THE SUBCELLULAR LOCALIZATION OF ACETYLCHOLINESTERASE A N D ITS MOLECULAR FORMS IN PIG CEREBRAL CORTEX C. H. S. MCINTOSH'a n d D. T. PLUMMER Department of Biochemistry, Chelsea College, Manresa Road, London. SW3 6LX, England (Receiivd 3 October 1975. Revised 30 January 1976. Accepted 9 February 1976)

Abstract-The distribution of acetylcholinesterase among the subcellular fractions of pig cerebral cortex was determined. The crude mitochondria1 and microsomal fractions obtained by differential centrifugation accounted for 750; of the enzyme, with the remainder divided between the crude nuclear and soluble fractions. The occurrence and distribution of the multiple molecular forms of AChE was the same in all four fractions with the dominant species of molecular weights 350,000. 270,000 and 60,000. Further purification of the mitochondria1 fraction by density gradient centrifugation gave a series of membrane fractions with very similar multiple forms. The one possible exception was the fraction containing the purified synaptosomal membranes where one band of mol wt 270,000 predominated, although the other molecular weight entities were present. The electrophoretic pattern of AChE present in the fractionated microsomes was the same as in the crude preparation. The content and pattern of the multiple molecular forms of AChE was therefore the same in all fractions of pig brain. apart from that containing the purified synaptosomal membranes. ACETYLCHOLINESTERASE AChE (acetylcholine hydro- was to see if these multiple molecular forms were to lase. EC 3.1.1.7) is present in the nervous system in be found equally in all membranes o r if each subcelluhigh activity a n d its subcellular localization within lar fraction could be characterized by the number a n d neurons has been determined by light and electron distribution of molecular forms present. A knowledge microscopy. AChE is found in both presynaptic a n d of the nature of these forms, their relationship to each LORENZO, 1961 ; TORACK other and their association with specific membranes postsynaptic membranes (DE & BARRNETT. 1962) of supposedly cholinergic should produce a greater understanding of some of synapses. T h e enzyme has also been demonstrated the molecular mechanisms involved in synaptic within the cisternae of the endoplasmic reticulum a n d transmission. the nuclear envelope in both rat brain (LEWISer a/., 1965) a n d in the neurons of rat spinal cord (NAVARATN A M & LEWIS.1970). In the latter case, activity was MATERIALS AND METHODS frequently observed in the axonal membranes although the plasma membrane of the neuronal perikAssoy qf acet~lcholinesrerasr.AChE activity was determined at 3 0 C and pH 7.9 by a titrimetric method using aryon showed n o enzymic activity, apart from that of the synapse. Almost identical results were obtained a pH-stat (Radiometer. Copenhagen, Denmark). The standard conditions for the assay procedure were first deterby KOKKOer a/. (1969) with cerebral cortex. T h e caumined on a crude brain homogenare and fhe enzyme wludate nucleus contains the highest activity of AChE bilized with I", Titron X-100. The detergent-solubilized a n d here the enzyme is mainly localized a t the enzyme showed a flat pH optimum in the region of pH synapses (LEWIS& SHUTE. 1964). AChE activity is not 7.5 to 8.5 whereas that of the homogenate gave a narrower present in vesicles or mitochondria. optimum range from pH 7.X to X.5. At pH 7.9. the substrate Reports from this and other laboratories have optimum was 2mh+ for the Triton extract and 1 . 5 m ~for shown that AChE exists in a number of multiple the homogenate. After these experiments. the following forms of different molecular weights probably arising standard assay procedure was adopted: A sample of from subunit aggregation (CHANct a/.. 1972; HO & 0.1 -1.0 ml was added to sufficient reaction mixture ELLMAN.1969: M C ~ T O S & H PLClMMl R. 1973: WthiT- (0.15 M-N~CI+ 1.3 m#-MgCI,) to give a total bolume of HOLU et d.. 1974). T h e a i m of this present study 7.7 ml and the pH adjusted automatically to 7.9 by the addition of 10 mM-NaOH. Homogenates often showed spontaneous acid production and this was followed for Present Address: 34 Gottingen Kliniken der Univer- 5 min before adding 0.3 ml of acetylcholine iodide solution sitht Gottingen, Hormon Labor. Humboldtalle, W Ger- (26.67 mM) to start the reaction. The enzymically catalysed rate of hydrogen ion production was followed by titration many. Ahhreriatious wed: GDH. glutamate dehydrogenase: with 30 mM-NaOH for a further 5 to 10 min and the rate LDH. lactate dehydrogenase: SDH. succinate dehydrogen- corrected by subtraction of any spontaneous activity and non-enz)mic hydrolysis of substrate. ase. 449

