Individual Myofibrillar Proteins Research Articles

Proteomic evaluation of myofibrillar carbonylation in chilled fish mince and its inhibition by catechin

The present study investigates the susceptibility of individual myofibrillar proteins from mackerel (Scomber scombrus) mince to undergo carbonylation reactions during chilled storage, and the antioxidant capacity of (+)-catechin to prevent oxidative processes of proteins. The carbonylation of each particular protein was quantified by combining the labelling of protein carbonyls by fluorescein-5-thiosemicarbazide (FTSC) with 1-D or 2-D gel electrophoresis. Alpha skeletal actin, glycogen phosphorylase, unnamed protein product (UNP) similar to enolase, pyruvate kinase, isoforms of creatine kinase, aldolase A and an isoform of glyceraldehyde 3-phosphate dehydrogenase (G3PDH) showed elevated oxidation in chilled non-supplemented mince. Myosin heavy chain (MHC) was not carbonylated in chilled muscle, but an extensive MHC degradation was observed in those samples. The supplementation of catechin reduced protein oxidation and lipid oxidation in a concentration-dependent manner: control>25>100≈200ppm. Therefore, the highest catechin concentrations (100 and 200ppm) exhibited the strongest antioxidant activity. Catechin (200ppm) reduced significantly carbonylation of protein spots identified as glycogen phosphorylase, pyruvate kinase muscle isozyme, isoforms of creatine kinase. Conversely, catechin was ineffective to inhibit the oxidation of actin and UNP similar to enolase. These results draw attention to the inefficiency of catechin to prevent actin oxidation, in contrast to the extremely high efficiency of catechin in inhibiting oxidation of lipids and other proteins.

Myofibrillar protein turnover: The proteasome and the calpains1,2

Metabolic turnover of myofibrillar proteins in skeletal muscle requires that, before being degraded to AA, myofibrillar proteins be removed from the myofibril without disrupting the ability of the myofibril to contract and develop tension. Skeletal muscle contains 4 proteolytic systems in amounts such that they could be involved in metabolic protein turnover: 1) the lysosomal system, 2) the caspase system, 3) the calpain system, and 4) the proteasome. The catheptic proteases in lysosomes are not active at the neutral pH of the cell cytoplasm, so myofibrillar proteins would have to be degraded inside lysosomes if the lysosomal system were involved. Lysosomes could not engulf a myofibril without destroying it, so the lysosomal system is not involved to a significant extent in metabolic turnover of myofibrillar proteins. The caspases are not activated until initiation of apoptosis, and, therefore, it is unlikely that the caspases are involved to a significant extent in myofibrillar protein turnover. The calpains do not degrade proteins to AA or even to small peptides and do not catalyze bulk degradation of the sarcoplasmic proteins, so they cannot be the only proteolytic system involved in myofibrillar protein turnover. Research during the past 20 yr has shown that the proteasome is responsible for 80 to 90% of total intracellular protein turnover, but the proteasome degrades peptide chains only after they have been unfolded, so that they can enter the catalytic chamber of the proteasome. Thus, although the proteasome can degrade sarcoplasmic proteins, it cannot degrade myofibrillar proteins until they have been removed from the myofibril. It remains unclear how this removal is done. The calpains degrade those proteins that are involved in keeping the myofibrillar proteins assembled in myofibrils, and it was proposed over 30 yr ago that the calpains initiated myofibrillar protein turnover by disassembling the outer layer of proteins from the myofibril and releasing them as myofilaments. Such myofilaments have been found in skeletal muscle. Other studies have indicated that individual myofibrillar proteins can exchange with their counterparts in the cytoplasm; it is unclear whether this can be done to an extent that is consistent with the rate of myofibrillar protein turnover in living muscle. It seems that both the calpains and the proteasome are responsible for myofibrillar protein turnover, but the mechanism is still unknown.

Protein extractability and thermal gel formability of myofibrils isolated from skeletal and cardiac muscles at different post‐mortem periods

AbstractThe effects of the post‐mortem ageing period on the extractability of myofibrillar proteins from pork cardiac and rabbit skeletal muscles under various conditions of pH and ionic strength were studied with particular reference to the changes in the solubility of individual myofibrillar proteins and their denaturation characteristics. The ultimate influence of these changes on the heat‐induced gel forming ability of myofibrils isolated from cardiac and skeletal muscles at different post‐mortem stages was also investigated.Results showed that pork cardiac myofibrils always exhibited lower solubility than those from rabbit skeletal muscles under identical conditions of pH, ionic strength and temperature. SDS‐PAGE profiles indicated several quantitative differences in the relative proportion of individual protein species present in cardiac and skeletal myofibrils. The solubility of various proteins present in myofibrils was also affected differently on heating in 0‐1 and 0‐6 M NaCl solution at various pH values. Thermal denaturation of cardiac myofibrils occurred at about 10°C higher than that of skeletal myofibrils as revealed by differential scanning calorimetry. Cardiac myofibrils formed much weaker heat‐induced gels than those produced by skeletal myofibrils under identical conditions of temperature, pH, ionic strength and protein content.

