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Latency differences of lysosomal enzymes in cardiac and skeletal muscles of male and female mice
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LATENCY DIFFERENCES OF LYSOSOMAL IN CARDIAC AND SKELETAL MUSCLES AND FEMALE MICE A. SALMINEN, Muscle
Research
Laboratory,
K.
HXNNINEN
and V.
ENZYMES OF MALE
VIHKO
Department of Cell Biology, University of Jyvbskyll, 10, Finland. Telephone: 35841-291-211 (Receitwi
12
December
SF-40100
Jyvlskyll
1984)
Abstract-l. The purpose of this study was to compare the latencies of lysosomal enzymes (/I-glucuronidase. b-N-acetylglucosaminidase, arylsulphatase and acid ribonuclease) in heart and in red and white skeletal muscle of male and female mice (Mus musculus). The unsedimentable, free activities together with releasable (Triton X-100, hypotonic shock and freeze-thawing treatments) and unreleasable, bound activities were assayed. 2. The distribution of acid hydrolases to different fractions was strikingly heterogeneous. The most distinct differences occurred between the distributions of b-glucuronidase and /?-N-acetylglucosaminidase. 3. The differences between muscle types occurred in the activity levels of lysosomal enzymes, rather than in the fractional distributions. 4. Sex-related differences were small and occurred mainly in the activity levels of heart muscle (higher in female mice). 5. The results suggest that the heterogeneous distribution of IysosomaI enzymes originates in the compartmental differences of iysosomal enzymes in muscle cells, rather than the differences in cell populations of different muscle types.
INTRODUCTION
The specific activities of lysosomal enzymes are low in muscular tissues, especially in skeletal muscles, although particles showing latent acid hydrolase activities have been sedimented in skeletal muscles (Poliack and Bird, 1968; De Bernard and Stagni, 1970; Bird, 1975). Morphological studies have shown primary iysosomes in cardiac myocytes (Topping and Travis, 1974), but in skeletal muscle fibers iysosomes and autophagic vacuoles are infrequent (Pellegrino and Franzini, 1963; Bird, 1975; Salminen and Vihko, 1984). Histochemical studies, however, demonstrate the activity granules of several lysosomai enzymes in skeletal muscle fibers, especially in those of red oxidative type (Lojda and Gutmann, 1976; Vihko et ul., f978a). The cells of interstitial tissue contain lysosomes and lysosomal enzyme activities, which affect the results of biochemical studies. Canonico and Bird (1970) and Etherington and Wardaie (1982) have shown that the greatest part of iysosomai enzyme activities originate in skeletal muscle fibres. Relatively, the unsedimentable activities for lysosomai enzymes are high in muscle tissues, as compared, for example. to those of liver. This reflects the rupture of primary lysosomes and other lysosomal compartments during homogenization. The latency of iysosomai enzymes can also be released by detergents (such as Triton X-100), hypotonic shock and freeze-thawing (De Duve and Wattiaux, 1966). Interestingly, the results of studies on muscle tissues suggest that the latency properties of iysosomal enzymes are different (Poilack and Bird, 1968; De Bernard and Stagni, 1970; Arcangeii et al., 1973), which the specialized compartreflect may mentalization of the enzymes of lysosomal systems.
The purpose of the present study was to determine the iatencies of several iysosomai enzymes in different muscle tissues of male and female mice to verify the heterogenous compartmentalization of lysosomal enzymes in muscular tissues. MATERIALS
AND METHODS
Animals
Fjve-month-old
male and female NMRI
mice (Mus 111us-
cuius) were used for the experiments.
Mice lived under normal cage conditions, 68 mice to each cage (Scanbur Type IV, Denmark), with free access to tap water and solid food pellets (R,, Astra Ewos, Sweden). Temperature (2&22”C) and daylight rhythm (12 hr light/l2 hr dark) were controlled. The weight of male mice (N = 16) was 36.2 f0.4g (SE) and that of female mice (N = 16) 29.3 f 0.7 g (SE). The number of muscle samples in the experiments was eight, because the muscle samples from two animals were combined.
