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The localization of enzyme activity in the electric organ of Electrophorus electricus (L)
Experimental
1
Cell Research 30, l-7 (1963)
THE LOCALIZATION OF ENZYME ACTIVITY IN THE ELECTRIC ORGAN OF ELECZ’ROPHORUS ELECTRICUS (L) A. G. E. PEARSE’ Institute
de Biojsica,
and DARCY Universidade
F. DE ALMEIDAe
do Bras&
Rio de Janeiro,
Brazil
Received June 12, 1962
CONDUCTINGcells integrate a variety of vital processes for which the utihzation of chemical energy is of fundamental importance. The innervated portions of these cells constitute the trigger system for the conductance changes underlying physiological activity. Bioelectric currents represent the result of such changes in the electric organs of electric fishes. The very high degree of specialization of these effecters in Electrophorus electricus is indicated not only by the magnitude of the resulting electrical phenomena, but also by the anatomical and morphological characteristics of the electroplate. Such properties made possible the isolation of a single electroplate from the electric organ [13] and, more recently, an attempt has been made to separate the active “posterior half” of the electroplate [l]. It was felt that progress in this held required a topographical knowledge of the biochemical properties of the electric organ in order to establish a correlation between its structure and function. The present paper describes investigations on the specific distribution of the enzymes involved in providing the chemical energy necessary for the production of the electrical discharge.
MATERIAL
AND
METHODS’
Small fragments were taken from the Sachs organ of E. electricus, mounted in a few drops of water on a microtome chuck and immediately frozen with solid CO,. Four to 12 p sections were cut on a cold microtome at -20X, mounted on coverslips and dried in air. For the demonstration of phosphorylase, thicker sections (up to 24 ,a) were used. For the demonstration of the various dehydrogenases, the following stock solution r British Council Visiting Lecturer. Present address: Department of Pathology, Postgraduate Medical School, London, England. 2 Research Fellow of the Conselho National de Pesquisas do Brasil. 3 The following abbreviations are used: MTT, 3(4.5-dimethylthiazolyl-2)-2.5-diphenyl tetrazolium bromide; Tris, tris (hydroxymethyl)aminomethane; Vit. I<, (menadione), 2-methyl-1:4naphthoquinone; DPN, diphosphopyridine nucleotide (Coenzyme I), and TPN, triphosphopyridine nucleotide (Coenzyme II). 1 - 631809
Experimental
Cell Research 30
2
A. G. E. Pearse and Darcy F. de Almeida
was prepared: 2.5 ml MTT (1 mg/ml); 2.5 ml 0.2 M Tris buffer, pH 7.4; 0.5 ml 0.5 &f cobalt nitrate; and 3.5 ml distilled water. This solution was stored frozen at - 30°C. Shortly before use, the substrate incubating solutions were prepared. The following enzymes were studied: DPNH diaphorase [ 121; DPN-linked malate, lactate and a-glycerophosphate dehydrogenases 171; mitochondrial a-glycerophosphate dehydrogenase [S]; succinate dehydrogenase [ll]; TPN-linked malate and glucose-6-phosphate dehydrogenases [ 71. Cytochrome oxidase activity was shown by the method of Burstone [2]. The addition of cytochrome c (5 x IO-6 M) was found necessary for the reaction to take place. For the demonstration of succinate and a-glycerophosphate dehydrogenases, the incubating solution was saturated with 2-methyl-l,4-naphthoquinone (vitamin K,, menadione). The method of Takeuchi [14, 151 was applied for the demonstration of phosphorylase. Incubation was carried out at room temperature, for 15 to 30 min. In the case of phosphorylase, incubation times were as high as 120 min. Subsequently the sections were rinsed in distilled water, fixed for a few minutes in formalin-saline, washed in water and mounted in glycerine jelly. RESULTS
