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Metabolism in calcified tissues: Pyridine nucleotidases of the rabbit femur
ARCHIVES
OF
BIOVIIEMISTRY
Metabolism
AND
BIOI’iIYSiC:K
in Calcified of the
From
/he Ikntal
93,
2~P-244
Tissues: Rabbit
(1961)
Pyridine
Nucleotidases
Femur’
Division, :\:c~val Mediml Research Institrcte, Naval Medical (‘enter, Ijeth.esdu, Maryy1an.d
:\‘ational
Received Octohcr 12, 19M The dest,ruct.ion of the pyridine rmchwtidex, I II’S and TI’IG, by preparatiozls frozn the epiphysial-mctaphysiai area of t,hc rahhit feznur ar~i frozn the mid-shaft marrow was investigated. The epiphysiai-mctqhysiai preparation was purified h-y differential cent,rifugation making we of the fact that t,he nucieotidasen are associated wit,h the insoluble particles. The purified enzyme was quit,e similar to the heef spicer~ enzyme in t,hat it had a pH optimum at (LiJOlit ncutraiity, wan inhibited by 10-a M nicot,inamide, and split. i)I’N at a faster rat.e t.han ‘WN. Assays on crude preparations from t.hc epi~)h~~ais-niet:~~)h~sis indicated t,hat. t,hc breakdown of i)l’N and TPN was almost. cnt.ire1.v al, the nicotizzalrzitle-riilose linkage whereas t,hc mid-shaft, marrw of t.he you11g rabbi1.s appeared !O have a pyrophosphatase component also. Studies 011adult, rabbits indic!at,ed that, the mid-shaft marrow had somewhat higher activity t,han znarrow from young animals. Thcrc was litt,ie difference in activit,y between the head of the feznur from adult raht)it,s and the cpphysial-metaphysial area of young nzlimais. INTROi)UCTION
The destruction of diphosphopyridine nucleotidc (DPS) has been demonstrat,ed to occur under t.hr influcnw of tissue preparations from a variety of plant,s and animals. Two species of reactions involving the breakdown of DPK have been described, namely, a nuctleotidc pyrophosphatase aetivit.y (1) and a splitting at, the nicot.inamiderihose linkage (2). The latter reaction has been &died extensively in brain (3), spleen (4)) and Neurospora (5). Triphosphopyridine nucleotide (TPS) is attacked also hy these preparations and, in the cast of spleen, is destroyed at 50 % of the rate of DPN. So data were available in t,h(l literature wncerning the DPKase and TPNase ac’tivity of hone preparations, although such informat,ioll would be intcrcsting in view of (a) t.hc 1 The opinions or assertions are the private Ones of the writer cozlat~rued as official or rcflect.ing Ssvy i)cpsrtment or t.hc r~av:d
coubained hcrein and are not to bc the views of i.hc service at large. 242
high lcvcls of citric! acid which arc found in calcified areas, and (11)t,hc limited respiration demonstrated by calcified tissues.
Assays of 1)I’Na.w and 1’I’Nasc The procedure used involved the determination of t,he disappearazlcc of 1)1’S or Ti’N during an incuhat~ion period of 10 min. The enzyme preparatiozz WLS diiut.ed to 2.0 ml. with 0.1 M Tris buffer, pH 7.2, rwtl after a prcincubation at. 37°C. izl a i)ulmoff met.alwiic shaker, 0.2 ml. of O.Wfi M III’N or Ti’N (SifyI:i Chemical Co., %5-100C~~pure) was added. At t.ho cntl of the reaction tizne, the flask was placed izi :i I~oiiizig water bath for 2 min. lo StOlJ the reaction, then cooied :wd t,he corlte111.s cent,ri fuged. The hcxt,i ng procedure was shctwzz to destroy t hc ezlzyme imt not 1.0 affect the 1)1’S concent rat ion. Cont.roi fiusks cant ainizlg the conpicte system were heated imznediatcly after the cocnzyme addit ion. The i)l’X or ‘TI’N concent rw ti(m of the supcrnatazlts w:w dcterznined cit,her 1)~
DPSase Tissue
prepa
ations
E(‘S
r:d~l~it I~:l,il~h~~is-n~,~t:~l~hynis
assay
TPN%C Enzymr
assay
)mlles
roerrryre
KCS splil:/rr.,~..~rnlr.~l~
w.say
Ihzyme
assay
lis.wc
Sc~~lg
Mitl-sh:tft
‘4.4
f 1.8 ( 12) IN.3 f 7.9 (12)
mtrrow
Adult r:rld)it. Hcwl of Ff?llllll NitI-shaft,
11Nunll,ersin
26.1 !)I.0
nliirrow
parent
bows
reprcscwt
f (6) f (6)
L’4.!J f 1.1 (1.2) !)5.1 f 6.4 (IS)
18.1
f
2.2
f 6.0 (!J)
I.0
14.5
1.3
54.2
f 1.3 (6) f I.2 (ti)
t 1~ qwiide reaction of Colowick, Kilpl:111, :tIltl (li0t.t i (6) or by the alcohol tlehy,lrogt:tl:~se ILWS:L> Iti) in t hc (!itse of I)I’?i,* or 1vit.h gl~~1!ose-li-~)horph:~tp ,l~~hy,lroger~:~sr! (7) in the wsc of TI’S.’
