484 •. c. WEINBACH, W. J. BOWEN ~,'OL. 30 (I958)

(1o -3 M). The e thylenediaminete t raaceta te-s t imulated enzyme was much more sensit ive to inhibi tory action of PCP than the calcium-activated enzyme. The effect of pentachlorophenol is in terpre ted as possible interference with the binding of ATP to the muscle protein.

R E F E R E N C E S

1 E. C. WEINBACH, Proc. Natl. Acad. Sci. U.S., 43 (1957) 393- 2 A. SZENT-GYORGYI, Chemistry of Muscular Contraction, 2nd Ed., Academic Press, Inc., New York,

1951. 3 R. E. RANNEY, J . Appl. Physiol., 6 (1954) 513 • 4 t{. LAKI AND W. J. BOWEN, Biochim. Biophys. Acta, 16 (1954) 3 ol . 5 E. C. W'EINBACH, J. Biol. Chem., 221 (1956) 609. 6 G. A. LEPAGE, in V. R. POTTER, 2VIethods in 3ledical Research, Vol. I, Yearbook Publishers, Inc.,

Chicago, 1948, p. 337. 7 R. L. DRYER, A. R. TAMMES AND J. I. RouTs , J. Biol. Chem., 225 (1957) 177. 8 A. ~VEBER AND VV'. HASSELBACH, Biochim. Biophys. Acta, 15 (1954) 237. 9 Vq. J. BowEN AND T. D. KERWlN, Bioehim. Biophys. Acta, 18 (1955) 83.

10 y . TONOMURA AND S. •ITAGAWA, Biochim. Biophys. Acta, 26 (1957) 15. Xl G. GESKE AND H. H. WEBER, Arch. exptl. Pathol. Pharmakol., 230 (i957) 3 lo. 12 M. F. MORALES AND J. BOTTS, Arch. Biochem. Biophys., 37 (1952) 283. 13 E. T. FRIESS, Arch. Biochem. Biophys., 51 (1954) 17. 14 W. W. I{IELLEY AND L. g. BRADLEY, J . Biol. Chem., 218 (1956) 653. 15 j . B. CHAPPELL AND S. "V. PERRY, Biochim. Biophys. Acta, 16 (1955) 285. le E. T. FRIESS, M. F. MORALES AND \V. J. BOWEN, Arch. Biochem. Biophys., 53 (1954) 311- 17 G. D. GREVlLLE AND E. REICH, Biochim. Biophys. Acta, 20 (1956) 44 o.

Received March 3rd, 1958

THE EFFECT OF DINITROPHENOL ON MAGNESIUM-ACTIVATED

ADENOSINETRIPHOSPHATASE*

CECIL COOPER**

Department o[ Biochemistry, Western Reserve University School o[ Medicine, Cleveland, Ohio ( U.S.A .)

INTRODUCTION

Previous work has demonstrated that particles obtained from rat-liver mitochondria following treatment with digitonin are capable of carrying out oxidative phosphoryl- ation, the exchange of inorganic phosphate and ADP*** with ATP, and the hy- drolysis of ATP which may be activated by uncoupling agents and a number of divalent cations 1, 2,3. It has generally been considered that the activation of ATPase by DNP and other uncoupling agents on the one hand and by Mg++ and certain

* This invest igat ion was suppor ted in pa r t by a research grant RG-53o2 f rom the National Ins t i tu tes of Health.

** Senior Research Fellow, SF-2, U.S. Public Heal th Service. *** The following abbrevia t ions are used: ADP, adenosine diphosphate ; ATP, adenosine tri-

phosphate ; ITP, inosine t r iphospha te ; DNP, 2,4-dinitrophenol ; PCP, pentachlorophenol ; ATPase, adenosine t r iphosphatase ; GTP, guanosine t r iphosphate ; UTP, uridine t r iphosphate ; CTP, cytidine t r iphosphate ; PCMB, parachloromercur ibenzoate; tris, t r i s (hydroxymethyl )amino- methane.