All the enzymes studied were found to be localized exclusively in the electroplate. The material outside the plate presented no activity, except for a few scattered cells in the ground substance of the electrogenic unit. Thin sections provided excellent fine localization of enzymes, as illustrated in Fig. 2, but somewhat thicker sections (12 to 24 ,u) were needed for a general view of their topographical distribution inside the electroplate. DPN diaphorase and succinate dehydrogenase were strongly active (Figs. 1 and 2), and a very high activity of malate (Figs. 3 and 4) and lactate (Fig. 5) dehydrogenases was also present. Figs. 1, 2, 3 and 5 show that activity of the various enzymes is limited to the papillae of the electroplate syncytium, where the nuclei and mitochondria are concentrated. The mitochondria are always situated around the nuclei [5], a fact which explains the perinuclear distribution of the formazan pigment (Figs. 1 and 4). Both n-glycerophosphate dehydrogenases were less active than the preceding enzymes; their arrangement in the electroplate was essentially similar, however (Fig. 6). Cytochrome oxidase, on the other hand, was found to be highly active (Fig. 7), but only after the addition of cytochrome c to the incubating solution. Rather similar patterns of distribution were thus found for all the oxidative enzymes which we studied. Their activity was restricted to the papillae of the electroplate, being in every case much higher in the posterior (innervExperimental
Cell Research 30
Localizaiion
of enzyme activity
Fig. l.-DPNH diaphorase. The reaction is very strong in the papillae of the posterior surfa ce. Weaker activity in the anterior papillae. 12 y section. x 345. Fig. 2.-Succinate dehydrogenase, (with added Vitamin K,) 4 /J section. The final product of enzymic activity is shown as formazan dots concentrated in the posterior surface of the elect roplate (to the right). x 390. Fig. 3.-DPN-linked malate dehydrogenase. General view of the distribution of the enzyme in the electroplate of Sachs organ. Fig. 4.-DPN-linked malate dehydrogenase. Detail of the activity in the papillae of the elect roplate. Enzyme highly active at the mitochondrial sites near the nuclei, which are unreacti ve. x 300. Experimental
Cell Research 30
A. G. E. Pearse and Marcy F. de Almeidu
Fig. S.-Lactate dehydrogenasc. tion (above). x 180.
The enzyme is most active in the papillae
Fig. 6.-Mitochondrial cc-glycerophosphate dehydrogenase. of distribution as the other dehydrogenases. x 220. Fig. 7.-Cytochrome of the electroplate;
following
por-
the same pattern
oxidase. Activity apparent only near the anterior and posterior surfaces the posterior membrane (to the right) shows stronger activity. x 230.
Fig. X.-Phosphorylase. of the anterior papillae x 35. Experimental
Activity
of the posterior
24 y section. Enzyme activity is sharply localized of the electroplate (to the left). The posterior portion
Cell Research 30
in the inner portion (right) is unreactive.
Localization of enzyme activity
5
ated) portion. Whether in the anterior or the posterior portion, most of the activity was localized in the perinuclear region of the cytoplasm, outlining clearly the unreactive nuclei (Figs. 1 and 4). The small dot-like deposits of the final reaction product were sharply limited to narrow strip of cytoplasm near the anterior and posterior surfaces of the electroplate. The innermost part of the electroplate was devoid of all activity and it appears as a clear broad zone between the active regions (Figs. 1, 4 and 5). The longitudinal and the transverse membranes, for the boundaries of the electrogenic unit, were always unreactive. Phosphorylase, the only transglycosylase we were able to study, differed in its distribution from the oxidative enzymes described above. Activity in this case was present only in the papillae of the anterior portion, those of the posterior portion being inactive. The reaction extended somewhat into the inner portion of the electroplate, but always without reaching the posterior surface. In the anterior portion, the enzyme was neatly localized in the core of the papillae (Fig. 8). No TPN-linked dehydrogenase could be demonstrated in the Sachs organ. DISCUSSION
Though we are aware that some differences may exist in the relative metabolic activities of Sachs organ and main organ of E. electricus, as shown by Hoskin [9] in relation to the utilization of the glucose-l-phosphate, Sachs organ was chosen for these studies on account of its widely separated electroplates. These provided a favourable background for the localization of the enzymes. Few biochemical data are available on the oxidative enzyme activity of the electric organ. Eisenberg [6] has shown the presence of strong DPNH oxidase and succinic oxidase activities in this material, a finding supported by Miranda [lo] in our laboratories. These results have been confirmed in the present study. However, contrary