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    CUN . CHEM . 24 /3 , 486 -470 (1978 )

    466 CLINI CAL CHEMISTRY, Vol. 24, N o . 3 , 1978

    Pu rifica tio n o f H um an P ros ta tic A c id Phospha tase b y A ffin ity

    C h rom a tog raphy and Iso e lec tric Focus ing . P a rt I.

    P irkko V ih ko , M atti K on ttu r i, and L . K a lev i K orhonen

    The main isoenzyme of hum an pro s ta tic ac id phospha -tases w as purified b y a ffin ity ch rom a tog raphy on L(+)-ta rtra te lin ked to aga rose and by i so el ec tr ic f oc us in g . T heenzym e was a s ing le p ro te in when exam ined by po lyac -ry lam ide ge l e le c tro pho re ses , e ithe r a s a na tive p ro te ino r in th e presence o f so dium d od ecy l su lfa te . Th e analyt ica lrecove ry o f enzym e activ ity was 19% . The s pe cific a ctiv itywas 4018 Mm o l/(m in ) m g) fo r h yd ro lys is o f 5 .5 m m o l/lite rp-n itropheny l p ho spha te a t pH 4 .8 and 3 7 # {1 76 }C .h e p urifi-cat ion fa c to r was as g rea t as 1900 . T he m o le cu la r w e igh to f th e enzym e as m easu red by ge l filtra tio n w as 109 000a n d b y p o ly ac ry la m id e ge l e le c tro ph o res is in th e p rese n ceo f sod ium dodecy l su lfa te 54 000 , in d ica ting th a t th e en -z yme ha d been iso la te d in the d im er fo rm . B y th is m e thodw e have ach ie ved the bes t pu rifica tion o f hum an p ros ta ticac id phospha ta se so fa r.Add I t Iona l Keyphras. : pu rifica tion and ch arac te riza tio n o f a nl soenzyme p ros ta tic d iseas e

    E stim ation o f ac id ph osph atase activ ity [o rtho -ph osph oric -m onoes ter ph osp hohydro lase (a cid o p ti-m um ), EC 3 .1 .3 .21 in se rum has se rv ed in the d iag nos iso f the p ro s ta t ic ca rc inom a sin ce the repo rt b y G u tm ane t a l . in 1 93 8 (2) . A n inc rea sed to ta l a c id pho sph ata seactiv ity in se rum , esp ec ia lly the re la tive a s w ell a s ab -so lu te inc rea se o f ta rtra te-in h ib ited ac id ph osph a tase ,ha s b een ob serv ed in sera o f p atien ts w ith p ro s ta t icc ance r (3) . H ow eve r, inco ns is ten t an d la rge ly co n tra-d ic to ry ob serv ations h av e been rep o rted co ncern ing theac tiv itie s in se ra , p ro s ta t ic se c re tio ns , an d tissu e itse lfin re la tion to ag e, p hy sio lo g ic a l a tro ph y , an d p ath o -log ica l co nd itio ns (4). O ne reaso n fo r th is m ay b e theac tua l v aria tions o f enzym e ac tiv ity , bu t th e o th erp oss ib ility is tha t th e p ros ta tic enzym e is n o t specifica l lyd e tec ted by the m e tho ds p re sen tly ava ilab le .

    W e now repo rt a s im p le m e tho d fo r th e iso la t ion o fa h ig h ly pu rified p ros ta tic a cid p ho spha ta se fo r fu rth ers tu d ies o f the enzym e , and fo r u se in im m unob ioche -m ica l ap p lic a tion s in th e d iagn os is o f p ro s ta tic d ise a s-es .

    D epartm en ts o f A na tom y and Su rgery ,1 U n iversity o f O ulu , K a-jaan in t ie 5 2 A , S F-9 0220 Ou lu 22 , F in lan d .

    A pre lim inary report o f th e resu lts has been presen ted (1) .R ece ived N ov . 8 , 1977; a ccep ted D ec. 1 9 , 1 97 7 .