of :tss:~ys.
* An c5l.inc.tiolt coeffic.icnt of 5.1.5 W:LS oniplo~cd for dir r~2uiidt~-I )1’S :m11 cy:initlt~-‘PI’S w111pl0sw :I1 340 mp. An rstiwtioli roc4lic:iriit of (i.22 wix iiwtl for I)I’NH :~ntl TI’NH :lt 340 Inp.
villllcs
1 .i
:II’P f~llo~d
32.2
f 6.1 (9 1
i)y st:~nd:~r(l
The originul cxtrwt of the bont~ had an of 1.7 unit,s/mg. protein, whorws the final preparation had 11.5 units of acrtivity/mg. protein. This rcprctsrut,s tlbout. a sevenfold purification of DPSasc from I hc (Arude homogenate. The final wzymc! prrparation split TPS at, nbout 70 % of r:Lt.v ilt. which it split DPN. The tirnc wxuw of the rc?uot,ion under t hc condit,ions listed ws linear whiln approsimnt,ely 23 of t,hc subst,rat,c was drstroyotl. For the routinr nualysc~s, an inwbat.ion pcriod of 10 min. was uwd and an amount of cnzymc amployed so that lrss than 60 % of thr substrate wtls utilized. The :wt.ivity w:ls also found to be linear with (wzynw vonwntr:km under t:hrsr wnditions. Data wnwriiing the c+at~ of pII on thp rpiphysinl-mct,:Lphysi:il DPXaw avt,ivit y suggest.ed a pH opt,imum wound (i.5-‘7.2. 111 this rrsprvt, t.hc cnzymtl is cluito similar to thr bcctf spleen and pig brain cwzyme (2) hut, is mnrkcdly dificrcnt from the Nr~rtrosporcL wzym(’ whwh tlws not haw a ~~l~~w-~~utoptimum :md whkh shows similar wtivity from pH 3 to !I. acitvity
YOIIII~ rabbits ww~ swrificetl by :L Idow on the IIP:I~. The femurs ww rcn~ovotl r:q,idl~ :ind ,+:111,x1 of I1lllsrle. tcIldoIls , :tntl the periost.cnni. ‘lk rl)il)hq’“i:tl-,n(!t:Il,tl?‘si:tl :LWLP from t hc ho:ds of the fonmrs wrr ren~wctl :~nd cmshetl in :L marI :w. ‘I’hr I~mr fr:tgrnent H NWF est r:lrt.cd with five I itws their weight. of eol,I 0.1 dl ‘I& IdTcr, pH 7.2. The hmvy wdinwnt w:is allowi~d lo rettlc, :Lnd t hcb r~~pcrt~:tt:t~it. wis p011rc:tl off. ‘I%, CW raction of the rcsitliir W:IS relmrtotl taiw. :tritl t hc conil~itied ml r:~ct,s were cwitrifugid at io0 X (1 for 10 min. at 5Y’. ill :iIl Itit~rri:tti,m:il l’lt-2 rcwtrifiig(~. Thr wpwm:~t:mt was 1 hurl ernt rifugd at 25.000 X (1 for 15 min. with :I high-slwctl :~tt:whnl,:,lt The wsidiw was resuslm,~,lc:l in :,,I c,liiiv:dclit vol,,mr of ‘l’ris. :LII~ the rnistum w:w writ rifugerl st iO0 X g for I5 min. ‘l’hc? sulwrn:tt:tnt w:~x then Spun at 25.000 X 9 for 15 min., illltl the rwitliir from this writ rifiigat ion w:w s~~n~~c~ntl~d in Tris iuid t Ii,, :wt ivit y t cart cd. ‘I’hr lwot ,lin wmtcnt of t hr v:lriolls fr:lc*t ions WLW tl~~t.rrmim:tl 1)~ the met hod of l,owr> PI tel. (8). A iinil of wlivit?. is tlefi~ic~l as lhtt :tnio~itit which will c*lwvc? 1 pm010 I )I’X/hr. iintli~ 1 hcb w)mlitions given iindcr Jltr/sricl/.u fret/ Mrlhodx.