to the findings of Eisenberg [6] who described the succinate dehydrogenase activity in the single electroplate as predominantly localized in the anterior (non-innervated) mamebrane of the cell, we found this enzyme mostly concentrated in the posterior (innervated) membrane. It can be suggested that the use of thin sections makes both membranes readily accessible to the substrate and to the tetrazolium salt used. In the performance of the method on intact electroplates free diffusion of substrates into the enzyme sites may be uneven. The presence of u-glycerophosphate dehydrogenase, as shown by Eisenberg [6] in homogenates of the main organ, has been confirmed histochemicExperimental
Cell Research 30
6
A. G. E. Pearse and Darcy F. de Almeida
ally for the electric organ. Further information in this field is now presented, concerning the presence of high levels of malate and lactate dehydrogenases, and also of cytochrome oxidase. The demonstration of cytochrome oxidase is strictly dependent on the addition of cytochrome c to the medium, as had already been demonstrated by Miranda [lo] in homogenates of electric tissue. This evidence points to the presence of very low concentrations of cytochrome c in the organ. The most interesting finding is that the oxidative enzyme activities, whether concerned with the tricarboxylic acid cycle or the glycolytic pathway, have similar distribution patterns and are concentrated in the posterior portion of the electroplate. Associated with the fact that the innervated membrane is the active surface of the electroplate, these results support the suggested utilization of the isolated posterior half in electrophysiological experiments [l],. In fact, this kind of preparation may well represent the material of choice for: the study of the biochemical processes underlying electrical activity. The only exception to this general rule of distribution is provided by the phosphorylase activity, which was found exclusively in the anterior portion of the electroplate. This enzyme can be directly related to the utilization of stored glycogen to provide the energy required for the metabolic processes. Glycogen can be shown to be the major storage form of carbohydrate in the anterior portion of the electroplate as indicated by Couceiro [3], who demonstrated the presence of a glycogen-like type of material in the electroplate. This is to be distinguished from the acid mucopolysaccharide found in the compartment outside the electroplate. We therefore consider that glycogen is the main source of energy in the electric organ and that its breakdown occurs in the anterior portion of the electroplate. The resulting product (glucose-lphosphate) may then diffuse through the protein of the inner portion to the posterior surface. Here it may be directly utilized as fuel or fed into the Embden-Meyerhof pathway. Indirect evidence that the latter may be functioning is provided by the high concentration of lactate dehydrogenase in the posterior surface, as described above. Our failure to detect any TPNlinked enzyme offers additional evidence for the non-existence of a shunt for the glycolytic process. The findings of Eisenberg [B] are also compatible with these assumptions. We were unable to carry out tests for the presence of glycogen synthesizing enzymes (uridine diphosphate glucose-glycogen transglucosylase) in the anterior portion of the electroplate, but these will be carried out as soon as possible. It is to be expected that the glycogen synthetase enzymes will be present only in this region. Experimental
Cell Research 30
Localization of enzyme activity
7
SUMMARY
The main oxidative enzyme activities in the Sachs organ of Electrophorus related to the tricarboxylic acid cycle or to the glycolytic pathways, are concentrated in the posterior portion of the electroplate. No evidence could be found for the existence of an alternative (pentose cycle) pathway of glucose utilization. Phosphorylase is present exclusively in the anterior portion of the electroplate. It is suggested that the neutral polysaccharide (glycogen) present in the anterior portion of the electroplate acts as the main source of energy for maintenance of electric potential in the membrane. Its breakdown product (glucose-l-phosphate) could diffuse from the anterior portion across the electroplate to be utilized at the active posterior membrane.
