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Molecular forms of butyrylcholinesterase in rat brain microvessels
46
Neuroscience Letters, 120 (1990)46-49 ElsevierScientificPublishers Ireland Ltd.
NSL 07317
Molecular forms of butyrylcholinesterase in rat brain microvessels R. E d g a r d o C a t a l h n l, A n a M. M a r t i n e z 2, M a r i a D. A r a g o n 6 s 2 a n d F61ix H e r n h n d e z 1 IDepartamento de Biologia Molecular, Centro de Biologia Molecular ( C SIC- UA M ) , Universidad Autbnoma de Madrid, Madrid (Spain) and 2Departamento de Bioquimica y Biologia Molecular I, Universidad Complutense de Madrid, Madrid (Spain)
(Received23 June 1990;Revisedversion received3 August 1990;Accepted6 August 1990) Key words:
Butyrylcholinesterase;Molecularform; Brain microvessel;Blood-brain barrier
Molecular forms of butyrylcholinesterase(BuChE) were studied in microvesselsisolated from rat brain, using sedimentation analysis in sucrose gradients. Three forms, G~, G2 and G4, were found with sedimentation coefficientsclose to 3S, 6S and 9S, respectively.The relative proportion of the 3 forms was 19% for the monomer, 33% for the dimer and 46% for the tetramer. This sedimentation pattern of BuChE forms appears to be characteristicof cerebral microvesselsand may representdistinct functionalfeaturesof the blood-brain barrier.
Brain microvessels are unique with respect to their barrier mechanism, preventing access to a large extent to many substances from the blood. It has been described that butyrylcholinesterase (BuChE; EC 3.1.1.8) is present in high concentrations in the wall of all those microvessels that are equipped with a morphological bloodbrain barrier (BBB) [8, 9, 11], and we have reported some biochemical data on both acetylcholinesterase (ACHE) and BuChE in rat brain microvessels [5, 6]. Although the precise physiological function of BuChE remains unclear, not only in cerebral microvessels but also in the other tissues in which it is found, different roles for the enzyme have been proposed, related and not related to neurotransmission, i.e. hydrolysis of neuropeptides and fatty acid metabolism [7]. Furthermore, an involvement of this enzyme in the BBB function has been suggested [12, 19]. Both acetylcholinesterase (ACHE; EC 3.1.1.7) and BuChE have been shown to exist in a number of molecular forms particularly the globular monomer (G0, dimer (G2) and tetramer (G4), and the collagen-tailed A4, A8 and A12 forms [7]. Although the significance of the different molecular forms remains unclear, their tissue-specific distributions are thought to reflect the physiological functions of the different tissues [7]. Whereas BuChE has been extensively studied in the human plasma, where its role in the metabolism of anesthetics is of considerable clinical significance [4], its presence within the brain has received little attention. Correspondence: R.E. Catalfin, Departamento de BiologiaMolecular, Centro de BiologiaMolecular (CSIC-UAM), Universidad Aut6noma de Madrid, E 28040, Madrid, Spain.
0304-3940/90/$03.50 © 1990ElsevierScientificPublishers Ireland Ltd.
