Markers for Gene Expression in Cultured Cells
from the Nervous System

(I~eceived for pul~licaIion, December 1X, 1971)

SUMMARY

Methods are presented for preparation of extracts from
cultured cells from the nervous system and for study of
choline acetyltransferase,  acetylcholinesterase, glutamate
decarboxylase, and catechol 0-methyltransferase activities.
These enzyme activities are markers that can be used for
studying gene expression in neurons.  The methods are
sufficiently sensitive so that all assays can be performed with
protein harvested from one Petri dish. Activities of the
marker enzymes were assessed in surface cultures of new-
born mouse brain cells, and in glial and nonbrain cell lines.
Low activities of choline acetyltransferase, acetylcholines-
terase, and glutamate decarboxylase were detected in all
the cells tested. All of these activities, and particularly
glutamate decarboxylase, were higher in cultured brain cells
from newborn animals than in non-neuronal cell lines.
Glutamate decarboxylase activity in glial cells and in brain
cells was inhibited more than 95 % by 1 mM amino-oxyacetic
acid.

`l'eclulicllies IN\-e beeit tlevelopetl in this l:~l~or:rtor~ and others
for cwlture of diflereiitinted iieuroti~ (1LlO). .Icti\-ities of em
zyii~ex iilrlx)rtallt iu iieuroi~al ~41 inetalwlisii~ are useful ]xnxn-
ettw for following i.ell mrtulation ;ind eslhiii~ htel)i ii) differ-
eliti:itiotI iiL huch culture*.

`he ,"lqw~e of tlii.3 ~cliirrrlluii~xtioir i> to dewilw :I set of
itietllocl~ wet1 to esl)lore tire es~~rw~ioii of genes tllat deterniiile
the metnbolim of ~rwlecule~ involved iu iute~cellular coniri~uni-
c7itic)ll ill the llerVou8 S;?steni.  Siuil)le, coliveiiieiit. methods are
~~re~e~rted for iwel)will~ c*ell-Tree extrwt, from iurf:i~e crtltut~er

and for :~wiyh of ac,et)l(~llolillester:l~e (EC' 3. 1. 1. i), cxtr?lloI
O-illetli?.ltrUlififer:~~e (ICC 2.1 1. I), choline acet!-lt,r:tn,.fernhe
(EC' 2.3. I .6), aud glutamate dewrborylase (EC ' 4 1.1.15)
activities.  The assay:: presented are esteiisive moclitic*atioll~ c~f
l)reCou+l\- Ihliahed procedures.

A\ctivities of the marker enzymes were aasestied itI ;t variety of
&I aiitl noihrain rell lilies iw well RS iu surface cultluw IIf lien-
borir moire braiii cells. The results document l)rolwrt ie- ;~tld
uhefuliie+ of the metllod~ preset+4 and alro SIWW that Irrwtwrn
hain cells in surface wlture attained level.5 OI ~lut~t.m:~tr cle-
carhos~~luse acti\-itT\- Aguifiwlltly hi&w tlulll in I~~-II~II~~II:I~
cell lillei.  .\ l)relinliiiary relwrt of this work ha5 tleeti ~~twel~tetl
01).

JIE'IIIODS

~3Iwle hiins from newborn IMb/c (Saticmal Ifiqtittites OF
Healtl~ stwk) mice were placed in Solution 1) (137 m?rz ~n(`l,
5.4 UIU fX1, O.lT 1~1~ SasHPO+ 0.22 IIIM KH#Oa, 5.5 ,,1.\1 +
case, and 5.0 nml sucrose) pII i.2, 340 tt~oshf (luotlifietl l'uck's
111 ~olutioii (12)) 3t O-4", weighed, wahbed hevera tinlw wit11
Solutioll 11, autl minced to about 1-mr11~ l)iecw with iris .t+aor<.
l'he nriwed tihaue was subjected to t\vo 15.miu tre;\tnietrt,q wit11
0.25(, crude trylhiu (?rTutritioilal Iliocl~eniical~ I :%O or IXfco
1 :300) in Solutioll 1) (100 rrrl l)er a of tissue) at 37" \Vittl (`01).
hut mild swirlilig.  After each treatment, ti>-ue [)iec+ ~vere
allowed to sediment and the di~swiated rells that rern:tilwcl 5~`;~
pentled were collected by decatltation.  `I'ryptic actlvit>- in both
the cell ~uspen&n and uudisswiated tiaue W:I~ illllit)itecl by tile
:itlditiou of an equal volume of growth medium (`otit:iiflillp 10' (,
(V/V) fetal l)oville xrum (Colorado Seruni C'o.).  The uudi?-
so&ted tiwie \v:is then triturated fi\-e time* 1)~ qr:lvitv flo\\
through a lO-ml serological pillette aiitl caell~ were :IX:I~ rrc*c)verecl
11s decnntation. Cells were lwoled aud ~edimente~i :lt 2.X x
gorax for 10 mill nt 3" :ind resuqxwtled iii Solution I).  C `ell 11uu~
I)er and viability were determined I>\- wuuting in ;I hrmcw!.tou-
eter \vitli 0.57; (\v/v) nipwiii (-Ulied (`llemicxl, ~IoI~~~..~(JIvI~,
?;. .J.).  Ouly tlwe cells wIGI eschtled the (Iye \vere (.oIl..i(lerrcl
\-iahle. ITsual cell recoveries for (liSwci;Ltioii ~1' tie\vt)orIi t)r;riI)
\vere 00 to 100 X 10E caellh lwt' a of tiwie (9X to 100 , vial,lr).
(`ell:: were illocuhted at 10' \ iable (*ells l)er 150.mr11 F:~lcor~
lwl\-styretIe Petri dish (145 ~11' durl'aw area) iii 20 nil of I )\IIC.\I

3159


(!10' ( I)uDwco-Vogt trrotlificatioti of Kaglr's medium, 10' c f&l
tw\,ittrA ietuttt tvitlt 50 uttita (J(' socliuIrt-l)etiicilliit alld 10 pg of
stl~e~`t',llj~~ill-~o~ l'er ml).  I)isltes were ttiuitttuitrrd at 3i" iu
:,*I Ittttrtitlifirtl :tttitohl~ltere (II' IO:& (Y&90", air. Cttatlges of
tlrrtli,tttt \vcxre lwrl'ortttrtt 011 ttte 3rd day of culture, ever)- 2tttl
day tltetwfter, ;tiitl 16 to 20 Iroui~~ tjrforr enzyme asw~`.

`I'ltr (`-6 rat ahtrocytottla c*ell litte uxs obtained from the .\iiirr-
icxti `I`yl'e ( `ult~ue (`ollwtiotr (So. (`CL 107).  IIeLa cells were
frottt FIo\v I,:ll)c,i,:~tol,irs, l~ockville, I\ld. The 3'1'3 id I%alb/C
3'1`3 cell lines \vere from l)r. George Todaro.  The RGlT9 line
\v;ts :I Iternxttirirt (~11 litte ot~tttitietl after tttultiple passageh of
t)r:ritt (*ells front ;i-tlaywld Fi~lier rats.  The hut11at1 nstt'ocytolll:t
(`lll( :t1~1 rat glionw (`2, wre provided by 1)r. S. I'feiffer and
lh. I I. Scheitr, rehpwtively.  The c'2, cell was originally de-
\-elot"`cl 1'2. lh. G. Ib3ltl:L.

f'repuration 0s Iloncogenates

Sttr,fccce Cultures-The procedure WI,* designed to wash cells
free of' serttttt protein tLtl(l to recover cell enzymes reproducibly
iI1 ltiglr yield.  Growth ttredium was removed from Prt,ri dishes
and tliwnrded :d the cell t1lO~l~J~ayet' was gently wasl~rd twice
with IO ml of Solut,iott 1) vontainittg 0.14 mbf Ca(`l? and once
lvittt 10 nil of Solution U.  The Petri dish was draitled for 90
s at ;I 45" angle, then the dish was scraped with a spatula 5 cm
\yitlr (the end of :t Ilesible plastic ruler) covered with disposable
l`rllot~ tttlte (Scietttific Sltecialties Services, Inc., Kandallstowtt,
AI( C:ells were recoveretl by asl)irutiott with a large bore
ttlicroltiltette aid tr:nAwwl to a polyullomer tube, 3 X 0.5
itt(*lt. The .surface of the tli& was washed twice with 0.1 to
I ..i-ml port~ions of IMfer A\ (50 m&l I)otassiuni phosp1itLte I)uffer,
1'11 6.S; 1 tttiv l!X)TA\, l~otassirtti~ salt) at 3". The aniount~ of
I\uRer X acltletl was acljusted sw that the final protein concentra-
tion was 2 to 10 mg per ml of homogenate.  Cells conCAned in
the washes were rechovered and cwmbined with the scraped cells.
`he tubes were stoltprretl attd placed in cold HrO in the chamber
of an ultrasonic oscillator (Raytheon, model No. DFlOl) ; cells
were Iysed by aotli(xfioti at 1" for 5 min.  Homogenate volutnes
wew trleasuretl; each liotnogetiate was divided into small por-
tions which were frozen quickly in a I)ty Ice-acetone bath and
storrtt in the valxw phase of a liquid nitrogen freezer.

