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Histochemical demonstration of acetylphosphate phosphatase in normal and dystrophic human muscle
ARCHIVES
OF
BIOCHEMISTRY AND BIOPHYSICS 86, 109-114 (1959)
Histochemical Demonstration of Acetylphosphate Phosphatase in Normal and Dystrophic Human Muscle Geoffrey H. Boume and Nelly Golarz From the Department
of Anatomy, Received
Emory
February
University,
Atlanta,
Georgia
26, 1959
Lipmarm (3) demonstrated the presence of a very active acetylphosphatase in animal tissues, and this paper gives the results of a study, using histochemical techniques, of the distribution of this enzyme in normal and dystrophic human muscle. MATERIAL
AND
METHODS
Biopsy specimens of human muscle were obtained from ten patients of young ages. Two of these were between 30 and 40 years, one was 25 years, and the remaining seven were all under 15 years of age. All were suffering from progressive muscular dystrophy. Normal muscle was obtained from three healthy male medical students approximately 22 years of age. The latter, although they do not constitute an ideal control group, are reasonably close in age and provide criteria for comparison. It is of interest that no fundamental difference in distribution or intensity of the reaction could be seen with age in the dystrophic group. The biopsies (removed under local anesthesia) were obtained either from the flexor digitorium muscles or the gastrocnemius. No difference was observed in the reactions given by the muscles from these two sites. The samples of muscle were placed, immediately on removal, in ice-cold acetone and kept in an ice box for 24 hr. Following this they were placed in methyl bensoate until clear and then embedded in wax. Sections were cut 10 ~1thick and mounted upon glass slides. After drying for 24 hr. in a desiccator, they were brought to water in the conventional way and incubated for 18 hr. at 39” C. in the following solution. Substrate Mixture. 0.2 M tris(hydroxymethyl)aminomethane buffer pH 7.0 20 ml. 2% calcium chloride (6HzO) 20 ml. 10% magnesium chloride (6H20) 0.5 ml. Acetylphosphate (with no free phosphate) 20 mg. At the conclusion of this period the sections were washed and treated with cobalt chloride and ammonium sulfide to visualize the calcium phosphate deposited as a result of the enzyme hydrolysis of the acetylphosphate. RESULTS
The reaction is present in both muscle fibers and the supporting connective tissue. In normal muscle the fibers have a higher activity than the
FIG. 1. Acetylphosphatase in transverse section of normal muscle. Note the reaction is oriented to the same side of each muscle fiber. This is the effect of the fixative and suggests that the enzyme is in the soluble fraction of the muscle fiber. X 200 FIG. 2. Acetylphosphatase in dystrophic muscle. Note diffuse reaction in muscle fiber and more strongly positive connective tissue. X 250 FIG. 3. Acetylphosphatase in dystrophic muscle. Note positive muscle fiber with cross striations on left. Central degenerating fiber shows negative areas at each end of photo. A positive capillary appears to be within the fiber. Strongly positive connective tissue can be seen. X 375 FIG. 4. Acetylphosphatase in dystrophic muscle. Note negative muscle fibers and nositive connective tissue and blood vessels. X 375 110
ACETYLPHOSPHATE
PHOSPHATASE
111
connective tissue, and in most of the muscular dystrophy specimens the activity is higher in the connective tissue. A reaction is also present in the nuclei of both the muscle and the connective tissue cells. The most striking feature of the histochemical preparations is that the reaction is concentrated to one side of the individual muscle fiber. In most cases all the fibers in a particular section showed this reaction localized on the same side (Fig. 1). This is an artifact and is undoubtedly produced by the fixative; as it penetrates through the fibers it displaces the enzyme. A similar effect can be seen in many preparations of liver glycogen-the glycogen having been swept to one side of the hepatic cell by the fixative as it penetrates. This observation concerning acetylphosphatase is of special interest since it suggests that the enzyme is not incorporated in the fibrils or into any of the other formed particles; furthermore, in transverse sections of the fibers the reaction is obviously in the spaces between the fibrils (Fig. 8). However, in both normal and dystrophic muscle the reaction could be seen in some fibers (in longitudinal sections) to be localized in the regions of the A bands, and in some cases it was obviously associated with the sarcosomes (Fig. 9). In other fibers it appeared quite diffuse (Fig. 5). The most satisfactory interpretation of these facts is that the enzyme is present in the sarcoplasm, and the various localizations demonstrated are simply artifacts induced by the violent changes in the disposition of the sarcoplasm as a result of the penetration of the acetone fixative. That the enzyme is not attached to the structural components of the fiber is also suggested by Lipmann’s (4) observations that the acetylphosphatase present in animal tissues is very easily extracted in solution (see also Fig. 7). In addition to the reaction in the muscle fibers, the connective tissue of both normal and dystrophic muscle shows an appreciable reaction (Figs. Z-4). This appears to be present in both cells and the fibers. In addition, the walls of the small blood vessels give a strong positive reaction. Shapiro and Wertheimer (5) have shown no acetylphosphatase activity in the blood FIG. 5. Acetylphosphatase reaction in dystrophic muscle. Note strongly positive reaction in muscle fibers and in nuclear membranes of muscle nuclei. Mg ions in substrate mixture. X 200 FIG. 6. Acetylphosphatase reaction from adjacent section of same piece of muscle as Fig. 5. ?Jote virtually negative reaction by muscle fibers but positive muscle nuclei. No Mg ions in substrate mixture. X 250 FIG. 7. Note ragged end to acetylphosphatase reaction (A) indicating that it is localized in the sarcoplasm. X 1000 FIG. 8. Transverse section of muscle showing acetylphosphatase reaction in sarcoplasm around fibrils which are completely negative. X 800 FIG. 9. In one case of muscular dystrophy the acetylphosphatase reaction appeared to be localized largely in the sarcosomes-shown here. X 1840 FIG. 10. Section of blood clot. Acetylphosphatase appears to be associated with erythrocyte membranes. X800
112
BOURNE AND GOLARZ
serum, but it is of interest that in our preparation a strong positive reaction is associated with the membranes of the red blood cells (Fig. 10). In general the reaction in the connective tissue is stronger in dystrophic muscle than in normal. This is in keeping with other results obtained in this laboratory in which it has been found that many phosphatases are increased in activity or appear de novo in the peri- and endomysial tissue in muscular dystrophy. The muscle biopsies removed from the ten muscular dystrophy patients were not in the same stage. Seven of them showed a proliferation of connective tissue (endo- and perimysium) characteristic of the disease with little change in the muscle fiber. It was in this stage that the connective showed its most intense acetylphosphatase activity (Figs. 3 and 4). In three of the muscle biopsies there was considerable destruction of the muscle fibers and apparent invasion of the fibers by connective tissue. In these cases the intensity of the acetylphosphatase reaction was great,ly reduced in the muscle fibers (Fig. 3). Other phosphatases are increased under these circumstances. DISCUSSION
Very little work has been carried out on acetylphosphate or its enzymic hydrolysis since Lipmann’s review (4). At that time he claimed that the role of this substance in animal tissues was obscure although it apparently played an important part as an intermediary substance in bacterial metabolism. Acetylphosphate possesses a high-energy phosphate bond, but it has not been found to take the place of adenosine triphosphate (ATP) in reactions which use the energy provided by hydrolysis of the phosphate groups of the latter compound. Furthermore, the investigation of the role of acetylphosphate has been hindered by the widespread occurrence in animal tissues of this phosphatase which is the subject of the present paper. Lipmann himself stressed the intensity of the reaction of animal acetylphosphatase, and our preparations show a reaction more intense than with any phosphate ester so far used in our studies. It is the interest that Harary (1) and Grisolia et al. (2) have shown that acetylphosphatase attacks both 1,3-diphosphoglyceric acid and carbamyl phosphate. However, our studies have shown that when either of these two substrates is used, either no hydrolysis occurs under histochemical conditions, as with phosphoglyceric acid, or a fundamentally different histochemical distribution of the reaction occurs, as with carbamyl phosphate (e.g., in some muscle the reaction is confined to the sarcosomes). Perhaps some cofactor for acetylphosphatase is necessary for the hydrolysis of these two substrates, and this factor is destroyed by the fixation-embedding procedures used in making sections.
