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Inhibition of deoxycytidylic and deoxyguanylic acid kinase activity by adenosine triphosphate and deoxyadenosine triphosphate in pneumococci
SHORT COMMUNICATIONS
26X
BBA93339 Inhibition of deoxycyCidyllc and deoxyguanylic acid kinase activity by adenosine triphosphate and de~xye~enosine triphosphate in pneumococci We have recently observed tha~ dATP itthibited the activity of dCMP and dGM'P Idnases in cell-free extracts of encapsulated pneumococoi containing ATP as the phesphoryl donor, The activities of the remaining dcoxyribonucleotide kinases (thymidylate and dcoxyadenylate) were unaffected by dATP at the concentrations employed. In view of the close structural st;hilarity between dATP and ATP and the fact that little is known concerning saturation levels of phosphoryl donors for dCMP and dGMP kJnases, it beamm.~of interest to characterize the inhibitory effects to a greater extent using a more pur~[fied enzyme preparation. Alse, it was o{ interest to determine wheth~ such inhibitory effects could be duplicated by other tTiphosphates~ including high levels of ATP, and whether the inhibition could be of significanco ~:~rico, Using an en~psuL~ted strain of Di~lococc~s #neumoniae (type III, A66, maintained on Difco Brain heart infusion agm p]ates #~us 5 °/o defibrinated rabbit blood), dCMl? and dGMP kinases were extracted from cell suspensions prepared as described previouslyt by a combination of severzd meth~Ks z-'s. dCMP kinase was purified 5on-fold while dGMP kinase was purified zro-fold. The enzymes catalyze the following reactions, respectively. dCMP+ ATP ~ dCDP+ADP dGMP+ ATP ~ ~GDP+ ADP 1~O]lucleo~ide diphosphate ldnasa activity was present in the dCMP kinase preparation while only 5 % diphosphate kinase activity was detected in the dGM'P kinase fsseporation. Dcoxyribonncleotide kinase activity ~vas assayed as described previouslyG using [14C]dCMP and ['-4C]dGI~fPas substrams (Schwarz Bioreseareh). The assay mixture (made up to o,6 ml) contained the following components: Tris (pH 7.5), ~o?*moles; ATP, 5.o/~moles; iCfgClz.6HiO, 9.o/*moles; ~x~C~deoxyribonuclcotide, o.7 pmoles (z.4 pC/O.OT#rnole isotope #lus carrier}; kinase, o.8/*g for dCMP ldnase; 4.SPg for dGMP kinase On o.ox M potassium phosphate buffer, pH 7-5). In some experiments, creatine phosphate and creatine phospho!dnase were added to the assay mixture at concentrations of 8.o/*moles and In/*g. respectively. Specific activity of the kinase was defined as mpmoles 14C product formed per h per mg protein. Protein was determined, by the m*thod of LowRY a cd.7. A survey of the effects of vm~ous ribe- and dcoxyribonucleeside triphosphates on dCMP and dGMP kinase activity of pneumocceci wi~h ATP as the phosphoryl donor showed (Table x) that of all the triphosphatc"3 tested only the purine deoxyrlbonucleeside derivatives exerted inhibitory effects on both dCMP and dGMP kinases. dATP produead the great~*t inhibition, while both guanine deriva-~ives {ribo- and deoxyribo-) were approximately one third as effective as dATP. dCTP and its riboderivative elicited some inhibitory effects against dCMP kinase activity, but the effects were smaller than those of the "purine'" t riphosphatc~, and no inldbitory effects were observed, against dGMP kJnase activity,
Bioahtn*, Biophy.~. Aaa, 16fi (t968} 26i-a64
~6~
SHORT COMMUNICATION5
TABLE I gFIcECTS OF RIBO- AND DEOXVRIBONUCLEOSID~'~RIPHOSPHATESON dCMP AND dOMP EI~ASg ^cTlvil~/ Percent inhibition was determined by calculating the 1~e~centd¢crea=¢in sprcific activlty (mpmoles uC product formedfll Imr mg protein × Ios) of the inhibited level from the control level. Con. centrations of various trlphosphates are given in the table, Additions to assay raixtur¢
Conch. ~moles/o.6 ~ n l )
% Inhibition o/ kinase o~vity /~'om eont~'ol fmlu~
a/tt~3o-min i~ubalion dUMP CTP or d C T P
dGMP
0.05 LO
3.0 6,0 GTP or dGTP
o,o5
o
o
z
i,o
z
!