450

C H S MClNTosH and D T P L L M W R

0.3 ml oi sodium-/~-gl!cerophosphate ( I 50 mM) and 0.2 ml . 4 ~ \ 1 .\i jlor n i i r r h tw:ynit'.\ I I I whcellirlur frucrioii~.Unless otherwise stated. asaayi were carried out at 30 C in a Perof diluted enzyme sample a a s preincubated at 30 C for IOmin. Adenoqine deaminase 10.1 ml of 12.5 units,ml) H B S kin Elmer recording sprctrophotometer fitted with a conadded to the mixture and the reaction initiated wlth 0.3 ml stant temperature cell housing and automatic cell interof AMP ( I mM). The decrease in extinction at 265 nm was changer. The kinstics of each enzyme in pig brain were investigated to determine the optimum conditions for followed for 2-5 min. .Aku/ine phospliut(t.w (orr/ioph(i.;p/ioric w i d nioiit~i~,~rc~r assay. Some enzymes were found to be inhibited by high ph~,.\pliolil.dr~il[ise ? . I .3.1). Alkaline phosphatase is probably concentrations of sucrose and this was corrected b! either a plasma membrane enzyme in liver (EMMELOT & BOS. diluting to a constant sucrose concentration (0.25 M ) or 1966: RAY. 1970). and in brain synaptosome subfractions centrifuging the samples at 1OO.ooOg for I h and resusthe enzyme uctivit) closel) follo\rs the diatrihution o i N a pending the pellet in distilled water. (COTMAN & MATTHIWS.1971). The hydrolhL(rrtute ~leh\~drt~qenosr (~-l(ictate-N.4Dt ~ . ~ i ~ l ~ ~ r i ~ ~ l i ~ K i ~ t'-ATPase ii.si~. EC 1.1.1.27). Lactate dehjdrogenase ( L D H ) is a marker sis of p-nitrophenyl phosphate was measured by following eiiijme Tor the cytoplasm i n brain as in other tissues the appearance of p-nitrophenol, which absorbs at 400 nm. (WRIGHT er al.. 1972). A reaction mixture containing 2.5 ml (JOHNSON. 1960) and it has a subsidiary function as a of 0.1 M-NaHCO,-Na,CO, buffer. pH 10, 0.1 ml of synaptosomal marker enzyme in its occluded form within MgSO, ( 1 0 0 m ~ and ) 0.3 ml of p-nitrophenyl phosphate E S . The free actnit!, of these structures ( M A R C H H A ~ 1967). LDH was determined by adding 0.1 ml of tissue fraction ('5 mM) was equilibrated at 30 c for 10 min. A sample and 0.1 ml of NADH (2.5 mgiml) to 2.7 ml of 0.1 M (0.1ml) of the diluted tissue fraction was then added and sodium-potassium phosphate buffer. pH 7.4 previously the release of p-nitrophenol followed by the change in equilibrated at 30'C. The absorbance at 340 n m was then extinction at 400nm. NADH :c!,rochronicc reductax. rurenorit.-intrnsiru-e ( N 4followed for 2 min to determine any non-specific oxidation for NADH. When a constant extinction was observed the DH:ryrochronie c oxidorediicrnse 1.6.99.3) aid N A D P H : ( N A D P H :c'vtoreaction was initiated by the addition of 0.1 ml of sodium cjtochronie c reducttrsr. rotelionr-irtsoiritf~e pyruvate (10 mM) and the change in extinction measured dironie c o.~idor~.ducrase1.6.99.11. The NADH-linked enzyme is associated with the outer mitochondrial memover a time period of about 5 min. The occluded activity was determined by allowing the brane and also the microsomal fraction of rat liver (SOTTOfree assay to proceed for 2 min then adding 0.3 ml of lo",, CASA 1'1 td.. 1967). and the NADPH requiring e n q m e is (w/v) Triton X-100 to the mixture and stirring: the change a marker for the smooth endoplasmic reticulum ( M O R G A ~ in absorbance was then followed for a further 3 min. The et [ I / . , 1971). These locations are. however. controversial measured activity before and after the addition of the and will be considered in more detail in the Discussion. detergent represents the free and total activity respecti\ely. The assay used was based on that of SOTTOCASAet a / . The ratio occluded to total activity is then: 1 - (free activi- (1967) in which the reduction of cytochrome c is followed at 550nm. A mixture of 0.1 ml of tissue extract. 