Life Sciences, Vol, 23, pp . 2665-2674 Printed in the U .S .A .
Pergamon Press
LOCALIZATION OF NORADRENALINE AND DOPAMINE-ß-HYDROXYLASE IN 01300 MOUSE NEUROBLASTOMA A BIOCHEMICAL AND ELECTRONMICROSCOPIC STUDY(1) W . P. De Potter, N. H. Fraeyman, J. W . Palm(Z) and A, F. De Schaepdryver Heymane Institute of Pharmacology, University of Ghent Medical School, De Pintelaan 135, B-9000 Cheat, Belgium (Received in final form November 6, 1978) Summary Investigations were carried out in order to obtain biochemical and electronmicroscopic information on the storage of noradrenaline (NA) and dopamine-ß-hydroxylase (DßH) in 0 mouse neuro1300 bla atoms . By application of two different gradient centrifugation methods only one population of NA vesicles, i. e. heavy NA containing vesicles could be demonstrated. Electronmicroscopic investigation of the tumor revealed the presence of large dense cored vesicles . Electronmicroscopic investigation of gradient fractions further showed that large dense cored vesicles were only present in those fractions of the gradient containing heavy vesicles . The distribution of DßH in relation to the distribution of NA after gradient centrifugation differs from what has been observed in peripheral noradrenergic neurone, In C mouse neuroblastoma the larger part of the enzyme is associaté~with the plasma membrane . Electronmicroecopic studies of human neuroblastoma have shown that one of the characteristic ultraetructural features of these tumors is the presence of large dense cored vesicles which are thought to contain catecholamines (1 ). This view is rather well supported by the results of differential centrifugation experiments which showed that NA ie associated with subcellular structures (2, 3, 4), thus suggesting the presence of NA containing vesicles . More direct evidence for their presence has been obtained by two groups of workers (3, 4) who, in using density gradient centrifugation, were able to characterize NA vesicles . These vesicles had a similar density as the heavy vesicles found in bovine sympathetic ganglia (5, 6) sad eplenic nerves (7, 8, 9, 10). (1) Aided by a research grant from the A. S, i~ . K. -Cancer Fund (Belgium) . (2) Heymaas Foundation Research Fellow, on leave from the Department of Pharmacology, College of Medicine, Ohio State University, Columbus, Ohio, U. S . A, 0300-9653/78/1231-2665$02,00/0 Copyright (c) 1978 Pergamon Press
2666
Noradrenaline Storage
Vol, 23, Nos . 27 & 28, 1978
A thorough biochemical study of the NA storage vesicles in neuroblastoma as done for adrenergic nerves (10) - ie made difficult by the rarity of this tumor. It was therefore felt that the CL~00 mouse neuroblastoma, a round cell transplantable tumor of sympa ehc cells (11, 12, 13) which grows relatively rapidly and which is also able to synthesize and store NA (14), might be a good substitute . Electronmicroscopic studies of C 13p~ mouse neuroblastoma have already revealed the presence of dense cored"vesicles (15, 16, 17), Although the composition of these vesicles is so far completely unknown (1 7), there ie biochemical evidence that a large part of the NA in the tumor ie associated with eubcellula.r structures (12) . The present study was performed in order to characterize the NA storing organelles and to investigate whether these NA containing structures correspond to the morphologically demonstrated large dense cored vesicles . Materiale and Methode 1, Biochemical. The tissue used in this study was the solid mouse 01300 neuroblaetoma . The tumor was obtained from Jackson Laboratories (Bar Harbor, Maine, U. S. A, ) and was further maintained in our laboratory by subcutaneous transfer in 4 to 10 weeks old A~J mice of both sexes. About two weeks after the inoculation, the mice were killed by cervical dislocation and the tumor dissected free from the surrounding tissue . The tissue was rinsed three times in ice-cold 0.25 M sucrose, chopped finely with a knife, suspended in 4 volumes of ice-cold 0. 25 M sucrose buffered with 5 mM trisHCl pH 7. 