.I/ottse Bruin-Brain tisstte from Mb/c mice was washed
\vit Ii Solution I), blotted, mitlced iii al~prosimat~ely 7 volumes of
l{rtffer ,\, and homoget\ized at I" with a Potter-Xlvehjenl ho-
ntogrnizer atId then I,)- sonicatiott as described above.  The
lrotirogertnte was centrifuged at, 35,000 X g,,,:,x for 15 min at 1';
the supernatant fraction (4 to 12 rng of protein per ml) n'as
tli\-idetl into 0.05.nil lx~rtiotts, quick frozen, and stored at - 100".

I'roteitl concentrtttion was det,ermined by a modification of
the tttethotl of Lowry et al. (13) with 3 t,o 20 pg of protein per
rwc~tion.  I)K.\ was determined by the spectrofluorophoto-
tttrtric method of Iiissane and Robbins (14). Histochemical
trst> were kindly performed by I jr. Lloyd Cuth.

Ilomogelintes in Buffer A were thawed shortly before use and
porticttts were dilut,ed with appropriate modifications of Buffer
A to adju& the homogettates to the specific conditions of each
awl\-. Homogenates were added to reactions last. Each ho-

rnogetlatr v-as aihayed at four coticelitrtltioits; valuea were used
only if tlte rate of retictioti \\-a~ ~~rol~ortiot~al to the hottiogeti:ite
concentratioti.  Triplicxtr hotiio~rttntes IYew lwe~~:iretl and
xwnyetl routitrety; the average \xlues nre Ao\~t1.

.\c~et~l~lioliliestertt.~e activity n-a? swayed by a ttiodification
of thett~ethodhof Reed et nl. (15) atrcl l%etrpreis el trl. (16).  Sew
robla~tomtt cslotie S-IX honiogett:~tr> were thawed ittitiirtlintrl~
before use ant1 trdjustetl to the aih:ty cottditions t+- mising 4
\olutiies of Iiotnogeti:~te with 1 \olutne of IZuffer .\ wtttaittitig
1.0 11 NaC'l ant1 2.5:; (x-/v) Triton X-100. The radioactive
~ubsttxte, 12-"H] ace T (3 10 me chloride, 250 mCi ~ter ttttnole
         t-.1 I 1'
(-~iuershnttt-Se:lrle) or [ I-tlC']acet~lcholirle iodide, 2.4 tn(`i per
innwle (Xew England Suclear) \vas dissolved in Hz0 ntrd ly-
ophilizetl for 16 hours to remove posAble volatile coiitt~niilttittt~.
The cation eschatige resin Ai(; 5OU-XX (H+ forttt, 100 to 200
~ttesl~, l%io-Rad Laboratories) \vas con\-ertecl to the r\;;t- form
with 2 s -\JaOIl at 25" for 45 rnin and then washed with II,0
until the pH of the effluent was 6.0.  Colutnlls (0.5 x 5.0 cm)
of resitt nrre formed over small phtgs of glass wool in S-inch
disposable Pasteur ltiljettes and washed with H,O.

Each rewtiott contained the following components in :t fit la1
volume of 50 pl unless stated otherwise: 2.8 mu [2-W]acetyI-
cholitie chloride (0.15 pCi per reaction, 1.08 mCi per mmole),
200 mxr SaCl, and 0.5', Trit,on X-100 in Buffer .1; and 0 to 40
pl of neuroblastoma homogenate, or 1Suffer Ai containing 200 nip
SaCl and 0.5:`; Triton X-LOO, or both.  Reavtiow in dieposable
glass tubes, 10 x 75 n,n,, were inwbuted for 10 niitt at 37", then
transferred to an ice-water bath and diluted rapidly by the utltli-
tion of 1.0 in1 of II20 at 1".  Each diluted reaction was immetli-
ately ljassed over a column of the catiotl exchange twin; the
tube was \vashrtl with two l.O~ml portions of H?O at l", and the
washes also were pushed through the rolunrn.  The column
effluent was collected in n glass scintillation vial, then 10 nit of
Tritott S-100.toluene-Liquifluor (333 g:666 ml :55 ml) >cintilla-
tion fluid were added and radioactivit>- was determined with a
scintillation counter (32?,, counting efficiency).

Since reactiotts are not deproteinized, the column step must be
l,erfornied rapidly. The rate of acetylctiolittesterwe nctivit)
after dilution at 1" was al)proxituately 1.5"; that. of undiluted
reactiotir; at 37".  Therefore, the assay was performed itt batches
of 20 tubes or les:: so that all renctiolw could be passed through
colunnts within 3 min.  In sottte instaticea distinction was made
betweet "true" and "pseudo" cholinesterases by employing
10-j M I<Tl- 284(151 dibromide.

Cateckol O-.llethyltrarls~erase Assay

The a<hay described \vas a modification of t'he method of
Sikodejevic et nl. (1'7). Keuroblastoma homogenates v-ere ad-
justed to the conditions of the cat'echol 0-meth?-ltrarlaferase
assay immediately before use by mixing 9 volumes of homogenat'e
with 1 volume of Buffer h containing 50 IIIM MgCls.  The L-S-
ndet1os~l[nlefkyZ-1"C]Iiirtltiottitte, 58 mCi per mmole (obtained
from A%nter~hsm-Searle in approximately 0.001 K II,SOI), was
extracted twice with 10 ml of toluene; the aqueous phase was
lyophilized before use.  Kottradioactive L-S-adeno~~linet~iiorlirie
iodide and 3,4-dihgdrosybenzoic acid were obtained from Cal-
biochem Corp.

Each reaction contained the following componettts in a final


volrllllr of 50 pl rscq,t \vllrlY~ stlLtet1: 0.55 nlM L-S-adenosyl-
(r~~e/lr~/~-l"(`~ilretllioiliile iotlide (0.318 pL(`i l)er reactioii, 11.55 niCi
l)er il~ii~ole), 2.5 in?,1 dih?-tli,os?-l)eiizoi~~ acaid, aild 5 nm AIg(`lr iii
IMTrr .\; :llKl 0 to 10 pl ol llr~l~~o~,l:~stol~la llollrogrll;1tr, or 1SufftY
.\ c*oikt:Alliirg 5 11131 A\Ig($, or l)oth.  Ii>ac*h reavtioii x:18 iiwuhated
ii1 :I I%-trll volliv:il glash c~eirtrif1lgr tube n-itll gmuid glass stoplwr
for 20 illiii at 37, t,lleii tr:uwl'erretl to ai1 ice-miter hth aid 0.2
nrl ol 1 .O s II(`1 alltl 10 1111 of tolue~w were atitled.  `I'ubrs \VPL'c:
sll:lkt~ll vigorousl~~ for 1 iiliii, xtltl c*ei~trifuged for 3 iniit at 250 X
g.  `I'hri~ 9 nrl of the tolririle ptiasr were ~~inoved :LII~ tmisferred
to a .sciiltillatioii \-ial contaiiiiiig 5 1111 of tolu~iie-LiquiLluo~ (958
nil:42 1111) rc~iiitillation solutioir niid radioactivity \v:is deteriniiied
(8i'; wuiit,iiig efhcieiivy).  .I cwrrectioll VW al)lllied so t,hat)
each \.alrit: wlwrtrd represented the eiltire toluerie phase.  Ah-
t,lieiitic* :~-[`4C]r~ietliox~--2-li~dro~~l~eiizoi~ .
                         acid  wld 4[I"( `line-
t.llos\-8-ll)d~o\-?-l,rllzoic acid \vere prq~a~~~ with lmrified rat liver
c:ltec*liol U-iiietli\.ltl.airh~ei,:ise ol)t:~ii~ed front 1)~. (`. R. (`reveling.