References p. 49-r.

VOL. 30 (1958) DNP aND ATPAsE 485

other divalent cations on the other hand, involves two distinct ATPases. These will be referred to as DNP-ATPase and Mg-ATPase respectively. The suggestion of two varieties of ATPase has been based on differences in: (I) susceptibility to o.3 M sucrose, (2) total activity, (3) stability on aging, (4) nucleotide polyphosphate speci- ficity, and (5) pH optima. The DNP-ATPase was completely inhibited in the presence of o.3 M sucrose whereas the Mg-ATPase was only inhibited IO-2O % under these conditions. The Mg-ATPase usually had 3 to 4 times the total activity of the D N P - ATPase. The DNP-ATPase was completely inactivated after aging the enzyme for ioo min at 37 ° whereas the Mg-ATPase still exhibited 7 ° % of its original activity. The DNP-ATPase was specific for ATP whereas the Mg-ATPase could use other nucleotide triphosphates as substrates with the relative activities being: ATP, IOO; ITP, 6o; GTP, 35; UTP, IO; CTP, 5. The DNP-ATPase had an optimal pH of 7-7.5 whereas that of the Mg-ATPase was approximately 92.

The activation of ATPase has been investigated further and it has been found that DNP and other uncoupling agents can stimulate Mg-ATPase. The properties formerly attr ibuted to a DNP-ATPase can be mainly reconciled with an effect by DNP on Mg-ATPase. These findings make it unnecessary to invoke the existence of an ATPase specifically activated by uncoupling agents and it is suggested that magnesium, either bound or free, is required for a demonstration of ATPase activity under all conditions.

METHODS

The rat-liver particles were prepared as previously described t. The liberation of inorganic phos- pha te was measured by the method of MARTIN AND DOT¥ ~. Protein was determined by the method of LOWRY et al. 5. This method was found to give protein values in good agreement with those determined by the Kjeldahl method assuming a nitrogen content of 16 %.

ATP was obtained f rom the Pabs t Laboratories. I T P was a product of the Sigma ChemicM Co. and was found to be free of detectable adenine nucleotides. This was determined by chromato- graphing an acid hydrolysate of the I T P in two different solvents under conditions tha t should have permi t ted the detection of a contaminat ion in excess of 1-2 %6.

RESULTS

In order to obtain further information on the ATPase enzymes, a study was made of the relationship of the total ATPase activity with increasing amounts of Mg ÷+ in the presence and absence of DNP. Fig. I shows the results of a typical experiment with Mg ++ alone (Curve A), Mg++ plus DNP (Curve B) and also a calculated curve (Curve C) showing the sum of the activities with Mg ++ alone plus that of DNP in the absence of added Mg ++, i.e., the activation of DNP alone is the difference between Curves C and A. The values recorded have been corrected for the liberation of in- organic phosphate which occurs in the absence of DNP and Mg ++ (o.o3 to o.06/,moles). I t is seen that the effect produced by the addition of DNP to various concentrations of Mg ++ is clearly a reflection of the concentration of the Mg ++ added. At low levels of Mg ++ there is very little ATPase activity in the absence of DNP. As the concen- trat ion of Mg ++ is increased, the activity with Mg ++ plus DNP becomes greater than the calculated sum of the two activities measured separately. The activity over and above that calculated by the summation of the two individual activities (Curve B - - Curve C) will hereafter be referred to as the "st imulation" of Mg-ATPase by DNP.

Re]erences p. 49 r .

486 c. COOPER VOL. 30 (1958)

This is to be distinguished from the activity evoked by DNP alone in the absence of added Mg ++ which will be termed "activation" by DNP. At higher levels of Mg ++ the reverse situation obtains and the activity observed in the presence of DNP plus Mg ++ is less than additive. This decreased activity observed at higher levels of Mg in the presence of DNP (Curve C minus Curve B) will be referred to as the "inhibition" of Mg-ATPase by DNP. At still higher Mg ++ concentrations this "inhibition" by DNP becomes very marked and the hydrolysis observed in the presence of DNP plus Mg++ is less than that obtained with Mg ++ alone.

