An Alanine-dependent, Ribonuclease-inhibited Conversion of AMP to

[CONTRIHUTIOX FROM THE nIVIsION OF BIOLOGY, C.4LIFORNIA INSTIT UTE OF TECHSOLOGY]. An Alanine-dependent, Ribonuclease-inhibited Conversion ...
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ROBERTW. HOLLEI.

compound. The yicld of the monohydrate was 15.5 g., [ a ]z4D -5.9” (in water). Chromatographic analysis, as described for the D,L-forni above, revealed only a single spot. .4naZ. Calcd. for ClaHziO5Ns.H20: C , 46.3; H , 8.1; N, 19.3. Found: C,45.8; H , 8 . 2 ; N,18.8. In order t o ascertain t h a t the above compound was, in f a c t , an epimeric mixture, a sample of 7.27 g. was refluxed with 6 ?Jhydrochloric acid for 24 hr. Paper chromatograms, as above, showed only a single spot corresponding t o octopine (or isooctopine). After removal of chloride ions with .ilver carbonate, the filtrate yielded 2.12 g. of D-octopine a n t i 0.42 g. of L-allooctopine, isolated via their respective picrate and flavianate derivatives. Although such data indeed indicatcd the presence of an epimeric mixture, the ratio of “D-octopinj-1”- t o “L-allooctopiny1”-L-valine could not properly be determined from the final yields of n-octopilie and L-allooctopine subsequently secured, since large losse.; occur during the isolation of the flavinnate of thi.; latter compound.

[CONTRIHUTIOX FROM

THE n I V I s I O N O F

111. Rotatory Dispersion Measurements.-The rotatory dispersion of (+)-octopine and ( +)-isooctopine was determined at 589, 578, 546, 435, 405 and 365 mp, as l‘;;, solutions in water and 5 N HCl, with a Rudolph photoelectric polarimeter. The procedure employed was similar t o that described earlier .lg Replicate readings viere reproducible t o +0.003° (Table I). IV. Kinetic Measurements.-Separate solutions of I.-(?bromopropionic acid (632.4 mg.) in water (4.14 nil.) a ~ l t l of L-arginine.HC1 (435.6 mg.) in 2 -V sodium hytlrosidt~ (4.14 ml.) were allowed to equilibrate for 1 hr. in a wntcrbath at 39’. The solutions were then mixed and t n o 0.2ml. samples removed immediately for Volliard titr:ition ani1 for Van Slyke ninhydrin-C02 determination. Siinil:ir sampling was effected at succeeding 30-n~inuteinterval.; f o r 5.5 hr., by which time about 5570 of the arginine liatl i l i + appeared. Results were as shown i n Tat)lt. 111. ~IE:TIIEST)A 14, ~ I A R Y L A S I J

BIOLOGY, C.4LIFORNIA

INSTITUTE O F T E C H S O L O G Y ]

An Alanine-dependent, Ribonuclease-inhibited Conversion of AMP to ATP, and its Possible Relationship to Protein Synthesis BY ROBERT157. HOLLEY’ RECEIVED.4UGlXT 3 , 1956 An enzyme preparation from the soluble fraction of r a t liver homogenate brings nbuut conversion of radioactive -IMP (adenosine 5/-monophosphate) into A T P (adenosine 5’-triphosphate) in the presence of L-alanine. Other amino acids, potassium acetate and sodium pvruvate have no effect. The L-alanine-dependent conversion of A M P to A T P is inhihitell by ribonuclease.

There is considerable evidence t h a t the first step in the synthesis of proteins from amino acids is an activation of the amino acid carboxyl groups by ATP.2 The work of Hoagland3 and Hoagland, Keller and Zamecnik4 has suggested a detailed mechanism for this activation (eq. 1). These workers found t h a t an enzyme preparation from the soluble fraction of rat liver homogenate brings about a rapid exchange between pyrophosphate and the terminal phosphates in A T P if amino acids are present. The amino acid activating enzymes could also be studied by demonstrating the formation of amino acid hydroxamic acids when the reaction was run in the presence of a high concentration of hydroxylamine. The activated amino acids have been formulated as enzyme-bound amino acylALfP derivatives3s4

the L-leucyl derivative of A M P (thought to be t h e mixed anhydride of L-leucine and AMP) ‘ind showed that in the presence of amino acid activating enzyme obtained from E . colt this compound reacts with pyrophosphate t o form ATP. Similar results were obtained by Berg and Newton6 using a methionine activating enzyme obtained fro111 yeast. If it is assumed that amino acid activation of this type is the first step in protein synthesis, the question immediately arises as t o the nature of the next step in protein synthesis. Presumably, the activated amino acid must react with something, or be transferred to something. As a working hypothesis, it was assumed that the activated amino acid reacts with snnie u n k n o ~ ~ imaterial i with liberation of XLIP

