Effect of vitamin E-deficiency on the activity of some lysosomal and non-lysosomal proteases in rabbit muscles.

The activity of different cathepsins and neutral proteinases was measured in normal and vitamin E-deficient rabbit muscles using specific substrates. Among the changes of enzyme activities in dystrophy caused by vitamin E-deficiency the increase in the activity of cathepsin B is the most striking. The activity of cathepsin H, both in the fast and slow muscles and that of MMP-ase in the slow muscle remains practically unchanged. Activities of other proteases significantly increase. The change in the activity of proteolytic enzymes in striated muscle of vitamin E-deficient rabbits seems to be selective. As a rule the increase in the activity is higher in fast than in slow muscles.

Proteolytic enzyme activities in skeletal muscle of high jumping rats.

Physical activity can be studied by various kind of exercises. Many works have been published in field of proteolytic enzyme activities in skeletal muscle during endurance training. In this work we used high jumping as a dynamic force velocity training to study the changes in proteolytic enzyme activities during this type of exercise. The activity of cathepsin D, cathepsin L and ATN-ase (Acetyl-Tyrosine-paranitroanilide-splitting enzyme) in vastus lateralis muscle was measured after one, 3, 7 or 11 weeks of high jumping exercise. The results demonstrated that proteinase activity began to increase when the load, i.e. number of jumping and the weight put on the rat's back was too much for their muscles. They could carry out the task consuming the energy originating from muscle tissue in the first period of the experiment, but in the second period (after 7 weeks) the type of training with this load became equal with an endurance training.

Activities of some serum enzymes in halothane reacted and non-reacted pigs.

Eight- and nine-week-old Hungarian Landrace pigs were tested with halothane as described by Laky et al. (1985). Immediately after the test blood samples were taken for determination of the activity of serum creatine kinase (CK), creatine kinase MB (CK-MB) isoenzyme, aldolase (ALD), lactate dehydrogenase (LDH) and alpha-hydroxybutyrate dehydrogenase (alpha-HBDH). Elevated creatine kinase, creatine kinase MB isoenzyme and aldolase activities indicating enhanced susceptibility to stressors were found in 92% of the halothane reacted and 16% of the halothane non-reacted animals. In these individuals the activities of lactate dehydrogenase and alpha-hydroxybutyrate dehydrogenase were also high. Data of the literature show a close relationship between enhanced susceptibility to stressors and halothane reaction in pigs. It was suggested, therefore, that determination of the activity of appropriate serum enzymes might be used for detecting this enhanced susceptibility.

[Effect of short-term space flights on physiological properties and composition of myofibrillar proteins of the skeletal muscles of rats]. / Vliianie kratkovremennykh kosmicheskikh poletov na fiziologicheskie svoistva i sostav miofibrilliarnykh skelethykh myshts krys.

Contractile properties of preparations of glycerinated myofibers and subunit composition of myofibrillar proteins of skeletal muscles were studied using rats flown on Kosmos-1514 (pregnant females) and Kosmos-1667 (males). After the 5- and 7-day flights the strength and velocity of contraction of myofibers decreased, although this change was not correlated with functional differentiation of muscles. The myosin population tended to vary in terms of the proportion of fast and slow isoforms. It is concluded that physiological properties of skeletal muscles at an early stage of orbital flights deteriorated primarily due to a decline in the functional activity of the excitation-contraction conjugation system of myofibers.

Vanadate stimulated NADH oxidation in sarcoplasmic reticulum membrane.

Vanadate stimulated NADH oxidation was detected in sarcoplasmic reticulum membrane preparations. The reaction showed enzymatic character, with half maximal activating concentration of 1.2 mM vanadate and maximal NADH oxidation 50 nmol/mg protein/minute. Acidic pH, micromolar free Ca2+ concentration and decavanadate addition increased the rate of NADH oxidation. The described enzyme activity is similar to the ones observed in erythrocyte liver and cardiac plasma membranes. The vanadate stimulated NADH oxidation in sarcoplasmic reticulum preparations does not seem to originate from other contaminating membrane elements. The presence of this enzyme activity in the sarcoplasmic reticulum should be taken into consideration when planning experiments with vanadate, especially when measuring ATPase activity through NADH oxidation with coupled enzymatic assay.

Effects of disuse on the function of fragmented sarcoplasmic reticulum of rabbit M. gastrocnemius.

A preparation method has been described to obtain a relatively pure and functionally intact fragmented sarcoplasmic reticulum (SR) vesicles fraction from normal and atrophied muscles. Purified SR preparations from rabbit gastrocnemius muscle atrophied by disuse showed similar protein composition (gel electrophoresis; Laemmli 1970) and similar vanadate induced crystallization (Dux and Martonosi 1983) properties of Ca2+-ATPase as those of control preparations. In the early period of atrophy (1-2 weeks) both the Ca2+-ATPase activity and Ca2+ uptake showed a 2-3-fold increase (from 3.42 +/- 0.24 to 7.34 +/- 0.25 mumol Pi X mg-1 prot X min-1 and from 1.26 +/- 0.10 to 3.36 +/- 0.22 mumol/l Ca2+ X min-1 X mg-1 prot. respectively).