`I'l~e :~ssay dewrihed \vas a rrlodifiratioll of the nlethotl of
Schrier and Xhuster (18).  l\Iouse brain est,rac%s were t,h:iwed
inmediately lwfore use and adjusted to the choline acetgltiwis-
ferase assay m~ditions by iuixiiig 4 volumes of brain eztiwt. with
1 \ohuue of 13uffer .I co!lt:rinillg 1.0 M NaCl and 2.57; Triton
X-100.  The [l-"(`lac~et~l-Co~~ (50 in(`i per mrnole) (?r'ew Eng-
laiitl Yuclear) was 1yol)hilized for 16 hours before use. I-ii-
1:lbrtetl acetyl-(`0.k (trilithirun salt,, trihydrate, A grade) and
iirostignriiir n~etliylsulfate \VPI`~ I'ronl (`:~lbiocheni; choliiie iodkle
\vas ~IYII~I Schwarz.  `I`he anion esvha~~ge resill AiG 1-X8, (Cl-
form, 100 to 200 mesh, Bio-Rat1 Laboratories) was washed with
HZ0 until the effluent was plI 5.5, aild colurn~ls of this resin for
each assay were lwepared as described for the acetylcholines-
twase al-say.

li,acli reaction ~oiitainetl the follo~viiig components in :I final
~oluiiie of -50 pl, ewept where &rted: 0.21 ml* [lJT]aeetyl-Co;
(6 ni(`i per nmiole), 2 II~M vhohue iodide, 200 iii~ Kac'l, 0.1 nnf
Iieostigi~ririe nietliylsulf:~te, aird 0.5';, Triton X-100 iii Buffer .I;
mid 0 to 40 pl of Ironrogeii;~te, or IIuEer A containing 200 nnr
KaCl :~iirl 0.5';, Tritoii X- 100, or both. Reactiolls were incu-
babed ilk glass tubes, IO x 75 IMII, for 10 nlin at W, then trans-
ferred to an ice-water bath and diluted by the atlditiou of 1.0 in1
of 11~0 at lo.  The contents of eac~li tube were lmssed t,hrough
a11 allion rxchmge wluniri; the tube was washed with two 1.0.ml
portiollh of II& at I", and the washes were also passed through
ttltb c~)lriillii.  The colunlii effluent was collected in a glass scin-
tillatioli vial, then 10 ml of Tritoil-toluelie-Liquifiuor sciutillation
nlisture (see above) were added and radioactivity was deter-
uiiiietl ;it a wluitiiig efficiency of 877;.

`I'Ko tuodifications of the niethod of 11'illgo and Awspara (19)
were employed.

.llethocl rc-Homogenates lvere thawed llrior to use and ad-
justed to the conditions of the glut,arnate decarbosylase assay by
misiilg 9 volumes of homogenate with I yoluine of Isuffer A% COII-
taining 10 nm 2-iliercaptoethailol, 5y0 Tritou X-100, and 5 IBM
pyridosnl ~~hosphate.  The L-[l-`4C]glut,anlic acid, obtained from
<`albio~hern, was neutralized with KOH a11d lyophilized before
use.  ~\riiiiio-oxYacet,ic acid was obtained from Sigma.
1~::1cli reaction wnt~aiued the followiilg components iii a final

\-oluiue of 50 pl, except n-here <tated: 5 imf L-[l-"(`]glutaliiic acid,
Imtassiim salt (1.04 &`i l)er reaction, 4.16 niC'i ]Jer innrole),
1 iwu 2-nlerral)toethnilol, 0.5 ni~l p\~ridosal phosphate nioi~ol~y-
drate, and 0.5(`; Triton X-100 in IWfer ;\; and 0 to 40 pl of
Iioinogen:~t,e, or I<uffer h co!rtaiiliiig 0.5c, Tritoii X-100, 1 III~I
2-liierc,:il)toet,llallol, and 0.3 niv l~~ridos:d phosphate, or hotli.
Reaction mixtures ii1 glass tubes, 10 X 37 inni, were sealed \vitlt
tightl\- fittiilg rubber
lvitll ;l Ileetlle.     <topper5 that c~nild be e:isil\- l)erforatetl
         Renctiollh \vere incubated for 10 mill at 37" atrtl
theii transferred to :iii ice-\vater bath. Eacli tube was then
l~lacecl iI1 a glass s~iiitillation vial containing 5 ml of llyamiiw
toIuelIe~I,irluifluor scintillation solutiotl (288 ml of 1 M Hyamine
hydroxide in methanol to 640 nil to 42 ml).  The vial was sealed
lvith the reaction tube stopper; then 0.2 ml of a solution of 10
inhr acetic acid in nirthanol was injected through t,he rubber
stoplw into the reaction ill the tube with a hypodermic syringe.
The injection needle was used to dislodge the glass reaction tube
from the rubber Aopper PO that the tube fell to the bottom of the
.seintillation vial.  The stoppered vial was incubat,ed for 30 min
at 24" to allow ahsorption ot' l4CO.1 br the Hvarninc solution.
                          _
Then the reaction tube was removed f;om the"scintillatioll vial
with forceps and the vial was capped with a scintillation vial top.
Radioactivity was determined by scintillation counting at, 807;
efficieiwy.

JletM b--This alterllnte method differed only in the technique
of `"C'O~ collection.  Etlch reaction was perfornled ill the &m111
diameter center well of a. 10-1111 Erleimieyer Ha& (Koiites)
equipl)e'tl with a tightly fitting rubber stopper.  Hydroxide of
Hyannine at 37" (Packard Instruments, J)owner's Grove, Ill.)
was plaved in the outer portion of the flask and reaction compo-
nents were added to the center well.  The flask was sealed with
the stopper and incubated at 37" for 11 min.  The reactiotr was
terminated hy injection of 0.2 ml of 10 niiv acetic acid in meth-

T.WLE I

Range and sensitivity of assays

Choline :tce-
ty1traw
fcrase
Glutamate
decarhox-
J&Se
7
(,`atechol O-
methyl-
Irnnsfer-
itse

-

Ncuroblas-
t,onia clone
K-18

Incu- ,
hation Radioactive product
time

10  [l(C]Acetyl

choline

Neuroblas-
toma clone
T-18

20 3.[`*C]Me-
   thoxy-4.hy-
   droxybenzoic
     acida

pmoirs ' PnlolPs
1,000~75,000
14,000
7% 75,000
14,000
  5m 1,500
700


20-   150
3,000


  lo-    75
1,200

a Approximately 54; of the `"C-product, is 4.[l*C]methoxy-3-
hydroxyheuzoic arid.


316"

a1101 tluwugh the stopper iltto tile crtiter well.  .\fter att addi-
tioiial 90 inin at 37" an aliquot of the Il~-ai~ine-~"CO~ solution was
tratlrferred to a sc~itttillwtiot~ \-ial cwtitaitlittg 10 1111 of tolttette-
Liqui~luor xit~tillat~ioti misttire.  Ik~tlionctivity was detwmit~etl
l)y scititillatiott coutitilig at 87';; eficienq.

Tlte usu~i1 range and setisitivity ol)ver\-ed for the four etizyme
assays iire sliowti in Table 1.  In ev:iIttntiotl of t,Ite asnays tieuro-
hlastoma clotle N- 18 hotnogettat,e 1~:~s used f'or acet)-IclioIities-
terase and catecltol O-ttietli~Itlatis~er:t~e :~ss:iys; mouse hraitl es-
tr:lct-; were used for evaluation of tile w5;:iyh for choline acetyl-
traitsfw~he :md glutamate decarbosylnre, although these assays
jvere :rlso ullltlicable to cell culture extract>.  The ratige of liuear-
ity bitli 1)roteitt for Ileuroblastoma cltoliue ~rcet?-ltratl;jt'eru,~e was
about half t,Iint ol~taitied with mouse brain homogenates (20).