Nevertheless, BuChE has been reported to be associated with glial cells in white matter regions and with neuronal structures in grey matter regions [7]. The purpose of the present study was to determine the distribution of BuChE molecular forms in rat brain microvessels, where capillary endothelial cells contain high concentrations of this enzyme. The experiments were carried out on 30 male Wistar rats two months of age, and freely fed with standard pellets and tap water. Brain microvessels were obtained as previously described [5, 6]. The fraction collected from 50 pm mesh was used. The purity of brain microvessels was assessed by light microscopy and by the measurement of alkaline phosphatase and y-glutamyl transpeptidase as marker enzymes [17]. Presence of erythrocytes in the sample was negligible. Microvessels were resuspended and homogenized in a l0 mM Tris-HC1 buffer, pH 7.0, containing 1 M NaCl, 50 mM MgC12 and, unless stated otherwise, 1% Triton X-100. Homogenates were centrifuged at 17,000 g for 20 min. Supernatants were layered on sucrose density gradients (5-20% w/v) according to Rieger and Vigny [18]. Alkaline phosphatase from bovine intestine (6.1S) and catalase from beef liver (11.3S) were added to the enzyme samples as sedimentation markers. The samples were centrifuged for 20h at 40,000 rpm (130,000 gmax) in a Beckman SW 40 rotor, at 4°C. Approximately 50 fractions were collected and assayed for BuChE activity, as described previously [5]. BW2845C51, in a concentration of 30 pM, was included in the assay mixtures as a specific AChE inhibitor; hence, any residual activity in the presence of this inhibitor must be due to BuChE. Identification of the
47
0.45
0.BO
O.15 rn
I
i
O
2o
10
~o
,'o
~o
Fraction number
Fig. 1. Representative distribution of BuChE molecular forms in cerebral microvessels. Arrows indicate the positions of (from left to right) catalase (11.4S) and alkaline phosphatase (6.0S). Actual amounts of sample loaded were derived from microvessel homogenates containing the equivalent of 0.5 mg of protein.
different molecular forms was made on the basis of sedimentation coefficient, which was calculated relative to the positions in the gradient of the marker enzymes. Protein was determined by the method of Lowry et al. [15] using albumin as standard. The distribution of the different molecular forms of BuChE in brain microvessels is shown in Fig. 1, and an analysis of the data is presented in Table 1. The recovery from the gradient of the BuChE activity loaded (total activity prior to centrifugation/total activity recovered following centrifugation) was generally > 95 %. The loss of activity is probably due to loss of enzyme activity during centrifugation [1]. Our results indicate that 3 molecular forms of BuChE are present in brain microvessels, Gh G2 and G4 with sedimentation coefficients close to 3S, 6S and 9S, respectively. The sedimentation coefficients, although slightly lower are in good agreement with those obtained in other sources [1, 2]. The difference found in our study could be due to the detergent utilized [3]. The G4 form accounted for the majority of the BuChE activity, approximately 46% of the activity recovered. The 3.4S form accounted for 33% of total activity, and the 6S for only about 19%. The predominance for the tetrameric form of BuChE described here is in agreement
TABLE I MOLECULAR FORMS OF BuChE IN BRAIN MICROVESSELS Values are means ___S.D. of 5 determinations. Molecular forms Sedimentation coetticients (S) Percentage of total BuChE activity
GI
G2
3.4+0.2
6.0+0.3
33.4+2.5
18.9__ 1.8
G4 8.9__+0.3 46.0+2.9
with that reported by other authors for different biological sources, such as plasma, cerebrospinal fluid, and different areas of the central nervous system [1, 2, 16]. However, in these cases, the relative contribution of the G4 form to the total BuChE activity was higher (6994 %) than that observed by us in cerebral microvessels. The analysis of slower sedimentation peaks of BuChE (6.0S and 3.4S) indicates that the contribution of G1 species is slightly lower than that of G4 form, and it constitutes the majority of the non-G4 BuChE activity, in agreement with previous data reported for cerebrospinal fluid [2]. On the other hand, we must point out the considerable