Studies of a calcium-activated neutral protease from chicken skeletal muscle. II. Substrate specificity.

Calcium-activated neutral protease purified from chicken skeletal muscle hydrolyzed myofibrillar proteins, tubulin, spectrin, and oxidized insulin B chain, but hardly hydrolyzed synthetic or natural peptides.     

Endocrine and metabolic regulation of muscle growth and body composition in cattle

Muscle metabolism (in interaction with other organs and tissues, including adipose tissue) plays an important role in the control of growth and body composition. Muscle ontogenesis has been described in different genotypes of cattle for myofibres, connective tissue and intramuscular depots. The ontogenesis or the action of putatively important factors controlling muscle development (IGF-II expression, IGF receptors, growth hormone (GH) receptor, myostatin, basic fibroblast growth factor, transforming growth factor-β1, insulin and thyroid hormones) has also been studied on bovine foetal muscle samples and satellite cells. The glucose/insulin axis has been specifically studied in both the bovine adipose tissue and heart. Clearly, cattle, like sheep, are mature species at birth based on their muscle characteristics compared to other mammalian or farm animal species. The different myoblast generations have been well characterised in cattle, including the second generation which is liable to be affected by foetal undernutrition at least in sheep. Interesting genotypes, for example, double-muscled genotype, have been characterised by an altered metabolic and endocrine status associated with a reduced fat mass, specific muscle traits and different foetal characteristics. Finally, the recent development of genomics in cattle has allowed the identification of novel genes controlling muscle development during foetal and postnatal life. Generally, a high muscle growth potential is associated with a reduced fat mass and a switch of muscle fibres towards the glycolytic type. The possibility and the practical consequences of manipulating muscle growth and, hence, body composition by nutritional and hormonal factors are discussed for bovines based on our current biological knowledge.     

Age-related changes in the incorporation of [14-C] leucine into myofibrillar and sarcoplasmic proteins of red and white muscles of chicks.

Studies on the incorporation of DL-[1- 14-C] leucine into myosin, total myofibrillar protein and total sarcoplasmic protein have shown age-dependent alterations in the rate of synthesis of these protiens in red and white skeletal muscles of chicks. During the early phase of ex ovo development white muscle synthesizes significantly higher amounts of myofibrillar proteins, especially myosin, in comparison with red muscle. The rate of sarcoplasmic protein synthesis in red and white muscles one day after hatching is almost identical. The red muscle shows a markedly higher rate of sarcoplasmic protein synthesis from 10 days after hatching. The incorporation of amino acid into various protein fractions of both the muscle types decreases with advancing age. In adult chicks red muscle displays a higher ability to synthesize sarcoplasmic and myofibrillar proteins.     

Soluble and particulate forms of muscle alkaline proteinase show differential sensitivity to endogenous inhibitor(s)

Membrane-free washed myofibrils derived from rat skeletal muscle homogenates contained a chymostatin-sensitive protease(s) which acted on associated myofibrillar proteins, at an optimum pH of 8.5, much less rapidly at low ionic strength (insoluble myofilaments) than at high salt concentrations (solubilized proteins). When the myofibrillar fraction was added to the particle-free cytosol prepared from the muscle extracts, proteins of the cytosol were also degraded, but the activity in this case was much more pronounced at low ionic strength. This was because inhibitor(s) of the proteinase present in the cytosol fraction were only effective at high ionic strength when all the myofibrillar (and associated) proteins were in solution. The protease was separated from the bulk of the myofibrillar proteins by gel chromatography at high ionic strength. On dialysis against a low-salt buffer, part of the enzyme was precipitated. The putative cytosolic inhibitor(s) were again only effective on the soluble enzyme at high ionic strength.     