2.2ml ty)/total activity. G/iit(illiarca di4iI~dro[/anasr. GDH (~-{//irratilar~, . V A D ( P ) of sodium-potassium phosphate buffer. pH 7.4 (50 mw). osidorcducrase dc~aniinariiiy, EC 1.4.13.1.This enzyme is 0.3 ml of KCN (9 mM) and 10 pg of rotenone in 10 pl of widely used as a marker for mitochondria and is present ethanol was incubated at 30 C for IOmin. Following this, in the mitochondrial matrix (BEAUFYer a/., 1959). In brain 0.3 ml of cytochrome c (5 mg,ml) was added and the rcacit follows the same distribution as succinate dehydrogenase tion initiated with 0.1 ml of NADH or NADPH (SALGANICOFF & DE ROBERTIX1965). Latency is observed (2.5 mg,'ml). The increase in extinction at 550 nm was then with intact mitochondria so the mitochondrial membrane measured. Na+-K'-A TPaw (.4TP pliosphoh~drolts~,.EC 3.6.1.3). was disrupted prior to assay by a final concentration of 0.07", Triton X-100.All solutions and dilutions were made The localization of ouabain-sensitive Na'-K '-ATPase is in 0.1 M-sodium-potassium phosphate buffer, pH 7.4 and frcquently used as a markcr for the plasma membrane adjusted to pH 7.4 if necessary. The diluted tissue sample although there is no reliable data as to its difkrential locali(0.2 ml) was added to a mixture of 2.2 ml of the phosphate zation in glial and neuronal cells. The enzyme was assayed using a coupled system whereby the ADP produced reacts buffer containing 0.1% Triton X-100. 0.21111 of EDTA (30mM) and 0.2 ml of ammonium acetate (0.75 M) and incu- with phosphoenolpyruvate i n the presence of pyruvate kinase to give pyruvate and ATP. NADH and LDH are bated at 30°C for 10min. After the addition of 0.1 ml NADH (2.5 mg/ml). any non-enzymic oxidation was bl- also present and the measurement actually recorded IS the lowed for 5 min by the decrease in absorbance at 340 nm. fall in absorbance of NADH at 340 nm. In brain, optimal The reaction was then initiated by the addition of 0.1 ml activity of the enzyme has been demonstrated with l00mM-Na' and 30mM-K+ and the activity i s 85-90',,, of sodium-2-oxoglutarate and the decrease in absorbance inhibited with I mM-ouabain (SKOU. 1964; MCILWAIN& followed for a further 5 min. BACHELARD, 1971). s'-Nurleotidase (5'-rihonucleoride phosphoh~drolasr,EC The following reaction mixture was prepared and incu3.1.3.5.). On the basis of an extensive review of the litera(1970) bated at 20 C Tor 10 m i n . 2.3 ml of TrwHCI buffer. pH ture on the plasma membranes. STECK& WALLACH. concluded that 5'-nucleotidase is the most specific and gen- 7.4 (100 mM). 0.3 ml of NaCl KCI (1 M'ZOOmM). 0.05 ml of eral plasma membrane marker known. The enzyme was NADH i10mgiml in buffer). 0.025ml of pyruvate kinase assayed by the method of IPATA (1968) in which the reac- (5000 unitsiml). 0.01 ml of LDH (25.000 units/ml). 0.05 ml tion is coupled to the deamination of the adenosine formed of phosphoenolpyruvate (0.1 M i n water). and 003 ml of in the presence of adenosine deaminase. A high concen- KCI ( I M). A baseline was established at 340nm then 1@50pl of sample added and the volume made up to tration of sodium-b-glycerophosphate was included in the reaction medium to direct any alkaline phosphatase acti- 2.9ml with water. The reaction was initiated with 0.1 ml vity away from AMP hydrolysis. A mixture or 2.0ml of ATP sodium salt (0.1M in buffer). Assays were carried out in the presence and absence of ouabain and the differTris-HCI buffer, pH 7.4 (33 mM), 0.1 ml of MgCI, (0.3M).