3 and homogenized in a motor driven Potter-Elvehjem homogenizer (Kontes, Vinela.ad, New Jersey, U. S. A, ) using a Teflon pestle (clearance 0. 12 mm). The homogenization was continued until the pestle had been passed up and down once . The homogenate was submitted to differential centrifugation in two subsequent steps : 3, 000 g for 10 min (Sorvall) followed by S8, 000 g for 45 min (Spinco L50 ) . This procedure yields three fractions, indicated by sediment 1 (mainly composed of unbroken material, nuclei and the larger mitochondria), sediment 2 (or microsomal fraction) and a supernatant. Sediment 2 was subfractionated using two types of gradient centrifugation . Both gradients were spun in the SW 40 rotor of the Beckman ultracentrifuge . Differen_tial~radient centrif_u~ation. Sediment 2 was reeuspended in the 0.25 M éucrose solution ~0. 5 ml%g original tissue) and applied oa top of a linear 0 . 3 to 0, 8 M sucrose gradient . The bottom of the tube contained 1 . 0 ml of 2, 0 M sucrose. The gradient contained 5 mM trie -HCl pH 7. 3 and was centrifuged for 40 min at 198, 000 g av' Ec~uilibr_ium deneit~!radient centrifu~atio_n. Sediment 2 was re suspended in 0, 4~ M sûcrôéè ~0 . 5 ml]g ôriginâf tiseue~ and 0, 75 ml of the suspension was layered on top of a 0, 5 to 1 . 7 M sucrose gradient which was constructed on top of a 0. 5 ml cushion of 2 M sucrose (all solutions contained 5 mM trie -HCl pH 7 . 3), The total volume (sample + gradient + cushion) was 12 . 5 ml, The gradient was spun for 150 min at 198, 000 g av' After centrifugation, 12 fractions of 1 ml each were obtained by pumping a 2 M sucrose solution through the bottom of the tube and collecting by overflow (ISCO - model 640 density gradient fractiona.tor), Aliquots of these fractions were used for the determination of noradrenaline (1 S), ß-glucuronidase (1 q), monoamine oxidaee (MAO) (20), glucose-6-phosphatase (21), inosine diphosphataee (22), alkaline phoephatase (23), '5-nucleotidase (24) and proteins (25) . DßH was routinely determined ae follows
Vol, 23, Nos . 27 & 28, 1978
Noradrenaline Storage
2667
The incubation mixture contained 1, 5 mM tyramine, 6 mM ascorbic acid, 6 mM fumaric acid, 0, 8 mM tranylcypromine sulfate, 0, 1 mM p-chloromercuribenzoate (PCMB), 3, 200 unite of catalane, 0. 2 °f° (W~V) Triton X-100, 50 mM tria-HCl buffer (pH b) and a 200 Wl sample in a total volume of 0 . 4 ml . The tyramine, aecorbate and fumarate solutions were adjusted to pH 6 with NaOH . After 40 min of incubation at 37° C the reaction was stopped by addition of 100 ~~1 of 12 . 5 °f° (W~V) trichloroacetic acid and after centrifugation at 2, 000-3, 000 rev. min for 10 min, the supernatant extracted with 2 ml of ethylether (saturated with water) . The extraction (to remove trichloroacetic acid) was repeated four times, The remaining ethylether wan then removed by bubbling a stream of nitrogen through the mixture, The second step of the method was initiated by adding 200 ~.l of thin treated sample to a mixture containing 100 N1 of purified PNMT, 2, 7 ~IVI S-adenosyl-L- [ methyl 14 0 1 methionine (0, 1 ~.Ci) and 0. 5 M trio-HCl buffer (pH 8. 6) . The final volume was of 400 N1 . The mixture wan incubated at 3 7° C for 30 min and the reaction stopped by addition of 1 ml f 0, 5 M sodium-borate buffer (pH 10) and 3 g of sodiumchloride . The [~ 4C ] -labeled N-methyl octopamiae (synephrine) was extracted with 6 ml of a toluene-ieoamylalcohol mixture (3~2, V~V), After centrifugation 4 ml of the organic phase wan transferred to a vial for radio activity determination. In addition to a blank, standard values were obtained with each assay by adding 40 ng and 80 ng of octopamine to a complete assay mixture and these samples were carried through the procedure, In some cases the method of Molinoff et al, (26), in which CuS04 ie used to inactivate endogenous inhibitors, has been used, The distribution of NA and enzyme activities ie obtained by expressing the value of each fraction as a percentage of the sum of all fractions of the gradie¢, 2, Electronmicroscopy. Tumors were excised at different times after inoculation and small pieces immediately immersed into ice-cold 6 °Jo glutaraldehyde in sodium phosphate buffer (pH 7. 