EXPERIMENTAL
NEUROLOGY
57, 1042-1048 (1977)
RESEARCH The
Subcellular
Distribution in Mouse
of S-Nucleotidase Cerebellum
E.
of rlmtomy
Dcpartwccnt
Rcccivcd
26, 1977;
Activity
MARANI
and Embryology, Lcidm. The January
NOTE
University Netherlauds revision
of Leidefl,
rcccived
May
Wasse~zaarsewcg
62,
12, 1977
Because the peculiar localization of 5’-nucleotidase in longitudinal bands in the molecular layer of the cerebellum of mouse (Fig. 1A) and rat (1 l13, 18) has not been correlated with the histologic structure of the molecular layer, Pilcher and Jones (17) used fractionation techniques to determine the subcellular distribution of this enzyme in the mouse. They suggested that the distribution of S-nucleotidase in the various fractions of nlousc cerebellum, prepared according to Gray and Whittaker (3), reflects that of an axoplasmic location (17) and of specific synapses containing S-nucleotidase. They indicate that it may be possible to distinguish two t!-pes of synapses, those with and without 5’-nucleotidase (17). In the course of a study of the ultrastructural localization of 5’-nucleotidase in the cerebellum of the mouse, the fractionation experiments of Pilcher and Jones ( 17) were repeated using a slightly different technique (20). Our differential centrifugation results do not entirely support their suggestion of a localization of 5’-nucleotidase in a specific type of synapse in the molecular layer. but are not contradictory to their results (17). *Adult (6 months to 1 year of age) female mice (USA, Naval Medical Research Institute strain) were killed by chloroform anesthesia and the cerebella were rapidly dissected. All stages of the fractionation procedure were performed at 0 to 4°C. Each homogenate contained three pooled cerebella (175 mg) in 3.5 ml 0.32 M sucrose solution. The cerebella were pottered and fractions were prepared according to the method of Whittaker Abbreviation
: AMP-adenosine
S-phosphate. 1042
Copyright All rights
(9 1977 by Academic Press. Inc. of reproduction in any form reserved.
ISSN
0014-4886
CEKEBELLAR
IiUCLEOTIDASE
ACTIVITY
10-43
ct ul. (20 j . Gradient centrifugation was done according to the method used with by Van der Krogt (7, 16, 19), using a Spinco L2-65B ultracentrifuge a SW 27 rotor, at 82.5009 and 4°C. Sucrose solutions at PH i, prepared by adding some crystals of Tris, were used. Addition of the Tris crystals did not coacervate particles in P2 (unpublished results, Van Dijke). The SF crude synaptosomal-mitochondrial fraction ( 1.5 ml) was used. (;ratlients were collected in about 20 fractions, each of 1.5 ml. Protein cleterminntions were made according to Lowry c,t nl. (10). The S-nucleotidase activit! was measured after Persijn ct al. (14, 15) : incubation time was 1 11. L,actate dehydrogenase, a cytoplasmic marker, was assayed according to Kornberg (5). The monoamine oxidase activity was determined with kynuramine as substrate, according to the Kroon modification (S, 9) of the method of Kraml (6). Electron microscopic procedures were carried out by perfusion-fixation at a velocity of 15 ml/min. Paraformaldehyde, 40/o, in 0.16 M cacodylate buffer, PH 7.3, osmolality 1550, was perfused for 2 min. followed by a 2-min perfusion-fixation with 2.5 % glutaraldehyde in 0.16 M cacodylate buffer, to which was added 5.4 g/liter sucrose (final PH 7.3, osmolality 570 1. These steps were followed by an incubation perfusion of 5 min, at the same velocity, and 37°C with the medium of Scott (18). The cerebellum was dissected free, and the tissue was cut in sections 200 pm thick and su11sequently incubated 0.5 h in the incubation medium. After incubation, the sections were fixed 0.5 h in the glutaralclehyde solution and prepared in the routine manner for 0~0~ fixation and Epon embedding. To exclude a possible influence of sucrose, 5’-nucleotidase activity \vas determined in two homogenates using different sucrose concentrations. It was found that sucrose does not influence the cerebellar 5’-nucleotidase activity. Adenosine 5’-phosphate (AMP) deaminase can influence the Persijn t~f al. determination (14, 15). It is important to note that this activity in the cerebellum is of the order of 8 units per liter (unpublished results), which is very low compared to the 5’-nucleotidase activity (see Table 1) . The main difference between our investigations and those of Pilcher and Jones (17) is the use of the method of Persijn ct al. (13, 15) for the determination of 5’-nucleotidase activity, instead of the phosphate method of Fiske and SubbaRow (2) used by Pilcher and Jones and others (1). According to Bartlett (1) the Fiske-SubbaRow method is unsatisfactory because it is dependent on its reagents, having a narrow range of permissible H,SO, concentration, and it also produces an unstable color. Moreover, our preliminary experiments with this method showed it to be time and temperature dependent. The elaborate precautions necessarv to obtain reproducible results with the Fiske-SubbaRow method (2) caused us to
1044
E.
