- Email: [email protected]
A sensitive micro assay for inorganic pyrophosphatase
.tNALYTICAL
BIOCHEMISTRY
34, 66-70
(1070)
Inorganic pyrophosphatase ~1~yropl~oel~l~att1~l~o~l~l~ohydrolase, EC 3.6.1.1) is generally determinetl by the appearance of phospl1at.e (Pi), which is detect.ed by modifications of the calorimetric method of Fiske and SubbaRom (I ). Thus, .Jow~ (21 reports a method suitable for the measurement of the hydrolysis of 0.01 to 0.12 pmole pyrophosphate (PPi) in 15 min at 37” in an incubation mixture of 0.3 rd. The prwcnw of exogenous Pi from buffer salts interffbres with this calorimetric assay. This communication presents a micro assay which can dekct. the enzymic hytlrolysis of 0.03-10 in~~i~iolcs of ““P-~~yrol~lios[~lintc in aliquots of 5 ,pl. Higher concentrations than 10 m31 hart not II~YJII tested. Thus, in acldition t,o its acnsitivit8y, this method enal)lw one to study the efl’ect of an excess of the products on the enzymic reaction. The present method is especially helpful for dctccting enzymic activity in fractions following chromat~ography in which phosl~hatc hufifm are cmploycd. MATEHL11,S
AND
METHOI)S
“‘P-Sodium pyrophoq~h:~te was obt~aineclfrom n’ew England Nuclear Corp., Boston, Massachuaetta, an11diluted in a 4ock solution of 0.0231 sodium pyrophosphate to a specific activity of 0.2 mC’i,/mmolc. T\‘o “‘Pi could be deteckd in this solution, employing the thin-layer t,c~chniquc described. Plastic sheets of 20 X 20 cm”, coat4 with polyethylencimine ccllulow wcrc made according to the method of Ranclerath and Rantlerath (3). Commercial plates distributed by Brinkmann Instruments, Inc., 1l’cstbury, New York (IIn’-polygrwm Ccl 300 PEI for thin-layw chroby ~fa~~lwr~~y,Nag<4 a1111 (‘o., JX~WII, Wc’st matography, 111:11111f;lCf,lll.(lfl (ki*mniiy) or 1)~ .I. P. U:kcr (‘h(wivaI (‘o., l?l~illi~~~l~u~~g, iYf9 .If~tWy, (‘l’nl)lv I ). A protein ftxrtioli clf~l+vcvl from arc also satisfartorj 1 Publication Yo. 1364 of thr Cancer (‘onxnission of Harvard Univwity. I’ Prewnt ~ddw~s : T’m’t Hoff I~hor:~tory, Stc,rrrnhos 19, The= Ncthcrlandr. ,’ .4~ltlrc5s lo whil*ll rc~l)rint. tvcllwsts shoul~l hc- wnl. 66
1
The cotttpout~tls were elut8cd by asccnclitig cliroti~atography in 1 iI1 LiCl (3). The plat’es were dried in a stream of cold air, and were examined in short-wave ultraviolet light where AMP was used as a marker. When Pi was used as a marker, the plates were treated with a HCl/HCl@, atnmoniuill molybdate spray according to Hanes and Isherwood (4) with developtnent of the color in ultraviolet light (5). The arcas containing the markers were cut, from t)he PEI-ccllulosc plat,or. They wcrc usually rectangular pieces about, 1.5 X 2.0 cm containing the AJIP or Pi in tltc center. They were mouttt,cd in l~lattchcts wit11 glue and counted using a Nuclear-Chicago cttd-window gas-flow counter. Aliquots of solutions of PP were plat’ed on sitnilar pieces of PEI plate attd counted in the samt’ manner in order t,o get tlte specific activity of the original PP solution. RESULTS
AND
DISCUSSIOK
The tnethod makes use of the separation of PP, and Pi by means of thin-layer chromatography, using plastic shccbts coated with polycthyleneimine cellulose (3). Unlabeled Pi, unless otherwise present, is added as a carrier and its location on the tlcvelopcd plates is dctermincd as ment’iortcd under “Materials and Methods.” Alternatively, advantage may be taken of the fact that,, in the system ctnployed, Y-AMP and Pi have approxitna,tely the same K, value. Thus, when AMP is added as a marker, the position of Pi can bc detected by examining the layer in short wave ultraviolet light. The Pi area is subsequently cut) out, and its radioactivity measured. In order to stop the cnzymic reaction, cthylenediaminetetraacetate (EDTA) is added in excess of the divalent cation concentration in the react,ion mixture. Alkaline pyrophosphat.ases from all sources studied have bectt shown t.o be dependent on the prescsttccb of divaltttt cations, particularly oti Mg++ or in some (2, 6) but not, all (,7) cases on CO’+, Mn++, or Ztt++. Acid pyrophosphatascs, on the other hand, do not require the prescncc of cations, as discussecl by Naganna et al. (7). In this case, heat trcatmcttt or t.he acldition of :i suitable inhibitor such as Cu++ is suggested for the termination of the enzymic rcxtion. With the chromatographic systetn employed PPi remains t~oar the origin and is well separated front Pi. It orerlal)s part’ially with ATP when the latter is also added as a marker (Table 1). On plates maclc in our laboratory (3), AMP and Pi travel wit11 approximately the same rate of migration when elutetl with 1 111I,iCl. On some commercial plates, however, ALIP and Pi were slightly separated (Table 1, C). In this context, it should bc pointed out that the pH of the PEI solution used in and slight tliffcrcttcc~~ iu K, preparing the PEI cellulose is important, values can occur from batch to batch of plntvs.
