a 58-kDa molecular weight protein, possibly vimentin, and 5 mM ZnCl2 completely blocked these changes. Results from this study indicate that the degradation by the lysosomal extract is far more extensive than the degradation that occurs with normal postmortem storage and that possibly a non-cysteine protease is present that is capable of hydrolyzing some myofibrillar proteins under this in vitro condition, because Zn2+ did not block all proteolysis. However, similar changes were induced by m-calpain incubation and postmortem storage.  相似文献   

11.
Three chief cell types were studied in 8 canine mixed mammary tumors--luminal epithelial cells, myoepithelial cells, and filament cells, the last being the "stellate" cells recognized by light microscopists. Most cytoplasmic filaments in the myoepithelial cells were 5 to 8 nm thick, whereas another type of filaments were scattered, 8 to 10 nm thick. Almost all cytoplasmic filaments in the filament cells were 8 to 10 nm thick. In the adenomatous parts of the tumors, myoepithelial cells were chiefly seen, along with luminal epithelial cells. Filament cells were rarely observed without intercellular accumulation of mucoid. The filament cells were largely confined to the myxomatous and chondromatous parts of the tumors. The presence of cells of transitional forms in adenomatous areas suggested that the filament cells were derived from the myoepithelial cells.  相似文献   

12.
1. The effect of lactic acid marination at 5°C on post mortem changes in breast muscle pectoralis major of spent layer Tsaiya duck was studied.

2. Myofibrils were prepared from 0.1 M and 0.2 M lactic acid marinated muscle and control (non‐marinated samples) sampled at 0, 1, 3, 7 and 14 d post mortem.

3. Changes in myofibril fragmentation index (MFI), myofibrillar proteins and Z‐line structure were examined.

4. Marination of duck breast muscle in lactic acid at 5°C enhanced fragmentation of myofibrils and degradation of myofibrillar proteins and Z‐line structure as compared to control samples.

5. In summary, lactic acid marination at 5°C can accelerate the post mortem degradation of myofibrils in Tsaiya duck breast muscle.  相似文献   


13.
The calpain protease system, in particular, μ-calpain is involved in the disassembly of specific myofibrillar proteins, resulting in tenderization of meat postmortem. Given the size, complexity, and integral nature of titin to the structure of the sarcomere, it is plausible that the length of a sarcomere may alter the susceptibility of various domains of titin to cleavage by the calpains. Therefore, we hypothesized titin degradation differs in a sarcomere-length-dependent manner in beef. After slaughter, beef carcasses were split and sides were either suspended by the Achilles tendon (normal suspension, NS) or by the aitchbone (hip suspension, HS). Immediately after suspension, samples were dissected from the LM, psoas major (PM), and semitendinosus (STN) muscles to serve as 0-d controls. After 24 h, 4 steaks were removed from each muscle and randomly assigned to 1-, 4-, 7-, or 10-d aging treatments. After the assigned aging period, myofibrils were purified for determination of sarcomere length. Warner-Bratzler shear force analysis was also performed to evaluate differences in tenderness. Muscle proteins were solubilized and subjected to SDS-VAGE (vertical agarose gel electrophoresis) to evaluate titin degradation. Sarcomere lengths differed (P < 0.0001) between contralateral muscles of NS and HS carcasses. Quantification of SDS-VAGE gels revealed less (P < 0.05) intact titin in the PM muscle of NS carcasses at each aging period compared with the PM of HS carcasses. No significant differences (P > 0.05) were detected in the disappearance of intact titin among suspension methods in the LM or STN. These data demonstrate that suspension method alters proteolysis of titin and suggest an increase in sarcomere length may contribute to the susceptibility of titin to postmortem proteolysis in beef.  相似文献   

14.
To assess both quantitative and qualitative differences between the slow‐ and fast‐type muscles, masseter (slow) and semitendinosus (fast) from four Holstein cows were analyzed by two‐dimensional difference gel electrophoresis (2D DIGE) and mass spectrometry. The proteome analysis identified 27 spots as 20 proteins in the whole protein fraction extracted with 8 mol/L urea solution, and 16 spots were identified as 11 proteins in the water‐soluble protein fraction. Two slow‐type myofibrillar proteins (myosin light chain‐1 slow‐b and myosin light chain‐2 slow), and aconitase‐2 mitochondria were present at higher levels in the masseter muscle (P < 0.05). Four fast‐type myofibrillar proteins (myosin light chain‐1 fast, myosin light chain‐2 fast, myosin light chain‐3 fast and tropomyosin‐1), and three enzymes of glycolytic pathway (enolase‐3, aldolase‐A and triosephosphate isomerase), were present at higher levels in the semitendinosus muscle (P < 0.05). Our proteome analysis showed that the composition of sarcoplasmic proteins as well as myofibrillar proteins was clearly different between slow‐ and fast‐type muscles.  相似文献   

