Yeast alcohol dehydrogenase

VOL..23

(1957)

YEAST ALCOHOL DEHYDROGENASE I

58I

SUMMARY The effect of pyridine derivatives on yeast alcohol dehydrogenase has been described. These compounds have been found to compete with DPN or DPNH for the active site on the enzyme. Three classes of compounds have been studied : pyridine bases, Nl-methylpyridinium salts, and analogues of DPN. A number of pyridine compounds have been found to show inhibitory powers different for the DPN reduction than for the DPNH oxidation. The inhibition of pyridine bases appears to be due to their ionized forms. Although pyridine bases inhibit the yeast alcohol dehydrogenase reaction, some of these compounds could actually, at low concentrations, stimulate the rate of reaction. A partial reaction sequence and an enzyme-coenzyme model are proposed to explain the experimental data. REFERENCES 1 J. VAN EYS, Federation Proc., 15 (1956) 374.

2 H. TIiEORELL AND a . K. BONNICHSEN, Acta Chem. Scan&, 5 (1951) 11o5. 3 j . E. HAYES, JR., AND S. V. VELICK, J . Biol. Chem., 207 (1954) 225. 4 A. P. NYGAARD AND H. THEORELL, Acta Chem. Scan&, 9 (I955) I3 oo. 5 A. P. NYGAARD AND H. THEORELL, Acta Chem. Scan&, 9 (1955) I551. * H. VON EULER, Ber. deut. Chem. Ges., 75 (1942) 1876. N. G. BRINK, Acta Chem. Scan&, 7 (1953) lO9O. 8 p. FEIGELSON, J. N. WILLIAMS, JR. AND C. A. ELVEHJEM, J. Biol. Chem., 198 (1951) 361. 9 L. J. ZATMAN, N. O. KAPLAN, S. P. COLOWICK AND M. M. CIOTTI, J. Biol. Chem., 2o9 (1954) 453. 10 N. O. KAPLAN AND M. M. CIOTTI, J. Biol. Chem., 221 (1956) 823. 11 N. O. KAPLAN, M. M. CIOTTI, F. E . STOLZENBACH AND N. R. BACHUR, J, Am. Chem. Soc., 77 (1955) 815. 12 M. E. PULLMAN, S. P. COLOWlCK AND N. O. KAPLAN, J. Biol. Chem., 194 (1952) 593. 13 H. H. JAFFI~ AND G. O. DOAK, J. Am. Chem. Soc., 77 (1955) 4441. 14 L. P. HAMMETT, Physical Organic Chemistry, i 9 4 o, McGraw-Hill B o o k Co., N e w York, C h a p t e r VII. 15 H. H. JAFFf~, Chem. Revs., 53 (1953} 191. xe H. LINEWEAVER AND D. BURK, J. Am. Chem. Soc., 56 (1934) 658. 17 N. O. KAPLAN, M. M. CIOTTI AND F. E. STOLZENBACH, J. Biol. Chem., 221 (1956) 833. is N. O. KAPLAN, S. P. COLOWlCK, L. J. ZATMAN AND M. M. CIOTTI, J. Biol. Chem., 2o 5 (1953) 31. 1, j. VAN EYS, M. M. CIOTTI AND N. O. KAPLAN, Biochim. Biophys. Acta, 23 (1957) 581. 20 E. S. GUZMAN BARRON AND S. LEVlNE, Arch. Biochem. Biophys., 41 (1952) 175. ~1 B. L. VALLEE AND F. L. HOCH, J. Am. Chem. Soc., 77 (1955) 821.

Received July 28th, 1956.

YEAST ALCOHOL DEHYDROGENASE* II. P R O P E R T I E S OF T H E CATALYTICALLY ACTIVE SITE" J A N V A N EYS, M A R G A R E T M. C I O T T I A N D N A T H A N O. K A P L A N

The McCollum-Pratt Institute, The Johns Hopkins University, Baltimore, Md. (U.S.A.)

