- Email: [email protected]
Plasminogen activator is enriched in the synaptosomal plasma membranes
Brain Research, 248 (1982) 129-139 Elsevier Biomedical Press
129
Plasminogen Activator is Enriched in the Synaptosomal Plasma Membranes NAVA ZISAPEL, RUTH MISKIN, MOSHE LAUDON and HERMONA SOREQ* Dept. of Biochemistry, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Rarnat-Aviv; (R.M.) Dept. of Biochemistry, Weizmann Institute of Science, Rehovot and (H.S.) Dept. of Neurobiology, Weizmann Institute of Science, Rehovot 76100 (Israel)
(Accepted February 25th, 1982) Key words: plasminogen activator - - bovine brain cortex - - synaptosomes - - membrane association
The intracellular localization of the serine protease plasminogen activator was analyzed in homogenates of bovine brain cortex using differential fractionation procedures. The distribution of the enzymewas clearly different from that of cytosol and mitochondrial markers, and was similar to that of plasma membrane proteins and of the muscarinic acetylcholinereceptor, which is a specific marker for the synaptic membrane. The specific activity of plasminogen activator was increased in fractions enriched in intact synaptosomes. Most of the enzyme in intact synaptosomes was found to be firmly associated with the synaptosomal membrane, and could be solubilized by high concentrations of salt or by non-ionic detergent. Purified synaptic vesicles, however, did not contain large amounts of plasminogen activator. Bovine brain synaptosomes were shown to contain two species of the enzyme, having apparent molecular weights of 80,000 and 55,000. The presence of plasminogen activator in the synaptosomal membrane may indicate its possible involvement in the functioning of nerve terminals. INTRODUCTION The serine proteases plasminogen activators (PAs) are found in different vertebrate organs such as kidney, lung and brain, as well as in body fluids like plasma, urine and cerebrospinal ftuidS,lo, 32. A variety of cell types in culture can be induced by physiological and non-physiological inducers to synthesize and secrete large amounts of P A 25. Plasminogen activators have also been detected in neuroblastoma cells of mouse 11 and of human origin2, 26, 37 The enzymatic activity of PA is limited to the specific conversion of the zymogen plasminogen into the trypsin-like protease plasmin, which is the physiological fibrinolytic enzyme 4. In addition to its thrombolytic function, the plasminogen activation system appears to play a major role in general extracellular proteolysis, necessary for cell migration and rearrangement of tissues ca. PA involvement in the processing of prohormones to hormones has also
* To whom correspondence should be addressed. 0006-8993/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
been recently suggested 36. Similar functions can be envisioned for PA in the developing and the mature brain, and a detailed intracellular localization of the brain enzyme may aid to the understanding of its function(s) in the nervous system. In the few cell types analyzed so far for intracellular localization of the enzyme, most of the PA activity has been confined to membranous elements 7,1e,
19,24,27,34. In frozen brain sections, P A activity was recently localized to regions and cell layers rich in neuronal cell bodies. This analysis was carried out by autoradiography of radiolabeled proteo]ytic products cross-linked to the surface of cells in active sections (ref. 30). When applied to viable differentiated neuroblastoma cells in culture, this technique revealed extensive labeling in growth cones and on the surface o f the extended processes, in addition to perikarya ~9. These observations raised the possibility, that PA might also be present in synapses and involved with neuronal functioning.
130 In the present work, which was aimed at extending the subcellular localization of PA to who!e brain tissue, the distribution of PA activity was determined during the course of preparation of synaptosomes from bovine brain cortex, a source in which the properties of purified synaptosomes were well documented as. PA activity was compared with that of known markers for cytosol, mitochondria and membrane, as well as with markers specific for synaptosomes. We now report that a large fraction of PA in bovine brain cortex appears to be membrane associated, that the enzyme is found in preparations enriched with synaptosomes, and that it appears to be firmly associated with the synaptosomal membrane. MATERIALS AND METHODS
Preparation andfractionation of synaptosomes Two procedures were employed as follows. (a) Slices of cerebral cortex were obtained from newly removed bovine brain and homogenized (7 passages at 1200 rpm) in a teflon-glass homogenizer in 10 ml per g tissue of 0.32 M sucrose adjusted to pH 6.8 with NaOH. Synaptosome-rich fraction was subsequently obtained following the procedure described by Whittaker and Barker 3s. All purification steps were carried out at 4 °C. The homogenized material (H) was centrifuged (104 g-min). The resulting pellet (P1) was washed with 1 vol. of the homogenization medium followed by centrifugation (104 g-rain) and discarded. The supernatants ($1) were combined and centrifuged (2 × 105 g-min) to yield a crude synaptosomal pellet (PD, which was resuspended by homogenization (one stroke at 150 rpm) using a teflon-glass homogenizer and loaded onto a sucrose step gradient, composed of three layers of 1.2 M, 0.8 M and 0.5 M sucrose. After centrifugation (105 g-rain) in a Beckman rotor SW 25.1, 6 fractions were carefully collected with a pasteur pipette, starting at the lightest fraction. Synaptosomes were collected from the 0.8 M/1.2 M sucrose interface. (b) The procedure displayed by Michaelson and Sokolovsky22 was adapted as follows: slices of bovine cerebral cortex were homogenized (10 ml per g tissue) in 0.4 M glycine buffer pH 6.8 (7 passages at 1200 rpm in a teflon-glass homogenizer), and synaptosome-enriched fraction (P2), subsequently ob-
tained following the procedure described by Whittaker and Barker 3s, was resuspended by homogenization and loaded onto a density gradient. Density gradient centrifugation (6 × 106 g-min) was performed with a discontinuous gradient in an SW40 rotor. The loaded gradient tubes contained 6 layers of equal volume of the following concentrations (M) of sucrose: 0, 0.15, 0.3, 0.55, 0.8, 1.2. Layers 1 and 2 also contained glycine, so that their osmolarity was equal to that of the homogenization buffer. After centrifugation the material which separated at the interfaces and in the layers of the gradient was carefully collected using Pasteur pipettes, starting at the lightest fractions as described in Fig. 2. Fraction 11 included material which occasionally centrifuged to the bottom of the tube after centrifugation, as well as some contaminating remnants of fraction 10 which, due to its high viscosity, remained in the tube after collection.
Isolation of plasma membranes Synaptosomal membranes were prepared by the procedure of Van Leeuwen et al. 35, as modified by Zisape] et al. a9. Crude synaptosomal fraction (P2) or purified synaptosomes obtained by procedure b were subjected to hypoosmotic shock in 60 mM sucrose, and centrifuged to yield a membrane enriched pellet (Wp). The pellet was resuspended in 50 mM MgC12, mixed with 2 vol. of 51 ~ (w/v) sucrose to a final concentration of 0.6 M and overlaid on 0.8 M sucrose. A 0.4 M sucrose solution was then pipetted on top and was overlaid finally with buffer (0 M sucrose). After centrifugation (6 × 106 g-min) the membrane-rich fraction was collected from the 0.60.8 M sucrose interface (Fig. 2).
Enzyme assays The following marker enzyme activities were determined spectrophotometrically using a Cary 118 recording spectrophotometer: acetylcholinesterase (EC 2.1.1.7) 8, lactate dehydrogenase (EC 1.1.2.7) 13 and cytochrome c oxidase (EC 1.9.2.1) 31. Horse heart cytochrome c (Sigma) was reduced by sodium dithionite and excess reductant was eliminated by gel filtration through a column of Biogel P-4. ATPase and 5'-nucleotidase activities were measured by the liberation of inorganic phosphate from ATP and AMP 21. Muscarinic acetylcholine recep-
131 tors were measured by means of the binding assay of 3H-labeled N-methylpiperidyl-benzilatO7. Dopamine synthesis was measured by the liberation of labeled 14COz from L-[14C]tyrosinO6. Plasminogen activator was assayed as previously described by the plasminogen-dependent liberation of fibrin degradation products from ~25I-labeled fibrin3°. Urokinase (UK) (Leo Pharmaceutical Products, Denmark) was added to each assay, and results were expressed as Ploug units of UK per mg of sample protein. Preparation of extracts for PA analyses was as described (ref. 30). Acetylcholine was determined by a bioassay using guinea pig ileum31. Protein was removed from the samples by precipitation with 10~ trichloroacetic acid and centrifugation. The acid was extracted with diethyl ether saturated with water. Protein was assayed as described by Lowry et al. 2°.
Electrophoretic analysis of PA activity Samples of fractionated homogenates were electrophoretically separated in SDS-polyacrylamide gels containing casein, according to Huessen and Dowdle 9. Following electrophoresis, SDS was extracted from the gel, which was then incubated at 37 °C to allow proteolysis. Dark staining of the gel visualized clear zones of proteolytic activity.
Preparation of synaptic vesiclefraction Slices of cerebral cortex were obtained from freshly removed bovine brain and homogenized in 10 ml per g tissue of 0.32 M sucrose adjusted to pH 6.8, using a teflon-glass homogenizer (7 passages at 1200 rpm). Vesicles were isolated as described by Zisapel and Zurgil4°. Fraction P2 obtained from homogenized slices of bovine cerebral cortex was suspended in water adjusted to pH 6.8 and subjected to mild homogenization in a glass-teflon homogenizer (1 passage, 150 rpm). The suspension was centrifuged (106 g-min) to yield a vesicle-rich supernatant fraction (Ws) and a membrane-rich pellet (Wp) which was further used for membrane preparation. The vesicle-rich fraction was laid on a sucrose-step gradient composed of 1.2 M, 0.6 M and 0.4 M sucrose layers and centrifuged 1.7.5 × 106 g-min) (Fig. 1). The synaptic vesicles obtained from 0.4 M sucrose layer were further purified by gel filtration on a Sepharose 6B column.