MARANI
TABLE 5’-Nucleotidase
Fractions
H Pl P2 S2 P3 s3
Mean,
107 23.6 47.6 38.5
Recovery
1
Activity in Various Fractions Obtained Centrifugation Techniques at pH 7.2” Expt
f f zk f
l-6
2.6 0.6 2.0 2.6
Expt
10
Expt
11
Expt
12
by Differential
Mean, Expt 10-12
Mean specific activity per milligram protein
15 39
14 36
15 39
15 38
3.3 6.5
15 30
14 35
15 30
15 32
6.2 7.1
103 y0
I’ The activities are expressed as absolute total activity (Expt 10-12) in units per II-PAC, and WCC).
Recovery values (Expt l-6) liter (Commission
70$0
or as percentages of the on Clinical Chemistry,
prefer the method of Persijn et ~2. (14, 15). The extra advantage of this method is in the substrate retention; therefore, no corrections have to be made for unspecific phosphatase activity. The results of six fractionation experiments summarized in Table 1 show that the specific activities of the subcellular fractions Pl, P2, and S2 are reproducible, Our measurements of the percentage activity of S-nucleotidase in the Pl fraction (Table 1) mainly containing nuclei and debris (20) are in the same range as those of Pilcher and Jones (17). As shown in Table 1, only 36 to 4470 of the total activity was recovered in the P2 fraction compared to 67 to 787 0 in Pilcher and Jones’s experiments (17). Conversely, the percentage of activity in the supernatant S2 is high in our experiments (36%) and low in those of Pilcher and Jones (17). It seems unlikely that this difference is caused only by the use of a different method for preparing the P2, although Whittaker et al. (20) stated that this difference resulted in a lower yield of synaptosomes. The difference can rather be explained by the solubility of the 5’-nucleotidase, also known from previous experiments ( 12, 13)) and the problem of accurate 5’-nucleotidase determination [measuring the total AMP ASE activity and subtracting the p-glycerophosphatase activity at pH 7.2 (17) versus the substrate retention method at PH 7.2 (14,15)1. In the experiments shown in Table 1 the relative activities of the P3 fraction are nearly the same as in Pilcher and Jones’s experiments. However, nearly 32% was recovered in our soluble fraction S3, whereas those
CEREBELLAR
NUCLEOTIDASE
ACTIVITY
1045
authors (17) did not find any activit) in the supernatant of pellet P3 although the same speed and time of centrifugation were applied. Considering the relative distribution of the 5’-nucleotidase activity over the sum of the activity of the different fractions (see Table 1)) it is obvious that the P2 and S3 fractions have the same S-nucleotidase activity and that the P3 fraction contains only half that of the P2 or S3 fraction. The comparison of the mean specific activities of the fractions (Table 1) does not indicate a difference between P2. P3. and S.3 ratios per milligram protein. To check whether or not the S-nucleotidase activity in the synaptosomal fraction P2 is bound to a special type of synaptosome. we used the linear sucrose-gradient technique on crude P2 fractions. The results of three experiments, shown in Fig. 2, indicate that S-nucleotidase is not bound to all mitochondria, because there is a difference in the peaks for S’-nucleotidase and monoaminoxidase. The peak of the cytoplasmic marker, lactate dell!