.i\ctivity
f (1%)
4ll.X
t hr n~rn~l~er
l!J.li
i10)
Morrosporn enzyrno is twt, sciisitivc t.0 iiicotiasmidc up to cotlccnt,rnt.ioils as high as 3.3 X IO-’ ill. The mhbi t. femur prcparat,ioli wil:: srnsit,iw to ni~ot,in:~midc. ICmployillg the assay syst,cm drscribcd under Matwials and .Ilr~fhorls, wrious ~oiicaiitrut,ioi~s of iiiwtiilumidr wcrc inwrporatcd in t.hc flasks :uld ihr DI’Saw wti\~it.y was d&rmiiird using a IO-min. inwl)ut.ioti at. 37°C. Ii. WM forn~~ that at IO-:’ 31 tlic~otiil:lmitlr, t.hr rnaytnr was inhil)it,td hy ~3 %. (lomplcrtc inhihitioii occl1rrcd at IO-” II iii~ot.iil:linidc Ilid pr:ictic*:llly ii0 iiihihitioii at 1W4 Al.
linktqc?, sinrc t,hc destruction of the cofwtors as mcusurcd by t,hc qunidra rr:wt,ion was :~pproximst.el.y the same ac: hy t.ho calxymc assays. The mid-shaft marrow wnt,a&d considerahly mow uc:t.ivity than t hc hmd of the frmur wit,h lwth DPS and ‘I‘PN bring dcgrudvd I)y t,hc marrow at mow th:m twiw, i,hc rate of the cpiphysial-mctaphysisl prcparations. Mid-shaft murow also appears t,o cont,ain pyrophosphat:w wtivit,y as wrll as splitjtillg niwt,inumidc from the c*ocnaymes. ITsing thv results from the vy:widc> rcwf,ion, the cpiphysial-mct:lphysial pwparat.iou split: ‘LTS at. i5 % of t.hcxratv of I)PS splitting. ‘I’hc mid-shaft marrow drgwdrd TPS at. 020%:of the t’atc of I)I’S. Prrlimimwy studies ww pwformcd to Similar studirs wrc prrformcvl using dctcrminc the rxtcnt. t,o which t.hc cnxymc mutro’c rahhits which wrc over 2 :ycars old wuld lw cstrwtcd from t hc 1imrw. I’I’c~LLI’:L- :wd no longer showd z111 cbpiphys;lulplate. tioii of cwde hoii~ogc~liat,c!s thin t,h ftwiiir 1S1up~~ pwpar:&ons from t.hr hwd of the mid-shaft m:wrow prwcnt,rd no prohlcms frmur and mid-shaft m:~~ow wcrc tmdc as except for the removal of fnl,t y mat,wiul at)o\:e and wstlyrd by t hr cyuuidc rcw1 ioll which ~1s ~~Ic:~r~dfrom t.hcasw&wc of the (‘l’ahlc 1). Thc~ I,rc!akdo\vn of IMPS t,y the prcpwations aft.er homogrlliz:lbioli 1vit.h :1 propartitions from thv hcucl of the fcwur wts Ten Ibocck 1issrir griiidrr. To ~ssiiro IcaLpon- upprosimatc4y thv sum: :LSfor the similar nhly wmplrtc c~xtrwt.ion of t ho owymc from arca in t hc young mbhit. A slightly rc~ducctl the how prrpuratiow, swial rst.ractions of tlcgrutlation of ‘I’PN was ohsrrvcd. ‘I’htr midlww c&hipswrc cmploycd, a11dthe wmhincd shaft. marrow from the old :tninml appcarcd cxtrwts wrc ussnycd for uotivit y. hpproxito tlrs;t.roy 1,ot.h 1)1’S and TPS at a fustcr m:ltcly 60 7%of the tot~ulcxt~rwt~ahlc acbivity r& th:m m:wrow from thr yormg :ulimnl. was ohtnincd ill thv first, treat mcnt with five times the tissw’s weight of Tris hrifl’cr, 20 % in the wwnd Cxtrwfion, IO % in t,hcat.hirtl cxtmrtioii, aiid only minor act i\-it,& iii sill+ 1. seclwatj cxtr:wtions. ‘l’hc routiac 1~ of t.hn!c 2. snwcssivc~rxt ructions was adoptrd. l’hc wtivitits of t.hcnr c~l~lr prcpamt,ions tit’c show1 ill ‘l’ahlc 1. Assays for 1)1’S alltl TI’K 3. hrcakdowu wrc ~nadr by two metjhods. 