Solubilization and lysis experiments Allquots of the synaptosomal preparations were suspended in 2 ml ef(a) 0.15 M glycine-0.15 M sucrose containing 0.5 ~ Triton X-100, or (b) 0.155 M or 1.0 M NaCI, adjusted to pH 6.8. Samples were incubated for 10 rain at room temperature and then centrifuged (2 × 106 g-min). The supernatant and the pellet were collected, and pellet was resuspended in an equal volume of fresh incubating solution. Aliquots from the resuspended pellets and supernatants were assayed for plasminogen dependent fibrinolytic activity30.
Electron microscopy Samples were fixed in 0.15 M cacodylate buffer pH 7.4 which contained 2.5~o glutaraldehyde. The material was postfixed with osmium tetroxide, dehydrated and embedded in epon. Sections were stained on the grids with lead citrate and observations were carried out using a Jeol 1008 electron microscope. RESULTS
Distribution of PA in subcellular fractions The subcellular distributiort of PA in bovine brain cortex was determined by differential centrifugation of brain cortex homogenates, and compared with the distribution of the cytoplasmic enzyme lactate dehydrogenase (LDH) and the synaptic membrane component acetylcholine receptor (AChR) (Table I). In the supernatant fraction Sa, over half of each of the analyzed activities was recovered, together with the cytosol components and subcellular structures known to appear in this fraction, such as synaptosomes, microsomes, mitochondria and myelin. The remaining components, consisting of tissue debris, nuclei and part of the myelin, sedimented at low velocity into the P1 pellet. It should be noted that the recovery of PA activity at this step was reproducibly low (about 50 ~ of that of the homogenate activity). However, the further purification steps did not cause losses in PA yield. After centrifugation at high velocity, about 60 of PA activity as well as 6 0 ~ of the muscarinic AChR sedimented into the crude synaptosomal pellet, Pz, known to contain synaptosomes, microsomes, mitochondria and myelin3s. Both PA and the AChR displayed specific activities which were al-
132 TABLE I Distribution of enzymatic markers in synaptosomepreparations
PA activity is expressed in urokinase Ploug units; acetylcholine receptor content is expressed in pmol; lactate dehydrogenase activity is expressed in #mol/min. All activities (A) are given per g original tissue and all specificactivities (SA) are given per mg protein. Data represent averages of three experiments, which varied by less than 30 %. Fraction
H $1 P1 S~ P~
Plasminogen activator
Acetylcholine receptor
Lactate dehydrogenase
A
SA
A
SA
A
SA
54.0 16.0 8.8 4.9 7.4
0.53 0.31 0.18 0.19 0.35
73.5 36.8 32.7 12.6 20.2
0.72 0.72 0.67 0.47 0.96
154 151 29 99 29
1.5 2.9 0.6 3.7 1.4
most 2-fold higher in the crude synaptosomal pellet than in the supernatant fractions ($2) obtained at high velocity. The reverse situation was observed with L D H , 77 % of which remained in the supernatant. This seems to suggest the association of the major part of PA activity in the bovine cerebral cortex with membranal structures rather than with cytosol components. Association o f PA with bovine cortex synaptosomes The association of PA with bovine cortex synapA
PLASMINOGEN -----
ACTIVATOR
ACETYLCHOLINESTERASE
4-0-c C 0
I
tosomes, prepared by discontinuous sucrose gradient from crude synaptosomal pellet as, was determined by comparison to the distribution of markers of cytosol (LDH), mitochondria (cytochrome c oxidase) and synaptosomes (acetylcholinesterase). The sucrose gradient fractions mostly enriched with PA were found to be the 0.8 M sucrose fraction and the 0.8/1.2 M sucrose interface (Fig. 1A). These fractions are also enriched with acetylcholinesterase, in agreement with Whittaker and Barker as who have reported the preparation of synaptosomes from these fractions. According to this preparation procedure, L D H mostly remains in the 0 M sucrose fraction, whereas mitochondria segregate to the 1.2 M sucrose fraction, as revealed by the migration of cytochrome c oxidase (Fig. 1B).
o. 2 0 E 0 0
> o o ID
B - -
LACTIC -
DEHYDROGENASE
CYTOCHROME
OXIDASE
II
a: 4 0 -
I
I I I
I I I
~ 2o-
I L
--
L__ i. . . . I
I
2
I
3
I
4
I
5
6
Fraction
Fig. 1. Distribution of plasminogen activator and of enzymic markers in synaptosomal preparations separated on a discontinuous sucrose density gradient by procedure a (one out of three experiments is displayed (see Materials and Methods for details). Height of blocks refers to the amount of component in each fraction expressed as a percentage of total recovered component.
Characterization o f enriched synaptosomes prepared in glycine buffer To further investigate the association of PA with synaptosomal.enriched fractions, and to improve the separation of these fractions from mitochondria, we also prepared synaptosomes by procedure b (see Methods), based on a modified enrichment technique 2z. Homogenization of bovine brain cortex and subsequently the preparation of crude synaptosomal pellet was performed in this procedure in isoosmotic glycine buffer instead of the 0.32 M sucrose. Following this step, the synaptosomal pellet was fractionated by sucrose density centrifugation (Fig. 2). The fractions obtained were assayed for protein content (Fig. 3) and for several markers of known subcellular distribution (Figs. 3 and 4).
I33
Purification of
I
Bovine Cortex Synaptosomes Homogenate I spun at IO~-rnin
Pellet P,
Suspended in water, spun at 106g-rnin
I Pellet Wp
Distribution of molecular markers in synaptosomal preparations
I
30-
I Spun at IO~-min
5' Nucleotidase - - - Protein
20 -
~
Sup. Sj
J
I
I Sup.Ws
Separation by density gradient at Z5xIOs
sJp,
Pellet Pz Suspend in J homogenization buffer I Separafeon I density gradient at 6xl0 6 J g-min ~, 3
{::: 4'
ATPase ~A c e3t0yVl c h °-l-i "n e s t e r ° s e > ~
.15MI--HSynaptosomesJ """'1
i
j
55M I
:.
g.o 2°I--
7
r-'l II iI i t
o
I "--7
I0--
/
r-1
I
r-!
--4
I
.-~-~ I ~ i i L-~ L-J
I
i
I
-
F-~
I I
I
I
t -
I
g-min 0 1
II
gel filtration D r ~ 00~ Sel:tDaros e I
I~
I
lZ
Suspend in water Separate:densitygradient at 6x IOSg-min
30 20
OIV
I0
).41
lPloe.~ ~mbra,~sl~
061
60
_c
,r-] Plosminogen Activator - - - Acetylcholine Receptor
-
~
z-]D r-'-t
Fig. 2. Preparation of synaptosomes, synaptosomal membranes and synaptic vesicles from brain cerebral cortex. The details (procedure b) are given in Materials and Methods.
The distribution of PA on the sucrose density gradient used for the preparation of synaptosomes obtained by procedure b was compared with that of the other markers. PA distribution was generally similar to that of the membrane markers, and differed from that of cytosol and mitochondrial constituents6, a4. This was evident whether PA activity was expressed as percent of recovered activity (Fig. 3) or as specific activity (Fig. 4). High levels of PA activity were detected in the fractions enriched with intact synaptosomes, as well as in otber fractions containing membranous markers. The distribution of intact synaptosomes prepared by procedure b was indicated by the content of occluded acetylcholine and by the activity of the muscarinic AChR. Acetylcholinesterase, 5'-nucleotidase and Na, K-ATPase served as markers of
40 20
I I
i .I
L,,_
- - D o p a m i n e Synthesis -Acetylcholine
! I
-I i --
"-J, 2
I
I
i ,~-i-d. 4
... 6 Fraction
,_.r'r, 8 I0
Fig. 3. Distribution of molecular markers from crude synaptosomal fraction derived from bovine cerebral cortex, as obtained following sucrose step gradient centrifugation (procedure b; one out of three experiments; see Materials and Methods for details). Height of blocks refers to the amount of component in each fraction expressed as a percentage of total recovered component.
synaptosomal external membrane and of microsomal plasma membrane. L D H and dopamine synthase served as markers of the cytosol fraction, and the presence of mitochondria was determined by measuring the distribution of cytochrome c oxidase. The distribution of 5'-nucleotidase, acetylcholinesterase and ATPase on the sucrose gradient used for
134 the preparation of synaptosomes according to procedure b indicated membrane enrichment in fractions 3-8 and 10, 11 (Fig. 3A, B), a finding which was further supported by the similar distribution of Ca2+-activated alkaline phosphatase (not shown). AChR generally partitioned, when procedure b was employed, like the other membranal markers, and exhibited its highest specific activity in fractions 3-6 and 9, 10 (Fig. 4A). Acetylcholine was detected mainly in fractions 3 and 4. The low acetylcholine content of other fractions might be a consequence of exposure of non-occluded acetylcholine to acetylcholinesterase. Moreover, substances such as histamine or epinephrine, present in these fractions may interfere with the guinea pig ileum bioassay1. Acetylcho[ine would therefore be detected primarily in intact synaptosomes, whereas the receptor distribution indicates the presence of both intact and disrupted synaptosomes. Thus, it appears that intact synaptosomes, when prepared in glycine buffer, tend to accumulate mainly in fractions 3 and 4. Sucrose Gradient Centrifugation of Bovine Cortex Synaptosomes
S
I --AChR !
I l'~I r
~I
L_~
I I
Fill
~
I
[
--~8 ~I
-
'o.8F I I
.
L__,__J '--F-,-o.4 a
in -
~
o ~
~I
FI I I',
I ! I--J
Im
i i
2
I I
4
6 Fraction
8
I t
0.2:_~ s <
I0
Fig. 4. Specific activities of plasminogen activator and of enzymatic markers in synaptosomal preparation separated on a discontinuous sucrose density gradient according to procedure b (see text for details). Height of blocks refers to the activity of component per mg protein content of the corresponding fraction. Units of activity (average of duplicate assays) are expressed as follows: LDH, /~mol/min; cytochrome c oxidase, 10-a0 mol/min; AChR, pmol; PA, urokinase Ploug units.