(`otiditious were selected so th:it a homogenate wuld be ye-
paretl 1)~ hnrrestitlg a single I'etri tliih ;itld stored frozen fol
future \I>?.  .\l)out 30 nig of 1)roteitl could be liarve4ed Iron1 a

MINUTES               MINUTES

.Ss slio\vtl in Fig. 1, the rate of each reac~tiott \vas lmportion;~l
to concentration of honiogenate i)roteiu withit the range >trttlied.
Conditions were adjuht.ed so that the Importion of radioactive
substrate coiiverted t,o i,toduct \vas < 5 ' ; it1 blie case of choline
ncet,?-ltr:tnsfel,~ise and < 104: with the other three enzymes.
The relntioii hetweell time of ilwuhation and renctioli rate i.s

SIIOWI in Fig. 2. CTnder tile conditio~~s employed, reactions
catalyzed by glutnntate decnrbosyl:we, cxtechol @tnetti~ltr:itw
ierase, cltolitle iicetvltrati~:fera~e, atld :icet~Irliolitiester~i~e were
linear for 60, 98, 20, and 90 min, respectkely.

The relation between react~iott niirtrue pII and enz>-me a&\-it>
is showt~ it1 Fig. 3.  AAt l)H 6.8 tlte activities of I?;lutnnratr tlcc*;tr-
twsylase atld wtechol O-nietli\-ltralisi~r~i~e )I-ere ewelltially ina~i-
ma1 (the latter w;w ilihihited I)\- `I'ris) ; however, choline acetyl-
transferase and acet)-lcholirieste~~lse activities mere 60 to 6.5'1, of
the observed niasinm.  ;\lthough tltehe latter two activities wtx
opt,intaI betweetl pII 8 and 8.5, the four enzymes were rotttittely
wsayett at 1)lI 6.8 l)ewiw acetyl-Co;\ attd acetylcholit1e are more
stable att pIIlL thatI :tt pII s.  In :itldition, the snme homogeii:rte
could be u$etl to detertl~ine tile activity of each enzynw n-it,liout
ndjus;tmetit of 1tI1.

1
9


1'::trh rcxctiott c'otlt aitlcd thp corrl~otwttts dcscritxd uttdet
",\lcthods" wld 40, 15. Xti, ok (Xl pg of protein for choline acetyl-
I r:tllsfc~rasc , :lcct~l(,tlolil1estrr:tse~ gl11tarnate decarl~oxylase, a~td
wtechcll 0.methylt r:lt1sfer:m reactions, respectively. The
:tn~ot~~~t of r:Ldiwtctive prod11ct for1ned (picomoles) per complete
re:cctiol1 c'orrcspo11ding to lOO(,; were 010, 11,300, MO, and 114 for
choli11r acet?-ltlwllsfer;\sr,  :lcct~lcholillester~~e, gl11t~tmatr dr-
c:lr~,os~l:w, :111d catcchol O-rnethylt ra1Lsferase reactions, re-
spect ively.  (~1~1tarnate drc*:trt)oxylas:(: was determilled by ~lcthc,d
a (SCC "~lethods"). I'rodt1ct recovery wits tested by adding the
followi1lcr conlnol111ds to react iotls it1 nlace of radioactive sill)-

I<nch rr:rctioll contnil1ed the compo11et~1s described 1111de1
"Rlethods," except :IS noted, nlld the following: 40, 15. X6, a11d 60
pg of homoge11:ite protein for the choli1w acetylt ransferase,
ncct~lcllolirlcstcr~~se, glutamate decarboxylnse, and catechol O-
met hylt r:t11sl'cr:we  reactiol1.i,  rcspectivcly. l~:uzynie specific
activities, it1 the order stated atjove were: 1,480, (i4,400, 140. :111d
8X pmolcs of prc,dr1c*t per mill per mg of protei1l. (;111t amate
decarh~~syl:~se \v:w nw:~s~~red 1)~ Llethod a wii h [l-~lC]glrlt:tn~ic~
arid RS Slll)Str:1te.

.~ITIOU~I~ 0i
radioactive   Per

                    1      . "
(i3.4 X lo3 dpm) ;11ld 0.015 Ilrnole of 4. ~`1C]1nethc~s~-3-11~drox~--   Complete      
twllzoic acid (360 dpttt).                         ,\littrts etizytne (*~IOIIR~~ I)rnitr).
                                           Minus choline.

I~:11zynw st,:tbilit>
<Io~~iplete react io1L.
Lli11us enzyme.  
1<1lzyme froze11 :t1ld thaw-cd
  three times.   
1211~yme held at 1" for 2 -3 hrs
ls:tlzyme held at 100" for 10 mill

                         100    100    100
                           2     9   ~ 100    Minus enzyme (1ie11roblastorii:t~
                                             Millus Trito11 S-100..
                   8~ 0     Minr1s N&l.
                                               ?*li1111s I+:l)TA.     
                          9x    0'1 ~ 109    10%    Mi1lus El)TA, plus 1 mu I\lgCl,.
                         100    100 116    104     PlllS 10-j Y HW "XK51
                           2    9    x     s   Glut amate der~~rt~c~sylaae
l<r:wt iota i11cu~~:ttetl at 1".    9    31  10~ 7    Complete
l'rodlict rec~~very                            hlinus e1izyme (mouse br:iiu).
1~:rtlio:active product added ill-                    hlirms pyridos:tl phosphate.
  st cad of snhstratr   !I5   110 i  94 !   83     LIi1irls l+:l)TA..
-~                                    PIlinus %-mercaptoeth:\11(,1
                                             Min11s Triton S-10()  
lll:ltrl~ zo(,`; of that fou11tl at Yi".  For t,his reason acetylcl1olii1es-   Plus 1 rnM MgCl:.  
tewse reactiotrs \vere l1etfornrrtl iii btttc*hes (< 20 tubes) sn tlmt,    Plus 10 ~11~ iodoacet:rmide.
rrwtions could he cl1illrd i111nrrrli:~tel~- after incubation, diluted,   Plus 10 mu hydroxylamine.
                                                  ,,, 1              : .I

    Gluta- `Caterhol    MinL1s N&l.
4cetyl-
cholin-   mate   O-meth-
     decar-
`StemSe boxy,nse  yltrans-    Xlinr1s Triton S-100.

           fcrase    l'lr1s 2 mill Mg(`l,   
            Aret?lcholillrster~~~e
5'     5     5%     Complete

64

      2
   40
   61
  59

1130

 105
   i50
 1140
   1 lli0

 , 1130
_.../  135

1~:1clio:Lctive l)roduct n-:18 :ttltlrd to reactions iti~te:1tl of 5111,.
stratr to drt,erini11e t,he l~e1wntagr of product recovered.  Greater
tlL:lll 94:;) of the ratlioactivr lnwlwts of the choline awtyltr:r1is-
ierase, ;1~et~~lcl1oli1iestet~asr, :urd glutmnat~e decat+~osyl:ise rear-
tions :iittf XSC,l of the r:itlio:lctivr product of the c~atec~liol O-meth-
yltr:11wfer:~se re:wtinn \vere rrc~overetl.

5.7
0.48
0.09

The effect of twctiorr w11l~o11ents aid other cwn1l1out~ds on the
rate of' enzyme :wtivity is slion-II in Table III.  C'liolittr acetyl-
tr:it1sl'erase from ntousp lwai11 was conq~letely del)endent upon
c~holiiie for activity :ttid XV:I~ stirtrulat~pd by 200 iml ?;a(`l.  E:r1-
zj'trre activity was not :rffec*teci :1plnwial~l~ I,\- oniission of `l'ritotl
X-100 or by the addition of 2 IIIM A\IgCl,.

Se~1t~oblasto111a :~cet~l1~lroIi11ester:~s~ wtivity \v:~s reduced in
lhr :il~~ence of `I'riton X-100; however, on&ion of SnC'l or E 1)`1',\
iit tl1e lnwrnce or ahhe11ce of lIg(`l? was without effect.  JIarked
it1hibition was ohrprvpd by IO-: M l3W 28W51, :1 potet1t i1111ihitor
(11' :tc.et?-lcholinr~ter~~~~ (I<(' 3. 1 1 .T).

