Histochemistry 46, 131--137 (1976) 9 by Springer-Verlag 1976
Electron Microscopic Localization of Monoamine Oxidase in the Rat Liver* B. Y. Yoo Department of Biology, University of New Brunswick Fredericton, N. B., Canada L. Oreland Department of Pharmacology, University of Ume~, Umes Sweden Received September 19, 1975 Summary. Two methods have been employed to localize monoamine oxidase activity in the cells of rat liver, using either 2-(2'-benzothiazolyl)-5-stryl-3-(4'-phtalhydrazidyl) tetrazolium chloride (BSPT) or ferricyanide as electron acceptor. With both methods monoamine oxidase activity was found both in the inner and the outer mitochondra] membrane, although the outer membrane appeared the most probable location. In addition the BSPT method but not the ferricyanide method, revealed monoamine oxidase activity in the endoplasmatic reticulum. The results obtained by the two methods have been compared and are discussed in view of available biochemical data on monoamine oxidase. Introduction Two methods have previously been used to localize the activity of monoamine oxidase (MAO) (monoamine-oxygen oxidoreductase. E.C. 1.4.3.4.) by electron microscopy. 1) The formation of osmiophilic formazans by reduction of a variety of tetrazolium salts (Shannon et al., 1974). I n this method, to avoid introducing staining artifacts (Seligman et al., 1967), it is desirable to use a nonosmophilic tetrazolium salt, which u p o n reduction produces an osmophilic formazan. Using such a tetrazolium salt; 2-(2'-benzothiazolyl)-5-stryl-3-(4'-phtalhydrazidyl) tetrazolium chloride (BSPT) (referred to as " t h e B S P T m e t h o d " ) , Shannon etal. (1974) have recently demonstrated MAO activity in the cells of various organs in rats and guinea pigs b y electron microscopy. 2) The formation of copper ferrocyanide (Hatchett's brown) b y using ferricyanide as an electron aeceptor. The original sites of t t a t c h e t t ' s brown deposits are t h e n amplified b y the oxidative polymerization of 3.3'-diaminobenzidine (DAB), and subsequent osmication of the polymer formed yields an electrondense " o s m i u m b l a c k " deposit (Bloom et al., 1972; Ha~lker et al., 1973). Because of the solubility and diffusibility of H a t c h e t t ' s brown (Lukaszyk, 1971), Shannon et al. (1974) have questioned the usefulness of ferricyanide in the localization of MAO as described b y H a n k e r et al. (1973) (referred to a s " the ferricyanide m e t h o d " ) . I n this communication we compare these two methods and examine their usefulness in the localization of MAO. * Supported by research grants from the National Research Council of Canada (A 3651), The Swedish Medical Research Council (4145) and M. Bergwall's Foundation, Stockholm.
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Materials and Methods Adult Sprague-Dawley rats of both sexes weighing 200-250 g were killed with an i.p. dose (10 mg/100 g) of pentobarbital sodium. The liver was then fixed at room temperature in situ by perfusing the liver with freshly depolymcrized 2% paraformaldehyde in phosphate buffer (0.1 M), pH 7.4, as described by Hanker et al. (1973). We have previously studied effects of formaldehyde fixation on the MAO activity in preparations of rat liver mitochondria (u et al., 1974), and by extrapolating these data it can be assumed that it would take less than 40 rain to inactivate MAO in rat liver mitochondria under the conditions described above. Keeping this in mind, we fixed the liver in situ for about 10 min at room temperature. Subsequent cytochemical manipulations were performed according to the method of Hanker et al. (1973), "ferricyanide method", or to Shannon et al. (1974), "BSPT method", depending on the electron acceptor used. Controls consisted of 1) omission of the substrate (tryptamine) and 2) addition of MAO inhibitors (pargyline, nialamide) at a concentration of 1 mM prior to and/or concomittant with incubation. When the BSPT method was used also omission of ]3SPT from the incubation medium served as a control. After dehydration in acetone, all tissues were embedded in epomaraldite resin. Thin sections were examined with or without counterstaining in lead citrate, using a Philips 200 electron microscope operated at 60 kV. All electron micrographs shown in this communication, however, were taken without counterstaining.
