Crude Muscle Homogenates Research Articles

Triclosan Impairs Swimming Behavior and Alters Expression of Excitation-Contraction Coupling Proteins in Fathead Minnow (Pimephales promelas)

Triclosan (TCS), a high volume chemical widely used in consumer products, is a known aquatic contaminant found in fish inhabiting polluted watersheds. Mammalian studies have recently demonstrated that TCS disrupts signaling between the ryanodine receptor (RyR) and the dihydropyridine receptor (DHPR), two proteins essential for excitation-contraction (EC) coupling in striated muscle. We investigated the swimming behavior and expression of EC coupling proteins in larval fathead minnows (Pimephales promelas) exposed to TCS for up to 7 days. Concentrations as low as 75 μg L(-1) significantly altered fish swimming activity after 1 day; which was consistent after 7 days of exposure. The mRNA transcription and protein levels of RyR and DHPR (subunit CaV1.1) isoforms changed in a dose and time dependent manner. Crude muscle homogenates from exposed larvae did not display any apparent changes in receptor affinity toward known radioligands. In nonexposed crude muscle homogenates, TCS decreased the binding of [(3)H]PN20-110 to the DHPR and decreased the binding of [(3)H]-ryanodine to the RyR, demonstrating a direct impact at the receptor level. These results support TCS's impact on muscle function in vertebrates further exemplifying the need to re-evaluate the risks this pollutant poses to aquatic environments.

Effects of prolonged exercise and recovery on sarcoplasmic reticulum Ca2+ cycling properties in rat muscle homogenates.

To examine the effects of exercise and exercise plus active and passive recovery on sarcoplasmic reticulum (SR) Ca2+-handling properties. Crude muscle homogenates were prepared from adult rat gastrocnemius muscle from two experiments. In one experiment, the muscle was extracted immediately after prolonged treadmill running (RUN), after a 45 min period of reduced exercise intensity (RUN+) following RUN and compared with controls (CON). In the second experiment, muscle was extracted during passive recovery following the same run protocol at 10 min (REC10), 25 min (REC25) and 45 min (REC45) and compared with CON. Sarcoplasmic reticulum Ca2+-uptake was 31% higher (P < 0.05) in RUN+ compared with CON and RUN. Higher values (P < 0.05) were also found in REC25 (48%) and REC45 (50%) compared with CON. Maximal Ca2+-ATPase was increased by 23% (P < 0.05) in RUN+ compared with CON and RUN and by 65-68% (P < 0.05) in REC25 and REC45 compared with CON. A higher (P < 0.05) Hill coefficient for Ca2+-ATPase activity was observed in RUN+ (2.3 +/- 0.2) compared with CON (1.7 +/- 0.2) or RUN (1.6 +/- 0.2), but not for any REC conditions. In addition, the coupling ratio (Ca2+-uptake/Ca2+-ATPase activity) was higher (P < 0.05) in RUN+ (2.2 +/- 0.10) compared with CON (1.9 +/- 0.05) and RUN (1.9 +/- 0.08). It is concluded that in crude homogenates, SR Ca2+-uptake and Ca2+-ATPase activity are elevated in recovery following prolonged running and that the elevation in these properties is more pronounced during passive compared with active recovery.

Effects of ischemia on sarcoplasmic reticulum Ca2+uptake and Ca2+release in rat skeletal muscle

