l -arabinose metabolism by cell-free extracts of Penicillium chrysogenum

l -arabinose metabolism by cell-free extracts of Penicillium chrysogenum

BIOCHIMICA ET BIOPHYSICA ACTA L-ARABINOSE METABOLISM BY CELL-FREE OF P E N I C I L L I U M 271 EXTRACTS CHRYSOGENUM CHING CHIANG AND S. G. KNIGHT...

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BIOCHIMICA ET BIOPHYSICA ACTA

L-ARABINOSE METABOLISM BY CELL-FREE OF P E N I C I L L I U M

271

EXTRACTS

CHRYSOGENUM

CHING CHIANG AND S. G. KNIGHT

Department o] Bacteriology, University o] Wisconsin, Madison, Wisc. (U.S.A.) (Received June I4th, 196o)

SUMMARY

The enzymic conversion of L-arabinose to both L-ribulose and L-xylulose b y the formation of an intermediate, L-arabitol, has been observed in cell-free extracts of Penicillium chrysogenum. All enzymic products involved in these enzymic steps have been isolated and characterized. This finding emphasizes a common enzymic sequence for the initiation of pentose metabolism by this mold.

INTRODUCTION

During the course of studies on D-xylose metabolism by Penicillium chrysogenum in this laboratory, it was found that the n-xylose was converted to D-xylulose with the formation of D-xylito1 as an intermediate in the enzymic sequence. The specific enzyme for the reduction of D-xylose to D-xylitol was partially purified and we believe that this is the enzyme which initiates the metabolism of the free pentose b y this mold1, *. This metabolic characteristic, different from that in the bacteria which metabolize xylose, has been found in other species of fungi that can utilize xylose as a carbon source; a detailed account of this work will appear elsewhere. This paper describes a somewhat similar pattern of initial steps in the degradation of L-arabinose b y cell free extracts of P. chrysogenum when the cells have been grown on a L-arabinose -mineral salts medium. It appears that L-arabinose is reduced to L-arabitol by a TPNH-linked reductase followed b y the oxidation of L-arabitol to both L-xylulose and L-Iibulose by two independent DPN-linked dehydrogenases. EXPERIMENTAL TECHNIQUES

Preparation of cell free extracts Cultures of P. chrysogenum strain I95I-B25 were grown in the previously described mineral salts medium with I °/o L-arabinose as the sole carbon source ~. The spore suspension was inoculated directly into the liquid media. After 72-h incubation at 3 °0 on a rotary shaker, the mycelia were harvested and washed thoroughly in cold Abbreviations : TPN and TPNH, oxidized and reduced triphosphopyridine nucleotide ; DPN and D P N H , oxidized and reduced diphosphopyridine nucleotide; glucose-6-P, glucose-6-phosphate; Tris, tris (hydroxymethyl) aminomethane; TCA, trichloroacetic acid.