Stimulation of protein synthesis by both insulin and amino acids is unique to skeletal muscle in neonatal pigs

In neonatal pigs, the feeding-induced stimulation of protein synthesis in skeletal muscle, but not liver, can be reproduced by insulin infusion when essential amino acids and glucose are maintained at fasting levels. In the present study, 7- and 26-day-old pigs were studied during 1) fasting, 2) hyperinsulinemic-euglycemic-euaminoacidemic clamps, 3) euinsulinemic-euglycemic-hyperaminoacidemic clamps, and 4) hyperinsulinemic-euglycemic-hyperaminoacidemic clamps. Amino acids were clamped using a new amino acid mixture enriched in nonessential amino acids. Tissue protein synthesis was measured using a flooding dose of L-[4-(3)H]phenylalanine. In 7-day-old pigs, insulin infusion alone increased protein synthesis in various skeletal muscles (from +35 to +64%), with equivalent contribution of myofibrillar and sarcoplasmic proteins, as well as cardiac muscle (+50%), skin (+34%), and spleen (+26%). Amino acid infusion alone increased protein synthesis in skeletal muscles (from +28 to +50%), also with equivalent contribution of myofibrillar and sarcoplasmic proteins, as well as liver (+27%), pancreas (+28%), and kidney (+10%). An elevation of both insulin and amino acids did not have an additive effect. Similar qualitative results were obtained in 26-day-old pigs, but the magnitude of the stimulation of protein synthesis by insulin and/or amino acids was lower. The results suggest that, in the neonate, the stimulation of protein synthesis by feeding is mediated by either amino acids or insulin in most tissues; however, the feeding-induced stimulation of protein synthesis in skeletal muscle is uniquely regulated by both insulin and amino acids.     

Toxic impact of phosphamidon on protein degradation in phasic and tonic muscles of marine prawn, Penaeus indicus (H. Milne Edwards).

Levels of various protein fractions, (sarcoplasmic, myosin, actin, non-collagen and collagen) and the rate of their degradation by proteases were studied in phasic and tonic muscles of marine prawn, Penaeus indicus following acute (2 d) and chronic (15 d) exposure to sublethal concentration of phosphamidon. During exposure, greater decrease in sarcoplasmic protein fraction was observed in phasic muscle as compared to other myofibrillar proteins. But the sarcoplasmic protein content showed an elevation in tonic muscle. The changes in protein fractions were more pronounced during acute exposure than chronic exposure both in phasic and tonic muscles. These changes were correlated with the elevation of the acidic, neutral and basic protease activities during acute and chronic exposure. Free amino acids were increased during acute exposure, while they showed a significant decrease during chronic exposure in both the muscles. These results indicate that protein metabolism in both phasic and tonic muscles was significantly altered following phosphamidon exposure. These differential responses observed at acute and chronic exposure indicate the operation of compensatory mechanisms to mitigate the phosphamidon toxic stress.     

Preservation of differences in the degree of polymerization of actin, isolated at different stages of ontogenesis after its purification]

Actin preparation from skeletal muscles of new-born, 10 days old and adult rabbits, containing less than or equal to 5% of inactivated actin and 1-2% of other myofibrillar proteins, were studied by means of flow birefringence and viscosimetry. It is found, that, like earlier studied crude preparations, purified actin preparations, isolated at different ontogenesis stages, differ in their intrinsic viscosity and extinction angle. These differences retain after the additional trypsin treatment. Non-polymerizing fraction of Straub F-actins, isolated from rabbit muscles of all ontogenesis stages studied, practically does not affect the polymerization in the course of ontogenesis, which is due to changes in its stucture.     

Effect of tumour necrosis factor-alpha on total myofibrillar and sarcoplasmic protein synthesis and polysomal aggregation in rat skeletal muscles

The total sarcoplasmic and myofibrillar protein synthesis was reduced in incubated fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus of rat after in vivo tumour necrosis factor-alpha treatment at 50 micrograms/kg/day for 5 days. The rate of protein synthesis in the myofibrillar fraction was inhibited more severely (41% in EDL and 34% in soleus) than that in the sarcoplasmic fraction (23% in EDL and 14% in soleus). Sucrose density gradient centrifugation analysis indicated that TNF-alpha treatment impaired polysomal aggregation in rat diaphragm muscle. Compared with the control muscles, the ratio of 40S and 60S subunits to polysomes was higher in TNF-alpha treated muscles. These findings suggest a role for TNF-alpha in the translational regulation of protein synthesis in rat skeletal muscle.     