Mice were killed by cervical dislocation. The heart was first removed, opened and washed free from blood with ice-cold homogenization buffer. Only ventricles were studied. The skeletal muscle samples were excised from the hind legs. The red muscle sample was composed of the red proximal parts of quadriceps femoris muscle and of the deep red parts of gastrocnemius and tibialis anterior muscles. Soleus and plantaris muscles were also included in the red muscle sample. The white muscle sample was composed of the white distal parts of quadriceps femoris muscle and of the white superficial parts of ~strocnemius muscle. The heart sample from the male mice (two hearts combined) weighed 292 t_ 6mg (SE) and that of the female mice 230 + IOmg (SE). The red muscle samples weighed 395 k 17 mg (from male mice) and 433 k I3 mg (from female mice). Correspondingly, the weights of white samples
930 e-Glucuronidase 4.0.
Heart
4
Red
e-m-Acetylglucotaminidase Heart Red
White
White
Male 2 0.
Female
H FR B
H
F
R
B
Ii
F
k
B
H
Arylsulphatase
H
F
R
A
B
tl
F
A
B
Ii
F
R
B
R
8
Acid ribonuclease Red
Heart
t
F
B
Ii
F
White
R
B
HFRB
80
Heart
1
H
F
White
Red
R
B
Ii
F
R
B
Ii
F
Fig. I. Distribution of specific activities of lysosomal enzymes in heart and red and white skeletal muscles of male and female mice. Enzyme activities are given as nkat,!kg protein. Abbreviations: H (homogenate), F (free. unsedimentable fraction). R (releasable fraction). B (bound fraction). Values are means + SE.
were 504 k 32mg mice).
and 561 k 24mg
(from male and female
After preparation. the muscle samples were cut into small pieces with scissors and homogenized in ice-cold buffer (0.25 M sucrose, I mM EDTA and 1OmM TRIS;HCI. homogenized in a The samples were pH 7.2). Potter-Elvehjem homogenizer. with a Teflon pestle, by two sequences of 2-3 up-and-down strokes (670rpm). The homogenates of hearts were made up to 3”,, (w/v) and those of skeletal muscles up to 5”,, (w/v). A small portion of the homogenates was frozen and the total activities of the lysosomal enzymes were assayed in incubation mixtures containing 0.2”/, Triton X-100. The homogenates were weighed and centrifuged at 105,OOOg (5 C) for 60min with a swing-out rotor (MSE. Superspeed 50). The unsedimentable. free activities of lysosomal enzymes were assayed from the supernatant. The pellets were resuspended in the original weight of the homogenate with distilled water containing 0.2”,, Triton X-100 and thereafter the tubes were frozen and thawed three times and centrifuged at 105,OOOg for 60 min. These treatments (hypotonic shock, detergents and freeze-thawing) are known to release the latencies of the lysosomal enzymes (De Duve and Wattiaux, 1966). The activities of lysosomal enzymes in the supernatant fraction were referred to as the releasable activities. The pellets were resuspended in distilled water in the original weight of homogenate and the activities of lysosomal enzymes were referred to as the bound activities. All fractions and the homogenates were frozen and stored at -2o’C until analyzed.
The activities of /I-N-acetylglucosaminidase (EC 3.2.1.30), fl-glucuronidase (EC 3.2.1.31). acid ribonuclease (EC 3. I .27.5) and arylsulphatase (EC 3.1.6. I) together with the protein concentrations, were assayed from the homogenates and fractions. as described earlier (Barrett. 1972:
Vihko er ~1.. lY78b). The incubation times and the fraction volumes used in different assays were tested before the measurements.
Means and standard errors (SE) were calculated. The significance of the differences between the means were tested by Student’s f-test.
RESULTS
The distribution of specific enzyme activities to the different fractions varied greatly between various lysosomal acid hydrolases, as well as the activity levels in different muscle types (Fig. 1). The highest activities of /I-N-acetylglucosaminidase, specific /?-glucuronidase and arylsulphatase occurred in the releasable fractions, whereas the highest acid ribonuclease activities occurred in the free fractions. The specific activity of arylsulphatase was also high in the free fractions of every muscle type. The specific activities of fl-N-acetylglucosaminidase were very low in the free fraction, but considerably higher in the bound fraction of different muscle types. Instead, the specific activity of p-glucuronidase was very low in the bound fractions (Fig. 1). The differences between various muscle types occurred in the activity levels of lysosomal enzymes, rather than in the distribution to the different fractions (Fig. 1). The total activities of P-glucuronidase, fi-N-acetylglucosaminidase and arylsulphatase were higher in the red than in the white muscle. Correspondingly, the total activities of arylsulphatase and acid ribonuclease were higher in the heart than in the skeletal muscles. The differences in the total activities
931
Lysosomal latencies in muscle tissues 8.N-Acetylglucosaminidase Frsa
Rels*S.ble
Bound
%t
Female
HRW
HRW
H
RW
H
RW
tl
RW
H
RW
HRW
%
T
40
Female 20
HRW
HRW
H
RW
HRW
“RW
Fig, 2. Percentage distribution of lysosomal enzyme activities in free, releasable and bound fractions in heart and red and white skeletal muscles of male and female mice. Abbreviations: H (heart), R (red skeletal muscle), W (white skeletal muscle). Values are means k SE.