amount of the G2 form found in cerebral microvessels. This feature seems to be a characteristic of cerebral microvessels, since no discrete peaks of activity associated with any molecular form of BuChE other than the G4 and G1 species were detected by us in brain (unpublished data), in agreement with Atack et al. [1] and Muller et al. [16]. Furthermore, in plasma and cerebrospinal fluid, Gl and G4 forms were the only molecular species observed [2]. The possibility that the G2 form corresponds to AChE activity must be ruled out, since the assays were performed in the presence of an AChE inhibitor as described above. Therefore, the distribution of BuChE molecular forms reported here may be related to distinct anatomical and functional features of the BBB. Thus, the relatively high proportion of the lighter molecular form could suggest that the rate of conversion of G~ to G4 is slow and, therefore, a relatively large amount of the total BuChE is present as the Gh and G2 forms. However, this assumption seems to be unlikely since it has been reported that different mRNA species contribute to the formation of the various molecular forms of cholinesterase [20]. Further studies are required to resolve whether this fact takes place in brain microvessels. We have previously reported a monophasic Arrhenius plot of BuChE from brain capillaries [5], suggesting that it is unlikely that this enzyme interacts with membrane lipids. Nevertheless, the possibility that this interaction occurs and does not affect the catalytic activity of BuChE must be considered. In order to confirm whether BuChE molecular forms are membrane-bound, additional experiments, using different concentrations of Triton X-100 were performed. Table II shows the percentage of molecular forms extracted in the presence of Triton X-100, concentrations ranging from 0% to 1%. As can be seen, at least 0.1% Triton X-100 is necessary to completely solubilize the 3 forms. At concentrations as low as 0.01%, or without Triton X-100, only traces of G4 were detected in the supernatant. The analysis of the respective sediments confirmed that all molecular forms remained in the particulate fraction (unpublished data).
48 TABLE II EFFECT OF TRITON X-100 ON SOLUBILIZATION OF BuChE MOLECULAR FORMS IN CEREBRAL MICROVESSELS Results are expressed as percentage of activity of each molecular form considering as 100% the activity obtained after solubilization with 1% Triton X- 100. Triton X- 100 concentration (%)
Extraction (%)
0 0.01 0.05 0.1 1
Gi
G2
G4
0 0 93 99 100
0 0 85 97 100
12 12 66 95 100
These findings suggest t h a t the enzyme is w e a k l y a n c h o r e d to the m e m b r a n e , possibly by one o r m o r e t r a n s m e m b r a n e h y d r o p h o b i c sequences. This a s s u m p tion is s u p p o r t e d by the results o b t a i n e d when the molec u l a r forms were solubilized with 1% T r i t o n X-100, a n d then subjected to density g r a d i e n t c e n t r i f u g a t i o n in the absence o f d e t e r g e n t (Fig. 2). In these c o n d i t i o n s , the m a j o r i t y o f the non-G4 B u C h E activity is f o u n d at the b o t t o m o f the gradient, suggesting that an a g g r e g a t i o n by the h y d r o p h o b i c sequences takes place. O n the o t h e r hand, it has been suggested t h a t the m o l e c u l a r forms o f B u C h E c o r r e s p o n d to different locations: G i a n d G2 at intracellular sites, chiefly in the r o u g h e n d o p l a s m i c retic u l u m where they are synthesized for the G j form, a n d in the p l a s m a m e m b r a n e for the G4 f o r m where they migrate quickly [13]. In a d d i t i o n , F l u m e r f e l t et al. [9], have f o u n d in rat b r a i n capillaries, B u C h E activity in the i n t e r m e m b r a n o u s space o f the e n d o t h e l i a l nuclear envelope, the e n d o t h e l i a l e n d o p l a s m i c reticulum, a n d the e n d o t h e l i a l b a s e m e n t m e m b r a n e . Therefore, it seems likely t h a t these different localizations c o r r e s p o n d to
0.30 0 < 0.20
# > ,j o
0.10
rn
%--/,'
io
~
4'o
~o
Fraction number
Fig. 2. Distribution of BuChE molecular forms in cerebral microvessels extracted with 1% Triton X-100 and subjected to density gradient centrifugation in the absence of detergent.