    M ate r ia ls and M ethodsSam p le Hand lin g P ro cedu re

    F re sh h um an pro sta tic tissue , ob ta ined from theU ro log ic al C lin ic o f the U n iv ers ity C en tra l H o sp ita l,O u lu (F in lan d ), d u rin g su rge ry fo r p ros ta tic hy pe rtro -phy , con st itu ted the so u rce o f the enzym e. A fte r ex ci-s ion , the g lan d w as im m ed iate ly fro zen and s to red a t-2 0 #{176}Cr -70 #{176}C .ll th e p ro ced u re s w ere ca rried o u ta t +4 #{176}C.he frozen p ros ta te g land s w e re s lic ed an dhom ogen iz ed (200 m g w et w t/m l) w ith an U ltra -T urrax(th ree 1 0 -s b u rs ts ) in d ist illed w a te r co n ta in ing 1 g o fTw een 80 pe r li ter . T he h om og en a te w as allow ed tostan d w ith occa s ion al s ti rr ing fo r 30 m m , re-h om og e-n iz ed , an d cen trifu ged a t 1 5 000 X g fo r 30 m m . T h esu pe rna tan t flu id w as used . T he sup ern ate (4 5 m l) w as30% sa tu rated w ith so lid am m onium su lfa te an d cen -trifug ed (15 000 X g , 2 0 m m ). T he p rec ip ita te w as d is -ca rded , an d the su pe rna tan t flu id w as 70% sa tu ratedw ith am m on ium su lfa te . A fter cen trifug ation fo r 30 m mat 15 000 )( g , the p rec ip ita te w as d isso lved in 17 m l o fd is tilled w a te r and su bsequ en tly d ia ly zed fo r 1 6 hag ain st 8 lite rs o f sod ium ace ta te bu ffer (5 0 m m o l/li ter ,pH 5 .0 ). T he sm a ll am oun t o f p recip ita te fo rm ed w asrem ov ed by cen trifug ation fo r 20 m m a t 1 5 000 X g.Coup ling o f L (+ )-Ta rtra te to Am ino e th y l A g a rose

    L (+ )-T a rtra te (E . M erck , D arm stad t) w as cou p ledto am in oe thy l aga rose (A H -Seph aro se 4B ; Ph arm ac iaF ine C h em icals A B , U pp sala , Sw eden ) acco rd ing to th ein s truc tio ns o f the m anufac tu re r and C ua tre ca sas (5) ,a s fo llow s: 0 .5 g o f L (+ )-ta rtra te w as ad ded to 5 0 g o f w e tge l. F o r th e fo rm ation o f am ine bounds w e used 1 -e thy l-3 (3 -d im e thy lam in op rop y l)-c arb od iim ide hy -d roch lo ride (S igm a C hem ica l C o ., S t. L ou is, M o . 63 178)a t a fina l co ncen tra t ion o f 0 .1 m ol/lite r . T he ge l w asw ash ed w ith N aH C O 3 bu ffer (0 .1 m o l/lite r , pH 9 .0 ) andsod ium ace ta te bu ffer (0 .1 m o l/liter , pH 4 .0 , and co n-ta in in g 1 m o l o f N aC1 pe r lite r) , a l tern ate ly . T h erea fter,th e ge l w as packed in to a g la ss co lum n (P ha rm acia , 45X 16 m m ). T he con cen tra tio n o f L (+ )-ta r tra te used w as79 iLm ol/m l o f A H -Seph aro se 4B .A ffin ity C h ro m ato gra ph y