Subcellular localization of AChE ence in actibity with and without ouahain taken as the Na'-K +-ATPase activity. . s i i l l i l i u r L ~ d14l l'ilro~/L~llu,se (,ul~~illor ll\ ~ ~ r:o l l l r ~ o r l lc, ~ 0 \ i , / , l r t d i i ~ ~ r c i . sEC c ~ 13.99. I).Succinate dehydrogenase ISDH) is gcncr;illy considcred to he a marker for the inner mitoer ol.. 1971). The method chondrial membrane (MORGAN uscd was that of KUROKAWA ct a!.. (1965) with slight modifications. A sample (0.1 ml) of diluted tissue fraction was mixed with 1.9 ml of sodium-potassium phosphate buffer, , ml of Triton X-100 (0.3", v/v), 0.1 ml pH 7.4 ( 5 0 m ~ )0.1 of KCN (9 mM) and 0.1 ml of CaCI, (12 mM) all in buffer and equilibrated for 10 min at 30'C. Cytochrome c (0.3 ml of 5 mg/ml) was then added and after following any nonspecific reduction of cytochrome c for 2 min the reaction was started with 0.4 ml of sodium succinate (0.25 M in buffer. pH 7.4) and the change in extinction at 550m followed for 2-5 min. €vprc.\.\ioii oftw:,riiic urrii3if;es. All activities were calculated in international units (pnol of substrate transformed/ miti). The activity of the enzymes in the cell fractions were expressed as a percentage of the total recovered activity and also the 'relative specific activity'. This latter value was obtained by dividing the percentage recovered activity by the percentage of the total recovered protein in that fraction. C / i ~ ~ i i i iLI.\WIJ.\. ~ u / Ribonucleic acid was assayed using the orcinol reactions and protein was determined by a biuret method (PLUMMER, 1971). The sucrose concentration in the gradients was determined with a refractometer. Difereiiriul centrfugorion of pig bruin. Pig brains were obtained from the slaughterhouse about 20 min after death. The tissue was then placed in a polythene bag in crushed ice and transported to the laboratory. The total time taken from the death of the pig to homogenization was approx I+h. All subsequent operations were carried out in the cold room (4 C). The brain was now stripped of its external membranes. washed with ice-cold 0.32 M-sucrose and the cerebral cortex dissected out. As much as possible of the white matter was removed by scraping with a blunt scapel and the cortex finely chopped before homogenization. Pofrer-Elcelijem Iioimgriii~er. The brain was hornogenized by 6 complete strokes of the pestle and then subjectzd to the fractionation scheme outlined in Fig. I Aldridyr rype o/ homogeiii;er (ALDRIDGE er ol.. 1960). This consisted of a precision bore tube and nylon pestle driven by an electric motor at 1500 rev.;min. The number of passes could be automatically pre-set and the pressure between stationary and rotating parts at the bottom of each pass was reproducible. The "optimal' number of thrusts uas found to be three series of 6 thrusts and this h a s adoptcd as the standard for homogenization. Different i a l Iractionation was then carried out a s i n Fig. 1. P r t p ~ r u r i o i iof piirifiecl syiioprir plusimi mmibntries. The & MATTHFWS(1971) aa modified by method of COTMAN (1972) wa5 slightly modified for MCBRIDF& V A N TASSELL. pig brain (Fig. 3). Initially very poor separation of components bas obtained and this was found to be due to incomplete lysis of the synaptosomes. Optimum resolution and purification was only obtained iF_the following precautions were taken: ( I ) All sucrose and Ficoll solutions were precooled and their pH adjusted to 7.4. (11) Gradients were left for 2 4 h at 4 C before use. (iii) The crude mitochondrial pellet was washed 3 times. (I\) The precipitate P2 was gcntlj suspended in 2",, Ficoll then homogcnized w i t h a loose-fitting pestle. ( v ) The synaptosome preparation \ \ a s osmotically lysed by resuspension of the