3.0
z5 3z
Z6 30
0.05 I,o 3.o 6.0
o
o
o.o 5 LO 3.o 6.0
o 5 55 too
62 88
6.0
UTP or TTP
3 14 19
dATP
o
o~ 0,7 O~ 0,5
u/v
OA 0,~
~
0,2
I
0.1 o,~::: c "L'2 -1
E.
0.11
0
0.11
0.22 0.33 0,44 0 . . ~ 0,60
~/ATPor ~/dArP (~ma.,/O.~m,)
Fig, L L[Egw~AVnR-BURKpl0t of dCMF k i r l ~ ¢ activity a~ x ~ur~tion ol tho concentration ol ATP or dATP, z]~ was calculated from the specific ~Lctlvltyobtail~d with ¢~¢h ¢o1~.~ntf~tlon of ATP @~@ or dATP. O - - 0 . Bivchim. Biophy.~, Acla, 166 (x968) 261-~¢64
SH~T
COMMUNICATIONS
~3
Further stndie~ showed that concentrations of ATP which were equivalent to those of dATP { ~ s ATP also resulted in an inhibition of kinase activity from peak levels but the total iahibitory effects were not as great as those of ATP ~bl~ dATP. A determination of the fate of dATP and ATP after 3c~min incubation revealed that small percentages of both tripbosphates (0.43 % for ATP, o.23 % dATP) were convetted to their diphosphate derivatives. From a partial stolehiometry calculation of the reaction, it was found that the conversion of triphosphate to diphosphate was balanced by the appearance of the diphospbate product of dCMP kioase activity. Since some dATP was converted to dADP during incubation and since several investigators~ showed that dATP could serve as a phosphoryl donor for dCMP and dGMP kinases in other organisms, a determination was made as to whether dATP and other triphosphates could act as phosphoryl donors for dCMP and dGMP kinases extracted from pneumococeL It was found that dATP was capable of servingasa phosphoryl donor for both dCMP and dGMP kinases with about two thirds the efficiency of ATP up to a concentration of 6.o/~moles/o.6 ml (io-z M). At higher levels, in a manner similar to that of ATP alone, a depression of activity from the peak level was observed. Lra~WsAwn-BuR~s plots of r/v as a function of 1/rATP] or I/[dATP] (with dCMP kinase) showed a typical substrate inhibition curve (Fig. I). The apparent Kj concentration for both dATP and ATP derived from the curve was 5.z ~moles/o.6 ml. None of the other triphosphates, including the guanine derivatives which produced some inhibition of kiuase activity" in the presence of ATP (~ee Table I), were capable of acting as phosphoryl donors for dCMP mid dGMP kinases. Inereaslag the concentration of other components in the assay" mixture (Mgz÷, deoxyribonucJeotide substrates) did not affect the substrate inhibition by" excess triphosphate. However, addition of a phosphate regenerating system (creatine phesphate and ereatina phosphokinase) decreased the concentration of ATP (and dATP) required to inhibit kinase activity 3-fuld (2.o/~moles/o.6 ml, 3.3' ro-S M) and at this new level of saturation, produced a 3-fuld stimuJation of klnase activity in comparison to assay mixtures without the regenerating system. The stimulatery effects did not seem to be related to maintenance of the pbosphoryl donor in the triphosphate state, since given the presence of o.7/~mole deoxyribonucleotide substmte in the assay mixture initially (with little or no phosphatase activity), it is possible to convert only 40 % of the added phosphoryl donor theoretically (o.7pmole out of ~.o /~moles added~, Experimentally, the expectation would be much lower. One possibility is that creatine phosphate is an activator of the kinase, binding to it and increasing the affinity of the ATP substrate, in which case a smaller amount of triphosphate might be required to saturate the enzyme. In considering the extent to which such an inhibition could occur i*t vlro, a determination was made of the levei,oof various ribo- and deoxyribonucleoside triphosphates in pneumocoecal cells that inhibit dCMP and dGMP ldnase activity {ATP, dATP, GTP, dGTP) by the method of N'EUHARD~.In four replicate experiments, it was found that there was an average of z.8/*males of triphosphate per g dry weight, approx. 