Effect of caffeine on active Ca2+ ion transport in a homogenate of skeletal muscles and myocardium

A Ca2-selective electrode was used to study active transport of Ca2+ by sarcoplasmic reticulum fragments of rabbit skeletal muscle and myocardium homogenates. The specific Ca2+ transport activities (mumol Ca2+/min/mg tissue) are 40 = 60 and 3 = 5 units for fast and slow muscles and the myocardium, respectively. Caffeine (5 mM) exerts a powerful inhibitory influence on Ca2+ transport in skeletal muscle homogenates. For fast muscles, the degree of inhibition exceeds 50%. The rate of Ca2+ transport in the myocardium homogenate increases in the presence of creatine phosphate. The latter produces no effect on Ca2+ transport in skeletal muscle homogenates. The high sensitivity of Ca2 transport to caffeine, a specific blocker of Ca2+ transport to the terminal cisterns of the sarcoplasmic reticulum, suggests that the terminal cisterns, apart from being a reservoir for Ca2+ needed for contraction trigger, may play an essential role in muscle relaxation.     

Purification and characterization of UDP-GalNAc:polypeptide N-acetylgalactosaminyl transferase from swine trachea epithelium

It is well established that periods of increased contractile activity result in significant changes in muscle structure and function. Such morphological changes as sarcomeric Z-line disruption and sarcoplasmic reticulum vacuolization are characteristic of exercise-induced muscle injury. While the precise mechanism(s) underlying the perturbations to muscle following exercise remains to be elucidated, it is clear that disturbances in Ca²⁺ homeostasis and changes in the rate of protein degradation occur. The resulting elevation in intracellular [Ca²⁺] activates the non-lysosomal cysteine protease, calpain. Because calpain cleaves a variety of protein substrates including cytoskeletal and myofibrillar proteins, calpain-mediated degradation is thought to contribute to the changes in muscle structure and function that occur immediately following exercise. In addition, calpain activation may trigger the adaptation response to muscle injury. The purpose of this paper is to: (i) review the chemistry of the calpain-calpastatin system; (ii) provide evidence for the involvement of the non-lysosomal, calcium-activated neutral protease (calpain) in the response of skeletal muscle protein breakdown to exercise (calpain hypothesis); and (iii) describe the possible involvement of calpain in the inflammatory and regeneration response to exercise.     

Regulation of myofibrillar protein turnover during maturation in normal and undernourished rat pups

The study tested the hypothesis that a higher rate of myofibrillar than sarcoplasmic protein synthesis is responsible for the rapid postdifferentiation accumulation of myofibrils and that an inadequate nutrient intake will compromise primarily myofibrillar protein synthesis. Myofibrillar (total and individual) and sarcoplasmic protein synthesis, accretion, and degradation rates were measured in vivo in well-nourished (C) rat pups at 6, 15, and 28 days of age and compared at 6 and 15 days of age with pups undernourished (UN) from birth. In 6-day-old C pups, a higher myofibrillar than sarcoplasmic protein synthesis rate accounted for the greater deposition of myofibrillar than sarcoplasmic proteins. The fractional synthesis rates of both protein compartments decreased with age, but to a greater degree for myofibrillar proteins (-54 vs. -42%). These decreases in synthesis rates were partially offset by reductions in degradation rates, and from 15 days, myofibrillar and sarcoplasmic proteins were deposited in constant proportion to one another. Undernutrition reduced both myofibrillar and sarcoplasmic protein synthesis rates, and the effect was greater at 6 (-25%) than 15 days (-15%). Decreases in their respective degradation rates minimized the effect of undernutrition on sarcoplasmic protein accretion from 4 to 8 days and on myofibrillar proteins from 13 to 17 days. Although these adaptations in protein turnover reduced overall growth of muscle mass, they mitigated the effects of undernutrition on the normal maturational changes in myofibrillar protein concentration.     