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distilled water in a suction funnel. The cell extracts were prepared by grinding the wet mycelia and an equal weight of sea sand with a mortar and pestle, followed by extraction with 2 volumes of o.I M phosphate buffer at pH 7.2, and the removal of the cell debris and sand by centrifugation at 2,000 × g for IO rain. The supernatant adjusted to contain io mg protein/ml was the source of the enzymes. Occasionally, the extracts were dialyzed against o.I M phosphate buffer at pH 7.2 for 8 to 12 h before use. Analytic methods All spectrophotometric measurements were made with Beckman spectrophotometers, either model DU or the DK-2 automatic recorder. In various colorimetric analyses, aliquots removed from the enzymic reactions were quenched with I.O ml 5 % Z n S Q . 7H~O and I.O ml 0.3 N Ba(OH)~, the reagents having been adjusted to a final pH 7.2. Aldopentose was measured by the orcinol reaction modified by HORECKER et al. 3 and the ketopentoses were determined by cysteine-carbazole color test of DISCHE AND BORENFREUNDi. In the case of ribulose, color development was completed after 15 min at room temperature with L-ribulose-o--nitrophenyl-hydrazone as the standard; while with xylulose or a mixture of xylulose and ribulose, 2 h incubation at 37 ° was employed for full color development with I)-xylulose monoacetone as the standard. The determination of pentitol was made by the periodate oxidation of RAPOPORT AND WEST5. The protein content was determined by the method of LOWRY et al. e. Separation of free sugars was accomplished with a Dowex 1-borate column 7. Paper chromatograms for identification of the ketopentoses were developed on Whatman No. I filter paper with descending water saturated phenol at 3°0 for 20 h. The sugars were detected by spraying with the orcinol-TCA reagent of BEVENUE AND WILLIAMS 8, and the combination of orcinol-TCA and aniline hydrogen phthalate as described by HOCHSTER9. The melting points were determined with a Fisher-Jones melting point apparatus. Polarimetric measurements were made with Haensch polarimeter No. 52-b, reading to o.oi ° with a semimicro tube having a light path of I dm. Materials L-arabinose was a Pfanstiehl product and L-arabitol was from Nutritional Biochemical Corporation. D- and L-xylulose and L-ribulose wele prepared by isomerization of I)- and L-xylose and L-arabinose respectively in boiling pyridine 1°, 11, and further purified by bromine oxidation according to the method described by HOCHSTER9. All the ketopentose solutions were stored frozen. D-xylulose monoacetone and L-ribulose-o-phenylhydrazone were gifts of Dr. W. A. Wool) of the Michigan State University. TPNH, DPNH, TPN, DPN, glucose-6-P and glucose-6-P dehydrogenase were from the Pabst Laboratories and the Sigma Chemical Company. RESULTS

Reduction of L-arabinose with T P N H The reduction of L-arabinose by cell extracts with T P N H was demonstrated by measuring spectrophotometrically the decrease in O.D. at 34 ° m/~. Fig. I illustrates the rapid oxidation of T P N H by the cell extracts in the presence of L-arabinose. D P N H also was oxidized at a considerable rate, but with no difference in rate in the 13iochim. Biophys. Acta, 46 (1961) 271-278

L-ARABINOSE METABOLISM BY P e n i c i l l i u m

chrysogenum

273

presence or absence of L-arabinose; thus indicating the specific requirement for T P N H as the coenzyme in the reduction of L-arabinose. No oxidation of T P N H was observed if the cell extracts were inactivated b y heating in boiling water for 5 min. I t has been impossible to demonstrate the presence of an arabinose isomerase or an arabinokinase. The cell free extracts also oxidized T P N H fairly rapidly with D-xylose, L-xylose and D-glucuronic acid; slowly with D-arabinose, D-ribose and D-galacturonic acid; but not with D-glucose or D-mannose. A competition for T P N H oxidation with a TABLE I THE

SPECIFICITY

AND

COMPETITION

FOR

THE

REDUCTION

OF L - A R A B I N O S E

AND

D-XYLOSE

T h e r e a c t i o n m i x t u r e w a s a d i a l y z e d cell e x t r a c t c o n t a i n i n g o.o7 m g prot e i n, o.16 /~mole T P N H a n d 0.05 M Tris buffer a t p H 7.4, in a t o t a l v o l u m e of I.O ml. The v e l o c i t y w a s r e c o r d e d b y m e a s u r i n g t h e decrease in O.D. a t 34 ° m/~ pe r m i n u t e Addition

Final (M) concentration

L-arabinose

D-xylose

L-arabinose + D-xylose L-arabinose + D-xylose

Velocity

0.04 0.08

0.05 ° 0.087

O.IO

O.10 5

o.04 0.08 o.io o. i o 0.04 o. I o 0.O8

0.o22 o.o36 0.040 0.086 O.O72

* E n d o g e n o u s o x i d a t i o n of T P N H was negligible. TABLE II STOICHIOMETRY