INORGBNIC
I?{ V:dr~es of AAlP, Polyrthyleneimine
AI)I’,
PTROPHOPI’HATASE
69
ASSAY
‘I’ABLK 1 ATP, I’;, :nrd Pl’i (‘elluloar, Kmploying
,bn I )ifTrrent, Thin Layers 1 !I[ LiC:l for Eh~tion
of
A, plate prepared in this laboratory 13 ). B, commercial plate distrihllled by Brinkmann isee text). C, commercial plate distrihrlted by Baker. 5 ~1 of a misture containing 5 mpmoles of each nucleotide and 15 mpmoles each of Pi and PP, were spotted at the origin of each plate. The sample was elated with 1 ~11 I,iC’l. The nucleutides were detected with short-xave ultraviolet light, and Pi 2tnd PPi were visrlalizrd k)y qxying (4 :tnd developing r.5) tllcx pl:+te- 31: clrsA)ed ill the text.
In this csperimental prowdure tlw colitlitions for the e11zyrnic reactions arc arl)itrary. The mctl1d (‘~II he :1lq)livcl nhc11 tl1cs I)!l1,ol)bo~l)llatnre rewtiu11 ih stuclicbtl at, tliffcw11t. l)H \-:tluw;. hul)strnte coiicc~litrntions, teInpcsr:1turcs, or c~o11coniit:111tly nit11 otlior (~tizynii(~ rcxctions it1 wliicli PI?, is :L pro(luctt. 111\rstigntio11 of miscvl ci~zyllic~ sptcms iilay tw pvrti11(~11t to tlics ~~i~ch~~ilic~cl (2. 8) l)iologic*:il sipiific:11lw~ of i11org:mic ~~~~0~~110~~~11:1ta~~~ i11 such iinl)ort:11it rt~:ic*tioii s :w tl1caI’(~l!-111c’t,iz:lt,io11of 1)9A a11c1 RKX ~1~1 in :rmi110 aritl nc?i\.:1tioil.
REFERENCES 1. FISKE. C. H.. .+su SVBR.ARUW, 2. JOSSFC, J., J. Biol. C/cm. 241, 3.
~I.~NI)I:IL\TI~,
1. H\xs. ,5.
I~ANI)KRSKI.
I<.,
.ANN
C. S., AXI) K. S.,
~NIXRATII,
ISHEl~\VOOl). AXI)
AX~~LROD,.
Y.. J. n;oc. 1938 (1966). E..
./.
C”~I~~WI. 66, 375 (1925). (‘/iro,rtrrloq.
F. A.. .\‘,rl/itx B.. J. Rio/.
164, (‘hem.
22, 110 (1966). 1107 (1049). 193, 40.5 (1951).
70
BLOEMERS,
STEPHENSON,
AND
ZAMECNIK
M., ASLI ROBBINS, I’. W., in “Ttw Enzyme-s” (R. II. Boyr,r, H. K. Myrblck, cds.), Vol. 5, 11. 169. Awdrmic Press, Sew York, 1961. 7. NAGASNA, B.. RAMAN, A., VENCGOPAL. B., A~UD SHIHATHI, C. E., Biochenz. 215 (1955). 8. KOHNBERG, A., in “Horizons in Biochemistry” (M. Knsha and B. Pullman, p. 251. Academic Press, SW York. 1962.
