Synthesis and properties of O6-methyldeoxyguanylic acid and its copolymers with deoxycytidylic acid

770

Biochimica et Biophysica Acta, 521 ( 1 9 7 8 ) 7 7 0 - - 7 7 8 © E l s e v i e r / N o r t h - H o l l a n d Biomedical Press

BBA 9 9 3 3 3

SYNTHESIS AND PROPERTIES OF O6-METHYLDEOXYGUANYLIC ACID AND ITS COPOLYMERS WITH DEOXYCYTIDYLIC ACID

J I T E N D R A R. M E H T A a n d D A V I D B. L U D L U M

Department of Pharmacology and Experimental Therapeutics, The Albany Medical College of Union University, Albany, N. Y. 12208 (U.S.A.) (Received May 1 8 t h , 1 9 7 8 )

Summary This paper describes the synthesis of O6-methyldeoxyguanosine triphosphate (m6dGTP) and its copolymerization to a high molecular weight polymer with deoxycytidylic acid. The monomer, m6dGTP, was synthesized from deoxyguanosine first protected by acetylation of the sugar hydroxyls, and then chlorinated in the 6-position with POC13. The product, 6-chloro-3',5'-di-Oacetyl deoxyguanosine, was converted to O6-methyldeoxyguanosine with sodium methoxide and phosphorylated in the 5' position with carrot phosphotransferase. Monophosphate was converted chemically to the triphosphate and copolymerized with dCTP by terminal deoxynucleotidyl transferase. The resulting template, which contained O~-methylgnanine, was tested for its ability to direct RNA synthesis by bacterial RNA polymerase. The presence of O6-methylguanine was shown to lead to the misincorporation of UMP in the product polymer, thus strengthening the hypothesis that O~-methylguanine is a promutagenic base.

Introduction Many chemical carcinogens react directly or after metabolic activation with nucleic acids. Since such reactions may alter the informational content of these macromolecules, it is widely assumed that they can cause somatic mutations and hence malignancies. Substituted bases such as O6-methylguanine [1], O4-methylthymine [2], 3-methylcytosine [3,4], and others [5,6] which are formed in relatively small amounts, may be more important in this process than NT-methylguanine, even though it is formed in greater amounts. Abbreviations: m6dGuo, O6-methyldeoxyguanos/ne; m6dGMP° O6-methyldeoxyguanosine monophosphate; m6dGTP, O6 -me thyldeox yguanos/ne trlphosphate; poly(dC), polydcoxycytidylic acid; poly(dC, dG), copolymer of deoxycytidylic and deoxyguanylic acids; poiy(dC, m6dG), copolymer of deoxycytidylic and O6-methyldeoxyguanylic acids; d(pT)3, trimer of deoxythymidyllc acid.

771 After Loveless suggested that alkylation of the O6-position of guanine might be especially significant [1], numerous observations have been reported which would support this hypothesis. In particular, O6-alkylguanine has been found in the nucleic acids of animals treated with alkylating carcinogens [7--9], and there is some [10] evidence that this lesion is repaired slowly. In order to obtain direct information on the effects of this base modification, Gerchman and Ludlum [11] synthesized synthetic polyribonucleotide templates which contained O6-methylguanine. When these polynucleotides were used as templates for RNA polymerase, O6-methylguanine conveyed misinformation in the replicative process. Although these studies indicated that O6-methylguanine did not pair normally in an RNA template, they raised the question whether modification of guanine in a polydeoxyribonucleotide would have a similar effect. Accordingly, we have synthesized polydeoxynucleotides which contain O6-methylguanine and have used these polymers as templates for RNA polymerase. The synthesis of the required monomer, O6-methyl dGTP, and its polymerization to high molecular weight copolymers are described below together with data on the template properties of these polymers.

Experimental methods Deoxyguanosine was purchased from Calbiochemical; unlabeled nucleoside triphosphates, from P-L Biochemicals or Swartz Bioresearch; and 14C-labeled nucleoside triphosphates f r o ~ w England Nuclear. All of these nucleotides were purified before use bY column chromatography. Snake venom phosphodiesterase and alkaline ~hosphatase (Escherichia coil) were obtained from Worthington. Other compounds were standard reagent grade materials.