Partial characterization of an endogenous inhibitor of a calcium-dependent form of insulin protease

Insulin protease activity has resisted high-yield purification to homogeneity, due to its low amount in tissues, its instability, and its erratic recovery from several types of chromatography. This report outlines the preliminary characterization of a naturally-occurring insulin protease inhibitor that accounts for some of these problems in rat skeletal muscle. In these experiments, inhibitory activity was assayed by its effect upon hydrolysis of 125I-(A14)-insulin by the partially purified insulin protease activity of rat skeletal muscle cytosol. During Sephadex G-200 chromatography of cytosol at pH 7.5, inhibitory activity copurifies with insulin protease activity, and the incomplete resolution of the two activities contributes to the impression that insulin protease exists in distinct 180,000-dalton and 80,000-dalton forms. By contrast, during DEAE-Sephacel chromatography of cytosol at pH 7.5, inhibitory activity and insulin protease activity are resolved by eluting the resin with 50 mM NaCl and 200 mM NaCl, respectively. Post-DEAE-Sephacel inhibitor has an Mr(app) of 67,000 daltons or 80,000-120,000 daltons, as determined by high-performance liquid chromatography or Sephadex G-150 chromatography, respectively. Post-DEAE-Sephacel insulin protease activity exhibits a Km for insulin of 15 nM and resides in a 200,000-dalton neutral thiol protease which requires 50 micromolar calcium for its maximum insulin-degrading activity. The inhibitor reduces the enzyme's activity reversibly, nonprogressively, and non-competitively with respect to insulin, but it does not alter the enzyme's sensitivity to calcium ion. These observations suggest that calcium and an endogenous protease inhibitor may influence cellular degradation of insulin via previously unrecognized effects upon cytosolic insulin protease activity.     

Differential catabolism of muscle protein in Garden Warblers (Sylvia borin): flight and leg muscle act as a protein source during long-distance migration

Samples of flight and leg muscle tissue were taken from migratory garden warblers at three different stages of migration: (1) pre-flight: when birds face an extended flight phase within the next few days, (2) post-flight: when they have just completed an extended flight phase, and (3) recovery: when they are at the end of a stop-over period following an extended flight phase. The changes in body mass are closely related to the changes in flight (P<0.001) and leg muscle mass (P<0.001), suggesting that the skeletal muscles are involved in the protein metabolism associated with migratory flight. From pre- to post-flight, the flight and the leg muscle masses decrease by about 22%, but are restored to about 12% above the pre-flight masses during the recovery period. Biochemical analyses show that following flight a selective reduction occurred in the myofibrillar (contractile) component of the flight muscle (P<0.01). As this selective reduction accounts only for a minor part of the muscle mass changes, sarcoplasmic (non-contractile) and myofibrillar proteins of both the flight and leg muscle act as a protein source during long-distance migration. As a loss of leg muscle mass is additionally observed besides the loss in flight muscle mass, mass change seems not to be strictly associated with the mechanical power output requirements during flight. Whereas the specific content of sarcoplasmic proteins in the flight muscle is nearly twice as high as that in the leg muscle (P<0.001), the specific content of myofibrillar proteins differs only slightly (P < 0.05), being comparably low in both muscles. The ratio of non-contractile to contractile proteins in the flight muscle is one of the highest observed in muscles of a vertebrate.     

Identification of a myofibril-bound serine protease and its endogenous inhibitor in mouse skeletal muscle

Myofibrillar proteins, like all other intracellular proteins, are in a dynamic state of continual degradation and resynthesis. The proteolytic system responsible for degrading myofibrillar proteins in skeletal muscle is not well defined. A proteolytic activity associated to myofibrils was found in mouse skeletal muscle, as show electrophoretic patterns, and denominated by us, as protease M. During incubation of whole myofibrils at 37 degrees C, myosin heavy chain, alpha actinin, actin and troponin T suffered degradation. These effects were inhibited selectively by serine protease inhibitors (soybean trypsin inhibitor, di-isopropyl phosphofluoridate, phenylmethanesulfonyl fluoride). Using myofibrils as protease M source, azocaseinolytic activity was also detected. Endogenous inhibitor and various compounds effects on protease M activity were also quantified by trichloroacetic acid soluble products formation, using radiolabeled myofibrils. An endogenous trypsin inhibitor isolated from the muscle cytoplasmic fraction could inhibit protease M activity on myofibrillar proteins and on azocasein. While K(+) increased protease M activity, the presence of Ca(2+) did not show any effect. Data presented in this study suggest that reported protease M may be implicated in myofibrillar degradation in vivo and isolated endogenous inhibitor may provide a mechanism to control its action in mouse skeletal muscle.     