Enzyme Amino Acid ATP Enzyme-Amino Acyl-AMP

~117vnie-Xmino~

+

+

+ Pyrophosphate

(1)

The assumption t h a t this type of enzymatic activation of amino acids is the first step in protein synthesis rests primarily on the discovery of the enzymes in a fraction which is required for the in d r o incorporation of radioactive amino acids by rat liver m i c r o ~ o m e s . ~ ~ ~ Strong support for the above formulation of the activation of amino acids has been furnished by DePvLoss, Genuth and Novelli“ who synthesized (1) New York State Agricultural Experiment Station. Cornell Kniversitg, Geneva, hT.Y. John Simon Guggenheim hfemorial I’oundation Fellow, 1955-1956. (2) H. Borsook, J . Cellular Comp. Physiol., 47, Suppl. 1, 35 (1946). ( 3 ) S I . B. Hoagland, Biochim. Biophys. Acta, 16, 288 (1955). (4) M B. Hoagland, E. R. Keller and P. C . Zamecnik, J . R t o l . (‘h~m., 218, 345 ( 1 9 3 ) . (5) J. A . Dehfoss, S. M. Genuth and (;. I.). Novelli, Proc. S t i l i . A c a d . S c i , 42, 325 (1956).

C

I --IMP J L

Enzyme

x

e

+ Amino A i c l l - S + AhlP

i?

Such a reaction might be reversible arid, if cornbincd with the pyrophosphate exchange reaction (eq. 1 ), it might be detected by assaying for exchange of AMP into ATP. A procedure for assay of AMP exchange w x therefore devised Crude en7yme preparations were incubated with radioactive AMP under conditions in which pyrophosphate was known to e\ change with ATP. -it the end of the incubation period, the rlTP was precipitated as the bariuiii salt and its radioactivity was determined The assay was first applied to extracts of spmarll acetone powder, hog pancreas acetone powder a n t 1 lyophilized Lactob~icill~i Y plantarurn cells, extract? ( b ) P nerg and

(>

‘irntrin

f , d , , d x i ) 1’)

J

1 5 , 21‘1 \ l c l i O ’

known to contain amino acid activating enzymes.’ I n the presence of these extracts, radioactive AMP is converted rapidly to A T P in the absence of added amino acids. This conversion may be due to the presence of adenylic kinase, which would bring about exchange between A M P and ATP: AMP A T P 8 2=1DP. Whatever the mechanism, the amino acid-independent conversion in these systems is very rapid and obscures any amino acid-dependent exchange which may be present. These results suggested that it might be difficult to find a crude preparation of amino acid activating enzymes which was sufficiently free of “adenylic kinase” to be used in the AMP exchange assay procedure. Since Hoagland, Keller and Zamecnik4 reported that their amino acid activating enzyme preparation from rat liver failed to exchange AMP with ATP, the A M P exchange assay procedure was tried with their enzyme preparation. Using material prepared according to their directions for the “pH 5 e n ~ y m e , ”it~was found that approximately 10% of the added radioactive AMP was converted to A T P in 5 min. a t 37” in the absence of added amino acids, indicating the probable presence of adenylic kinase. However, even in the presence of this high amino acid-independent incorporation, there was a slight increase in the radioactivity of the ATP when amino acids were added to the incubation mixture. Assays on the various fractions from the preparation of the “pH 5 enzyme” showed that the supernatant from the precipitation of the “pH 5 enzyme’’ contained a high concentration of “adenylic kinase,” and suggested that a second precipitation a t PH 5 might reduce the “adenylic kinase” activity. This was found to be the case. Using an enyme preparation which had been precipitated twice a t pH 5.1-5.2, the addition of amino acids to the incubation mixture increased the conversion of radioactive AMP into ATP approximately 100%. With fresh enzyme preparations this amino acid-dependent conversion (“exchange”8) of AMP into ATP consistently represented 2-3% of the added AMP (see Fig. 1 and Table I), Evidence that the radioactive material formed in the presence of added amino acids is A T P is as follows. The radioactivity precipitates with the barium salt of ATP. It chromatographs on paper as i l T P . Conversion of the barium salt of A T P to the triacridine salt of ATP, followed by recrystallization of the triacridine salt, causes no significant change in specific radioactivity. By studying the effect of individual amino acids it was found that L-alanine accounted for all of the activity of the mixture of 15 amino acids which had been used in the initial experiments. (DAlanine is inactive.) Other natural amino acids, tested individually, did not cause significant conversion of A M P to ATP. This finding suggested that perhaps the conversion of A M P to -4TP resulted from conversion of L-alanine to pyruvate,

