- Email: [email protected]
Differential effects of putrescine, cadaverine, and glyoxal-bis-(guanylhydrazone) on DNA- and nucleohistone-supported DNA synthesis
SHORT COMMUNICATIONS
2~I
BBA93335
Differential effects of putrescine, cadaverlne, and glyoxolobis(guanylhydrazone) on DNA- and nncleohistone~supported D N A synthesis Aliphatic polyamines, at least in pars by virtue of their ability to complex with and stabilize po]yanchotides, invoke a diversity of both in vi~o and in vitro biochemical and biophysical effects1. Among these diverse effects are their ability to influence the synthesis of DN'A in vitro. Thus B e ~ n AND RUSCRI report stimulation by spermine of DNA polymerase (deoxynucleos~detdphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) oi isolated nuclei of Physarum polycephalum supplied with exogenous source of DNA and deoxynucleeside tripbosphates. On the other hand O'BRms, OLEmCK AND HAHSa report an inhibition by spermine of the activity of partially purified DNA polymerase in the presence of native DNA primer. In the present communication we report that putrescine and cadaverine, but not glyoxal-bis0guanylhydrazone) (LI'-[(ethanediylidene)dinitrilo]diguanidine) inhibit DNA-dependent DNA synthesis by Escherichia coli polymerase, Glyoxal-bis(guanylhydrazone) and its methyl, derivatives have been proposed as anticareinogens~s. By contrast, the three above-mentioned pclyamines stimulate DNA-polymerase activity with nueleohistones or ehromatin from various sources as primers. DNA polymerase was prepered from frozen late log-phase E. colt according to a modification of the method of ~CHWIMMER AI~D ARON$ON6 and RICHARDSONel ~ 7 through step 7, and it bad a specific activity of 8,)o units/rag of protein using activated DNA as primer and about I8o units/rag protein with native calf thymus DN'A as primer, The chromatin ot the pea nuclei (var. Alaska) w~,sprepared from 4o h embryonic axes according to the procedure of HLTANGANDBONm~R8. Nuclcohistene was prepared from the cbromatin by shearing and differential ¢cntrifugation~. Chromatin can serve as primer for both RNA and DNA polymerase but only DNA polymerase will utilize nucleohistone as primere, s,lo. Isolation of native uncomplexed DNA front the chromatin traction and characterization of these DNA .fracrions have been described previously6,~. Rat-liver chromatin was prepared according to the procedure of MARUSHIGE AND ]~ONNER11. Thymus nucleohistone was a preparation used in a study of the use of reconstituted nucleohistone as primers for DNA poiymerase~. Each ml of the DNA-polymerase reaction contained: potassium phosphate buffer, 4o#moles; MgCI~, 4pmoles; 2-mercaptoethanol, o.6pmole; triphosphates of deoxythymidine, deoxycytidine and deoxyadenosine (SH-labeled, Seh~-arz, xo pC per pmole), o.oz pmole each; DNA polymerase, 1.3 units (native calf thymus DNA as primer). The concentrations of pea embryo primers in nucleotide e(tnivalents of DNAI were o,o2 and o.~9/~mole/ml of DNA- and nueleohistc, ne-primed enzyme reaction mixture, respectively~ DNA synthesized was measured by the incorporation of SH into perchloric acid-insoluble material. Controls consisted of adding enzyme to the reaction mixture after incubation of the other cemponei~ts. The rates of DNA synthesis of pea embryo DNA- and nucleohistone-primed reactions in the absence of added polynmine were 9.0 and 5-4 mpmoles of DNA syntilesized per mI of reaction mixture in :30 min at 37 °, respectively, The Folyamiues used in this study were obtained from Nutritional Biochemicals. Btochim. Biop~ys. A¢la, z66 (I968) ~5z-~54
~Z
SNORT COMMUNICATIONS
NH2
y
~NH
HN-I-I-.NH
HN~C
/
s,.. N
I II HN~'~N ~ C? H
potvaMiN~ MmoJ