,~2.0

J t.5

o

co 1.0

~o.~

t0-~ 10-4 10-3 CONC MgCt a

Fig. 2. Effect of DNP on Mg ATPase. Curve A, Mg++ alone; Curve B, observed Mg ++ plus 4" IO4 3~ DNP; Curve C, calculated Mg +4 plus 4"1o-~ M DNP. System contained o.oi M tris pH 7.0, o.o06 M ATP pH 7.0, and o.12 mg enzyme protein in a final volume of 0.50 ml. Incubated 20 min

at 2 3 ° .

Three possible explanations of the "stimulation" are suggested. These a r e : ( I )

DNP is affecting the Mg-ATPase, (2) Mg ++ is affecting the DNP-ATPase, or (3) there exists a complex interrelationship between two enzymes and the simultaneous activation of both results in a greater total activity.

The first of these possibilities, namely that Mg-ATPase may be affected by DNP, can be tested by taking advantage of the fact that the DNP-ATPase may be com- pletely inhibited without producing a large decrease in activity of the Mg-ATPase. In the presence of o.3 M sucrose, where presumably only the Mg-ATPase is active, it might be anticipated that the effect of DNP on Mg-ATPase could be determined directly. Fig. 2 shows the results of such an experiment. I t is readily apparent that in spite of the presence of o.3 M sucrose, both the "stimulation" and "inhibition" may still be observed. This result would appear to support the contention that the DNP does in fact affect the Mg-ATPase. A second test of this thesis was made by inhibiting the DNP-ATPase completely by the addition of I- IO ~ M PCMB. Under these circumstances the total activity of the Mg-ATPase is unaffected and in some cases slightly increased. The results obtained by superimposing DNP on this system are identical to those obtained with o.3 M sucrose. At still higher concentrations of PCMB, 5 . I o - 5 M , the "stimulation" is enhanced and the "inhibition" greatly diminished as may be seen in Fig. 3. These findings indicate that DNP stimulates the Mg-ATPase.

The question arose whether the DNP "stimulation" of the Mg-ATPase represents a different action by DNP than does the "activation" produced in the absence of added Mg ÷+. In order to obtain information on this question, properties that had been considered characteristic of the DNP-ATPase were investigated to determine

Refere~wes p. 49I.

VOL. a 0 ( I 9 5 8 ) D N P AND A T P A S E 487

if they applied to the "st imulation" of the Mg-ATPase by DNP. The first such property, the loss of activity in the presence of 0. 3 M sucrose, has already been shown not to apply. A second property of the DNP-ATPase was a much lower total activity than that of the Mg-ATPase. I t may be seen in Fig. i that the effect of DNP is to cause the production of maximum Mg-ATPase at a lower concentration of Mg+%

~2.0

# ,.c

~o.51

~ A , C

~a~ /c /

i0-~ i0 -4 i0-~ GONG MgC/2 m

Fig. 2. Effect of sucrose on " S t i m u l a t i o n " by DNP. Curves A, B, and C same as in Fig. i . Sys t em con ta ined o.o[ 3q t r i s p H 7.o, o.oo6 ll, I ATP p H 7.0, 0.3 21/I sucrose and o . ] i mg enzyme p ro t e in in a final vo lume of o.50 ml. I n c u b a t e d 2o rain

a t 25 °.