LaTect101 u-,,leT1i~ltrii11sIel`ilse
Complete........  ,........
Minus e1lzynie l1it~l1rot,li1sto1na) ,
Minrls dih~dros?-l,etlzoic acid. ~
Minrls dihydrosybe11zoic acid, ~111s 1 marl p- ~
  ll~drox~~he11~1:1c~etic~ acid.
Plt1s 1 nw IJ-h?dros~phet1?-lacctic acid.
Minus hIgC1,. _.
Min11s l*:l)TA..   
Minus hIgC1, 1tl1d E1)T.i..
I'lr~s 0.5"( (v/v) Tritun S-100..
Plr1s 1 ml\1 L'-nlercnpt',etl':\llol
Plus 0.25 111~ dihydroxymet hoxyhenzoic acid.
Plus 1 IllM tropolone.  

0.70
0.5
0.98
5.i
3.0
6.6
7 !I
1.3
0.94

13
115
17
100
-`)
17,
140
2. 3
17

`I'riton X-100 or 1)~ the additiotr of 1 IIIRI Algc`l,.  .\I:trkrd itt-
liibitiorr \van oberved in the lnwetwe of iodoacetan1itle, hydras-
ylamine or 3n1i11o-os;vncetic acid, a~ eslwted (21).

`1'1~ act&$ of glutmn:ite dwarbosylase from mouhe bin was   The activity of 11euloblnrto1un catechol O-nleth~ltr:tIlxfela~~
red11wd Rlightly in the :tbw11~e of lyridosal phosphate hut was  w-as aln1ost wtnpletely depe11dent on the sub&ate, tlih~tlros~-
not :1ffrcted :ipprecial~ly 1)~ otnisaion of mT,i 0~' 2.n~erc:ipto-  benzoic acid. p-H~dt~os~l~llet1?-lncetic acid did 11ot serve :LS R
ctll:tttc~l. Tltp rettctiott \\`;I* ~tiirlttlat~ed ?;ligltt,ly by ottlksioit of  suMrate for the ruzy~t~r :tud did 11ot, itlhihit methyl trntwfer to

100
3
3
G"
94
91

100
!I
60
101
102
100
1"

100
s
8i
110
96
140
1'1'0
2! 1
1"
1"


               ~~~~~ - . .___
Ch,,otrcatoy,,ccphic  s!/slrnr~s-Solvelrt 1,  ethatlol-H,O-colIcen-
trated NH,011 (X:1:1) (Keferellce 2?), thin layer chrornntograph)
with .\IN I'olygram Cel 300; .S:olvent 2, 1-b~~tarlol-cycltrhex:nlc-
<>thyletxs gl~vrol-I-l,O-cortcc?lltr:ttc~d NII,OH-c?-clohexylamitte (30:
30:10:3.7:0.07:0.05), thill layer chromatography with hIN Poly-
gram Gel 300; Solvellt 3, 1.propa~~ol-0.1 pr' arctic acid (:$:I) (l<ef-
erertce 23), Whatman 3AlhI paper; Solvellt 4, l-propatlol~f(,rn~i(.
acid-H& (8:l:l) (Iiefcrettce B), Whatman 3MRZ paper; Solvent
5, 1-~,rlt;trlol-l-propallol.~~~O (4:": 1) (Reference 23), MN Polygram
Gel 300 thilt layer; Solvent 6, 1-propatlol-benzyl alcohol-H&
(5:2:2) (Reference 23), XIN t'olygratn Cel 300 thill layer;
Solveltt 7, 2-prol.):tlrol~collcelitrated TH,OH-H,O (X:1:1), Silica
(;el (; thitl layer; Solvent, 8. 1-bntanol-formic acid-benzene-IIs0
(15:%5:5:1:1.5), Silica (:el (+ thin layer; Solvent 9. henzene-gln-
cial acetic acid-tnethxnol (10:1:5), Silica Gel (; thin layer.

;lcctylch,o/inesterase- The reaction contained the rompottents
described ttnder "Methods" except that 2.78 mrx [2-W]acetyl-
rholine chloride (0.G PCi) and 48 pg of neuroblastoma qlone N-18
protein were present) (61,200 pmoles per mg of protein per mitt).
The column effluent was collected and NaOH was added to :t fillal
conretltratiolt of 0.1X M. Authentic sodium [`"C]acetate was
added and portions were applied to thin layer chromatograms.
The chromatogrnms were developed by ascending rhrotnat.ogra-
phy, dried, rovered with cellophane tape, cut into 0.5~cm hori-
zontal strips, and each was placed ilt a scintillation vial with 20
ml of tolttene-Liqttifuor scitltillation solution (see "Methods")
for determination of radioactivity at efficiencies of 5471 (I%) and
16';;. ("11).

Ckoline .tce(yll~(l,ns~erarnsr-The reaction cr)tLtained the compo-
nents described ttnder "Methods" except, that> 8.1 ~lmoles of
["-"H]acetyl-CoA (1.24 Ci per mmole) were used as srtbstrate,
choline chloride was added in place of choline iodide, 16% pg of
homogelutte protein from tnottse brain were added, and the rear-
tiort u-as incubated for 20 min at 37" in a final volume of 0.1 ml.
Altthetlt.ic I'4C]at:et~lrholille chloride was added to the reaction
after illcuhstion.  Acetic acid was added to the column efficient
to :t final cottcentration of 0.06 M.  Portions of the cohlrnll efflttent
were subjected to ascending paper or thin layer chromatography.
1)eveloped chromatograms were cut illto 1.0. or 0.5.cm strips and
c:Lcah was placed it1 a scintillatiott vial containing 20 ml of tolttetle-
I,iq\liflttor scintillation soltttiotl for determination of radioactivity
as described above.

Culwhol 0-.~~ethyllransferase-The reaction mixture contained
the cornpotlents described under "Methods" in a final volume of
- ~__________

0.25 ml with 750 fig of nerlrc~hlastr)rrt:~ N-1X homogetlate protrill.
The reaction was itlcrlbated for 60 mill ntld extracted with tollt(atle;
the tolttenc phase was evaporated to dryl~rss ttndcr :I strrant of
tlitrogen at 24" wtld the residttc theu was dissolved it1 0.X ml of
methanol.  Of the radioactivity origitlally preset11 in thr tclllwtle
extrart , X1(:; was recovert ,d in the mcthatlc~l solutioll.  :llllhrltric
:~-mrthox3--4-hydro~~betl~oi~ acid  atld 4-methox~-3-h~dr~)~~-
I)cnzoic acid were added and portion9 of the extract were srlbjrctcd
to asrctlding thill layer c~hrorrlatogrnph?-.  Chrornatogrxms were
dried at 24" atld marker rclntpottl~d s were located by c.x~~os~~~P t I)
ttltraviolet light Chromatogrxms were cut illto strips, 1 X 3
cm, atld each was placed itt a scintillation vial cotltaining 10 ml
of Tritotl-toluclle.Liclrlifluor scintillation solution for drrerrnitla-
tion of radioactivity.

Choline acet~ltraIlsfer:tsc

Catechol 0-mct,hyltratls-
fernse

Cxtechol 0-methyltratls-
ferase

-

      Percentage
       of radiu-
Jhromato-   active

1
`2

product
with RF of
authentic
compound
1"
S";l?"rn
[Wlacetate
/ iz
   90
[W]`4C.&+
choline
chloride

   80
) ;;
103

x-l
   i3
   76
4.[`C-
Methoxy-
.?-hydroxy-
benzoic ack

i Z
   5

E

I-

i

73
90

8"
81
87
104

92
7x
Xl





92
TX
81

0.70
0.25

0. tici
0.7i
0.49
0.X9
O.&v

0.x
0.91
0.86





0.76
0.75
o.i5

n With Solvent (i two peaks of radioactivity were fottnd, both with attthelltic [`"C]acet?rl(:holitle chloride and with the tritiated
r~ct io11 prcdtlc'i. This phettomellon has been attribttt,ed to the effect of salts 011 the rhroln:ltogr:cplrir tnobility of acet!-lrholine
(23).