In the previous paper a reaction scheme and a model of the enzyme-coenzyme complex of yeast alcohol dehydrogenase*" were proposed 1. In the development of the theoretical rate in the presence and absence of a pyridinium ion, it was assumed t h a t * C o n t r i b u t i o n No. 161 of t h e M c C o l l u m - P r a t t I n s t i t u t e . S u p p o r t e d in p a r t b y g r a n t s f r o m t h e Rockefeller F o u n d a t i o n a n d t h e N a t i o n a l Cancer I n s t i t u t e of t h e N a t i o n a l I n s t i t u t e s of H e a l t h (Grant No. C-2374C ). ** A b b r e v i a t i o n s used in t h i s p a p e r are: A D H : alcohol d e h y d r o g e n a s e ; D P N a n d D P N H : oxidized a n d r e d u c e d d i p h o s p h o p y r i d i n e nucleotide, respectively; P y - 3 A 1 D P N a n d P y - 3 A I D P N H : oxidized a n d r e d u c e d a n a l o g s of D P N of pyridine-3-aldehyde, respectively; p C M B : para-chloromercuribenzoate.

Re]erences p. 587. 38

582

J. VAN EYS, M. M. CIOTTI, N.O. KAPLAN

VOL. 2 3

(1,957)

the enzyme has multiple sites, all of which are active and independent in the oinding of the coenzymes and in the reaction catalysis. Under these circumstances the relative rate in the presence and absence of a pyridinium ion should be inversely proportional to the concentration of the pyridinium ion. Experimentally, however, the relative rate is proportional to the logarithm of the concentration of the pyridinium ion. This can be the result of one of three possibilities : (i) The sites are not equal and independent, (2) there is more than one pyridinium ion required per site, and (3) there is more than one site for binding, but only a few of these sites are active at one time in catalyzing the enzymic reaction. In the studies of HAYES AND VELICK ~ it has been shown that there are four equal and independent sites for binding DPN and DPNH. This is in agreement with VALLEE'S observation that there are four zinc atoms per molecule of enzyme a. However, there is no evidence that all four sites are independently active. In this paper evidence will be presented which suggests that only one site is active in catalyzing the reaction of alcohol oxidation. Furthermore, some indications as to the chemical nature of the reactive center will be reported. MATERIAL AND METHODS The source and p r e p a r a t i o n of the enzyme and coenzymes has been described 1. The ultracentrifugal s e p a r a t i o n analysis of coenzyme binding was performed according to VELICK et al. 4. E n z y m e concentration was measured spectrophotometrically using a molar extinction coefficient at 28o m # of 1.89" lO5 ~. The extinction of D P N H was t a k e n to be 6.22. lO 3 at 34 ° m/~S; t h a t for PY-3A1 D P N H at 355 m/z 7.o" lO 36. Sulfhydryl groups were determined with pCMB as described b y BOYER 7. W h e n the reduction of 2,6-dichlorophenolindophenol was measured, a molar extinction of 1,9. lOS at 61o m # (pH 7.0) was used 8. At the isosbestic point (520 m/t) an extinction of 8.1. lO4 was employed.

Coenzyme binding

RESULTS

Yeast ADH, as commercially obtained, contains an appreciable amount of ethanol which cannot be dialyzed out without excessive inactivation of the enzyme. Consequently, when the ultracentrifugal analysis of HAYES AND VELICK~ was tried, no reliable results could be obtained with DPN, since a significant amount was converted to DPNH. However, the binding of D P N H could be easily verified. By the same technique it could also be shown that Py-3A1DPNH was bound to the enzyme. Analysis of the data as described 2 gave again four coenzyme molecules per molecule of enzyme for either D P N H or Py-3A1DPNH. The dissociation constant for both complexes was approximately 1.5-1o -5 moles/liter.

Spectrophotometric evidence/or enzyme-coenzyme complex As noted in the previous paper I the yeast enzyme differs from the horse liver alcohol dehydrogenase, in that D P N H does not show a spectral shift with the yeast protein. This spectral shift does occur when the reduced coenzyme is bound to the liver enzyme. This difference holds true for A P D P N H and Py-3A1DPNH.* Similarly, the DPNhydroxylamine complex observable with the mammalian enzyme9 does not form with the yeast enzyme. When one uses, however, Py-3A1DPN and hydroxylamine, a complex is observed with yeast ADH. This complex has a maximum at 315 m/z (Fig. I). These results indicate that the liver and the yeast enzymes may have some similarity in the formation of a ternary complex. * N. O. KAPLAN and M. M. CIOTTI unpublished observations.

Re[erences p. ,587.