Changes in exopeptidase activities in skeletal muscles during disuse.

Some aminopeptidase activities, dipeptidase-, tripeptidase-, and carboxypeptidase activities were measured in two different types of skeletal muscle in rabbit soleus muscle as a slow oxidative, and gastrocnemius muscle as a fast glycolytic type after immobilization in full extension with a plaster cast for 1, 2, 4, 7, 14 or 28 days. In correlation to the higher protein turnover in red muscles, the activities except of leucine and alanine aminopeptidase were higher in the normal soleus muscle than in the gastrocnemius muscle. Much higher activities of the tested enzymes were obtained in the immobilized soleus muscle than in the normal one after 2 weeks of immobilization. In the gastrocnemius muscle the tested enzyme activities generally did not change or decrease. The results demonstrate that the peptidases play a role in the process of protein breakdown in normal and disused skeletal muscles.

The effect of chronic alcohol ingestion on the contractile proteins of the rat heart.

With increasing duration of alcohol consumption the amounts of total myofibrillar proteins in CFY rats decreased slightly, but significantly (from 63.3 +/- 5.7 mg X g wet muscle weight to 54.9 +/- 5.9 mg X g-1 after 12 weeks on alcohol). Similar slight changes could be observed in the case of sarcoplasmic proteins. No significant changes were observed in the composition of the myofibrillar proteins. The densitiometrically calculated percentage ratios of myosin/actin, myosin light chains (LC1/LC2) and troponin components remained the same in the alcoholic animals. The same distribution of native myosin isoenzymes was found in the ventricles of the alcoholic animals as in the controls. We found no electrophoretically detectable evidence that a change in the composition of the myofibrillar proteins is responsible for the decreased contractility of the rat heart following chronic alcohol ingestion.

The effect of 4-aminopyridine-induced increased neuromuscular activity on the metabolism of developing muscles in chick embryos.

Chick embryos were treated with 4-aminopyridine (4 X 100 micrograms) during a critical stage of muscle development, and the effect of enhanced neuromuscular activity upon energy metabolism was studied in two fast-twitch muscles and a slow-tonic muscle. In the slow-tonic muscles of treated embryos, the specific activities of creatine kinase (CK) and lactate dehydrogenase (LDH) were reduced by 11 and 21%, respectively, compared with control values, whereas the ratios of the CK-MB isoforms and the LDH-H subunits increased to 125 and 135% of the control values, respectively. No significant changes could be shown in the enzymatic pattern of fast muscles. These results indicate that a moderate increase in neuromuscular activity of the chick embryo primarily influences the metabolism of developing slow muscles, promoting the development of an enzyme profile characteristic of slow oxidative fibres.

Enzyme activity changes in rat heart after chronic alcohol ingestion.

The effect of a chronic intake of dietary alcohol upon myocardial enzymes was studied in rats. Alcohol, comprising more than 40% of the dietary calorie content, was administered to rats for 6 or 12 weeks. To assess the metabolic changes in the myocardium, the following enzymes were measured: lactate dehydrogenase (LDH), malate dehydrogenase (MDH), aldolase (ALD), isocitrate dehydrogenase (ICDH), creatine kinase (CK) and glutamate-pyruvate transaminase (GPT). The activity of CK was decreased (4.79 +/- 0.99 U X mg-1 protein) after 6 weeks on alcohol and was significantly different from that of the controls (5.98 +/- 1.44 U X mg-1 protein). After 12 weeks the CK activity of alcoholic rats had recovered to 5.99 +/- 1.08 U X mg protein-1 and approached the value found in the normal myocardium. A pronounced decrease was found in the activity of MDH: 8.26 +/- 0.69 U X mg protein-1 in the controls, and 6.78 +/- 1.07 U X mg protein-1 and 5.79 +/- 0.85 U X mg protein-1 in the alcoholic rats after 6 and 12 weeks, respectively. The LDH activity decreased to a lesser extent, but significantly: 2.45 +/- 0.18 U X mg protein-1 in the controls, and 2.11 +/- 0.07 U X mg protein-1 and 2.06 +/- 0.29 U X mg protein-1 after 6 and 12 weeks on test. Only slight, not significant, changes were observed for the other enzymes investigated (ICDH, ALD, GPT).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of weightlessness on myofibrillar proteins of rat skeletal muscles with different functions in experiment of biosatellite "Cosmos-1129".

The composition of contractile and regulatory proteins was studied in rat muscles with different functions. The rats were exposed to weightlessness for 18.5 days during a space journey in biosatellite Cosmos-1129. Under the effect of weightlessness the myosin light chain composition changed, the quantity of myosin LC-3 subunits increased in the soleus and extensor digitorum longus (EDL) while it decreased in the triceps and brachialis muscles. The experiments showed changes in the subunit composition of the TN-TM complex, too. The results obtained are in favour of a possible adequate transformation of fibril phenotypes of some (antigravitational) muscles under the effect of spaceflight.