6. KUNITZ,
Lardy,
and
J. 60, cds.),
BIOCHEMISTRY
34, 66-70
(1070)
Inorganic pyrophosphatase ~1~yropl~oel~l~att1~l~o~l~l~ohydrolase, EC 3.6.1.1) is generally determinetl by the appearance of phospl1at.e (Pi), which is detect.ed by modifications of the calorimetric method of Fiske and SubbaRom (I ). Thus, .Jow~ (21 reports a method suitable for the measurement of the hydrolysis of 0.01 to 0.12 pmole pyrophosphate (PPi) in 15 min at 37” in an incubation mixture of 0.3 rd. The prwcnw of exogenous Pi from buffer salts interffbres with this calorimetric assay. This communication presents a micro assay which can dekct. the enzymic hytlrolysis of 0.03-10 in~~i~iolcs of ““P-~~yrol~lios[~lintc in aliquots of 5 ,pl. Higher concentrations than 10 m31 hart not II~YJII tested. Thus, in acldition t,o its acnsitivit8y, this method enal)lw one to study the efl’ect of an excess of the products on the enzymic reaction. The present method is especially helpful for dctccting enzymic activity in fractions following chromat~ography in which phosl~hatc hufifm are cmploycd. MATEHL11,S
AND
METHOI)S
“‘P-Sodium pyrophoq~h:~te was obt~aineclfrom n’ew England Nuclear Corp., Boston, Massachuaetta, an11diluted in a 4ock solution of 0.0231 sodium pyrophosphate to a specific activity of 0.2 mC’i,/mmolc. T\‘o “‘Pi could be deteckd in this solution, employing the thin-layer t,c~chniquc described. Plastic sheets of 20 X 20 cm”, coat4 with polyethylencimine ccllulow wcrc made according to the method of Ranclerath and Rantlerath (3). Commercial plates distributed by Brinkmann Instruments, Inc., 1l’cstbury, New York (IIn’-polygrwm Ccl 300 PEI for thin-layw chroby ~fa~~lwr~~y,Nag<4 a1111 (‘o., JX~WII, Wc’st matography, 111:11111f;lCf,lll.(lfl (ki*mniiy) or 1)~ .I. P. U:kcr (‘h(wivaI (‘o., l?l~illi~~~l~u~~g, iYf9 .If~tWy, (‘l’nl)lv I ). A protein ftxrtioli clf~l+vcvl from arc also satisfartorj 1 Publication Yo. 1364 of thr Cancer (‘onxnission of Harvard Univwity. I’ Prewnt ~ddw~s : T’m’t Hoff I~hor:~tory, Stc,rrrnhos 19, The= Ncthcrlandr. ,’ .4~ltlrc5s lo whil*ll rc~l)rint. tvcllwsts shoul~l hc- wnl. 66
1
The cotttpout~tls were elut8cd by asccnclitig cliroti~atography in 1 iI1 LiCl (3). The plat’es were dried in a stream of cold air, and were examined in short-wave ultraviolet light where AMP was used as a marker. When Pi was used as a marker, the plates were treated with a HCl/HCl@, atnmoniuill molybdate spray according to Hanes and Isherwood (4) with developtnent of the color in ultraviolet light (5). The arcas containing the markers were cut, from t)he PEI-ccllulosc plat,or. They wcrc usually rectangular pieces about, 1.5 X 2.0 cm containing the AJIP or Pi in tltc center. They were mouttt,cd in l~lattchcts wit11 glue and counted using a Nuclear-Chicago cttd-window gas-flow counter. Aliquots of solutions of PP were plat’ed on sitnilar pieces of PEI plate attd counted in the samt’ manner in order t,o get tlte specific activity of the original PP solution. RESULTS
AND
DISCUSSIOK
The tnethod makes use of the separation of PP, and Pi by means of thin-layer chromatography, using plastic shccbts coated with polycthyleneimine cellulose (3). Unlabeled Pi, unless otherwise present, is added as a carrier and its location on the tlcvelopcd plates is dctermincd as ment’iortcd under “Materials and Methods.” Alternatively, advantage may be taken of the fact that,, in the system ctnployed, Y-AMP and Pi have approxitna,tely the same K, value. Thus, when AMP is added as a marker, the position of Pi can bc detected by examining the layer in short wave ultraviolet light. The Pi area is subsequently cut) out, and its radioactivity measured. In order to stop the cnzymic reaction, cthylenediaminetetraacetate (EDTA) is added in excess of the divalent cation concentration in the react,ion mixture. Alkaline pyrophosphat.ases from all sources studied have bectt shown t.o be dependent on the prescsttccb of divaltttt cations, particularly oti Mg++ or in some (2, 6) but not, all (,7) cases on CO’+, Mn++, or Ztt++. Acid pyrophosphatascs, on the other hand, do not require the prescncc of cations, as discussecl by Naganna et al. (7). In this case, heat trcatmcttt or t.he acldition of :i suitable inhibitor such as Cu++ is suggested for the termination of the enzymic rcxtion. With the chromatographic systetn employed PPi remains t~oar the origin and is well separated front Pi. It orerlal)s part’ially with ATP when the latter is also added as a marker (Table 1). On plates maclc in our laboratory (3), AMP and Pi travel wit11 approximately the same rate of migration when elutetl with 1 111I,iCl. On some commercial plates, however, ALIP and Pi were slightly separated (Table 1, C). In this context, it should bc pointed out that the pH of the PEI solution used in and slight tliffcrcttcc~~ iu K, preparing the PEI cellulose is important, values can occur from batch to batch of plntvs.
