ENZYMATIC DEAMINATION OF ADENOSINE DERIVATIVES*

BY NATHAN 0. KAPLAN, SIDNEY P. COLOWICK, AND

MARGARET M. CIOTTI

(From the McCollum-Pratt Institute, The Johns Hopkins University, Baltimore, Maryland)

(Received for publication, July 18, 1951)

Enzymes capable of deamination of adenosine and adenylic acid have been found in a number of animal tissues and in microorganisms. A specific deaminase from muscle acting only on 5-adenylic acid was dis- covered by Schmidt (l), and more recently has been further purified by Kalckar (2). A specific adenosine deaminase also exists in animal tissues (2). Mitchell and McElroy (3) have described and partially purified a deaminase from taka-diastase which acts on adenosine. Adenylic acid was also found to be deaminated by this Aspergillus preparation; however, a potent phosphatase was associated with the deaminase, and, therefore, no conclusions could be made as to the direct deamination of the nucleo- tide. The present communication deals with the further purificatibn of the taka-diastase enzyme. Evidence is presented demonstrating not only the direct deamination of adenylic acid by this enzyme, but also the direct deamination of a number of adenylic acid derivatives, such as DPN, ATP, ADP, and ADPR.

Materials and Methods

Desamino DPN was prepared essentially according to the procedure of Schlenk et al. (4). 200 mg. of DPN were dissolved in 2 M acetic acid (20 ml.) and 1.6 gm. of NaNOz in 6 ml. of water were added dropwise. After standing at room temperature for 4 hours, the mixture was neutral- ized to phenol red with 5 N NaOH. 1.0 ml. of 25 per cent barium acetate and 1 volume of alcohol were added. The resulting precipitate was re- moved and found to contain only small amounts of the desamino com- pound. Alcohol was then added to 90 per cent, and this precipitate collected and washed with alcohol and ether. The yield of barium salt was 133 mg.

* Contribution No. 14 of the McCollum-Pratt Institute. Supported by a grant from the American Cancer Society, as recommended by the Committee on Growth of the National Research Council.

The following abbreviations will be used: ATP adenosinetriphosphate, ADP ad- enosinediphosphate, ADPR adenosinediphosphate ribose, ITP inosinetriphosphate, IDP inosinediphosphate, DPN diphosphopyridine nucleotide, TPN triphosphopyri- dine nucleotide.

579

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580 DEAMINATION OF ADENOSINE DERIVATIVES

ITP was prepared from ATP by treatment with nitrous acid. 100 mg. of ATP (free acid, Schwarz) were dissolved in 5 ml. of 2 N acetic acid and 800 mg. of NaN02 in 3 ml. of Hz0 were added dropwise. The mixture was allowed to stand at room temperature for 3 hours, and then neutral- ized to phenol red with 5 N NaOH. After addition of 0.5 ml. of 25 per cent barium acetate and 1 volume of alcohol, the barium salt of ITP was centrifuged, washed with alcohol and ether, and dried over PZOs. The yield of barium salt was 119 mg.

The DPN samples used in the present experiments were a commercial preparation of 50 per cent purity and a preparation of 80 per cent purity isolated by chromatography from sheep liver. TPN of 75 per cent purity was also prepared from sheep liver by an unpublished method of Kornberg and Horecker. Adenosinediphosphate ribose was prepared as described previously (5). ADP was obtained from the Sigma Chemical Company. 5-Adenylic acid was obtained from the Ernst Bischoff Company; adenylic acids a and b were kindly supplied by Dr. Waldo E. Cohn.

Alcohol dehydrogenase was prepared by the procedure of Racker (6). The yeast hexokinase was a preparation of approximately 50 per cent purity, and was prepared by the procedure of Berger et al. (7). Prostatic phosphatase was generously furnished by Dr. Gerhard Schmidt. Myo- kinase and the Neurospora DPNase were prepared as described in previous publications (5, 8). The deaminase was assayed by measuring the rate of deamination of adenosine; the decrease of optical density at 265 rnp was used as the spectrophotometric determination of the deamination. 3 ml. of 8 X 10M5 M adenosine in 0.1 M phosphate of pH 6.8 were used in the test system for the enzyme. An optical density change of 0.01 for the period from 15 seconds to 120 seconds after the addition of enzyme was used as a unit of the enzyme activity. Protein was determined by a modification of the Herriott method (9).