each o f the three m o l e c u l a r forms r e p o r t e d by us. It is also p r o b a b l e t h a t G 4 f o r m is secreted a n d a s s o c i a t e d with the extracellular matrix, which has been involved in the cell g r o w t h a n d differentiation [10]. This idea is supp o r t e d by previous findings d e m o n s t r a t i n g a strong correlation between B u C h E a n d cell p r o l i f e r a t i o n [14]. Finally, based on the characteristic d i s t r i b u t i o n o f B u C h E m o l e c u l a r forms in cerebral microvessels, we suggest t h a t a m o r e detailed study o f this e n z y m a t i c activity m a y lead to a better u n d e r s t a n d i n g o f certain p a t h ological c o n d i t i o n s where the BBB is altered. The a u t h o r s are i n d e b t e d to Mr. J. Palacin for his v a l u a b l e assistance. This w o r k was s u p p o r t e d in p a r t by g r a n t s from the F u n d a c i 6 n R. Areces, F I S a n d D G I CYT. 1 Atack, J.R., Perry, E.K., Bonham, J.R., Candy, J.M. and Perry, R.H., Molecular forms of acetylcholinesterase and butyrylcholinesterase in the aged human central nervous system, J. Neurochem., 47 (1986) 263-277. 2 Atack, J.R., Perry, E.K., Bonham, J.R. and Perry, R.H., Molecular forms of acetylcholinesterase and butyrylcholinesterase in human plasma and cerebrospinal fluid, J. Neurochem., 48 (1987) 18451850. 3 Brimijoin, S., Molecular forms of AChE in brain, nerve and muscle: nature, localization and dynamics, Prog. Neurobiol., 21 (1983) 291~22. 4 Brown, S.S., Kalow, W., Pilz, W., Whittaker, M. and Woronick, C.L., The plasma cholinesterase: a new perspective. Adv. Clin. Chem., 22 (1981) 1-123. 5 Catal~m, R.E. and Hern~mdez, F., Temperature effects on cholinesterase from rat brain capillaries. Biosci. Rep., 6 (1986) 573-577. 6 Catalan, R.E. and Hernandez, F., Acetylcholinesterase and butyrylcholinesterase in rat brain capillaries, Med. Sci., 15 (1987) 291292. 7 Chatonnet, A. and Lockridge, O., Comparison of butyrylcholinesterase and acetylcholinesterase, Biochem. J., 260 (1989) 625~34. 8 Djuricic, B.M. and Mrsulja, B.B., Enzymatic activity of the brain microvessels vs. total brain homogenate, Brain Res., 138 (1977) 561 564. 9 Flumerfelt, B.A., Lewis, P.R. and Gwyn, D.G., Cholinesterase activity of capillaries in the rat brain. A hght and electron microscopic study, Histochem. J., 5 (1973) 67-77. 10 Goldstein, G.W. and Betz, A.L. Recent advances in understanding brain capillary function, Ann. Neurol., 14 (1983) 389-395. 11 Jo6, F. and Csillik, B., Topographic correlation between the hematoencephalic barrier and the cholinesterase activity in brain capillaries, Exp. Brain Res., 1 (1966) 147-151. 12 Jo6, F., Varkonyi, T. and Csillik, B., Development alterations in the histochemical structures of brain capillaries: histochemical, contributions to the problem of the blood-brain barrier, Histochemie, 8 (1967) 140-148. 13 Koelle, G.B., Massouli6, J., Eugene, D., Melone, M.A.B. and Boulla, G., Distribution of molecular forms of acetylcholinesterase and butyrylcholinesterase in nervous tissue of the cat, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 7749-7752. 14 Mayer, P.G. and Sporn, O., Spatiotemporal relationship of embryonic cholinesterase with cell proliferation in chicken brain and eye, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 284-288.