Results As reported before b y others ( S h a n n o n et al., 1974; R e i t h a n d Schuler, 1972), we f o u n d t h a t both B S P T a n d D A B have v e r y limited p e n e t r a t i o n into tissue blocks. Therefore, t h i n sections were cut close to the surface of each tissue block. Because of a p p a r e n t poor perfusion i n some experiments the i n s i t u fixation was i n a d e q u a t e to p r e v e n t the m i t o c h o n d r i a from swelling a n d only a few cristae could be seen in some mitoehondria. BSPT M e t h o d . Fig. 1 shows t h e a c t i v i t y of MAO oa the m i t o m e m b r a n e s as detected b y the B S P T method. A l t h o u g h the electron-dense deposits appear to be localized on t h e outer m e m b r a n e (see Fig. 6), it was n o t possible, however, to certainly discern whether the a c t i v i t y resided on the outer m e m b r a n e , the i n n e r m e m b r a n e , or i n the i n t e r m e m b r a n e space, even at a high degree of magnification (Fig. 6). All MAO a c t i v i t y in the m i t o c h o n d r i a l m e m b r a n e s disappeared after t r e a t m e n t with pargyline or nialamide (Figs. 4 a n d 5). No MAO a c t i v i t y was f o u n d in the n u c l e a r m e m b r a n e . I n the endoplasmatic r e t i c u l u m (ER) some a c t i v i t y was f o u n d (Fig. 7). Some of this a c t i v i t y p a r t l y survived t h e effect of t h e two MAO inhibitors pargyline a n d nialamide (Figs. 4 a n d 5). E v e n i n the absence of the exogenous s u b s t r a t e t r y p t a m i n e some s t a i n i n g was f o u n d in the E R (arrows in Fig. 2), b u t in the absence of B S P T no such staining was detected (Fig. 3).
Fig. 1. MAO acti~Tityon the mitomembrane as demonstrated by the BSPT method. • Fig. 2. Substrate control of the BSPT method. Incubation was performed without the MAO substrate, tryptamine. No electron-dense deposit was found on the mitomembrane, but some activity was found in the ER (arrows) : x 22,300 Fig. 3. BSPT control. Incubation in MAO medium was performed without BSPT. No reaction was found anywhere in the cell. • 22,300 Fig. 4. Incubation in MAO medium (BSPT method) with 1 mM nialamide. • 22,300
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Fig. 10. MAO activity on the mitomembrane demonstrated b y the ferricyanide method. Note the absence of electron-dense deposits on the nuclear m e m b r a n e . N Nucleus. M Mitochondria. • 13,400
Fig. 5. Incubation in MAO medium (BSPT method) with 1 mM pargyline. X 22,300 :Fig. 6. MAO activity on the mitomembrane localized b y the B S P T method. Electron-dense deposits are found on the outer membrane. Also note the activity in the ER. X 84,500 Fig. 7. MAO activity in the E R localized b y the BSPT method. • Fig. 8. MAO activity demonstrated b y the s method. The activity appears to be localized on the inner membrane (single arrow) as well as the outer membrane (double arrows) and on the eristae, x 64,000 Fig. 9. MAO activity on the mitomembrane as shown b y the ferricyanide method. As in Fig. 8, the activity appears on the inner (single arrow) as well as the outer membrane (double arrows) and on the cristae. X 84,500
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ferricyanide Method. With this method not only the mitomembrane, but also the mitochondrial cristae were stained with electron-dense deposits (Fig. 8, 9 and 10). MAO activity was found both in the inner and the outer mitomembrahe. Even in the same mitochondrion, in some parts the outer mitomembrane seemed to contain the activity (double arrows in Figs. 8 and 9), while in other parts the opposite was indicated (single arrows in _Figs. 8 and 9). Neither the nuclear membrane nor the E R showed any MAO activity with the fcrrieyanide method (Fig. 10). Pargyline and nialamide completely inhibited the MAO activity (EM photographs are not shown). Discussion
Not all mitochondria in the same section showed electron-dense reaction products. This may be due to a functional heterogenity of mitochondria as is the case with other mitoehondrial enzymes, e.g. suceinic dehydrogenase (Ogawa et al., 1968), or to partial and differential inactivation of mitochondral MAO during the fixation in situ. The electron-dense deposits found with the ferricyanide method on the mitochondrial cristae, on the imler membrane as well as on the outer membrane (Figs. 8 and 9) can be interpreted either as a localization of MAO on all those mitochondrial structures or as an artifact e.g. caused by the diffusion of copper ferroeyanide as Shannon et al. (1974) have cautioned. A widespread localization of the enzyme in the cell would be in contrast to most previous biochemical studies in which MAO seems to be localized mainly to the mitochondrial outer membrane and possibly also to the endoplasmatic reticulum (De Duve et al., 1962; Schnaitman et al., 1967; Beattie, 1968). However, Gorkin (1970)have reported of the presence of the enzyme also in other structures with the highest specific activity in the nuclear membrane, a localization which could not be confirmed in this investigation (Fig. 10). If, on the other hand, the present result is partly artifactual, diffusion of copper ferrocyanide seems to be a too simple explanation since according to Lukaszyk (1971) copper ferrocyanide is soluble only at pH higher than 8, and in the ferricyanide method used in the present study the pH of incubation media was kept far below 8 during the eytochemical manipulations. Since it was difficult also with the BSPT method to exclude a localization of MAO activity to other parts of the mitochondrion than the outer membrane, it may be concluded that unless there is a localization also to other parts, the histoehemical techniques available at present are not fully adequate in this respect. As regards the ER, MAO activity was found only with the BSPT method (Fig.7). Another possibility would, however, be that the staining found in the E R with the BSPT method does not reflect MAO activity. Considering the electrondense deposits found in the E R in the absence of the substrate, tryptamine (Fig. 2) and the preparation treated with MAO inhibitors (Figs. 4 and 5) another enzyme with endogenous substrates present might have caused some of this staining. If this was the case the question about the presence of MAO in the E R (see Schnaitman et al., 1967) still remains to be settled.