The capacity of the sarcoplasmic reticulum for phospholipid synthesis: a developmental study

The activities of three enzymes involved in phospholipid synthesis, sn-glycerol-3-phosphate acyltransferase (EC 2.3.1.15), cholinephosphate cytidylyltransferase (EC 2.7.7.15), and cholinephosphotransferase (EC 2.7.8.2), were assayed in adult skeletal muscle. The acyltransferase and cholinephosphotransferase were concentrated in the sarcoplasmic reticulum, where their specific activities were 80 and 33%, respectively, of the specific activity in liver microsomes. Cytidylyltransferase activity was distributed throughout the cell with most of the activity in the cytosol. Its activity in muscle was only 10% of liver activity. Functional sarcoplasmic reticulum was isolated by density gradient centrifugation after calcium loading in the presence of phosphate. The specific activities of these enzymes wee undiminished in the calcium-loaded fraction, suggesting that these enzymes are intrinsic components of the sarcoplasmic reticulum. In developing muscle (2 and 6 days postnatal) acyltransferase and cholinephosphotransferase activities were also present in a calcium-loaded microsomal subfraction at the same level as in the adult. Cytidylyltransferase activity, on the other hand, was 8-fold higher in developing muscle. In addition, developing muscle had a 3-fold increase in the proportion of cytidylyltransferase associated with the microsomal fraction. These data suggest that sarcoplasmic reticulum has the capacity for phospholipid synthesis in mature and developing muscle, and that the rate of phosphatidylcholine synthesis may be regulated by the levels of cytidylyltransferase and by translocation of this enzyme between the sarcoplasmic reticulum and the cytosol.     

Development of different types of muscle fiber in the postnatal ontogeny of guinea pigs

As demonstrates estimation of myosin ATPase and SDG activity, the guinea pig is already born with differentiated muscle fibers (MF), and the first histochemical differences between them take place in the uterine 10 days before birth. Tonic oxidative fibers of the first type, arranging hexagonally, develop especially quickly at early stages of postnatal ontogenesis. Their relative contents up to the end of the observations (185 days) do not change, and area of their transversal section increases but slightly in comparison to the phasic fibers. The main age changes of the muscle tissue are connected with formation and rearrangement of the phasic fibers. The most intensive reconstructions of the phasic fibers coincide with the period of game activity and sex maturation. In mixed muscles the part of the glycolytic fibers increase during the postnatal ontogenesis. In the process of ontogenesis the soleus muscle fully consists of oxidative fibers. The definitive level of the MF development is established after the guinea pigs have reached their sex maturation. Comparing the results of the given investigation with the previous data on development of MF in rats, it is possible to conclude that term and premature animals have various rates in development of the muscle system, however, main stages of myogenesis coincide, though they are connected with various phases of ontogenesis.     

Human red blood cell insulin-degrading enzyme and rat skeletal muscle insulin protease share antigenic sites and generate identical products from insulin.