"~ 2.0

.g 1.5 Fig. 3. Effect of PCMB on " S t i m u l a t i o n " c~

by DNP. Curves A, B, and C same as in 0. Fig. i . Sys t em con ta ined o .o i M t r i s p H ~ i.o 7.o, o.oo6 31 ATP p H 7.o, 5 ' lO-5 .}I PCMB, and o.13 mg enzyme pro te in in a "~ 0.5 final vo lume of o.5o ml. I n c u b a t e d 2o rain

a t 2 3 °- 10-e

/ , K / " ~ - f ~ . ~ "B A.C

/6 /

/ /

./a j

i0-~ lO-S CONC MgCI 2 m

The total activity evoked however is the same in the presence and absence of the DNP. The stability to aging has been investigated and some of the changes are shown in Fig. 4. I t may be seen that : (a) the "activation" of ATPase elicited by DNP alone was gradually lost, until after IOO min of aging at 37 ° no response could be obtained, (b) the response to Mg++ was diminished initially with aging, and (c) the maximum "st imulat ion" by DNP was unaffected by aging. Another effect may be seen in Fig. 5. On aging, the "st imulation" occurs over an increasing range of concentrations of Mg ++ whereas the "inhibition" evoked by DNP at higher levels of Mg++ is gradually abolished over the range of concentrations of Mg ++ customarily employed. The plot represents the difference between the phosphate liberation obtained in the presence of Mg++ plus DNP (Curve B) and that calculated by the summation of the activities in the presence of Mg ++ alone and DNP alone (Curve C). These results demonstrate that DNP, in the presence of added Mg++, can still produce "st imulation" of ATPase after prolonged aging despite the fact that DNP alone can no longer "ac t iva te" ATPase.

A fourth property cited previously in favor of the two ATPases is that of speci- ficity differences. The Mg-ATPase gave maximal activity with ATP but also caused the liberation of inorganic phosphate from other nucleoside triphosphates whereas the DNP-ATPase appeared to be absolutely specific for ATP. When the assay was carried out with I T P as the substrate it was found that there was no enhancement of the hydrolysis by DNP alone. However, in the presence of Mg++, DNP was found

Re/erences p. 49;:.

488 c. COOPER VOL. 30 (I958)

to produce a " s t imu la t i on" and " inh ib i t ion" of the hydrolys is of ITP. The m a g n i t u d e of this " s t imu la t i on" was small bu t significant. In tests wi th ATP i t was found t ha t when the concent ra t ion of subs t ra te was o.o2 to 0.04 M ins tead of the 0.o06 M cus tomar i ly employed, D N P caused a considerably greater " s t imu la t ion" of ATPase and the " s t imu la t i on" pers is ted over the ent ire range of Mg ++ cus tomar i ly employed. The exper iment wi th I T P was therefore repea ted with 0.04 M I T P as the subs t ra te .

o 1.5! iJJ

5 113

o.l.O a .

o :~O.5

o 2b

"STIMULATION"

~ .~ACTIVATION" ~ x

So do e"S" ~6o AGING T~ME~M,.~

--z 0_+0.4

<z

F- m.m uJ- 8 o

-0 .2 v-

~ - 0 . 4

-O.E i0 -s

Fig. 4- Effect of aging. System contained o.oi M tris pH 7.o, o.oo6 M ATP pH 7.o and o.12 mg enzyme protein in a final volume of o.5o ml. DNP present at 4" lO-4 M where indicated. In- cubated 2o rain at 23 °. Enzyme aged for in- dicated times at 38°. Curve with Mg ++ alone

represents activity at 0.0o 4 M Mg ++.

tO'" I0 -s CONC MgCt2 m

Fig. 5. Effect of aging on " S t i m u l a t i o n " by DNP. Same system as in Fig. 4. Curves re- present the differences between the observed and calculated activity in the presence of Mg ++ plus 4" I°-42~I DNP at the indicated periods

of aging.

The resul ts of this exper iment are shown in Fig. 6. I t is obvious t ha t a ve ry sub- s t an t i a l " s t imu la t i on" of phospha te l ibera t ion can be effected b y D N P with I T P as the subs t ra te . GREVILLE AND I~EICH 7 have repor ted t ha t D N P accelerates the hydro lys i s of ATP and CTP b y L-myosin, and SACKTOR AND COCHRAN 8 have repor ted an accelera t ion of the hydro lys i s of all nucleoside t r iphospha tes b y D N P using insect f l ight muscle mi tochondr ia .