Results

Purification of Enzyme-The initial purification of the enzyme was car- ried out as described by Mitchell and McElroy (3).’ The final step in the procedure of these authors, which is a 65 per cent alcohol precipitation, was used as our starting material. From 100 gm. of taka-diastase, 300 ml. of solution were obtained with a specific activity of 12 units per mg. of protein. Acetone was added at 0” to this solution to 23 per cent con- centration and the precipitate removed by centrifugation at 0”. The so- lution was then brought to 40 per cent acetone concentration; this precipi-

1 The taka-diastase powder was kindly supplied by Parke, Davis and Company. We wish to thank Dr. W. D. McElroy for his suggestions on the purification of the enzyme and for his interest in this work.

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KAPLAN, COLOWICK, AND CIOTTI 581

tate contained most of the deaminase activity. A 50 per cent acetone fraction contained more of the enzyme, but the purity of the enzyme was much less than in the 40 per cent fraction.

The 40 per cent acetone precipitate was dissolved in 100 ml. of HzO, and showed a 4-fold purification with a 30 per cent yield (see Table I). 70 ml. of 95 per cent alcohol were added at 0” and the precipitate which contained only a small amount of activity was discarded. The super- n&ant was then chilled to -12” and, as a result, a second precipitation took place. This precipitate was centrifuged at -12” and dissolved in 25 ml. of HzO. This step resulted in a S-fold purification.

The - 12’ alcohol fraction was then brought to 70 per cent ammonium sulfate saturation. The precipitate contained a good deal of the deaminase activity, but also contained dark material and was not as pure as higher

TABLE I Summary of Purification

Total units Units per mg. Per cent reccwery

Starting material, 65yo alcohol ppt. of Mit- chell and McElroy (3). . . . . 84,000 12

40% acetone ppt.. . 26,500 53 30.8 -12” alcohol “ . . . 10,250 146 12.2 Ammonium sulfate ppt., O-70.. . 3,650 108 4.4

L‘ ‘< <c 70-90. . . 3,070 242 3.1 “ I‘ <c 9~100. 1,800 290 2.1

ammonium sulfate fractions and was discarded. Fractions were obtained between 70 and 90 per cent saturation and also between 90 and 100 per cent saturation. These fractions were either devoid of phosphatase or contained only traces of this enzyme. The purification scheme, as out- lined in Table I, has been carried out several times; although the yields varied considerably, the purity in the ‘final ammonium sulfate fractions could be duplicated. The 70 to 90 and 90 to 100 per cent ammonium sul- fate fractions were used in most of the experiments described beldw.

Action of Deaminase on DPN-The action of the taka-diastase deaminase on DPN results in a decrease in absorption at 265 rnp, indicating a removal of the amino group from the adenine ring (Fig. 1). The experiments shown in Fig. 1 were carried out by incubating DPN with the enzyme in phos- phate buffer (pH 6.8) and removing aliquots for analyses at 265 rnl.c at different time intervals. At the same periods, samples were taken for DPN analyses by yeast alcohol dehydrogenase. As can be seen from Fig. 2, incubation of the DPN with the deaminase results in a decreased rate of response of the deaminated DPN to the alcohol dehydrogenase;

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582 DEAMINATION OF ADENOSINE DERIVATIVES

the longer the incubation time with the deaminase, the slower was the rate of reduction of the pyridine nucleotides, although the total extent

0.8 -

0.4 -

I I I I 15 30 45 60

TIME IN MINUTES FIG. 1. Deamination of DPN by taka-diastase deaminase. 3 PM of DPN were

incubated with 50 units of deaminase in phosphate buffer (pH 6.8) in a total volume of 1 ml. Aliquots were removed at the various time intervals and fixed with per- chloric acid for spectrophotometric determination at 265 rnp.