(in the homogenate) were also reflected as the higher specific activities in the different fractions (Fig. 1). Sex-related differences were small and occurred mainly in the activity levels (Fig. 1). Significant differences occurred in the specific activities of /I-N-acetylglucosaminidase, arylsulphatase and acid ribonuclease in the heart, where the activities were higher in the female than in the male mice. In the skeletal muscles the differences were insignificant. The protein concentration in the heart of male mice was 214 + 4 g/kg wet weight and that in female mice 203 k 3 g/kg wet weight. The difference is significant (P < 0.01). The protein concentration in the red muscle of male mice was 222 IfI 4 g/kg wet weight and that in the white muscle 214 + 6 g/kg wet weight. Sex-related differences were insignificant in skeletal muscles. The proportional distribution of protein to the free, releasable and bound fractions was in the male heart 21.2kO.3, 28.7kO.7 and 50.1 &0.7x, respectively. Sex-related differences did not occur. The corresponding distribution of protein in the red muscles of male mice was 21.3 f 0.4, 21.5 + 0.5 and 57.2 k 0.6% and in the white muscle 23.4 f I .l, 21.4 k 0.5 and 55.2 f 1.4%. respectively. Sex-related differences were insignificant. The proportional distribution (per cent) of the lysosomal enzyme activities to different fractions did not change the differences between various lysosomal enzymes, although the proportional values in the
bound fraction increases due to the high protein level (Fig. 2). For instance, 55.4 f 1.5% of the total /?-N-acetylglucosaminidase activity occurred in the bound fraction and only 3.0 f 0.4% in the free fraction in the red skeletal muscle of the male mice. The relation of free activity to releasable activity, suggesting the location of acid hydrolases in fragile structures, showed that the highest proportions occurred in the activities of acid ribonuclease and arylsulphatase and the lowest in the activities of /?-N-acetylglucosaminidase in every muscle type (Fig. 2). The differences in the proportional distribution of lysosomal enzyme activities were few between different muscle types, as well as between male and female mice (Fig. 2). In the white muscle the percentage proportion of /?-glucuronidase activity, but not the activities of other lysosomal enzymes, was much higher in the free fraction and correspondingly lower in the releasable fraction, than in the red muscle or in the heart. The percentage recoveries of proteins were 100 + 3 (white muscle), I08 _t 3 (red muscle) and I 14 f 2 (heart muscle) in male mice. No differences occurred between male and female mice. The recoveries of arylsulphatase, /J’-N-acetylglucosaminidase and ribonuclease activities were significantly lower in heart (86-94%) than in skeletal muscles (106129%). The recovery of fi-glucuronidase activity was 91 i: 4 for