The distribution of cytosol and mitochondrial markers, which clearly differed from that of synaptosomal markers, further supports this conclusion. Minor fractions of the original activities of cytoplasmic enzymes dopamine syntbase and LDH remained in the crude synaptosomal P2 pellet. These activities accumulated primarily in fractions 1 and 2, and apparently originated from residual cytesol as well as from synaptosomes and microsomes which were disrupted in the course of preparatien (Figs. 3D and 4B). Mitochondria, on the other hand, centrifuged down to the 1.2 M sucrose layer or precipitated into the pellet, as indicated by the high specific activity of cytochrome c oxidase in fractions 10 and 11 (Fig. 4B). Minute amounts of both cytoplasmic and mitochondrial markers can, however, be detected in fractions 3 and 4 (Figs. 3 and 4), further indicating the presence of sealed membranal elements within these fractio,s. Electron microscopic examination of samples from fractions 3 and 4 revealed rounded sac-like structures characteristic of nerve endings which contain synaptic vesicles, as well as occasional mitochondria (Fig. 5). This observation indicates that the enriched synaptosomes as prepared in glycine buffer are relatively free of non-occluded cytoplasmic elements, membrane fragments and mitochondria, and further confirms the biochemical analysis.
Distribution of PA in synaptosomal fractions To further examine the association of PA with membranous structures, membranes were prepared from crude synaptosomal pellet (P2) and from purified synaptosomes (fractions 3 and 4 of the sucrose gradient). The specific activit~ of PA was measured in these preparations, and the extent of enrichment in PA compared with that obtained for markers of synaptosomal plasma membranes (Table II). The results indicate an approximately 2-fold enrichment of acetylcholinesterase, AChR, alkaline phosphatase and PA activities in intact purified synaptosomes as compared with crude synaptosomes. Preparations of membranes from crude synaptosomes resulted in a ~light increase in PA specific activity, thus confirming its association with the membranous component of the Pz pellet. A similar increase was observed in the specific activities of alkaline phosphatase and of acetylcholinesterase,
135
Fig. 5. Electron micrographs of various magnifications of the synaptosome preparation obtained from isoosmotic sucrose density gradient centrifugation (procedure b, fractions 3 and 4) (for details see text).
136 TABLE II Enrichment of PA and enzymatic markers in synaptic plasma membranes and crude synaptosomal pellet
All enzyme activity values are presented per nag protein. Enzyme
AChE (/~mol/min) AChR (pmol) Alk. Phos. (nmol/min) PA (urokinase Ploug units)
Fraction Crude synaptosomes
Purified synaptosomes
Membranes from crude S.
Membranes from purified S.
Sp. act.
F
Sp. act.
F
Sp. act.
F
Sp. act.
F
44 0.96 9.1 0.35
1 1 1 1
85 2.1 20 0.65
1.93 2.17 2.20 1.86
69 0.94 13.5 0.55
1.57 0.98 1.48 1.57
179 2.50 23.80 2.16
4.08
while the specific activity of the muscarinic receptor for acetylcholine remained apparently unchanged. The membrane fractions obtained from purified synaptosomes were found to be further enriched with PA, as well as with other synaptosomal membrane markers. This enrichment of synaptosomal markers is consistent with earlier observations on synaptosomes from ratlZ, 23, chick 35 and bovine brainZS. The nature of PA interaction with the synaptosomal plasma membranes was investigated by means of solubilization experiments aimed at removing attached membrane proteins is. Purified synaptosomes f r o m fractions 3 and 4 were suspended in salt or in non-ionic detergent solution, and the precipitable material collected by centrifugation as described in Materials and Methods. Following incubation in glycine buffer containing 0.15 M sucrose, in 0.155 M NaC1 or in 0 . 5 ~ Triton X-100, most of the membranal PA activity (80-95 ~ ) remained attached to the synaptosomal membrane. A moderate release of 20-40 ~ was observed following incubation in 1.0 M NaC1. A firm association of the majority of PA molecules with the synaptosomal membrane is thus indicated. Synaptic vesicles, which constitute a minor fraction of the synaptosomal protein content but contain occluded neurotransmitters 40, are putative candidates for storage of the minor fraction of PA activity which is not associated with the synaptosomal plasma membrane. We therefore determined the specific activity of PA in purified synaptic vesicles, obtained f r o m bovine cortex by centrifugation
2.58 2.62 6.20
in sucrose density gradients (Fig. 1). The relatively low specific activity, 0.45 U K units per mg protein, indicates that synaptic vesicles do not contain large quantities of PA activity, either in a soluble or in a membrane-associated form. Synaptosomal PA displays two active species having apparent molecular weights of about 80,000 and 55,000 following SDS-polyacrylamide gel electrophoresis in a casein-containing gel (Fig. 6). The estimated molecular weights of PA species in bovine cortex synaptosomes therefore appear to be close to those of the two species of PA found in the mouse za and rat brain 3°.
Fig. 6. Molecular species of PA from bovine cortex synaptosomes (fractions 3 and 4). A 6/zl sample of purified synaptosomes containing 20 #g protein (2) was separated by gel electrophoresis in parallel to a 20/~1 sample (10:1, v/w) of rat brain extract (1), prepared as described previously3°. Proteolytic activity is visualized as destained zones on the darkly stained background.
137 DISCUSSION The subcellular distribution of PA activity has been examined to date in several cell types grown in culture. The major part of intracellular PA has been found in most cases to be associated with the particulate fraction. We now extend this finding to bovine brain cortex. The distribution of brain PA between the various subcellular fractions differs somewhat from that observed with fractions of cells in culture. In bovine brain homogenate about half of the PA activity is found in the low speed centrifugation pellet, whereas a much smaller fraction of the enzyme is found in this pellet when cultured cell homogenates are similarly centrifuged2a. In cortical homogenate about 40 9/ooof PA activity remains in the cytosol supernatant following high speed centrifugation, a higher fraction than that obtained upon similar centrifugation of cultured cell homogenate. The subcellular distribution of brain PA is similar, however, to that of brain AChR and is clearly different from that of cytoplasmic brain proteins such as LDH. Both these established markers dispersed between the different subcellular fractions in a manner fully consistent with other reports on fractionation of brain homogenatesaT,3s. It therefore appears that the less distinct subceUular distribution of brain PA, as compared with PA from cells in culture, is due to the biochemical and structural complexity of the brain tissue. The yield of PA activity in brain cortex homogenate is reduced to half following low speed centrifugation, a reduction which does not occur in centrifuged cell homogenates23. This decrease appears to be rather specific for PA as tile muscarinic receptor for acetylcholine and the cytoplasmic enzyme LDH both retain full activities following this step. The existence of specific protease inhibitor(s) in brain tissue has been reported 3. The initial centrifugation step may enhance the association of PA with such inhibitors and thereby reduce the recovered activity. Further fractionation of the P2 pellet revealed a distribution of PA activity which resembled that of membranous components and differed from that of cytosol and mitochondrial constituents, under both centrifugatien methods employed. High specific activity was obtained for PA in the 0.8 M sucrose
fraction according to procedure a, and in fractions 3, 4, 7 and 9 of procedure b gradients, in parallel to the increase in specific activity of acetylcholinesterase and of the acetylcholine receptor in these fractions. Both biochemical and ultrastructural analyses have shown these fractions to be enriched with intact synaptosomes. The high specific activity of plasminogen activator in synaptosome-enriched fractions may reflect the presence of higher amounts of this enzyme in synaptosomal membranes, as compared with other membrane fractions. Another possibility is that the synaptosomal membranes may have lower quantities of endogenous proteinase inhibitors than are found in the other membrane fractions, such as those obtained from cell bodies. In both cases, PA appears to be an integral component of the synaptosomal structure. PA specific activity in purified synaptic vesicles was significantly lower than that found in synaptosomal membranes. The major part of the synaptosomal PA is associated with the synaptosomal membrane, as indicated by the increase in PA specific activity upon preparation of membranes from purified synaptosomes. Most of the enzyme remains attached to purified synaptosomal membranes upon treatment with 0.155 M salt of 0.59/00 non-ionic detergent, but part of its is released by 1.0 M NaC1. A firm association of PA with the synaptosomal membrane is indicated by the finding that the enzymatic activity remains membrane-attached in 0.5 9/00 Triton X- 100. Cell-associated as well as secreted forms of PA have been found in a variety of cell types in culture, but only a few have been analyzed for the intracellular localization of the enzyme. In all these cell types most of the intracellular PA appears to be firmly associated with plasma membrane-like particlesT, 19, 24,2L The only exception is confluent 3T3 cells, in which the enzyme appears to be preferentially associated with a membranous fraction of high density, different from that of the plasma membrane. Unlike growing and SV-40 transformed 3T3 cells, the confluent cells do not secrete PA, and it has been suggested that these two unique characteristics are correlated, and that the presence of PA in the plasma membrane might be related to the secretion process 12. If this is indeed the case, our findings may indicate that within the bovine brain cortex, PA is
138 secreted f r o m nerve cells as well as f r o m their t e r m i nals into the s u r r o u n d i n g extracellular fluid. It has been speculated t h a t m e m b r a n e - a s s o c i a t e d P A in t r a n s f o r m e d cells is involved in a l t e r a t i o n s o f the cell surface which a c c o m p a n y cellular t r a n s f o r m a t i o n 24. It has in fact been d e m o n s t r a t e d that the p l a s m i n o g e n a c t i v a t i o n systems affects the t u r n o v e r o f the nicotinic A C h R , a distinct m e m b r a n e c o m p o nent o f chick e m b r y o muscle cells 15. A similar role cart be envisioned for P A in nerve cell p l a s m a m e m b r a n e a n d terminals, where this enzyme might p l a y a p a r t in the a l t e r a t i o n o f m e m b r a n e c o m p o s i t i o n involved in intercellular c o m m u n i c a t i o n . A n o t h e r f u n c t i o n which might be a t t r i b u t e d to s y n a p t o s o m a l P A is its involvement in the processing o f p r e c u r s o r s for p e p t i d e h o r m o n e s a n d / o r n e u r o t r a n s m i t t e r s , similarly t o the f o r m a t i o n o f insulin f r o m p r o i n s u l i n in the p a n c r e a t i c fl-islets3~.