2 0. 1 M) for 1 hr . Postfixation was carried out with 1 °f° osmium tetroxide, The fixed tissue was dehydrated in an alcohol series and propylene oxide and embedded in a Spurr Low Viscosity Embedding Medium (SEM). Semithin and ultrathin sections were cut with a LKB ultrotome III. Samples from gradient centrifugations were diluted with ice-cold 6 °f° phosphate buffered glutaraldehyde and centrifuged (88, 000 for 60 min) . Further gav treatment of the resulting pellet was as for the tiesues . The sections were viewed in a J. E, M. 100 B (JEOL) electronmicroacope . Re culte Differential centrifugation . Homogenates of 0 mouse neuroblastoma 1300 contain NA and several enzymes which have been shown to be characteristic for different aubcellular particles or structures (Table I), Thie table gives the NA and enzyme contents of the tumor as well as the distribution of these constituents amongst the aubcellular fractions obtained by differential centrifugation . It can be seen that a large part of NA sediments in fractions 1 and 2 (81, 6 _+ 5 . 7 °fe), which is in agreement with previous observations on human neuroblastoma (4). The sedimeatable form of NA moat probably represents the NA storage vesicles whose distribution appears to be different from that of marker enzymes ouch as ~ -glucuronidase (lysosomea), MAO (mitochondria), glucose-b-phoephatase (endoplaemic reticulum) and alkaline phosphatase (plasma membranes),
2668
Vol . 23, Nos . 27 $ 28, 1978
Noradrenaline Storage
TABLE I Distribution of Constituents of Cf3 Mouse Neuroblastoma Among Fractions 00 Obtained Upon Differential Centrifugatidn of Tumor Homogenates . Enzyme
No . of Experimente
NA
7
ß -Glucuronidase
3
MAO
6
Glucose-6Pass
3
Alkaline phosphatase
6
DßH
3
Proteine
7
Absolute Value in Total Homogenate
Distribution (%) Fraction 2
1
+
1, 4 0,2
+
17. 1 2,8
+
+
0, 04 0, O1
+
60, 8 6, 5
+
+
0. 54 0. 07
+
67. 7 6. 0
27, 6 + 2. 4
+
45 . 2 5. 6
+
+
5. 1 0, 4
+
1 92, 7 4S . 4
+
721, 5 70, 7
+
114 . 0 5. 8
46 . 7 + 3. 9 + +
30, 7 6, 3 51, 6 0, 9
Recovery ( °fo)
64 . 5 5 .0 7, 1 0, 9
3 18 . 9 + 1 .5
+
79 . 0 4.6
+
32, 1 7, 2
+
80, 3 7, 4
+
4, 5 1. 9
+
90 . 6 2, 2
34 . 4 1, 6
20 . 0 + 4. 3
105, 6 + 22 . 4
2 7. 9 + 2, q
25 . 4 ± 1, 8
+
90, 9 4. 4
38 . 5 + 4. 1
+
103 . 2 12, 0
39 . 6 0, 8
+
95 . 1 1.8
30, 8 + 2. 3 +
B. 8 0. 9
+
Units : NA : F~g~g wet weight of tissue Enzymes : units~g wet weight of tissue Proteins : mg~g wet weight of tissue Results are the mean value _+ S. E, M. DßH activity is determined using the above described PCMB method . DßH is also present in a sedimentable form, at least 61, 5 _+ 8. 6 °fo under the present centrifugation conditions ; and it is noteworthy that, comparatively to NA, a larger percentage of DßH is present in fraction 1, Gradient centrifugation . Gradient centrifugatione were carried out in order to further characterize these NA storage vesicles . Upon equilibrium density gradient centrifugation (Fig . 1) NA appears to be associated with particlee which are slightly less dense than lyeosomes (marked by ß-glucuroaidase), about as dense ae mitochondria (marked by MAO) and more dense than endoplasmic reticulum (marked by glucose-6-phoephataee and inosine diphosphatase) and plasma membranes (marked by alkaline phoephatase and ' S-nucleotidaee), Figure 1 further shows that the largest part of DßH is found in the low density region of the gradient where it almost completely parallels the distribution of alkaline phosphatase and '5-nucleotidase . However, it ie well known that light NA vesicles - which are mainly found in adrenergically innervated organs - also equilibrate in this region of the gradient . There ie no indication for the existence of such light NA vesicles in this tumor but the possibility still exists that DßH may be associated with light - eventually (relatively) empty - NA vesicles,
Vol . 23, Nos . 27 $ 28, 1978