    The co lum n con ta in in g L (+ )-ta rtra te link ed to aga -

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    CL IN I C AL CHEM I ST RY , Vo l. 24 , N o . 3 , 1978 467

    ro se w as equ ilib ra ted w ith sod ium aceta te b u ffe r (50mmol / l i t e r , pH 5 .0 ) an d 15 m l o f th e d ia lyza te con -tam in g 180 m g of p ro te in pe r m illili ter w as app lied o n tothe co lum n , w h ich th en w as w ashed w ith 2 0 m l o f theequ ilib ra t ing bu ffe r. T he en zym e w as then elu teds tepw ise w ith 2 0 -m l p o rtio ns o f th e sam e buffe r con -ta in in g 10 , 25 , o r 40 m m ol o f L (+ )-tartra te pe r liter .F rac tio ns o f 2 m l w ere co lle c ted a t a flow rate o f 3 0 m I/h .F rac tio ns co rre sp on d in g to the second peak of p ro te inw ith h igh enzym e ac tiv ity w e re po o led . O f th e po o ledp rep ara tio n , 10 m l w as ap p lied o n to a S ep hadex G -200co lum n (Ph a rm acia , 25 X 325 m m ), w h ich w as eq u ili-b ra ted and e lu ted w ith tris(h yd rox ym ethy l)m e th y i-am ine ace ta te bu ffer (50 m m o l/liter , pH 7 .0 ). F rac tio nso f 3 m l w ere co lle c ted a t a flow ra te o f 2 0 m l/h . T hosefra ct ion s rep resen ting the h igh es t sp ec ific ac tiv ity o fac id p ho sp ha ta se w e re p oo led an d su b je c ted to iso -e le ctric fo cu sing .Iso elec tr ic Fo cu sin g

    A su cro se g rad ien t in an A m pho line 81 00 -1 -co lum n(LK B -P rod uk te r A B , S to ck ho lm ) w as u sed . T h e pHrang ed from 3 .5 to 10 .0 . A con s tan t v o ltage o f 30 0 Vdurin g th e firs t 4 h and 800 V during th e nex t 44 h w asu sed . T h irty m illil ite rs o f sam ple w as app lied on to th eco lum n w ith a h eavy su c rose so lu tio n an d the cath od ew as used as th e up pe r po le (6 ). F ra ct ion s o f 2 .5 m l w ereco llec ted . F ract ion s o f h igh e st ac tiv ity w ith iso ele c tricp o in t 4 .9 w e re po o led and app lied o n to a S ep hadex G -5 0co lum n , to rem ove A m pho line and su cro se . T h e co lum n(Ph arm ac ia , 15 X 335 m m ) w as p acked w ith S ep hadexG -50 , equ ilib ra ted w ith th e tris(h yd rox ym ethy l)m e -th y lam ine ace ta te bu ffer, and e lu ted w ith equ ilib ra tin gb u ffe r. T h e sam p le w as app lied in 5 m l, and 4 -m l fra c-tion s w ere co llec ted ; the f low ra te w as 25 m l/h . F rac tio nsw ith h ig h acid p ho sph ata se activ ity w ere p oo led , con -cen tra ted by u ltra filtra tion unde r n itrog en p ressu re(M em b ran e D ia flo P M 10; A m ico n C orp ., L ex ing ton ,M ass . 0 21 73 ), aga in p assed th ro ug h a Sephad ex G -5 0co lum n, and recon cen tra ted .As s ays o f Enzym e A c tiv ity

    A cid phosph atase ac tiv ity w as a ssay ed a t 3 7 # {176}Cit h5 .5 m m ol/lite r p -n itroph eny l pho sph ate (B oehring erM annh eim G m bH , M annh eim ) a s sub s tra te in citra teb u ffe r (50 m m o lflite r , pH 4 .8 ). A fte r an incub ation o f1 5 m m , th e re ac tio n w as s top ped by add ing 2 m l o f a0 .02 m ol/lite r so lu tio n o f N aO H , and th e am oun t o fp -n itro ph en o l lib era ted w as m easu red at 4 00 nm (7 ).T h e m ethod w as ad ap ted fo r a to ta l vo lum e of 2 20 (200 d of sub stra te in b u ffe r an d 20 o f sam p le ). T h eenzym e activ ity w as ex p re ssed in m icrom o le s o f p-ni-trop heno l sp lit o ff by 1 m l o f enzym e so lu tio n a t 3 7 #{176}Cw ith in 1 m m [m oV (m in . m l)] . T he sp ecific a ctiv ity w asex p re ssed as m o l/(m in m g o f p ro te in ).