In this study, we investigated the hypothesis that prolonged ischemia would impair both sarcoplasmic reticulum (SR) Ca(2+) uptake and Ca(2+) release in skeletal muscle. To induce total ischemia (I), a tourniquet was placed around the upper hindlimb in 30 female Sprague-Dawley rats [wt = 256 +/- 6.7 (SE) g] and inflated to 350 mmHg for 4 h. The contralateral limb served as control (C). Immediately after the 4 h of ischemia, mixed gastrocnemius and tibialis anterior muscle was sampled from both limbs, and both crude muscle homogenates and SR vesicles were prepared. In another 10 control animals (CC), muscles were sampled and prepared exactly the same way, but immediately after anesthetization. Ca(2+) uptake and Ca(2+) release were measured in vitro with Indo-I on both homogenates and SR vesicles. As hypothesized, submaximal Ca(2+) uptake was lower (P < 0.05) in I compared with CC and C, by 25 and 45% in homogenates and SR vesicles, respectively. Silver nitrate (AgNO(3))-induced Ca(2+) release, which occurred in two phases (phase 1 and phase 2), was also altered in I compared with CC and C, in both muscle homogenates and SR vesicles. With ischemia, phase 1 peak Ca(2+) release was 26% lower (P < 0.05) in SR vesicles only. For phase 2, peak Ca(2+) release was 54 and 24% lower (P < 0.05) in SR vesicles and homogenates, respectively. These results demonstrate that prolonged skeletal muscle ischemia leads to a reduced SR Ca(2+) uptake in both homogenates and SR vesicles. The effects of ischemia on SR Ca(2+) release, however, depend on both the phase examined and the type of tissue preparation.

Exercise-induced translocation of Na(+)-K(+) pump subunits to the plasma membrane in human skeletal muscle.

Six human subjects performed one-legged knee extensor exercise (90 +/- 4 W) until fatigue (exercise time 4.6 +/- 0.8 min). Needle biopsies were obtained from vastus lateralis muscle before and immediately after exercise. Production of giant sarcolemmal vesicles from the biopsy material was used as a membrane purification procedure, and Na(+)-K(+) pump alpha- and beta-subunits were quantified by Western blotting. Exercise significantly increased (P < 0.05) the vesicular membrane content of the alpha(2)-, total alpha-, and beta(1)-subunits by 70 +/- 29, 35 +/- 10, and 26 +/- 5%, respectively. The membrane content of alpha(1) was not changed by exercise, and the densities of subunits in muscle homogenates were unchanged. The ratio of vesicular to crude muscle homogenate content of the alpha(2)-, total alpha-, and beta(1)-subunits was elevated during exercise by 67 +/- 33 (P < 0.05), 23 +/- 6 (P < 0.05), and 40 +/- 14% (P = 0.06), respectively. It is concluded that translocation of subunits is an important mechanism involved in the short time upregulation of the Na(+)-K(+) pumps in association with human muscle activity.

The isolation of a plasma membrane-rich fraction from the skeletal muscle of two species of marine crab, Carcinus maenas and Cancer pagurus

1. 1. A rapid method for the isolation of a plasma membrane-rich fraction from crab leg muscle, with high purity and yield recovery was developed. 2. 2. The method is based on sodium iodide extraction of the crude homogenate, followed by centrifugation on Percoll self-creating gradient. 3. 3. ( Na + K +)ATPase and alkaline phosphodiesterase I were used as marker enzymes for the plasma membrane and revealed levels of purification of approximately 13-fold and yields recovery of the total activity in the crude muscle homogenate of approximately 18%, for both species of crab studied.

Studies of glutamine uptake across human skeletal sarcolemma

In the human body skeletal muscle is the largest store of glutamine, an important amino-acid in whole body nitrogen balance. Glutamine transport was measured in purified human skeletal muscle sarcolemmal vesicles (HMSV). The activity of sarcolemmal marker enzymes (K +-stimulated nitrophenylphosphatate (K pNPPase) and 5′-nucleotidase) was increased ∼ 14-fold in the sarcolemmal fraction (SF) compared to the crude muscle homogenate (CH). Glutamine transport in HMSV was Na +-dependent (initial rate of 1 μM glutamine in the presence of 0.1 M NaCl = 7 (± 1.7) × 10 −3 pmol.mg −1 protein.s −1 compared to 1.5 (± 0.3) × 10 −3 pmol mg −1 protein.s −1 in the presence of 0.1 M Choline Cl). The rate of glutamine uptake into HMSV was increased in the presence of an inside negative membrane potential.