-clrogenase, coincides with the %nucleotidase peak. The sharpness of the peak for S-nucleotidase and the overlap with the top of the lactate dehydrogenase peak can be seen as an argument for the binding of part of the 5’-nucleotidase activity to a certain particle within the synaptosome pool. At low sucrose molarities in the gradients a large portion of S’-nucleotidase is found that is interpreted as S-nucleotidase which may correspond to a certain soluble form of S-nucleotidase ( 12, 13) or which is bound to ruptured structures. \\%en our results are compared to the measurements of Israel and Fran&on-Mastour (4) in fractions of the cerebral cortex of the rat, prepared according to the same technique as in our experiments (20), their values for Pl, P2, and P3 are found to be in good accordance with our determinations in mouse cerebellum. However, their value for S3 (9%) is substantially lower than that in our experiments, but higher than that in the Pilcher and Jones measurements [OR, (17)]. This indicates that a part of the 5’-nucleotidase activity is soluble in rat cerebral cortex also. The higher values for the S3 in our experiments could mean that the 5’-nucleotidase is loosely bound to structures in the cerebellum. As a consequence of this study and the comparisons made with the work of others (4, 17)) one cannot decide on a uniform location of S-nucleotidase in the synaptosomes. The results found in our experiments for the P2 fraction are more in agreement with the results of Israel and FrachonMastour (4) in the rat cortex than with the results of Pilcher and Jones ( 17). Therefore any conclusion based on these differential centrifugation results about a distribution that reflects that of specific synapses containing 5’-nucleotidase is premature. In conclusion one can suggest that the 5’-nucleotidase is not very strongly bound to one or more structure(s) that
1046
E.
MARANI
FIG. 1. Light and electron photomicrographs of results after incubations according to Scott (18). A-Detail of the light microscopic location of 5’-nucleotidase in uvula (IX) and pyramis (VIII). X 20. B-Electron microscopic location of 5’-nucleotidase reaction products in the subsurface cisternae of a Purkinje cell dendrite. X 30,000.
CEREBELLAR
NUCLEOTIDASE
0.86
1.2
1047
ACTIVITY
04
M.
FIG. 2. Linear sucrose gradients from crude P2 fractions. Each point in the figure is the mean of three experiments (Expt. 7-9). Open circles represent the S-nucleotidase activity. Asterisks represent the monoamine oxidase activity and crosses indicate the protein determinations. Filled squares indicate the lactate dehydrogenase activities. All points are represented as percentages of the fractions with the highest content of enzyme activity or protein. The recoveries are : protein, 104% ; S-nucleotidase, 95% ; monoamine oxidase, 92% ; and lactate dehydrogenase, 75%.
possibly are present both in the synaptosome fractions and, less, in the microsome fraction. A detailed description of the localization of 5’-nucleotidase in an electron microscopic investigation in the mouse cerebellum is in Ilroqess.
Preliminary
results
are already
lntblished
(1 I ) which
are
in
agreement with these results. Summarizing these studies, we may say that our electron microscopic results indicate a distribution of S’-nucleotidase in the subsurface cisternae of Purkinje cell dendrites (Fig. 1Rj (11) and in the spine apparatus of Purkinje cell dendrite spines (Fig. 1C j On the other hand, S-nucleotidase activity is also found in parallel fibers and in parallel fiber synapses on Purkinje cell spines (Fig. 1D j. This electron microscopic location of the reaction products of S-nucleotidase is in good accordance with the data gathered by differential centrifugation techniques.
C-Electron microscopic location of 5’-nucleotidase in the spineapparatusof Purkinje cell dendritic spines (see asterisks). X 60,000. D-Electron microscopic, location of 5’-nucleotidase reaction products in the parallel fiber bouton, synapsing on a Purkinje cell dendritic spine (asterisk). X 60,000.
E. MARANI
REFERENCES 1. BARTLETT, G. R. 1959. 2.