,500

0 0 2 4 6 8 IO 12 14

TIME IN MINUTES FIG. 2. Reaction of deaminase product of DPN with crystalline yeast alcohol

dehydrogenase. The various curves represent the time intervals of Fig. 1; the alcohol dehydrogenase tests were carried out in 0.5 M alcohol and 0.1 M tris(hydroxy- methyl)aminomethane (pH 10) in a volume of 3 ml. The reaction was started by addition of the alcohol dehydrogenase.

of reduction remained almost unchanged. Schlenk et al. (4) have pre- viously shown that chemically prepared d&amino DPN reacts at a slower rate with yeast alcohol dehydrogenase. The fact that the product from

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KAPLAN, COLOWICK, AND CIOTTI 583

the deaminase reaction also reacts at a similarly slow rate is indicative that the product is desamino DPN and that the deaminase promotes the direct deamination of DPN. Evidence demonstrating that the deaminase which deaminates DPN is the same enzyme that attacks other adenine derivatives is given in Fig. 3. By measuring the formation of desamino DPN as determined by the rate of reaction of the yeast alcohol dehy- drogenase system, the inhibition of DPN deamination can be ascertained. Adenosine and adenylic acid inhibit the deamination of DPN, as indicated by the faster rate of reaction to the alcohol dehydrogenase system after

0 2 4 6 8

TIME IN MINUTES FIG. 3. Effect of adenosine and adenylic acid on deamination of DPN as measured

by alcohol dehydrogenase. All tubes contained 2 X 1OW M DPN in phosphate buffer of pH 6.7 with or without 2 X 10-B M adenosine or 2 X 1OW M adenylic acid. 50 units of enzyme. Final volume 1 ml. Incubation period 20 minutes at 37”. Alcohol dehydrogenase tests were carried out as in Fig. 2. 10 y of alcohol dehydrogenase so- lution were used for assay.

incubation with the deaminase in the presence of these two substances. This then is indicative that less desamino DPN is formed and that ade- nosine and adenylic acid competitively inhibit the deamination of DPN. The inhibition can be correlated to the affinities of the various compounds for the deaminase (see Table III).

Reduced DPN is also deaminated by the taka-diastase enzyme. Evi- dence that reduced desamino DPN is formed can be obtained by the slower rate of reaction of the product with acetaldehyde and alcohol de- hydrogenase. It is of interest to mention that only enzymatically reduced DPN is deaminated by the enzyme. Chemically reduced DPN does not react; this is probably due to inhibition by sulfite.

Deamination of TPN-In Fig. 4 the activity of the enzyme on ade-

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584 DEhMINATION OF ADENOSINE DERIVATIVES

nosine derivatives is compared. ADP, ATP, and ADPR, as well as DPN, are deaminated by the enzyme. However, TPN is not attacked.

Kornberg and Pricer (10) have recently reported that the difference between TPN and DPN is in an additional phosphate group on the ribose

a w" 0

2 6 IO 14 18 22 26 30 L

34

TIME IN MINUTES FIG. 4. Deamination of adenylic acid derivatives by taka-diastase deaminase.

All substrate concentrations 8 X 1OP M in 3 ml. of phosphate buffer (pH 6.8). 30 units of purified deaminase added in each case to initiate reaction.

of the adenylic portion of TPN. This additional phosphate group has a monoester linkage.

Schmidt et al. (11) have shown that prostatic phosphatase will act only on monoester groupings and not on phosphate diesters. We have found that the prostatic phosphatase will cleave the monoester linkage in TPN and yield DPN, which can be measured by yeast alcohol dehydrogenase (Table II). Table II also shows that the action of the prostatic enzymes makes the TPN subject to deamination. It is therefore evident that the presence of the monoester group in TPN prevents the action of the taka-diastase enzyme.