OF T H E

REDUCTION

OF L - A R A B I N O S E

TO P E N T I T O L

The T P N H - g e n e r a t i n g s y s t e m in b o t h e x p e r i m e n t s was c o m p o s e d of 3 ° /2moles of glucose-6-P, I /*mole of T P N , o.2 m g of glucose-6-P d e h y d r o g e n a s e , a n d IO / *mol e s of MgC1 v I n E x p t . i , und i a l y z e d cell e x t r a c t s e q u i v a l e n t to 8. 7 m g protein, 23. 7 #*moles of L-arabinose a n d 2 4 0 / z m o l e s of p h o s p h a t e buffer a t p H 7.5 were m i x e d in a t o t a l v o l u m e of 3.o ml. A n o.5-ml a l i q u o t w a s r e m o v e d a t t i m e i n t e r v a l s . I n E x p t . 2, d i a l y z e d cell e x t r a c t s of IO m g prot e i n, 32.3 /*moles of L-arabinose a n d 2 0 0 / * m o l e s of Tris buffer a t p H 7.5 were m i x e d in a t o t a l v o l u m e of 2.0 ml. A n o.2-ml a l i q u o t w a s r e m o v e d a t t h e t i m e i n t e r v a l s . All r e a c t i o n s were i n c u b a t e d a t 3 °o . Time

(rain)

#moles of penlose (as L-arabinose)

Total

Difference

Itmoles of pentitol (as L-arabitol)

Ratio Pentitol

Total

Difference

PenIose

o 3° 60 12o

Expt. i 23. 7 -18.8 - - 4-9 16. 7 - - 7.o 13. 5 - - lO.2

16.9 21.6 24.0 27.o

-+ 4.7 + 7.1 + IO.I

-0.96 I.Oi 0.99

o IO 3° 6o

Expt. 2 32.3 -26.4 - - 5.9 18.3 --14.o 12. 3 --20.0

3.6 9.2 17.2. 23.9

-+ 5.6 + 13.6 + 20. 3

-o.95 o.97 I.Oi

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C. CHIANG, S. G. KNIGHT

mixture of L-arabinose and D-xylose was observed, indicating that one enzyme system probably acts on these aldopentoses. In a comparison of L-arabinose and D-xylose reduction, it was found that the apparent Ks and Vmax were o.21 M and o.318 for D-arabinose, and o.12 M and 0.088 for D-xylose, respectively. This enzyme has a comparatively high specificity for the reduction of L-arabinose and the highest reaction rate was obtained with this substrate (Table I). Another measurement of the enzymic reduction of L-arabinose was made by simultaneously determining the disappearance of L-arabinose and the formation of pentitol. Table II shows two typical experiments performed in different conditions; however, a stoichiometric relationship of approximately a I : I ratio in pentitol and pentose strongly supports the existence of a one-step enzymic reduction. There was no change recorded if the T P N H generating system was omitted from the reaction mixture. Preparation and characterization of L-arabitol A substantial amount of pentitol has been prepared for identification from an enzymic reaction containing 500 ~moles L-arabinose, 500 ~moles glucose-6-P, 3 mg glucose-6-P dehydrogenase, IO mg TPN, 50 ~moles MgCI~, and 123 mg dialyzed cellextract pzotein in 20 ml of o.I M (final concentration) Tris buffer, pH 7.5- Analysis of aliquots for pentose indicated that a 50 % conversion had occurred after 4 h incubation. The reaction was stopped by heating in boiling water for 5 min, and the solution was acidified to pH 4.5 by adding a few drops of 6 N HC1; the precipitated protein was removed by centrifugation. The supernatant was deionized by passing over a two-layer column (2.5 × 60 cm) composed of equal parts of IR-I2o(H) and IR-45(OH). The eluate from the deionizing column was concentrated in vacuo and the yellow syrup obtained was dissolved into 5 ml of o.o05 M K,B407 and then

I

I

TPNH E~O~ENOU~

\

o

m .2|

i

L'~ITOL .I."