~llt~c,ttt:lto~t~:~ltlte(l lvitlt :~rttltentic~ ,`S~t~retltos~~4-l~~~l~o~~l~rttzoic~
:Ic*i(l; 4 to 5' , of the rr;tc?iotr I)rodut.t rsttibit~rtl the c*ttroltiato-
gtxl)lti(* *rlctbj;itv of 4-rtietItos~-~~-lt~~lro~~l~ettz~~i~~ x+1.  C:tY~atel~
illilll %`,;a of the alq>lit~tl txtlicxlcative nxttrrial xv;t~ rewveretl after
c~llt~c,ttt:~togral~tt~.  So tliscrrte c,otttatltiltaltt--; were detected.
(`ltatxcterizatiott of tlte I)rc)(lttc'ts 01' the glutamate decnrbosyl-
asr twc*tiott is slio\vt~ it1 `I':tl)lr \`.  Itt addition, the amnrttlts of
y-;itrtitloll'-`"(`ItJut?-ric acaicl att(l t'(Y)., forrid tlttrittg ittcttlxttiotr
                          _
xritlt L-Jl~-tl(`]~i\tt:ttiti~ acitl as huhstrate are conipuwxl.  'LIr Ye-
httlts show tll:lt y-~tti~itto~l~-`4(~]l~ttt~ri~~ acid xvas fornwd tlurittg
itlc~ttlxttiotr ;t~i(l :tIso that hinAtr anioutits of y-:Lmittobtttyri~ wit1
af~tl (`Or ivere formed.  I IOWPL\.~T, a8 shown below, this 1 : I ratio
of l~roclticts \V:IS not foutitL \Yith 311 &ties :we>setl.  13ec:iusr of
the lwssibility that `"00~ tiray itot :iccurately reflect production
of y-:niriltol,tttyri~ acid, quantitative idrtitifiwt~iott of the tatter
product is required to validate tlte glutamate dec:irl~os~lnse
:l,%WY.

The relatiotts between substrate c`ottcetttratiot~s and enzyme
activities are sllolvlt in Fig. 4.  The apparettt ~`Iicltsrlis (WII-
statits, determined by the method of Linenearer and ISurk (24),
were as follows: ac~rt~t~ltolittes~erase, 9.1 X 1OP M acetylcltolittr-
chloride, cltoline a~rt~ltr:ttlsfer:Ise, 1.5 X 1OP 1\1 acetyl-(`o&L
(lvitli 2 X lOPa 81 choline iodide), and 9.1 X 1OV M choline iodide
(with 2.1 X 1OP M acetyl-(`oA) ; glutamate decarbosylnse, 3.7 X
1OP 11 L-glutatnic acid; and cxtechol 0-methyltralwferase, 2.3 X
lo-" ~1 diliydrox~brttzoic' acid (wit,11 5.5 X 10e4 M L-S-adettosyl-
tnethiotlitte), mid 4.0 X 10-j 3i r,-S-:itler~os~lmetltiollille (wit11
2.5 X 10e3 M dil-tydrosybeitzoic acid).  Substrate inhibition wai
detect,ed ody itt the case of uc,et~lcholittestersse, similar to ob-
servations that have been reported with that, enzyme from other
tissues (25).

III st:titd:trd reactions (Fig. 1) substrates were presettt, iu COIL-
wttttxtions well above their respective ettzymr K, values and
1~:; tll:tn 10::; of the txlioactive substrates were converted to
proctwt during reactions.  Iligh substr:tt,e concentrations were
used to maititaitt zwo order kiuetiw aid to reduce error due to
ettcloget~ous subhtrates or inttibitors atitl ottter reactions cornpet,-
itlg for hub&rate.

Recoverer of Protein nnd hzyttw declivity from Petri LAsIws
Ttte effectiveness of the harvesting procedure was esuminetl

IJ~ comparing the protein yield obtained by liarvestittg replicate
plates by the scrape-wash method nit,lt the amount of protein
recovered by the addition of 0.1 s n'a0I-I to dishes (Table VI).
The results show that 95 y. of the protein was recovered by the
standard ltarvesting procedure.  In experiments witlt a variety
of cells in c&we the reproducibility between replicate plates was
*15S;x with reslleet to the specific activity of the four enzymes
nt~tl +25% \vit,tt respect to the yield of proteilt recovered per
1JklW.

Ttte recovery of etizyrtre xtivity and cell protein also Jvas
st.utlied by adding to Petri disltes known quantities of a mixture
ol' ~teuroblustonm N-18 altd braitt homogenates which had preri-
ottsly been cltaracterizetl wittt respect to enzyme activity and
lxotriit c,otIc~rtitratioit.  l'lte homogenate was then recovered by,
tltr ttsual procedure.  A 1Jortiott was set aside for future assay of
ettzgnre activity and proteitt wncerttration; tlte remainder was
adtletl to a fre& Petri dish and recovered again.  Wie harvesting
c.y~te \vas performed four times.  The results, presented itt Fig. 5,

(156 pntoles of llCOr formed per tnin per rrtg of protcitt) :tnd tllr>
cotnpot~f~~~fs dewrit)ed tulder "~Ielhods" escept that t,-[IT-*4C-
gllttamatc WRS crnpl~~~ed as sltbstratr :urtl the film1 volrunc was
0.25 ml. It~clthnliotl was for 45 mitl at 37". The rractiotl was
trtmittatccl by the additiotl of 0.175 ml of 10 mix% acetic avid itt
methaltol.  The evolved `~C02 was collected for fi0 min by, 3Irt I~od
(I. The acidified reactiott then was rentrifttgrd xt 15.000 x y
f'or 10 mitt :t~ld the strperttat:tttt fraction was collected.  Unlabrlrd
7, -:ttnittol)ulyric ticid, glutnmic acid, and glllt:ttnittP were added
:~ttd portiotrs were applied to thin layer chromatogrxns of cellulosc~
;\Ix l'ol~gr;tm Gel X00 and subjected to :tscending thiu lager
chrolnatoaraph?i~gr~~~l~~ with Solvent Systems l? 2, and 4 (two-dimetl-
siolutl chromatogrttphy with System 3) and to Whatman :SRIRI
paper for high voltage electrophoresis (45 mitt at 01 volts per cm
and 1X0 nla) with 8ulveut 4.  Solvent systems were: Solvent, 1,
2.propattol~methaIlol-corlcetttrated TU'H&II (`3:7:4); Solvent L',
phenol saturated with 6 Y NII,OII; Solvent 3, L'-propano.I-
btttatto-1 N HCl (6: 1.5:%.5) for the first, dimension and phenol
sat,urated with H& for the second dimension; and Solvent 4:
p)-ridine-acetic arid-II$O, pI1 3.95 (1.X:5.0: 144.5). Chromato-
grams :ttld electropherogranls were dried, stained with 0.F;.
ninhydritt in acetone, and cut into 0.5- or 1.0~cm strips.  Each
strip W:LS placed it1 a scintillat~ion vial with 10 ml of Tritowtolu-
cne-Liqrtifittor scintillation solution and radioactivity was deter-
mined. Specific activities of "C02 ttnd r-amirro[U-`"C]l,~ltS-ric
arid reaction products were 1,580 and 6,320 dpm per nmole, respcc-
t ively.

altow that recovery for each harvest cycle was 967; for protein,
877; for glutamate decnrbosytase, 95';,; for cholitte acetgltrarts-
ferase, 83'; for ratechol 0-methyltratwferase, and 88':;. for acetyl-
c+tolinesterase. Thus, enzyme activity released front cells dur-
ing the scraping procedure would be recovered in high yield atttl
with titt,le alternt,ion of enzyme-sl)ecific activit)..

.Von-neuronnl Cells in Cullure-In order to determine the
specificity of the marker enzymes, established cell lines front
brain :t1~1 other tissues were grown in surface culture until several
days after cwufluency and then tested for presence of the enzyme
activities. As shown in Table VII, choline acet,yltrat~sfer:tse,
:t~et\-lchotinester~~se, and glutamate decarbosylase &ivit ies were
l)resent, it1 all of the cell lines examined.  A1(?ivities of the three
enzymes in established cell lines were considerably loner than
corresponding activities in newborn brain.