2oo

t L-A LAN I NE

+

(7) Unpublished work (a) J . M. Clark, Jr., with spinach; (b) R. S. Schweet with hog pancreas; (c) R . W. Holley, with L . p l n n l a r ~ m . (8) Presumably t h e radioactivity of t h e A T P results from a twoway exchange process between AMP and A T P , b u t since this has not been proved the process is termed simply a conversion of AMP into ATP.

0

5

IO

15

MINUTES Fig. 1.-Effect of L-alanine on the conversion of radioactive A M P into A4TPat 37”. T h e .4TP was counted as the barium salt.

with subsequent formation of acetyl coenzyme A and involvement of the acetate activation enzyme system.6s9 Such a possibility was excluded by the finding that pyruvate, acetate and coenzyme A have no effect. Of particular interest is the finding that the Lalanine-dependent conversion8 of AMP to A T P is sensitive to ribonuclease (see Table I). The addition of 0.1 pg, of ribonuclease a t the start of the 10 min. incubation significantly reduces the Lalanine-dependent incorporation of radioactivity. The addition of 1 pg. suppresses the L-alaninedependent incorporation completely. Ribonuclease has no effect on pyrophosphate exchange (eq. 1). As controls, to determine if the action of ribonuclease might be attributed to its basicity, lo the basic proteins histonell and protamine sulfatell were added a t 1 and 10 pg. levels and were found to have no effect. Thus far, attempts to increase the conversion of AMP to ATP by the addition of ribonucleic acid preparations or preparations of microsomes have been unsuccessful. These experiments are summarized in the Experimental Part. The extent of incorporation of radioactive A M P into ATP was compared with the incorporation of radioactive pyrophosphate into ,4TP. Using identical reaction mixtures, except for the use of radioactive AMP in one instance and radioactive pyrophosphate in the other, the incorporation of AMP is approximately 9y6 of the incorporation of pyrophosphate. If pyrophosphate exchange (eq. 1) is an intermediate step in the incorporation of (9) P. Berg, THISJOWRNAL, 17, 3163 (1955). (10) J. Brachet, Biockim. Biophrs. Acta, 19, 583 (1956). (1 1) Purchased from Nutritional Biochemicals Corp.

660

ROBERTW. HOLLEY TABLE I CONVERSION O F RADIOACTIVE AMP

INTO

ATP

Counts/min. in BaZATP

Con v ersion of A M P into ATP, %

Complete system‘ 152 f 27 5 L-Alanine omitted 74 i 13 2.5 icj Controlb 20 i 1 0.7 ( d ) As (a), plus 1 pg. ribonucleasec 73 rt 18 2.5 ((.) As (b), plus 1 pg. ribonuclease 71 f 20 2.5 ( I ) As (a), plus 0.1 pg ribonuclease 115 f 30 4 The complete system contained 100 pmoles of tris buffer, pH 7.5, !0 pmoles of potassium ATP, PH 7.0, 5 pmoles of potassium pyrophosphate, pH 7.5, 5 pmoles of tnagnesium chloride, 10 pmoles of potassium fluoride, 0.5 pmole of AMP-8-C1* containing 3000 counts/min., 2 pnioles of L-alanine, and approximately 2 mg. of protein, in 1 ml. The results given are for 10 min. incubation a t 37’, and are averages of several experiments, with standard deviations. I n over 20 experiments, the smallest increase in radioactivity of the A T P brought about by the addition of I.:hnirie (increase of (a) over ( b ) ) was 60%. Trichloroacetic acid was added immediately after addition of the enzyme solution. Crystalline, salt-free ribonuclease (TVorthington Biochemical Corp.). iu)