06-methyldeoxyguanosine This nucleoside was synthesized according to the outline in Fig. 1. DeoxyO

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VT Fig. I . Synthetic route of O6-methyl dGTP. Details of each step are deserlbed in the text.

Car,ct,0,os.~l'o~,a,~sfe,ase ...... i:l,enylp~ospl~aLe

772 guanosine (I) was converted by the method of Schuller et al. [12] to the acetylated derivative, 3',5'-di-O-acetyldeoxyguanosine (II}. One gram of deoxyguanosine (3.7 mmol) was reacted with an excess of acetic anhydride (10 ml) in pyridine (50 ml) for three days at room temperature. The product was filtered, washed first with warm pyridine (50 ml) and then with dry ether (50 ml), and finally vaccum dried at 75°C for 2 days before the next step. Chlorination of the acetylated deoxyguanosine was accomplished by the method of Gerster et al. [13] with some modifications to avoid decomposition due to the acid and heat lability of the glycosidic linkage. All of the glassware was dried in vacuo at 75°C, and the POCI3 was freshly distilled immediately before use. Acetylated deoxyguanosine {1.31 g, 3.7 mmol) was added with stirring to a solution of POC13 (75 ml) and N,N-diethylaniline (1.5 ml) at room temperature. The suspension was heated until all of the solid was dissolved and a clear yellowish solution was obtained. (If any moisture was present at this stage, the glycosidic linkage was cleaved resulting in a dark, decomposed solution.} Excess POCla was removed in vacuo, and the resulting syrup was stirred a drop at a time into excess ice until hydrolysis was complete. Then, the solution was extracted three times with 100 ml of dichloromethane. Organic extracts were combined, washed three times with 100 ml of cold 1 N HC1, and finally with cold H20 until neutral. After the dichloromethane solution was dried with anhydrous magnesium sulfate, the solvent was evaporated under vacuum to leave a thin oil. A few extractions with dry ether gave a crude, yellow, solid product of 6-chloro-3',5'-di-O-acetyl deoxyguanosine (III, 0.217 g, 0.56 mmol). This product was dissolved in 50 ml of freshly prepared sodium methoxide solution (250 mg of sodium metal in 50 ml of dry methanol} and heated at 40°C for 2 h. The solution was then cooled in an ice bath and carefully neutralized with cold 6 N HC1. Sodium chloride was removed by filtration and the solution was evaporated to leave dry O6-methyldeoxyguanosine (IV, 0.157 g, 0.56 mmol}.

06-Methyldeoxyguanosine monophosphate O6-Methyldeoxyguanosine was phosphorylated with carrot phosphotransferase using p-nitrophenyl phosphate as the phosphate donor. This enzyme, which is specific for the 5'-position, was isolated according to the procedure of Strider et al. [14] with the improvements suggested by Harvey et al. [15]. 75 mg of nucleoside (0.261 mmol) were incubated in 50 ml of 0.1 M sodium acetate buffer, pH 5, with 5 mmol of p-nitrophenyl phosphate and enzyme for 24 h at 37°C. A preliminary separation of this mixture was achieved on an AG 1 X 8 (200--400) column as described for the ribonucleotide by Gerchman et al. [16]. The column was eluted first with 0.01 M Tria-HC1, pH 5, to remove unreacted nucleoside and then with 0.01 N HC1 to remove contaminating O6-methyl GMP. O6-Methyl dGMP was eluted with 0.03 N HC1, neutralized with NH,OH, lyophilized, desalted and purified as before [16].