VOL. 23 (1957)

YEAST ALCOHOL DEHYDROGENASE II

583

The molar extinction of the Py-3A1DPN/hydroxylamine complex at 315 m ~ is 6.0. lO8. When the data were treated in the manner of HAYES AND VELICK, i.e.: r = n --

(I)

K(r/Cf)

w h e r e r s t a n d s f o r t h e n u m b e r of c o m p l e x e s b o u n d t o t h e e n z y m e , n t h e n u m b e r of b i n d i n g s i t e s p e r e n z y m e , a n d C f t h e c o n c e n t r a t i o n of free c o e n z y m e , a p l o t is o b t a i n e d a s i l l u s t r a t e d i n F i g . 2. rr LLI {3.. X

,,,

I00

._1

(3.

:E

0 (.j ILl

~.4 O3 Z

c~ "1- Z

zL'J 50

.J


ro

~o

4o

r,

< o

i ,

.I

~

N3

13.

m

~00 325 350 WAVELENGTH ( m p )

0

W ..../

J .0025

"X 0075

.0100

?-/Cf

o

Fig. i. The spectrum of enzyme and enzymePy-3A1DPN-hydroxylamine complex, O = free enzyme, • = enzyme plus Py-3A1DPN. The coenzyme was also added to the blank cuvette. • = difference spectrum. The spectra were taken in o.i M phosphate buffer, p H 7.5. E n z y m e concentration 13o m/~ M, hydroxylamine 0.02 M, final volume I.O ml.

J .0050

Fig. 2. A plot of Py-3A1DPN/hydroxylamine complex bound per mole of enzyme vs. this ratio over concentration of free Py-3A1DPN. Phosphate buffer, 0.04 M, p H 7.o, h y d r o x y l a m i n e 0.02 M, enzyme: 14o m/z M. Final volume i.o ml. The intercept corresponds to the m a x i m a l n u m b e r of complexes bound per mole of enzyme, at infinite coenzyme concentration.

The intercept n equals o.8o, or one mole of complex is formed per mole of enzyme. This value did not change over the whole p H range of 5.2-8.2. The dissociation constant (K) was the same at all p H values (lO -3 moles/liter). It must be concluded that not all sites are capable of accepting or forming this complex, even though four moles of coenzyme are bound. If one assumes that this complex is similar to the DPN-ethanol-enzyme complex ~, it appears that only one site is involved in forming the activated ternary complex.

Enzyme denaturation Apart from complex-formation, the active site participates in other reactions, as is indicated by the acid denaturation of the enzyme. When yeast A D H is placed in a buffer of p H 5.0 or lower, a slow precipitation is observed. The rate of precipitation can be followed by observing the increased turbidity in the Beckman spectrophotometer at an arbitrary wavelength. Usually 550 m/, was chosen. This denaturation is illustrated in Fig. 3. The denaturation was relatively independent of the buffer used. The reaction sequence responsible can be pictured as follows: E + n . H + \ K \ E-nil References

p. 587.

~1 ~ E a x

k8 ~ E d ~

> ....

kx > E a x

(2)

584

J. VAN EYS, M. M. CIOTTI, N. O. KAPLAN

VOL. 23 (I957)

where Edx is the xth m e m b e r of a sequence of d e n a t u r e d forms of the enzyme. The rate of d e n a t u r a t i o n can be described as: (dEd~/dt) = (k 1.k2 . . . .

kx) K (E) (H+)n

(3)

A plot of log (dEdJdt) = - - n p H + log (k1.k 2 . . . . .

k~) K (E)

(4)

will yield as slope the n u m b e r of hydrogen ions involved. W h e n the rate of d e n a t u r a tion is plotted vs. pH, a n i n d i c a t i o n of the dissociation of the proton acceptor involved 03 6.0 uJ I-5.0

6 w ~ .5 z

.5

~

~ 4O ~M

4

o to to3 Ld <3 .2

c,J

~3.0 ff'l

w 2.0 <3

~.2 w

0 I

0

I

5 10 TIME(MINUTES) Fig. 3. The rate of denaturation of yeast ADH. Phosphate buffer 0.04 M, pH 4.0. Enzyme concentration: 13 m#M. Final volume 3.0 mL The increase in optical density is a measure of increased turbidity.