(11)

Vol. 79

which directly involve an amino acid, A T P and ribonuclease-sensitive material. Because of the evidence that ribonucleic acid is involved in soiiie way in protein synthesis,3 studies of the nature of the ribonuclease-sensitive material and elucidation of its role in the present enzyme system will be of special interest. Experimental Part

AMP Exchange Assay Procedure.-Assays were normally run using a volume of 1.0 ml. in 13 X 100 m m . test-tubes. In each tube was placed 0.10 ml. of 0.05 h!l MgCl? in 0.A M tris (tris-(hydroxymethyl)-an~inornethane)buffer, pl-I 7.5, 0.10 ml. of 0.1 ,If dipotassium A T P (crystalline, Pabst) neutralized t o pH 7.0-7.2 with either sodium hydroxide or potassium hydroxide, 0.10 ml. of 0.1 A I potassium fluoride in 0.05 ~1!l potassium pyrophosphate solution neutralized to pH 7 . 5 , and 0.05 ml. of 0.010 M AMP-8-C14 containing approximately 3000 counts/min.12 Amino acids or other compounds were added a t the concentrations indicated elsewhere, and the volume was made up to 0.50 ml. by the addition of water. T o this solution was added 0.50 ml. of cold enzyme solution, and the mixture was incubated, with shaking, in a water-bath a t 37’. At the end of the incubation period, the tubes were cooled in ice and 1.0 nil. of l2Yc trichloroacetic acid was added immediately t o each tube. (If necessary, the tubes can be stored briefly in the cold a t this stage.) The tubes were centrifuged and the supernatant trichloroAMP into A T P (eq. 1 and 2 combined), it is clear that AMP incorporation could not exceed pyro- acetic acid solutions were decanted into centrifuge tubes. T h e protein precipitates were washed with 1.0 ml. of water. phosphate incorporation. each, and the washes were added to the trichloroacetic acid The results described in this paper are compat- solutions. Each trichloroacetic acid solution was neutralible with a combination of eq. 1 and a more de- ized t o pH 7-8 by the addition of a measured volume (approximately 0.75 ml., determined by prior titration of an tailed formulation of eq. 2 as aliquot of t h e 12% trichloroacetic acid solution) of 1 N SOdium hydroxide. The A T P in each tube was precipitated Enzyme L-Alanine ATP by the addition of 0.50 ml. of 1 M barium acetate solution. Enzyme-L-Alanyl-AMP Pyrophosphate (1’) The tubes were left in a refrigerator 20 min. or longer, and then the precipitates were collected by centrifugation. Iliiz~irie-~-Alanpl-iihlP Each precipitate was washed twice with 1 ml. of water and Cofactors? Enzymes? twice with 0.5 ml. of water, and was finally suspended in s z Enzyme f 0.15 ml. of water and transferred t o a metal planchet, by means of a small pipet. T h e centrifuge tube and pipet were Includiiig ril)oniiclccl~crinsed with a few drops of water. The suspensions of solid \en\itiVe material were evaporated t o dryness, by means of an infrared lamp. (In order to obtain residues