Regional differences in the in vivo synthesis and degradation of myosin subunits in rabbit ventricular myocardium.

To test for regional differences in the rates of synthesis and degradation of contractile proteins during normal physiological growth of the heart in vivo, fractional rates of protein accumulation and synthesis were assessed for total protein, myosin heavy chain, myosin light chain, phosphorylatable myosin light chain, and actin in the right and left ventricular free walls of growing, New Zealand White rabbits. Fractional rates of protein accumulation were determined from regional protein content growth curves of total and individual myofibrillar proteins measured in 82 animals ranging from 1 day to 9 weeks of age. In vivo right and left ventricular fractional rates of protein synthesis were determined by the [3H]leucine constant infusion method in 9-week-old rabbits. At this age, right and left ventricular fractional accumulation rates for total protein, myosin subunits, and actin were found to be identical, thus allowing for the comparison of the effect of regional hemodynamic load on protein degradation independent of its effect on growth. Total protein and individual myofibrillar protein fractional degradative rates were then derived as the difference between fractional rates of synthesis and accumulation. The results indicate increased fractional synthesis and degradation of myosin heavy chain and light chains, but not actin, in the left compared to the right ventricular free wall. Accelerated left ventricular replacement of myosin subunits may be related to regional differences in hemodynamic load. These results help explain the apparent increase in the in vivo rate of myocardial protein degradation observed in several experimental models of ventricular hypertrophy.

Quantitative analysis of myofibrillar protein subunits: Demonstration of large molar excesses of myosin light chains in rabbit ventricular myocardium

We describe a method for the complete solubilization and quantitative analysis of individual myofibrillar proteins in whole tissue homogenates of ventricular myocardium using gradient dodecyl sulfate polyacrylamide gel electrophoresis and staining with 125I-labeled Coomassie brilliant blue. The procedure allows for the simultaneous quantification of myosin heavy chain, myosin light chain, phosphorylatable myosin light chain and actin from as little as 50 mg of tissue. Within-assay and between-assay variations range from 8.0% to 12.6% for each protein subunit. The method was applied to the determination of the subunit stoichiometry of purified myosin, and to the measurement of myosin and actin concentrations in the neonatal and adult rabbit heart. Furthermore, we provide quantitative biochemical evidence for the existence of large molar excesses of myosin light chains in tissue homogenates of both neonatal and adult rabbit ventricular myocardium.

Contents of myofibrillar proteins in cardiac, skeletal, and smooth muscles.

The in situ contents of myosin, actin, alpha-actinin, tropomyosin, troponin, desmin were estimated in dog cardiac, rabbit skeletal, and chicken smooth muscles. Whole muscle tissues were dissolved with 8 M guanidine hydrochloride and subjected to two-dimensional gel electrophoresis, which is a nonequilibrium pH gradient electrophoresis (Murakami, U. & Uchida, K. (1984) J. Biochem. 95, 1577-1584) with some modification. The amount of protein in a spot on a slab gel was determined by quantification of the extracted dye. Dye binding capacity of individual myofibrillar proteins was determined by using the purified protein. Myosin contents were 82 +/- 7 pmol/mg wet weight in cardiac muscle, 105 +/- 10 pmol/mg wet weight in skeletal muscle, and 45 +/- 4 pmol/mg wet weight in smooth muscle. Actin contents were 339 +/- 15 pmol/mg wet weight in cardiac muscle, 625 +/- 27 pmol/mg wet weight in skeletal muscle, and 742 +/- 13 pmol/mg wet weight in smooth muscle. The subunit stoichiometry of myosin in the three types of muscles was two heavy chains and four light chains, and there was one light chain 2 for every heavy chain. The molar ratio of actin to tropomyosin was 7/1 in the three types of muscles. Striking differences were seen in the molar ratio of myosin to actin: 1.0/4.1 in cardiac muscle, 1.0/6.0 in skeletal muscle, and 1.0/16.5 in smooth muscle.

