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 Iac*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 . 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$etl to detertl~ine tile activity of each enzynw n-it,liout ndjus;tmetit of 1tI1. 1 9 1'::trh rcxctiott c'otlt aitlcd thp corrl~otwttts dcscritxd uttdet ",\lcthods" wld 40, 15. Xti, ok (Xl pg of protein for choline acetyl- I r:tllsfc~rasc , :lcct~l(,tlolil1estrr:tse~ gl11tarnate decarl~oxylase, a~td wtechcll 0.methylt r:lt1sfer:m reactions, respectively. The :tn~ot~~~t of r:Ldiwtctive prod11ct for1ned (picomoles) per complete re:cctiol1 c'orrcspo11ding to lOO(,; were 010, 11,300, MO, and 114 for choli11r acet?-ltlwllsfer;\sr, :lcct~lcholillester~~e, gl11t~tmatr dr- c:lr~,os~l:w, :111d catcchol O-rnethylt ra1Lsferase reactions, re- spect ively. (~1~1tarnate drc*:trt)oxylas:(: was determilled by ~lcthc,d a (SCC "~lethods"). I'rodt1ct recovery wits tested by adding the followi1lcr conlnol111ds to react iotls it1 nlace of radioactive sill)- I thyletxs gl~vrol-I-l,O-cortcc?lltr:ttc~d NII,OH-c?-clohexylamitte (30: 30:10:3.7:0.07:0.05), thill layer chromatography with hIN Poly- gram Gel 300; Solvellt 3, 1.propa~~ol-0.1 pr' arctic acid (:$:I) (l 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