932
A. SALMINEN et al.
red muscle and I 17 k 4 for heart and 120 f 5 for white muscles of male mice. Sex-related differences in the recoveries of enzyme activities did not occur. DISCUSSION
The lysosomal enzymes of muscular tissues showed a strikingly heterogeneous pattern of latency properties. Acid ribonuclease showed the highest percentage distribution in the unsedimentable, free fraction, while only a slight part of b-N-acetylglucosaminidase occurred in this fraction. The releasable fraction contained approx. 40”/, of the total activities, although the releasable proportions of b-glucuronidase hand. other were over 607;. On the fl-N-acetylglucosaminidase showed the highest percentage distribution in the unreleasable, bound fraction. The heterogeneous distribution could be due, for example, to the different distributions of lysosomal enzymes between muscular cell populations, differences in the compartmentalization of lysosomal enzymes in muscle fibres, or coprecipitation of lysosomal enzymes, e.g. with myosin molecules. The considerably different interstitial cell populations in heart and skeletal muscles, as compared to considerably uniform distribution of lysosomal enzymes in different latency fractions, suggest that the latency patterns observed are typical for muscle cells and that the majority of lysosomal enzymes originate in muscle cells. The compartmentalization of lysosomal enzymes in skeletal muscle fibers, as well as in cardiomyocytes, is still largely unknown. The results of Stauber and Bird (1974) and Weglicki et al. (1975) suggest that a considerable proportion of the lysosomal enzymes is linked to the vesicles of sarcoplasmic reticulum. A part of this linkage could be mediated by specialized receptors, as observed in the microsomes of several tissues (Sly and Fischer, 1982; Shepherd er al., 1983). We have recently observed the presence of the phosphomannosyl-enzyme receptors of lysosomal enzymes in the membranes of skeletal muscle (Salminen, 1984). The occupancy of these receptors with endogenous enzymes increases during the repair of necrotic exercise injuries. In the present study, a part of receptor-bound enzymes could be distributed in the releasable fraction, because Triton X-100 releases the receptors from the membranes (Sahagian et cd., 1982). However, it is very likely that the greater part of lysosomal enzymes is located inside the membranelimited lysosomes, without the receptors. The heterogeneous distribution of lysosomal enzymes could reflect the morphological variety and varying enzyme contents of lysosomal particles, both in skeletal muscle fibers and in cardiomyocytes. Koenig and his collaborators (1980, 1982) have shown that the activities of lysosomal enzymes are higher in cardiac and skeletal muscles of male than female mice. They also showed that this sex-related difference was due to the androgen regulation. The results of the present study do not verify those of Koenig et al. (1980 and 1982). By contrast, the aryla-N-acetylglucosaminidase, activities of sulphatase and acid ribonuclease were higher in the cardiac muscles of female mice than those of male ones. The sex-related differences did not occur in
skeletal muscles. The discrepancy in the results could be due to the differences, e.g. in the strain of mice, in the age of mice. or in other experimental conditions, such as in cage conditions and in the composition of food pellets. Ac,knoM,lrdg~ments--This study was supported by the Academy of Finland and the Research Council Education and Sport (Ministry of Education.
for Phvsical Finlanb).
REFERENCES Arcangeli P., Del Soldato P.. Digiesi V. and Melani F. (1973) Changes in the activities of lysosomal enzymes in striated muscle following ischemia. Lifr Sri. 12, 13-23. Barrett A. J. (1972) Lysosomal enzymes. In Llsosome.v. u Laboratory Handbook (Edited dy Dingle j. T.). pp. 46126. North-Holland Pub.. Amsterdam. Bird J. W. C. (1975) Skeletal muscle Ivsosomes. In Lysosomes in Biology and Patho1og.v (Edited by Dingle J. T. and Dean R. T.). Vol. 4. DD. 75-109. Elsevier/North-Holland, Amsterdam. Canonico P. G. and Bird J. W. C. (1970) Lysosomes in skeletal muscle tissue. Zonal centrifugation evidence for multiple cellular sources. J. Cell Biol. 45, 321-333. De Bernard B. and Stagni N. (1970) Lysosomes in muscle tissues. In Muscle Diseases (Edited by Walton J. N.. Canal N. and Scarlato G.). pp. 212-221. Excerpta Medica, Amsterdam. De Duve C., Wattiaux R. (1966) Functions of lysosomes. A. Rev. Physiol. 