    T he e lu tion o f p ro te in s from co lum n s w as m on ito redby m easu rin g th e abso rb an ce o f the effluen t a t 280 nm .Q u an tita tiv e p ro te in m easu rem en ts w e re pe rfo rm ed bythe m e tho d of L ow ry et a l . (8) , w ith c ry sta lline bov in eserum alb um in as s tan da rd .

    Othe r Me thod sD is c e le ct ro ph o re si s of th e en zym e w as d on e b y us in g

    7 .5% poiyac ry lam ide ge ls w ith a tris (h ydroxym e thy l)- .m ethy lam in e -g ly cin e b u ffe r, pH 8 .4 (9 , 10) . The en zy -.m a tic ally ac tiv e ban ds in the ge l w e re s ta in ed w ith a -n ap h th y l p ho sph ate co up led to F a s t G a rne t G B C ;p ro te in s w e re s ta ined w ith C oom ass ie B ril lian t B lue .D isc e lec tro ph ore sis o f th e dena tu red en zym e in th ep resence o f sod ium dodecy l su lfa te w as a s de scrib ed byW ebe r an d O sbo rn (11) .

    Molecu lar w eigh t d eterm ina tio ns by g el filtra tionw e re pe rfo rm ed on a Seph ad ex G-200 co lumn acco rd ingto A nd rew s ( 12 ) . The d im ens ion s o f th e co lum n w ere16 m mX 300m m, and tris (hyd rox ym ethy i)m eth y lam ineace ta te bu ffer (50 m m o l/li ter , pH 7 .0 ) w as u sed a t a flowra te o f 2 0 m l/h , co llec tin g frac tio ns o f 1 m l. T h e co lum nw as s tan da rd iz ed w ith 2 m g of B lue D ex tran (Pha rm a-c ia , m o l. w t. 2 00 0 000), 2 m g of hum an -y -g lob u lin(S igm a , m o l. w t. 20 5 000 ), 0 .5 m g of p ep sino gen (S igm a,m o l. w t. 4 0 4 00 ), an d 2 m g o f ch ym otryp sin og en (S igm a,m o l. w t. 23 0 00 ). T h ese s tan da rds w e re d isso lved in 2 m lo f sam ple an d th is m ix tu re w as ap p lied o n to th e co l-u m n .

    Resul tsEva lu atio n o f the L (+ )-T a rtra te -AH -S epha ro se 4BCo lumn

    The ab ility o f th e co lum n to b ind h um an p ros ta tica cid ph osp ha tase w as in itia lly te s ted in exp erim en tsw ith a co lum n hav ing a bed vo lum e of 8 m l. T he spec ificactiv ity and purifica tio n co effic ien t o f th e en zym e w erea lm ost th e sam e w hethe r w e used 1 .4 m l o r 1 5 m l o fam m onium su lfa te d ia ly sa te w ith a p ro te in con ten t o f18 0 m g/m i. T he enzym e cou ld b e e lu ted w ith a sod iumace ta te bu ffer (50 m m o l/lite r , pH 5 .0 ) from th e co lum no f A H -Seph aro se 4B w ithou t L (+ )-ta rtra te . N aC l, 1m ol/ lite r , had no e ffe ct o n elu tio n . T h e sp ec ific ac tiv ityan d pu rific a tion o f the enzym e e lu ted from the A H -S epha rose 4B co lum n w ere p oo r, ab ou t 0 .05 o f tha to b ta in ed b y u se o f the L (+ )-tartra te -A H -S epha rose 4Bco lum n . W e a lso tes ted a 10 0 m m o i/iite r p hospha tebuf fe r , pH 6 .0 and pH 7 .0 , w ith 1 m ol/ lite r N aC 1 , ase lu t ion b uffe rs . T hey g av e poor resu lts as com paredw ith the aceta te bu ffe r, w ith 10 m m ol/i ite r L (+ )-tar-tra te . T he co lum n lo s t som e o f its capac ity a fte r 18elut ions .