3.0

4.0

5.0

0

pH

1.0

2.0

5.0

4.0

50

60

pH

Fig. 4. A. A plot of the rate of denaturation versus pH. Enzyme concentration: I8 m/~M. Acetate buffer o.osM. Final volume 3.o ml. B. A plot of the logarithm of the rate of denaturation vs. pH. O = enzyme concentration : 18 m/~M, • = enzyme concentration: 9 m/~M. Acetate buffer o.05 M.

c a n be obtained. These graphs are presented in Fig. 4. F r o m the data the n u m b e r of hydrogen ions i n v o l v e d was found to be 0. 9 , which indicates t h a t one proton reacts with one enzyme molecule. The proton acceptor on the p r o t e i n appeared to have a p K between 3.0-3.5. Reduction o/2,6-dichlorophenolindophenol and denaturation 2,6-Dichlorophenolindophenol has been f o u n d to react with sulfhydryl groups of proteins 10. Although it is a two electron dye, 2,6 dichlorophenolindophenol will react in a one to one ratio with each sulfhydryl grouping s, 10,11. Yeast ADH, however, was a n exception to this rule. The dye did not react with the protein unless a low p H was employed, even t h o u g h the dye is more easily reduced at higher p H values. The rate of r e d u c t i o n at p H 4 was twice t h a t at p H 5, while at p H 7.0 a n d higher very little r e d u c t i o n occurred. The e x t e n t of reduction was b u t 8.5 moles of dye reduced per mole of protein. The correlation between the rate of dye reduction at different p H values a n d the d e n a t u r a t i o n rate at these p H values suggested t h a t the p o t e n t i a l proton acceptor on the protein was a n u n r e a c t i v e sulfhydryl group. W h e n the total n u m b e r of sulfh y d r y l groupings was measured with pCMB a value of 18.5 moles S - H was found per mole protein. This value is i n fair agreement with the d a t a of BARRONTM. Sile o / d e n a t u r i n g groups Acid d e n a t u r a t i o n of the yeast A D H could be prevented, or at least slowed b y the Re/erences p. 587.

VOL. 23 (1957)

YEAST ALCOHOL DEHYDROGENASE II

585

presence of DPN*, D P N H , Py-3A1DPN or beta-picoline. If the sites on the e n z y m e are equal a n d i n d e p e n d e n t , a n d if each site yields, after a d d i n g a proton, a n e n z y m e equally susceptible to d e n a t u r a t i o n , a protective agent (P) m u s t cover all n sites to be effective i n protecting the enzyme. Thus the e q u i l i b r i u m between protected e n z y m e ( E - P ) a n d susceptible e n z y m e (E~) can be w r i t t e n as: (5)

(Es) + n. (P) \ K \ (E-P)

Solving for E s a n d s u b s t i t u t i n g E t -

E s for E - P we get:

Es = Et/( K (p)n + I)

(6)

where E, stands for t o t a l e n z y m e concentration. S u b s t i t u t i n g e q u a t i o n (6) in the rate e q u a t i o n for d e n a t u r a t i o n , i.e., e q u a t i o n (3), we get: (dEd,/dt) = K(H+)(Et)/(K(P) n + i)

(7)

For the relative rate v, of d e n a t u r a t i o n in the presence a n d absence of a protecting agent (equation (7) divided b y e q u a t i o n (3)) we thus get: v, = ~ / ( K ( P ) * + ~)

(8)

log[(I/Vr) - - I ] = nlog (P) + log K

(9)

which can be rewritten as: W h e n the d a t a for beta-pieoline protection are plotted in this fashion, we get the plot of Fig. 5. The d a t a agree well with the predicted linear relationship. Different experim e n t s yielded a value for n r a n g i n g from 2.4 to 3.3.

S~

I0

jS

znC, I

05 I IDPN

>~ o.o

o

0 0 t t ~ . +NH 3 - P ' O - P - N+ S 0 ° "0 S

-0.5

//////////////7 Z n ++\

-I0 I

t.l S ~.

SH

*l

/////I/////////

I

-2.5 - 2D -I.5 L06 MOLARITY~. PICOLINE Fig. 5. The effect of beta-picoline on the acid DENATURATION denaturation of yeast ADH. Enzyme concentra- Fig. 6. The nature of the active site: the two tion: 9 m/zM. The plot is the logarithm of the sulfhydryl groupings are in non-ionized comreciprocal of the relative rate minus one vs. the bination with the zinc. logarithm of the concentration of beta-picoline. See text for details. The slope is an indication of the number of beta-picolineions per mole of enzyme. DISCUSSION

The picture which suggests itself for the active site of yeast ADH is one that contains unreactive sulfhydryl groups. That sulfhydryl groups are present on the active site * At low concentrations DPN actually somewhat accelerates the denaturation reaction. This is in line with the low affinity of the enzyme for DPN. Once the Zn-S complex is broken, the enzyme is susceptible to denaturation (see Fig. 5). Re]erences p. 587.