Fig, L Effect of polyamines on DIgA-polymcrase activity with pea embryo DNA (C)) or pea embryo nucleohistone (O) as primer. Activities in absence of polyamine set at ion, Actual valueB are given in text• Abbreviations: GAG, glyoxal-bis(guanylhydrazone); PUT, putrescine; CAD, cadgverine. Fig. ~. Possible internal hydrogen bonding in glyoxal-bis(gu~nyLhydrazooe), Fig. x shows t h a t both putreseine a n d cadaverine inhibited pea e m b r y o D N A primed DIgA polymerase. 5° % inhibition occurred at about xo -s M. At a conCentration of z ' zo - s M, only about 25 % of the activity remained. Glyoxal-bis(guanylhydrazone) did not inhibit DN'A polymerase at concentrations u p to zo -8 M. On the other band, all the polyamines accelerated the rate of p e a e m b r y o nucleohistnnep r i m e d synthesis. Glyoxaf-bis{guanylhydrazone) prr, ved to be the ~ o s t effective stimulator of nneleohistone-primed DN'A polymerase activity of the three polyamines tested. Thus, interpalated values for millimolar concentrations giving 5o % stimulation of activity are o.5, LX a n d L3 for giyoxabbis(guanylhydrzaone), cadaverine and putreseine, respectively. I n addition to the experiments cited above, less extensive d a t a were obtained on the effect of putrescine a n d eadaverine on the activity of D N A polymerase primed with DIgA in the form ol free DNA, nueleohistone and ebromatin prepared from calf thymus, rat liver, a n d pea embryo. T h e results in Tabl e I show t h a t the TABLE I EVICT OF PUTR~SeI~E A-~D CADAVI~INB (O,OOI M) ON DNA-~oLY~ERAS~ ACTIVITYIN TIt~ PRESBNCN,OF VARIOUSPRIMIg~
DNA, pea DNA, thymu~ DNA, liver Nueleo~stone, pea Nucleohi~tone, thymus Chrornatin, pea Cl~omatin, [iv~
ConcH. (pg DNAIml r#action)
Activity"
Activity in presence o/'" Putrescine Cadaverine
xo 15 -So t4o 11o --
L3 z,l 4.3 I,z o,6 x.9 0.8
58 49 64 155 lI 5 -:t~z
4z 43 57 I~ -lzz l~tt
Rate expressed ~ rn/~mole~ of dAMP incorporated into acid*insoluble rrtateriat in 3o rain at 37° in absence of polyp.mines. Varioul amounts of E, ¢ofi D~A polymeric were used, "' Rate in absence of lmlyamirtes set at zoo. Biochim. Biophys~ ACl~, 166 (1068) 25I--254
SHORT COMMUNICATIONS
25~
simple aJkyl diamines inhibited DNA polymerase in the presence of thymus and liver D NA as well as in the presence of pe~ embryo D N'A. On the other hand, these die mines caused an apparent augmentation in the rate of DN'A-pelymerase activity in the presence of pea and thymus nucle,~histone, as well as in the presence of pea and liver ehromatin. The inhibition of DNA-primed DMA polymerase by simple dis mines is in accord with the observation of O'BRIEN, OLENICKANDI - L ~ s that spermlne inhibits D~IAprimed DN'A polymerase. The observed stimulation by both uliphatic diamines and by giyoxal-bis(guanylhydrazone) of nuclenhistone- and chromatin-primed DNA polymerase is qualitatively similar to the stimulation by spermine of the DNA-polymera~ activity of Phy~m'mn nuclei 2. This stimulation is also reminiscent of that achieved by adding an endonuclease to a nueleohistone-dependent DNA-pulymerase reaction mixture after maximum DN'A synthesis has taken placea. The simplest explanation of the diamine stimulation is that the latter partially displaces histone from the DNA with added assumption that histones exert a greater inhibitory effect than do the amines. The fact that the stimulation by the uliphatic amine is not as great as that exerted by glyoxal-bis(guanylhydrazone) (which does not inhibit the free Dl~YA-supperted reaction), is in agreement with this concept. Furthermore, in the presence of the highest concentration of glyoxal-bis(guanylliydrazone) used (xo-8 M) the rate with nuclechistone as primer approaches that of the rate in the presence of dehistonized DN'A. The observation that this stimulation occurs with primers as diverse as reconstituted nucleehistone and with liver chromxtin (see Table I), also suggests such a rather non-specific effect. An argument against this simple concept of competition between histone and diamine for DNA is that one would expect, at sufficiently high concentrations of diamine, a reversal of the stimulation etiect on the nucleohistane supported reaction. However, no such reversal was observed at concentration as high as 2" xo-s M at which there was a 70-80 % iuldbitian of the DNA-primed reaction. The previoksly mentioned data of B~WER ANY RUSC:~ points to the release or activation by spermine of a "latent" DNA polymerase in Pliysarum nuclei. Yatelpretation of the kinetics and action of nuclease on nucleoli/stone-dependent DNA l~olymerase led to the suggestion that the polymerase is reversibly bound in complex s~th product DN'A in a manner such that it is no longer available for further catalysise,~. Thns~ one possible function of the diamines would be to uncouple this complex and then allow DNA synthesis to proceed. The overall effect would appear to be an increase in the activity of the DNA polymerase, This uncoupling could be accomplished by: the afore-said competition with histone; by combination with the historic-associated RNA2s, or perhaps by activation of a latent dcoxyribonucJease. Alth3ugil the polymcrase preparation had no nucleolytio activity, as measured by increase in acid-~omble nucleotides, and although the action of massive doses of exanuclease on the nuclcehlstone primer as measured by this procedure is sllght e, it is quite conceivable that subtle hydrolysis of strategic pliosphodiester bonds of the DIqA of the nucleohistone by small amount of endogenous naclease activated by the polyamines (both inhibition and activation of nucleases has been reportedt) could ]ead to release and "activation" of the DMA polymerase. The relationship o:[ deoxyribonueleases to D]gA synthesis has been recently revieweda, Eiochim. Biophys. Acla, z6o (x968} 25z-254
~54
SHORT COMMUNICATIONa
It is surprising that glyoxal-bis (guanylhydrazone), whose methyl derivative has been shown to comp, ex with sarcoma DNA ~, does not inhibit DNA-supported DNA-polymerase activity but stillstimulates the nucleohistonc-supported reaction. We suggest, on the basis of molecular models, thai internal hydrogen bonding (Fig. z) shapes this polyamine in such a way that its complexing with DNA does not interfere with the action of the polymerase. In this configuration, the compound would presumably interact with DNA as do monoamines which are considered not to confer the stability on polynucleotides and enzyme-influencing properties effected by polyaminesL However, binding of glyoxal-bis (guanylhydrazone) to nucleohistone, perhaps by complexing with historic-bound R N A ta prevents historic from interfering with DNA-polymerase activity. The substitution of glyoxal hydrogen by a methyl group to give methylglyoxal-bis{guanylhydrazone) would prevent this hydrogen bonding, The methyl derivative could then bind to DNA as do other polyamines and interfere with DNA synthesis t~. This work was conducted under a cooperative agreement between the Division of Biology, California Institute of Technology and the Western Utilization Research and Development Division, Agricultural Research Service. U.S. Department of Agriculture. The work was supported in part by U.S, Public Health Serv',ce Grants GM-o3977, GM-osx4~. AM-o3xoz. Reference to a company or product name does not imply approval or recommendation of the product by the U,S, Department of Agriculture to the exclusion of others that may be suitable. Wec~em Regional Research Lab6rat,o¢y, Agricultural Research Service, U.S. Departmen~ o] Agriculture, Albarvy, Cali[, 947xo, and Di~isio~ o[ Biology, Cali/ornia Institute o/Technology, Pasadena, Calf[. (U.S.A.)