ACETYLPHOSPHATE PHOSPHATASE
113
The significance of the increased activity of the acetylphosphatase in connective tissue in muscular dystrophy is of interest but of obscure significance at the moment. Identity of the Enzyme Biochemical studies have demonstrated that the activity of acetylphosphatase is dependent upon the presence of Mg ions. All the preparations described above were made using traces of magnesium chloride in the reaction mixture (see Fig. 5). Preparations incubated without the magnesium chloride showed only a slight reaction in the muscle fibers, but the reaction in the nuclei of both muscle and connective tissue cells was much less diminished (see Fig. 6)-so one must consider the possibility that there are two (possibly related) acetylphosphatases in muscle-one Mg-dependent and the other not. The difference in reaction is not a pH effect since the sections were incubated at pH’s varying from 3.6 to 9.0, and the optimum reaction was obtained at a pH of 7.0 although a fairly strong reaction was also obtained at pH 6.0. This optimum was the same for the muscle fibers, the connective tissue, and the nuclei. It should also be noted that a number of other phosphatases are present in both the muscle fibers and the connective tissue, in particular of dystrophic muscle. A histochemical study of the enzymic hydrolysis of over 30 phosphate esters has been made in both normal and dystrophic muscle, and only acetylphosphate gives the results illustrated in this paper. We feel therefore that the results obtained in this work are due to the presence of a specific acetylphosphatase. ACKNOWLEDGMENTS This work was carried out under grants from the Muscular Dystrophy Associations of America Inc., and a U. S. Public Health Service Grant No. B 2038 from the Natl. Institutes of Health. Some of the apparatus used was donated by the National Foundation for Muscular Dystrophy. We are greatly indebted to Dr. H. Dale Richardson for performing the biopsies. SUMMARY
A phosphatase hydrolyzing acetylphosphate which is magnesium-sensitive and which has the optimum activity at pH 7.0 has been demonstrated histochemically in human muscle. It is concluded that this enzyme is localized in the sarcoplasmic part of the muscle fiber. It is present in the connective tissue as well as in muscular elements. The activity of the enzyme is increased in both sites in progressive muscular dystrophy. Muscle and connective tissue nuclei show acetylphosphatase activity,
114
BOURNE AND GOLARZ
and although this reaches its optimum at pH 7.0 it is not as sensitive to the absence of Mg as the sarcoplasmic enzyme. REFERENCES 1. 2. 3. 4. 5.
HARARY, GRISOLIA, LIPMANN, LIPMANN, SHAPIRO,
I., Biochim. et Biophys. Acta 26, 434 (1957). S., Biochim. et Biophys. Acta 29, 432 (1958). F., J. Biol. Chem. 140, Proc. (1940). F., Advances in Enzymol. 6, 231 (1946). S., AND WERTHEIMER, E., Nature 166, 690 (1945).
OF
BIOCHEMISTRY AND BIOPHYSICS 86, 109-114 (1959)
Histochemical Demonstration of Acetylphosphate Phosphatase in Normal and Dystrophic Human Muscle Geoffrey H. Boume and Nelly Golarz From the Department
of Anatomy, Received
Emory
February
University,
Atlanta,
Georgia
26, 1959
Lipmarm (3) demonstrated the presence of a very active acetylphosphatase in animal tissues, and this paper gives the results of a study, using histochemical techniques, of the distribution of this enzyme in normal and dystrophic human muscle. MATERIAL
AND
METHODS
Biopsy specimens of human muscle were obtained from ten patients of young ages. Two of these were between 30 and 40 years, one was 25 years, and the remaining seven were all under 15 years of age. All were suffering from progressive muscular dystrophy. Normal muscle was obtained from three healthy male medical students approximately 22 years of age. The latter, although they do not constitute an ideal control group, are reasonably close in age and provide criteria for comparison. It is of interest that no fundamental difference in distribution or intensity of the reaction could be seen with age in the dystrophic group. The biopsies (removed under local anesthesia) were obtained either from the flexor digitorium muscles or the gastrocnemius. No difference was observed in the reactions given by the muscles from these two sites. The samples of muscle were placed, immediately on removal, in ice-cold acetone and kept in an ice box for 24 hr. Following this they were placed in methyl bensoate until clear and then embedded in wax. Sections were cut 10 ~1thick and mounted upon glass slides. After drying for 24 hr. in a desiccator, they were brought to water in the conventional way and incubated for 18 hr. at 39” C. in the following solution. Substrate Mixture. 0.2 M tris(hydroxymethyl)aminomethane buffer pH 7.0 20 ml. 2% calcium chloride (6HzO) 20 ml. 10% magnesium chloride (6H20) 0.5 ml. Acetylphosphate (with no free phosphate) 20 mg. At the conclusion of this period the sections were washed and treated with cobalt chloride and ammonium sulfide to visualize the calcium phosphate deposited as a result of the enzyme hydrolysis of the acetylphosphate. RESULTS