28, 435492. Etherington D. J. and Wardale R. J. (1982) The mononuclear cell population in rat leg muscle: its contribution to the lysosomal enzyme activities of whole muscle extracts. J. Cell Sci. 58, 139-148. Koenig H., Goldstone A. and Lu C. Y. (1980) Androgens regulate mitochondrial cytochrome C oxidase and lysosomal hydrolases in mouse skeletal muscle. Biochem. J. 192, 349-353. Koenig H., Goldstone A. and Lu C. Y. (1982) Testosteronemediated sexual dimorphism of the rodent heart. Ventricular lysosomes, mitochondria and cell growth are modulated by androgens. Circula!ion Res. 50, 782-787. Lojda 2. and Gutmann E. (1976) Histochemistry of some acid hydrolases in striated muscles of the rat. Histoc,hemistry 49, 337-342. Pellegrino C. and Franzini C. (1963) An electron microscope study of denervation atrophy in red and white skeletal muscle fibers. J. Cell Biol. 17, 327--349. Pollack M. S. and Bird J. W. C. (1968) Distribution and particle properties of acid hydrolase in denervated muscle. Am. J. Phpsiol. 215, 71&722. Sahagian G. G., Distler J. J. and Jourdian G. W. (1982) Membrane receptor for phosphomannosyl residues. In Methods in En:ymoiog_v (Edited by Colowick S. P. and Kaplan N. O.), Vol. 83. pp. 392-396. Academic Press. New York. Salminen A. (1984) Latencies and phosphomannosylenzyme receptors of lysosomal enzymes during the appearance and reDair of exercise injuries in mouse skeletal muscles. E.up. molec. Puthol. 41, b09-418. Salminen A. and Vihko V. (1984) Autouhagic resuonse _. to strenuous exercise in mouse skeletal muscle fibers. Virchobvs Arch. (Cell. Pathol.) 45, 97-106. Shepherd V., Schlesinger P., Stahl P. (1983) Receptors for lysosomal enzymes and glycoproteins. In Membrane Receptors (Edited by Kleinzeller A.), .pp. . 3 I7- 338. Academic Press, New York. Sly W. S. and Fischer H. D. (1982) The phosphomannosyl recognition system for intracellular and intercellular transport of lysosomal enzymes. J. cell. Biochem. 18, 67-85. Stauber W. T. and Bird J. W. C. (1974) S-p Zonal fraction-
Lysosomal
latencies
ation studies of rat skeletal muscle lysosome-rich fractions. Biochim. biophys. Acta 338, 234-245. Topping T. M. and Travis D. F. (1974) An electron cytochemical study of the mechanisms of lysosomal activity in the rat left ventricular mural myocardium. J. ultrastruct. Res. 46, 1-22. Vihko V., Rantamaki .I., Salminen A. (1978a) Exhaustive physical exercise and acid hydrolase activity in skeletal muscle. A histochemical study. Histochemistry 57, 237-249.
in muscle
tissues
933
Vihko V., Salminen A., Rantamaki J. (1978b) Acid hydrolase activity in red and white skeletal muscle of mice during a two-week period following exhausting exercise. Pfliigers Arch. Eur. J. Physiol. 378, 99-106. Weglicki W. B., Ruth R. C., Gottwik M. G., McNamara D. B. and Owens K. (1975) Lysosomes of cardiac and skeletal muscle: resolution by zonal centrifugation. In The Cardiac Sarcoplasm (Edited by Roy P.-M. and Harris P.). pp. 503-517. University Park Press, Baltimore.
LATENCY DIFFERENCES OF LYSOSOMAL IN CARDIAC AND SKELETAL MUSCLES AND FEMALE MICE A. SALMINEN, Muscle
Research
Laboratory,
K.
HXNNINEN
and V.
ENZYMES OF MALE
VIHKO
Department of Cell Biology, University of Jyvbskyll, 10, Finland. Telephone: 35841-291-211 (Receitwi
12
December
SF-40100
Jyvlskyll
1984)
Abstract-l. The purpose of this study was to compare the latencies of lysosomal enzymes (/I-glucuronidase. b-N-acetylglucosaminidase, arylsulphatase and acid ribonuclease) in heart and in red and white skeletal muscle of male and female mice (Mus musculus). The unsedimentable, free activities together with releasable (Triton X-100, hypotonic shock and freeze-thawing treatments) and unreleasable, bound activities were assayed. 2. The distribution of acid hydrolases to different fractions was strikingly heterogeneous. The most distinct differences occurred between the distributions of b-glucuronidase and /?-N-acetylglucosaminidase. 3. The differences between muscle types occurred in the activity levels of lysosomal enzymes, rather than in the fractional distributions. 4. Sex-related differences were small and occurred mainly in the activity levels of heart muscle (higher in female mice). 5. The results suggest that the heterogeneous distribution of IysosomaI enzymes originates in the compartmental differences of iysosomal enzymes in muscle cells, rather than the differences in cell populations of different muscle types.