3.
4.
5.
6. 7. 8. 9. 10. 11.
12. 13. 14.
15. 16. 17. 18. 19. 20.
Phosphorous assay in column chromatography. J. Biol. Chcnc. 234 : 466-468. FISKE, C. H., AND Y. SUBBAROW. 1925. The calorimetric &termination of phosphorus. J. Biol. Chcm. 66: 375-400. GRAY, E. G., AND V. P. WHITTAKER. 1962. The isolation of nerve endings from brain: an electronmicroscopic study of cell fragments derived by homogenization and centrifugation. J. nlzat. 96 : 79-88. ISRAEL, M., AND P. FRANCHON-MASTOUR. 1970. Fractionnement du cortex &&bra1 du rat, distribution subcellulaire de la 5’-nucleotidase et des cholinest&ases. .4rch. Amt. Microsc. 59 : 383-392. KORENBERG, A. 1955. Lactic dehydrogenase of muscle. Pages 441-443 in s. P. COLOWICE( AND N. 0. KAPLAN, Eds., IMetRods ix En,-ymology, Vol. I. Academic Press, New York. KRAMI, M. 1965. A rapid microfluorimetric determination of monoamineoxidase. Biochem. Phannacol. 14 : 1684-1686. KROGT, J. A. VAN DER. 1974. Localisatie valt Enzymen van het CatccholalrtincmVabolisnae in Rattehcrsenen. Thesis. Leiden. KROON, M. G. 1972. Mo,zoa+nim Oxidase. Een Onderzock over Eigcnschappen en Lokalisatie in Rattchcrsencn. Thesis. Leiden. KROON, M. G., AND H. VELDSTRA. 1972. Multiple forms of rat brain mitochondrial monoamine oxidase subcellular localization. Febs Left. 24 : 173-176. LOWRY, 0. H., N. J. ROSENBROUGH, A. L. FARR, AND R. J. RANDALL. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chent. 193: 265-275. MARANI, E., AND J. VOOGD. 1973. Some aspects of the localization of the enzyme 5’-nucleotidase in the molecular layer of the cerebellum of the mouse. Actn Morpltol. Neerl. Stand. 11: 365-367. MARANI, E. 1977. Enzyme histochemistry 5. Comparison to biochemistry irz R. LAHUE, Ed., Methods in Ncnrobiology. Plenum, New York (in press). MARANI, E. 1977. Enzyme histochemistry 5.4 Isoenzymes i+z R. LAHUE, Ed., Methods in fzczbrobiology. Plenum, New York (in press). PERSIJN, J. P., W. VAN DER SLII(, AND C. J. TIMMER. 1969. On the determination of serum 5’-nucleotidase activity in the presence of beta-glycerophosphate. C/in. Biochms. 2 : 398-402. PERSIJN, J. P., AND W. VAN DER SLII\-. 1970. A new method for the determination of serum 5’-nucleotidase. 2. Klin. Chem. Kliu. Biochem. 8: 398-402. PETERSON, E. A., AND H. A. SOBER. 1959. Variable gradient device for chromatography. ,4+tal. C~keWZ. 31 : 857-862. PILCHER, C. W., AND D. G. JONES. 1970. The distribution of 5’-nucleotidase in subcellular fractions of mouse cerebellum. Brain Res. 24: 143-147. SCOTT, T. G. 1965. The specificity of 5’-nucleotidase in the brain of the mouse. J. Iiistochent. Cytochem. 13 : 657-667. STOLS, A. L. H. 1964. Tzhrnip Yellow Mosaic Virzls. Interacties met Kwaternairc Anzmoniumsouten. Thesis. Leiden. WHITTAKER, V. P., J. A. MICHAELSON, AND R. J. H. KIRKLAND. 1964. The separation of synaptic vesicles from nerve-ending particles (‘synaptosomes’) . Biochem. J. 90 : 293-303.