The mechanisms of cellular insulin degradation remain uncertain. Considerable evidence now exists that the primary cellular insulin-degrading activity is a metallothiol proteinase. Two similar degrading activities have been purified and characterized. Insulin protease has been purified from rat skeletal muscle and insulin-degrading enzyme from human red blood cells. Whereas the two degrading activities share a number of similar properties, significant differences have also been reported; and it is not at all established that they are the same enzyme. To examine this, we have compared antigenic and catalytic properties of the two enzymatic activities. Monoclonal antibodies against the red blood cell enzyme adsorb the skeletal muscle enzyme; and on Western blots, the antibodies react with an identical 110-kDa protein. Immunoaffinity-purified enzymes from both red blood cells and skeletal muscle degrade [125I]iodo(B26)insulin to the same products as seen with purified insulin protease and with intact liver and kidney. Chelator-treated muscle and red blood cell enzymes can be reactivated with either Mn2+ or Ca2+. Thus, insulin-degrading enzyme and insulin protease have similar properties. These results support the hypothesis that these activities reside in the same enzyme.     

Lysosomal enzyme activities in muscle following starvation and refeeding in the saithe Pollachius virens L

Saithe (Pollachius virens L.) were starved for 66 days at 10 degrees C and activities of aryl sulfatase, acid proteinase, beta-glucuronidase, RNAase and acid phosphatase measured in homogenates prepared from fast and slow myotomal muscles. In fed fish, hydrolase activities were generally higher in slow than fast muscles. With the exception of acid proteinase activity in slow muscle, the activities of all the lysosomal enzymes increased by 70 to 100% during starvation. In general, there was a proportionally larger increase in the hydrolase activities in fast than in slow muscle. In a second experiment, fish were starved for 74 days, and refed for up to 52 days. The increases in aryl sulfatase and acid proteinase activity produced in fast muscle with starvation were found to be rapidly reversed by refeeding. Lysosomal enzyme activities in fish sampled after 10 days refeeding were not significantly different from fed controls. Membrane fractions enriched in aryl sulfatase activity were prepared from the fast muscle of 66-day starved fish. These were capable of degrading both myosin heavy chains and actin to lower molecular weight peptides at acid (pH 5.0), but not at neutral pH. The results suggest a role for lysosomal enzymes in the breakdown of myofibrillar proteins during starvation.     

Myofibrillar protein turnover. Synthesis rates of myofibrillar and sarcoplasmic protein fractions in different muscles and the changes observed during postnatal development and in response to feeding and starvation.

Measurement of rates of synthesis of skeletal-muscle proteins in adult rats shows that the faster overall rate of turnover in diaphragm and soleus muscles compared with several other, more glycolytic, muscles is also exhibited by the myofibrillar proteins, since the ratio of sarcoplasmic to myofibrillar protein synthesis is similar for all muscles. Further, throughout postnatal development, when the overall turnover rate falls with age, parallel changes occur for the myofibrillar proteins, as indicated by a constant ratio of sarcoplasmic to myofibrillar protein synthesis (2.06) in the steady state after overnight starvation. Only in the youngest (4 weeks old) rats is a slightly lower ratio observed (1.72). These results indicate that, when changes in the overall turnover rate of muscle proteins occur, the relative turnover of the two major protein fractions stays constant. However, measurements in the non-steady state during growth and after starvation for 4 days show that the relative synthesis rates of the two fractions change as a result of a disproportionate increase in myofibrillar protein synthesis during growth and decrease during starvation. Thus the synthesis rate of the slower-turning-over myofibrillar protein fraction is more sensitive to nutritional state than is that of the sarcoplasmic protein. It is suggested that such responses may help to maintain constant tissue composition during non-steady-state conditions of growth and atrophy.     

Studies on the Metabolic Activity of Muscle Proteins

The time-course of changes in total amount of proteins of sarcoplasmic, myofibrillar and stromal fractions in muscle of the rats fed a protein-free diet for 8, 16, 24 and 32 days, together with the referential data of those changes in the rats fed a protein-free diet up to time of death and a 60% casein diet for 12 days was determined respectively. The results were as follows: (1) The sarcoplasmic and the myofibrillar fractions decreased much more than the stromal fraction in the earlier stages of protein depletion following the same pattern as seen in reserve proteins. (2) The sarcoplasmic fraction decreased slightly more than the myofibrillar fraction as early as 8 days of the depletion, but the relative proportion between these two fractions was thereafter almost the same as that of the standard diet group. (3) In rats fed a 60% casein diet, the sarcoplasmic fraction increased markedly than the others.