INTRODUCTION
The specific activities of lysosomal enzymes are low in muscular tissues, especially in skeletal muscles, although particles showing latent acid hydrolase activities have been sedimented in skeletal muscles (Poliack and Bird, 1968; De Bernard and Stagni, 1970; Bird, 1975). Morphological studies have shown primary iysosomes in cardiac myocytes (Topping and Travis, 1974), but in skeletal muscle fibers iysosomes and autophagic vacuoles are infrequent (Pellegrino and Franzini, 1963; Bird, 1975; Salminen and Vihko, 1984). Histochemical studies, however, demonstrate the activity granules of several lysosomai enzymes in skeletal muscle fibers, especially in those of red oxidative type (Lojda and Gutmann, 1976; Vihko et ul., f978a). The cells of interstitial tissue contain lysosomes and lysosomal enzyme activities, which affect the results of biochemical studies. Canonico and Bird (1970) and Etherington and Wardaie (1982) have shown that the greatest part of iysosomai enzyme activities originate in skeletal muscle fibres. Relatively, the unsedimentable activities for lysosomai enzymes are high in muscle tissues, as compared, for example. to those of liver. This reflects the rupture of primary lysosomes and other lysosomal compartments during homogenization. The latency of iysosomai enzymes can also be released by detergents (such as Triton X-100), hypotonic shock and freeze-thawing (De Duve and Wattiaux, 1966). Interestingly, the results of studies on muscle tissues suggest that the latency properties of iysosomal enzymes are different (Poilack and Bird, 1968; De Bernard and Stagni, 1970; Arcangeii et al., 1973), which the specialized compartreflect may mentalization of the enzymes of lysosomal systems.
The purpose of the present study was to determine the iatencies of several iysosomai enzymes in different muscle tissues of male and female mice to verify the heterogenous compartmentalization of lysosomal enzymes in muscular tissues. MATERIALS
AND METHODS
Animals
Fjve-month-old
male and female NMRI
mice (Mus 111us-
cuius) were used for the experiments.
Mice lived under normal cage conditions, 68 mice to each cage (Scanbur Type IV, Denmark), with free access to tap water and solid food pellets (R,, Astra Ewos, Sweden). Temperature (2&22”C) and daylight rhythm (12 hr light/l2 hr dark) were controlled. The weight of male mice (N = 16) was 36.2 f0.4g (SE) and that of female mice (N = 16) 29.3 f 0.7 g (SE). The number of muscle samples in the experiments was eight, because the muscle samples from two animals were combined.
Mice were killed by cervical dislocation. The heart was first removed, opened and washed free from blood with ice-cold homogenization buffer. Only ventricles were studied. The skeletal muscle samples were excised from the hind legs. The red muscle sample was composed of the red proximal parts of quadriceps femoris muscle and of the deep red parts of gastrocnemius and tibialis anterior muscles. Soleus and plantaris muscles were also included in the red muscle sample. The white muscle sample was composed of the white distal parts of quadriceps femoris muscle and of the white superficial parts of ~strocnemius muscle. The heart sample from the male mice (two hearts combined) weighed 292 t_ 6mg (SE) and that of the female mice 230 + IOmg (SE). The red muscle samples weighed 395 k 17 mg (from male mice) and 433 k I3 mg (from female mice). Correspondingly, the weights of white samples
930 e-Glucuronidase 4.0.
Heart
4
Red
e-m-Acetylglucotaminidase Heart Red
White
White
Male 2 0.
Female
H FR B
H
F
R
B
Ii
F
k
B
H
Arylsulphatase
H
F
R
A
B
tl
F
A
B
Ii
F
R
B
R
8
Acid ribonuclease Red
Heart
t
F
B
Ii
F
White
R
B
HFRB
80
Heart
1
H
F
White
Red
R
B
Ii
F
R
B
Ii
F
Fig. I. Distribution of specific activities of lysosomal enzymes in heart and red and white skeletal muscles of male and female mice. Enzyme activities are given as nkat,!kg protein. Abbreviations: H (homogenate), F (free. unsedimentable fraction). R (releasable fraction). B (bound fraction). Values are means + SE.
were 504 k 32mg mice).