The final ind ica t ion of two different enzymes was the p H op t imum. The Mg- ATPase has been repor ted to have an o p t i m u m p H of a p p r o x i m a t e l y 8.5-99, 2 whereas t ha t of the D N P - A T P a s e is in the ne ighborhood of 7-7.52. MEYERS AND SLATER 10 have concluded as a result of p H studies t ha t there are three D N P - A T P a s e s having o p t i m a of 6.3, 7.4 and 8.5 respect ively. These three D N P - A T P a s e s require successively higher concent ra t ions of D N P for full ac t iva t ion . At p H 6. 3 the enzyme m a y be a c t i v a t e d b y 5" I ° -5 M DNP, at p H 7.4 b y IO -4 M D N P whereas the enzyme having an op t ima l p H of 8.5 requires IO ~ M D N P for ac t iva t ion . Fig. 7 shows the effect of p H on the " s t imu la t i on" of ATPase b y D N P in the presence of Mg++. The curve represents the m a x i m a l " s t imu la t ion" ob ta ined as a funct ion of pH. The observed p H o p t i m u m for the " s t imu la t i on" of ATPase b y D N P is therefore in agreement wi th the r epor ted values for the " a c t i v a t i o n " of ATPase b y D N P in the absence of a d d e d Mg++. This low p H o p t i m u m for the " s t imu la t i on" of ATPase b y D N P m a y represen t the resu l tan t of the t rue op t ima l p H of the Mg-ATPase and the dissociat ion s t a t e of the DNP. The enzyme in the presence of Mg++ alone has only negligible

ReJerences p. 49 ±.

VOl~. 30 (1958) DNP .~ND ATPasE 489

activi ty below pH 6.0 and only approximately 45 % of the maximal activity at pH 7.02. I t has been suggested n that the undissociated form of DNP is required to produce uncoupling of oxidative phosphorylation. If the undissociated form is also required as an ATPase activator, it is understandable that DNP as an activator has a lower pH opt imum than does Mg ++. The curve under these circumstances would represent a balance between the increasing activity of the enzyme itself as the pH increases and the decreasing concentration of the undissociated form of DNP as the pH increases. MEYERS AND SLATER 9 have suggested that it is the dissociated form of DNP that is responsible for ATPase activation. However, their observations and deductions in this regard are subject to other interpretations.

2.5

2.0 r~ hJ

-J 1.5

d ~0.5

0.4

IO-S 10 -4 i0-* CONC. MgCIz M

Fig. 6. H y d r o l y s i s of ITP. Curves A, B and C same as in Fig. i . Sys t em con ta ined o .o i M t r i s p H 7.o, 0.o4 ~ I I T P p H 7.o, 4" lO-4 M D N P and o. i I mg enzyme p ro t e in in a final vo lume of 0.50 ml. I n c u b a t e d 2o m in a t 22 °.

aB / / t l

# oTA,C Y .~ .-

~a ~ j a ~0.2

"------ --~-7-~'T, ,. , ,,,

0.1

5D 6,0 7.0 8~ pH

Fig. 7. Effect of p H on " S t i m u l a t i o n " by DNP. Sys t em con ta ined O.Ol M t r i s and 0.006 M ATP a t the ind ica t ed pH's , and o.I 5 nag e n z y m e p ro t e in in a final vo lume of 0.50 ml. I n c u b a t e d 2o min a t 23 ° . The curve represen ts the m a x i m u m differences be tween the observed and ca lcu la ted

a c t i v i t y in t he presence of Mg ++ plus 4" lO-4 M D N P a t the ind ica ted pH's .