Rapid separation and quantification of myofibrillar proteins by high performance gel permeation chromatography

A gel permeation chromatography method was developed to rapidly separate and quantify myofibrillar proteins from bovine and porcine muscle samples. Muscle samples were homogenized with one of three salt buffers (phosphate buffered saline, 0·015 m NaCl, 0·02 m Na 2HPO 4, pH 7·4; potassium phosphate, 0·03 m KH 2PO 4, pH 7·4; or phosphate buffered potassium iodide, 1·1 m KI, 0·01 m KH 2PO 4, pH 7·4), filtered and separated by the chromatography method developed. Samples were eluted from the column with 0·5 m KCl, 20 m m Tris buffer, pH 6·0 at 0·6 ml/min and detected at 280 nm. A set of standards was dissolved in elution buffer and was used to create a standard curve for molecular weight determinations. Actomyosin, myosin, tropomyosin, actin, myosin light chains and troponin peaks were identified by spiking the samples with standards and by comparison to Phastgel sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Quantitation was achieved either by using regression equations which described the relationship between peak area and concentration or by expression of the protein in the fraction collection representing the peak as a percent of the total protein injected. The method described here was capable of separating the major proteins from a crude muscle homogenate in thirty minutes with very good reproducibility.

Quantitative determination of Ca2+-dependent Mg2+-ATPase from sarcoplasmic reticulum in muscle biopsies

The possibility of quantifying the total concentration of Ca2+-dependent Mg2+-ATPase of sarcoplasmic reticulum was investigated by measurement of the Ca2+-dependent steady-state phosphorylation from [gamma-32P]ATP and the Ca2+-dependent 3-O-methylfluorescein phosphatase (3-O-MFPase) activity in crude muscle homogenates. The Ca2+-dependent phosphorylation at 0 degree C (mean +/- S.E.) was 40.0 +/- 2.5 (n = 6) and 6.2 +/- 0.7 (n = 4) nmol/g wet wt. in rat extensor digitorum longus (EDL) and soleus muscle, respectively (P less than 0.001). The Ca2+-dependent 3-O-MFPase activity at 37 degrees C was 1424 +/- 238 (n = 6) and 335 +/- 56 (n = 4) nmol/min per g wet wt. in rat EDL and soleus muscle, respectively (P less than 0.01). The molecular activity calculated from these measurements amounted to 35 +/- 5 min-1 (n = 6) and 55 +/- 10 min-1 (n = 4) for EDL and soleus muscle respectively. These values were not different from the molecular activity calculated for purified Ca2+-ATPase (36 min-1). The Ca2+-dependent 32P incorporation in soleus muscle decreased in the order mice greater than rats greater than guinea pigs. In EDL muscles from hypothyroid rats at a 30% reduction of the Ca2+-dependent phosphorylation was observed. The Ca2+-dependent phosphorylation in vastus lateralis muscle from three human subjects amounted to 4.5 +/- 0.8 nmol/g wet wt. It is concluded that measurement of the Ca2+-dependent phosphorylation allows rapid and reproducible quantification of the concentration of Ca2+-dependent Mg2+-ATPase of sarcoplasmic reticulum. Since only 20-60 mg of tissue is required for the measurements, the method can also be used for biopsies obtained in clinical studies.

Effects of exercise of varying duration on sarcoplasmic reticulum function

Sarcoplasmic reticulum (SR) Ca2+ uptake and Ca2+-Mg2+-ATPase activity were examined in muscle homogenates and the purified SR fraction of the superficial and deep fibers of the gastrocnemius and vastus muscles of the rat after treadmill runs of 20 or 45 min or to exhaustion (avg time to exhaustion 140 min). Vesicle intactness and cross-contamination of isolated SR were estimated using a calcium ionophore and mitochondrial and sarcolemmal marker enzymes, respectively. Present findings confirm previously reported fiber-type specific depression in the initial rate and maximum capacity of Ca2+ uptake and altered ATPase activity after exercise. Depression of the Ca2+-stimulated ATPase activity of the enzyme was evident after greater than or equal to 20 min of exercise in SR isolated from the deep fibers of these muscles. The lowered ATPase activity was followed by a depression in the initial rate of Ca2+ uptake in both muscle homogenates and isolated SR fractions after greater than or equal to 45 min of exercise. Maximum Ca2+ uptake capacity was lower in isolated SR only after exhaustive exercise. Ca2+ uptake and Ca2+-sensitive ATPase activity were not affected at any duration of exercise in SR isolated from superficial fibers of these muscles; however, the Mg2+-dependent ATPase activity was increased after 45 min and exhaustive exercise bouts. The alterations in SR function could not be attributed to disrupted vesicles or differential contamination in the SR from exercise groups and were reinforced by similar changes in Ca2+ uptake in crude muscle homogenates.(ABSTRACT TRUNCATED AT 250 WORDS)