Acknowledgement. This work was initiated at the Department of Pharmacology, University of Umes Sweden, during the sabbatical leave of B. Y. Yoo in 1972.
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References Beattie, D. S. : Enzyme localization in the inner and outer membranes of rat liver mitoehondria. Bioehem. biophys. Res. Commun. 31, 901 907 (1968) Bloom, F . E . , Sims, K . L . , Weitsen, G.A., Hanker, J . S . : Cytochemical differentiation between monoamine oxidase and other neuronal oxidases. In: Monoamine oxidases - New Vistas. Advances in biochemical Psyehopharmaeology. (Costa, E., Sandler, M. eds,) vol. 5, p. 243-262. New York: Raven Press 1972 De Duve, C., Wattiaux, R., Baudhuin, P.: Distribution of enzymes between subcellular fractions in animal tissues. Advanc. Enzymol. 24, 291-358 (1962) Gorkin, W. Z. : Monoamine oxidase activity in membrane structures of rat liver cell. Experientia (Basel) 27, 30 (1970) Hanker, d.S., Kusyk, C. J., Bloom, E. E., Pearse, A. G. E.: The demonstration of dehydrogenases and monoamine oxidase by the formation of osmium blacks at the sites of Hatchett's brown. Histochemie 83, 205~30 (1973) Lukaszyk, A.: A method for histochemieal demonstration of alpha-glycerophosphate-ferricyanide oxidoreduetase activity. Eolia histochem, eytochem. 9, 167-186 (1971) Ogawa, K., Saito, T., Mayahara, H. : The site of ferricyanide reduction by reductases within mitochondria as studied by electron microscopy. J. Histochem. Cytochem. 16, 49-57 (1968) Reith, A., Schulcr, B. : Demonstration of cytochemieal oxidase activity with diaminobenzidine. A biochemical and electron microscopic study. J. Histochem. Cytochem. 20, 583 589 (1972) Sehnaitman, C., Erwin, V. G., Greenwalt, J. W. : The submitoehondrial localization of monoamine oxidase. An enzymatic marker for the outer membrane of rat liver mitochondria. J. Cell Biol. 112, 719-735 (1967) Seligman, A.M., Ueno, H., Morizono, Y., Wasserkrug, H . L . , Katzoff, L., Hanker, J. S.: Electron microscopic demonstration of dehydrogenase activity with a new osmiophilic ditetrazolium salt (TC-NBT). J. Histoehem. Cytoehem. 15, 1-13 (1967) Shannon, W. A. Jr., Wasserkrug, H . L . , Seligman, A . M . : The ultrastructural localization of monoamine oxidase (MAO) with tryptamine and a new tetrazolium salt 2-(2'-benzothiazolyl)-5-stryl-3-(4'phtalhydrazidyl) tetrazolium chloride (BSPT). J. Histochem. Cytochem. 22, 170-182 (1974) Yoo, B . Y . , Oreland, L., Persson, A. : Effects of formaldehyde and glutaraldehyde fixation on the monoamine oxidase activity in isolated rat liver mitochondria. J. Histochem. Cytoehem. 22, 445-446 (1974) Prof. Lars Oreland University of Umes Department of Pharmacology S-90187 Umeh, Sweden
Electron Microscopic Localization of Monoamine Oxidase in the Rat Liver* B. Y. Yoo Department of Biology, University of New Brunswick Fredericton, N. B., Canada L. Oreland Department of Pharmacology, University of Ume~, Umes Sweden Received September 19, 1975 Summary. Two methods have been employed to localize monoamine oxidase activity in the cells of rat liver, using either 2-(2'-benzothiazolyl)-5-stryl-3-(4'-phtalhydrazidyl) tetrazolium chloride (BSPT) or ferricyanide as electron acceptor. With both methods monoamine oxidase activity was found both in the inner and the outer mitochondra] membrane, although the outer membrane appeared the most probable location. In addition the BSPT method but not the ferricyanide method, revealed monoamine oxidase activity in the endoplasmatic reticulum. The results obtained by the two methods have been compared and are discussed in view of available biochemical data on monoamine oxidase. Introduction Two methods have previously been used to localize the activity of monoamine oxidase (MAO) (monoamine-oxygen oxidoreductase. E.C. 1.4.3.4.) by electron microscopy. 