firmly bound t o the planchcts, L-Alanyl-X A M P (2’) t o permit easy handling, discs of lens tissue were cemented IVhether this formulation is correct, or not, only on the surface of the planchet< b y means of thinned Tygoil paint .) T h e radioactivities of the dried precipitates were iurther work will tell. using a thin-mica-rindow Geiger-Muller tube or As has been indicated, it is suggested, as a work- ameasured flow counter. ing hypothesis, that eq. 2 (or 2’) describes the Preparation of the Enzyme System from Rat Liver.-(The second step in the synthesis of proteins from amino procedure as far as resuspension of the first pH 5 precipitate x i d s . If this postulate is correct, amino acids is essentially t h a t of Hoagland, ~t al.,4 but is described here their reDort t h a t A M P fails t o exchange with ATP other than L-alanine should undergo a similar type because suggests t h a t the slight modifications may be important.) of reaction. It is known t h a t enzymes active with White rats were stunned and decapitated, and the liver.; ceveral amino acids in pyrophosphate exchange (1%-t.4-12 g . ) were removed and immediately chilled in an (eq. 1) are present in the rat liver p r e p a r a t i ~ n . ~ice-bath. The livers were homogenized individually. Unotherwise indicated all operations were carried out in a Therefore, it might have been anticipated that less cold room a t 2 ” . In a typical preparation, an 8-g. liver was .l?rIP exchange, if it took place with this enzyme minced quickly with scissors in 4 ml. of 0.05 N potassium preparation, would not be limited to one amino chloride solution and the mixture, with 8 ml. of additional acid. One possible explanation for the limitation potassium chloride solution, was homogenized in a PotterElvehjem type homogenizer with a Teflon pestle (A. H. of activity to L-alanine in the AMP exchange assay Thomas C a t . No. 4288-R, size C, diameter of pestle 0.4 would be that essential factors (“X,” etc. in eq. 2’) mm. less than the diameter of the glass tube). The pestle for the other amino acids are lost or inactivated was turned by a motor a t approximately 75r.p.m. Homogenization was continued only long enough for all the mate(luring preparation of this enzyme system. Since rial to pass between the pestle and tube. The mixture was it is thought t h a t an amino acid must find its diluted with 14 ml. more 0.05 N potassium chloride solution, specific place on the “coding system” or “tem- and homogenization was continued very briefly, t o mix the plateJJ2before it is incorporated into a protein, i t material. T h e homogenate (homogenates of several livers would not be surprising to find t h a t individual fac- can be combined) was centrifuged for 50 min. a t 40,000 r.p.m. (108,000 X g, average) in a refrigerated S p h c o tors are involved in the pathways of individual Model I, preparative ultracentrifuge. T h e red supernatant