60 % of which was ATP. The equivalent amount of pneumocecci present in each assay tube from which the kinases were extracted was calculated to be o.o9-o,i g on a dry weight basis. Thus, in the absence of a regenerating system (a situation which is not likely to exist in rive), pneumococci would have to synthesize or accumulate approx. ~x-23 times as much triphasphate as that ordinarily presl~ioc~im, Biophys. Acta, x66 {x96S}26t-264
264
SHORTCOMMUNICATIONS
ent ~ ~/vu before saturation levels were obtained for the two kinases. However, under conditions which are more likely to exist in ~/~o (i.¢, in the presence of an ATP regenerating system), the cells would have to synthesize or accumulate approx. 7 times as much trJphasphate in order for saturation levels to be reached. Several observations with pneumococcl and other organisms suggest that in v~vo (at least in the presence of an ATP regenerating sy~'tem) it might be possible to reach or exce~ the saturation level for dCMP and dGMP kina~s. (z) The level of dAMP ldnase activity in pneumocoeci has been found to be 4o-6o tlmes greater than those of the other kinases 1. (z) It has been possible to enhance dGMP klnase activity 5--6-laid in cell suspensions of pneumococei by adding certain naturally occurxingstimuhtors a. (3) 6-7-foldincreasesin"nermal"dATPleveIshavebeenobserved by NEUHa~° in Escherichia co~i cultures under conditions where DNA synthesis was inhibited. However, since the levels of other components in the assay mixture were not determined in rive (deoxyribonucleotide substrates, ATP regenerating components, Mg~+), and since an "unnatural" buffer (Tris) was employed in the assay mixture, it cannot be stated with certainty m v/~reconditions approximate those present/n rive. Experiments are new underway to determiHe the levels of other components of the a.~ay system ~n v/~o in order to approximate such conditions as closely as possible in future assays. This work was supported by a grant from the National Cancer Institute, CA-o6343. The senior author is the recipient of a Career Development Award from the Public Health Service. De~arlment o/Biology, Wesleyan Uni~ersily, Middletown, Conn. (U.S,A.)
WILLIA~t FIRSHm~ THOMASR. BnOKEX*
W. FIRSHEIN, J, Ba~t$~ol,, 90 (1965)327, 2 I. R. ].BIr~iAN, M, J. B£;SMAI~, E, 5. SIMMSANDA. Konuezaa, J. Biol. Cicero, 233 (19iS) 163,
3 F. MAL~YASD5. OcuoA, f , Biol. Chem., 2~3 (t958) xS38. 4 M. P, O~CHc3~I~RANDM. J, BESSMAU,J. Biol, Chore,, 24t (i966) 545z. Y. 5IIGINO, H* TERAOKAANDH* 5HIMONO,J, B~0LC~r~., ~41 (I966) 96I, 6 W. FiRmlEru,S. J. BEnBYANDM, 5Wlt¢DL~XURST.B~ocfdtB, BJopt~y$. Aae, i49 (t967) x9o. 7 0 . H, LowRy, N, J, ROS~BROUGff,A, L, FAREANDR, J. RAIqDALL,f, BdOl.C~eff$,, ~[93(I951 ) 265. $ H. LIUeWEAVgRa2~OD. BUNK,f , Hm. Ckem, 5oc., ~6 (t934) 658, 9 J. NZU~ARD,Biockim, Biophys, Aaa, r45 (~t967)r, Received April ~:znd, x968
" Present address: Departmentof Biochc~mi~tty,Stanford University,Palo Alto,Calif.U,5,A, Bio~him. Biopl~y$. A¢la, t06 (1968) ~61-Z64
26X