Biochemical characterization and cellular localization of serine protease in myopathic hamster

Purified serine protease was obtained from skeletal and heart muscle of 150-day-old myopathic hamsters. The skeletal muscle enzyme showed identical molecular weight and characteristics toward various reagents and inhibitors when compared with the cardiac muscle enzyme. In addition, no antigenic differences were detected between the two enzymes using double-immunodiffusion test. Further characterization of the properties of the two enzymes was carried out by using individual myofibrillar proteins as substrates. Both enzymes were capable of degrading myosin, tropomyosin and troponin but not actin. The divalent cations Ca 2+ or Mg 2+ were able to protect the myosin light chain 2 against proteolytic cleavage. The cellular localization of the serine protease of skeletal and cardiac muscle was studied by immunohistochemical techniques. The observations indicate that serine protease is contained within the cytoplasmic granules of cardiac and skeletal muscle mast cells of myopathic and control hamsters. The number of labeled mast cells is about three-fold greater in myopathic than in control tissues. This increase correlates with the increase in enzyme activity as detected by biochemical assay in tissue homogenates.

Vacuum Mixing Influence on Characteristics of Sectioned and Formed Beef Steak

ABSTRACTChoice, Yield Grade 3 chucks were purchased vacuum packaged within 48 hr of slaughter. They were randomly allocated to treatments of vacuum or no vacuum mixing and 6 or 12 min mixing periods. The meat was formulated into sectioned and formed steaks. A trained sensory panel evaluated all samples and color scores were assigned to each steak. Instron adhesion measured binding of meat pieces, Kramer shear indicated tenderness, mg exudate/cm2 measured binding proteins. Reflectance spectrophotometry measured oxymyoglobin and metmyoglobin. Electrophoresis was used to separate and quantitate individual myofibrillar proteins. Sensory analyses indicated vacuum processed steaks had superior bind (P < 0.01) while juiciness, flavor and tenderness remained unchanged. Instron analyses indicated no difference in tenderness or bind strength due to vacuum. Subjective color analyses indicated less desirable color for vacuum mixing (P < 0.05) and the amount of oxymyoglobin and metmyoglobin were not changed due to treatment. There was less exudate at the bond site for vacuum mixing (P < 0.01). Sarcoplasmic and myofibrillar proteins were similar over vacuum treatment.

ACTION OF CRUDE PAPAIN ON ACTIN AND MYOSIN HEAVY CHAINS ISOLATED FROM CHICKEN BREAST MUSCLE

ABSTRACTThe individual myofibrillar proteins, myosin and actin, were digested with crude papain (1:125 w/w for 0–3 min). The sodium dodecyl sulfate electrophoretic patterns and the rate of proteolytic hydrolysis were observed. Actomyosin, natural actomyosin and both contracted and uncontracted glycerinated myofibrils were also studied. It was found that the gel pattern of the actin proteolytic digestion suggested that two populations of actin exist; that although actin had the highest rate of hydrolysis in the pH stat, the gels suggested only a very limited proteolysis; that actomyosin is less rapidly digested than myosin; and that the proteins of the myofibrils, especially when contracted, are rapidly cleaved.

Protein turnover in skeletal muscle of piglets.

1. Rates of protein synthesis and catabolism were measured in longissimus dorsi and hind-limb muscles of suckling piglets.2. Half-lives for synthesis and catabolism for mixed sarcoplasmic proteins were 4.8 and 9.4 d respectively. The corresponding values for mixed myofibrillar proteins were 5.7 and 16.4 d.3. The half-lives for synthesis of sarcoplasmic proteins were significantly different from those of myofibrillar proteins and were not confounded by contamination of the sarcoplasmic protein fraction with plasma proteins of higher specific activity.4. Individual myofibrillar proteins were synthesized and catabolized at rates which were not statistically significantly different. Intramuscular connective tissue also appeared to turnover rapidly, the half-life for synthesis being 8 d and that for catabolism 20 d.5. Values obtained for the specific radioactivities of aspartate + glutamate in mixed plasma proteins support the view that, in so far as the young of animals larger in mature body size than rats or mice are concerned, muscle assumes a more important role relative to liver in regulating whole body amino acid metabolism.

Synthesis rates of protein in the Langendorff-perfused rat heart in the presence and absence of insulin, and in the working heart.

The synthesis rates of total heart protein and of sarcoplasmic and myofibrillar protein fractions have been determined by perfusion of isolated rat hearts with [(14)C]tyrosine at constant specific radioactivity. In hearts perfused without insulin, both myofibrillar and sarcoplasmic proteins were synthesized at a fractional rate of 10-11% per day. This corresponds to a half-life for synthesis of about 7 days. The effect of added insulin was to increase the rate of heart-protein synthesis to a half-life of 3-4 days. With hearts perfused via the left atrium and performing external work, there was a rise in the specific radioactivity of intracellular free tyrosine, and the half-life for synthesis of proteins was 3-4 days. The extent of labelling of individual myofibrillar proteins was estimated after polyacrylamide-gel electrophoresis of solubilized myofibrils in the presence of sodium dodecyl sulphate. No particular protein showed an unusually high or low specific radioactivity after labelling in perfusion. Insulin caused a general increase in labelling of all the proteins analysed.