.EC

~

ADOEL PfliL~

/

.10 TPNH a .0!

.Oe

0

TIME

Z

IN

3

MINUTES

¢

5

Fig. I. O x i d a t i o n of T P N H in t h e presence of L-arabinose. The s y s t e m c o n t a i n s 20 /~moles L-arabinose w h e n added, o . 0 6 / , m o l e T P N H or DPNH, d i a l y z e d cell e x t r a c t s c o n t a i n i n g 0.2 m g pro tein, a n d i o o # m o l e s Tris buffer of p H 7.4 in a t o t a l v o l u m e of i . o ml. The r e a c t i o n was i n i t i a t e d b y m i x i n g in T P N H or D P N H ; t h e t e m p e r a t u r e w a s 28 ° .

S

TI M

4

IN

6 Ill MINUTES

Fig, 2.-Reduction of D P N in the presence of L-arabitol. The i . o - m l reaction m i x t u r e contained 20 /*moles L - a r a b i t o l w h e n added, o. 5 /*mole D P N , d i a l y z e d cell e x t r a c t s cont a i n i n g o.2 m g p r o t e i n , a n d IOO # m o l e s Tris buffer of p H d e s i g n a t e d . The r e a c t i o n w a s initiated by mixing in D P N and the temperat u r e w a s m a i n t a i n e d a t 28 ° .

Biochim, Biophys. Acta, 46 (1961) 271-278

L-ARABINOSE METABOLISM BY

Penicillium chrysogenum

275

placed on a Dowex i-borate column (I.I × 15 cm). The peak positive to periodate oxidation but negative to the orcinol and cysteine-carbazole color tests was eluted after 430 ml of o.o15 M KzB~07; it was collected to be deionized b y treatment with Dowex 50 (H) and freed of borate ions with absolute methanol; the residue was concentrated to a pale yellow oil. This compound was characterized from its pentaacetate derivative. 25 mg of the oily syrup was acetylated with 0.3 ml acetic anhydride b y shaking overnight in the presence of 0.3 ml dried pyridine. Since no precipitate appeared after the addition of 3 ml of cold water, the m i x t m e was dried and freed of pyridine by three distillations with benzene. The residue was taken up into 0.5 ml of ether and the insoluble material was then centrifuged off. The supernatant was kept cold (6 °) and after several hours a shiny whit e flaky solid gradually crystallized along the side of the tube at the liquid surface until a small portion of oil was left. The crystals were collected with a spatula, recrystallized twice from ether, and dried over anhydrous CaC1v This product weighed 2.8 mg and melted at 76-77 ° as did L-arabitol-pentaacetate prepared in the same manner from known L-arabitol. This indicates that the pentitol which was produced from the enzymic reduction of Larabinose was L-arabitol.

Oxidation of L-arabi~ol with DPN Since L-arabinose was found to be reduced to L-arabitol by an enzyme in the extracts, attempts were made to explore the degradation of this latter compound. Similar to the pattern of D-xylitol utilization by this organism, L-arabitol also is oxidized b y cell extracts with an increase in reduction of DPN measured at 340 m/z, as shown in Fig. 2. The enzymic reaction in vitro proceeds to completion at p H 8.5; while at p H 7.4, an equilibrium was reached after 5 min. Therefore, most subsequent studies were carried out at p H 8.5. T P N also was reduced; however, there was no stimulation in the reduction of T P N over the endogenous reduction even though 4 ° tmaoles of L-arabitol was added. The enzyme system showed a specific requirement for D P N as the coenzyme in this reaction. Under the conditions described the cell extracts oxidized D-xylitol at a rate 50 % of that for L-arabitol. Efforts to show stoichiometry between DPN reduction and the oxidation of L-arabitol were unsuccessful berause the D P N H oxidation system interfered with the D P N H measurement. TABLE III FORMATION OF KIgTOPENTOSES FROM THE OXIDATION OF L-ARABITOL T h e r e a c t i o n s y s t e m c o n t a i n e d 4 ° /*moles L-arabitol, 2o # m o l e s D P N , u n d i a l y z e d cell e x t r a c t s c o n t a i n i n g 17. 4 m g protein, a n d 3 6 0 / * m o l e s Tris buffer of p H 8. 5 i n a t o t a l v o l u m e of 4.0 ml. A n o.6-ml a l i q u o t w a s w i t h d r a w n for a n a l y s i s a t t h e t i m e i n t e r v a l s . All r e a c t i o n s w e re i n c u b a t e d a t 3o °. pmotes of ketopentose Time (h)