49 15 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 16 Muller, F., Dumez, Y. and Massouli6, J., Molecular forms and solubility of acetylcholinesterase during the embryonic development of rat and human brain, Brain Res., 331 (1985) 295-307. 17 Pardridge, W.M., Eisenberg, J. and Yang, J., Human blood-brain barrier insulin receptor, J. Neurochem., 45 (1985) 1771-1778. 18 Rieger, F. and Vigny, N., Solubilization and physicochemical characterization of rat brain acetylcholinesterase: Development and
maturation of its molecular forms, J. Neurochem., 27 (1976) 121129. 19 Shimon, M., Egozi, Y., Kloog, Y., Sokolovsky, M. and Cohen, S., Vascular cholinesterase and choline uptake in isolation of rat forebrain microvessels: A possible link, J. Neurochem., 53 (1989) 561565. 20 Soreq, H., Zevin-Sonkin, D. and Raz6n, N., Expression of cholinesterase gene(s) in human brain tissue: translational evidence for multiple mRNA species, EMBO J., 3 (1984) 1371-1375.
Neuroscience Letters, 120 (1990)46-49 ElsevierScientificPublishers Ireland Ltd.
NSL 07317
Molecular forms of butyrylcholinesterase in rat brain microvessels R. E d g a r d o C a t a l h n l, A n a M. M a r t i n e z 2, M a r i a D. A r a g o n 6 s 2 a n d F61ix H e r n h n d e z 1 IDepartamento de Biologia Molecular, Centro de Biologia Molecular ( C SIC- UA M ) , Universidad Autbnoma de Madrid, Madrid (Spain) and 2Departamento de Bioquimica y Biologia Molecular I, Universidad Complutense de Madrid, Madrid (Spain)
(Received23 June 1990;Revisedversion received3 August 1990;Accepted6 August 1990) Key words:
Butyrylcholinesterase;Molecularform; Brain microvessel;Blood-brain barrier
Molecular forms of butyrylcholinesterase(BuChE) were studied in microvesselsisolated from rat brain, using sedimentation analysis in sucrose gradients. Three forms, G~, G2 and G4, were found with sedimentation coefficientsclose to 3S, 6S and 9S, respectively.The relative proportion of the 3 forms was 19% for the monomer, 33% for the dimer and 46% for the tetramer. This sedimentation pattern of BuChE forms appears to be characteristicof cerebral microvesselsand may representdistinct functionalfeaturesof the blood-brain barrier.
Brain microvessels are unique with respect to their barrier mechanism, preventing access to a large extent to many substances from the blood. It has been described that butyrylcholinesterase (BuChE; EC 3.1.1.8) is present in high concentrations in the wall of all those microvessels that are equipped with a morphological bloodbrain barrier (BBB) [8, 9, 11], and we have reported some biochemical data on both acetylcholinesterase (ACHE) and BuChE in rat brain microvessels [5, 6]. Although the precise physiological function of BuChE remains unclear, not only in cerebral microvessels but also in the other tissues in which it is found, different roles for the enzyme have been proposed, related and not related to neurotransmission, i.e. hydrolysis of neuropeptides and fatty acid metabolism [7]. Furthermore, an involvement of this enzyme in the BBB function has been suggested [12, 19]. Both acetylcholinesterase (ACHE; EC 3.1.1.7) and BuChE have been shown to exist in a number of molecular forms particularly the globular monomer (G0, dimer (G2) and tetramer (G4), and the collagen-tailed A4, A8 and A12 forms [7]. Although the significance of the different molecular forms remains unclear, their tissue-specific distributions are thought to reflect the physiological functions of the different tissues [7]. Whereas BuChE has been extensively studied in the human plasma, where its role in the metabolism of anesthetics is of considerable clinical significance [4], its presence within the brain has received little attention. Correspondence: R.E. Catalfin, Departamento de BiologiaMolecular, Centro de BiologiaMolecular (CSIC-UAM), Universidad Aut6noma de Madrid, E 28040, Madrid, Spain.
0304-3940/90/$03.50 © 1990ElsevierScientificPublishers Ireland Ltd.
