Histochemistry 44, 307--312 (1975) 9 by Springer-Verlag 1975
Fine Structural Localization of Arylsulfatase B Activity in the Rabbit Blood Platelets F u s a y o s h i M u r a t a , T e t s u j i N a g a t a , a n d Samuel S. Spieer Department of Anatomy, Shinshu University School of Medicine, 5Iatsumoto, Japan and Department of Pathology, Medical University of South Carolina, Charleston, S. C., U.S.A. Received June 9, 1975 Summary. Fine structural localization of arylsulfatase in the rabbit blood platelets has been investigated in this study. Among many cell organellae, reaction products were exclusively observed in the alpha granules of the platelets. Whithin the alpha granules, arylsulfatase activity appeared to locMize in variable patterns, i.e. reaction products confined mainly at the peripheral region in many granules, while they deposited heavily throughout the granule matrices in some others. In a blood platelets, each alpha granule showed the different staining pattern which indicated more variable functional heterogeneity in the granules.
Introduction Since t h e i n t r o d u c t i o n of t h e concept of lysosome b y de D u v e (1963), the s t u d y of this field has d e v e l o p e d extensively. HistochemieMly, acid p h o s p h a t a s e is v e r y f r e q u e n t l y used for t h e m a r k e r of lysosomes. A r y l s u l f a t a s e A a n d B are other e n z y m e s which localize in the lysosomes. Using p - n i t r o c a t e e h o l sulfate as a s u b s t r a t e , t h e t e c h n i q u e in d e m o n s t r a t i n g a r y l s u f a t a s e a c t i v i t y in the electron microscopic level has been d e v e l o p e d b y two groups (Goldfischer, 1965; t I o p s u H a v u et al., 1967). I n the h e m a t o l o g i c a l field, these enzymes were f o u n d to localize in p r i m a r y , a n d t e r t i a r y granules of r a b b i t h e t e r o p h i l s (Bainton a n d F a r q u h a r , 1968; M u r a t a a n d Spicer, I973a). t~abbit eosinophils a n d m o n o c y t e s were also k n o w n to c o n t a i n a r y l s u l f a t a s e positive granules (Bainton a n d F a r q u h a r , 1970; Nicholas et al., 1971). This p a p e r deals w i t h the a r y l s u l f a t a s e B a c t i v i t y of the r a b b i t blood platelets.
Material and Methods Healthy albino rabbits of both sexes were used for this experiment. Twenty to 25 ml of peripheral blood was drawn into a heparinized syringe by cardiac punctures. To make the bully coat specimen, a modified Anderson's method was used (Murata et al., 1973b). Minced bully coat was prefixed 1 hr to 3 hrs at 4~ in 3% glutaraldehyde (Sabatini et al., 1963) buffered with 0.1 M cacodylate buffer (pH 7.2). They were kept in 0.1 M cacodylate buffer containing 7.5% sucrose overnight. Forty ~z thick sections were cut with a cryostat. They were incubated in Goldfischer's medium for 60-90 minutes at 37~ (Goidfischer, 1965). After incubation, sections were washed thoroughly with 0.1 M veronal acetate buffer (pH 7.4) and lead sulfate was converted to lead sulfide by dipping these carriers into 2 % ammonium sulfide
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solution buffered with 0.1 IV[veronal acetate buffer (pH 7.4) for 5-15 minutes at room temperature, and thereafter rinsed thoroughly through several changes of 0.1 1V[veronal acetate buffer (pH 7.4). As a control, 40 ~m thick sections were incubated in the substrate free medium for 60-90 minutes at 37~ and the conversion of lead sulfate to lead sulfide was done as described above. Thick sections were dehydrated through a graded series of alcohol and propylene oxide and embedded in Epon (Luft, 1961). Thin sections were cut with a Porter Blum MT-1 microtome and were examined with a Hitachi HS-8-1 electron microscope usually without any electron staining.