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KAPLAN, COLOWICK, AND CIOTTI 585

Deamination of Various Adenylic Acids-In Fig. 4 the rates of deamina- tion by the enzyme of various adenylic acids are compared, when equal concentrations of the substances are used. 5-Adenylic acid is dean&rated at a rate approximately one-half that of adenosine. Adenylic acid b (3- adenylic acid) is also deaminated by the enzyme, the rate being somewhat

TABLE II Deanzination of TPN and DPN

Reaction mixture in a volume of 1 ml. in acetate buffer of pH 5. Incubation period, 30 minutes at 37”. Appropriate aliquots were then taken for alcohol dehy- drogenase and deaminase determinations.

DPN, measured by Deamination alcohol dehydro- by takadiastase

genase deaminase

IrM w

20 LLLM TPN + boiled prostatic phosphatase.. . . . 0 0 20 (( “ + prostatic phosphatase. . . . . . . 9.8 10.5

0.300

z w” 0.200 a

0 I 00

0 2 4 6 8 IO 12 0 2 4 6 8 IO 12 0 2 4 6 8 IO 12 TIME IN MINUTES

FIG. 5. Effect of prostatic phosphatase on rates of deamination of various adenylic acids. Treated samples were incubated with 0.05 ml. of purified prostatic phospha- tase in 0.1 M acetate buffer (pH 5.8) for 15 minutes. After incubation 2.7 ml. of 0.1 M phosphate buffer (pH 6.8) were added. 10 units of purified deaminase were added to start the reaction. Untreated samples were incubated with 0.05 ml. of boiled prostatic phosphatase.

slower than that of the muscle adenylic acid. However, adenylic acid a (2-adenylic acid?) is not attacked by the deaminase. Yeast adenylic acid, which is a mixture of a and b forms, is deaminated at a rate slower than the b isomer. The data in Fig. 4 also illustrate that the yeast prepa- ration is deaminated to an extent of only approximately 60 per cent. This would be in good agreement with the view that yeast adenylic acid is a mixture of the two isomers.

In Fig. 5, the rates of deamination of the various adenylic acids before and after treatment with prostatic phosphatase are compared. It is evi-

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586 DEAMINATION OF ADENOSINE DERIVATIVES

dent that incubation with the phosphatase converts all the adenylic acids into products which are deaminated at identical rates and at a rate which is comparable to the action of the enzyme on adenosine. This would seem to suggest that the position of the phosphate is the factor which governs the rate of reaction of the three adenylates to the deaminase.

&@&es of Various Substrates-Table III summarizes data comparing the affinities of a number of adenine derivatives for the deaminase. The Michaelis constants (K,) for the substances are roughly of the same order of magnitude, although adenosine, 5-adenylic acid, and ADP have the highest affinities for the enzyme. The K, for DPN, ADPR, and adenylic acid b is approximately twice that of adenosine. These differences in

TABLE III

Afinities of Various Substrates for Deaminase

The data were obtained by measuring the initial rates of deamination (10 min- utes) with several concentrations, at 37”. Deamination determined by change at 265 mH. The K,,, of each substrate was determined from a graphic plot.

Substance Approximate Km

ATP .................................................... DPN ................................................... 5-Adenylic acid. ....................................... ADP ................................................... Adenylic acid b ......................................... ADPR. ................................................. Adenosine ..............................................

dl x 10-a

1.2 1.8 0.8 0.7 1.7 1.5 0.6

affinity account in part for the rate differences found at low substrate concentrations (Fig. 4).