O~-Methyldeoxyguanosine triphosphate Using the same procedure as that used for the ribonucleotide [16], 10 mg

773 (0.03 mmol) of m6dGMP was dissolved in 20% aqueous pyridine and converted to the pyridinium salt b y passage through a small D o w e x 50 column in the pyridinium form. Water was removed b y repeated evaporations of dry pyridine, 0.01 ml of tributyl amine was added to convert the pyridinium salt to the tributylammonium salt, and this was dried further b y repeated evaporations of dimethylformamide. The anhydrous tributylammonium m6dGMP was dissolved in 0.25 ml of dry dimethylformamide and reacted with 32 mg of carbonyl diimidazole (0.2 mmol) in 0.4 ml of dry dimethylformamide. After 4 h at room temperature, methanol was added to destroy excess carbonyl diimidazole. 0.5 h later, a solution o f 0.2 mmol tributylammonium pyrophosphate in 2 ml of dimethylformamide was added and allowed to react under anhydrous conditions for 24 h at room temperature. The mixture was concentrated, dissolved in 50 ml of 0.05 M triethylammonium bicarbonate, pH 7.8, and applied to a DEAE-Sephadex A-25 column (1.5 × 30 cm, HCO~ form). On elution with a linear triethylammonium bicarbonate gradient (0.05 to 1.0 M, pH 7 . 8 , 2 0 0 ml of each) peaks appeared in the nucleoside and mono-, di-, tri- and tetra-phosphate regions. Triphosphate was the main product and accounted for 25% of the m6dGMP.

Synthesis of poly(dC, m6dG) and poly(dC, dG) R a n d o m copolymers of dCMP and dGMP or m6dGMP were synthesized in various compositions. Polymerization mixtures contained per ml: 200 pmol of potassium cacodylate buffer (pH 7.5); a total of 2 ~mol of purified substrate consisting of dCTP and variable amounts of dGTP or m6dGTP to 0.4 pmol; 1 gmol of mercaptoethanol; 1 gmol of CoC12; 10 nmol of d(pT)3 as an initiator; and 50 gg of terminal deoxynucleotidyl transferase from calf thymus [17]. After 24 h at 37°C the reaction mixture was deproteinized with a mixture of isoamyl alcohol and chloroform { 1 : 3 ) , and purified on a G-100 column (1.2 × 2 8 c m ) .

Copolymer composition Copolymer compositions were determined by high pressure liquid chromatography after digestion to the nucleoside level. Approximately 10 optical density units of c o p o l y m e r were dissolved in 0.5 ml of 0.1 M Tris-HCl buffer, pH 8.9, which was 9 mM in Mg 2÷. 50 pg of snake venom phosphodiesterase and 300 ~g of E. coli alkaline phosphatase were added, and the solution was incubated for 21 h at 37°C. Digestion was complete under these conditions as tested by passage through a small Sephadex G-100 column. The nucleosides from each digested copolymer were separated on a high pressure liquid chromatography system utilizing a ~-Bondapak C18 column, 4 mm × 30 cm, from Waters Associates {Milford, MA.). This column was protected by a small guard column and was eluted at a flow rate of 0.9 ml/min with 1 mM formic acid, pH 3.5, for 10 min, followed b y a linear gradient of acetonitrile {0--10% in 1 mM formic acid) for 80 min. Elution profiles were followed at the appropriate maximum for each nucleoside on a Perkin Elmer LC-55 ultraviolet s p e c t r o p h o t o m e t e r and recorded by a Perkin Elmer Sigma 10 Data System. Compositions were calculated from the total area corrected b y

774 the following extinction coefficients at pH 3.5:e279 (dCyd) = 10.84, e:s3 (dGuo) = 8.71, and e2s2 (m6dGuo) = 9.83 per nmol.