Further data on the androgenic dependency of the skeletal musculature: the effect of prepubertal castration on the structural development of the skeletal muscles.

The effect of prepubertal castration was investigated in the soleus, semimembranosus and levator ani muscles of rat, using light and electronmicroscopic morphometric methods. After castration the semimembranosus and the levator ani muscles showed significant morphometrical alterations. The weight of the muscles diminished, the diameter of the white fibers decreased. The ultrastructure of these muscles showed a well expressed myofibrillar atrophy. The changes in the semimembranosus muscle were less severe. The slow oxidative soleus muscle did not show any similar alterations. The changes in the levator ani and semimembranosus muscles could be prevented or moderated by testosterone substitution.

Experimental studies on hypokinesis of skeletal muscle with different functions. VI.

In rabbits, the right hind limb was immobilized by means of plaster cast for 1, 2, 4 or 6 weeks and the activities of some metabolic enzymes: GOT, GPT, LDH, aldolase and acid phosphatase (in part of lysosomal origin), were examined in the slow m. soleus, and in the fast m. gastrocnemius. The former muscle is known to have mainly an oxidative, while the latter mainly a glycolytic type of metabolism. The activities of enzymes highly involved in the metabolism of the muscle diminished for a certain time during atrophy, then a relative rise occurred. Acid phosphatase activity likewise decreased after an initial relative increase. Reduction of enzymatic activities is explained by the activation of proteolytic enzymes, on the basis of measurements performed in these experiments and of results published by others. The decrease of enzymatic activity was more marked in the muscle which in normal state exhibits higher activity than in the other type of muscle studied. Thus, in the gastrocnemius a high rate of degradation of glycolytic enzymes was observed, while in the soleus degradation of oxidative enzymes prevailed. This phenomenon leads to the dedifferentiation of the muscle cell during immobilization.

Proteases and proteinase inhibitors in experimental glucocorticosteroid myopathy.

The unknown enzymatic mechanism of enhanced protein breakdown in steroid myopathy was studied in functionally and biochemically different muscles of rabbits treated with dexamethasone for three weeks. After glucocorticoid administration the fast-twitch glycolytic semimembraneous muscle of treated animals was atrophied, whereas the weight of the slow-twitch oxidative soleus muscle was not altered. The specific activity of the lysosomal endo- and exopeptidases (cathepsin D, E, B and L, lysosomal carboxypeptidase A and dipeptidylpeptidase I) was increased about 2-fold in the atrophied white muscle. The activity of the cytosol enzyme Ca++-activated neutral proteinase was also elevated, whereas that of the other cytosol endopeptidase, chymotrypsin-like enzyme, was unaltered. The level of alanine aminopeptidase was only slightly increased. On the other hand, there were no unequivocal changes in protease activity in the soleus muscle. These findings are in agreement with the known differences in glucocorticoid-sensitivity of the various muscles. Our results suggest that the lysosomal proteolytic system and the Ca++-activated neutral proteinase may play an important role in the glucocorticoid-induced intracellular protein catabolism in muscle. The inhibitor capacities of cathepsin B and trypsin detectable in muscle cytosol were not altered after steroid treatment. Consequently, the increase in cathepsin B activity was not due to the loss of its inhibitor.

Experimental investigations on the hypokinesis of skeletal muscles with different functions, V.

The composition of myofibrillar proteins was studied in the soleus and gastrocnemius muscles of rabbit hind limbs immobilized by plaster cast in experiments lasting 1--4 weeks. The amounts of actin, M protein and C protein increased relative to the normal composition. The ratio of the light chain peptides of the fast muscle myosin changed from 1 : 2 : 1 to 1 : 2 : 0.5 as a result of 4 weeks of disuse. The LC-1 : LC-2 ratio of slow myosin did not change considerably while the amount of fast LC-3 peptide, hardly detectable in soleus muscle, increased more than tenfold. The amount of tropomyosin decreased significantly in both muscles. The submolecular composition of troponin changed, mostly in the slow muscle; TN--C and TN--I decreased significantly, whereas there was an increase in the TN--T values. It is concluded that the phenotype of the structural proteins of muscles with different functions is de-differentiated by disuse, while the genetic functions of the muscle cells is reprogrammed to the synthesis of contractile proteins (e.g. myosin) characteristic of the other type of muscle.

The value of simple lipid stains for typing skeletal muscle fibres.

Skeletal muscle fibre types can be distinguished rapidly with simple lipid stains. Comparative studies showed that Sudan Black B is superior to Oil Red O for this purpose and that optimum staining is obtained using unfixed section or sections fixed in calcium-glutaraldehyde. Factors that possibly influence the staining reaction, such as freeze-thawing, are considered. The stained lipids were identified by thin layer chromatography.