SIGMUND ScnwIM~f~R
z H. TABORANYC. W. T^nOR, Pharm¢¢ol. Rev.,16 (~964) ~45. E. N. BI~VCBRa ~ H P, Rvsc~, Biochem, Biopkys. Re~. Commun,, 25 0966) 579, 3 R. L, O'BwmN, J. Cr, OLI~I~ICKAND F. E. Ht,nN, Pro~. Natl. Acad. S¢L U.S., 55 (:96o) IStL 4 B. L. FRE~DLANDERANDF. A. FRENCh/,Cancer ReS,, 3 (t958) 360. 5 E. Mmtc, Cancer Res., z 3 (I963) t375. 6 S. SCHW~MmER AND A. ARO~SON, Bio~h~m. Bioplvy#. Aura, I34 (t967) 59. 7 C. C. BICMARDSON,C. L. SCHILDKBAUT,H, V, APOSHIANAND A. KORNBERG,J. BiOl, Chem., *39 (1964) zZ2. 8 R, C, C. HUAN~A~;DJ. BONN~!%Pr~, ~ValLAe~d. SoL U.$, 48 (X962) t~Z6. 9 J, BO~NF,R aND R. C. C. HV^~G, J, MoL Biol,, 6 (i063) x69. IO S. SCHWIMMER AND J. ~ONNER, Bioehim, Biophy*, ,$¢ta. to$ ft965) 67, xt K. M^RUSmGEAzV J, BOI*UgR,J, Mol,/~D/., t5 (1966) t6"~ x~ $. Sc~wmHe,, Lile Sa., 4 (x965) z~47. I 3 J. BONNERAND J. WIDHOLR,~e6. Natl. Acad. S~i. U,S., 57 (x967) [379, 14 I. R. LEHMAN. A~n. BeY. B~ochem.. 36 (I967} 645, z5 A. C. SARzomzLLX.A. T, IAtczorl, B, A, BCO~H.iv. H, SC~INI~tDEI~,J. R, BZRTL'~OANYD, G. JOHNS, Bio~hlm, Biopl~y#. ,4~ta, IO~ (1965) I74, Received April z9th , x968
Bioehlm. Biophys.A¢la, t66 (x968) ~51-z54
2~I
BBA93335
Differential effects of putrescine, cadaverlne, and glyoxolobis(guanylhydrazone) on DNA- and nncleohistone~supported D N A synthesis Aliphatic polyamines, at least in pars by virtue of their ability to complex with and stabilize po]yanchotides, invoke a diversity of both in vi~o and in vitro biochemical and biophysical effects1. Among these diverse effects are their ability to influence the synthesis of DN'A in vitro. Thus B e ~ n AND RUSCRI report stimulation by spermine of DNA polymerase (deoxynucleos~detdphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) oi isolated nuclei of Physarum polycephalum supplied with exogenous source of DNA and deoxynucleeside tripbosphates. On the other hand O'BRms, OLEmCK AND HAHSa report an inhibition by spermine of the activity of partially purified DNA polymerase in the presence of native DNA primer. In the present communication we report that putrescine and cadaverine, but not glyoxal-bis0guanylhydrazone) (LI'-[(ethanediylidene)dinitrilo]diguanidine) inhibit DNA-dependent DNA synthesis by Escherichia coli polymerase, Glyoxal-bis(guanylhydrazone) and its methyl, derivatives have been proposed as anticareinogens~s. By contrast, the three above-mentioned pclyamines stimulate DNA-polymerase activity with nueleohistones or ehromatin from various sources as primers. DNA polymerase was prepered from frozen late log-phase E. colt according to a modification of the method of ~CHWIMMER AI~D ARON$ON6 and RICHARDSONel ~ 7 through step 7, and it bad a specific activity of 8,)o units/rag of protein using activated DNA as primer and about I8o units/rag protein with native calf thymus DN'A as primer, The chromatin ot the pea nuclei (var. Alaska) w~,sprepared from 4o h embryonic axes according to the procedure of HLTANGANDBONm~R8. Nuclcohistene was prepared from the cbromatin by shearing and differential ¢cntrifugation~. Chromatin can serve as primer for both RNA and DNA polymerase but only DNA polymerase will utilize nucleohistone as primere, s,lo. Isolation of native uncomplexed DNA front the chromatin traction and characterization of these DNA .fracrions have been described previously6,~. Rat-liver chromatin was prepared according to the