451

75,000 g pellet in distilled water (1 ml/g tissue) and forcing the suspension through a 14 gauge needle attached to a 25ml syringe. The suspension was then transferred to a glass homogenizer and subjected to 5 hand strokes with the Teflon pestle. Sub-frucrionation of the microsom/ fiacrion (P3). The microsomal pellet prepared at 170.000g for I h was suspended in 0.32 M-SUCIOSB and subfractionated on a discontinuous gradient (BACHELARD, 1967). In some experiments 5 or 10 mM-MgC1, was incorporated into all the sucrose solutions. Gradienr gel elccrrophoresis. 'Gradipore' gels in the form slabs 70 m x 70 mm x 3 mm were used with a concave gradient of polyacrylamide from 4 to 26% (w/v) (Universal Scientific Ltd., London, E.17, U.K.). Prior to electrophoresis gels were run for 1 h without the sample at 100 V in a buffer of 88 mM-Tris-82 m~-borate-2.5mht-EDTA (pH 8.3). Samples (30 pI in sucrose) containing 2-3 nmol/min of AChE activity were applied using a plastic spacer and electrophoresis carried out at room temperature for 24 h at 1M)V (constant voltage). For molecular weight estimations the gels were stained for AChE activity and the distances migrated by the bands compared with those of standard proteins run under identical conditions. Staining of AChE acririty. Regions of AChE activity were detected by incubating the gels with acetylthiocholine iodide a s substrate by the method of LEWIS & SHUTE (1966). When eserine ( 1 0 ~or~ ethopropazine ) ( 3 0 ~were ~ ) included in the incubation mixture. the gels were initially presoaked in a solution of the inhibitor for I h then exposed to the incubation mixture. RESULTS

Differential certrrifugation Th e major drawback to t h e Potter-Elvehjem homogenizer is its lack of reproducibility. In 9 experiments, 4 gave satisfactory results with 9% of the H a m o g e n a t c ~ l O O ~ ~ ~inw0 / v32 l u-sucrose

I

IOOOg IOmin

I

I

Crude nuclear pellet washed twice in 0 32 M-sucrose and recentrifuged a t 1000 g for 10 min

1

lurge myelin fragments

I

1O.OOOg 2 0 m t n washed 3 times

I

P 2 (crude mitochondrial pellet) mitochondria. synaptosomes. small myelin and membrane fragments