586

J. VAN EYS, M. M. CIOTTI, N. O. KAPLAN

VOL. 2 3

(1957)

is inferred from the observations of BARRON12 a n d from the fact t h a t the hydroxyla m i n e - Py-3A1DPN complex can be broken u p with pCMB. These sulfhydryl groups m u s t be b o u n d in such a way t h a t acid will release the grouping. A possible reason for this lack of r e a c t i v i t y is illustrated in Fig. 6 where the sulfhydryl groups are b o u n d to the zinc. The d e n a t u r a t i o n can be pictured as a sequence of reactions: the proton adds to one of the sulfhydryl groups, yielding a free - S H grouping which is susceptible to oxidation. Thus the d e n a t u r e d e n z y m e p r o b a b l y is the result of the formation of r a n d o m l y crosslinked disulfide groupings. B r e a k i n g up of one sulfhydryl zinc linkage would t e n d to s t r e n g t h e n the second zinc sulfide bond*. The native enzyme appears to be completely reduced. This picture is compatible with the previously postulated enzyme-coenzyme complex 1. It appears probable t h a t only one site on the enzyme is at one time engaged in alcohol oxidation. The reasons for this possibility are as follows. First, all sites are equal a n d i n d e p e n d e n t for coenzyme binding, yet only one forms the Py-3A1DPNh y d r o x y l a m i n e complex. Secondly, in the e v e n t of acid d e n a t u r a t i o n all four sites are equally effective i n accepting a proton, a n d thus all four sites m u s t be occupied for the e n z y m e to be protected. Therefore, four moles of beta-picoline will be required per mole of enzyme to completely protect it, if the sites are equal for beta-picoline. E x p e r i m e n t a l l y , the measure is m a x i m a l l y 3.3 ions per four sites. Thus, only one pyrid i n i u m ion is required pel site. However, the effect of the p y r i d i n i u m ions on the rate of alcohol oxidation is logarithmically related to the c o n c e n t r a t i o n of the p y r i d i n i u m ionL It is clear from the above t h a t this relationship is not the result of the fact t h a t the sites are n o t equal a n d i n d e p e n d e n t for the p y r i d i n i u m ion. F u r t h e r m o r e , the exp e r i m e n t a l d a t a show b u t one beta-picoline ion per site. The logarithmic proportionality, then, is most logically explained b y the a s s u m p t i o n t h a t only one site is active at a given time. I t m u s t be m e n t i o n e d t h a t this does n o t i m p l y t h a t only one D P N is required per e n z y m e molecule, b u t r a t h e r only one D P N out of four reacts with a n e t h a n o l molecule at a time. These postulations require a n obligatory reaction sequence: enzyme ~ e n z y m e - c o e n z y m e - - ~ e n z y m e - c o e n z y m e - s u b s t r a t e . This sequence does not involve a s u b s t r a t e enzyme complex, a n d is in accord with our previous proposaP. The same reaction sequence appears to be operating for heart muscle lactic dehydrogenase 13 a n d hence m a y not be u n i q u e for the yeast ADH. SUMMARY Yeast alcohol dehydrogenase binds four moles of the reduced pyridine-3-aldehyde analog of 1)PN (Py-3A1DPN) or DPNH per mole of enzyme. No DPN-hydroxylamine complex is formed with yeast ADH. However, one mole of Py-3A1DPN-hydroxylamine complex is formed per mote of enzyme. \Vhen acid denaturation of the enzyme is measured, pyridine derivatives inhibit the rate. For inhibition close to four moles of pyridinium ions are required per mole of enzyme, but only one hydrogen ion is required to give an enzyme species susceptible to denaturation. Evidence is presented which indicates that yeast ADH contains unreactive sulfhydryl groupings on the active sites; the nature of this unreactive sulfhydryI group is discussed. Discussion of the possibility that one of the four DPN binding sites of the enzyme at a time is engaged in the conversion of DPN to DPNH, is presented. * That at least two sulfhydryl groups are present per active site can be calculated from the data of I~ARRONTM. It is also indicated by the observation that deamino DPN does not protect against the acid denaturation (J. VAN EYS, M. M. CIOTTI and N. O. KAPLAN, to be published). Free pyridine derivatives do inhibit the denaturation. Thus at least two different sulfhydryl groupings are implied. R e f e r e n c e s p. 55'7 .