procedure of MARUSHIGE AND ]~ONNER11. Thymus nucleohistone was a preparation used in a study of the use of reconstituted nucleohistone as primers for DNA poiymerase~. Each ml of the DNA-polymerase reaction contained: potassium phosphate buffer, 4o#moles; MgCI~, 4pmoles; 2-mercaptoethanol, o.6pmole; triphosphates of deoxythymidine, deoxycytidine and deoxyadenosine (SH-labeled, Seh~-arz, xo pC per pmole), o.oz pmole each; DNA polymerase, 1.3 units (native calf thymus DNA as primer). The concentrations of pea embryo primers in nucleotide e(tnivalents of DNAI were o,o2 and o.~9/~mole/ml of DNA- and nueleohistc, ne-primed enzyme reaction mixture, respectively~ DNA synthesized was measured by the incorporation of SH into perchloric acid-insoluble material. Controls consisted of adding enzyme to the reaction mixture after incubation of the other cemponei~ts. The rates of DNA synthesis of pea embryo DNA- and nucleohistone-primed reactions in the absence of added polynmine were 9.0 and 5-4 mpmoles of DNA syntilesized per mI of reaction mixture in :30 min at 37 °, respectively, The Folyamiues used in this study were obtained from Nutritional Biochemicals. Btochim. Biop~ys. A¢la, z66 (I968) ~5z-~54
~Z
SNORT COMMUNICATIONS
NH2
y
~NH
HN-I-I-.NH
HN~C
/
s,.. N
I II HN~'~N ~ C? H
potvaMiN~ MmoJ
Fig, L Effect of polyamines on DIgA-polymcrase activity with pea embryo DNA (C)) or pea embryo nucleohistone (O) as primer. Activities in absence of polyamine set at ion, Actual valueB are given in text• Abbreviations: GAG, glyoxal-bis(guanylhydrazone); PUT, putrescine; CAD, cadgverine. Fig. ~. Possible internal hydrogen bonding in glyoxal-bis(gu~nyLhydrazooe), Fig. x shows t h a t both putreseine a n d cadaverine inhibited pea e m b r y o D N A primed DIgA polymerase. 5° % inhibition occurred at about xo -s M. At a conCentration of z ' zo - s M, only about 25 % of the activity remained. Glyoxal-bis(guanylhydrazone) did not inhibit DN'A polymerase at concentrations u p to zo -8 M. On the other band, all the polyamines accelerated the rate of p e a e m b r y o nucleohistnnep r i m e d synthesis. Glyoxaf-bis{guanylhydrazone) prr, ved to be the ~ o s t effective stimulator of nneleohistone-primed DN'A polymerase activity of the three polyamines tested. Thus, interpalated values for millimolar concentrations giving 5o % stimulation of activity are o.5, LX a n d L3 for giyoxabbis(guanylhydrzaone), cadaverine and putreseine, respectively. I n addition to the experiments cited above, less extensive d a t a were obtained on the effect of putrescine a n d eadaverine on the activity of D N A polymerase primed with DIgA in the form ol free DNA, nueleohistone and ebromatin prepared from calf thymus, rat liver, a n d pea embryo. T h e results in Tabl e I show t h a t the TABLE I EVICT OF PUTR~SeI~E A-~D CADAVI~INB (O,OOI M) ON DNA-~oLY~ERAS~ ACTIVITYIN TIt~ PRESBNCN,OF VARIOUSPRIMIg~
DNA, pea DNA, thymu~ DNA, liver Nueleo~stone, pea Nucleohi~tone, thymus Chrornatin, pea Cl~omatin, [iv~
ConcH. (pg DNAIml r#action)
Activity"
Activity in presence o/'" Putrescine Cadaverine
xo 15 -So t4o 11o --
L3 z,l 4.3 I,z o,6 x.9 0.8
58 49 64 155 lI 5 -:t~z
4z 43 57 I~ -lzz l~tt
Rate expressed ~ rn/~mole~ of dAMP incorporated into acid*insoluble rrtateriat in 3o rain at 37° in absence of polyp.mines. Varioul amounts of E, ¢ofi D~A polymeric were used, "' Rate in absence of lmlyamirtes set at zoo. Biochim. Biophys~ ACl~, 166 (1068) 25I--254