DISCUSSION

A number of arguments have been previously marshalled in favor of the existence of two separate and distinct ATPases. The properties of the DNP-ATPase have been re-examined and the differences in the "st imulation" and "act ivat ion" of ATPase by DNP may be mainly reconciled on the basis of an effect of DNP on Mg-ATPase. The production of maximum ATPase activity requires the addition of Mg ++ to the medium. I t is not known with any degree of certainty how Mg ++ functions in this respect. Most ATPase preparations that have been investigated show some latent activity in the absence of any added agents. This suggests that the Mg++ may be necessary in forming an active Mg-enzyme complex. I t may however, also be required for the formation of a Mg-ATP chelate which may be the preferred substrate for the reaction. A number of reactions requiring Mg ++ and ATP have been shown to operate optimally when the ratio of concentrations of Mg++: ATP is onem12-15. However, GRIFFITHS et al . 15 have shown in studies with the enzyme arginine phospho- kinase that, while this same relationship obtains in this case, the true role of Mg ++ appears to be the formation of a Mg-enzyme complex.

Re/erences p. 49z.

49 ° C. COOPER VOL. 30 (I958)

If the activation of ATPase requires the formation of a Mg-enzyme complex, the "activation" by DNP will be limited by the amount of magnesium present in the enzyme preparation. Since the enzyme is obviously not saturated with magnesium, i.e., the addition of Mg ++ produces a large increase in activity, it is understandable that the "activation" by DNP cannot be maximal. Only when sufficient Mg ++ is added may the total activity be demonstrated. It may be seen in Fig. I that the "stimulation" of Mg-ATPase by DNP enables maximum activity to be obtained at a lower concentration of added Mg ++ but the total activity is the same. KIELLF, Y AND BRONK TM have found that sub-initochondrial preparations obtained by sonic treatment of rat-liver mitochondria exhibit no "activation" of ATPase by DNP in the absence of added Mg++.

The differences with respect to aging are also readily understood on this basis. If the bound magnesium is released on aging, the "activation" by DNP would be lost, but the "stimulation" by DNP would not be. The apparent difference of nucleo- tide specificity may be a reflection of the inability of bound Mg ++ to function in the hydrolysis of ITP. No difference has to be resolved with respect to pH optima since they are the same for both the "stimulation" and "activation" by DNP. The only difference that may be difficult to reconcile is that of the effect of 0.3 M sucrose. The reason for the inhibition of ATPase "activation" by sucrose is unknown. It is apparently not due to a tonicity effect since KC1 does not produce the same result 2. The sucrose also produces some inhibition of Mg-ATPase. If the sucrose in some way prevents the utilization of the magnesium already bound to the enzyme then the "stimulation" of ATPase by DNP upon the addition of Mg ++ to the medium would be understandable.

In light of the effect of DNP on Mg-ATPase, it may not be necessary to invoke the existence of a separate enzyme "activated" by DNP. With tile possible exception of the preparation employed by KIELLEY AND BRONK 15, all other systems used to study DNP-ATPase most probably contain either bound magnesium or had Mg ++ added to the test system. This does not of course exclude the existence of a second ATPase but it is suggestive that the effects of DNP alone may be due to the presence of ample levels of Mg bound to the enzyme in the proper manner.

These findings raise the interesting question of what is required in order to obtain an "activation" of ATPase by DNP alone in the absence of added Mg ++. The present experiments indicate that free sulfhydryl groups may be necessary. In the presence of I . lO -5 M PCMB there was no "activation" of ATPase by DNP although the Mg-ATPase was unaffected or occasionally slightly increased under these conditions. A second requirement may be the presence of at least some magnesium bound in the proper manner.

An even more pertinent question is whether the "stimulation" of ATPase by DNP has anything in common with the enzymic reactions which lead to the synthesis of ATP and the exchange of inorganic phosphate with ATP. The reasons for believing that DNP-ATPase and ATP synthesis may involve a common mechanism were: (I) the specificity for adenine nucleotides in each of the reactions, (2) all three activi- ties were lost at the same initial rate on aging, and (3) all of the agents capable of producing uncoupling also block the exchange of inorganic phosphate with ATP and "activate" ATPase~, 3. With respect to the effect of DNP on Mg-ATPase, substrate specificity has been shown not to apply, and in the following paper it will be shown

Re/ere~ces p. 49I.