Isolation of Desamino DPN from Deaminase Reaction Mixture-Isolation of the desamino product from the enzymatic reaction was carried out as follows: 100 mg. of DPN (dissolved in 2 ml. of water) were brought to pH 6.8 with 0.1 N NaOH, and then 1 ml. of 0.2 M glycylglycine (pH 6.8) plus 0.4 ml. of enzyme (160 units) was added. After a 40 minute incuba- tion period, the reaction mixture was adjusted with NaOH until pink to phenolphthalein and 0.5 ml. of 25 per cent barium acetate added; this was followed by the addition of a volume of alcohol. The procedure was carried out at room temperature. After removal of the precipitate, the supernatant fluid was placed in an ice bath, and, as a result, a second flocculent precipitate was obtained, which contained nearly all of the de- aminated product of DPN. This precipitation, in the cold, resembles that observed with ADPR and DPN, which has been noted previously (5). Addition of 2 more volumes produced a further precipitation, but

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KAPLAN, COLOWICK, AND CIOTTI 587

this product contained only a small amount of the desamino DPN. In the preparation of the chemical desamino DPN more alcohol was required to precipitate the barium salt; this is most likely due to the presence of nitrite.

After removal of barium, the amount of nicotinamide riboside linkage was determined by the cyanide reaction.2 The yield of the deaminated product was found to be approximately 65 per cent. The absorption

A ENZYMATIC INOSINE TRIPHOSPHATE A CHEMICAL INOSINE TRIPHOSPHATE

o ENZYMATIC o ENZYMATIC DESAMINO DESAMINO DPN DPN . CHEMICAL DESAMINO DPN . CHEMICAL DESAMINO DPN

240 260 280 300

MI1 FIG. 6. Absorption spectra of chemically and enzymatically prepared desamino

DPN and ITP. Desamino DPN concentration based on absorption of cyanide complex. (Millimolar extinction of 6.3 at 325 rnp (12).) Concentration of ITP based on labile P analyses. Spectra determined in neutral solution.

spectrum of the enzymatic preparation is compared to the synthetic des- amino DPN in Fig. 6 and indicates the close similarity of the two prepara- tions. Both have absorption maxima at 249 rnp.

The activity of the synthetic desamino compound on the yeast alcohol dehydrogenase system is identical to that of the enzymatic product. Both compounds have much lower activities with this enzyme. Further verifi- cation of the identity of the chemically and enzymatically prepared com-

2 The pyridine nucleoside linkage remains intact after treatment with the deami- nase; this is indicated by the fact that the cyanide reaction of DPN (12) is not af- fected by incubation with the enzyme.

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588 DEAMINATION OF ADENOSINE DERIVATIVES

pounds is indicated by the much slower rate of splitting of these compounds by the purified DPNase from Neurospora (Fig. 7).

Isolation of ITP-Inosinetriphosphate has been isolated as a reaction product of the action of deaminase on ATP. For the isolation, 100 mg. of ATP (free acid, Schwarz) were dissolved in 3 ml. of 0.2 M glycylglycine

100

e 0

g 80- o DPN

F A ENZYMATIC DESAMINO DPN I- 60- . SYNTHETIC DESAMINO DPN

IO 20 30 40 50 6(

TIME IN MINUTES

J

FIG. 7. Action of Neurospora DPNase on desamino DPN. The reaction mixtures contained 0.2 PM of each nucleotide and 100 units of purified Neurospora DPNase in 0.1 M KHzPOd in all cases. Appropriate aliquots were removed at different times and amount of splitting determined by cyanide reaction.

TABLE IV Activities of Chemically and Enzymatically Prepared ITP in Hexokinase and

Myokinase Reactions

All samples contained 0.01 M MgClp, 0.05 M glucose, 0.05 M NaHC03 in a total volume of 1 ml. 13 units of hexokinase and 0.05 ml. of myokinase were used in the determinations. Time of incubation, 60 minutes; temperature 37”.

Additions ATP Chemical ITP Enzymatic ITP

JIM 7 w&i% P JIM 7 min. P PM 7 min. P

None . 5.1 4.4 4.6 Hexokinase 3.0 2.5 2.9

‘I + myokinase 0.6 2.6 2.9

buffer of pH 6.8 and 200 units of purified deaminase were added. The final volume was 6 ml., and the pH was adjusted to pH 6.8 with NaOH. After incubation for 90 minutes at 37”, the mixture was placed in a boiling water bath and then neutralized to phenol red with alkali. 0.5 ml. of 25 per cent barium acetate was added, along with a volume of alcohol, and the precipitate removed by centrifugation. The precipitate was washed with 50, 95, and 100 per cent alcohol, and finally with ether. After dry- ing, the yield was 164.9 mg. The absorption spectrum of the enzymatic

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KAPLAN, COLOWICK, AND CIOTTI 589

product is compared to that of the chemically prepared inosinetriphosphate in Fig. 6; the two spectra are identical.