BBA93339 Inhibition of deoxycyCidyllc and deoxyguanylic acid kinase activity by adenosine triphosphate and de~xye~enosine triphosphate in pneumococci We have recently observed tha~ dATP itthibited the activity of dCMP and dGM'P Idnases in cell-free extracts of encapsulated pneumococoi containing ATP as the phesphoryl donor, The activities of the remaining dcoxyribonucleotide kinases (thymidylate and dcoxyadenylate) were unaffected by dATP at the concentrations employed. In view of the close structural st;hilarity between dATP and ATP and the fact that little is known concerning saturation levels of phosphoryl donors for dCMP and dGMP kJnases, it beamm.~of interest to characterize the inhibitory effects to a greater extent using a more pur~[fied enzyme preparation. Alse, it was o{ interest to determine wheth~ such inhibitory effects could be duplicated by other tTiphosphates~ including high levels of ATP, and whether the inhibition could be of significanco ~:~rico, Using an en~psuL~ted strain of Di~lococc~s #neumoniae (type III, A66, maintained on Difco Brain heart infusion agm p]ates #~us 5 °/o defibrinated rabbit blood), dCMl? and dGMP kinases were extracted from cell suspensions prepared as described previouslyt by a combination of severzd meth~Ks z-'s. dCMP kinase was purified 5on-fold while dGMP kinase was purified zro-fold. The enzymes catalyze the following reactions, respectively. dCMP+ ATP ~ dCDP+ADP dGMP+ ATP ~ ~GDP+ ADP 1~O]lucleo~ide diphosphate ldnasa activity was present in the dCMP kinase preparation while only 5 % diphosphate kinase activity was detected in the dGM'P kinase fsseporation. Dcoxyribonncleotide kinase activity ~vas assayed as described previouslyG using [14C]dCMP and ['-4C]dGI~fPas substrams (Schwarz Bioreseareh). The assay mixture (made up to o,6 ml) contained the following components: Tris (pH 7.5), ~o?*moles; ATP, 5.o/~moles; iCfgClz.6HiO, 9.o/*moles; ~x~C~deoxyribonuclcotide, o.7 pmoles (z.4 pC/O.OT#rnole isotope #lus carrier}; kinase, o.8/*g for dCMP ldnase; 4.SPg for dGMP kinase On o.ox M potassium phosphate buffer, pH 7-5). In some experiments, creatine phosphate and creatine phospho!dnase were added to the assay mixture at concentrations of 8.o/*moles and In/*g. respectively. Specific activity of the kinase was defined as mpmoles 14C product formed per h per mg protein. Protein was determined, by the m*thod of LowRY a cd.7. A survey of the effects of vm~ous ribe- and dcoxyribonucleeside triphosphates on dCMP and dGMP kinase activity of pneumocceci wi~h ATP as the phosphoryl donor showed (Table x) that of all the triphosphatc"3 tested only the purine deoxyrlbonucleeside derivatives exerted inhibitory effects on both dCMP and dGMP kinases. dATP produead the great~*t inhibition, while both guanine deriva-~ives {ribo- and deoxyribo-) were approximately one third as effective as dATP. dCTP and its riboderivative elicited some inhibitory effects against dCMP kinase activity, but the effects were smaller than those of the "purine'" t riphosphatc~, and no inldbitory effects were observed, against dGMP kJnase activity,
Bioahtn*, Biophy.~. Aaa, 16fi (t968} 26i-a64
~6~
SHORT COMMUNICATION5
TABLE I gFIcECTS OF RIBO- AND DEOXVRIBONUCLEOSID~'~RIPHOSPHATESON dCMP AND dOMP EI~ASg ^cTlvil~/ Percent inhibition was determined by calculating the 1~e~centd¢crea=¢in sprcific activlty (mpmoles uC product formedfll Imr mg protein × Ios) of the inhibited level from the control level. Con. centrations of various trlphosphates are given in the table, Additions to assay raixtur¢
Conch. ~moles/o.6 ~ n l )
% Inhibition o/ kinase o~vity /~'om eont~'ol fmlu~
a/tt~3o-min i~ubalion dUMP CTP or d C T P
dGMP
0.05 LO
3.0 6,0 GTP or dGTP
o,o5
o
o
z
i,o
z
!