VOL. 30 (1958) D N P ANI) ATPASE 491

that other uncoupling agents behave in a manner that appears to be similar to DNP. Although the initial loss of activity on aging at 30 ° is very similar in each of the three reactions, the exchange reaction and oxidative phosphorylation are found to be lost completely after 35 min of aging at 37 ° whereas IOO min of aging are required to inactivate completely the stimulation of ATPase by D N P in the absence of added Mg ++~7. The present results therefore lend no support to the thesis that the "stimu- lation" of ATPase by D N P is a manifestation of reactions which are pertinent to the process of oxidative phosphorylation or the exchange of inorganic phosphate with ATP.

ACKNOWLEDGEMENT

The author is indebted to K S E N I J A DIMITROV a n d DONALD VINICOR for skilful techni- cal assistance.

S U M M A R Y

The ev idence for the ex i s tence of tw o different ATPases, one s t i m u l a t e d by Mg ~+ and the second by DNP, has been re - inves t iga ted . I t has been found t h a t in con t ras t to the resul ts ob ta ined wi th D N P alone, D N P in the presence of v a r y i n g concen t r a t i ons of Mg ++ can:

I. s t i m u l a t e ATPase in the presence of 0. 3 M sucrose or 1- 5" lO -5 M PCMB; 2. evoke the same m a x i m u m a c t i v i t y as is o b t a i n e d w i t h Mg ++ alone ; 3. p roduce an increase in ATPase af ter p ro longed ag ing ; 4. s t i m u l a t e the hyd ro ly s i s of ITP. I t is concluded t h a t i t m a y no t be necessary to p o s t u l a t e the ex i s tence of a second ATPase,

specif ical ly " a c t i v a t e d " by DNP, in order to exp la in the effects of D N P on the hydro lys i s of ATP. The s ignif icance of these f indings is discussed.

R E F E R E N C E S

1 C. COOPER AND A. L. LEHNINGER, J. Biol. Chem., 219 (1956) 489. 2 C. COOPER AND A. L. LEHNINGER, J. Biol. Chem., 224 (1957) 547. 3 C. COOPER AND A. L. LEHNINGER, J. Biol. Chem., 224 (1957) 561. a j . B. MARTIN AND D. M. DOTY, Anal. Chem., 21 (1949) 965. 5 D. H. LOWRY, N. J. ROSEBRAUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265.

G. R. WYATT, in E. CHARGAFF AND J. N. DAVIDSON, The Nucleic Acids, Vol. I, Academic Press Inc., New York, 1955, p- 252.

7 G. D. GREVlLLE AND E. REICH, Biochim. Biophys. Acta, 20 (1956) 44 o. 8 ]~. SACKTOR AND D. G. COCHRAN, J. Biol. Chem., 226 (1957) 241. o W. W. KIELLEY AND R. K. KIELLEY, J. Biol. Chem., 200 (1953) 213.

10 D. K. MEYERS AND E. C. SLATER, Biochem. J., 67 (I957) 558. 11 R. H. DE DEKEN, Biochim. Biophys. Acta, 17 (1955) 494. 12 H. G. HERS, Biochim. Biophys. Acta, 8 (1952) 424 . 13 C. LIEBECQ, Biochem. J., 54 (1953) xx i i . 14 S. A. KUBY, L. NODA AND H. A. LARDY, J. Biol. Chem., 21o (1954) 65. 15 D. E. GRIFVlTHS, J. E. MORRISON AND A. N. ENNOR, Biochem. J., 65 (t957) t53. 16 \V. ~V. KIELLEY AND J. R. BRONK, Proc. Intern. Enzyme Symposium, Tokyo, 1957, in the press. 17 C. COOPER, unpub l i shed observa t ions .

Received April 2Ist, 1958