1t. has IMYY~shown that the cyanide rcaation 4. will dvtcct the rupt,ure of the niwt.ianmidcrihow linkage but. not the ruptwo of the 5. pyrophosphat,c link:qc of t,hr co(wzymcs. Ii. ‘rhc :&who1 dchydrogcuuso and gl~ww-(iphosphate dchydrogcnasc assaysarc spwifio 7. for t.hr intact co~~n~ymwSOthat split.ting at ally site. renders thr cwcnzymr inactive. The splitting of I>PS and TPS hy prcparatioils from the rpiphysinl-met.aphysial 8. arc:, of young rnhhit. fcmws was shown to lw nlmost rilt,ircly at the ilic,otiilamidr-rit)o~e
OF
BIOVIIEMISTRY
Metabolism
AND
BIOI’iIYSiC:K
in Calcified of the
From
/he Ikntal
93,
2~P-244
Tissues: Rabbit
(1961)
Pyridine
Nucleotidases
Femur’
Division, :\:c~val Mediml Research Institrcte, Naval Medical (‘enter, Ijeth.esdu, Maryy1an.d
:\‘ational
Received Octohcr 12, 19M The dest,ruct.ion of the pyridine rmchwtidex, I II’S and TI’IG, by preparatiozls frozn the epiphysial-mctaphysiai area of t,hc rahhit feznur ar~i frozn the mid-shaft marrow was investigated. The epiphysiai-mctqhysiai preparation was purified h-y differential cent,rifugation making we of the fact that t,he nucieotidasen are associated wit,h the insoluble particles. The purified enzyme was quit,e similar to the heef spicer~ enzyme in t,hat it had a pH optimum at (LiJOlit ncutraiity, wan inhibited by 10-a M nicot,inamide, and split. i)I’N at a faster rat.e t.han ‘WN. Assays on crude preparations from t.hc epi~)h~~ais-niet:~~)h~sis indicated t,hat. t,hc breakdown of i)l’N and TPN was almost. cnt.ire1.v al, the nicotizzalrzitle-riilose linkage whereas t,hc mid-shaft, marrw of t.he you11g rabbi1.s appeared !O have a pyrophosphatase component also. Studies 011adult, rabbits indic!at,ed that, the mid-shaft marrow had somewhat higher activity t,han znarrow from young animals. Thcrc was litt,ie difference in activit,y between the head of the feznur from adult raht)it,s and the cpphysial-metaphysial area of young nzlimais. INTROi)UCTION
The destruction of diphosphopyridine nucleotidc (DPS) has been demonstrat,ed to occur under t.hr influcnw of tissue preparations from a variety of plant,s and animals. Two species of reactions involving the breakdown of DPK have been described, namely, a nuctleotidc pyrophosphatase aetivit.y (1) and a splitting at, the nicot.inamiderihose linkage (2). The latter reaction has been &died extensively in brain (3), spleen (4)) and Neurospora (5). Triphosphopyridine nucleotide (TPS) is attacked also hy these preparations and, in the cast of spleen, is destroyed at 50 % of the rate of DPN. So data were available in t,h(l literature wncerning the DPKase and TPNase ac’tivity of hone preparations, although such informat,ioll would be intcrcsting in view of (a) t.hc 1 The opinions or assertions are the private Ones of the writer cozlat~rued as official or rcflect.ing Ssvy i)cpsrtment or t.hc r~av:d
coubained hcrein and are not to bc the views of i.hc service at large. 242
high lcvcls of citric! acid which arc found in calcified areas, and (11)t,hc limited respiration demonstrated by calcified tissues.