SHORT COMMUNICATIONS
25~
simple aJkyl diamines inhibited DNA polymerase in the presence of thymus and liver D NA as well as in the presence of pe~ embryo D N'A. On the other hand, these die mines caused an apparent augmentation in the rate of DN'A-pelymerase activity in the presence of pea and thymus nucle,~histone, as well as in the presence of pea and liver ehromatin. The inhibition of DNA-primed DMA polymerase by simple dis mines is in accord with the observation of O'BRIEN, OLENICKANDI - L ~ s that spermlne inhibits D~IAprimed DN'A polymerase. The observed stimulation by both uliphatic diamines and by giyoxal-bis(guanylhydrazone) of nuclenhistone- and chromatin-primed DNA polymerase is qualitatively similar to the stimulation by spermine of the DNA-polymera~ activity of Phy~m'mn nuclei 2. This stimulation is also reminiscent of that achieved by adding an endonuclease to a nueleohistone-dependent DNA-pulymerase reaction mixture after maximum DN'A synthesis has taken placea. The simplest explanation of the diamine stimulation is that the latter partially displaces histone from the DNA with added assumption that histones exert a greater inhibitory effect than do the amines. The fact that the stimulation by the uliphatic amine is not as great as that exerted by glyoxal-bis(guanylhydrazone) (which does not inhibit the free Dl~YA-supperted reaction), is in agreement with this concept. Furthermore, in the presence of the highest concentration of glyoxal-bis(guanylliydrazone) used (xo-8 M) the rate with nuclechistone as primer approaches that of the rate in the presence of dehistonized DN'A. The observation that this stimulation occurs with primers as diverse as reconstituted nucleehistone and with liver chromxtin (see Table I), also suggests such a rather non-specific effect. An argument against this simple concept of competition between histone and diamine for DNA is that one would expect, at sufficiently high concentrations of diamine, a reversal of the stimulation etiect on the nucleohistane supported reaction. However, no such reversal was observed at concentration as high as 2" xo-s M at which there was a 70-80 % iuldbitian of the DNA-primed reaction. The previoksly mentioned data of B~WER ANY RUSC:~ points to the release or activation by spermine of a "latent" DNA polymerase in Pliysarum nuclei. Yatelpretation of the kinetics and action of nuclease on nucleoli/stone-dependent DNA l~olymerase led to the suggestion that the polymerase is reversibly bound in complex s~th product DN'A in a manner such that it is no longer available for further catalysise,~. Thns~ one possible function of the diamines would be to uncouple this complex and then allow DNA synthesis to proceed. The overall effect would appear to be an increase in the activity of the DNA polymerase, This uncoupling could be accomplished by: the afore-said competition with histone; by combination with the historic-associated RNA2s, or perhaps by activation of a latent dcoxyribonucJease. Alth3ugil the polymcrase preparation had no nucleolytio activity, as measured by increase in acid-~omble nucleotides, and although the action of massive doses of exanuclease on the nuclcehlstone primer as measured by this procedure is sllght e, it is quite conceivable that subtle hydrolysis of strategic pliosphodiester bonds of the DIqA of the nucleohistone by small amount of endogenous naclease activated by the polyamines (both inhibition and activation of nucleases has been reportedt) could ]ead to release and "activation" of the DMA polymerase. The relationship o:[ deoxyribonueleases to D]gA synthesis has been recently revieweda, Eiochim. Biophys. Acla, z6o (x968} 25z-254