REFERENCES 1 Aprison, M. H. and Nathan, P., Determination of acetylcholine in small samples of fresh brain tissue, Arch. BIOchem., 66 (1957) 388-395. 2 Becherer, P. R. and Wachsman, J. T., Increased neurite development and plasminogen activator expression by exposure of human neuroblastoma cells to a plasminogendeficient growth medium, J. cell. Physiol., 104 (1980) 47-52. 3 Brecher, A. S. and Quinn, N. H., The occurrence of a trypsin inhibitor in brain, Biochem. J., 102 (1967) 120-121. 4 Christman, J. K., Acs, G., Silagi, S. and Silverstein, S. C., Plasminogen activator: biochemical characterization and correlation with tumorigenicity. In E. Reich, D. B. Rifkin and E. Shaw (Eds.), Proteases and Biological Control, Cold Spring Harbor Laboratory, New York, 1978, pp. 827-839. 5 Christman, J. D., Silverstein, S. C. and Acs, G., Plasminogen activators. In A. J. Barret (Ed.), Proteinases in Mammalian Cells and Tissues, Elsevier, Amsterdam, 1977, pp. 91-149. 6 Cotman, C. W. and Matthews, D. A., Synaptic plasma membranes from rat brain synaptosomes: isolation and partial characterization, Biochim. biophys. Acta, 249 (1971) 380-394. 7 Dvorak, H. F., Orenstein, N. S., Rypysc, J., Colvin, R. B. and Dvorak, A. M., Plasminogen activator of guinea pig basophilic leukocytes: probably localization to the plasma membrane, J. lmmunol., 120 (1978) 766-773. 8 Ellman, G. L., Courtney, P. K., Andres, V. and Flatherstore, R. M., A new and rapid colorimetric determination of acetylcholinesterase activity, Biochem. Pharmacol., 7 (1961) 88-95. 9 Huessen, C. and Dowdle, E. B., Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulfate and copolymerized substrates, Analyt. Biochem., 102 (1980) 196-202.
U l t r a s t r u c t u r a l labeling studies w o u l d be r e q u i r e d to d e t e r m i n e u n a m b i g u o u s l y w h e t h e r P A is highly enriched within the synapse itself. A n a u t o r a d i o graphic p r o c e d u r e has recently been d e v e l o p e d to detect active P A in frozen cell layers o f organ sections a0. Electron m i c r o s c o p i c r e s o l u t i o n o f labeled cells in b r a i n sections m a y p r o v i d e the necessary m e t h o d o l o g y for the fine u l t r a s t r u c t u r a l localization o f this enzyme. ACKNOWLEDGEMENTS W e t h a n k Ms. O f r a D e u t s c h for excellent technical assistance. This w o r k was s u p p o r t e d b y grants f r o m the U n i t e d S t a t e s - I s r a e l B i n a t i o n a l SOence F o u n d a t i o n ( B S F ) Jerusalem, Israel (to R . M . ) a n d f r o m the Israeli C o m m i s s i o n for Basic R e s e a r c h (to H.S.). H e r m o n a Soreq is a n i n c u m b e n t o f a Charles R e v s o n C a r e e r D e v e l o p m e n t Chair.
10 Glas, P. and Astrup, T., Thromboplastin and plasminogen activator in tissues of the rabbit, Amer. J. Physiol., 219 (1970) 1140-1146. 11 Granelli-Piperno, A. and Reich, E., A study of protease and protease-inhibition complexes in biological fluids, J. exp. Med., 148 (1978) 223-234. 12 Jacken, S. and Black, P. H., Differences in intracellular distribution of plasminogen activator in growing, confluent and transformed 3T3 cells, Proc. nat. Acad. Sci. U.S.A., 76 (1979) 246-250. 13 Johnson, M. K., The intracellular distribution of glycolytic and other enzymes in rat brain homogenates and mitochondrial preparations, Biochem. J., 77 (1960) 610618. 14 Jones, D. H. and Matus, A. I., Isolation of synaptic plasma membrane from brain by combined flotation sedimentation density gradient centrifugation, Biochim. biophys. Acta, 356 (1974) 276-287. 15 Hatzfeld, J., Miskin, R. and Reich, E., Acetylcholine receptor: Effect of proteolysis on receptor metabolism, J. Cell Biol., 62 (1982) 176-182. 16 Kapatos, G. and Zigmond, M. J., Regulation of dopamine synthesis in striated synaptosomes during depolarization, Brain Research, 170 (1979) 299-312. 17 Kloog, Y. and Sokolovsky, M., Muscarinic acetylcholine receptor interactions: competition binding studies with agonists and antagonists, Brain Research, 134 (1977) 167-172. 18 Leblond, C. and Bennett, G., In B. Brinkley and K. Porter (Eds.), International Cell Biology, Rockefeller Univ. Press, New York, 1977, pp. 526-567. 19 Loskutoff, D. J. and Edgington, T. S., Synthesis of a fibrinolytic activator and inhibitor by endothelial cells, Proc. nat. Acad. Sei. U.S.A., 74 (1977) 3903-3907. 20 Lowry, O. H., Rosenbrough, N. J., Farr, A. L. and Randall, R. J:, Protein measurements with the Folin phenol reagent, J. biol. Chem., 193 (1951) 205-215. 21 Medzihazdsky, R., Nadeaszi, P. S., Idoyapa-Yorgas, V.
139 and Sellinger, O. Z., A comparison of ATPase activity of the glial cell fraction and the neuronal perikaryal fraction isolated in bulk from rat brain cerebral cortex, J. Neurochem., 18 (1971) 1599-1603. 22 Michaelson, D. M. and Sokolovsky, M., Induced acetylcholine release from active, purely cholinergic Torpedo synaptosomes, J. Neurochem., 30 (1978) 217-230. 23 Morgan, I. G., Wolf, L. S., Mandel, P. and Gombos, G., Isolation of plasma membranes from rat brain, Biochim biophys. Acta, 241 (1971) 737-757. 24 Quigley, J. P., Association of a protease (plasminogen activator) with a specific membrane fraction isolated from transformed cells, J. Cell Biol., 71 (1976) 472486. 25 Reich, E., Activation of plasminogen: a widespread mechanism for generating localized extracellular proteolysis. In R. W. Ruddon (Ed.), Biological Markers of Neoplasia: Basic and Applied Aspects, Elsevier, Amsterdam, 1978, pp. 491-500. 26 Seeger, R. C., Rayner, S. A., Banerjee, A., Chung, H., Lang, W. E., Neustein, H. B. and Benedict, W. F., Morphology, growth, chromosomal pattern, and fibrinolytic activity of two new human nenroblastoma cell lines, Cancer Res., 37 (1977) 1364-1371. 27 Solomon, J. A., Chou, I. G., Schroder, E. W. and Black, P. H., Evidence for membrane association of plasminogen activator activity in mouse macrophages, Biochem. biophys. Res. Commun., 94 (1980) 480-486. 28 Soreq, H. and Miskin, R., Screening of the protease plasminogen activator in the developing mouse brain. In U. Z. Littauer et al. (Eds.), Neurotransmitters and their Receptors, Wiley, London, 1980, pp. 559-563. 29 Soreq, H., Miskin, R., Zutra, A. and Littauer, U. Z., Modulation of plasminogen activator in differentiating neuroblastoma cells, Proc. of X l l l FEBS Meeting, 1980, p. 113. 30 Soreq, H. and Miskin, R., Plasminogen activator in the
rodent brain, Brain Research, 216 (1981) 361-374. 3l Sottocasa, G. L., Kuylenstierna, B., Ernster, L. and Bergstrand, A., An electron transport system associated with the outer membrane of liver mitochondria, J. Cell Biol., 32 (1967) 415438. 32 Takashima, S., Koga, M. and Tanaka, K., Fibrinolytic activity of human brain and cerebro-spinal fluid, Brit. J. exp. Path., 50 (1969) 533-539. 33 The Edinburgh Staff, Pharmacological Experiments on lsolated Preparations, Livingstone, London, 1970. 34 Unkeless, J. C., Gordon, S. and Reich, E., Secretion of plasminogen activator by stimulated macrophages, J. exp. Med., 139 (1974) 834-850. 35 Van Leeuwen, C., Stand, H. and Oestreicher, A. B., Isolation and partial characterization of chick brain synaptic plasma membranes, Biochim. biophys. Acta, 436 (1976) 53-67. 36 Virgi, M. A. G., Vassalli, J. D., Estensen, R. D. and Reich, E., Plasminogen activator of islets of Langerhans: modulation by glucose and correlation with insulin production, Proc. nat. Acad. Sci. U.S.A., 77 (1980) 875-879. 37 Wachsman, J. T. and Biedler, J. L., Fibrinolytic activity associated with human neuroblastoma ceils, Exp. Cell Res., 86 (1974) 264-268. 38 Whittaker, V. P. and Barker, L. A., The subcellular fractionation of brain tissue with special reference to the preparation of synaptosomes and their component organelles. In Methods in Neurochemistry, Vol. 2, Marcel Dekker, New York, 1972, pp. 1-52. 39 Zisapel, N,, Levi, M. and Gozes, I., Tubulin: An intergral protein of mammalian synaptic vesicle membranes, J. Neurochem., 34 (1980) 26-32. 40 Zisapel, M. and Zurgil, N., Studies on synaptic vesicles in mammalian brain. Characterization of highly purified synaptic vesicles from bovine cerebral cortex, Brain Research, 178 (1980) 297-310.