NEUROLOGY
57, 1042-1048 (1977)
RESEARCH The
Subcellular
Distribution in Mouse
of S-Nucleotidase Cerebellum
E.
of rlmtomy
Dcpartwccnt
Rcccivcd
26, 1977;
Activity
MARANI
and Embryology, Lcidm. The January
NOTE
University Netherlauds revision
of Leidefl,
rcccived
May
Wasse~zaarsewcg
62,
12, 1977
Because the peculiar localization of 5’-nucleotidase in longitudinal bands in the molecular layer of the cerebellum of mouse (Fig. 1A) and rat (1 l13, 18) has not been correlated with the histologic structure of the molecular layer, Pilcher and Jones (17) used fractionation techniques to determine the subcellular distribution of this enzyme in the mouse. They suggested that the distribution of S-nucleotidase in the various fractions of nlousc cerebellum, prepared according to Gray and Whittaker (3), reflects that of an axoplasmic location (17) and of specific synapses containing S-nucleotidase. They indicate that it may be possible to distinguish two t!-pes of synapses, those with and without 5’-nucleotidase (17). In the course of a study of the ultrastructural localization of 5’-nucleotidase in the cerebellum of the mouse, the fractionation experiments of Pilcher and Jones ( 17) were repeated using a slightly different technique (20). Our differential centrifugation results do not entirely support their suggestion of a localization of 5’-nucleotidase in a specific type of synapse in the molecular layer. but are not contradictory to their results (17). *Adult (6 months to 1 year of age) female mice (USA, Naval Medical Research Institute strain) were killed by chloroform anesthesia and the cerebella were rapidly dissected. All stages of the fractionation procedure were performed at 0 to 4°C. Each homogenate contained three pooled cerebella (175 mg) in 3.5 ml 0.32 M sucrose solution. The cerebella were pottered and fractions were prepared according to the method of Whittaker Abbreviation
: AMP-adenosine
S-phosphate. 1042
Copyright All rights
(9 1977 by Academic Press. Inc. of reproduction in any form reserved.
ISSN
0014-4886
CEKEBELLAR
IiUCLEOTIDASE
ACTIVITY
10-43
ct ul. (20 j . Gradient centrifugation was done according to the method used with by Van der Krogt (7, 16, 19), using a Spinco L2-65B ultracentrifuge a SW 27 rotor, at 82.5009 and 4°C. Sucrose solutions at PH i, prepared by adding some crystals of Tris, were used. Addition of the Tris crystals did not coacervate particles in P2 (unpublished results, Van Dijke). The SF crude synaptosomal-mitochondrial fraction ( 1.5 ml) was used. (;ratlients were collected in about 20 fractions, each of 1.5 ml. Protein cleterminntions were made according to Lowry c,t nl. (10). The S-nucleotidase activit! was measured after Persijn ct al. (14, 15) : incubation time was 1 11. L,actate dehydrogenase, a cytoplasmic marker, was assayed according to Kornberg (5). The monoamine oxidase activity was determined with kynuramine as substrate, according to the Kroon modification (S, 9) of the method of Kraml (6). Electron microscopic procedures were carried out by perfusion-fixation at a velocity of 15 ml/min. Paraformaldehyde, 40/o, in 0.16 M cacodylate buffer, PH 7.3, osmolality 1550, was perfused for 2 min. followed by a 2-min perfusion-fixation with 2.5 % glutaraldehyde in 0.16 M cacodylate buffer, to which was added 5.4 g/liter sucrose (final PH 7.3, osmolality 570 1. These steps were followed by an incubation perfusion of 5 min, at the same velocity, and 37°C with the medium of Scott (18). The cerebellum was dissected free, and the tissue was cut in sections 200 pm thick and su11sequently incubated 0.5 h in the incubation medium. After incubation, the sections were fixed 0.5 h in the glutaralclehyde solution and prepared in the routine manner for 0~0~ fixation and Epon embedding. To exclude a possible influence of sucrose, 5’-nucleotidase activity \vas determined in two homogenates using different sucrose concentrations. It was found that sucrose does not influence the cerebellar 5’-nucleotidase activity. Adenosine 5’-phosphate (AMP) deaminase can influence the Persijn t~f al. determination (14, 15). It is important to note that this activity in the cerebellum is of the order of 8 units per liter (unpublished results), which is very low compared to the 5’-nucleotidase activity (see Table 1) . The main difference between our investigations and those of Pilcher and Jones (17) is the use of the method of Persijn ct al. (13, 15) for the determination of 5’-nucleotidase activity, instead of the phosphate method of Fiske and SubbaRow (2) used by Pilcher and Jones and others (1). According to Bartlett (1) the Fiske-SubbaRow method is unsatisfactory because it is dependent on its reagents, having a narrow range of permissible H,SO, concentration, and it also produces an unstable color. Moreover, our preliminary experiments with this method showed it to be time and temperature dependent. The elaborate precautions necessarv to obtain reproducible results with the Fiske-SubbaRow method (2) caused us to
1044
E.