3.0
z5 3z
Z6 30
0.05 I,o 3.o 6.0
o
o
o.o 5 LO 3.o 6.0
o 5 55 too
62 88
6.0
UTP or TTP
3 14 19
dATP
o
o~ 0,7 O~ 0,5
u/v
OA 0,~
~
0,2
I
0.1 o,~::: c "L'2 -1
E.
0.11
0
0.11
0.22 0.33 0,44 0 . . ~ 0,60
~/ATPor ~/dArP (~ma.,/O.~m,)
Fig, L L[Egw~AVnR-BURKpl0t of dCMF k i r l ~ ¢ activity a~ x ~ur~tion ol tho concentration ol ATP or dATP, z]~ was calculated from the specific ~Lctlvltyobtail~d with ¢~¢h ¢o1~.~ntf~tlon of ATP @~@ or dATP. O - - 0 . Bivchim. Biophy.~, Acla, 166 (x968) 261-~¢64
SH~T
COMMUNICATIONS
~3
Further stndie~ showed that concentrations of ATP which were equivalent to those of dATP { ~ s ATP also resulted in an inhibition of kinase activity from peak levels but the total iahibitory effects were not as great as those of ATP ~bl~ dATP. A determination of the fate of dATP and ATP after 3c~min incubation revealed that small percentages of both tripbosphates (0.43 % for ATP, o.23 % dATP) were convetted to their diphosphate derivatives. From a partial stolehiometry calculation of the reaction, it was found that the conversion of triphosphate to diphosphate was balanced by the appearance of the diphospbate product of dCMP kioase activity. Since some dATP was converted to dADP during incubation and since several investigators~ showed that dATP could serve as a phosphoryl donor for dCMP and dGMP kinases in other organisms, a determination was made as to whether dATP and other triphosphates could act as phosphoryl donors for dCMP and dGMP kinases extracted from pneumococeL It was found that dATP was capable of servingasa phosphoryl donor for both dCMP and dGMP kinases with about two thirds the efficiency of ATP up to a concentration of 6.o/~moles/o.6 ml (io-z M). At higher levels, in a manner similar to that of ATP alone, a depression of activity from the peak level was observed. Lra~WsAwn-BuR~s plots of r/v as a function of 1/rATP] or I/[dATP] (with dCMP kinase) showed a typical substrate inhibition curve (Fig. I). The apparent Kj concentration for both dATP and ATP derived from the curve was 5.z ~moles/o.6 ml. None of the other triphosphates, including the guanine derivatives which produced some inhibition of kiuase activity" in the presence of ATP (~ee Table I), were capable of acting as phosphoryl donors for dCMP mid dGMP kinases. Inereaslag the concentration of other components in the assay" mixture (Mgz÷, deoxyribonucJeotide substrates) did not affect the substrate inhibition by" excess triphosphate. However, addition of a phosphate regenerating system (creatine phesphate and ereatina phosphokinase) decreased the concentration of ATP (and dATP) required to inhibit kinase activity 3-fuld (2.o/~moles/o.6 ml, 3.3' ro-S M) and at this new level of saturation, produced a 3-fuld stimuJation of klnase activity in comparison to assay mixtures without the regenerating system. The stimulatery effects did not seem to be related to maintenance of the pbosphoryl donor in the triphosphate state, since given the presence of o.7/~mole deoxyribonucleotide substmte in the assay mixture initially (with little or no phosphatase activity), it is possible to convert only 40 % of the added phosphoryl donor theoretically (o.7pmole out of ~.o /~moles added~, Experimentally, the expectation would be much lower. One possibility is that creatine phosphate is an activator of the kinase, binding to it and increasing the affinity of the ATP substrate, in which case a smaller amount of triphosphate might be required to saturate the enzyme. In considering the extent to which such an inhibition could occur i*t vlro, a determination was made of the levei,oof various ribo- and deoxyribonucleoside triphosphates in pneumocoecal cells that inhibit dCMP and dGMP ldnase activity {ATP, dATP, GTP, dGTP) by the method of N'EUHARD~.In four replicate experiments, it was found that there was an average of z.8/*males of triphosphate per g dry weight, approx. 