and 561 k 24mg
(from male and female
After preparation. the muscle samples were cut into small pieces with scissors and homogenized in ice-cold buffer (0.25 M sucrose, I mM EDTA and 1OmM TRIS;HCI. homogenized in a The samples were pH 7.2). Potter-Elvehjem homogenizer. with a Teflon pestle, by two sequences of 2-3 up-and-down strokes (670rpm). The homogenates of hearts were made up to 3”,, (w/v) and those of skeletal muscles up to 5”,, (w/v). A small portion of the homogenates was frozen and the total activities of the lysosomal enzymes were assayed in incubation mixtures containing 0.2”/, Triton X-100. The homogenates were weighed and centrifuged at 105,OOOg (5 C) for 60min with a swing-out rotor (MSE. Superspeed 50). The unsedimentable. free activities of lysosomal enzymes were assayed from the supernatant. The pellets were resuspended in the original weight of the homogenate with distilled water containing 0.2”,, Triton X-100 and thereafter the tubes were frozen and thawed three times and centrifuged at 105,OOOg for 60 min. These treatments (hypotonic shock, detergents and freeze-thawing) are known to release the latencies of the lysosomal enzymes (De Duve and Wattiaux, 1966). The activities of lysosomal enzymes in the supernatant fraction were referred to as the releasable activities. The pellets were resuspended in distilled water in the original weight of homogenate and the activities of lysosomal enzymes were referred to as the bound activities. All fractions and the homogenates were frozen and stored at -2o’C until analyzed.
The activities of /I-N-acetylglucosaminidase (EC 3.2.1.30), fl-glucuronidase (EC 3.2.1.31). acid ribonuclease (EC 3. I .27.5) and arylsulphatase (EC 3.1.6. I) together with the protein concentrations, were assayed from the homogenates and fractions. as described earlier (Barrett. 1972:
Vihko er ~1.. lY78b). The incubation times and the fraction volumes used in different assays were tested before the measurements.
Means and standard errors (SE) were calculated. The significance of the differences between the means were tested by Student’s f-test.
RESULTS
The distribution of specific enzyme activities to the different fractions varied greatly between various lysosomal acid hydrolases, as well as the activity levels in different muscle types (Fig. 1). The highest activities of /I-N-acetylglucosaminidase, specific /?-glucuronidase and arylsulphatase occurred in the releasable fractions, whereas the highest acid ribonuclease activities occurred in the free fractions. The specific activity of arylsulphatase was also high in the free fractions of every muscle type. The specific activities of fl-N-acetylglucosaminidase were very low in the free fraction, but considerably higher in the bound fraction of different muscle types. Instead, the specific activity of p-glucuronidase was very low in the bound fractions (Fig. 1). The differences between various muscle types occurred in the activity levels of lysosomal enzymes, rather than in the distribution to the different fractions (Fig. 1). The total activities of P-glucuronidase, fi-N-acetylglucosaminidase and arylsulphatase were higher in the red than in the white muscle. Correspondingly, the total activities of arylsulphatase and acid ribonuclease were higher in the heart than in the skeletal muscles. The differences in the total activities
931
Lysosomal latencies in muscle tissues 8.N-Acetylglucosaminidase Frsa
Rels*S.ble
Bound
%t
Female
HRW
HRW
H
RW
H
RW
tl
RW
H
RW
HRW
%
T
40
Female 20
HRW
HRW
H
RW
HRW
“RW
Fig, 2. Percentage distribution of lysosomal enzyme activities in free, releasable and bound fractions in heart and red and white skeletal muscles of male and female mice. Abbreviations: H (heart), R (red skeletal muscle), W (white skeletal muscle). Values are means k SE.
(in the homogenate) were also reflected as the higher specific activities in the different fractions (Fig. 1). Sex-related differences were small and occurred mainly in the activity levels (Fig. 1). Significant differences occurred in the specific activities of /I-N-acetylglucosaminidase, arylsulphatase and acid ribonuclease in the heart, where the activities were higher in the female than in the male mice. In the skeletal muscles the differences were insignificant. The protein concentration in the heart of male mice was 214 + 4 g/kg wet weight and that in female mice 203 k 3 g/kg wet weight. The difference is significant (P < 0.01). The protein concentration in the red muscle of male mice was 222 IfI 4 g/kg wet weight and that in the white muscle 214 + 6 g/kg wet weight. Sex-related differences were insignificant in skeletal muscles. The proportional distribution of protein to the free, releasable and bound fractions was in the male heart 21.2kO.3, 28.7kO.7 and 50.1 &0.7x, respectively. Sex-related differences did not occur. The corresponding distribution of protein in the red muscles of male mice was 21.3 f 0.4, 21.5 + 0.5 and 57.2 k 0.6% and in the white muscle 23.4 f I .l, 21.4 k 0.5 and 55.2 f 1.4%. respectively. Sex-related differences were insignificant. The proportional distribution (per cent) of the lysosomal enzyme activities to different fractions did not change the differences between various lysosomal enzymes, although the proportional values in the
bound fraction increases due to the high protein level (Fig. 2). For instance, 55.4 f 1.5% of the total /?-N-acetylglucosaminidase activity occurred in the bound fraction and only 3.0 f 0.4% in the free fraction in the red skeletal muscle of the male mice. The relation of free activity to releasable activity, suggesting the location of acid hydrolases in fragile structures, showed that the highest proportions occurred in the activities of acid ribonuclease and arylsulphatase and the lowest in the activities of /?-N-acetylglucosaminidase in every muscle type (Fig. 2). The differences in the proportional distribution of lysosomal enzyme activities were few between different muscle types, as well as between male and female mice (Fig. 2). In the white muscle the percentage proportion of /?-glucuronidase activity, but not the activities of other lysosomal enzymes, was much higher in the free fraction and correspondingly lower in the releasable fraction, than in the red muscle or in the heart. The percentage recoveries of proteins were 100 + 3 (white muscle), I08 _t 3 (red muscle) and I 14 f 2 (heart muscle) in male mice. No differences occurred between male and female mice. The recoveries of arylsulphatase, /J’-N-acetylglucosaminidase and ribonuclease activities were significantly lower in heart (86-94%) than in skeletal muscles (106129%). The recovery of fi-glucuronidase activity was 91 i: 4 for