Template properties of copolymers with RNA polymerase RNA polymerase was isolated from Micrococcus luteus b y the procedure of Nakamoto et al. [18] as described previously [19]. Since the composition of all polynucleotide templates was entirely or mostly dCMP, the amount of template added to an incubation mixture was determined from the reported extinction coefficient for poly(dC) of 5.3 • 103 at 260 nm at pH 7.5. Incubations with R N A polymerase were performed as before [19] except that a total volume of 0.125 ml was used. Tubes contained enzyme, Tris-HC1 buffer at pH 7.5, Mn 2÷, polynucleotide template, one 14C-labeled nucleoside triphosphate, and other unlabeled nucleoside triphosphates at the concentrations given in the figure legends. These tubes were incubated at 30°C for 30 min, and then 0.1 ml aliquots were withdrawn, applied to 2.3 cm discs of Whatman 3 MM paper, and quenched in a cold solution of 5% trichloroacetic acid which contained 1% sodium pyrophosphate. The discs were washed free of acid-soluble radioactivity, dried, counted, and the amounts of product determined as described previously [ 19]. Results and Discussion

Monomer synthesis The lability of the purine-deoxyribose bond makes the synthesis of O~-methyl dGTP more difficult than the corresponding synthesis of O6-methyl GTP. The overall synthetic scheme is shown in Fig. 1. A yield of 100% was achieved in the acetylation reaction (Step I) and, by observing strictly anhydrous conditions, a yield of 15% was obtained in the chlorination reaction (Step II). 6-Chloro-3',5'-di-O-acetyl deoxyguanosine was converted in 100% yield to O6-methyldeoxyguanosine (Step III). Although yields ranged from 30 to 40% in the enzymatic phosphorylation and subsequent steps to the triphosphate, unconverted material was recovered and recycled at each step by performing the column separations in a cold room. Purity of the product was tested at the nucleoside level by high pressure liquid chromatography on a ~-Bondapak CI8 column (4 mm × 30 cm), which was eluted with 0.05 M KH2PO4, pH 4.5. The eluent was monitored at 254 nm and a major peak o f O6-methyldeoxyguanosine appeared which was contaminated with a small a m o u n t (approximately 5%) of O6-methylguanosine. This contaminant was removed at the nucleotide level by chromatography on AG-1. Further purification of the mono- and triphosphates followed on DEAESephadex. Spectroscopic properties of the purified monomers are given in Table I. In general, the ultraviolet absorption maxima and minima o f the deoxyribose derivatives are rather similar to the values for the corresponding ribose derivatives [16]. The retention times for thin layer chromatography on 300-PEI paper in 0.3 M KH2PO4 (pH 4.5) were m~dGMP, 1.00; m6dGDP, 0.44; m6dGTP, 0.22. As before, this m e t h o d o l o g y proved useful in following the conversion of the

775 TABLE I ULTRAVIOLET SPECTRAL DATA FOR DEOXYGUANOSINE DERIVATIVES Compound

Di-O-acetyl d G u o 6-Cl-di-O-acetyl d G u o m6dGuo rn6dGMP m6dGTP

pH 1 *

p H 7 **

p H 11 ***

kmax

kmin

kmax

kmin

kmax

kmin

258 315 241 287 238 288 245 287 247

229 272 234 259 -263 -261 233

255 307 244 282 247 281 249 280 248

225 268 234 262 228 263 231 262 229

262 306 -282 249 280 250 280 247

233 --262 237 264 234 262 237

* p H 1, 0.1 N HCI. ** p H 7, 0 . 0 5 M Tris-HCI. *** p H 110 0 . 0 1 N N a O H .

m o n o p h o s p h a t e to the di- and triphosphates. It also provided a useful check on purity at this stage of the synthesis.

Polymer syn thesis In general terminal deoxynucleotidyl transferase [17] will polymerize deoxynucleoside triphosphates to high molecular weight polymers in the presence of an initiator and an appropriate divalent metal ion. We used d(pT)3 as initiator and obtained a good yield of poly(dC) in the presence o f Co 2÷. However, we were unable to obtain the h o m o p o l y m e r of O6-methyl dGMP either under these polymerization conditions or with Mg 2÷ in place of Co s*. Copolymers suitable for testing the base pairing properties of O6-methyl deoxyguanosine were obtained with relative ease, although these experiments confirmed that O6-methyl dGTP is a poor substrate for the polymerization enzyme. This is shown in Fig. 2, which relates polymer yield to the amount of dGTP or m6dGTP added. The yield was decreased by only a small fraction when dGTP was added b u t the addition o f O6-methyl dGTP resulted in a marked decrease. As shown in Fig. 3, the composition of the polymer was directly related to the composition of the polymerization mixture in both cases. Under these polymerization conditions, the percent of deoxyguanosine in poly (dC, dG) was close to the percent of dGTP in the polymerization mixture. However, the percent of O6-methyldeoxyguanosine in poly (dC, m 6 d G ) w a s considerably less than the percent of O6-methyl dGTP in the polymerization mixture. This again indicates that O6-methyl dGTP is an indifferent substrate for this enzyme.