Results Observing the rabbit platelets, reaction products composed of particles of high electron density were observed in the alpha granules. Though several to m a n y granules being discernible in a blood platelet, like acid phosphatase activity of the blood platelet, not all granules showed a positive reaction. Reaction products were observed only in a few granules (Figs 1, 2). The distribution p a t t e r n of the reaction product in a granule was variable. I n some granules, reaction products were observed at the peripheral rim of the granule (Figs. 3a, b), in others, a quarter to one third of whole rim contained depositions of reaction product (Figs. 3c-e), sometimes the reaction p r o d u c t was discernible all t h r o u g h the peripheral granule rim (Fig. 3f). A quarter to one third of the granule matrix were deposited with reaction products in some granules (Figs. 3g-j). Nucleoid regions of the granules were especially heavily deposited with lead sulfide in others (Fig. 3h). I n the strongest reaction, the deposition of reaction products was observed to locahze t h r o u g h o u t the granules (Figs. 3k, 1). I n the cytoplasm, one to several serotonin containing granules were usually found. However, these granules did not show a n y arylsulfatase activity (Figs. 1, 2). No reaction p r o d u c t was found in the mitochondria, endoplasmic retieulum or cytoplasmic matrix. Reaction p r o d u c t in the Golgi apparatus has not been ascertained hitherto. I n the control, omitting the substrate, no reaction product was observed t h r o u g h o u t the platelets.
Discussion I n a former experiment, it has been shown t h a t the platelet extracts of small laboratory animals, like the guinea pig and the rabbit, had ten times higher activity of arylsulfatase t h a n the h u m a n (Pols and Rotrekl, 1967). I n the human, acid phosphatase activity of the platelet was strong, wherease arylsulfatase activity was almost negligible. Acid phosphatase activity was approximate]y one
Fig. 1. Electron photomicrograph showing ultrastructural localization of arylsulfatase B activity in the rabbit blood platelet. A cap shaped deposit of the reaction product was observed at the margin of the upper left granule, while very faint reaction product was noticed at the peripheral rims of the other granules. Several serotonin containing granules were free of reaction product. No electron staining. • 51250 Fig. 2. Electron photomicrograph of arylsulfatase B activity in the rabbit blood platelets. Only the peripheral regions of the granules were deposited with reaction products. Several electron dense serotonin containing granules were lacking of reaction product. No electron staining. • 41250
Arylsulfatasc B Activity in Rabbit Blood Platelets
309
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F. Murata et al.
Fig. 3a--1. Arylsulfatase B activity in the rabbit blood lolatelet granules, showing many different staining patterns of the activity, starting weak reaction (a-d), medium reaction (e-i) and strong reaction (j-l). No electron staining. Magnifications are (a) • 41250, (b) • 55 000, (c) • 35000, (d) • 51250, (e) • (f) • 51250, (g) • 38000, (h) • 41250, (i) • 51250, (j) • 51250, (k) • 33000, (1) • 51250
thousand times higher than arylsulfatase in the human (Day et al., 1968). Therefore, the rabbit was selected for the experimental animal in the present study. To the best of our knowledge, no report on the fine structural localization of arylsulfatase activity of the human has been published up to the present time. The reason for this m a y chiefly be attributable to its very low activity. Even in the rabbit, the demonstration of arylsulfatase B activity was much more difficult than acid phosphatase. The extraneous reaction was sometimes inevitable. In m a n y cell organellae of the rabbit blood platelets, the reaction products were almost exclusively found in the granules. However, the staining pattern of the granules was very variable. In one blood platelet, staining pattern ranged from no reaction deposit to very heavy staining. Since alpha granules and serotonin containing granules arc only two main types of granules in the rabbit blood platelets, this variety would reflect