Kleinzeller (13) has reported that ITP is active in the hexokinase system, but reacts at a much slower rate than does ATP. This has been confirmed by using limiting amounts of hexokinase; both the chemical and enzymatic inosinetriphosphate preparations have been found to react at similar slow rates. Kleinzeller has also indicated that inosinediphosphate is not ac- tive in the myokinase reaction. We have also confirmed this observation (Table IV). It can be seen that addition of myokinase promotes a marked increase in phosphate transferred from ATP in the presence of a large excess of hexokinase, whereas addition of myokinase under the same con- ditions caused no changes with the chemical ITP or the enzymatic prepa- ration. This, therefore, is further verification that the product of de- aminase action on ATP is ITP.

DISCUSSION

Enzymatic deamination of ATP and DPN suggests the possibility that the deaminated derivatives of these compounds may have some rble in normal cellular metabolism. Evidence has been obtained which indicates that desamino DPN is as active as DPN in a number of dehydrogenase systems, whereas in other dehydrogenase reactions the deaminated co- enzyme has little activity or is completely inactive.3

Kleinzeller (13) has found that myosin ATPase attacks inosinetriphos- phate at as rapid a rate as ATP. However, the deaminated product does not function in muscle contraction and has not been detected in muscle. As yet, deamination of ATP and DPN has been found only with the taka-diastase preparation.

The taka-diastase deaminase is unique in its relative non-specificity when compared to other deaminases which have been studied. The muscle ade- nylic acid deaminase has been found to be specific for 5-adenylic acid, whereas the adenosine deaminases of animal tissues appear to be specific for adenosine (2, 14).

There is still some uncertainty as to the exact structures of adenylic acids a and b. Some evidence has been reported indicating that the two compounds have the phosphate groupings on different positions in the ribose, the a isomer containing phosphate on the 2nd carbon, and the b isomer being linked in the 3 position (15-17). Other evidence has been presented by Doherty (18), which suggests that the two isomers have the phosphate grouping in the same position and that the difference between the two isomers is in the configuration of the glycosidic linkage.

Our evidence would tend to support the view that the difference between

3 Pullman, M. E., Colowick, S. P., and Kaplan, N. O., in preparation.

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590 DEAMINATION OF ADENOSINE DERIVATIVES

the two adenylates is in the position of the phosphate grouping. This conclusion is based on the fact that TPN, which is not deaminated by the taka-diastase enzyme, can be converted to DPN by prostatic phosphatase, which is attacked by the deaminase. Since the conversion of TPN to DPN involves only a removal of the monoester phosphate grouping, it is highly likely that the two pyridine nucleotides have identical glycosidic linkages. Furthermore, adenylic acid a, which is not deaminated by the taka-diastase enzyme, can be deaminized after treatment with the pros- tatic phosphatase. Adenylic acid b is attacked by the deaminase, but at a rate considerably slower than that of adenosine. However, after removal of the phosphate grouping, the rate of deamination becomes much faster and comparable to that of adenosine and that of the dephosphoryl- ated adenylic acid a and 5-adenylic acid. Since all adenylic acid isomers have identical rates of deamination after removal of the phosphate groups, the differences in activity to the deaminase of the intact compounds appear to lie in the position of the phosphate groups. An enzyme catalyzing the formation of TPN from ATP and DPN has been described by Korn- berg (19). This can also be considered as evidence supporting the view that the two yeast adenylates differ in the position of the phosphate group- ings, unless mutarotation occurs in removal or addition of the phosphate group. Although the above evidence strongly points to the structure of adenylic acids a and b as being 2- and 3-phosphoadenosine, final verifica- tion of the structures must await the isolation of the corresponding ribose phosphates.