electricus, whether
We should like to express our gratitude to Dr. J. N. Bica for the permission to use the cryostat and his laboratory facilities, and to Dr. M. Miranda for helpful discussions. REFERENCES 1. DE ALMEIDA, D. F.. DE OLIVEIRA CASTRO, G., MIRANDA, M. and CHAGAS, C., To be published. 2. BURSTONE, M. S., J. Histochem. Cyfochem. 7, 112 (1959). 3. COUCEIRO, A., An. Aead. Brasil. Ci. 12, 447 (1950). 4. COUCEIRO, A. and AKERMAN, M., ibid. 20, 383 (1948). 5. COUCEIRO, A., CHAGAS, C. and MEYER, H., ibid. 27, 52 (1955). 6. EISENBERG, M. A., Arch. Biochem. Biophys. 74, 372 (1958). 7. HESS, R., SCARPELLI, D. G. and PEARSE, A. G. E., J. Biophys. Biochem. Cytol. 4, 753 (1958). 8. HESS, R. and PEARSE, A. G. E., Nature 191, 718 (1961). 9. HOSKIN, F. C. G., Arch. Biochem. Biophys. 81, 330 (1959). 10. MIRANDA, M., Memorias sobre a atividade do Instituto de Biofisica. Universidade do Brasil, Rio de Janeiro, 1960. 11. PEARSE, A. G. E., Histochemistry, Theoretical and Applied. Little, Brown and Co. Boston, 2nd ed. 1960. 12. SCARPELLI, D. G., HESS, R. and PEARSE, A. G. E., Biophys. Biochem. Cyfol. 4, 747 (1958). 13. SCHOFFENIELS, E., Biochim. Biophys. Acta. 26, 585 (1957). 14. TAKEUCHI, T., J. Histochem. Cytochem. 4, 84 (1956). 15. TAKEUCHI, T. and KURIAKI, H., ibid. 3, 153, 1955.
Experimental
Cell Research 30
1
Cell Research 30, l-7 (1963)
THE LOCALIZATION OF ENZYME ACTIVITY IN THE ELECTRIC ORGAN OF ELECZ’ROPHORUS ELECTRICUS (L) A. G. E. PEARSE’ Institute
de Biojsica,
and DARCY Universidade
F. DE ALMEIDAe
do Bras&
Rio de Janeiro,
Brazil
Received June 12, 1962
CONDUCTINGcells integrate a variety of vital processes for which the utihzation of chemical energy is of fundamental importance. The innervated portions of these cells constitute the trigger system for the conductance changes underlying physiological activity. Bioelectric currents represent the result of such changes in the electric organs of electric fishes. The very high degree of specialization of these effecters in Electrophorus electricus is indicated not only by the magnitude of the resulting electrical phenomena, but also by the anatomical and morphological characteristics of the electroplate. Such properties made possible the isolation of a single electroplate from the electric organ [13] and, more recently, an attempt has been made to separate the active “posterior half” of the electroplate [l]. It was felt that progress in this held required a topographical knowledge of the biochemical properties of the electric organ in order to establish a correlation between its structure and function. The present paper describes investigations on the specific distribution of the enzymes involved in providing the chemical energy necessary for the production of the electrical discharge.