either the difference of total enzyme content of each granule or the difference of limiting membrane changes due to fixation. Therefore, these results indicate more variable functional heterogeneity in the alpha granules. Arylsulfatase B activity has been reported to localize not only in the lysosome but also in mitochondria of rat kidney proximal covoluted tubule epithelial cells (Makita and Sandborn, 1971). They perfused the rat kidney, and used DMSO as an additive to the fixative and found arylsulfatase B activity in the space between the inner and outer membanes and the intracristal spaces of the mitochondria. In the present study, we have not yet encountered a positive arylsulfatase activity in the mitochondria of the blood platelet. It is very probable
Arylsulfatase B Activity in Rabbit Blood Platelets
311
that the perfusion fixation with DMSO might have changed the permeability of the membrane system and brought about the different results. In heterophil leukocytes and epiphyseal plates, arylsulfatase activity was found in the Golgi apparatus (Bainton and Farquhar, 1968; Thyberg, 1972). No arylsulfatase activity was reported in the Golgi apparatus of thyroid follicle cells (Seljelid and Helminen, 1968). Since blood platelets had poorly developed Golgi apparatus or no Golgi apparatus, arylsulfatase activity was not ascel~ained yet in the Golgi apparatus in this experiment. As it was pointed out before, the lysosomal concept requires the concomitant presence of several hydrolytic enzymes with acid pH optimum within a membrane bound vesicles (de Duve, 1963; Beck and Llyod, 1969). Although the association of hydrolytic enzyme activities with the platelet granules has been reported (Marcus et al., 1966), the claim was proposed that human blood platelet alpha granules were not typical lysosomes (Siegel and Liiseher, 1967). Among acid phosphatase, beta-glucuronidase, and cathepsin, only acid phosphatase showed a higher activity in granule fraction, while the other two enzymes showed higher activity in microvesicles than granule fraction. It is a well known fact that beta-glucuronidase activity localize not only lysosome but in microsome as well (Ide and Fishman, 1969). Histoehemically, acid phosphatase in the rabbit platelet alpha granules was reported formerly (Bak et al., 1969) and arylsulfatase B activity has been demonstrated in this experiment. Therefore, the result obtained in this experiment would support the concept that alpha granules of rabbit platelets are typical lysosomes. Fixation time for some tissues was a critical factor to demonstrate the arylsulfatase activity in the electron microscopic level (Goldfischer, 1965). On the other hand, it is interesting to note that in thyroid follicle cells, as to the intensity of the precipitates ions, no clear cut differences were reported for the tissues fixed for various period of times (Seljelid and Helminen, 1968). In this experiment, extraneous reaction was observed sometimes, which could possibly be attributable to the fixation time. Heparin ions were used as an anticoagulant in this experiment. A slight increase of arylsulfatase activity with polyanionic anticoagulant heparin has been reported (Austin and Bischel, 1961). On the contrary, no inhibitory effect on arylsulfatase activity with heparin and EDTA has been reported by another group (Smutka and Brunning, 1969). Though the exact investigation has not been undertaken as the effect of heparin on the arylsulfatase activity, from the observation of repeated experiments, it would be beyond dispute to neglect the effect of heparin on the arylsulfatase activity in these experiments. Acknowledgements. This study was supported in part by research grants No. AM-10956 and AM-11028 from National Institutes of Health, U.S.A. and No. 901047 from the Basic Scientific Research Fund of the Ministry of Education, Ja.pan. The authors are grateful to 1VImes.Betty J. Hall and Nancy M. Smythe for the technical assistances during the course of this study. References Austin, J.H., Bischel, M. A. : Histochemical method for sulfatase activity in heroic cells and organ imprints. Blood 17, 212-214 (1961)
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F. Murata et al.