Nevertheless, BuChE has been reported to be associated with glial cells in white matter regions and with neuronal structures in grey matter regions [7]. The purpose of the present study was to determine the distribution of BuChE molecular forms in rat brain microvessels, where capillary endothelial cells contain high concentrations of this enzyme. The experiments were carried out on 30 male Wistar rats two months of age, and freely fed with standard pellets and tap water. Brain microvessels were obtained as previously described [5, 6]. The fraction collected from 50 pm mesh was used. The purity of brain microvessels was assessed by light microscopy and by the measurement of alkaline phosphatase and y-glutamyl transpeptidase as marker enzymes [17]. Presence of erythrocytes in the sample was negligible. Microvessels were resuspended and homogenized in a l0 mM Tris-HC1 buffer, pH 7.0, containing 1 M NaCl, 50 mM MgC12 and, unless stated otherwise, 1% Triton X-100. Homogenates were centrifuged at 17,000 g for 20 min. Supernatants were layered on sucrose density gradients (5-20% w/v) according to Rieger and Vigny [18]. Alkaline phosphatase from bovine intestine (6.1S) and catalase from beef liver (11.3S) were added to the enzyme samples as sedimentation markers. The samples were centrifuged for 20h at 40,000 rpm (130,000 gmax) in a Beckman SW 40 rotor, at 4°C. Approximately 50 fractions were collected and assayed for BuChE activity, as described previously [5]. BW2845C51, in a concentration of 30 pM, was included in the assay mixtures as a specific AChE inhibitor; hence, any residual activity in the presence of this inhibitor must be due to BuChE. Identification of the
47
0.45
0.BO
O.15 rn
I
i
O
2o
10
~o
,'o
~o
Fraction number
Fig. 1. Representative distribution of BuChE molecular forms in cerebral microvessels. Arrows indicate the positions of (from left to right) catalase (11.4S) and alkaline phosphatase (6.0S). Actual amounts of sample loaded were derived from microvessel homogenates containing the equivalent of 0.5 mg of protein.
different molecular forms was made on the basis of sedimentation coefficient, which was calculated relative to the positions in the gradient of the marker enzymes. Protein was determined by the method of Lowry et al. [15] using albumin as standard. The distribution of the different molecular forms of BuChE in brain microvessels is shown in Fig. 1, and an analysis of the data is presented in Table 1. The recovery from the gradient of the BuChE activity loaded (total activity prior to centrifugation/total activity recovered following centrifugation) was generally > 95 %. The loss of activity is probably due to loss of enzyme activity during centrifugation [1]. Our results indicate that 3 molecular forms of BuChE are present in brain microvessels, Gh G2 and G4 with sedimentation coefficients close to 3S, 6S and 9S, respectively. The sedimentation coefficients, although slightly lower are in good agreement with those obtained in other sources [1, 2]. The difference found in our study could be due to the detergent utilized [3]. The G4 form accounted for the majority of the BuChE activity, approximately 46% of the activity recovered. The 3.4S form accounted for 33% of total activity, and the 6S for only about 19%. The predominance for the tetrameric form of BuChE described here is in agreement
TABLE I MOLECULAR FORMS OF BuChE IN BRAIN MICROVESSELS Values are means ___S.D. of 5 determinations. Molecular forms Sedimentation coetticients (S) Percentage of total BuChE activity
GI
G2
3.4+0.2
6.0+0.3
33.4+2.5
18.9__ 1.8
G4 8.9__+0.3 46.0+2.9
with that reported by other authors for different biological sources, such as plasma, cerebrospinal fluid, and different areas of the central nervous system [1, 2, 16]. However, in these cases, the relative contribution of the G4 form to the total BuChE activity was higher (6994 %) than that observed by us in cerebral microvessels. The analysis of slower sedimentation peaks of BuChE (6.0S and 3.4S) indicates that the contribution of G1 species is slightly lower than that of G4 form, and it constitutes the majority of the non-G4 BuChE activity, in agreement with previous data reported for cerebrospinal fluid [2]. On the other hand, we must point out the considerable