The reaction is present in both muscle fibers and the supporting connective tissue. In normal muscle the fibers have a higher activity than the
FIG. 1. Acetylphosphatase in transverse section of normal muscle. Note the reaction is oriented to the same side of each muscle fiber. This is the effect of the fixative and suggests that the enzyme is in the soluble fraction of the muscle fiber. X 200 FIG. 2. Acetylphosphatase in dystrophic muscle. Note diffuse reaction in muscle fiber and more strongly positive connective tissue. X 250 FIG. 3. Acetylphosphatase in dystrophic muscle. Note positive muscle fiber with cross striations on left. Central degenerating fiber shows negative areas at each end of photo. A positive capillary appears to be within the fiber. Strongly positive connective tissue can be seen. X 375 FIG. 4. Acetylphosphatase in dystrophic muscle. Note negative muscle fibers and nositive connective tissue and blood vessels. X 375 110
ACETYLPHOSPHATE
PHOSPHATASE
111
connective tissue, and in most of the muscular dystrophy specimens the activity is higher in the connective tissue. A reaction is also present in the nuclei of both the muscle and the connective tissue cells. The most striking feature of the histochemical preparations is that the reaction is concentrated to one side of the individual muscle fiber. In most cases all the fibers in a particular section showed this reaction localized on the same side (Fig. 1). This is an artifact and is undoubtedly produced by the fixative; as it penetrates through the fibers it displaces the enzyme. A similar effect can be seen in many preparations of liver glycogen-the glycogen having been swept to one side of the hepatic cell by the fixative as it penetrates. This observation concerning acetylphosphatase is of special interest since it suggests that the enzyme is not incorporated in the fibrils or into any of the other formed particles; furthermore, in transverse sections of the fibers the reaction is obviously in the spaces between the fibrils (Fig. 8). However, in both normal and dystrophic muscle the reaction could be seen in some fibers (in longitudinal sections) to be localized in the regions of the A bands, and in some cases it was obviously associated with the sarcosomes (Fig. 9). In other fibers it appeared quite diffuse (Fig. 5). The most satisfactory interpretation of these facts is that the enzyme is present in the sarcoplasm, and the various localizations demonstrated are simply artifacts induced by the violent changes in the disposition of the sarcoplasm as a result of the penetration of the acetone fixative. That the enzyme is not attached to the structural components of the fiber is also suggested by Lipmann’s (4) observations that the acetylphosphatase present in animal tissues is very easily extracted in solution (see also Fig. 7). In addition to the reaction in the muscle fibers, the connective tissue of both normal and dystrophic muscle shows an appreciable reaction (Figs. Z-4). This appears to be present in both cells and the fibers. In addition, the walls of the small blood vessels give a strong positive reaction. Shapiro and Wertheimer (5) have shown no acetylphosphatase activity in the blood FIG. 5. Acetylphosphatase reaction in dystrophic muscle. Note strongly positive reaction in muscle fibers and in nuclear membranes of muscle nuclei. Mg ions in substrate mixture. X 200 FIG. 6. Acetylphosphatase reaction from adjacent section of same piece of muscle as Fig. 5. ?Jote virtually negative reaction by muscle fibers but positive muscle nuclei. No Mg ions in substrate mixture. X 250 FIG. 7. Note ragged end to acetylphosphatase reaction (A) indicating that it is localized in the sarcoplasm. X 1000 FIG. 8. Transverse section of muscle showing acetylphosphatase reaction in sarcoplasm around fibrils which are completely negative. X 800 FIG. 9. In one case of muscular dystrophy the acetylphosphatase reaction appeared to be localized largely in the sarcosomes-shown here. X 1840 FIG. 10. Section of blood clot. Acetylphosphatase appears to be associated with erythrocyte membranes. X800
112
BOURNE AND GOLARZ
serum, but it is of interest that in our preparation a strong positive reaction is associated with the membranes of the red blood cells (Fig. 10). In general the reaction in the connective tissue is stronger in dystrophic muscle than in normal. This is in keeping with other results obtained in this laboratory in which it has been found that many phosphatases are increased in activity or appear de novo in the peri- and endomysial tissue in muscular dystrophy. The muscle biopsies removed from the ten muscular dystrophy patients were not in the same stage. Seven of them showed a proliferation of connective tissue (endo- and perimysium) characteristic of the disease with little change in the muscle fiber. It was in this stage that the connective showed its most intense acetylphosphatase activity (Figs. 3 and 4). In three of the muscle biopsies there was considerable destruction of the muscle fibers and apparent invasion of the fibers by connective tissue. In these cases the intensity of the acetylphosphatase reaction was great,ly reduced in the muscle fibers (Fig. 3). Other phosphatases are increased under these circumstances. DISCUSSION