Bainton, D.F., Farquhar, M.G.: Differences in enzyme content of azurophil and specific granules of polymorphonuelear leukocytes. II. Cytochemistry and electron microscopy of the bone marrow cells. J. Cell Biol. 89, 299-317 (1968) Bainton, D.F., Farquhar, M. G. : Segregation and packaging of granule enzymes in eosinophilic leukoeytes. J. Cell Biol. 45, 54-73 (1970) Bak, I.J., May, B., Hasler, 1%.: Electron microscopical demonstration of acid phosphatase in blood platelets. Z. Zellforseh. 96, 641-648 (1969) Beck, F., Llyod, J.B.: Lysosomes in biology and pathology. Histochemistry and electron microscopy of lysosomes, voh 2. p. 567-599. Amsterdam: North Holland Pubh 1969 Day, H.J., Holmsen, H., Hovig, T.: Subeellular particles of human platelets. Seand. J. Haematoh Suppl. No. 7, 1-35 (1968) de Duve, C. : General properties of lysosomes. The lysosome concept. Ciba Foundation Symposium: lysosomc (I~euek, A.V.S. and Cameron, M.P. eds.), p. 1-35, Boston, Mass., Little, Brown and Company 1963 Goldfischer, S. : The eytochemieal demonstration of lysosomal arylsulfatase activity by light and electron microscopy. J. Histoehem. Cytoehem. 11~,520-523 (1965) I-Iopus-Havu, V.K., Arstila, A.U., Helminen, H.J., Kalimo, H. 0., Glenner, G. G. : Improvements in the method for the electron microscopic localization of aryl-sulphatase activity. Histochemie 8, 54-64 (1967) Ide, H., Fishman, W. I-I.: Dual localization of fl-glucuronidase and acid phosphatase in lysosomes and microsomes. II. membrane associated enzymes. Histoehemie 20, 300-321 (1969) Luft, J.I-I. : Improvements in epoxy resin embedding method. J. biophys, biochem. Cytoh 9, 409414 (1961) Makita, T., Sandborn, F.B.: Ultrastruetural localization of arylsulfatase B in mitoehondria of epithelial cells of the proximal convoluted tubules of the rat kidney. Experientia (Basel) 27, 187-189 (1971) Marcus, A.J., Zucker-Franklin, D., Safier, L.B., Ullman, H.L. : Studies on human platelet granules and membrane. J. elin. Invest. 45, 14-28 (1966) Murata, F., Spicer, S.S.: Morphological and eytochemical studies of rabbit heterophilic leukoeytes: evidence for tertiary granules. Lab. Invest. 29, 65-72 (1973a) Murata, F., Hardin, J.H., Spicer, S. S. : Coexistence of acid phosphatase and acid mucosubstance in the nucleoid of human blood platelet granules. Histoehemie. 85, 319-329 (19735) Nicholas, B.A., Bainton, D.F., Farquhar, M. G.: Differentiation of monocytes: origin, nature, and fate of their azurophil granules. J. Cell Biol. 50, 498-515 (1971) Pols J., Rotrekl, B.: Aryl sulphatases of human blood platelets. Nature (Lend.) 214, 187-188 (1967) Sabatini, D.D., Bensch, K., Barrnett, l~.J.: Cytochemistry and electron microscopy: the preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. J. Cell Biol. 17, 19-58 (1963) Seljelid, 1~., Helminen, H. J. : The localization of arylsulfatase activity in thyroid follicle cells. J. Histoehem. Cytoehem. 16, 407412 (1968) Siegel, A., Liischer, E.F.: Non-identity of the ~ granules of human blood platelets with typical lysosomes. Nature (Lend.) 215, 745-747 (1967) Smutk~, P., Brunning, l%.D.: An evaluation of nuclear arylsulfatase activity in acute leukemias. Acta haemat. (Basel) 41, 290-295 (1969) Thyberg, J. : Ultrastructur~l localization of arylsulfatase activity in the epiphyseal plate. J. Ultrastruct. l~es. 38, 332-342 (1972) Fusayoshi Murata, M. D., Ph.D. Associate Professor of Anatomy Tetsuji Nagata, lV[.D., Ph.D. Professor of Anatomy First Department of Anatomy Shinshu University School of Medicine Matsumoto 390
Japan
Samuel S. Spicer, M. D. Professor of Pathology Department of Pathology Medical University of South Carolina Charleston, S. C. 29401 U.S.A.