MATERIAL
AND
METHODS’
Small fragments were taken from the Sachs organ of E. electricus, mounted in a few drops of water on a microtome chuck and immediately frozen with solid CO,. Four to 12 p sections were cut on a cold microtome at -20X, mounted on coverslips and dried in air. For the demonstration of phosphorylase, thicker sections (up to 24 ,a) were used. For the demonstration of the various dehydrogenases, the following stock solution r British Council Visiting Lecturer. Present address: Department of Pathology, Postgraduate Medical School, London, England. 2 Research Fellow of the Conselho National de Pesquisas do Brasil. 3 The following abbreviations are used: MTT, 3(4.5-dimethylthiazolyl-2)-2.5-diphenyl tetrazolium bromide; Tris, tris (hydroxymethyl)aminomethane; Vit. I<, (menadione), 2-methyl-1:4naphthoquinone; DPN, diphosphopyridine nucleotide (Coenzyme I), and TPN, triphosphopyridine nucleotide (Coenzyme II). 1 - 631809
Experimental
Cell Research 30
2
A. G. E. Pearse and Darcy F. de Almeida
was prepared: 2.5 ml MTT (1 mg/ml); 2.5 ml 0.2 M Tris buffer, pH 7.4; 0.5 ml 0.5 &f cobalt nitrate; and 3.5 ml distilled water. This solution was stored frozen at - 30°C. Shortly before use, the substrate incubating solutions were prepared. The following enzymes were studied: DPNH diaphorase [ 121; DPN-linked malate, lactate and a-glycerophosphate dehydrogenases 171; mitochondrial a-glycerophosphate dehydrogenase [S]; succinate dehydrogenase [ll]; TPN-linked malate and glucose-6-phosphate dehydrogenases [ 71. Cytochrome oxidase activity was shown by the method of Burstone [2]. The addition of cytochrome c (5 x IO-6 M) was found necessary for the reaction to take place. For the demonstration of succinate and a-glycerophosphate dehydrogenases, the incubating solution was saturated with 2-methyl-l,4-naphthoquinone (vitamin K,, menadione). The method of Takeuchi [14, 151 was applied for the demonstration of phosphorylase. Incubation was carried out at room temperature, for 15 to 30 min. In the case of phosphorylase, incubation times were as high as 120 min. Subsequently the sections were rinsed in distilled water, fixed for a few minutes in formalin-saline, washed in water and mounted in glycerine jelly. RESULTS
All the enzymes studied were found to be localized exclusively in the electroplate. The material outside the plate presented no activity, except for a few scattered cells in the ground substance of the electrogenic unit. Thin sections provided excellent fine localization of enzymes, as illustrated in Fig. 2, but somewhat thicker sections (12 to 24 ,u) were needed for a general view of their topographical distribution inside the electroplate. DPN diaphorase and succinate dehydrogenase were strongly active (Figs. 1 and 2), and a very high activity of malate (Figs. 3 and 4) and lactate (Fig. 5) dehydrogenases was also present. Figs. 1, 2, 3 and 5 show that activity of the various enzymes is limited to the papillae of the electroplate syncytium, where the nuclei and mitochondria are concentrated. The mitochondria are always situated around the nuclei [5], a fact which explains the perinuclear distribution of the formazan pigment (Figs. 1 and 4). Both n-glycerophosphate dehydrogenases were less active than the preceding enzymes; their arrangement in the electroplate was essentially similar, however (Fig. 6). Cytochrome oxidase, on the other hand, was found to be highly active (Fig. 7), but only after the addition of cytochrome c to the incubating solution. Rather similar patterns of distribution were thus found for all the oxidative enzymes which we studied. Their activity was restricted to the papillae of the electroplate, being in every case much higher in the posterior (innervExperimental
Cell Research 30
Localizaiion
of enzyme activity
Fig. l.-DPNH diaphorase. The reaction is very strong in the papillae of the posterior surfa ce. Weaker activity in the anterior papillae. 12 y section. x 345. Fig. 2.-Succinate dehydrogenase, (with added Vitamin K,) 4 /J section. The final product of enzymic activity is shown as formazan dots concentrated in the posterior surface of the elect roplate (to the right). x 390. Fig. 3.-DPN-linked malate dehydrogenase. General view of the distribution of the enzyme in the electroplate of Sachs organ. Fig. 4.-DPN-linked malate dehydrogenase. Detail of the activity in the papillae of the elect roplate. Enzyme highly active at the mitochondrial sites near the nuclei, which are unreacti ve. x 300. Experimental
Cell Research 30
A. G. E. Pearse and Marcy F. de Almeidu
Fig. S.-Lactate dehydrogenasc. tion (above). x 180.