The non-deamination of either adenylic acid a or TPN can be considered as confirming evidence of Kornberg and Pricer’s (10) observation on the position of the third phosphate in TPN. If adenylic acid a contains the ‘phosphate in the 2 position, the interesting possibility exists that the close proximity of the phosphate group to the adenine nucleus may lead to some interaction with the amino group, which might prevent the action of the deaminase. This would not be the case for the 3- and 5-adenylic acids.

SUMMARY

1. The deaminase from taka-diastase has been found to deaminate 5- adenylic acid, adenylic acid b (3-adenylic acid), DPN, ADPR, and ADP, as well as adenosine. Adenine, TPN, and adenylic acid a (2-adenylic acid ?) are not attacked by the enzyme.

2. Desamino DPN and ITP have been isolated and characterized as the products of the action of the enzyme on DPN and ATP.

3.. After removal of the monoester phosphate grouping of TPN with prostatic phosphatase, DPN is formed, which is deaminated by the taka-

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KAPLAN, COLOWICK, AND CIO!CTI 591

diastase enzyme. The non-activity of the enzyme on TPN and adenylic acid a is confirming evidence for the presence of adenylic acid a in TPN.

4. Details of the further purification of the enzyme are given. Data are also presented on the affinities of the various substrates for the de- aminase.

5. Evidence is presented which suggests that adenylic acids a and b differ in the position of esterification of the phosphate, .rather than in being (Y- and /I-riboside isomers.

BIBLIOGRAPHY

1. Schmidt, G., 2. Physiol. Chem., 179, 243 (1928). 2. Kalckar, H. M., J. Biol. Chem., 167,429,445,461,477 (1947). 3. Mitchell, H. K.,, and McElroy, W. D., Arch. Biochem., 10, 351 (1946). 4. Schlenk, F., Hellstrom, H., and von Euler, H., Ber. them. Ges., 71, 1471 (1938). 5. Kaplan, N. O., Colowick, S. P., and Nason, A., J. Biol. Chem., 191, 473 (1951). 6. Racker, E., J. Biol. Chem., 164, 313 (1950). 7. Berger, L., Slein, M. W., Colowick, S. P., and Cori, C. F., J. Gen. Physiol., 29,

379 (1946). 8. Kaplan, N. O., Colowick, S. P., and Barnes, C. C., J. BioZ. Chem., 191,461 (1951). 9. Herriott, R. M., Proc. Sot. Exp. BioZ. and Med., 46, 642 (1941).

10. Kornberg, A., and Pricer, W. E., Jr., J. BioZ. Chem., 186, 557 (1950). 11. Schmidt, G., Cubiles, R,, Zollner, N., Hecht, L., Strickler, N., Seraidarian, K.,

Seraidarian, M., and Thannhauser, S. J., J. BioZ. Chem., 192, 715 (1951). 12. Colowick, S. P., Kaplan, N. O., and Ciotti, M. M., J. BioZ. Chem., 191,

447 (1951). 13. Kleinzeller, A., Biochem. J., 36, 729 (1942). 14. Schaedel, M. L., Waldvogel, M. J., and Schlenk, F., J. Biol. Chem., 171, 135

(1947). 15. Cohn, W. E., J. Am. Chem. Sot., 72, 1471 (1950). 16. Carter, C. E., J. Am. Chem. SOL, 72, 1466 (1950). 17. Carter, C. E., and Cohn, W. E., Federation Proc , 8, 190 (1949). 18. Doherty, D. G., Abstracts, American Chemical Society, Division of Biological

Chemistry, 118th meeting, Chicago, 56C, Sept. (1950). 19. Kornberg, A., J. BioZ. Chem., 182, 805 (1950).

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Margaret M. CiottiNathan O. Kaplan, Sidney P. Colowick and

ADENOSINE DERIVATIVESENZYMATIC DEAMINATION OF

1952, 194:579-591.J. Biol. Chem. 

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