Fine Structural Localization of Arylsulfatase B Activity in the Rabbit Blood Platelets F u s a y o s h i M u r a t a , T e t s u j i N a g a t a , a n d Samuel S. Spieer Department of Anatomy, Shinshu University School of Medicine, 5Iatsumoto, Japan and Department of Pathology, Medical University of South Carolina, Charleston, S. C., U.S.A. Received June 9, 1975 Summary. Fine structural localization of arylsulfatase in the rabbit blood platelets has been investigated in this study. Among many cell organellae, reaction products were exclusively observed in the alpha granules of the platelets. Whithin the alpha granules, arylsulfatase activity appeared to locMize in variable patterns, i.e. reaction products confined mainly at the peripheral region in many granules, while they deposited heavily throughout the granule matrices in some others. In a blood platelets, each alpha granule showed the different staining pattern which indicated more variable functional heterogeneity in the granules.
Introduction Since t h e i n t r o d u c t i o n of t h e concept of lysosome b y de D u v e (1963), the s t u d y of this field has d e v e l o p e d extensively. HistochemieMly, acid p h o s p h a t a s e is v e r y f r e q u e n t l y used for t h e m a r k e r of lysosomes. A r y l s u l f a t a s e A a n d B are other e n z y m e s which localize in the lysosomes. Using p - n i t r o c a t e e h o l sulfate as a s u b s t r a t e , t h e t e c h n i q u e in d e m o n s t r a t i n g a r y l s u f a t a s e a c t i v i t y in the electron microscopic level has been d e v e l o p e d b y two groups (Goldfischer, 1965; t I o p s u H a v u et al., 1967). I n the h e m a t o l o g i c a l field, these enzymes were f o u n d to localize in p r i m a r y , a n d t e r t i a r y granules of r a b b i t h e t e r o p h i l s (Bainton a n d F a r q u h a r , 1968; M u r a t a a n d Spicer, I973a). t~abbit eosinophils a n d m o n o c y t e s were also k n o w n to c o n t a i n a r y l s u l f a t a s e positive granules (Bainton a n d F a r q u h a r , 1970; Nicholas et al., 1971). This p a p e r deals w i t h the a r y l s u l f a t a s e B a c t i v i t y of the r a b b i t blood platelets.
Material and Methods Healthy albino rabbits of both sexes were used for this experiment. Twenty to 25 ml of peripheral blood was drawn into a heparinized syringe by cardiac punctures. To make the bully coat specimen, a modified Anderson's method was used (Murata et al., 1973b). Minced bully coat was prefixed 1 hr to 3 hrs at 4~ in 3% glutaraldehyde (Sabatini et al., 1963) buffered with 0.1 M cacodylate buffer (pH 7.2). They were kept in 0.1 M cacodylate buffer containing 7.5% sucrose overnight. Forty ~z thick sections were cut with a cryostat. They were incubated in Goldfischer's medium for 60-90 minutes at 37~ (Goidfischer, 1965). After incubation, sections were washed thoroughly with 0.1 M veronal acetate buffer (pH 7.4) and lead sulfate was converted to lead sulfide by dipping these carriers into 2 % ammonium sulfide
308
F. Murata et al.
solution buffered with 0.1 IV[veronal acetate buffer (pH 7.4) for 5-15 minutes at room temperature, and thereafter rinsed thoroughly through several changes of 0.1 1V[veronal acetate buffer (pH 7.4). As a control, 40 ~m thick sections were incubated in the substrate free medium for 60-90 minutes at 37~ and the conversion of lead sulfate to lead sulfide was done as described above. Thick sections were dehydrated through a graded series of alcohol and propylene oxide and embedded in Epon (Luft, 1961). Thin sections were cut with a Porter Blum MT-1 microtome and were examined with a Hitachi HS-8-1 electron microscope usually without any electron staining.