~54
SHORT COMMUNICATIONa
It is surprising that glyoxal-bis (guanylhydrazone), whose methyl derivative has been shown to comp, ex with sarcoma DNA ~, does not inhibit DNA-supported DNA-polymerase activity but stillstimulates the nucleohistonc-supported reaction. We suggest, on the basis of molecular models, thai internal hydrogen bonding (Fig. z) shapes this polyamine in such a way that its complexing with DNA does not interfere with the action of the polymerase. In this configuration, the compound would presumably interact with DNA as do monoamines which are considered not to confer the stability on polynucleotides and enzyme-influencing properties effected by polyaminesL However, binding of glyoxal-bis (guanylhydrazone) to nucleohistone, perhaps by complexing with historic-bound R N A ta prevents historic from interfering with DNA-polymerase activity. The substitution of glyoxal hydrogen by a methyl group to give methylglyoxal-bis{guanylhydrazone) would prevent this hydrogen bonding, The methyl derivative could then bind to DNA as do other polyamines and interfere with DNA synthesis t~. This work was conducted under a cooperative agreement between the Division of Biology, California Institute of Technology and the Western Utilization Research and Development Division, Agricultural Research Service. U.S. Department of Agriculture. The work was supported in part by U.S, Public Health Serv',ce Grants GM-o3977, GM-osx4~. AM-o3xoz. Reference to a company or product name does not imply approval or recommendation of the product by the U,S, Department of Agriculture to the exclusion of others that may be suitable. Wec~em Regional Research Lab6rat,o¢y, Agricultural Research Service, U.S. Departmen~ o] Agriculture, Albarvy, Cali[, 947xo, and Di~isio~ o[ Biology, Cali/ornia Institute o/Technology, Pasadena, Calf[. (U.S.A.)
SIGMUND ScnwIM~f~R
z H. TABORANYC. W. T^nOR, Pharm¢¢ol. Rev.,16 (~964) ~45. E. N. BI~VCBRa ~ H P, Rvsc~, Biochem, Biopkys. Re~. Commun,, 25 0966) 579, 3 R. L, O'BwmN, J. Cr, OLI~I~ICKAND F. E. Ht,nN, Pro~. Natl. Acad. S¢L U.S., 55 (:96o) IStL 4 B. L. FRE~DLANDERANDF. A. FRENCh/,Cancer ReS,, 3 (t958) 360. 5 E. Mmtc, Cancer Res., z 3 (I963) t375. 6 S. SCHW~MmER AND A. ARO~SON, Bio~h~m. Bioplvy#. Aura, I34 (t967) 59. 7 C. C. BICMARDSON,C. L. SCHILDKBAUT,H, V, APOSHIANAND A. KORNBERG,J. BiOl, Chem., *39 (1964) zZ2. 8 R, C, C. HUAN~A~;DJ. BONN~!%Pr~, ~ValLAe~d. SoL U.$, 48 (X962) t~Z6. 9 J, BO~NF,R aND R. C. C. HV^~G, J, MoL Biol,, 6 (i063) x69. IO S. SCHWIMMER AND J. ~ONNER, Bioehim, Biophy*, ,$¢ta. to$ ft965) 67, xt K. M^RUSmGEAzV J, BOI*UgR,J, Mol,/~D/., t5 (1966) t6"~ x~ $. Sc~wmHe,, Lile Sa., 4 (x965) z~47. I 3 J. BONNERAND J. WIDHOLR,~e6. Natl. Acad. S~i. U,S., 57 (x967) [379, 14 I. R. LEHMAN. A~n. BeY. B~ochem.. 36 (I967} 645, z5 A. C. SARzomzLLX.A. T, IAtczorl, B, A, BCO~H.iv. H, SC~INI~tDEI~,J. R, BZRTL'~OANYD, G. JOHNS, Bio~hlm, Biopl~y#. ,4~ta, IO~ (1965) I74, Received April z9th , x968
Bioehlm. Biophys.A¢la, t66 (x968) ~51-z54