The enzyme is most active in the papillae
Fig. 6.-Mitochondrial cc-glycerophosphate dehydrogenase. of distribution as the other dehydrogenases. x 220. Fig. 7.-Cytochrome of the electroplate;
following
por-
the same pattern
oxidase. Activity apparent only near the anterior and posterior surfaces the posterior membrane (to the right) shows stronger activity. x 230.
Fig. X.-Phosphorylase. of the anterior papillae x 35. Experimental
Activity
of the posterior
24 y section. Enzyme activity is sharply localized of the electroplate (to the left). The posterior portion
Cell Research 30
in the inner portion (right) is unreactive.
Localization of enzyme activity
5
ated) portion. Whether in the anterior or the posterior portion, most of the activity was localized in the perinuclear region of the cytoplasm, outlining clearly the unreactive nuclei (Figs. 1 and 4). The small dot-like deposits of the final reaction product were sharply limited to narrow strip of cytoplasm near the anterior and posterior surfaces of the electroplate. The innermost part of the electroplate was devoid of all activity and it appears as a clear broad zone between the active regions (Figs. 1, 4 and 5). The longitudinal and the transverse membranes, for the boundaries of the electrogenic unit, were always unreactive. Phosphorylase, the only transglycosylase we were able to study, differed in its distribution from the oxidative enzymes described above. Activity in this case was present only in the papillae of the anterior portion, those of the posterior portion being inactive. The reaction extended somewhat into the inner portion of the electroplate, but always without reaching the posterior surface. In the anterior portion, the enzyme was neatly localized in the core of the papillae (Fig. 8). No TPN-linked dehydrogenase could be demonstrated in the Sachs organ. DISCUSSION
Though we are aware that some differences may exist in the relative metabolic activities of Sachs organ and main organ of E. electricus, as shown by Hoskin [9] in relation to the utilization of the glucose-l-phosphate, Sachs organ was chosen for these studies on account of its widely separated electroplates. These provided a favourable background for the localization of the enzymes. Few biochemical data are available on the oxidative enzyme activity of the electric organ. Eisenberg [6] has shown the presence of strong DPNH oxidase and succinic oxidase activities in this material, a finding supported by Miranda [lo] in our laboratories. These results have been confirmed in the present study. However, contrary to the findings of Eisenberg [6] who described the succinate dehydrogenase activity in the single electroplate as predominantly localized in the anterior (non-innervated) mamebrane of the cell, we found this enzyme mostly concentrated in the posterior (innervated) membrane. It can be suggested that the use of thin sections makes both membranes readily accessible to the substrate and to the tetrazolium salt used. In the performance of the method on intact electroplates free diffusion of substrates into the enzyme sites may be uneven. The presence of u-glycerophosphate dehydrogenase, as shown by Eisenberg [6] in homogenates of the main organ, has been confirmed histochemicExperimental
Cell Research 30
6
A. G. E. Pearse and Darcy F. de Almeida
ally for the electric organ. Further information in this field is now presented, concerning the presence of high levels of malate and lactate dehydrogenases, and also of cytochrome oxidase. The demonstration of cytochrome oxidase is strictly dependent on the addition of cytochrome c to the medium, as had already been demonstrated by Miranda [lo] in homogenates of electric tissue. This evidence points to the presence of very low concentrations of cytochrome c in the organ. The most interesting finding is that the oxidative enzyme activities, whether concerned with the tricarboxylic acid cycle or the glycolytic pathway, have similar distribution patterns and are concentrated in the posterior portion of the electroplate. Associated with the fact that the innervated membrane is the active surface of the