932
A. SALMINEN et al.
red muscle and I 17 k 4 for heart and 120 f 5 for white muscles of male mice. Sex-related differences in the recoveries of enzyme activities did not occur. DISCUSSION
The lysosomal enzymes of muscular tissues showed a strikingly heterogeneous pattern of latency properties. Acid ribonuclease showed the highest percentage distribution in the unsedimentable, free fraction, while only a slight part of b-N-acetylglucosaminidase occurred in this fraction. The releasable fraction contained approx. 40”/, of the total activities, although the releasable proportions of b-glucuronidase hand. other were over 607;. On the fl-N-acetylglucosaminidase showed the highest percentage distribution in the unreleasable, bound fraction. The heterogeneous distribution could be due, for example, to the different distributions of lysosomal enzymes between muscular cell populations, differences in the compartmentalization of lysosomal enzymes in muscle fibres, or coprecipitation of lysosomal enzymes, e.g. with myosin molecules. The considerably different interstitial cell populations in heart and skeletal muscles, as compared to considerably uniform distribution of lysosomal enzymes in different latency fractions, suggest that the latency patterns observed are typical for muscle cells and that the majority of lysosomal enzymes originate in muscle cells. The compartmentalization of lysosomal enzymes in skeletal muscle fibers, as well as in cardiomyocytes, is still largely unknown. The results of Stauber and Bird (1974) and Weglicki et al. (1975) suggest that a considerable proportion of the lysosomal enzymes is linked to the vesicles of sarcoplasmic reticulum. A part of this linkage could be mediated by specialized receptors, as observed in the microsomes of several tissues (Sly and Fischer, 1982; Shepherd er al., 1983). We have recently observed the presence of the phosphomannosyl-enzyme receptors of lysosomal enzymes in the membranes of skeletal muscle (Salminen, 1984). The occupancy of these receptors with endogenous enzymes increases during the repair of necrotic exercise injuries. In the present study, a part of receptor-bound enzymes could be distributed in the releasable fraction, because Triton X-100 releases the receptors from the membranes (Sahagian et cd., 1982). However, it is very likely that the greater part of lysosomal enzymes is located inside the membranelimited lysosomes, without the receptors. The heterogeneous distribution of lysosomal enzymes could reflect the morphological variety and varying enzyme contents of lysosomal particles, both in skeletal muscle fibers and in cardiomyocytes. Koenig and his collaborators (1980, 1982) have shown that the activities of lysosomal enzymes are higher in cardiac and skeletal muscles of male than female mice. They also showed that this sex-related difference was due to the androgen regulation. The results of the present study do not verify those of Koenig et al. (1980 and 1982). By contrast, the aryla-N-acetylglucosaminidase, activities of sulphatase and acid ribonuclease were higher in the cardiac muscles of female mice than those of male ones. The sex-related differences did not occur in
skeletal muscles. The discrepancy in the results could be due to the differences, e.g. in the strain of mice, in the age of mice. or in other experimental conditions, such as in cage conditions and in the composition of food pellets. Ac,knoM,lrdg~ments--This study was supported by the Academy of Finland and the Research Council Education and Sport (Ministry of Education.
for Phvsical Finlanb).
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