Glutamate decxrbosylase r;l)ec+ir activity ill I,-929 cells :rnti
it1 &al (sell liues C6, <`2,, and RG-IS9 when measured by y-anlino-
[`4C]tnttyric acid production was 10c; or less of that measured b?
]`c'O~ production.  The presence of 1 111~ y~ntnillobut?-l.ic acid in


3160

         I
ACETYLCHOLINE MOLARITY x IO4

II I  CHOLINE ACETY~TRA~SFER~SE  `I

SUBSTRATE MOLARITY x IO'             L-GLUTAMATE MOLARITY x IO'

FIG. 4. llelation between reaction velocity and substrate
concentration. Reactions for acetylrholillrsterase, choline acetyl-
tmnsferase. gllltamate decarboxylase. and catechol O-methyl-
transferase contained the components desrribed under "Meth-
ods" except for substrate concentrations as indicated and 15,
42.4. 188, and 200 ~g of protein, respectively.  In addition aretyl-

cholinesterase and catechol 0-methpltransferase reactions were
incubated for i and 15 min, respectively.  The data were plot I et1
according to the Lineweaver and Hurk method (24). Glutamate
decarhosylase ~-as determined by Jlethods n and b Method u is
shown)

     IZecwoery of ~11 prolein from Petri dishrs
Petri dishes (145 cm* sluface area) cont,aining Ilellrot)l:rstorrl:L
rIotIe N-18 cells were incllbatrd for 4 days alld then harvested
bp the procedure specified in the table.  Bach method was tested
ill triplicate; values show11 are averages.  Harvesting Alethod
la is the standard procedure described under "Met,hods." For
~lethod lb: 2.5 ml of 0.1 N NaOH was added to each dish that had
beet1 scraped :uld washed. The dish then was incubated at 4"
for 15 mill to dissolve protrill.  For Alethod 2, 10 ml of 0.1 N
NaOH was added to each dish containing a coIlfluent mollolayel
of cells alld the dish was agait illcubateti for 15 min at 1".

Method 01 harvesting protein

`!L-_i_j
I 2 3 40 I 2 3 4

TIMES HARVESTED

rrig

1. a, 1 )ish scraped and washed (standard method)   6.14
b, l'roteill remailking in dish that had been scraped
   :tlld washed was recovered by the addition of 0.1 ~
    s xao1r..  ~  0.30
  Total .I  ti.44
2.  0.1 in KaOTI added to l+tri dish cotl(aining a cell
   monolayer.       li.00

1'1~. 5. I-lecovery of homogenate protein and total enz)-nlc :IC-
tivities in multiple cycles of the scrape-nnsh procedure. Stalld-
ard homogenates for assnys of glt~tamate dccarbosylasc, catecho
O-methyltransferase-acetylcholinesterase, and choline. arrtyl-
transferase \vcre mixed in the proportion 10:1:1, added to cldturc
dishes, and repeatedly cycled t hrolq$ t hr scrape-wash reeovcr)
procedlue described in the test.  The starting homogenate VOII-
tained 20.3 mg of protein and enz>-me specific activities of 115,
4l.i, 31,850, and 118.4 pmoles of product formed per min per mg
of protein for gl[ltamate decurboxylase, catcchol O-methyltrana-
ferasc, acetylcholinesiernse, and caholillr acetyltransferasr, re-
spectively. Gllltamate decarbosylase u as determined by .\Iet hoc1
a.

rea~tioll mixtures had little influence on activity by eit,her mea;;-
uremelit.  `rile presence of 1 lllM nlniuO-Os~-:~cet~ic acid reduced
(`Or productiolr by only 10 to SOT&, yet, y-arninobutyric acid lxo-
ducntiolt \V:W retlu~rd 1)s. at lenst 95';.  Thus, it appeared that

I
0    2   4   6    8   IO   12   I
   SUBSTRATE MOLARITY x IO3

0 I 2 3#4


14



I2

do
m

n
v
A
w
oc

"0
-
   L

;


3168

               TABLE VII
Zhzyrne specijiic activities in newborn mouse brain untl
              cultured cells
Specific activit,ies of glutamate decarboxylase, choline acetyl-

transferase, and acetylcholinesterase were determined on extracts
of cultured cells as described under "Methods."  Ail unceib
trifuged homogenate of newborn Balb/c mouse brain was lised
for comparison. The maximum content of hom0genat.e protein
in the assays was: 237 pg for mouse brain homogenates, 110 rg
for C-C;, 906 rg for C21, 852 pg for CHB, 258 rg for HG-179, 1306 rg
for L-c329 (B-82 clone), 936 pg for HeLa, 533 pg for 3T3-S (Swiss
mouse 3T3), and 580 rg for mouse brain cells cultured 30 days.
Formation of -f-aminobutyric acid was determined in assays with
~,-[U-`4C]glut,amate as substrate, followed by electrophorrsis and
chromatography as described above, with both Methods a and
6 for collection and determination of evolved COZ. All deter-
minations on cultured cells were performed when cells were 7 to
20 days postconfluencg.  Glutamate decarboxylase act)ivity
shown for C-6 represents the highest activity (at, 31.5 mg of cell
protein per 150.mm dish) found among follr separate points on a
growth curve.

Determination

c:1ut armtte   decar-
  ho.xyln,sc

By CO, produc-
  tion.
By -j-Aminobut,yr-
  ate production.
Choline acetyltrans-
  ferase.
Aretylcholinest,er-
  ase

Cultured cells

                    13  8    93


49G  8 5~  1  3 0.2'     G G    86
                          86
~ ~                I I
75,  9     2,lO
             2,lO 3 3  5 5    1ti
                          1ti

!5,900
!5,900 732
  7321701;
   I 701,  280 iF25
      280 ~F25 299
                  299 10'1
                     10'1  1,120
                   1,120

on top of which were smaller cells that were dark in the phase
microscope (phase-dark cells) (Fig. CC). Also present) were
llhase-bright rells with large cell bodies and long processes (Fig.
GD).  A\s seen in Fig. GE, silver impregnation was detected (with
a modified Hodian stain) in (*ells with multiple long processes and
large cell bodies.

The efiect of time in culture on marker elw,yne act,ivities iu
sho~vt~ in Fig. 7.  Catecho O-tneth!-ltlallsfer:ise activity did not
re:ic*h measurable levels during culture.  Glutamate decarboql-
ahe sljevific activity (Fig. 7A) did not begin to rise until t,he 15th
thy ill cwlture, when increase in cell number had ceased.  111
roiitrxat to glial (sell lines (Table VII), cultured Iwain (sell extracts
Iwoducetl y-aminobutyric avid and (`O:! in equal amounts.
(`holine :~c~et~ltr:lusferas:r spec'ific artivity (Fig. iB) fell ill t,he
first, 3 days to less than 7 units (the minimum that could have
1w11 detSect,ed). The spec-ific activity t'hen itwreased I'NNU the
31~1 to t,he 15th dlay; this rise was cwincidellt in time and rate xvith
tile iii,-iwsr ii1 cell iiutrlber.  .\fter the culture had reached a
st:tt.ion:iry cell number, choline acetyltransferase specific :IcstiritJ
wiitinurvl to rise, but at :I slower rate.

`I'lir sl)rcsific' activities of cboliile ac,et!,ltr:lllsferase and glut:l-
niate drwrbo\;yl:~se attaiired in these cultures were 47 and 43 :A,
res~wctivrly, of the levels ill disswiat~ed newborn brai,, cell.<.

FIG. 7. Development of marker enzyme syrcific activities it)
surface cultures of newborn mouse brain cells.  Bach homogenate
of Fig. 0.4 was evaluated for the marker enzyme activities as
described Imder "Methods."  Changes in specific artivitics
(0, 0) and cell number (dashed Jines) with timcl are sho1v-n for:
8, glutamate decarboxylase (assessed by "CO* evollltion b>
Method 6); U, choline acetyltransferase; and (`, acetylvholinrs-
terase. One unit of activity is defined as 1 pmole of prodrlct
formed per min per mg ot homogenate protein.  Open and ~lr~sc~/
.s,ymbol.s are explained in the lepcnd to Fig. (i.

From the 3rd to the 30th day in vulture, t,otal activitjieh of tllrse
enzymes (picomoles per tnin per cult,ure dish) increased 1,~~ 4%
fold for choline ;Icet~ltratlsferwse atrd 21 -fold for glutamate tle-
carbosylase.  Specific activitjy of glutamate tlrcarbosylahe n:is
at least IO-fold higher in these cultures than in the nonbrain and
glial cell lines tested (Table VII).