15.
The latent form of multicatalytic proteinase complex (MCP) was purified to homogeneity from ovine skeletal muscle. The MCP ran as a single band (M(r) 600,000) on nondenaturing polyacrylamide gel (PAGE) and dissociated to a number of subunits (M(r) 21,000 to 31,000) under denaturing and reducing conditions (SDS-PAGE). The proteinase complex was activated reversibly by heating at 60 degrees C and in the presence of SDS. Maximum activation (18-fold) was observed after 2 min at 60 degrees C and there was rapid inactivation beyond 2 min. Maximum proteolytic activity (12.8-fold) occurred in the presence of .25 mM SDS and diminished rapidly at higher SDS concentrations. The MCP was maximally active at pH 7.5 to 8.0 and 45 degrees C using radiolabeled alpha-casein. These and other results (e.g., proteinase inhibitor profiling) indicate that ovine skeletal muscle does indeed contain MCP and that its biochemical properties are the same as MCP isolated from other sources. By using [14C]-casein as a substrate, the specific activities (milligrams of protein degraded/milligrams of proteinase) for mu-, m-calpain, and MCP were 44.0, 59.7, and 2.0, respectively. Purified ovine myofibrils were incubated with mu-calpain or MCP. Classical effects of calpains, which include degradation of Z-disks, titin, desmin, troponin-T, and troponin-I and removal of alpha-actinin, were observed. However, only troponin-C and myosin light chains-2 and -3 were degraded by MCP. Morphologically, MCP had no detectable effect on myofibrils. Results suggest that MCP is not involved in the initial steps of myofibril disassembly. However, its involvement in the degradation of myofilaments remains to be determined.  相似文献   

16.
The interaction between sarcomere length and postmortem proteolysis as related to meat tenderness is not clear. The extent of thick and thin filament overlap alters actomyosin binding and may alter substrate availability during aging-induced tenderization. The objective of this study was to determine the influence of sarcomere length on proteolytic degradation in beef. Strips from bovine semitendinosus were either stretched 40% and restrained or allowed to shorten unrestrained in an ice bath. After rigor completion, 0.6-cm cross sections were fabricated and were randomly assigned to 2, 4, 7, or 10 d of aging treatments. Myofibrils were isolated for sarcomere length determination. Samples were collected and frozen for shear force analysis, and muscle proteins were extracted for SDS-PAGE and Western blotting analyses to determine troponin T (TnT) proteolysis. Sarcomere length was greater (P < 0.01) in stretched muscle samples compared with shortened samples (2.57 vs. 1.43 microm, respectively). Correspondingly, shear force values were greater (P < 0.05) in shortened samples than stretched samples. Western blots revealed the presence of 3 major intact TnT bands that diminished with time postmortem and 4 bands (TnT degradation products) that accumulated during postmortem storage. Quantification of intact TnT showed increased (P < 0.05) proteolysis at 4 and 7 d postmortem in samples with long sarcomeres. By 10 d, only traces of the greatest molecular weight intact TnT band were evident in both shortened and stretched samples, suggesting this TnT band may be more susceptible to proteolysis than other intact TnT bands. Degradation products of TnT appeared earlier postmortem in samples with long sarcomeres. The 30-kDa TnT fragment appeared after 7 d of postmortem storage in samples with long sarcomeres but not until 10 d in muscle containing short sarcomeres. Collectively, these data show that postmortem TnT proteolysis is sarcomere length-dependent and suggest that thick and thin filament overlap may influence the postmortem aging process in beef.  相似文献   

17.
Tenderness is a key palatability trait influencing perception of consumers of meat quality and is influenced by a multitude of factors, including postmortem proteolysis. A fundamental understanding of this biological mechanism regulating tenderness is necessary to decrease variability and increase consumer satisfaction. However, reports regarding the enzyme systems involved in postmortem tenderization are conflicting. Therefore, the objective of this study was to determine if caspase-3 is responsible for the degradation of myofibrillar proteins during aging. Bovine semitendinosus muscles were removed from 2 carcasses. Muscle from the left side of each carcass was excised 20 min postmortem and utilized for in vitro analysis of protein degradation. Muscle strips were dissected from the semitendinosus, restrained to maintain length, and placed in a neutral buffer containing protease inhibitors. Upon rigor completion, myofibrils were isolated from each strip and sarcomere length was determined. Samples with similar sarcomere lengths were selected to minimize the effect of sarcomere length on proteolysis. Myofibrils were then incubated at 22°C with μ-calpain, caspase-3, or μ-calpain + caspase-3 for 0.25, 1, 3, 24, 48, or 72 h at optimum pH for enzyme activity. The semitendinosus from the right side of each carcass was excised 1 d postmortem, cut into 2.54-cm steaks, vacuum-packaged, and allowed to age for 2, 4, 7, or 10 d to evaluate normal protein degradation during beef aging. Proteolysis of troponin T, α-actinin, and desmin was monitored using SDS-PAGE and Western blotting techniques, whereas proteolysis of titin and nebulin was monitored using SDS-vertical agarose gel electrophoresis and Western blotting. Analysis of Western blots revealed no change in abundance of intact troponin T, desmin, titin, or nebulin over time in myofibrils incubated with caspase-3. However, abundance of these proteins subjected to digestion with μ-calpain and μ-calpain + caspase-3 revealed degradation patterns similar to in situ samples. No degradation of α-actinin was observed in in vitro or in situ samples. Results of this study indicate μ-calpain, not caspase-3, is responsible for the degradation of key myofibrillar proteins during beef aging.  相似文献   