60 % of which was ATP. The equivalent amount of pneumocecci present in each assay tube from which the kinases were extracted was calculated to be o.o9-o,i g on a dry weight basis. Thus, in the absence of a regenerating system (a situation which is not likely to exist in rive), pneumococci would have to synthesize or accumulate approx. ~x-23 times as much triphasphate as that ordinarily presl~ioc~im, Biophys. Acta, x66 {x96S}26t-264
264
SHORTCOMMUNICATIONS
ent ~ ~/vu before saturation levels were obtained for the two kinases. However, under conditions which are more likely to exist in ~/~o (i.¢, in the presence of an ATP regenerating system), the cells would have to synthesize or accumulate approx. 7 times as much trJphasphate in order for saturation levels to be reached. Several observations with pneumococcl and other organisms suggest that in v~vo (at least in the presence of an ATP regenerating sy~'tem) it might be possible to reach or exce~ the saturation level for dCMP and dGMP kina~s. (z) The level of dAMP ldnase activity in pneumocoeci has been found to be 4o-6o tlmes greater than those of the other kinases 1. (z) It has been possible to enhance dGMP klnase activity 5--6-laid in cell suspensions of pneumococei by adding certain naturally occurxingstimuhtors a. (3) 6-7-foldincreasesin"nermal"dATPleveIshavebeenobserved by NEUHa~° in Escherichia co~i cultures under conditions where DNA synthesis was inhibited. However, since the levels of other components in the assay mixture were not determined in rive (deoxyribonucleotide substrates, ATP regenerating components, Mg~+), and since an "unnatural" buffer (Tris) was employed in the assay mixture, it cannot be stated with certainty m v/~reconditions approximate those present/n rive. Experiments are new underway to determiHe the levels of other components of the a.~ay system ~n v/~o in order to approximate such conditions as closely as possible in future assays. This work was supported by a grant from the National Cancer Institute, CA-o6343. The senior author is the recipient of a Career Development Award from the Public Health Service. De~arlment o/Biology, Wesleyan Uni~ersily, Middletown, Conn. (U.S,A.)
WILLIA~t FIRSHm~ THOMASR. BnOKEX*
W. FIRSHEIN, J, Ba~t$~ol,, 90 (1965)327, 2 I. R. ].BIr~iAN, M, J. B£;SMAI~, E, 5. SIMMSANDA. Konuezaa, J. Biol. Cicero, 233 (19iS) 163,
3 F. MAL~YASD5. OcuoA, f , Biol. Chem., 2~3 (t958) xS38. 4 M. P, O~CHc3~I~RANDM. J, BESSMAU,J. Biol, Chore,, 24t (i966) 545z. Y. 5IIGINO, H* TERAOKAANDH* 5HIMONO,J, B~0LC~r~., ~41 (I966) 96I, 6 W. FiRmlEru,S. J. BEnBYANDM, 5Wlt¢DL~XURST.B~ocfdtB, BJopt~y$. Aae, i49 (t967) x9o. 7 0 . H, LowRy, N, J, ROS~BROUGff,A, L, FAREANDR, J. RAIqDALL,f, BdOl.C~eff$,, ~[93(I951 ) 265. $ H. LIUeWEAVgRa2~OD. BUNK,f , Hm. Ckem, 5oc., ~6 (t934) 658, 9 J. NZU~ARD,Biockim, Biophys, Aaa, r45 (~t967)r, Received April ~:znd, x968
" Present address: Departmentof Biochc~mi~tty,Stanford University,Palo Alto,Calif.U,5,A, Bio~him. Biopl~y$. A¢la, t06 (1968) ~61-Z64