129
Plasminogen Activator is Enriched in the Synaptosomal Plasma Membranes NAVA ZISAPEL, RUTH MISKIN, MOSHE LAUDON and HERMONA SOREQ* Dept. of Biochemistry, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Rarnat-Aviv; (R.M.) Dept. of Biochemistry, Weizmann Institute of Science, Rehovot and (H.S.) Dept. of Neurobiology, Weizmann Institute of Science, Rehovot 76100 (Israel)
(Accepted February 25th, 1982) Key words: plasminogen activator - - bovine brain cortex - - synaptosomes - - membrane association
The intracellular localization of the serine protease plasminogen activator was analyzed in homogenates of bovine brain cortex using differential fractionation procedures. The distribution of the enzymewas clearly different from that of cytosol and mitochondrial markers, and was similar to that of plasma membrane proteins and of the muscarinic acetylcholinereceptor, which is a specific marker for the synaptic membrane. The specific activity of plasminogen activator was increased in fractions enriched in intact synaptosomes. Most of the enzyme in intact synaptosomes was found to be firmly associated with the synaptosomal membrane, and could be solubilized by high concentrations of salt or by non-ionic detergent. Purified synaptic vesicles, however, did not contain large amounts of plasminogen activator. Bovine brain synaptosomes were shown to contain two species of the enzyme, having apparent molecular weights of 80,000 and 55,000. The presence of plasminogen activator in the synaptosomal membrane may indicate its possible involvement in the functioning of nerve terminals. INTRODUCTION The serine proteases plasminogen activators (PAs) are found in different vertebrate organs such as kidney, lung and brain, as well as in body fluids like plasma, urine and cerebrospinal ftuidS,lo, 32. A variety of cell types in culture can be induced by physiological and non-physiological inducers to synthesize and secrete large amounts of P A 25. Plasminogen activators have also been detected in neuroblastoma cells of mouse 11 and of human origin2, 26, 37 The enzymatic activity of PA is limited to the specific conversion of the zymogen plasminogen into the trypsin-like protease plasmin, which is the physiological fibrinolytic enzyme 4. In addition to its thrombolytic function, the plasminogen activation system appears to play a major role in general extracellular proteolysis, necessary for cell migration and rearrangement of tissues ca. PA involvement in the processing of prohormones to hormones has also
* To whom correspondence should be addressed. 0006-8993/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
been recently suggested 36. Similar functions can be envisioned for PA in the developing and the mature brain, and a detailed intracellular localization of the brain enzyme may aid to the understanding of its function(s) in the nervous system. In the few cell types analyzed so far for intracellular localization of the enzyme, most of the PA activity has been confined to membranous elements 7,1e,
19,24,27,34. In frozen brain sections, P A activity was recently localized to regions and cell layers rich in neuronal cell bodies. This analysis was carried out by autoradiography of radiolabeled proteo]ytic products cross-linked to the surface of cells in active sections (ref. 30). When applied to viable differentiated neuroblastoma cells in culture, this technique revealed extensive labeling in growth cones and on the surface o f the extended processes, in addition to perikarya ~9. These observations raised the possibility, that PA might also be present in synapses and involved with neuronal functioning.
130 In the present work, which was aimed at extending the subcellular localization of PA to who!e brain tissue, the distribution of PA activity was determined during the course of preparation of synaptosomes from bovine brain cortex, a source in which the properties of purified synaptosomes were well documented as. PA activity was compared with that of known markers for cytosol, mitochondria and membrane, as well as with markers specific for synaptosomes. We now report that a large fraction of PA in bovine brain cortex appears to be membrane associated, that the enzyme is found in preparations enriched with synaptosomes, and that it appears to be firmly associated with the synaptosomal membrane. MATERIALS AND METHODS
Preparation andfractionation of synaptosomes Two procedures were employed as follows. (a) Slices of cerebral cortex were obtained from newly removed bovine brain and homogenized (7 passages at 1200 rpm) in a teflon-glass homogenizer in 10 ml per g tissue of 0.32 M sucrose adjusted to pH 6.8 with NaOH. Synaptosome-rich fraction was subsequently obtained following the procedure described by Whittaker and Barker 3s. All purification steps were carried out at 4 °C. The homogenized material (H) was centrifuged (104 g-min). The resulting pellet (P1) was washed with 1 vol. of the homogenization medium followed by centrifugation (104 g-rain) and discarded. The supernatants ($1) were combined and centrifuged (2 × 105 g-min) to yield a crude synaptosomal pellet (PD, which was resuspended by homogenization (one stroke at 150 rpm) using a teflon-glass homogenizer and loaded onto a sucrose step gradient, composed of three layers of 1.2 M, 0.8 M and 0.5 M sucrose. After centrifugation (105 g-rain) in a Beckman rotor SW 25.1, 6 fractions were carefully collected with a pasteur pipette, starting at the lightest fraction. Synaptosomes were collected from the 0.8 M/1.2 M sucrose interface. (b) The procedure displayed by Michaelson and Sokolovsky22 was adapted as follows: slices of bovine cerebral cortex were homogenized (10 ml per g tissue) in 0.4 M glycine buffer pH 6.8 (7 passages at 1200 rpm in a teflon-glass homogenizer), and synaptosome-enriched fraction (P2), subsequently ob-
tained following the procedure described by Whittaker and Barker 3s, was resuspended by homogenization and loaded onto a density gradient. Density gradient centrifugation (6 × 106 g-min) was performed with a discontinuous gradient in an SW40 rotor. The loaded gradient tubes contained 6 layers of equal volume of the following concentrations (M) of sucrose: 0, 0.15, 0.3, 0.55, 0.8, 1.2. Layers 1 and 2 also contained glycine, so that their osmolarity was equal to that of the homogenization buffer. After centrifugation the material which separated at the interfaces and in the layers of the gradient was carefully collected using Pasteur pipettes, starting at the lightest fractions as described in Fig. 2. Fraction 11 included material which occasionally centrifuged to the bottom of the tube after centrifugation, as well as some contaminating remnants of fraction 10 which, due to its high viscosity, remained in the tube after collection.
Isolation of plasma membranes Synaptosomal membranes were prepared by the procedure of Van Leeuwen et al. 35, as modified by Zisape] et al. a9. Crude synaptosomal fraction (P2) or purified synaptosomes obtained by procedure b were subjected to hypoosmotic shock in 60 mM sucrose, and centrifuged to yield a membrane enriched pellet (Wp). The pellet was resuspended in 50 mM MgC12, mixed with 2 vol. of 51 ~ (w/v) sucrose to a final concentration of 0.6 M and overlaid on 0.8 M sucrose. A 0.4 M sucrose solution was then pipetted on top and was overlaid finally with buffer (0 M sucrose). After centrifugation (6 × 106 g-min) the membrane-rich fraction was collected from the 0.60.8 M sucrose interface (Fig. 2).
Enzyme assays The following marker enzyme activities were determined spectrophotometrically using a Cary 118 recording spectrophotometer: acetylcholinesterase (EC 2.1.1.7) 8, lactate dehydrogenase (EC 1.1.2.7) 13 and cytochrome c oxidase (EC 1.9.2.1) 31. Horse heart cytochrome c (Sigma) was reduced by sodium dithionite and excess reductant was eliminated by gel filtration through a column of Biogel P-4. ATPase and 5'-nucleotidase activities were measured by the liberation of inorganic phosphate from ATP and AMP 21. Muscarinic acetylcholine recep-
131 tors were measured by means of the binding assay of 3H-labeled N-methylpiperidyl-benzilatO7. Dopamine synthesis was measured by the liberation of labeled 14COz from L-[14C]tyrosinO6. Plasminogen activator was assayed as previously described by the plasminogen-dependent liberation of fibrin degradation products from ~25I-labeled fibrin3°. Urokinase (UK) (Leo Pharmaceutical Products, Denmark) was added to each assay, and results were expressed as Ploug units of UK per mg of sample protein. Preparation of extracts for PA analyses was as described (ref. 30). Acetylcholine was determined by a bioassay using guinea pig ileum31. Protein was removed from the samples by precipitation with 10~ trichloroacetic acid and centrifugation. The acid was extracted with diethyl ether saturated with water. Protein was assayed as described by Lowry et al. 2°.
Electrophoretic analysis of PA activity Samples of fractionated homogenates were electrophoretically separated in SDS-polyacrylamide gels containing casein, according to Huessen and Dowdle 9. Following electrophoresis, SDS was extracted from the gel, which was then incubated at 37 °C to allow proteolysis. Dark staining of the gel visualized clear zones of proteolytic activity.
Preparation of synaptic vesiclefraction Slices of cerebral cortex were obtained from freshly removed bovine brain and homogenized in 10 ml per g tissue of 0.32 M sucrose adjusted to pH 6.8, using a teflon-glass homogenizer (7 passages at 1200 rpm). Vesicles were isolated as described by Zisapel and Zurgil4°. Fraction P2 obtained from homogenized slices of bovine cerebral cortex was suspended in water adjusted to pH 6.8 and subjected to mild homogenization in a glass-teflon homogenizer (1 passage, 150 rpm). The suspension was centrifuged (106 g-min) to yield a vesicle-rich supernatant fraction (Ws) and a membrane-rich pellet (Wp) which was further used for membrane preparation. The vesicle-rich fraction was laid on a sucrose-step gradient composed of 1.2 M, 0.6 M and 0.4 M sucrose layers and centrifuged 1.7.5 × 106 g-min) (Fig. 1). The synaptic vesicles obtained from 0.4 M sucrose layer were further purified by gel filtration on a Sepharose 6B column.