Results Observing the rabbit platelets, reaction products composed of particles of high electron density were observed in the alpha granules. Though several to m a n y granules being discernible in a blood platelet, like acid phosphatase activity of the blood platelet, not all granules showed a positive reaction. Reaction products were observed only in a few granules (Figs 1, 2). The distribution p a t t e r n of the reaction product in a granule was variable. I n some granules, reaction products were observed at the peripheral rim of the granule (Figs. 3a, b), in others, a quarter to one third of whole rim contained depositions of reaction product (Figs. 3c-e), sometimes the reaction p r o d u c t was discernible all t h r o u g h the peripheral granule rim (Fig. 3f). A quarter to one third of the granule matrix were deposited with reaction products in some granules (Figs. 3g-j). Nucleoid regions of the granules were especially heavily deposited with lead sulfide in others (Fig. 3h). I n the strongest reaction, the deposition of reaction products was observed to locahze t h r o u g h o u t the granules (Figs. 3k, 1). I n the cytoplasm, one to several serotonin containing granules were usually found. However, these granules did not show a n y arylsulfatase activity (Figs. 1, 2). No reaction p r o d u c t was found in the mitochondria, endoplasmic retieulum or cytoplasmic matrix. Reaction p r o d u c t in the Golgi apparatus has not been ascertained hitherto. I n the control, omitting the substrate, no reaction product was observed t h r o u g h o u t the platelets.
Discussion I n a former experiment, it has been shown t h a t the platelet extracts of small laboratory animals, like the guinea pig and the rabbit, had ten times higher activity of arylsulfatase t h a n the h u m a n (Pols and Rotrekl, 1967). I n the human, acid phosphatase activity of the platelet was strong, wherease arylsulfatase activity was almost negligible. Acid phosphatase activity was approximate]y one
Fig. 1. Electron photomicrograph showing ultrastructural localization of arylsulfatase B activity in the rabbit blood platelet. A cap shaped deposit of the reaction product was observed at the margin of the upper left granule, while very faint reaction product was noticed at the peripheral rims of the other granules. Several serotonin containing granules were free of reaction product. No electron staining. • 51250 Fig. 2. Electron photomicrograph of arylsulfatase B activity in the rabbit blood platelets. Only the peripheral regions of the granules were deposited with reaction products. Several electron dense serotonin containing granules were lacking of reaction product. No electron staining. • 41250
Arylsulfatasc B Activity in Rabbit Blood Platelets
309
310
F. Murata et al.
Fig. 3a--1. Arylsulfatase B activity in the rabbit blood lolatelet granules, showing many different staining patterns of the activity, starting weak reaction (a-d), medium reaction (e-i) and strong reaction (j-l). No electron staining. Magnifications are (a) • 41250, (b) • 55 000, (c) • 35000, (d) • 51250, (e) • (f) • 51250, (g) • 38000, (h) • 41250, (i) • 51250, (j) • 51250, (k) • 33000, (1) • 51250
thousand times higher than arylsulfatase in the human (Day et al., 1968). Therefore, the rabbit was selected for the experimental animal in the present study. To the best of our knowledge, no report on the fine structural localization of arylsulfatase activity of the human has been published up to the present time. The reason for this m a y chiefly be attributable to its very low activity. Even in the rabbit, the demonstration of arylsulfatase B activity was much more difficult than acid phosphatase. The extraneous reaction was sometimes inevitable. In m a n y cell organellae of the rabbit blood platelets, the reaction products were almost exclusively found in the granules. However, the staining pattern of the granules was very variable. In one blood platelet, staining pattern ranged from no reaction deposit to very heavy staining. Since alpha granules and serotonin containing granules arc only two main types of granules in the rabbit blood platelets, this variety