amount of the G2 form found in cerebral microvessels. This feature seems to be a characteristic of cerebral microvessels, since no discrete peaks of activity associated with any molecular form of BuChE other than the G4 and G1 species were detected by us in brain (unpublished data), in agreement with Atack et al. [1] and Muller et al. [16]. Furthermore, in plasma and cerebrospinal fluid, Gl and G4 forms were the only molecular species observed [2]. The possibility that the G2 form corresponds to AChE activity must be ruled out, since the assays were performed in the presence of an AChE inhibitor as described above. Therefore, the distribution of BuChE molecular forms reported here may be related to distinct anatomical and functional features of the BBB. Thus, the relatively high proportion of the lighter molecular form could suggest that the rate of conversion of G~ to G4 is slow and, therefore, a relatively large amount of the total BuChE is present as the Gh and G2 forms. However, this assumption seems to be unlikely since it has been reported that different mRNA species contribute to the formation of the various molecular forms of cholinesterase [20]. Further studies are required to resolve whether this fact takes place in brain microvessels. We have previously reported a monophasic Arrhenius plot of BuChE from brain capillaries [5], suggesting that it is unlikely that this enzyme interacts with membrane lipids. Nevertheless, the possibility that this interaction occurs and does not affect the catalytic activity of BuChE must be considered. In order to confirm whether BuChE molecular forms are membrane-bound, additional experiments, using different concentrations of Triton X-100 were performed. Table II shows the percentage of molecular forms extracted in the presence of Triton X-100, concentrations ranging from 0% to 1%. As can be seen, at least 0.1% Triton X-100 is necessary to completely solubilize the 3 forms. At concentrations as low as 0.01%, or without Triton X-100, only traces of G4 were detected in the supernatant. The analysis of the respective sediments confirmed that all molecular forms remained in the particulate fraction (unpublished data).
48 TABLE II EFFECT OF TRITON X-100 ON SOLUBILIZATION OF BuChE MOLECULAR FORMS IN CEREBRAL MICROVESSELS Results are expressed as percentage of activity of each molecular form considering as 100% the activity obtained after solubilization with 1% Triton X- 100. Triton X- 100 concentration (%)
Extraction (%)
0 0.01 0.05 0.1 1
Gi
G2
G4
0 0 93 99 100
0 0 85 97 100
12 12 66 95 100
These findings suggest t h a t the enzyme is w e a k l y a n c h o r e d to the m e m b r a n e , possibly by one o r m o r e t r a n s m e m b r a n e h y d r o p h o b i c sequences. This a s s u m p tion is s u p p o r t e d by the results o b t a i n e d when the molec u l a r forms were solubilized with 1% T r i t o n X-100, a n d then subjected to density g r a d i e n t c e n t r i f u g a t i o n in the absence o f d e t e r g e n t (Fig. 2). In these c o n d i t i o n s , the m a j o r i t y o f the non-G4 B u C h E activity is f o u n d at the b o t t o m o f the gradient, suggesting that an a g g r e g a t i o n by the h y d r o p h o b i c sequences takes place. O n the o t h e r hand, it has been suggested t h a t the m o l e c u l a r forms o f B u C h E c o r r e s p o n d to different locations: G i a n d G2 at intracellular sites, chiefly in the r o u g h e n d o p l a s m i c retic u l u m where they are synthesized for the G j form, a n d in the p l a s m a m e m b r a n e for the G4 f o r m where they migrate quickly [13]. In a d d i t i o n , F l u m e r f e l t et al. [9], have f o u n d in rat b r a i n capillaries, B u C h E activity in the i n t e r m e m b r a n o u s space o f the e n d o t h e l i a l nuclear envelope, the e n d o t h e l i a l e n d o p l a s m i c reticulum, a n d the e n d o t h e l i a l b a s e m e n t m e m b r a n e . Therefore, it seems likely t h a t these different localizations c o r r e s p o n d to
0.30 0 < 0.20
# > ,j o
0.10
rn
%--/,'
io
~
4'o
~o
Fraction number
Fig. 2. Distribution of BuChE molecular forms in cerebral microvessels extracted with 1% Triton X-100 and subjected to density gradient centrifugation in the absence of detergent.