Solubilization and lysis experiments Allquots of the synaptosomal preparations were suspended in 2 ml ef(a) 0.15 M glycine-0.15 M sucrose containing 0.5 ~ Triton X-100, or (b) 0.155 M or 1.0 M NaCI, adjusted to pH 6.8. Samples were incubated for 10 rain at room temperature and then centrifuged (2 × 106 g-min). The supernatant and the pellet were collected, and pellet was resuspended in an equal volume of fresh incubating solution. Aliquots from the resuspended pellets and supernatants were assayed for plasminogen dependent fibrinolytic activity30.
Electron microscopy Samples were fixed in 0.15 M cacodylate buffer pH 7.4 which contained 2.5~o glutaraldehyde. The material was postfixed with osmium tetroxide, dehydrated and embedded in epon. Sections were stained on the grids with lead citrate and observations were carried out using a Jeol 1008 electron microscope. RESULTS
Distribution of PA in subcellular fractions The subcellular distributiort of PA in bovine brain cortex was determined by differential centrifugation of brain cortex homogenates, and compared with the distribution of the cytoplasmic enzyme lactate dehydrogenase (LDH) and the synaptic membrane component acetylcholine receptor (AChR) (Table I). In the supernatant fraction Sa, over half of each of the analyzed activities was recovered, together with the cytosol components and subcellular structures known to appear in this fraction, such as synaptosomes, microsomes, mitochondria and myelin. The remaining components, consisting of tissue debris, nuclei and part of the myelin, sedimented at low velocity into the P1 pellet. It should be noted that the recovery of PA activity at this step was reproducibly low (about 50 ~ of that of the homogenate activity). However, the further purification steps did not cause losses in PA yield. After centrifugation at high velocity, about 60 of PA activity as well as 6 0 ~ of the muscarinic AChR sedimented into the crude synaptosomal pellet, Pz, known to contain synaptosomes, microsomes, mitochondria and myelin3s. Both PA and the AChR displayed specific activities which were al-
132 TABLE I Distribution of enzymatic markers in synaptosomepreparations
PA activity is expressed in urokinase Ploug units; acetylcholine receptor content is expressed in pmol; lactate dehydrogenase activity is expressed in #mol/min. All activities (A) are given per g original tissue and all specificactivities (SA) are given per mg protein. Data represent averages of three experiments, which varied by less than 30 %. Fraction
H $1 P1 S~ P~
Plasminogen activator
Acetylcholine receptor
Lactate dehydrogenase
A
SA
A
SA
A
SA
54.0 16.0 8.8 4.9 7.4
0.53 0.31 0.18 0.19 0.35
73.5 36.8 32.7 12.6 20.2
0.72 0.72 0.67 0.47 0.96
154 151 29 99 29
1.5 2.9 0.6 3.7 1.4
most 2-fold higher in the crude synaptosomal pellet than in the supernatant fractions ($2) obtained at high velocity. The reverse situation was observed with L D H , 77 % of which remained in the supernatant. This seems to suggest the association of the major part of PA activity in the bovine cerebral cortex with membranal structures rather than with cytosol components. Association o f PA with bovine cortex synaptosomes The association of PA with bovine cortex synapA
PLASMINOGEN -----
ACTIVATOR
ACETYLCHOLINESTERASE
4-0-c C 0
I
tosomes, prepared by discontinuous sucrose gradient from crude synaptosomal pellet as, was determined by comparison to the distribution of markers of cytosol (LDH), mitochondria (cytochrome c oxidase) and synaptosomes (acetylcholinesterase). The sucrose gradient fractions mostly enriched with PA were found to be the 0.8 M sucrose fraction and the 0.8/1.2 M sucrose interface (Fig. 1A). These fractions are also enriched with acetylcholinesterase, in agreement with Whittaker and Barker as who have reported the preparation of synaptosomes from these fractions. According to this preparation procedure, L D H mostly remains in the 0 M sucrose fraction, whereas mitochondria segregate to the 1.2 M sucrose fraction, as revealed by the migration of cytochrome c oxidase (Fig. 1B).
o. 2 0 E 0 0
> o o ID
B - -
LACTIC -
DEHYDROGENASE
CYTOCHROME
OXIDASE
II
a: 4 0 -
I
I I I
I I I
~ 2o-
I L
--
L__ i. . . . I
I
2
I
3
I
4
I
5
6
Fraction
Fig. 1. Distribution of plasminogen activator and of enzymic markers in synaptosomal preparations separated on a discontinuous sucrose density gradient by procedure a (one out of three experiments is displayed (see Materials and Methods for details). Height of blocks refers to the amount of component in each fraction expressed as a percentage of total recovered component.
Characterization o f enriched synaptosomes prepared in glycine buffer To further investigate the association of PA with synaptosomal.enriched fractions, and to improve the separation of these fractions from mitochondria, we also prepared synaptosomes by procedure b (see Methods), based on a modified enrichment technique 2z. Homogenization of bovine brain cortex and subsequently the preparation of crude synaptosomal pellet was performed in this procedure in isoosmotic glycine buffer instead of the 0.32 M sucrose. Following this step, the synaptosomal pellet was fractionated by sucrose density centrifugation (Fig. 2). The fractions obtained were assayed for protein content (Fig. 3) and for several markers of known subcellular distribution (Figs. 3 and 4).
I33
Purification of
I
Bovine Cortex Synaptosomes Homogenate I spun at IO~-rnin
Pellet P,
Suspended in water, spun at 106g-rnin
I Pellet Wp
Distribution of molecular markers in synaptosomal preparations
I
30-
I Spun at IO~-min
5' Nucleotidase - - - Protein
20 -
~
Sup. Sj
J
I
I Sup.Ws
Separation by density gradient at Z5xIOs
sJp,
Pellet Pz Suspend in J homogenization buffer I Separafeon I density gradient at 6xl0 6 J g-min ~, 3
{::: 4'
ATPase ~A c e3t0yVl c h °-l-i "n e s t e r ° s e > ~
.15MI--HSynaptosomesJ """'1
i
j
55M I
:.
g.o 2°I--
7
r-'l II iI i t
o
I "--7
I0--
/
r-1
I
r-!
--4
I
.-~-~ I ~ i i L-~ L-J
I
i
I
-
F-~
I I
I
I
t -
I
g-min 0 1
II
gel filtration D r ~ 00~ Sel:tDaros e I
I~
I
lZ
Suspend in water Separate:densitygradient at 6x IOSg-min
30 20
OIV
I0
).41
lPloe.~ ~mbra,~sl~
061
60
_c
,r-] Plosminogen Activator - - - Acetylcholine Receptor
-
~
z-]D r-'-t
Fig. 2. Preparation of synaptosomes, synaptosomal membranes and synaptic vesicles from brain cerebral cortex. The details (procedure b) are given in Materials and Methods.
The distribution of PA on the sucrose density gradient used for the preparation of synaptosomes obtained by procedure b was compared with that of the other markers. PA distribution was generally similar to that of the membrane markers, and differed from that of cytosol and mitochondrial constituents6, a4. This was evident whether PA activity was expressed as percent of recovered activity (Fig. 3) or as specific activity (Fig. 4). High levels of PA activity were detected in the fractions enriched with intact synaptosomes, as well as in otber fractions containing membranous markers. The distribution of intact synaptosomes prepared by procedure b was indicated by the content of occluded acetylcholine and by the activity of the muscarinic AChR. Acetylcholinesterase, 5'-nucleotidase and Na, K-ATPase served as markers of
40 20
I I
i .I
L,,_
- - D o p a m i n e Synthesis -Acetylcholine
! I
-I i --
"-J, 2
I
I
i ,~-i-d. 4
... 6 Fraction
,_.r'r, 8 I0
Fig. 3. Distribution of molecular markers from crude synaptosomal fraction derived from bovine cerebral cortex, as obtained following sucrose step gradient centrifugation (procedure b; one out of three experiments; see Materials and Methods for details). Height of blocks refers to the amount of component in each fraction expressed as a percentage of total recovered component.
synaptosomal external membrane and of microsomal plasma membrane. L D H and dopamine synthase served as markers of the cytosol fraction, and the presence of mitochondria was determined by measuring the distribution of cytochrome c oxidase. The distribution of 5'-nucleotidase, acetylcholinesterase and ATPase on the sucrose gradient used for
134 the preparation of synaptosomes according to procedure b indicated membrane enrichment in fractions 3-8 and 10, 11 (Fig. 3A, B), a finding which was further supported by the similar distribution of Ca2+-activated alkaline phosphatase (not shown). AChR generally partitioned, when procedure b was employed, like the other membranal markers, and exhibited its highest specific activity in fractions 3-6 and 9, 10 (Fig. 4A). Acetylcholine was detected mainly in fractions 3 and 4. The low acetylcholine content of other fractions might be a consequence of exposure of non-occluded acetylcholine to acetylcholinesterase. Moreover, substances such as histamine or epinephrine, present in these fractions may interfere with the guinea pig ileum bioassay1. Acetylcho[ine would therefore be detected primarily in intact synaptosomes, whereas the receptor distribution indicates the presence of both intact and disrupted synaptosomes. Thus, it appears that intact synaptosomes, when prepared in glycine buffer, tend to accumulate mainly in fractions 3 and 4. Sucrose Gradient Centrifugation of Bovine Cortex Synaptosomes
S
I --AChR !
I l'~I r
~I
L_~
I I
Fill
~
I
[
--~8 ~I
-
'o.8F I I
.
L__,__J '--F-,-o.4 a
in -
~
o ~
~I
FI I I',
I ! I--J
Im
i i
2
I I
4
6 Fraction
8
I t
0.2:_~ s <
I0
Fig. 4. Specific activities of plasminogen activator and of enzymatic markers in synaptosomal preparation separated on a discontinuous sucrose density gradient according to procedure b (see text for details). Height of blocks refers to the activity of component per mg protein content of the corresponding fraction. Units of activity (average of duplicate assays) are expressed as follows: LDH, /~mol/min; cytochrome c oxidase, 10-a0 mol/min; AChR, pmol; PA, urokinase Ploug units.