    Purifica tio n o f H um an P ro s ta tic A c id P hospha ta seA seco nd pro te in p eak w ith h ig h en zym e activ itycou ld be elu ted from the L (+ )-ta rtra te -A H-Sepha rose4B co lu mn w ith the ace ta te b u ffe r, w ith 1 0 m m ol/lite rL (+ )-ta rtra te (F igu re 1 ). T he fra ct ion s w ith the h ig he s ta ct iv it ies w ere p oo led fo r th e S eph ad ex G-200 ch ro -m atog raphy , w h ich gave one peak o f ac tiv e en zym e(F ig u re 2 ), o f w h ich frac tio ns o f the h ig he s t spec ifica ct iv it ies w e re po o led fo r iso e lec tric fo cu sing . T h is gavetw o enzym e p eak s. T h e h ig he r ac tiv i ty w as at isoe le ctricp o in t 4 .9 , the low e r o ne a t p o in t 5 .5 (F ig u re 3 ). T h ep ro te in con cen tra tio n w as no t de te rm in ed b ecause o f

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    2 0 0 k 4100

    -3 -.0 , >,

    1 .0

    Ia.

    Frac t ion ru,be r Frac t ion rsberig . 1 . A ffin ity c h rom atog ra ph y o f hum an p ro s ta tic acid pho s -

    pha taso on i (+) - tar tra te lin ke d to A H-S eph arose 4B ge l

    Ic 0F a>.

    tI33F ra ct io n ru nb er

    46 8 CL IN ICA L CHEM ISTRY , V o l. 2 4 , N o . 3 , 197 8

    F ig . 2 . Sephadex 0 -200 ge l filtra tion a fte r a ff in ity ch rom atog -raphy o f poo led frac tion s 25 -30the A m pho line and su cro se p resen t . W e poo led frac tionsw ith th e h ighe s t enzym e ac tiv itie s . T o rem ove A m -pho line an d su cro se , w e u sed a Seph ad ex G -50 co lum n ,w h ich gave on e enzym e p ro te in p eak .

    T h irty en zym e ba tche s h av e b een purified b y th em e th od d e sc rib ed h ere . T h e p urific a tion o f a ty p ic a len zym e p rep ara tio n is p resen ted in T ab le 1 . T he specificac tiv ity o f the en zym e p rep ara tio ns w as abo u t 27 00mo l / (min X m g); in th e b e st p repa rat ion s it w as abo u t40 18 mo l / (min X m g ), an d th e an aly tic a l recov ery o fen zym e activ ity a lw ay s ex ceed ed 15% . T he purifica tio nfac to r w as ab ou t 1300 , in the be st frac tio ns ab ou t1 9 0 0 .

    F ig . 3 . Iso e lec tric fo cus ing o f poo led frac tIon s 31 -4 1 a fte r S o-phadex 0 -200 g el f iltra tionSom e P rope rties o f th e P urifie d H um an P ro s ta ticA cid P ho sph atase