MARANI
TABLE 5’-Nucleotidase
Fractions
H Pl P2 S2 P3 s3
Mean,
107 23.6 47.6 38.5
Recovery
1
Activity in Various Fractions Obtained Centrifugation Techniques at pH 7.2” Expt
f f zk f
l-6
2.6 0.6 2.0 2.6
Expt
10
Expt
11
Expt
12
by Differential
Mean, Expt 10-12
Mean specific activity per milligram protein
15 39
14 36
15 39
15 38
3.3 6.5
15 30
14 35
15 30
15 32
6.2 7.1
103 y0
I’ The activities are expressed as absolute total activity (Expt 10-12) in units per II-PAC, and WCC).
Recovery values (Expt l-6) liter (Commission
70$0
or as percentages of the on Clinical Chemistry,
prefer the method of Persijn et ~2. (14, 15). The extra advantage of this method is in the substrate retention; therefore, no corrections have to be made for unspecific phosphatase activity. The results of six fractionation experiments summarized in Table 1 show that the specific activities of the subcellular fractions Pl, P2, and S2 are reproducible, Our measurements of the percentage activity of S-nucleotidase in the Pl fraction (Table 1) mainly containing nuclei and debris (20) are in the same range as those of Pilcher and Jones (17). As shown in Table 1, only 36 to 4470 of the total activity was recovered in the P2 fraction compared to 67 to 787 0 in Pilcher and Jones’s experiments (17). Conversely, the percentage of activity in the supernatant S2 is high in our experiments (36%) and low in those of Pilcher and Jones (17). It seems unlikely that this difference is caused only by the use of a different method for preparing the P2, although Whittaker et al. (20) stated that this difference resulted in a lower yield of synaptosomes. The difference can rather be explained by the solubility of the 5’-nucleotidase, also known from previous experiments ( 12, 13)) and the problem of accurate 5’-nucleotidase determination [measuring the total AMP ASE activity and subtracting the p-glycerophosphatase activity at pH 7.2 (17) versus the substrate retention method at PH 7.2 (14,15)1. In the experiments shown in Table 1 the relative activities of the P3 fraction are nearly the same as in Pilcher and Jones’s experiments. However, nearly 32% was recovered in our soluble fraction S3, whereas those
CEREBELLAR
NUCLEOTIDASE
ACTIVITY
1045
authors (17) did not find any activit) in the supernatant of pellet P3 although the same speed and time of centrifugation were applied. Considering the relative distribution of the 5’-nucleotidase activity over the sum of the activity of the different fractions (see Table 1)) it is obvious that the P2 and S3 fractions have the same S-nucleotidase activity and that the P3 fraction contains only half that of the P2 or S3 fraction. The comparison of the mean specific activities of the fractions (Table 1) does not indicate a difference between P2. P3. and S.3 ratios per milligram protein. To check whether or not the S-nucleotidase activity in the synaptosomal fraction P2 is bound to a special type of synaptosome. we used the linear sucrose-gradient technique on crude P2 fractions. The results of three experiments, shown in Fig. 2, indicate that S-nucleotidase is not bound to all mitochondria, because there is a difference in the peaks for S’-nucleotidase and monoaminoxidase. The peak of the cytoplasmic marker, lactate dell!