Lactic Acidosis and Mitochondrial Myopathy Associated with Deficiency of Several Components of Complex III of the Respiratory Chain

Summary: We have studied a 17-year-old girl with lactic acidosis (3-18 mEq/liter) and progressive muscle weakness since 9 years of age. Morphological findings in muscle were of a typical ragged red myopathy with multiple collections of bizarre mitochondria, some containing paracrystalline inclusions. The carnitine content of serum and muscle was normal, as were the activities of carnitine palmitoyltransferase, carnitine octanoyltransferase, and carnitine acetyltransferase in the patient's muscle. Measurement of the enzymes of oxidative phosphorylation in both crude muscle homogenates and mitochondrial fractions showed close to normal activities of cytochrome c oxidase, succinate dehydrogenase, and ATPase. In contrast, succinate cytochrome c reductase activity was greatly reduced in the patient, being 0.035 μmol/min/g tissue in whole muscle (controls 1.16 ± 0.47 μmol/min/g tissue) and 8 nmol/min/mg protein in the mitochondria (control, 340 nmol/min/mg protein). Rotenonesensitive NADH-cytochrome c reductase was also undetectable in the patient's mitochondria. Spectral analysis of cytochromes showed decrease of reducible cytochrome b to 16% of the control. These results indicate a defect of ubiquinol-cytochrome c reductase or the cytochrome bc1 segment (complex III) of the electron transport chain. Antibody-binding studies of the individual components of complex III showed additional deficiencies of core proteins I and II and peptide VI, indicating a more widespread defect of complex III than was evident from spectral analysis and enzyme activity measurements alone. Urine organic acid analysis after fasting and following a medium chain triglyceride load showed unusually high levels of lactate and 3-hydroxybutyrate, lower than expected levels of acetoacetate and dicarboxylic acids, and the presence of several other metabolites suggesting a disturbed citric acid cycle and redox state. Thus, the defect in this patient may be more widespread than is apparent from the clinical presentation.

Neural regulation of cholinesterase in newt skeletal muscle. I. Distribution and characterization of the enzyme species

AbstractThe molecular forms of acetylcholinesterase (AChE) in triceps muscles of newts (Notophthalmus viridescensi were identified and their distribution in the muscle determined. Six forms of AChE were separated from crude muscle homogenates using velocity sedimentation on sucrose gradients. Their sedimentation coefficients were 16.5S, 1226, 9.8S, 7S, 5.5S, and 3.7S. They were identified as acetylcholinesterases by their ability to hydrolyze acetyl‐β‐methylcholine, but not butyrylthiocholine. The 7S form, however, has not been as thoroughly characterized as it can only be consistently identified in sucrose gradients of Tris‐Triton X‐100‐NaC1 extracts of muscle previously extracted with Tris‐Triton X‐100. The Tris‐Triton X‐100 extraction removes the 5.55 and 9.8S AChE forms which mask the 7S form in sucrose gradients of unfractionated material. The region of muscle containing the motor endplates was enriched in the 16.5S form, whereas the musculotendinous junction contained a high proportion of the 12.8S form. Contamination by AChE present in blood and nerve, did not account for the distribution of the 12.8S species within the muscle. The 3.7S and 5.5S forms were soluble in Tris buffer and are probably located in the cytoplasm. The 9.8S forms required 1 M NaCl for solubilization, indicating that it is associated with an osmotic shock‐resistant intracellular compartment, possibly the sarcotubular system. Triton X‐100 was required to solubilize the 16.5S, 12.8S and 7S forms indicating that they may be membrane bound. Crude muscle homogenates and each of the forms of AChE isolated on sucrose gradients yielded nine bands of AChE activity when subjected to polyacrylamide gel electrophoresis (PAGE). The bands of AChE were apparently monomers and multimers from which the larger forms of AChE were assembled.