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Pergamon Press
LOCALIZATION OF NORADRENALINE AND DOPAMINE-ß-HYDROXYLASE IN 01300 MOUSE NEUROBLASTOMA A BIOCHEMICAL AND ELECTRONMICROSCOPIC STUDY(1) W . P. De Potter, N. H. Fraeyman, J. W . Palm(Z) and A, F. De Schaepdryver Heymane Institute of Pharmacology, University of Ghent Medical School, De Pintelaan 135, B-9000 Cheat, Belgium (Received in final form November 6, 1978) Summary Investigations were carried out in order to obtain biochemical and electronmicroscopic information on the storage of noradrenaline (NA) and dopamine-ß-hydroxylase (DßH) in 0 mouse neuro1300 bla atoms . By application of two different gradient centrifugation methods only one population of NA vesicles, i. e. heavy NA containing vesicles could be demonstrated. Electronmicroscopic investigation of the tumor revealed the presence of large dense cored vesicles . Electronmicroscopic investigation of gradient fractions further showed that large dense cored vesicles were only present in those fractions of the gradient containing heavy vesicles . The distribution of DßH in relation to the distribution of NA after gradient centrifugation differs from what has been observed in peripheral noradrenergic neurone, In C mouse neuroblastoma the larger part of the enzyme is associaté~with the plasma membrane . Electronmicroecopic studies of human neuroblastoma have shown that one of the characteristic ultraetructural features of these tumors is the presence of large dense cored vesicles which are thought to contain catecholamines (1 ). This view is rather well supported by the results of differential centrifugation experiments which showed that NA ie associated with subcellular structures (2, 3, 4), thus suggesting the presence of NA containing vesicles . More direct evidence for their presence has been obtained by two groups of workers (3, 4) who, in using density gradient centrifugation, were able to characterize NA vesicles . These vesicles had a similar density as the heavy vesicles found in bovine sympathetic ganglia (5, 6) sad eplenic nerves (7, 8, 9, 10). (1) Aided by a research grant from the A. S, i~ . K. -Cancer Fund (Belgium) . (2) Heymaas Foundation Research Fellow, on leave from the Department of Pharmacology, College of Medicine, Ohio State University, Columbus, Ohio, U. S . A, 0300-9653/78/1231-2665$02,00/0 Copyright (c) 1978 Pergamon Press
2666
Noradrenaline Storage
Vol, 23, Nos . 27 & 28, 1978
A thorough biochemical study of the NA storage vesicles in neuroblastoma as done for adrenergic nerves (10) - ie made difficult by the rarity of this tumor. It was therefore felt that the CL~00 mouse neuroblastoma, a round cell transplantable tumor of sympa ehc cells (11, 12, 13) which grows relatively rapidly and which is also able to synthesize and store NA (14), might be a good substitute . Electronmicroscopic studies of C 13p~ mouse neuroblastoma have already revealed the presence of dense cored"vesicles (15, 16, 17), Although the composition of these vesicles is so far completely unknown (1 7), there ie biochemical evidence that a large part of the NA in the tumor ie associated with eubcellula.r structures (12) . The present study was performed in order to characterize the NA storing organelles and to investigate whether these NA containing structures correspond to the morphologically demonstrated large dense cored vesicles . Materiale and Methode 1, Biochemical. The tissue used in this study was the solid mouse 01300 neuroblaetoma . The tumor was obtained from Jackson Laboratories (Bar Harbor, Maine, U. S. A, ) and was further maintained in our laboratory by subcutaneous transfer in 4 to 10 weeks old A~J mice of both sexes. About two weeks after the inoculation, the mice were killed by cervical dislocation and the tumor dissected free from the surrounding tissue . The tissue was rinsed three times in ice-cold 0.25 M sucrose, chopped finely with a knife, suspended in 4 volumes of ice-cold 0. 25 M sucrose buffered with 5 mM trisHCl pH 7. 