Template properties of m~dGMP-containingcopolymers Using the m e t h o d o l o g y described above, it was possible to obtain copolymers of dCMP which contained a few percent of O6-methyl dGMP as well as the corresponding control polymers of dCMP and dGMP. These copolymers were then used as templates to direct the polymerization of nucleoside

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Fig. 4. D e p e n d e n c e o f G M P i n c o r p o r a t i o n o n t h e a m o u n t o f t e m p l a t e a d d e d . I n c u b a t i o n m i x t u r e s ( f i n a l v o l u m e , 0 . 1 2 5 m l ) c o n t a i n e d : Trla-HCl b u f f e r , p H 7 . 5 , 1 2 . 5 p n o l ; M n C I 2 , 0 . 1 2 5 p m o l : [ 1 4 C ] G T P ( 0 . 2 1 Ci/mol)0 0 . 1 5 p m o l ; U T P , 0 . 1 5 # t o o l ; e n z y m e , 3 1 p g ; a n d t e m p l a t e as i n d i c a t e d . ~, p o l y d C ; @, p o l y ( d C , d G ) c o n t a i n i n g 1.795 d G ; o , p o l y ( d C , d G ) c o n t a i n / r i g 5 . 0 % d G ; o. p o l y ( d C , m 6 d G ) c o n t a i n i n g 0.495 m 6 d G ; m, p o l y ( d C , m 6 d G ) c o n t a i n i n g 2% m 6 d G . Fig. 5. D e p e n d e n c e o f U M P i n c o r p o r a t i o n o n t h e a m o u n t o f t e m p l a t e a d d e d . I n c u b a t i o n m i x t u r e s as in Fig. 4, e x c e p t t h a t U T P w a s 1 4 C - l a b e n e d ( 0 . 2 1 C i / m o l ) i n s t e a d o f G T P . C T P ( 0 . 1 5 ~ m o l ) w a s a d d e d in t h e l o w e r p a n e l s (C a n d D ) , b u t a b s e n t in t h e u p p e r p a n e l s (A a n d B). S y m b o l s as in FiB. 4.

777

triphosphates by bacterial RNA polymerase. In such experiments, the question is whether the presence of O6-methyldeoxyguanosine can lead to the incorporation of a nucleotide other than the CMP called for by unaltered guanosine. Base line conditions for RNA polymerase incorporations were established as described earlier [ 20]. The overall capacity of the templates to direct RNA synthesis under these conditions is shown in Fig. 4. In these experiments, template was incubated with enzyme in the presence of ~4C-labeled GTP and unlabeled UTP for comparison with the misincorporation data shown in Fig. 5. Since each polymer consists mostly of cytosine, the level of GMP incorporation gives information on the comparative efficiency of the various templates. Although the amount of GMP incorporation increases as the amount of template is increased, it is clear that the purine-containing templates are less efficient than poly(dC). Since there is no adenosine in any of the copolymers, incorporation of UMP by these templates must be considered misincorporation. In order to test for this phenomenon, templates were incubated in the presence of enzyme, 14Clabeled UTP and unlabeled GTP. Such experiments are shown in the top panel of Fig. 5. It is clear from these data that both poly (dC, dG) (Fig. 5A) and poly (dC, m6dG) {Fig. 5B) lead to the incorporation of significant amounts of UMP in the absence of the preferred substrate, CTP. However, as shown in the bottom panel of this Figure, poly(dC, dG) does not cause any incorporation of UMP when CTP is present (Fig. 5C). In marked contrast, the misincorporation of UMP which is directed by poly(dC, m6dG) is not prevented by the presence of CTP as shown in Fig. 5D. This, of course, is what would be predicted if the normal base-pairing properties of guanosine were destroyed by substitution at the O6-position. The data reported here confirm our previous experiments with polyribonucleotide templates in which the presence of O6-methylguanosine led to the misincorporation of UMP in the product copolymer. Since very similar results are obtained regardless of whether the O6-methylguanine is in an RNA or DNA template, these results greatly strengthen the argument that it is the substituted base itself, O6-methylguanine, which leads to the misincorporation of uracil. This, in turn, strengthens the hypothesis that formation of O6-methylguanine is a promutagenic event.