+

+

+

+

+

~

+

amino acids. Whatever interpretation of the results is considered most plausible, i t is certain that the enzyme system described in this paper catalyzes reactions

(12) Radioactive A M P labelled with C I d In the 8-position was purchased from Schwarz Laboratories (0.7 rcuries/mg.) and was diluted approximately 10-fold w.ith non-radioactive AMP purchased from Pabst Laboratories.

Feb. 5, 1937

ALANINE-DEPENDENT CONVERSION OF A M P TO A T P

was removed with a pipet, avoiding, as much as possible, the scum of f a t t y material, and leaving approximately 1 ml. of solution in each centrifuge tube. T h e supernatant solution, in a beaker immersed in a n ice-bath, was adjusted t o PH 5.1-5.2 by the dropwise addition of approximately 1.7 ml. of 0.1 N hydrochloric acid. (A Beckman Model G pH meter was used in the Laboratory (room temperature) with the temperature adjustment at the lowest possible setting, which b y extrapolation was approximately 2 " . ) The precipitate was collected b y centrifugation for 5 min. a t 12,000 r.p.m. (10,000 X g average) in the refrigerated ultracentrifuge. T h e supernatant was decanted and the last drops were allowed t o drain from the tubes onto absorbcnt tissue. The tan-colored pellets were resuspended in a total of 14 ml. of cold 0.1 AI' (PH 7.5 at room temperature) tris buffer, homogeniied briefly, and centrifuged 5 min. a t 15,000 r.p.m. (16,000 X g, average) in the refrigerated ultracentrifuge. The tan-colored supernatant was frozen a t -20". With present knowledge, storage at -20" is best at this stage, and the second pH 5 precipitation is carried out immediately before the enzyme is used. Present data indicate t h a t the solution should be frozen before the second pH 5 precipitation is carried out. The above, "PH 5," enzyme preparation is thawcd and the cold solution is adjusted t o pH 5.1-5.2 by the addition of cold 0.1 N hydrochloric acid. The precipitate is collected by centrifugation for 5 rnin. at 12,000 r.p.m.. and the precipitate is resuspended in approximately eight-tenths of the original volume of 0.1 hf tris buffer at pH 7.5. After centrifugation for 5 min. at 15,000 r.p.m., the very-light-colored supernatant is ready for use in the A M P exchange assay. The solution contains 3-4 mg. of protein/ml., and its absorption maximum at 260 mp corresponds t o the presence of 0.3-0.4 mg. of nucleic acid/ml. Identification of the Radioactive Product as ATP.-To establish t h a t the radioactive material formed in the presence of added amino acid in the A M P exchange assay is actually A T P , samples of the barium salt were converted t o the potassium salt and these were chromatographed on 1Yhatman S o . 1 paper using the procedure of Cohn and Carter.13 After chromatography, the ratios of radioactivity to optical density (260 m p ) of the eluted A T P samples were determined. The results were the same as had been obtained by determining the radioactivity of the barium salts. As a further check on the identity of the radioactive material, samples of the barium salt of A T P from assays with and without added amino acids were treated v i t h an equivalent of dilute sulfuric acid and, after removal of the barium sulfate and addition of carrier ATP, ATP was precipitatcd as the triacridine salt.I4 The samples of triacridine salt were washed with ethanol and ether and dried. Aliquots were counted, and the remainder of each sample v a s recrystallized from Ivater and ethanol. The specific radioactivities of the samples of A T P remained constant, within experimental error. The radioactivities of the samples of recrystallized triacridine salt showed the same incrcase due t o the addition of L-alanine as had been found with the samples of barium salt. Pyrophosphate Exchange.-In order to compare AMP incorporation with pyrophosphate incorporation, two sets of reaction mistures were prepared according to the AMP exchange assay procedure (above), but with one set containing radioactive -4MP (3000 counts/min. in the 0.5 pmole present) and the other set containing radioactive pyrophosphateI6 (115,000 counts/min. in the 5 pmole present). .4t the end of the incubation, the tubes containing radioactive pyrophosphate were worked up according t o the method of Crane and Lipmann,I6 which involves adsorption of the A T P on charcoal. I n the presence of 2 pmoles of L-alanine, the .4TP contained 158 counts/min. from radioactive AMP and 4420 counts/min. from radioactive pyrophosphate. I n the absence of added amino acid the values were 67 and 700 counts/min ., respectively. Thus, the L-alanine-dependent radioactivity was 90 counts/min. from A M P and 3700 counts/min. from pyrophosphate. I n terms of pmoles of radioactive compound converted t o ATP, these values are (13) W. E. Cohn and C. E. Carter, THISJOURNAL, 72, 4273 (1950). (14) A. M , Michelson and A. R. Todd, J. Chem. Soc.. 2487 (1949). (15) Kindly furnished by J. M . Clark, Jr. Prepared by pyrolysis of

t h e dipotassium salt of Pa*-labeled orthophosphate obtained on allocation from the U . S Atomic Energy Commission. (1G) K . K. Crane and F. Lipmann, J . Btol. Chcm.. 201, 235 (1953).