1) The formation of osmiophilic formazans by reduction of a variety of tetrazolium salts (Shannon et al., 1974). I n this method, to avoid introducing staining artifacts (Seligman et al., 1967), it is desirable to use a nonosmophilic tetrazolium salt, which u p o n reduction produces an osmophilic formazan. Using such a tetrazolium salt; 2-(2'-benzothiazolyl)-5-stryl-3-(4'-phtalhydrazidyl) tetrazolium chloride (BSPT) (referred to as " t h e B S P T m e t h o d " ) , Shannon etal. (1974) have recently demonstrated MAO activity in the cells of various organs in rats and guinea pigs b y electron microscopy. 2) The formation of copper ferrocyanide (Hatchett's brown) b y using ferricyanide as an electron aeceptor. The original sites of t t a t c h e t t ' s brown deposits are t h e n amplified b y the oxidative polymerization of 3.3'-diaminobenzidine (DAB), and subsequent osmication of the polymer formed yields an electrondense " o s m i u m b l a c k " deposit (Bloom et al., 1972; Ha~lker et al., 1973). Because of the solubility and diffusibility of H a t c h e t t ' s brown (Lukaszyk, 1971), Shannon et al. (1974) have questioned the usefulness of ferricyanide in the localization of MAO as described b y H a n k e r et al. (1973) (referred to a s " the ferricyanide m e t h o d " ) . I n this communication we compare these two methods and examine their usefulness in the localization of MAO. * Supported by research grants from the National Research Council of Canada (A 3651), The Swedish Medical Research Council (4145) and M. Bergwall's Foundation, Stockholm.
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Materials and Methods Adult Sprague-Dawley rats of both sexes weighing 200-250 g were killed with an i.p. dose (10 mg/100 g) of pentobarbital sodium. The liver was then fixed at room temperature in situ by perfusing the liver with freshly depolymcrized 2% paraformaldehyde in phosphate buffer (0.1 M), pH 7.4, as described by Hanker et al. (1973). We have previously studied effects of formaldehyde fixation on the MAO activity in preparations of rat liver mitochondria (u et al., 1974), and by extrapolating these data it can be assumed that it would take less than 40 rain to inactivate MAO in rat liver mitochondria under the conditions described above. Keeping this in mind, we fixed the liver in situ for about 10 min at room temperature. Subsequent cytochemical manipulations were performed according to the method of Hanker et al. (1973), "ferricyanide method", or to Shannon et al. (1974), "BSPT method", depending on the electron acceptor used. Controls consisted of 1) omission of the substrate (tryptamine) and 2) addition of MAO inhibitors (pargyline, nialamide) at a concentration of 1 mM prior to and/or concomittant with incubation. When the BSPT method was used also omission of ]3SPT from the incubation medium served as a control. After dehydration in acetone, all tissues were embedded in epomaraldite resin. Thin sections were examined with or without counterstaining in lead citrate, using a Philips 200 electron microscope operated at 60 kV. All electron micrographs shown in this communication, however, were taken without counterstaining.