Acknowledgement This work was supported in part by Grants CA 20129 and 20292 from the National Cancer Institute Department of Health, Education, and Welfare. References 1 2 3 4 5 6 7 8

Loveless, A. ( ] 9 6 9 ) Nature 223, 206--207 Lawley, P.D., Orr, D.J., Shah, S.A., Farmer. P.B. and Jarman, M. (1973) Bioehem. J. 135, 193--201 L udlum, D.B. and Wilhelm, R.C. (1968) J. BioL Chem. 243, 2760--2753 Singer, B. and Fraenkel-Conrat. H. (1970) Biochemistry 9, 3694--3701 Singer. B. (1976) Nature 264, 333--339 Singer. B. (1975) Progress Nucleic Acid Res. Mol. Biol. 15, 219--332 Craddock, V. (1973) Biochim. Biophys. Acta 312, 202-- 210 O'Connor. P.J.. Capps` M.J. and Craig, A.W. (1973) Br. J. Cancer 2 7 , 1 5 3 - - 1 6 6

778 9 10 11 12 13 14 15 16 17 18 19 20

Kleihues, P. a n d Magee, P.N. ( 1 9 7 3 ) J. N e u r o c h e m 20, 5 9 , f r - 6 0 6 G o t h , R. a n d R a j e w s k y , M.F. ( 1 9 7 4 ) Proc. Natl. Acad. Sci. U.S. 7 1 , 6 3 9 - - 6 4 3 G e r c h r a a n , L.L. a n d L u d l u m , D.B. ( 1 9 7 3 ) Bioehim. Biophys. A c t a 308, 3 1 0 - - 3 1 6 Schuller, H., W e i m a n n , G.. Lerch, B. and K h o r a n a , H.G. ( 1 9 6 3 ) J. Am. Chem. Soc. 85, 3 8 2 1 - - 3 8 2 7 Gerster, J.F., Jones, J.W. and R o b i n s , R.K. ( 1 9 6 3 ) J. Org. Chem. 28, 9 4 5 - - 9 4 8 Strider. W., Harvey, C. and N u s s b a u m , A.L. ( 1 9 6 8 ) J. Med. Chem. 11, 5 2 4 - - 5 2 7 H a r v e y , C.L., Clericuzio, E. a n d N u s s b a u m , A.L. ( 1 9 7 0 ) Anal. Biochem. 36, 4 1 3 - - 4 2 1 G e r c h m a n , L.L., D o m b r o w s k i , J. and L u d l u m , D.B. ( 1 9 7 2 ) Biochim. Biophys. A c t a 272, 6 7 2 - - 6 7 5 Y o n e d a , M. a n d Bollum, F.J. ( 1 9 6 5 ) J. Biol. Chem. 240, 3 3 8 5 - - 3 3 9 1 N a k a m o t o , T., F o x , C.F. a n d Weiss, S.B. ( 1 9 6 4 ) J. Biol. Chem. 239, 1 6 7 - - 1 7 4 L u d l u m , D.B. ( 1 9 7 1 ) Biochim. B i o p h y s . A c t a 2 4 7 , 4 1 2 - - 4 1 8 L u d l u m , D.B. ( 1 9 7 1 ) Biochim. B i o p h y s . A c t a 247, 4 1 2 - - 4 1 8