would reflect either the difference of total enzyme content of each granule or the difference of limiting membrane changes due to fixation. Therefore, these results indicate more variable functional heterogeneity in the alpha granules. Arylsulfatase B activity has been reported to localize not only in the lysosome but also in mitochondria of rat kidney proximal covoluted tubule epithelial cells (Makita and Sandborn, 1971). They perfused the rat kidney, and used DMSO as an additive to the fixative and found arylsulfatase B activity in the space between the inner and outer membanes and the intracristal spaces of the mitochondria. In the present study, we have not yet encountered a positive arylsulfatase activity in the mitochondria of the blood platelet. It is very probable
Arylsulfatase B Activity in Rabbit Blood Platelets
311
that the perfusion fixation with DMSO might have changed the permeability of the membrane system and brought about the different results. In heterophil leukocytes and epiphyseal plates, arylsulfatase activity was found in the Golgi apparatus (Bainton and Farquhar, 1968; Thyberg, 1972). No arylsulfatase activity was reported in the Golgi apparatus of thyroid follicle cells (Seljelid and Helminen, 1968). Since blood platelets had poorly developed Golgi apparatus or no Golgi apparatus, arylsulfatase activity was not ascel~ained yet in the Golgi apparatus in this experiment. As it was pointed out before, the lysosomal concept requires the concomitant presence of several hydrolytic enzymes with acid pH optimum within a membrane bound vesicles (de Duve, 1963; Beck and Llyod, 1969). Although the association of hydrolytic enzyme activities with the platelet granules has been reported (Marcus et al., 1966), the claim was proposed that human blood platelet alpha granules were not typical lysosomes (Siegel and Liiseher, 1967). Among acid phosphatase, beta-glucuronidase, and cathepsin, only acid phosphatase showed a higher activity in granule fraction, while the other two enzymes showed higher activity in microvesicles than granule fraction. It is a well known fact that beta-glucuronidase activity localize not only lysosome but in microsome as well (Ide and Fishman, 1969). Histoehemically, acid phosphatase in the rabbit platelet alpha granules was reported formerly (Bak et al., 1969) and arylsulfatase B activity has been demonstrated in this experiment. Therefore, the result obtained in this experiment would support the concept that alpha granules of rabbit platelets are typical lysosomes. Fixation time for some tissues was a critical factor to demonstrate the arylsulfatase activity in the electron microscopic level (Goldfischer, 1965). On the other hand, it is interesting to note that in thyroid follicle cells, as to the intensity of the precipitates ions, no clear cut differences were reported for the tissues fixed for various period of times (Seljelid and Helminen, 1968). In this experiment, extraneous reaction was observed sometimes, which could possibly be attributable to the fixation time. Heparin ions were used as an anticoagulant in this experiment. A slight increase of arylsulfatase activity with polyanionic anticoagulant heparin has been reported (Austin and Bischel, 1961). On the contrary, no inhibitory effect on arylsulfatase activity with heparin and EDTA has been reported by another group (Smutka and Brunning, 1969). Though the exact investigation has not been undertaken as the effect of heparin on the arylsulfatase activity, from the observation of repeated experiments, it would be beyond dispute to neglect the effect of heparin on the arylsulfatase activity in these experiments. Acknowledgements. This study was supported in part by research grants No. AM-10956 and AM-11028 from National Institutes of Health, U.S.A. and No. 901047 from the Basic Scientific Research Fund of the Ministry of Education, Ja.pan. The authors are grateful to 1VImes.Betty J. Hall and Nancy M. Smythe for the technical assistances during the course of this study. References Austin, J.H., Bischel, M. A. : Histochemical method for sulfatase activity in heroic cells and organ imprints. Blood 17, 212-214 (1961)