18.
1. A procedure was developed to separate high and medium molecular weight myofibrillar proteins from chicken muscular tissue with a high resolution by flat bed sodium-dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent detection by either a general protein stain or Western blotting. These procedures were used to analyse the degradation process of cytoskeletal proteins in chicken breast and leg muscles during meat ageing. 2. This study demonstrates the degradation of all the examined cytoskeletal proteins: titin, nebulin and desmin as well as vinculin, a protein component of the costamere structure. All the examined proteins were found to be degraded during ageing of chicken breast and leg muscles. 3. Degradation of titin, nebulin and desmin started at 3 h post mortem in breast muscle. Intact titin and nebulin disappeared within 1 d. Intact desmin and vinculin were not detectable after 3 d post mortem. In leg muscle, the degradation process of all the examined proteins evolved much more slowly than in breast chicken muscles. 4. The changes observed in shear force, myofibrillar fragmentation and cooking loss were related to changes in cytoskeletal proteins and used to identify marker proteins or degradation products for the purpose of monitoring the development of meat ageing. The ageing process was faster in breast muscle than in leg muscle. 5. Significant correlations were found between degradation processes of titin, nebulin, and desmin and shear force, as well as myofibril fragmentation index of breast and leg muscles.  相似文献   

19.
Mammalian skeletal muscle expresses splice variants of neuronal nitric oxide synthase (nNOS). Skeletal muscles have a metabolically heterogeneous population of myofibers, and fiber composition in equine skeletal muscle is correlated with athletic ability in endurance events. In this study, we investigated whether nNOS expression in equine skeletal muscle is related to fiber type and endurance training. Biopsy samples obtained from the gluteus medius of sedentary- (SH) and endurance-trained (TH) horses were examined for the electrophoretic mobility of myosin heavy chain (MHC) and NOS activity. Serial tissue cross-sections were stained for myosin ATPase and nicotinamide adenine dinucleotide (NADH) reductase, and also immunostained for nNOS. The gluteus medius of TH had higher levels of nNOS expression and activity when compared to muscle from SH. In SH, nNOS was restricted to the subsarcolemmal area while in TH nNOS was also present at cytoplasmic sites. A splice variant of nNOS was heterogeneously distributed among the different myofibers, its expression being higher in fast-oxidative-glycolytic type IIA fibers than in fast-glycolytic type IIX fibers and absent in slow-twitch type I fibers. Trained horses had a significantly higher relative content of type IIA fibers, a greater oxidative capacity, and a lower percentage of type IIX fibers when compared with SH. The differences in muscle fiber typing between the 2 groups of horses reflected alterations that probably resulted from the endurance-training program. Overall, these results show that nNOS is differentially expressed and localized in the gluteus medius according to the fiber type and the athletic conditioning of the horses.  相似文献   

20.
Normal and well differentiated neoplastic canine tissues were immunohistochemically stained for keratin, vimentin and desmin intermediate filament proteins using commercially available monoclonal antibodies. Keratin was detected in 56 of 57 carcinomas, vimentin in 59 of 62 sarcomas and desmin in three of four muscle cell tumors. Most normal and neoplastic tissues expressed only one type of intermediate filament; exceptions were one hemangiosarcoma and one pulmonary carcinoma in which there was coexpression of vimentin and keratin proteins. Since immunohistochemical detection of intermediate filaments has tissue-specific distribution in the majority of well differentiated canine neoplasms, these stains may be useful in the differential diagnosis of anaplastic canine tumors. However, the monoclonal antibodies to cytokeratin which were tested in this study failed to detect intermediate filaments in liver, pancreas and salivary glands which suggests that these antibodies may also be unable to detect epithelial tumors derived from these tissues. In addition, in nine neoplasms, the normal tissues adjacent to neoplastic cells failed to stain for the intermediate filament normally expressed. When this occurs, evaluation of intermediate filament expression is invalid for the determination of tissue of origin of the neoplastic cells.  相似文献   

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