Results As reported before b y others ( S h a n n o n et al., 1974; R e i t h a n d Schuler, 1972), we f o u n d t h a t both B S P T a n d D A B have v e r y limited p e n e t r a t i o n into tissue blocks. Therefore, t h i n sections were cut close to the surface of each tissue block. Because of a p p a r e n t poor perfusion i n some experiments the i n s i t u fixation was i n a d e q u a t e to p r e v e n t the m i t o c h o n d r i a from swelling a n d only a few cristae could be seen in some mitoehondria. BSPT M e t h o d . Fig. 1 shows t h e a c t i v i t y of MAO oa the m i t o m e m b r a n e s as detected b y the B S P T method. A l t h o u g h the electron-dense deposits appear to be localized on t h e outer m e m b r a n e (see Fig. 6), it was n o t possible, however, to certainly discern whether the a c t i v i t y resided on the outer m e m b r a n e , the i n n e r m e m b r a n e , or i n the i n t e r m e m b r a n e space, even at a high degree of magnification (Fig. 6). All MAO a c t i v i t y in the m i t o c h o n d r i a l m e m b r a n e s disappeared after t r e a t m e n t with pargyline or nialamide (Figs. 4 a n d 5). No MAO a c t i v i t y was f o u n d in the n u c l e a r m e m b r a n e . I n the endoplasmatic r e t i c u l u m (ER) some a c t i v i t y was f o u n d (Fig. 7). Some of this a c t i v i t y p a r t l y survived t h e effect of t h e two MAO inhibitors pargyline a n d nialamide (Figs. 4 a n d 5). E v e n i n the absence of the exogenous s u b s t r a t e t r y p t a m i n e some s t a i n i n g was f o u n d in the E R (arrows in Fig. 2), b u t in the absence of B S P T no such staining was detected (Fig. 3).
Fig. 1. MAO acti~Tityon the mitomembrane as demonstrated by the BSPT method. • Fig. 2. Substrate control of the BSPT method. Incubation was performed without the MAO substrate, tryptamine. No electron-dense deposit was found on the mitomembrane, but some activity was found in the ER (arrows) : x 22,300 Fig. 3. BSPT control. Incubation in MAO medium was performed without BSPT. No reaction was found anywhere in the cell. • 22,300 Fig. 4. Incubation in MAO medium (BSPT method) with 1 mM nialamide. • 22,300
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Fig. 10. MAO activity on the mitomembrane demonstrated b y the ferricyanide method. Note the absence of electron-dense deposits on the nuclear m e m b r a n e . N Nucleus. M Mitochondria. • 13,400
Fig. 5. Incubation in MAO medium (BSPT method) with 1 mM pargyline. X 22,300 :Fig. 6. MAO activity on the mitomembrane localized b y the B S P T method. Electron-dense deposits are found on the outer membrane. Also note the activity in the ER. X 84,500 Fig. 7. MAO activity in the E R localized b y the BSPT method. • Fig. 8. MAO activity demonstrated b y the s method. The activity appears to be localized on the inner membrane (single arrow) as well as the outer membrane (double arrows) and on the eristae, x 64,000 Fig. 9. MAO activity on the mitomembrane as shown b y the ferricyanide method. As in Fig. 8, the activity appears on the inner (single arrow) as well as the outer membrane (double arrows) and on the cristae. X 84,500
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ferricyanide Method. With this method not only the mitomembrane, but also the mitochondrial cristae were stained with electron-dense deposits (Fig. 8, 9 and 10). MAO activity was found both in the inner and the outer mitomembrahe. Even in the same mitochondrion, in some parts the outer mitomembrane seemed to contain the activity (double arrows in Figs. 8 and 9), while in other parts the opposite was indicated (single arrows in _Figs. 8 and 9). Neither the nuclear membrane nor the E R showed any MAO activity with the fcrrieyanide method (Fig. 10). Pargyline and nialamide completely inhibited the MAO activity (EM photographs are not shown). Discussion
Not all mitochondria in the same section showed electron-dense reaction products. This may be due to a functional heterogenity of mitochondria as is the case with other mitoehondrial enzymes, e.g. suceinic dehydrogenase (Ogawa et al., 1968), or to partial and differential inactivation of mitochondral MAO during the fixation in situ. The electron-dense deposits found with the ferricyanide method on the mitochondrial cristae, on the imler membrane as well as on the outer membrane (Figs. 8 and 9) can be interpreted either as a localization of MAO on all those mitochondrial structures or as an artifact e.g. caused by the diffusion of copper ferroeyanide as Shannon et al. (1974) have cautioned. A widespread localization of the enzyme in the cell would be in contrast to most previous biochemical studies in which MAO seems to be localized mainly to the mitochondrial outer membrane and possibly also to the endoplasmatic reticulum (De Duve et al., 1962; Schnaitman et al., 1967; Beattie, 1968). However, Gorkin (1970)have reported of the presence of the enzyme also in other structures with the highest specific activity in the nuclear membrane, a localization which could not be confirmed in this investigation (Fig. 10). If, on the other hand, the present result is partly artifactual, diffusion of copper ferrocyanide seems to be a too simple explanation since according to Lukaszyk (1971) copper ferrocyanide is soluble only at pH higher than 8, and in the ferricyanide method used in the present study the pH of incubation media was kept far below 8 during the eytochemical manipulations. Since it was difficult also with the BSPT method to exclude a localization of MAO activity to other parts of the mitochondrion than the outer membrane, it may be concluded that unless there is a localization also to other parts, the histoehemical techniques available at present are not fully adequate in this respect. As regards the ER, MAO activity was found only with the BSPT method (Fig.7). Another possibility would, however, be that the staining found in the E R with the BSPT method does not reflect MAO activity. Considering the electrondense deposits found in the E R in the absence of the substrate, tryptamine (Fig. 2) and the preparation treated with MAO inhibitors (Figs. 4 and 5) another enzyme with endogenous substrates present might have caused some of this staining. If this was the case the question about the presence of MAO in the E R (see Schnaitman et al., 1967) still remains to be settled.