The distribution of cytosol and mitochondrial markers, which clearly differed from that of synaptosomal markers, further supports this conclusion. Minor fractions of the original activities of cytoplasmic enzymes dopamine syntbase and LDH remained in the crude synaptosomal P2 pellet. These activities accumulated primarily in fractions 1 and 2, and apparently originated from residual cytesol as well as from synaptosomes and microsomes which were disrupted in the course of preparatien (Figs. 3D and 4B). Mitochondria, on the other hand, centrifuged down to the 1.2 M sucrose layer or precipitated into the pellet, as indicated by the high specific activity of cytochrome c oxidase in fractions 10 and 11 (Fig. 4B). Minute amounts of both cytoplasmic and mitochondrial markers can, however, be detected in fractions 3 and 4 (Figs. 3 and 4), further indicating the presence of sealed membranal elements within these fractio,s. Electron microscopic examination of samples from fractions 3 and 4 revealed rounded sac-like structures characteristic of nerve endings which contain synaptic vesicles, as well as occasional mitochondria (Fig. 5). This observation indicates that the enriched synaptosomes as prepared in glycine buffer are relatively free of non-occluded cytoplasmic elements, membrane fragments and mitochondria, and further confirms the biochemical analysis.
Distribution of PA in synaptosomal fractions To further examine the association of PA with membranous structures, membranes were prepared from crude synaptosomal pellet (P2) and from purified synaptosomes (fractions 3 and 4 of the sucrose gradient). The specific activit~ of PA was measured in these preparations, and the extent of enrichment in PA compared with that obtained for markers of synaptosomal plasma membranes (Table II). The results indicate an approximately 2-fold enrichment of acetylcholinesterase, AChR, alkaline phosphatase and PA activities in intact purified synaptosomes as compared with crude synaptosomes. Preparations of membranes from crude synaptosomes resulted in a ~light increase in PA specific activity, thus confirming its association with the membranous component of the Pz pellet. A similar increase was observed in the specific activities of alkaline phosphatase and of acetylcholinesterase,
135
Fig. 5. Electron micrographs of various magnifications of the synaptosome preparation obtained from isoosmotic sucrose density gradient centrifugation (procedure b, fractions 3 and 4) (for details see text).
136 TABLE II Enrichment of PA and enzymatic markers in synaptic plasma membranes and crude synaptosomal pellet
All enzyme activity values are presented per nag protein. Enzyme
AChE (/~mol/min) AChR (pmol) Alk. Phos. (nmol/min) PA (urokinase Ploug units)
Fraction Crude synaptosomes
Purified synaptosomes
Membranes from crude S.
Membranes from purified S.
Sp. act.
F
Sp. act.
F
Sp. act.
F
Sp. act.
F
44 0.96 9.1 0.35
1 1 1 1
85 2.1 20 0.65
1.93 2.17 2.20 1.86
69 0.94 13.5 0.55
1.57 0.98 1.48 1.57
179 2.50 23.80 2.16
4.08
while the specific activity of the muscarinic receptor for acetylcholine remained apparently unchanged. The membrane fractions obtained from purified synaptosomes were found to be further enriched with PA, as well as with other synaptosomal membrane markers. This enrichment of synaptosomal markers is consistent with earlier observations on synaptosomes from ratlZ, 23, chick 35 and bovine brainZS. The nature of PA interaction with the synaptosomal plasma membranes was investigated by means of solubilization experiments aimed at removing attached membrane proteins is. Purified synaptosomes f r o m fractions 3 and 4 were suspended in salt or in non-ionic detergent solution, and the precipitable material collected by centrifugation as described in Materials and Methods. Following incubation in glycine buffer containing 0.15 M sucrose, in 0.155 M NaC1 or in 0 . 5 ~ Triton X-100, most of the membranal PA activity (80-95 ~ ) remained attached to the synaptosomal membrane. A moderate release of 20-40 ~ was observed following incubation in 1.0 M NaC1. A firm association of the majority of PA molecules with the synaptosomal membrane is thus indicated. Synaptic vesicles, which constitute a minor fraction of the synaptosomal protein content but contain occluded neurotransmitters 40, are putative candidates for storage of the minor fraction of PA activity which is not associated with the synaptosomal plasma membrane. We therefore determined the specific activity of PA in purified synaptic vesicles, obtained f r o m bovine cortex by centrifugation
2.58 2.62 6.20
in sucrose density gradients (Fig. 1). The relatively low specific activity, 0.45 U K units per mg protein, indicates that synaptic vesicles do not contain large quantities of PA activity, either in a soluble or in a membrane-associated form. Synaptosomal PA displays two active species having apparent molecular weights of about 80,000 and 55,000 following SDS-polyacrylamide gel electrophoresis in a casein-containing gel (Fig. 6). The estimated molecular weights of PA species in bovine cortex synaptosomes therefore appear to be close to those of the two species of PA found in the mouse za and rat brain 3°.
Fig. 6. Molecular species of PA from bovine cortex synaptosomes (fractions 3 and 4). A 6/zl sample of purified synaptosomes containing 20 #g protein (2) was separated by gel electrophoresis in parallel to a 20/~1 sample (10:1, v/w) of rat brain extract (1), prepared as described previously3°. Proteolytic activity is visualized as destained zones on the darkly stained background.
137 DISCUSSION The subcellular distribution of PA activity has been examined to date in several cell types grown in culture. The major part of intracellular PA has been found in most cases to be associated with the particulate fraction. We now extend this finding to bovine brain cortex. The distribution of brain PA between the various subcellular fractions differs somewhat from that observed with fractions of cells in culture. In bovine brain homogenate about half of the PA activity is found in the low speed centrifugation pellet, whereas a much smaller fraction of the enzyme is found in this pellet when cultured cell homogenates are similarly centrifuged2a. In cortical homogenate about 40 9/ooof PA activity remains in the cytosol supernatant following high speed centrifugation, a higher fraction than that obtained upon similar centrifugation of cultured cell homogenate. The subcellular distribution of brain PA is similar, however, to that of brain AChR and is clearly different from that of cytoplasmic brain proteins such as LDH. Both these established markers dispersed between the different subcellular fractions in a manner fully consistent with other reports on fractionation of brain homogenatesaT,3s. It therefore appears that the less distinct subceUular distribution of brain PA, as compared with PA from cells in culture, is due to the biochemical and structural complexity of the brain tissue. The yield of PA activity in brain cortex homogenate is reduced to half following low speed centrifugation, a reduction which does not occur in centrifuged cell homogenates23. This decrease appears to be rather specific for PA as tile muscarinic receptor for acetylcholine and the cytoplasmic enzyme LDH both retain full activities following this step. The existence of specific protease inhibitor(s) in brain tissue has been reported 3. The initial centrifugation step may enhance the association of PA with such inhibitors and thereby reduce the recovered activity. Further fractionation of the P2 pellet revealed a distribution of PA activity which resembled that of membranous components and differed from that of cytosol and mitochondrial constituents, under both centrifugatien methods employed. High specific activity was obtained for PA in the 0.8 M sucrose
fraction according to procedure a, and in fractions 3, 4, 7 and 9 of procedure b gradients, in parallel to the increase in specific activity of acetylcholinesterase and of the acetylcholine receptor in these fractions. Both biochemical and ultrastructural analyses have shown these fractions to be enriched with intact synaptosomes. The high specific activity of plasminogen activator in synaptosome-enriched fractions may reflect the presence of higher amounts of this enzyme in synaptosomal membranes, as compared with other membrane fractions. Another possibility is that the synaptosomal membranes may have lower quantities of endogenous proteinase inhibitors than are found in the other membrane fractions, such as those obtained from cell bodies. In both cases, PA appears to be an integral component of the synaptosomal structure. PA specific activity in purified synaptic vesicles was significantly lower than that found in synaptosomal membranes. The major part of the synaptosomal PA is associated with the synaptosomal membrane, as indicated by the increase in PA specific activity upon preparation of membranes from purified synaptosomes. Most of the enzyme remains attached to purified synaptosomal membranes upon treatment with 0.155 M salt of 0.59/00 non-ionic detergent, but part of its is released by 1.0 M NaC1. A firm association of PA with the synaptosomal membrane is indicated by the finding that the enzymatic activity remains membrane-attached in 0.5 9/00 Triton X- 100. Cell-associated as well as secreted forms of PA have been found in a variety of cell types in culture, but only a few have been analyzed for the intracellular localization of the enzyme. In all these cell types most of the intracellular PA appears to be firmly associated with plasma membrane-like particlesT, 19, 24,2L The only exception is confluent 3T3 cells, in which the enzyme appears to be preferentially associated with a membranous fraction of high density, different from that of the plasma membrane. Unlike growing and SV-40 transformed 3T3 cells, the confluent cells do not secrete PA, and it has been suggested that these two unique characteristics are correlated, and that the presence of PA in the plasma membrane might be related to the secretion process 12. If this is indeed the case, our findings may indicate that within the bovine brain cortex, PA is
138 secreted f r o m nerve cells as well as f r o m their t e r m i nals into the s u r r o u n d i n g extracellular fluid. It has been speculated t h a t m e m b r a n e - a s s o c i a t e d P A in t r a n s f o r m e d cells is involved in a l t e r a t i o n s o f the cell surface which a c c o m p a n y cellular t r a n s f o r m a t i o n 24. It has in fact been d e m o n s t r a t e d that the p l a s m i n o g e n a c t i v a t i o n systems affects the t u r n o v e r o f the nicotinic A C h R , a distinct m e m b r a n e c o m p o nent o f chick e m b r y o muscle cells 15. A similar role cart be envisioned for P A in nerve cell p l a s m a m e m b r a n e a n d terminals, where this enzyme might p l a y a p a r t in the a l t e r a t i o n o f m e m b r a n e c o m p o s i t i o n involved in intercellular c o m m u n i c a t i o n . A n o t h e r f u n c t i o n which might be a t t r i b u t e d to s y n a p t o s o m a l P A is its involvement in the processing o f p r e c u r s o r s for p e p t i d e h o r m o n e s a n d / o r n e u r o t r a n s m i t t e r s , similarly t o the f o r m a t i o n o f insulin f r o m p r o i n s u l i n in the p a n c r e a t i c fl-islets3~.