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YEAST ALCOHOL DEHYDROGENASE II

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REFERENCES 1 j. VA~ EYs AND N. O. KAPLAN, Biochim. Biophys. Acta, 23 (1957) 574. 2 j. E. HAYES JR., AND S. F. VELICK,J. Biol. Chem., 207 (1954) 225. 3 B. L. VALLEEAND F. L. HOCH,J. Am. Chem. Soc., 77 (1955) 821. 4 S. F. VELICK, J. E. HAYES JR. A N D J. HARTING,J. Biol. Chem. 203 (I953) 527. 5 A. I{ORNBERGAND B. L. HORECKER,Biochem. Preparations, 3 (1953) 23. 6 N. O. I{APLAN,M. M. CIOTTIAND F. E. STOLZENBACH,J. Biol. Chem., 221 (1956) 833. 7 p. D. BOYER AND H. L. SEGAL,in "W. D. ~¢[CELROYAND B. GLASS, The Mechanism o[ Enzyme Action, The Johns Hopkins Press, Baltimore, 1954, p. 528. s R. E. BASFORDAND F. M. HUENNEKENS,J. Am. Chem. Soc., 77 (1955) 3873• 9 N. O. KAPLANAND ~'~/[.M. CIOTTI,J. Biol. Chem., 211 (1954) 431. 10 G. RAFTERANn S. P. COLOWICK,Federation Proc., 14 (1955) 267. ll G. RAFTER, Federation Proc., 15 (1956) 334. 12 E. S. GUZMANBARRONAND S. LEVINE, Arch. Biochem. Biophys., 41 (1952) 175. 13 G. W. SCHWERTAND ~. TAKENAKA,Federation Proc., 15 (1956) 351. Received J u l y 28th, 1956

THE THE

INFLUENCE

INCORPORATION

OF DIETARY

PROTEIN

ON

O F 1 4 C - G L Y C I N E A N D 3~p I N T O T H E

RIBONUCLEIC

ACID OF RAT LIVER

c. M. CLARK, D. J. NAISMITH AND H. N. MUNRO Department o/Biochemistry, The University, Glasgow (Scotland)

Two d i e t a r y factors influence the course of ribonucleic acid (RNA) m e t a b o l i s m in the liver. P r o t e i n i n t a k e plays a d o m i n a n t role i n d e t e r m i n i n g the a m o u n t of R N A i n the liver 1, 2, whereas energy i n t a k e affects its rate of synthesis as measured b y 3~p incorp o r a t i o n 2, The influence which p r o t e i n i n t a k e exerts over the a m o u n t of R N A i n the liver does n o t appear to involve changes in the rate of R N A f o r m a t i o n ; thus the feeding of a protein-free diet causes a large reduction in the a m o u n t of R N A w i t h o u t affecting the absolute rate of ~2p u p t a k e b y R N A 3. A n a l t e r n a t i v e way in which the d i e t a r y supply of p r o t e i n could influence the a m o u n t of R N A in the liver is b y regulating its rate of b r e a k d o w n . I n the course of s t u d y i n g the influence of energy i n t a k e on 14C-glycine incorporation into RNA, we o b t a i n e d evidence strongly suggesting t h a t the rate of R N A b r e a k d o w n diminishes after a meal c o n t a i n i n g protein. This evidence emerged when we used rats which were t r a i n e d to eat p r o t e i n - c o n t a i n i n g or proteinfree diets at fixed times each d a y ; in consequence, it was possible to know with certaint y whether the a n i m a l s were in the post-absorptive state or were actively absorbing n u t r i e n t s at the time of injection with isotopes. W h e n the a n i m a l s were fasting after meals c o n t a i n i n g protein, there was evidence of a considerable b r e a k d o w n of RNA. After feeding protein, the picture r a p i d l y changed to one which we have i n t e r p r e t e d as i n d i c a t i n g cessation of breakdown. These findings have led us to conclude t h a t the i n t e n s i t y of p r o t e i n synthesis determines the s t a b i l i t y of liver RNA. This m a y have some b e a r i n g on the role of R N A in p r o t e i n synthesis. A p r e l i m i n a r y account of these experiments has already appeared 3. References p. 599.