o o.5 I.O 2.o 4.0

Complete system

L ambitol omitted

Tctal

Difference

Total

Difference

o.88 4.2o 4.96 5.88 6.1o

-3.32 4-°8 5.0o 5.22

o.92 I.OO i.o6 1.12 1.22

-o.08 o.I 4 o.2o o.30

B i o c h i m . B i o p h y s . A c t a , 46 (z96I) 271-278

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c. CHIANG, S. C. KNIGHT

Reasonable products from the oxidation of L-arabitol could be either L-ribulose or L-xylulose or both. A progressive increase of color intensity in the cysteinecarbazole reaction indicated the formation of ketopentoses from the oxidation of L-arabitol as shown in Table I I I . With dialyzed cell-free extracts, the reaction did not endogenously produce compounds positive to the cysteine-carbazole color test; however, the enzymic activity was decreased to 80 % of the original preparation.

Isolation and identification of ketopentoses Unfortunately, there are no chemical methods available to discriminate between ributose and xylulose in a mixture. However, the ketopentoses can be separated by means of fractionation on a Dowex 1-borate column. To minimize the endogenous materials which could produce a positive cysteine-carbazole test, the cell extracts were dialyzed before use. Then 60 /*moles L-arabitol and 60 /*moles DPN were incubated with cell extract containing 15 mg plotein in 3 ml, o.I M (final concentration) Tris, p H 8.5. After 4 h incubation the reaction mixture was treated in the same manner as described for the preparation of L-arabitol. A syrup containing approximately 5 /*moles of ketopentoses was placed on a Dowex 1-borate column (1.1 X 18. 7 cm) by dissolving in 5 ml of 0.005 M K , B 4 0 v After the column was eluted with 39 ° ml of 0.02 M K,B407, a peak positive to the orcinol and cysteine-carbazole color test and periodate oxidation appeared in an amount of 3.12 /*moles. The next peak started at 800 ml and was completed after 12oo ml of elution with 0.02 M K2B~O 7. This was apparently the residual L-arabitol which was positive to periodate oxidation only. The column then was subjected to elution with 0.03 M K2B~O 7, and another peak positive to the three tests, in an amount of 0.98/,mole, came off after 220 ml of elution. These two cysteine-carbazole positive peaks were separately pooled, freed of borate ion, and characterized by orcinol spectra and paper chromatography as the data in Table IV show. They were identified as xylulose and ribulose respectively. Neither was produced endogenously. 70.5/*moles xylulose isolated by this procedure had the L-configuration [(a)~ ----- + 34.05 °, c ---- 0.881, a = + 3 °0. the (~)~s of Lxylulose reported in the literature is + 33.15 ° to 34.8 ° (see ref. 12, 13)! ; while approx. 25/*moles of ribulose also obtained was dextrorotatory (no accurate reading in degree because of small amount of sample) indicating a L-configuration.