    T he pu rity o f th e enzym e w as exam in ed by d isce lec trop ho res is . In a ll en zym e p rep ara tio ns , o n S epha -dex G -50 passage on ly a sin g le b and w as seen in th eu pp er pa rt o f th e g el (F igu re 4 ,A an d B ). W hen the en -zym e w as exam ined by sod ium dodecy l su lfa te-p o ly -a cry lam id e g el e le ctrop hore s is , o n ly on e band w as seen(F igu re 4C) . The m ob il ity o f th is , w hen com pa red w iths tand ard s o f k now n m olecu la r w eig h ts , sug ge s ted am olecu la r w eig h t o f 54 000 , a s repo rted fo r th e su bun ito f th e enzym e (13) . The m ob ility o f the en zym e durin gg e l filtra tio n o n a S ephadex G -200 co lum n corre spo nd edto the m ob ility o f a g lo bu lar p ro te in o f m olecu la r w eigh t10 9 000 . T h is v alue ha s p rev iou s ly b een ob ta in ed fo r theen zym e by g el filtra tio n , w h ich is su gg es ted to b e th ed im er o f th is enzym e ( 13 -16 ) . The poo led p rep ara tio nso b ta in ed from th e affin ity ch rom a to g raph y and g elfi ltra t ion con ta in ed tw o en zym atica lly active p ro te in sw hen sub jec ted to g e l e lec trop ho res is (F ig u re 5 ). T h efa s te r m ov ing on e, c al led p ros ta tic ac id ph osp ha tase A ,w as sep ara ted from the slow er (B ). O n ly the ac idph osph atase A h as b een s tu d ied fu rthe r in th is in ve s-tiga tio n ; th e re su lts con ce rn ing th e isoenzym e B w ill bepu b lish ed sepa rate ly .Discuss ion

    A n app rox im a te ly 1 900-fo ld pu rific a tion and h ighspecific a ctiv ity o f th e m ain iso en zym e of h um an pro s-ta tic ac id p ho sph ata se , from an an im on ium su lfa te d i-a ly sa te o f p ro sta te t issu e , w as o b ta in ed in the p re sen t

    Tab le 1 . P u r IfIca tio n o f H u m an P ros t a tic A c id PhosphataseTota l To ta l Spec I f I c

    proteIn, acty , ac ty , Recovery . Purif ica t IonPurificatIon step m g m o I/m ln sm o l/(m ln X m g ) % factor

    (N H4 )2 S0 4, 3 0-7 0% sa tn . 2691 5757 2 .1 100 1AH-Sepha ro se 4B w ith L(+)-tartra te 15.9 2586 162 .6 45 77SephadexG - 200 11.1 2586 232 .9 45 11 1Is oe le ctric fo cu sin g - 1475 - 26 -Sephade xG -5 0 0 .4 1066 2665 19 1269

    Specific activity in th e b es t f ra ct io ns w as ab ou t 40 18 mo I / (m ln X m g)

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    A

    C L IN IC AL CHEM IS TRY , V o l. 24 , N o . 3 , 1978 469

    - /

    B CF ig . 4 . P o ly a cry la m id e g e l e le ct ro p ho re s is o f th e p urif ied h um anp ro sta tic a cid p ho sp ha ta seA , e nzym e sta in in g; B , p ro te in s ta in in g ; C, th e sam e en zym e In the p resenceof sod ium dodecy l sulfate