Assays of 1)I’Na.w and 1’I’Nasc The procedure used involved the determination of t,he disappearazlcc of 1)1’S or Ti’N during an incuhat~ion period of 10 min. The enzyme preparatiozz WLS diiut.ed to 2.0 ml. with 0.1 M Tris buffer, pH 7.2, rwtl after a prcincubation at. 37°C. izl a i)ulmoff met.alwiic shaker, 0.2 ml. of O.Wfi M III’N or Ti’N (SifyI:i Chemical Co., %5-100C~~pure) was added. At t.ho cntl of the reaction tizne, the flask was placed izi :i I~oiiizig water bath for 2 min. lo StOlJ the reaction, then cooied :wd t,he corlte111.s cent,ri fuged. The hcxt,i ng procedure was shctwzz to destroy t hc ezlzyme imt not 1.0 affect the 1)1’S concent rat ion. Cont.roi fiusks cant ainizlg the conpicte system were heated imznediatcly after the cocnzyme addit ion. The i)l’X or ‘TI’N concent rw ti(m of the supcrnatazlts w:w dcterznined cit,her 1)~
DPSase Tissue
prepa
ations
E(‘S
r:d~l~it I~:l,il~h~~is-n~,~t:~l~hynis
assay
TPN%C Enzymr
assay
)mlles
roerrryre
KCS splil:/rr.,~..~rnlr.~l~
w.say
Ihzyme
assay
lis.wc
Sc~~lg
Mitl-sh:tft
‘4.4
f 1.8 ( 12) IN.3 f 7.9 (12)
mtrrow
Adult r:rld)it. Hcwl of Ff?llllll NitI-shaft,
11Nunll,ersin
26.1 !)I.0
nliirrow
parent
bows
reprcscwt
f (6) f (6)
L’4.!J f 1.1 (1.2) !)5.1 f 6.4 (IS)
18.1
f
2.2
f 6.0 (!J)
I.0
14.5
1.3
54.2
f 1.3 (6) f I.2 (ti)
t 1~ qwiide reaction of Colowick, Kilpl:111, :tIltl (li0t.t i (6) or by the alcohol tlehy,lrogt:tl:~se ILWS:L> Iti) in t hc (!itse of I)I’?i,* or 1vit.h gl~~1!ose-li-~)horph:~tp ,l~~hy,lroger~:~sr! (7) in the wsc of TI’S.’
of :tss:~ys.
* An c5l.inc.tiolt coeffic.icnt of 5.1.5 W:LS oniplo~cd for dir r~2uiidt~-I )1’S :m11 cy:initlt~-‘PI’S w111pl0sw :I1 340 mp. An rstiwtioli roc4lic:iriit of (i.22 wix iiwtl for I)I’NH :~ntl TI’NH :lt 340 Inp.