Acknowledgement. This work was initiated at the Department of Pharmacology, University of Umes Sweden, during the sabbatical leave of B. Y. Yoo in 1972.
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References Beattie, D. S. : Enzyme localization in the inner and outer membranes of rat liver mitoehondria. Bioehem. biophys. Res. Commun. 31, 901 907 (1968) Bloom, F . E . , Sims, K . L . , Weitsen, G.A., Hanker, J . S . : Cytochemical differentiation between monoamine oxidase and other neuronal oxidases. In: Monoamine oxidases - New Vistas. Advances in biochemical Psyehopharmaeology. (Costa, E., Sandler, M. eds,) vol. 5, p. 243-262. New York: Raven Press 1972 De Duve, C., Wattiaux, R., Baudhuin, P.: Distribution of enzymes between subcellular fractions in animal tissues. Advanc. Enzymol. 24, 291-358 (1962) Gorkin, W. Z. : Monoamine oxidase activity in membrane structures of rat liver cell. Experientia (Basel) 27, 30 (1970) Hanker, d.S., Kusyk, C. J., Bloom, E. E., Pearse, A. G. E.: The demonstration of dehydrogenases and monoamine oxidase by the formation of osmium blacks at the sites of Hatchett's brown. Histochemie 83, 205~30 (1973) Lukaszyk, A.: A method for histochemieal demonstration of alpha-glycerophosphate-ferricyanide oxidoreduetase activity. Eolia histochem, eytochem. 9, 167-186 (1971) Ogawa, K., Saito, T., Mayahara, H. : The site of ferricyanide reduction by reductases within mitochondria as studied by electron microscopy. J. Histochem. Cytochem. 16, 49-57 (1968) Reith, A., Schulcr, B. : Demonstration of cytochemieal oxidase activity with diaminobenzidine. A biochemical and electron microscopic study. J. Histochem. Cytochem. 20, 583 589 (1972) Sehnaitman, C., Erwin, V. G., Greenwalt, J. W. : The submitoehondrial localization of monoamine oxidase. An enzymatic marker for the outer membrane of rat liver mitochondria. J. Cell Biol. 112, 719-735 (1967) Seligman, A.M., Ueno, H., Morizono, Y., Wasserkrug, H . L . , Katzoff, L., Hanker, J. S.: Electron microscopic demonstration of dehydrogenase activity with a new osmiophilic ditetrazolium salt (TC-NBT). J. Histoehem. Cytoehem. 15, 1-13 (1967) Shannon, W. A. Jr., Wasserkrug, H . L . , Seligman, A . M . : The ultrastructural localization of monoamine oxidase (MAO) with tryptamine and a new tetrazolium salt 2-(2'-benzothiazolyl)-5-stryl-3-(4'phtalhydrazidyl) tetrazolium chloride (BSPT). J. Histochem. Cytochem. 22, 170-182 (1974) Yoo, B . Y . , Oreland, L., Persson, A. : Effects of formaldehyde and glutaraldehyde fixation on the monoamine oxidase activity in isolated rat liver mitochondria. J. Histochem. Cytoehem. 22, 445-446 (1974) Prof. Lars Oreland University of Umes Department of Pharmacology S-90187 Umeh, Sweden