3 and homogenized in a motor driven Potter-Elvehjem homogenizer (Kontes, Vinela.ad, New Jersey, U. S. A, ) using a Teflon pestle (clearance 0. 12 mm). The homogenization was continued until the pestle had been passed up and down once . The homogenate was submitted to differential centrifugation in two subsequent steps : 3, 000 g for 10 min (Sorvall) followed by S8, 000 g for 45 min (Spinco L50 ) . This procedure yields three fractions, indicated by sediment 1 (mainly composed of unbroken material, nuclei and the larger mitochondria), sediment 2 (or microsomal fraction) and a supernatant. Sediment 2 was subfractionated using two types of gradient centrifugation . Both gradients were spun in the SW 40 rotor of the Beckman ultracentrifuge . Differen_tial~radient centrif_u~ation. Sediment 2 was reeuspended in the 0.25 M éucrose solution ~0. 5 ml%g original tissue) and applied oa top of a linear 0 . 3 to 0, 8 M sucrose gradient . The bottom of the tube contained 1 . 0 ml of 2, 0 M sucrose. The gradient contained 5 mM trie -HCl pH 7. 3 and was centrifuged for 40 min at 198, 000 g av' Ec~uilibr_ium deneit~!radient centrifu~atio_n. Sediment 2 was re suspended in 0, 4~ M sûcrôéè ~0 . 5 ml]g ôriginâf tiseue~ and 0, 75 ml of the suspension was layered on top of a 0, 5 to 1 . 7 M sucrose gradient which was constructed on top of a 0. 5 ml cushion of 2 M sucrose (all solutions contained 5 mM trie -HCl pH 7 . 3), The total volume (sample + gradient + cushion) was 12 . 5 ml, The gradient was spun for 150 min at 198, 000 g av' After centrifugation, 12 fractions of 1 ml each were obtained by pumping a 2 M sucrose solution through the bottom of the tube and collecting by overflow (ISCO - model 640 density gradient fractiona.tor), Aliquots of these fractions were used for the determination of noradrenaline (1 S), ß-glucuronidase (1 q), monoamine oxidaee (MAO) (20), glucose-6-phosphatase (21), inosine diphosphataee (22), alkaline phoephatase (23), '5-nucleotidase (24) and proteins (25) . DßH was routinely determined ae follows
Vol, 23, Nos . 27 & 28, 1978
Noradrenaline Storage
2667
The incubation mixture contained 1, 5 mM tyramine, 6 mM ascorbic acid, 6 mM fumaric acid, 0, 8 mM tranylcypromine sulfate, 0, 1 mM p-chloromercuribenzoate (PCMB), 3, 200 unite of catalane, 0. 2 °f° (W~V) Triton X-100, 50 mM tria-HCl buffer (pH b) and a 200 Wl sample in a total volume of 0 . 4 ml . The tyramine, aecorbate and fumarate solutions were adjusted to pH 6 with NaOH . After 40 min of incubation at 37° C the reaction was stopped by addition of 100 ~~1 of 12 . 5 °f° (W~V) trichloroacetic acid and after centrifugation at 2, 000-3, 000 rev. min for 10 min, the supernatant extracted with 2 ml of ethylether (saturated with water) . The extraction (to remove trichloroacetic acid) was repeated four times, The remaining ethylether wan then removed by bubbling a stream of nitrogen through the mixture, The second step of the method was initiated by adding 200 ~.l of thin treated sample to a mixture containing 100 N1 of purified PNMT, 2, 7 ~IVI S-adenosyl-L- [ methyl 14 0 1 methionine (0, 1 ~.Ci) and 0. 5 M trio-HCl buffer (pH 8. 6) . The final volume was of 400 N1 . The mixture wan incubated at 3 7° C for 30 min and the reaction stopped by addition of 1 ml f 0, 5 M sodium-borate buffer (pH 10) and 3 g of sodiumchloride . The [~ 4C ] -labeled N-methyl octopamiae (synephrine) was extracted with 6 ml of a toluene-ieoamylalcohol mixture (3~2, V~V), After centrifugation 4 ml of the organic phase wan transferred to a vial for radio activity determination. In addition to a blank, standard values were obtained with each assay by adding 40 ng and 80 ng of octopamine to a complete assay mixture and these samples were carried through the procedure, In some cases the method of Molinoff et al, (26), in which CuS04 ie used to inactivate endogenous inhibitors, has been used, The distribution of NA and enzyme activities ie obtained by expressing the value of each fraction as a percentage of the sum of all fractions of the gradie¢, 2, Electronmicroscopy. Tumors were excised at different times after inoculation and small pieces immediately immersed into ice-cold 6 °Jo glutaraldehyde in sodium phosphate buffer (pH 7. 