-clrogenase, coincides with the %nucleotidase peak. The sharpness of the peak for S-nucleotidase and the overlap with the top of the lactate dehydrogenase peak can be seen as an argument for the binding of part of the 5’-nucleotidase activity to a certain particle within the synaptosome pool. At low sucrose molarities in the gradients a large portion of S’-nucleotidase is found that is interpreted as S-nucleotidase which may correspond to a certain soluble form of S-nucleotidase ( 12, 13) or which is bound to ruptured structures. \\%en our results are compared to the measurements of Israel and Fran&on-Mastour (4) in fractions of the cerebral cortex of the rat, prepared according to the same technique as in our experiments (20), their values for Pl, P2, and P3 are found to be in good accordance with our determinations in mouse cerebellum. However, their value for S3 (9%) is substantially lower than that in our experiments, but higher than that in the Pilcher and Jones measurements [OR, (17)]. This indicates that a part of the 5’-nucleotidase activity is soluble in rat cerebral cortex also. The higher values for the S3 in our experiments could mean that the 5’-nucleotidase is loosely bound to structures in the cerebellum. As a consequence of this study and the comparisons made with the work of others (4, 17)) one cannot decide on a uniform location of S-nucleotidase in the synaptosomes. The results found in our experiments for the P2 fraction are more in agreement with the results of Israel and FrachonMastour (4) in the rat cortex than with the results of Pilcher and Jones ( 17). Therefore any conclusion based on these differential centrifugation results about a distribution that reflects that of specific synapses containing 5’-nucleotidase is premature. In conclusion one can suggest that the 5’-nucleotidase is not very strongly bound to one or more structure(s) that
1046
E.
MARANI
FIG. 1. Light and electron photomicrographs of results after incubations according to Scott (18). A-Detail of the light microscopic location of 5’-nucleotidase in uvula (IX) and pyramis (VIII). X 20. B-Electron microscopic location of 5’-nucleotidase reaction products in the subsurface cisternae of a Purkinje cell dendrite. X 30,000.
CEREBELLAR
NUCLEOTIDASE
0.86
1.2
1047
ACTIVITY
04
M.
FIG. 2. Linear sucrose gradients from crude P2 fractions. Each point in the figure is the mean of three experiments (Expt. 7-9). Open circles represent the S-nucleotidase activity. Asterisks represent the monoamine oxidase activity and crosses indicate the protein determinations. Filled squares indicate the lactate dehydrogenase activities. All points are represented as percentages of the fractions with the highest content of enzyme activity or protein. The recoveries are : protein, 104% ; S-nucleotidase, 95% ; monoamine oxidase, 92% ; and lactate dehydrogenase, 75%.
possibly are present both in the synaptosome fractions and, less, in the microsome fraction. A detailed description of the localization of 5’-nucleotidase in an electron microscopic investigation in the mouse cerebellum is in Ilroqess.
Preliminary
results
are already
lntblished
(1 I ) which
are
in
agreement with these results. Summarizing these studies, we may say that our electron microscopic results indicate a distribution of S’-nucleotidase in the subsurface cisternae of Purkinje cell dendrites (Fig. 1Rj (11) and in the spine apparatus of Purkinje cell dendrite spines (Fig. 1C j On the other hand, S-nucleotidase activity is also found in parallel fibers and in parallel fiber synapses on Purkinje cell spines (Fig. 1D j. This electron microscopic location of the reaction products of S-nucleotidase is in good accordance with the data gathered by differential centrifugation techniques.
C-Electron microscopic location of 5’-nucleotidase in the spineapparatusof Purkinje cell dendritic spines (see asterisks). X 60,000. D-Electron microscopic, location of 5’-nucleotidase reaction products in the parallel fiber bouton, synapsing on a Purkinje cell dendritic spine (asterisk). X 60,000.
E. MARANI
REFERENCES 1. BARTLETT, G. R. 1959. 2.
3.
4.
5.
6. 7. 8. 9. 10. 11.
12. 13. 14.
15. 16. 17. 18. 19. 20.
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