Very little work has been carried out on acetylphosphate or its enzymic hydrolysis since Lipmann’s review (4). At that time he claimed that the role of this substance in animal tissues was obscure although it apparently played an important part as an intermediary substance in bacterial metabolism. Acetylphosphate possesses a high-energy phosphate bond, but it has not been found to take the place of adenosine triphosphate (ATP) in reactions which use the energy provided by hydrolysis of the phosphate groups of the latter compound. Furthermore, the investigation of the role of acetylphosphate has been hindered by the widespread occurrence in animal tissues of this phosphatase which is the subject of the present paper. Lipmann himself stressed the intensity of the reaction of animal acetylphosphatase, and our preparations show a reaction more intense than with any phosphate ester so far used in our studies. It is the interest that Harary (1) and Grisolia et al. (2) have shown that acetylphosphatase attacks both 1,3-diphosphoglyceric acid and carbamyl phosphate. However, our studies have shown that when either of these two substrates is used, either no hydrolysis occurs under histochemical conditions, as with phosphoglyceric acid, or a fundamentally different histochemical distribution of the reaction occurs, as with carbamyl phosphate (e.g., in some muscle the reaction is confined to the sarcosomes). Perhaps some cofactor for acetylphosphatase is necessary for the hydrolysis of these two substrates, and this factor is destroyed by the fixation-embedding procedures used in making sections.
ACETYLPHOSPHATE PHOSPHATASE
113
The significance of the increased activity of the acetylphosphatase in connective tissue in muscular dystrophy is of interest but of obscure significance at the moment. Identity of the Enzyme Biochemical studies have demonstrated that the activity of acetylphosphatase is dependent upon the presence of Mg ions. All the preparations described above were made using traces of magnesium chloride in the reaction mixture (see Fig. 5). Preparations incubated without the magnesium chloride showed only a slight reaction in the muscle fibers, but the reaction in the nuclei of both muscle and connective tissue cells was much less diminished (see Fig. 6)-so one must consider the possibility that there are two (possibly related) acetylphosphatases in muscle-one Mg-dependent and the other not. The difference in reaction is not a pH effect since the sections were incubated at pH’s varying from 3.6 to 9.0, and the optimum reaction was obtained at a pH of 7.0 although a fairly strong reaction was also obtained at pH 6.0. This optimum was the same for the muscle fibers, the connective tissue, and the nuclei. It should also be noted that a number of other phosphatases are present in both the muscle fibers and the connective tissue, in particular of dystrophic muscle. A histochemical study of the enzymic hydrolysis of over 30 phosphate esters has been made in both normal and dystrophic muscle, and only acetylphosphate gives the results illustrated in this paper. We feel therefore that the results obtained in this work are due to the presence of a specific acetylphosphatase. ACKNOWLEDGMENTS This work was carried out under grants from the Muscular Dystrophy Associations of America Inc., and a U. S. Public Health Service Grant No. B 2038 from the Natl. Institutes of Health. Some of the apparatus used was donated by the National Foundation for Muscular Dystrophy. We are greatly indebted to Dr. H. Dale Richardson for performing the biopsies. SUMMARY
A phosphatase hydrolyzing acetylphosphate which is magnesium-sensitive and which has the optimum activity at pH 7.0 has been demonstrated histochemically in human muscle. It is concluded that this enzyme is localized in the sarcoplasmic part of the muscle fiber. It is present in the connective tissue as well as in muscular elements. The activity of the enzyme is increased in both sites in progressive muscular dystrophy. Muscle and connective tissue nuclei show acetylphosphatase activity,
114
BOURNE AND GOLARZ
and although this reaches its optimum at pH 7.0 it is not as sensitive to the absence of Mg as the sarcoplasmic enzyme. REFERENCES 1. 2. 3. 4. 5.
HARARY, GRISOLIA, LIPMANN, LIPMANN, SHAPIRO,
I., Biochim. et Biophys. Acta 26, 434 (1957). S., Biochim. et Biophys. Acta 29, 432 (1958). F., J. Biol. Chem. 140, Proc. (1940). F., Advances in Enzymol. 6, 231 (1946). S., AND WERTHEIMER, E., Nature 166, 690 (1945).