    s tu dy . P u rific a tion p rocedu res fo r p ro sta tic ac id ph os-p ha ta se h av e been d esc ribed by (e .g .) O strow sk i andRyba rs ka (17) , Paiseta l . ( 18 ) , L am e tal . ( 16 ) , and vanE tten an d S ain i (19) . L am e t a l. ( 16 ) show ed th ree acidph osp ha ta se iso en zym es in w a te r ex trac t from p ros ta te .A fte r p rec ip ita tion w ith am m onium su lfa te (55 -75%satn .) an d after do ub le D EA E -cellu lo se ch rom atog ra -ph y th ey o b ta in ed on e isoenzym e . Pu rifica tio n by oura ffin ity ch rom ato g raph y an d Sephad ex G -2 00 ge l fil -tra tio n gave ap pro x im ately th e sam e sp ec ific a ct iv ityas p rev io us ly ob ta in ed ( 16 -19 ) . H ow ev er, re co ve ry w asbe tte r by the p re sen t m e th od . T he en zym e pro te in sofar ob ta ined con sis ted o f tw o enzym es w ith d iffe ren te le ctrica l ch a rge s; th ese cou ld be sepa ra ted w ith the u seo f ge l e lec tro ph ore sis o r isoe le ctric fo cus ing . T he la tte rre su lted in the fin al pu rifica tio n o f a sing le enzym ep ro te in w hen exam in ed by po lyacry lam id e ge l e le ctro -phoresis , un de r n ond en atu rin g co nd itio ns an d in th ep resence o f so d ium dod ecy l su lfa te . Isoe lec tric fo cu s ingw as fa r b etter th an ch rom a to g raph y on C M - o rD EA E -cellu lo se a s the fina l s tep o f pu rifica tio n (1) . W ecou ld n o t com plete ly re so lve the se tw o isoenzym es byC M - o r D EA E -ce llu lo se ch rom atog rap hy (1) . G el fil-tra tio n and sod ium dod ecy l su lfa te ele c trop ho res is in -d ica ted th a t the enzym e h ad b een iso la ted in its d im e rfo rm , h av ing a m olecu la r w e ig h t o f abo u t 10 9 000 . T hem olecu la r w eigh t o f the su bun its o f the enzym e w as5 4000 , s im ila r to fig u re s repo rted p rev io us ly ( 13 ) . Thespec ific a ctiv ity an d reco ve ry o f the enzym e ob ta inedin th e p re sen t in ve st iga tio n are th e be s t y et d esc ribedin the lite ra tu re . Fu rther de ta i ls of th e isoenzym es and

    t

    c

    BA

    F ig . 5 . P o lyacry lam ide ge l e le c tropho res is o f hum an p ros ta ticac id phospha tase a fte r S ephadex 0 -200 ge l f iltra tionT he f as te r m ov in g I so en zy me (A ) w as p trif le d fL slh er . E n zy m e s t ai n in g .( + ). p ol ea t bo ttom . S ep ara tio n tim e in th is e xp e rim en t w a s 50 mmthe ir c lin ica l im portance a re u nd er inv es tig a tion . A pu reisoenzym e is im po rtan t fo r d ev elop ing h igh ly spec ifican tisera fo r rad io im m uno assay . A t le as t p a rt o f theele c tric a l cha rge o f h um an pros ta tic a cid p ho spha ta seis caused by n eu ram in ic ac id (19 , 20 ). This co u ld d e-term in e (e .g .) the location o f th e enzym e in the o rgan -e lle s o f the cell. F u rth er in ve s tig ation is need ed to showif the se isoenzym es have o th er d iffe ren ce s th an d ifferen tn um be rs o f n eg ative ele c trica l ch arg es .

    W e gratefully a ck now led ge the exp ert te chn ica l a ssista nce o f M rs .M a rja P alo n iem i. T he w o rk w as su pported in part b y gran t f rom theFinn ish Founda tion fo r C ancer R esearch .References1 . V ihk o , P ., P e lto nen , L ., an d K orhon en , L . K ., P rep ara tion ofhum an prosta tic ac id p hospha tase fo r rad io im munoassay . Sca nd . J .C lin . L ab . Invest. , Supp l . 147 , p 9 2 , A bstr. 10 5 (19 77 ).2 . G u tm an , A . B ., an d Gu tma n , E . A ., A n acid phosp ha tase oc-cu rring in the se rum of pa tien ts w ith m etastas iz ing carcinom a o f thepros tate g land . J . C lin . Invest. 17 ,4 73 (19 38 ).3. Bodansky, 0 ., A cid phospha tase . Ado . C lin . C hem . 15 , 43(1972) .4. Y am , L . T ., C lin ica l significance of the hum an ac id phosphatase.Am . J . Med . 56 ,604 (1974) .5 . C ua trecasas , P ., P ro te in purification b y a ff in it y ch romatography .D eriva tiza tions of agarose an d po lyacry lam ide beads . J . B io l. C hem .245 , 3059 (1970 ).