I I 100.000#

5 2 (supernotant) Ih

d

P3 (microsomal p e l l e t ) microsomes

53 ( s u p e r n a t a n t ) ribosomes. cytoplasm

FIG I Fractionation scheme for the ditterential centrifugation of pig brain

C. H. S. MCIVTOSHand D. T. PLUMMER

452

somes but only fraction D was taken for further study since this banded at the normal isopycnic position for synaptosomes from other sources. The plasma membrane markers 5'-nucleotidase and alkaline phosphatase showed a similar distribution but Na'-K'ATPase exhibited a higher relative specific activity in fraction B compared to D. The mitochondrial enzyme glutamate dehydrogenase ( G D H ) occurred mainly in fractions D, E and F with D the 'synaptosomal fraction' accounting for 30"" or the total activity. This first fractionation step was extremely effective at separating the pyridine nucleotide reductases between fractions B. D and F. Only lo", of the NADPH-linked enzyme and 22": of the NADH enzyme was found in D. Fraction B contained the highest amount (65",,) and the greatest RSA of Efect qf washing the 'mifochondria2'frciction P2 NADPH:cytochrome c reductase, while fraction F As a preliminary to the isolation of synaptosomes. had the highest percentage (46";) and RSA of NADHthe crude mitochondrial pellet (P2) was washed cytochrome c reductase. Subfractionation of the osmotically lysed 'synaptoseveral times to determine the optimal number of washes. A crude mitochondrial pellet (P2) was pre- somal' fraction D (Fig. 4 ) o n a sucrose gradient pared a s described and the supernatant assayed for resolved the enzyme markers even further (Fig. 5). total LDH. rotenone-insensitive NADPH :cyto- Acetylcholinesterase and 5'-nucleotidase showed simichrome c reductase and RNA. The pellet was then lar distributions with maximum relative specific actiresuspended in half the original volume of 0 . 3 2 ~ - vity in fraction H although appreciable amounts sucrose. centrifuged under the same conditions and occurred in all the other fractions apart from the pelthe supernatant assayed for the same markers. This let. Na+-K+-ATPase was most highly purified in procedure was then repeated several times (Fig. 2). fraction 1. but again large amounts were found in The optimum number of washes adopted resulted + C 0 0 Acetylcholinesterase from a compromise between the need to reduce mic- + 0 Total LDH rosomal contaminations to a minimum and the NADPH-cytochrorne c reductose avoidance of excessive synaptosome loss. Another imB, (rotenone insensitive) portant factor was the time taken for each wash (3S45min) and in view of this and the results obtained (Fig. 2). 3 washes was adopted a s standard practice.

recovered protein in rract~on PI and l X ' o in the microsomal fraction P!. In 3 other experiments. howof , the , protein remained in the ever. as much as '-I" PI fraction due to incomplete homogenization while in 1 other experiments 32"" of the protein appeared in the microsomal fraction P3 suggesting extensive disruption of cells and organelles. This latter conclusion was confirmed by the low activity of occluded LDH in the synaptosomal fraction P2 and the high total LDH activity in the supernatant S3. In view of this the Aldridge-type of homogenizer was used in further investigations when the reproducibility obtained was always within f49,. The percentage recoveries of protein and some enzyme markers are shown in Table I.

~

Pirrification of synaptic p/u.smn menlhranes The appearance of the bands on the Ficoll gradient is shown in the fractionation scheme (Fig. 3). The distribution of LDH showed a sharp localization in the two bands B and D with 7 2 9 of the enzyme occluded in B and 900,; in D (Fig. 4). This indicated the possible existence of two populations of synaptoT A B LI~ THEDlFFCRENTlAL

Enzyme or protein

Protein Acetylcholinesterase

Lactate dehydrogenase (free)

Lactate dehydrogenase (occluded)

No.

of

washings

FIG.2. The efficiency of washing the crude mitochondrial pellet (P2) Obtained from the differential centrifueation of pig brain.

CtNTKIFl~GATlOh OF PIG DRAIN HOMOGFNIZFD WITH AN

ALI~KII)C.I -TYPI

0 1 H O M O G I N171 R

Relative specific activity and 2,recovered component (in brackets) among the various crude fractions PI P2 P3 S2 (nuclear) (mitochondrial) (microsomal) (supcrnatant) ~

-

0 12

(53",) 0 72 (38"") 1 60 (84%) 0 18

(2"J

(9"A

(16",) 0 66

(1I0J

Glutamate dehydrogenase

c W

0 59 (190,) -

-

(23"")

(707")

~

(I2"J 2 96 (37"") 0 I9

(2:") 0 23 (3%) ~

(7"")

-

( 19"