66 1

0.015pmole from A M P and 0.16 pmole from pyrophosphate. Therefore, under these conditions, incorporation of radioactive A M P is approximately 9% of the incorporation of .. pyrophosphate. The addition of 1 pgram of ribonuclease did not inhibit the L-alanine-dependent incorporation of pyrophosphate into ATP. Addition of Various Preparations Containing Ribonucleic Acid.-Addition of the following materials did not increase the amino acid-dependent conversion of AMP to A T P in the standard A M P exchange assay. (A mixture of 2 pnioles of each of the following amino acids was used in this set of experiments: L-alanine, L-arginine, L-glycine, L-histidine, L-isoleucine, 1.-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, Ltyrosine and L-valine.) (1) Commercial yeast ribonucleic acid, 0.3 mg. added/ assay tube. ( 2 ) Freshly prepared beef liver ribonucleic acid,17 0.4 mg. or 0.04 mg. added/assay tube. (3) Freshly prepared rat liver microsomes@: the pellet obtained in one centrifuge tube was homogenized with 0.5 ml. of 0.1 M tris buffer, and 0.05 ml. of the mixture was added t o the assay mixture. (The addition of this r a t liver microsome preparation greatly increased the amino acid-independent conversion of A M P t o A T P . Abood and RomanchekIg have reported the presence of adenylic kinase in r a t liver microsomes.) (4) R a t liver microsomes treated with sodium desoxycholate's: the same fraction of a microsome pellet was used as in (3). (5) R a t liver microsomes treated u i t h sodium lauryl SUIfatez0: in the cold room, a microsome pellet was homogenized with 5 ml. of 0.1 M PH 7 . 5 tris buffer and 0.5 ml. of a 5y0 solution of sodium lauryl sulfate in 4570 ethanol was added. After 2 hr. a t 2", 320 mg. of sodium chloride was added, and the mixture was centrifuged 20 min. a t 25,000 g average. T o 4 ml. of the supernatant was added 8 mi. of cold 95% ethanol and the precipitate was collected 1 1 ~ centrifugation in a small centrifuge in the cold room. The precipitate was washed three times with cold acetone, and u a s allorTed to dry briefly in air. It was mixed with 0.5 nil. of 0.1 hf pH 7.5 tris buffer, and 0.050 ml. was added to the assay mixture. (6) R a t liver microsomes treated with phenol2': in tllc cold room, a microsome pellet was homogenized with 0.,5 ml. of 0.1 ill pH 7.5 tris buffer and the mixture was eatractfd four times with 0.5-1.0 ml. of phenol saturated with \ v a t u . The aqueous solution was extracted repeatedly with ether, the remaining ether was allowed t o evaporate into the air in the cold room, and 0.050 ml. of the solution was added to the assay mixture. ( 7 ) Freshly prepared pea microsomes22: 0.7 ing. or 0.07 rng. added/assay tube. Addition of Other Materials.-The following compounds were tested in the absence of added 1.-alanine in the standart1 AMP exchange assay procedure a t the concentrations indicated (number of pnioles added/assay tube is givcn in parctitheses) and had no significant effect on the conversion of radioactive A M P t o ATP, i.e., the radioactivity of the ATP at the end of the incubation was the same as if nothing had been added: amino acids (2 each), D-alanine, L-argininc, Laspartic acid, L;asparagine, L-cysteine, L-glutamic acid, Lglutamine, glycme, L-histidine, L-isoleucine, L-leucine,, Llysine, 1.-methionine, L-phenylalanine, L-prolinp, L-serine, L-threonine, L-tryptophan, L-tyrosine and L-valine; cornz y n e A (1); glucose ( 3 0 ) ; potassium acetate (2); soditnn propionate (2); sodium pyruvate (2); cytidine 5'-triphos(17) Kindly furnished by V. D. Burrows. Prepared according t o S. E. Kerr and K. Seraidarian, i b r d . . 180, 1203 (1949). (18) J. W. Littlefield, E . B. Keller, J. Gross and P. C. Zarnecnik. ibid., 217, 111 (1955). h u t homogenization was in 0.05 N potassium chloride.

(19) L. G. Ahood and L. Romanchek, Exfill. Cell Research, 8, 459 (1955). (20) Procedure patterned after t h a t of E. R. M. K a y and A. L. Dounce, THISJ O U R N A L , 75, 4041 (1953). (21) Procedure patterned after t h a t of A. Gierer and G. Schramm. N a f t r r c , 177, 702 (1950). (22) Kindly furnished b y P. 0. P. Ts'o. Prepared according t o P. 0. P. Ts'o,J. Bonner and J. Vinograd, J. Biophysic. Biochcm. C y f o l . , in press.

KENNETHD. Iiorrr,~

The Reaction of Amines with Amino Acid N-Carboxy Anhydrides BY KENNETH 1).KOPPLE RECEIVED SEPTEMBER 11, 1956 'l'hc i-cacli~illuf : t ~ i i i i i e iwith S-carl~osyaiili).dritlcsof a-amiiio acids priiccctih lo yicl:l citliel- J til-eido Lieid (attack d l 1 1 1 ~ 2-carhonyl of the oxazolidinedioiic ring) or a n amino amide (attack at the 5-carbuiiyl Tile actual course of the reactioii is tlependent on the steric requirenieiits of the aniiiie, its cunceiitration aiid its basicity. 21 iiuniber of iiew ureido acids arc re1)orted.

The reaction of an a-amino acid hi-carboxy anhydride (4-alkyl-2,5-oxazolidinedio1ic, I, abbreviated here to NCAj with an amine may, in principle, take either of two paths, X or B. r+ j A

KCH---CO

)o 3- K,1