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    6 . H ag lund , H ., Isoe lectric fo cu sing in pH g rad ien ts-a tech n iqu efo r frac tio na tio n and charac te riza tion of am pholy tes. In M ethods o fB io ch em ica l A na lys is , 19 , D . G lick , E d ., ln te rsc ience , N ew Y ork , N .Y .,1971 , pp 1 -.63 .7. Bessey , 0 . A ., Low ry , 0 . H ., and B ro ck , M . J ., M eth od fo r the ra pidde te rm in ation of alk alin e ph osph a tase w ith five cu b ic m illim ete rs o fse rum. J . B io l. C hem . 164 ,321(1946) .8 . L ow ry , 0 . H ., R oseb rough , N . J. , F arr , A . L ., and R anda ll, R . J .,P ro te in m easurem en t w ith the Fo lin pheno l reagen t. J . B io l. C hem .193 , 265 (1 951).9. O rn ste in , L ., D isc electrophoresis- ! , b ackg round and theory . Ann .N .Y . A ca d . S ci. 121 ,321 (1964) .10 . D avis, B . J ., D isc ele ctrop horesis-il , m ethod and app lica tio nto h um an serum p ro te in s. Ann . N .Y . A cad . S c i. 121 ,404 (1964) .11 . W eb er , K ., an d O sborn , M ., M olecu la r w eigh t de te rm ina tio ns onSD S gels . In T he P ro te in s, 1 , H . N eura th and R . L . H ill, E d s. A ca -dem ic P ress , N ew Y o rk , N .Y ., 1975 , pp 1 92-20 6 .12 . And r ew s , P ., E stim a tion of m olecu lar size an d m olecu la r w eig h tso f b io log ica l com pounds by g el fi ltra tio n . In M e tho ds o f B ioch em ica lAna lys is , 18 , D . G lick , E d ., In te rsc ien ce, N ew Y ork , N .Y ., 19 70 , pp1-450 .13 . L uch te r-W asy l, E ., and O strow sk i, W ., Su bun it struc tu re o fhum an prosta tic acid p hospha ta se . B ioch im . B ioph ys . A c ta , 365 ,349(1974) .

    14 . O strow sk i, W ., F ur the r c haracteriz atio n of acid phosphomo-noes terase o f h um an pros tate . A cta B ioch im . P o l. 15 , 2 13 (1 96 8).1 5. R yb ar sk a, J. , an d O strow sk i, W ., A c id phospha tase from hum anpro sta te: C hem ica l m odifica tio n and activ ity o f the en zym e. ActaB ioch im . P o l. 21 ,37 7 (1 974 ).16 . L am , K . W ., L i, 0 ., L i, Y . C ., an d Y am , L . T ., B iochem ica l prop-e rties o f hum an pros ta tic ac id phospha tase . Clin. Chem . 19 , 48 3(1973) .17 . Ostrow sk i, W ., and Ryb arska , J., S tu d ies on human pros tatic ac idpho sph om ono es te rase . F urthe r p urifica tion and m olecu la r w eigh to f th e en zym e. B io ch im . B iophys . A c ta 105 , 1 96 ( 19 65 ).18 . P ais , V . M ., M angold , A . W ., an d M ahoney , S . A ., F ract ion ationan d pu rif icat ion of prosta tic ac id p ho sph ata se . Inv est . U ro l . 12 , 13(1974) .1 9 . v an E tten , R . L ., and Sa in i, M . S ., P rep aration of hom ogeneou sh uman prosta t ic ac id ph osp ha tase us ing concanava lin A -S epharo se4B . B ioch im . B iophys . A c ta 484 , 48 7 (1977) .20 . D z iem bor, E ., G rasyk iew icz, J ., and O strow sk i, W ., P rop ertie s o fneuram in idase -treated ac id phosphatase of prosta te . A cta B io ch im .Po l . 1 8,4 19 (19 71).21 . O s trow sk i, W ., W azy l, Z ., W eber, M ., e t al., T he ro le o f neu ram in icac id in th e he te rogene ity o f ac id p hosphom onoeste rase from thehum an pro sta te g land . B ioch im . B ioph ys. A c ta 221 , 29 (1970 ).