each o f the three m o l e c u l a r forms r e p o r t e d by us. It is also p r o b a b l e t h a t G 4 f o r m is secreted a n d a s s o c i a t e d with the extracellular matrix, which has been involved in the cell g r o w t h a n d differentiation [10]. This idea is supp o r t e d by previous findings d e m o n s t r a t i n g a strong correlation between B u C h E a n d cell p r o l i f e r a t i o n [14]. Finally, based on the characteristic d i s t r i b u t i o n o f B u C h E m o l e c u l a r forms in cerebral microvessels, we suggest t h a t a m o r e detailed study o f this e n z y m a t i c activity m a y lead to a better u n d e r s t a n d i n g o f certain p a t h ological c o n d i t i o n s where the BBB is altered. The a u t h o r s are i n d e b t e d to Mr. J. Palacin for his v a l u a b l e assistance. This w o r k was s u p p o r t e d in p a r t by g r a n t s from the F u n d a c i 6 n R. Areces, F I S a n d D G I CYT. 1 Atack, J.R., Perry, E.K., Bonham, J.R., Candy, J.M. and Perry, R.H., Molecular forms of acetylcholinesterase and butyrylcholinesterase in the aged human central nervous system, J. Neurochem., 47 (1986) 263-277. 2 Atack, J.R., Perry, E.K., Bonham, J.R. and Perry, R.H., Molecular forms of acetylcholinesterase and butyrylcholinesterase in human plasma and cerebrospinal fluid, J. Neurochem., 48 (1987) 18451850. 3 Brimijoin, S., Molecular forms of AChE in brain, nerve and muscle: nature, localization and dynamics, Prog. Neurobiol., 21 (1983) 291~22. 4 Brown, S.S., Kalow, W., Pilz, W., Whittaker, M. and Woronick, C.L., The plasma cholinesterase: a new perspective. Adv. Clin. Chem., 22 (1981) 1-123. 5 Catal~m, R.E. and Hern~mdez, F., Temperature effects on cholinesterase from rat brain capillaries. Biosci. Rep., 6 (1986) 573-577. 6 Catalan, R.E. and Hernandez, F., Acetylcholinesterase and butyrylcholinesterase in rat brain capillaries, Med. Sci., 15 (1987) 291292. 7 Chatonnet, A. and Lockridge, O., Comparison of butyrylcholinesterase and acetylcholinesterase, Biochem. J., 260 (1989) 625~34. 8 Djuricic, B.M. and Mrsulja, B.B., Enzymatic activity of the brain microvessels vs. total brain homogenate, Brain Res., 138 (1977) 561 564. 9 Flumerfelt, B.A., Lewis, P.R. and Gwyn, D.G., Cholinesterase activity of capillaries in the rat brain. A hght and electron microscopic study, Histochem. J., 5 (1973) 67-77. 10 Goldstein, G.W. and Betz, A.L. Recent advances in understanding brain capillary function, Ann. Neurol., 14 (1983) 389-395. 11 Jo6, F. and Csillik, B., Topographic correlation between the hematoencephalic barrier and the cholinesterase activity in brain capillaries, Exp. Brain Res., 1 (1966) 147-151. 12 Jo6, F., Varkonyi, T. and Csillik, B., Development alterations in the histochemical structures of brain capillaries: histochemical, contributions to the problem of the blood-brain barrier, Histochemie, 8 (1967) 140-148. 13 Koelle, G.B., Massouli6, J., Eugene, D., Melone, M.A.B. and Boulla, G., Distribution of molecular forms of acetylcholinesterase and butyrylcholinesterase in nervous tissue of the cat, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 7749-7752. 14 Mayer, P.G. and Sporn, O., Spatiotemporal relationship of embryonic cholinesterase with cell proliferation in chicken brain and eye, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 284-288.
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