INORGBNIC
I?{ V:dr~es of AAlP, Polyrthyleneimine
AI)I’,
PTROPHOPI’HATASE
69
ASSAY
‘I’ABLK 1 ATP, I’;, :nrd Pl’i (‘elluloar, Kmploying
,bn I )ifTrrent, Thin Layers 1 !I[ LiC:l for Eh~tion
of
A, plate prepared in this laboratory 13 ). B, commercial plate distrihllled by Brinkmann isee text). C, commercial plate distrihrlted by Baker. 5 ~1 of a misture containing 5 mpmoles of each nucleotide and 15 mpmoles each of Pi and PP, were spotted at the origin of each plate. The sample was elated with 1 ~11 I,iC’l. The nucleutides were detected with short-xave ultraviolet light, and Pi 2tnd PPi were visrlalizrd k)y qxying (4 :tnd developing r.5) tllcx pl:+te- 31: clrsA)ed ill the text.
In this csperimental prowdure tlw colitlitions for the e11zyrnic reactions arc arl)itrary. The mctl1d (‘~II he :1lq)livcl nhc11 tl1cs I)!l1,ol)bo~l)llatnre rewtiu11 ih stuclicbtl at, tliffcw11t. l)H \-:tluw;. hul)strnte coiicc~litrntions, teInpcsr:1turcs, or c~o11coniit:111tly nit11 otlior (~tizynii(~ rcxctions it1 wliicli PI?, is :L pro(luctt. 111\rstigntio11 of miscvl ci~zyllic~ sptcms iilay tw pvrti11(~11t to tlics ~~i~ch~~ilic~cl (2. 8) l)iologic*:il sipiific:11lw~ of i11org:mic ~~~~0~~110~~~11:1ta~~~ i11 such iinl)ort:11it rt~:ic*tioii s :w tl1caI’(~l!-111c’t,iz:lt,io11of 1)9A a11c1 RKX ~1~1 in :rmi110 aritl nc?i\.:1tioil.
REFERENCES 1. FISKE. C. H.. .+su SVBR.ARUW, 2. JOSSFC, J., J. Biol. C/cm. 241, 3.
~I.~NI)I:IL\TI~,
1. H\xs. ,5.
I~ANI)KRSKI.
I<.,
.ANN
C. S., AXI) K. S.,
~NIXRATII,
ISHEl~\VOOl). AXI)
AX~~LROD,.
Y.. J. n;oc. 1938 (1966). E..
./.
C”~I~~WI. 66, 375 (1925). (‘/iro,rtrrloq.
F. A.. .\‘,rl/itx B.. J. Rio/.
164, (‘hem.
22, 110 (1966). 1107 (1049). 193, 40.5 (1951).
70
BLOEMERS,
STEPHENSON,
AND
ZAMECNIK
M., ASLI ROBBINS, I’. W., in “Ttw Enzyme-s” (R. II. Boyr,r, H. K. Myrblck, cds.), Vol. 5, 11. 169. Awdrmic Press, Sew York, 1961. 7. NAGASNA, B.. RAMAN, A., VENCGOPAL. B., A~UD SHIHATHI, C. E., Biochenz. 215 (1955). 8. KOHNBERG, A., in “Horizons in Biochemistry” (M. Knsha and B. Pullman, p. 251. Academic Press, SW York. 1962.
6. KUNITZ,
Lardy,
and
J. 60, cds.),