Acetylcholinesterase activity (Fig. iC) decreased until tire At11
day in culture, and remained csonstant for the remainder of the
culture period. In all cultures, including the established (~11
lines, esterase acstivities were inhibited X0 to 95C; 1,~ 1OP 11 I<W
284C51 dibromide, au inhibitor (25) of :~c~rt\lcllolil~e~t~e~~:~se
(EC 3. 1 1 7).  Hydrolysis of acetylcholine I,:- I~OUW brain n-as
also inhibited 95(/, hy t,his compound.

                 I)IscLTs91o4
                   L .

The results show that the I)rocedure for l)rel)arat ioil of rs-

tracts from cells in surface cwlturc is re~~rodwiblr anti gives e's-
cellent recovery of protein and the rnzynir activities nle:l.~;lu~etl.
Various other procedures for preparation of estrwts were not rx-
tensively studied; however, trypillizatiotr, 1x1 )`I'.\ treatmetlt, 01
scraping followed by washing of wll< rr~ultetl in lower recoveries
than the scraping procedure.  The cwzyriw assnys xre rapidI)
and easily performed and are applic~able for routine use wit11 a
large number of samples.  Inll)ortarit features ill :~l~~~lyiiig tile
assays are identifiration of reac-tiotl IwotlucLts :rl~tl dc,terlnitI:ltiol~
of the spec*ificity of the enzyme activities.  111 the g;hLtnmate cle-
carbosylase :is.q:ay, evolutioil of `Y`OL 1~:~s ilot always 11 n1c';,sui'e
of conconlit~ant pwductioti of y-:ririiiiohiit\-ri(~ ac+i by glutamate
decarboxylase. In the cholilre ;~~etvltr~~rlsfer:l~e away, rnzpe
@ems utilizing acetyl-(`0.1 may protlucr pr0tluc.t~ other than
wetylcholine that are recoverable in ion ewhange ~~oli~i~~ii efflw
riits (20).  There are siniilar l)otellti:rl diffic,ulties with tllc :tcartyl-
rholiiiesterase atid catecallol O-iiirtli~ltruusferasr :~.wiys.  I lPtrc*P,
methods for l)rod1lct identificatiolr alit1 enzyme ~li:llac,teriz:itioit
were 1)reseiited as iiltrgral l)arts of the :~ssays.

The enzyme activities studied were selected hecali.~e they ;~rc
important in neurot~raiistnitter nietabolism and al*o because awt-


hue of nray 25, 1972                   Wilson et al.                               3169

ylcholinesterase, choline acetyltransferase, and glutamate de-
carbosykse activities are higher in brain than in most other
tissues.  Relatively low levels of these enzyme activkies were
found, however, in several nonbrain and glial cell lines.  Thus,
the enzyme activities are markers for neurons only when specific
activit)ies are considerably higher than those of the non-neuronal
cell lines shown in Table VII.  The low levels of the activities
eshibited by these non-neuronal cell lines serve as a useful base-
line for assessment of nerve cell cultures.

Properties of growth of normal newborn brain cells in surface
culture were investigated as a first step toward establishing clonal
cell lines of mammalian neurons.  Primary surface cultures of
newborn brain cells contained cells that appeared to be differenti-
at,ed neurons both morphologically and histochemically.  Activi-
ties of choline acetyltransferase, acetylcholinesterase, and cate-
chol 0-methyltransferase did not reach levels that were signifi-
cantly higher than non-neuronal cell lines.  In contrast, brain
cultures attained levels of glutamate decarboxylase activity that
were at least lo-fold higher than non-neuronal cells, and the in-
crease in activity occurred after increase in cell number in the
cultures had ceased. Thus, primary brain cell cultures possess
several properties of differentiated neurons.
It is important t)o note that with improved culture techniques
and with cells from younger animals, brain cell cultures have been
obtained with activities of these enzymes which esceed those of
newborn brain homogenates1 (26).  In addition, other sensitive
radiochemical methods recently developed by Hildebrand et al.
(27`1 also may be applicable to cells in culture.

ilckxowledgments-We are grateful to Doctors 11. Wilcos,
D. R. Creveling, and J. W. Daly for helpful suggestions and t,o
Dr. J. llinna for providing cells used in some of the erperiments.

REFERENCES

1. WOLF, i%r. K. (I%%) J. Cell BiOl. 22, 259
2. KLIZBE, R. J., AND RurmLr,:, F. H. (1969) J. Cell Biol. 43, 69
3. Va~zos, S., AND KAIEO~S, C. W., JR. (1969) Brazn Res. 12, 180

1 B. Ii. Schrier, submit,ted for publicat.ion.

4.


5.


G


7.


8.

9.
10.


11.


12.


13.


14.


15.


16.


li.


18.
19.


20.


21.


22.


23.

A~GUSTI-TOCCO, G., ASD &TO, G. (1969) Proc. `Vat. Acad. Sri.
U. S. A. 64, 311

SCHUBERT, A., HVMPHREYS, S., B~RONI, C., AND COHN, RI.
(1969) Proc. n-at. Acad. Sci. U. S. A. 64, 316
NFLSON, P., RUFFNF,R, B. W., AND NIRENBERG, &!I. W. (1969)
Proc. ATal. Acad. Sci U. S. A. 64, 1004
WSRSER, I., PETEILSOX, G. R., AND SHUSTER, L. (1971) J.
Xeurochem. 18, 141

MIXK.I, J., NELSON, P., P~acocs, J., GLAZER, D.. AND NIREX-
BERG, M. (1971) Proc. Xat. Acad.`Sci. U s. A. 68, 234
FISCHBXH. G. D. (197Oi Science 169. 1331-1333
SHIM~D.~, iv., FISC~YI~`, D. A., AND MOSCONA, A. A. (19iO)
Proc. Nat. Acad. Sci. Cr. S. A. 62, 715
SCHRIER, B., ROSI:X~ERG, R., THOMPSOP~`, E., AND FARBER, J.
(1970) Fed. Proc. 29, 480

HAY, R. G.. END PUCK, T. T. (1962) Methods Enzym.ol. 5,
go-119

LOWRY, 0. H., HOSEIU~OUGH, N. J., F.~RR, A. L., RAR-DELL,
R. J. (1951) J. Biol. Chem. 193, 265-275
KIYSANE, J. M., ANI) ROBM~ZS, E. (1958) J. Biol. Chem. 233, 184-
188

REED, D. J., GOTO, K., ~KD WANG, C. H. (1966) Anal. Biochem.
16, 59-64

EHRESPREIS, S., CHIIZS.~, A., BIGO-GULLINO, &I., AND PATRICK.
P. (1967) Fed. Proc. 26, 296
XIICODEJEVIC, B., SENOH, S., Davy, J. W., AND CREVELING,
C. R. (1970) J. Pharmacol. Exp. Ther. 174.83
SCHRIER, B. Ii., AND SHUBTER, L. (1967) J. iVeurochem. 14,977
WISGO, W. J., AND AWAPARA, J. (1950) J. Biol. Chem. 187, 267-
271

AM.IXO, T., RICHELSOK, E., AND NIRENHERG, M. (1971) Fed.
Proc. 30, 1085

ROBERTS, E., .~ND QIMOSSBN, D. G. (1963) Biochem. Pharmacol.
12, 113

JONES, A. R., DOWLING, I<. J., AND YKR.~B.~, W. J. (1953) Anal.
Chem. 26, 394

WHITTAKER, V. P. (1963) in 0. EICHLER AND A. FARAH (Edi-
tors), Handbuch. der experimentellen pharmakologie, pp. 2-34,
Springer-Verlag, Berlin

24. LIXEWEIVER, H., AND BURK. D. (1934) J. Amer. Chem. Sot.

25.

66, 658         

AUGUSTINSSOK, K-B. (1963) in D. GLICK (Editor), Methods in
biochemical analwzs. D. 1, John Wilev and Sons, New York

26.
27.

SEEDS, N. W. (197i) I&. kat. Acad. &i. U. S. A. 68, 1858
HILDEBRSND, J. G., BARKER, D. L., HERBERT, E., AND
KR.IWTZ, E. A. (1971) J. Neurobiol. 2,231