REFERENCES 1 Aprison, M. H. and Nathan, P., Determination of acetylcholine in small samples of fresh brain tissue, Arch. BIOchem., 66 (1957) 388-395. 2 Becherer, P. R. and Wachsman, J. T., Increased neurite development and plasminogen activator expression by exposure of human neuroblastoma cells to a plasminogendeficient growth medium, J. cell. Physiol., 104 (1980) 47-52. 3 Brecher, A. S. and Quinn, N. H., The occurrence of a trypsin inhibitor in brain, Biochem. J., 102 (1967) 120-121. 4 Christman, J. K., Acs, G., Silagi, S. and Silverstein, S. C., Plasminogen activator: biochemical characterization and correlation with tumorigenicity. In E. Reich, D. B. Rifkin and E. Shaw (Eds.), Proteases and Biological Control, Cold Spring Harbor Laboratory, New York, 1978, pp. 827-839. 5 Christman, J. D., Silverstein, S. C. and Acs, G., Plasminogen activators. In A. J. Barret (Ed.), Proteinases in Mammalian Cells and Tissues, Elsevier, Amsterdam, 1977, pp. 91-149. 6 Cotman, C. W. and Matthews, D. A., Synaptic plasma membranes from rat brain synaptosomes: isolation and partial characterization, Biochim. biophys. Acta, 249 (1971) 380-394. 7 Dvorak, H. F., Orenstein, N. S., Rypysc, J., Colvin, R. B. and Dvorak, A. M., Plasminogen activator of guinea pig basophilic leukocytes: probably localization to the plasma membrane, J. lmmunol., 120 (1978) 766-773. 8 Ellman, G. L., Courtney, P. K., Andres, V. and Flatherstore, R. M., A new and rapid colorimetric determination of acetylcholinesterase activity, Biochem. Pharmacol., 7 (1961) 88-95. 9 Huessen, C. and Dowdle, E. B., Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulfate and copolymerized substrates, Analyt. Biochem., 102 (1980) 196-202.
U l t r a s t r u c t u r a l labeling studies w o u l d be r e q u i r e d to d e t e r m i n e u n a m b i g u o u s l y w h e t h e r P A is highly enriched within the synapse itself. A n a u t o r a d i o graphic p r o c e d u r e has recently been d e v e l o p e d to detect active P A in frozen cell layers o f organ sections a0. Electron m i c r o s c o p i c r e s o l u t i o n o f labeled cells in b r a i n sections m a y p r o v i d e the necessary m e t h o d o l o g y for the fine u l t r a s t r u c t u r a l localization o f this enzyme. ACKNOWLEDGEMENTS W e t h a n k Ms. O f r a D e u t s c h for excellent technical assistance. This w o r k was s u p p o r t e d b y grants f r o m the U n i t e d S t a t e s - I s r a e l B i n a t i o n a l SOence F o u n d a t i o n ( B S F ) Jerusalem, Israel (to R . M . ) a n d f r o m the Israeli C o m m i s s i o n for Basic R e s e a r c h (to H.S.). H e r m o n a Soreq is a n i n c u m b e n t o f a Charles R e v s o n C a r e e r D e v e l o p m e n t Chair.
10 Glas, P. and Astrup, T., Thromboplastin and plasminogen activator in tissues of the rabbit, Amer. J. Physiol., 219 (1970) 1140-1146. 11 Granelli-Piperno, A. and Reich, E., A study of protease and protease-inhibition complexes in biological fluids, J. exp. Med., 148 (1978) 223-234. 12 Jacken, S. and Black, P. H., Differences in intracellular distribution of plasminogen activator in growing, confluent and transformed 3T3 cells, Proc. nat. Acad. Sci. U.S.A., 76 (1979) 246-250. 13 Johnson, M. K., The intracellular distribution of glycolytic and other enzymes in rat brain homogenates and mitochondrial preparations, Biochem. J., 77 (1960) 610618. 14 Jones, D. H. and Matus, A. I., Isolation of synaptic plasma membrane from brain by combined flotation sedimentation density gradient centrifugation, Biochim. biophys. Acta, 356 (1974) 276-287. 15 Hatzfeld, J., Miskin, R. and Reich, E., Acetylcholine receptor: Effect of proteolysis on receptor metabolism, J. Cell Biol., 62 (1982) 176-182. 16 Kapatos, G. and Zigmond, M. J., Regulation of dopamine synthesis in striated synaptosomes during depolarization, Brain Research, 170 (1979) 299-312. 17 Kloog, Y. and Sokolovsky, M., Muscarinic acetylcholine receptor interactions: competition binding studies with agonists and antagonists, Brain Research, 134 (1977) 167-172. 18 Leblond, C. and Bennett, G., In B. Brinkley and K. Porter (Eds.), International Cell Biology, Rockefeller Univ. Press, New York, 1977, pp. 526-567. 19 Loskutoff, D. J. and Edgington, T. S., Synthesis of a fibrinolytic activator and inhibitor by endothelial cells, Proc. nat. Acad. Sei. U.S.A., 74 (1977) 3903-3907. 20 Lowry, O. H., Rosenbrough, N. J., Farr, A. L. and Randall, R. J:, Protein measurements with the Folin phenol reagent, J. biol. Chem., 193 (1951) 205-215. 21 Medzihazdsky, R., Nadeaszi, P. S., Idoyapa-Yorgas, V.
139 and Sellinger, O. Z., A comparison of ATPase activity of the glial cell fraction and the neuronal perikaryal fraction isolated in bulk from rat brain cerebral cortex, J. Neurochem., 18 (1971) 1599-1603. 22 Michaelson, D. M. and Sokolovsky, M., Induced acetylcholine release from active, purely cholinergic Torpedo synaptosomes, J. Neurochem., 30 (1978) 217-230. 23 Morgan, I. G., Wolf, L. S., Mandel, P. and Gombos, G., Isolation of plasma membranes from rat brain, Biochim biophys. Acta, 241 (1971) 737-757. 24 Quigley, J. P., Association of a protease (plasminogen activator) with a specific membrane fraction isolated from transformed cells, J. Cell Biol., 71 (1976) 472486. 25 Reich, E., Activation of plasminogen: a widespread mechanism for generating localized extracellular proteolysis. In R. W. Ruddon (Ed.), Biological Markers of Neoplasia: Basic and Applied Aspects, Elsevier, Amsterdam, 1978, pp. 491-500. 26 Seeger, R. C., Rayner, S. A., Banerjee, A., Chung, H., Lang, W. E., Neustein, H. B. and Benedict, W. F., Morphology, growth, chromosomal pattern, and fibrinolytic activity of two new human nenroblastoma cell lines, Cancer Res., 37 (1977) 1364-1371. 27 Solomon, J. A., Chou, I. G., Schroder, E. W. and Black, P. H., Evidence for membrane association of plasminogen activator activity in mouse macrophages, Biochem. biophys. Res. Commun., 94 (1980) 480-486. 28 Soreq, H. and Miskin, R., Screening of the protease plasminogen activator in the developing mouse brain. In U. Z. Littauer et al. (Eds.), Neurotransmitters and their Receptors, Wiley, London, 1980, pp. 559-563. 29 Soreq, H., Miskin, R., Zutra, A. and Littauer, U. Z., Modulation of plasminogen activator in differentiating neuroblastoma cells, Proc. of X l l l FEBS Meeting, 1980, p. 113. 30 Soreq, H. and Miskin, R., Plasminogen activator in the
rodent brain, Brain Research, 216 (1981) 361-374. 3l Sottocasa, G. L., Kuylenstierna, B., Ernster, L. and Bergstrand, A., An electron transport system associated with the outer membrane of liver mitochondria, J. Cell Biol., 32 (1967) 415438. 32 Takashima, S., Koga, M. and Tanaka, K., Fibrinolytic activity of human brain and cerebro-spinal fluid, Brit. J. exp. Path., 50 (1969) 533-539. 33 The Edinburgh Staff, Pharmacological Experiments on lsolated Preparations, Livingstone, London, 1970. 34 Unkeless, J. C., Gordon, S. and Reich, E., Secretion of plasminogen activator by stimulated macrophages, J. exp. Med., 139 (1974) 834-850. 35 Van Leeuwen, C., Stand, H. and Oestreicher, A. B., Isolation and partial characterization of chick brain synaptic plasma membranes, Biochim. biophys. Acta, 436 (1976) 53-67. 36 Virgi, M. A. G., Vassalli, J. D., Estensen, R. D. and Reich, E., Plasminogen activator of islets of Langerhans: modulation by glucose and correlation with insulin production, Proc. nat. Acad. Sci. U.S.A., 77 (1980) 875-879. 37 Wachsman, J. T. and Biedler, J. L., Fibrinolytic activity associated with human neuroblastoma ceils, Exp. Cell Res., 86 (1974) 264-268. 38 Whittaker, V. P. and Barker, L. A., The subcellular fractionation of brain tissue with special reference to the preparation of synaptosomes and their component organelles. In Methods in Neurochemistry, Vol. 2, Marcel Dekker, New York, 1972, pp. 1-52. 39 Zisapel, N,, Levi, M. and Gozes, I., Tubulin: An intergral protein of mammalian synaptic vesicle membranes, J. Neurochem., 34 (1980) 26-32. 40 Zisapel, M. and Zurgil, N., Studies on synaptic vesicles in mammalian brain. Characterization of highly purified synaptic vesicles from bovine cerebral cortex, Brain Research, 178 (1980) 297-310.