Activity of acid hydrolases in skeletal muscle of untrained, trained and detrained mice of different ages

The activities of p-nitrophenylphosphatase, beta-glucuronidase and beta-N-acetylglucosaminidase from crude skeletal muscle homogenates of 4 and 7 months old mice were assayed after short-term intensive and long-term moderate training and after terminated training. In the older untrained mice the activity of the hydrolases was higher than in the younger mice. The level increased with training and this increase was far more pronounced in the older animals. Cessation of training for 7 and 21 days decreased this activity in the older animals but it was again increased 42 days later and close to the level observed in the trained mice. In young mice 3 days' terminated training increased the activity of the acid hydrolases above the level of the trained animals but after additional 4 and 11 days' terminated training the activity decreased to slightly below that of the trained mice. The changes were most prominent in the activity of beta-glucuronidase and to a lesser extent in that of beta-N-acetylglucosaminidase while p-nitrophenylphosphatase activity was almost unaffected by training or terminated training. The effects of terminated training can be intepreted as representing altered catabolic processes in the turn-over of tissue components of skeletal muscle.

Isozymes of Creatine Phosphokinase and Myokinase in Human Heart and Skeletal Muscle

The isozyme patterns of creatine phosphokinase (CPK) and myokinase (MK) were investigated in biopsy material from human heart and skeletal muscle. The protein separation was performed on crude muscle homogenates by means of flat-bed isoelectric focusing. Two isozymes were demonstrated for each enzyme, irrespective of the sampling site. The pI values were, on the average, 9.8 (MK-1) and 8.9 (MK-2), 7.2 (CPK-1) and 6.9 (CPK-2), respectively. MK-1 and CPK-1 constituted an average of 81% and 70% of total staining density for each enzyme, respectively. The relative contribution of MK-1 differed, however. Heart muscle showed the highest values (mean 91%) and vastus lateralis the lowest (mean 74%). The mean value for soleus was 86% MK-1. Furthermore, the percentage of MK-1 present was negatively correlated with the percentage of fast twitch fibres in m. vastus lateralis (r = -0.67). No corresponding differences could be demonstrated for CPK isozyme distribution. In conclusion, it was demonstrated that MK and CPK each occurred as two isozymes in human heart and skeletal muscle, and that the relative distribution of MK isozymes, in contrast to CPK, was related to muscle fibre type composition, and thus to the metabolic profile of the muscle.

Immunological and ultrastructural observations on the role of lymphoid cells in the pathogenesis of polymyositis.

Muscle biopsies from two cases of polymyositis were examined with the electron microscope, particular attention being given to the inflammatory cells in the lesions. Lymphocytes which were enlarged and possibly “activated” were found in contact with the endothelium of small venules; they were also seen apparently traversing the vessel wall. Venular endothelial cells were often swollen with reduplication of the basement membrane. Perivascular and interstitial cells comprised small lymphocytes, larger transforming lymphocytes with distinctive morphological features, pleomorphic macrophages and a few plasma cells and granulocytes. In one case cells resembling primitive lymphoid cells were found internal to the basement membrane of several muscle fibres. Peripheral blood lymphocytes from both patients were stimulated on incubation with a crude muscle homogenate. The lymphocytes also appeared to be cytotoxic to cultures of foetal human striated muscle. The significance of these findings is considered in relationship to the pathogenesis of the muscle fibre damage in polymyositis.

Phosphoglucomutase activity in hereditary muscular dystrophy in mice.

SummaryA modified method of assaying maximum phosphoglucomutase activity in crude muscle homogenates is presented. Mutase activity is significantly decreased in leg muscles of mice with muscular dystrophy whether determined as total enzyme activity (at infinite coenzyme concentration) or as residual enzyme-coenzyme activity (in the absence of added coenzyme). Evidence is presented for a decrease in coenzyme content of dystrophic muscle and for a correlation of these enzyme changes with age of mice and severity of myopathy.