2 0. 1 M) for 1 hr . Postfixation was carried out with 1 °f° osmium tetroxide, The fixed tissue was dehydrated in an alcohol series and propylene oxide and embedded in a Spurr Low Viscosity Embedding Medium (SEM). Semithin and ultrathin sections were cut with a LKB ultrotome III. Samples from gradient centrifugations were diluted with ice-cold 6 °f° phosphate buffered glutaraldehyde and centrifuged (88, 000 for 60 min) . Further gav treatment of the resulting pellet was as for the tiesues . The sections were viewed in a J. E, M. 100 B (JEOL) electronmicroacope . Re culte Differential centrifugation . Homogenates of 0 mouse neuroblastoma 1300 contain NA and several enzymes which have been shown to be characteristic for different aubcellular particles or structures (Table I), Thie table gives the NA and enzyme contents of the tumor as well as the distribution of these constituents amongst the aubcellular fractions obtained by differential centrifugation . It can be seen that a large part of NA sediments in fractions 1 and 2 (81, 6 _+ 5 . 7 °fe), which is in agreement with previous observations on human neuroblastoma (4). The sedimeatable form of NA moat probably represents the NA storage vesicles whose distribution appears to be different from that of marker enzymes ouch as ~ -glucuronidase (lysosomea), MAO (mitochondria), glucose-b-phoephatase (endoplaemic reticulum) and alkaline phosphatase (plasma membranes),
2668
Vol . 23, Nos . 27 $ 28, 1978
Noradrenaline Storage
TABLE I Distribution of Constituents of Cf3 Mouse Neuroblastoma Among Fractions 00 Obtained Upon Differential Centrifugatidn of Tumor Homogenates . Enzyme
No . of Experimente
NA
7
ß -Glucuronidase
3
MAO
6
Glucose-6Pass
3
Alkaline phosphatase
6
DßH
3
Proteine
7
Absolute Value in Total Homogenate
Distribution (%) Fraction 2
1
+
1, 4 0,2
+
17. 1 2,8
+
+
0, 04 0, O1
+
60, 8 6, 5
+
+
0. 54 0. 07
+
67. 7 6. 0
27, 6 + 2. 4
+
45 . 2 5. 6
+
+
5. 1 0, 4
+
1 92, 7 4S . 4
+
721, 5 70, 7
+
114 . 0 5. 8
46 . 7 + 3. 9 + +
30, 7 6, 3 51, 6 0, 9
Recovery ( °fo)
64 . 5 5 .0 7, 1 0, 9
3 18 . 9 + 1 .5
+
79 . 0 4.6
+
32, 1 7, 2
+
80, 3 7, 4
+
4, 5 1. 9
+
90 . 6 2, 2
34 . 4 1, 6
20 . 0 + 4. 3
105, 6 + 22 . 4
2 7. 9 + 2, q
25 . 4 ± 1, 8
+
90, 9 4. 4
38 . 5 + 4. 1
+
103 . 2 12, 0
39 . 6 0, 8
+
95 . 1 1.8
30, 8 + 2. 3 +
B. 8 0. 9
+
Units : NA : F~g~g wet weight of tissue Enzymes : units~g wet weight of tissue Proteins : mg~g wet weight of tissue Results are the mean value _+ S. E, M. DßH activity is determined using the above described PCMB method . DßH is also present in a sedimentable form, at least 61, 5 _+ 8. 6 °fo under the present centrifugation conditions ; and it is noteworthy that, comparatively to NA, a larger percentage of DßH is present in fraction 1, Gradient centrifugation . Gradient centrifugatione were carried out in order to further characterize these NA storage vesicles . Upon equilibrium density gradient centrifugation (Fig . 1) NA appears to be associated with particlee which are slightly less dense than lyeosomes (marked by ß-glucuroaidase), about as dense ae mitochondria (marked by MAO) and more dense than endoplasmic reticulum (marked by glucose-6-phoephataee and inosine diphosphatase) and plasma membranes (marked by alkaline phoephatase and ' S-nucleotidaee), Figure 1 further shows that the largest part of DßH is found in the low density region of the gradient where it almost completely parallels the distribution of alkaline phosphatase and '5-nucleotidase . However, it ie well known that light NA vesicles - which are mainly found in adrenergically innervated organs - also equilibrate in this region of the gradient . There ie no indication for the existence of such light NA vesicles in this tumor but the possibility still exists that DßH may be associated with light - eventually (relatively) empty - NA vesicles,
Vol . 23, Nos . 27 $ 28, 1978
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