312
F. Murata et al.
Bainton, D.F., Farquhar, M.G.: Differences in enzyme content of azurophil and specific granules of polymorphonuelear leukocytes. II. Cytochemistry and electron microscopy of the bone marrow cells. J. Cell Biol. 89, 299-317 (1968) Bainton, D.F., Farquhar, M. G. : Segregation and packaging of granule enzymes in eosinophilic leukoeytes. J. Cell Biol. 45, 54-73 (1970) Bak, I.J., May, B., Hasler, 1%.: Electron microscopical demonstration of acid phosphatase in blood platelets. Z. Zellforseh. 96, 641-648 (1969) Beck, F., Llyod, J.B.: Lysosomes in biology and pathology. Histochemistry and electron microscopy of lysosomes, voh 2. p. 567-599. Amsterdam: North Holland Pubh 1969 Day, H.J., Holmsen, H., Hovig, T.: Subeellular particles of human platelets. Seand. J. Haematoh Suppl. No. 7, 1-35 (1968) de Duve, C. : General properties of lysosomes. The lysosome concept. Ciba Foundation Symposium: lysosomc (I~euek, A.V.S. and Cameron, M.P. eds.), p. 1-35, Boston, Mass., Little, Brown and Company 1963 Goldfischer, S. : The eytochemieal demonstration of lysosomal arylsulfatase activity by light and electron microscopy. J. Histoehem. Cytoehem. 11~,520-523 (1965) I-Iopus-Havu, V.K., Arstila, A.U., Helminen, H.J., Kalimo, H. 0., Glenner, G. G. : Improvements in the method for the electron microscopic localization of aryl-sulphatase activity. Histochemie 8, 54-64 (1967) Ide, H., Fishman, W. I-I.: Dual localization of fl-glucuronidase and acid phosphatase in lysosomes and microsomes. II. membrane associated enzymes. Histoehemie 20, 300-321 (1969) Luft, J.I-I. : Improvements in epoxy resin embedding method. J. biophys, biochem. Cytoh 9, 409414 (1961) Makita, T., Sandborn, F.B.: Ultrastruetural localization of arylsulfatase B in mitoehondria of epithelial cells of the proximal convoluted tubules of the rat kidney. Experientia (Basel) 27, 187-189 (1971) Marcus, A.J., Zucker-Franklin, D., Safier, L.B., Ullman, H.L. : Studies on human platelet granules and membrane. J. elin. Invest. 45, 14-28 (1966) Murata, F., Spicer, S.S.: Morphological and eytochemical studies of rabbit heterophilic leukoeytes: evidence for tertiary granules. Lab. Invest. 29, 65-72 (1973a) Murata, F., Hardin, J.H., Spicer, S. S. : Coexistence of acid phosphatase and acid mucosubstance in the nucleoid of human blood platelet granules. Histoehemie. 85, 319-329 (19735) Nicholas, B.A., Bainton, D.F., Farquhar, M. G.: Differentiation of monocytes: origin, nature, and fate of their azurophil granules. J. Cell Biol. 50, 498-515 (1971) Pols J., Rotrekl, B.: Aryl sulphatases of human blood platelets. Nature (Lend.) 214, 187-188 (1967) Sabatini, D.D., Bensch, K., Barrnett, l~.J.: Cytochemistry and electron microscopy: the preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. J. Cell Biol. 17, 19-58 (1963) Seljelid, 1~., Helminen, H. J. : The localization of arylsulfatase activity in thyroid follicle cells. J. Histoehem. Cytoehem. 16, 407412 (1968) Siegel, A., Liischer, E.F.: Non-identity of the ~ granules of human blood platelets with typical lysosomes. Nature (Lend.) 215, 745-747 (1967) Smutk~, P., Brunning, l%.D.: An evaluation of nuclear arylsulfatase activity in acute leukemias. Acta haemat. (Basel) 41, 290-295 (1969) Thyberg, J. : Ultrastructur~l localization of arylsulfatase activity in the epiphyseal plate. J. Ultrastruct. l~es. 38, 332-342 (1972) Fusayoshi Murata, M. D., Ph.D. Associate Professor of Anatomy Tetsuji Nagata, lV[.D., Ph.D. Professor of Anatomy First Department of Anatomy Shinshu University School of Medicine Matsumoto 390
Japan
Samuel S. Spicer, M. D. Professor of Pathology Department of Pathology Medical University of South Carolina Charleston, S. C. 29401 U.S.A.