villllcs
1 .i
:II’P f~llo~d
32.2
f 6.1 (9 1
i)y st:~nd:~r(l
The originul cxtrwt of the bont~ had an of 1.7 unit,s/mg. protein, whorws the final preparation had 11.5 units of acrtivity/mg. protein. This rcprctsrut,s tlbout. a sevenfold purification of DPSasc from I hc (Arude homogenate. The final wzymc! prrparation split TPS at, nbout 70 % of r:Lt.v ilt. which it split DPN. The tirnc wxuw of the rc?uot,ion under t hc condit,ions listed ws linear whiln approsimnt,ely 23 of t,hc subst,rat,c was drstroyotl. For the routinr nualysc~s, an inwbat.ion pcriod of 10 min. was uwd and an amount of cnzymc amployed so that lrss than 60 % of thr substrate wtls utilized. The :wt.ivity w:ls also found to be linear with (wzynw vonwntr:km under t:hrsr wnditions. Data wnwriiing the c+at~ of pII on thp rpiphysinl-mct,:Lphysi:il DPXaw avt,ivit y suggest.ed a pH opt,imum wound (i.5-‘7.2. 111 this rrsprvt, t.hc cnzymtl is cluito similar to thr bcctf spleen and pig brain cwzyme (2) hut, is mnrkcdly dificrcnt from the Nr~rtrosporcL wzym(’ whwh tlws not haw a ~~l~~w-~~utoptimum :md whkh shows similar wtivity from pH 3 to !I. acitvity
YOIIII~ rabbits ww~ swrificetl by :L Idow on the IIP:I~. The femurs ww rcn~ovotl r:q,idl~ :ind ,+:111,x1 of I1lllsrle. tcIldoIls , :tntl the periost.cnni. ‘lk rl)il)hq’“i:tl-,n(!t:Il,tl?‘si:tl :LWLP from t hc ho:ds of the fonmrs wrr ren~wctl :~nd cmshetl in :L marI :w. ‘I’hr I~mr fr:tgrnent H NWF est r:lrt.cd with five I itws their weight. of eol,I 0.1 dl ‘I& IdTcr, pH 7.2. The hmvy wdinwnt w:is allowi~d lo rettlc, :Lnd t hcb r~~pcrt~:tt:t~it. wis p011rc:tl off. ‘I%, CW raction of the rcsitliir W:IS relmrtotl taiw. :tritl t hc conil~itied ml r:~ct,s were cwitrifugid at io0 X (1 for 10 min. at 5Y’. ill :iIl Itit~rri:tti,m:il l’lt-2 rcwtrifiig(~. Thr wpwm:~t:mt was 1 hurl ernt rifugd at 25.000 X (1 for 15 min. with :I high-slwctl :~tt:whnl,:,lt The wsidiw was resuslm,~,lc:l in :,,I c,liiiv:dclit vol,,mr of ‘l’ris. :LII~ the rnistum w:w writ rifugerl st iO0 X g for I5 min. ‘l’hc? sulwrn:tt:tnt w:~x then Spun at 25.000 X 9 for 15 min., illltl the rwitliir from this writ rifiigat ion w:w s~~n~~c~ntl~d in Tris iuid t Ii,, :wt ivit y t cart cd. ‘I’hr lwot ,lin wmtcnt of t hr v:lriolls fr:lc*t ions WLW tl~~t.rrmim:tl 1)~ the met hod of l,owr> PI tel. (8). A iinil of wlivit?. is tlefi~ic~l as lhtt :tnio~itit which will c*lwvc? 1 pm010 I )I’X/hr. iintli~ 1 hcb w)mlitions given iindcr Jltr/sricl/.u fret/ Mrlhodx.
.i\ctivity
f (1%)
4ll.X
t hr n~rn~l~er
l!J.li
i10)
Morrosporn enzyrno is twt, sciisitivc t.0 iiicotiasmidc up to cotlccnt,rnt.ioils as high as 3.3 X IO-’ ill. The mhbi t. femur prcparat,ioli wil:: srnsit,iw to ni~ot,in:~midc. ICmployillg the assay syst,cm drscribcd under Matwials and .Ilr~fhorls, wrious ~oiicaiitrut,ioi~s of iiiwtiilumidr wcrc inwrporatcd in t.hc flasks :uld ihr DI’Saw wti\~it.y was d&rmiiird using a IO-min. inwl)ut.ioti at. 37°C. Ii. WM forn~~ that at IO-:’ 31 tlic~otiil:lmitlr, t.hr rnaytnr was inhil)it,td hy ~3 %. (lomplcrtc inhihitioii occl1rrcd at IO-” II iii~ot.iil:linidc Ilid pr:ictic*:llly ii0 iiihihitioii at 1W4 Al.