electroplate, these results support the suggested utilization of the isolated posterior half in electrophysiological experiments [l],. In fact, this kind of preparation may well represent the material of choice for: the study of the biochemical processes underlying electrical activity. The only exception to this general rule of distribution is provided by the phosphorylase activity, which was found exclusively in the anterior portion of the electroplate. This enzyme can be directly related to the utilization of stored glycogen to provide the energy required for the metabolic processes. Glycogen can be shown to be the major storage form of carbohydrate in the anterior portion of the electroplate as indicated by Couceiro [3], who demonstrated the presence of a glycogen-like type of material in the electroplate. This is to be distinguished from the acid mucopolysaccharide found in the compartment outside the electroplate. We therefore consider that glycogen is the main source of energy in the electric organ and that its breakdown occurs in the anterior portion of the electroplate. The resulting product (glucose-lphosphate) may then diffuse through the protein of the inner portion to the posterior surface. Here it may be directly utilized as fuel or fed into the Embden-Meyerhof pathway. Indirect evidence that the latter may be functioning is provided by the high concentration of lactate dehydrogenase in the posterior surface, as described above. Our failure to detect any TPNlinked enzyme offers additional evidence for the non-existence of a shunt for the glycolytic process. The findings of Eisenberg [B] are also compatible with these assumptions. We were unable to carry out tests for the presence of glycogen synthesizing enzymes (uridine diphosphate glucose-glycogen transglucosylase) in the anterior portion of the electroplate, but these will be carried out as soon as possible. It is to be expected that the glycogen synthetase enzymes will be present only in this region. Experimental
Cell Research 30
Localization of enzyme activity
7
SUMMARY
The main oxidative enzyme activities in the Sachs organ of Electrophorus related to the tricarboxylic acid cycle or to the glycolytic pathways, are concentrated in the posterior portion of the electroplate. No evidence could be found for the existence of an alternative (pentose cycle) pathway of glucose utilization. Phosphorylase is present exclusively in the anterior portion of the electroplate. It is suggested that the neutral polysaccharide (glycogen) present in the anterior portion of the electroplate acts as the main source of energy for maintenance of electric potential in the membrane. Its breakdown product (glucose-l-phosphate) could diffuse from the anterior portion across the electroplate to be utilized at the active posterior membrane.
electricus, whether
We should like to express our gratitude to Dr. J. N. Bica for the permission to use the cryostat and his laboratory facilities, and to Dr. M. Miranda for helpful discussions. REFERENCES 1. DE ALMEIDA, D. F.. DE OLIVEIRA CASTRO, G., MIRANDA, M. and CHAGAS, C., To be published. 2. BURSTONE, M. S., J. Histochem. Cyfochem. 7, 112 (1959). 3. COUCEIRO, A., An. Aead. Brasil. Ci. 12, 447 (1950). 4. COUCEIRO, A. and AKERMAN, M., ibid. 20, 383 (1948). 5. COUCEIRO, A., CHAGAS, C. and MEYER, H., ibid. 27, 52 (1955). 6. EISENBERG, M. A., Arch. Biochem. Biophys. 74, 372 (1958). 7. HESS, R., SCARPELLI, D. G. and PEARSE, A. G. E., J. Biophys. Biochem. Cytol. 4, 753 (1958). 8. HESS, R. and PEARSE, A. G. E., Nature 191, 718 (1961). 9. HOSKIN, F. C. G., Arch. Biochem. Biophys. 81, 330 (1959). 10. MIRANDA, M., Memorias sobre a atividade do Instituto de Biofisica. Universidade do Brasil, Rio de Janeiro, 1960. 11. PEARSE, A. G. E., Histochemistry, Theoretical and Applied. Little, Brown and Co. Boston, 2nd ed. 1960. 12. SCARPELLI, D. G., HESS, R. and PEARSE, A. G. E., Biophys. Biochem. Cyfol. 4, 747 (1958). 13. SCHOFFENIELS, E., Biochim. Biophys. Acta. 26, 585 (1957). 14. TAKEUCHI, T., J. Histochem. Cytochem. 4, 84 (1956). 15. TAKEUCHI, T. and KURIAKI, H., ibid. 3, 153, 1955.
Experimental
Cell Research 30