linktqc?, sinrc t,hc destruction of the cofwtors as mcusurcd by t,hc qunidra rr:wt,ion was :~pproximst.el.y the same ac: hy t.ho calxymc assays. The mid-shaft marrow wnt,a&d considerahly mow uc:t.ivity than t hc hmd of the frmur wit,h lwth DPS and ‘I‘PN bring dcgrudvd I)y t,hc marrow at mow th:m twiw, i,hc rate of the cpiphysial-mctaphysisl prcparations. Mid-shaft murow also appears t,o cont,ain pyrophosphat:w wtivit,y as wrll as splitjtillg niwt,inumidc from the c*ocnaymes. ITsing thv results from the vy:widc> rcwf,ion, the cpiphysial-mct:lphysial pwparat.iou split: ‘LTS at. i5 % of t.hcxratv of I)PS splitting. ‘I’hc mid-shaft marrow drgwdrd TPS at. 020%:of the t’atc of I)I’S. Prrlimimwy studies ww pwformcd to Similar studirs wrc prrformcvl using dctcrminc the rxtcnt. t,o which t.hc cnxymc mutro’c rahhits which wrc over 2 :ycars old wuld lw cstrwtcd from t hc 1imrw. I’I’c~LLI’:L- :wd no longer showd z111 cbpiphys;lulplate. tioii of cwde hoii~ogc~liat,c!s thin t,h ftwiiir 1S1up~~ pwpar:&ons from t.hr hwd of the mid-shaft m:wrow prwcnt,rd no prohlcms frmur and mid-shaft m:~~ow wcrc tmdc as except for the removal of fnl,t y mat,wiul at)o\:e and wstlyrd by t hr cyuuidc rcw1 ioll which ~1s ~~Ic:~r~dfrom t.hcasw&wc of the (‘l’ahlc 1). Thc~ I,rc!akdo\vn of IMPS t,y the prcpwations aft.er homogrlliz:lbioli 1vit.h :1 propartitions from thv hcucl of the fcwur wts Ten Ibocck 1issrir griiidrr. To ~ssiiro IcaLpon- upprosimatc4y thv sum: :LSfor the similar nhly wmplrtc c~xtrwt.ion of t ho owymc from arca in t hc young mbhit. A slightly rc~ducctl the how prrpuratiow, swial rst.ractions of tlcgrutlation of ‘I’PN was ohsrrvcd. ‘I’htr midlww c&hipswrc cmploycd, a11dthe wmhincd shaft. marrow from the old :tninml appcarcd cxtrwts wrc ussnycd for uotivit y. hpproxito tlrs;t.roy 1,ot.h 1)1’S and TPS at a fustcr m:ltcly 60 7%of the tot~ulcxt~rwt~ahlc acbivity r& th:m m:wrow from thr yormg :ulimnl. was ohtnincd ill thv first, treat mcnt with five times the tissw’s weight of Tris hrifl’cr, 20 % in the wwnd Cxtrwfion, IO % in t,hcat.hirtl cxtmrtioii, aiid only minor act i\-it,& iii sill+ 1. seclwatj cxtr:wtions. ‘l’hc routiac 1~ of t.hn!c 2. snwcssivc~rxt ructions was adoptrd. l’hc wtivitits of t.hcnr c~l~lr prcpamt,ions tit’c show1 ill ‘l’ahlc 1. Assays for 1)1’S alltl TI’K 3. hrcakdowu wrc ~nadr by two metjhods. 1t. has IMYY~shown that the cyanide rcaation 4. will dvtcct the rupt,ure of the niwt.ianmidcrihow linkage but. not the ruptwo of the 5. pyrophosphat,c link:qc of t,hr co(wzymcs. Ii. ‘rhc :&who1 dchydrogcuuso and gl~ww-(iphosphate dchydrogcnasc assaysarc spwifio 7. for t.hr intact co~~n~ymwSOthat split.ting at ally site. renders thr cwcnzymr inactive. The splitting of I>PS and TPS hy prcparatioils from the rpiphysinl-met.aphysial 8. arc:, of young rnhhit. fcmws was shown to lw nlmost rilt,ircly at the ilic,otiilamidr-rit)o~e