Oxidation of DPNH with ketopentoses Since two ketopentoses were produced simultaneously during the enzymic T A B L E IV IDENTIFICATION OF KETOPENTOSES I~ROM THE OXIDATION OF L-ARABITOL

First cysteine-carbazole positive p e a k Second c y s t e i n e - c a r b a z o l e Xylulose s t a n d a r d Ribulose s t a n d a r d

Orcinol spectrum E~,o ~o

Paper chrcmatogmphy RF

T CA -orcinol

TC A- ~rcinol and aniline hydrogen phthalate

o.4o o.86 o.46 0.89

o.54 0.63 0.56 0.64

Gray Yellowish b r o w n Gray Yellowish b r o w n

Purple Pink Purple Pink

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chrysogenum

277

oxidation of L-arabitol, the question can be raised as to whether a single enzyme reacts nonspecifically or two enzymes react independently on the hydroxyl groups of C2 and C4 of the carbon skeleton of L-arabitol. In experiments carried out in the presence of ketopentoses, the cell free extracts oxidized D P N H with either L-ribulose or L-xylulose and the reactions proceeded the most rapidly at pH 7.4. With nearly satulating amounts of L-ribulose (the apparent K8 for L-ribulose is 0.007 M) the addition of L-xylulose gave an additive effect for the oxidation of D P N H by cell free extracts, indicating that it is possibly a sum of reactions from two independent enzymes (Table V). TABLE

V

THE OXIDATION OF D P N H WITH HETOPENTOSES BY CELL FREE EXTRACTS OF penicillium chrysogenum T h e r e a c t i o n m i x t u r e c o n t a i n e d d i a l y z e d e x t r a c t s w i t h o . o 7 m g p r o t e i n , 0.2 # m o l e D P N H a n d t h e i n d i c a t e d k e t o p e n t o s e s i n a t o t a l v o l u m e of o . o 5 M T r i s b u f f e r a t p H 7.4. T h e v e l o c i t y is recorded as the decrease in O.D. at 34 ° mp per minute.

Addition

L-ribulose

L-xylulose L-ribulose + L-xylulose L-ribulose -- L-xylulose

Final concentration (m) 2 . 4 . I o -3 4.7" l O - 3 7.0. lO-3 1 . 2 . lO _2 1-5" I O-3 4.5" 10-3 1.2" 10 -3 1.5" l O - 3 1.2" lO -3 4.5" 1°-3

Velocity"

o.o24 o-o4o 0.052 0.o60 O.O40 O.120 o.ioo O.180

* A f t e r c o r r e c t i o n f o r e n d o g e n o u s o x i d a t i o n of D P N H .

DISCUSSION

These experimental results show that the degradation of L-arabinose by P. chrvsogenum, as studied in the cell-free extracts, is initiated with the reduction of L-arabinose to L-arabitol, followed by reoxidation of the latter compound to both L-xylulose and L-ribulose. There appears to be only one enzyme involved in the conversion of Larabinose to L-arabitol, since the enzyme is highly specific for L-arabinose and only L-arabitol could be isolated following the reaction. It is of interest that this enzyme is similar to D-xylose reductase which is induced when D-xylose is the carbon source. This mold has the genetic potential of forming a specific enzyme system for initiating the metabolism of a particular pentose. Furthermore, there seems to be a common enzymic route for pentose metabolism by this fungus. Although two independent enzymes simultaneously oxidized L-arabitol and produced two kinds of ketopentoses in vitro, the higher activity in the production of L-xylulose over L-ribulose indicates that L-xylulose may be the major product in physiological conditions. Recently, ANDERSON reported that a L-xylul0se kinase was found in the cell extracts of A erobacter aerogenes, and that the L-xylulose 5-phosphate thus formed was converted to the D-isomer by a 4-epimerase 14. It is possible that Biochim. Biophys. Acta, 4 6 (1961) 2 7 t - 2 7 8

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similar enzyme systems could exist in the cell extracts of P. chrysogenum. However, it is also likely that the L-xylulose formed from the oxidation of L-arabitol m a y be reduced to D-xylit01 and then oxidized to D-xylulose, as that was found in the animal cellslS, 16. ACKNOWLEDGEMENT

This investigation has been supported by grant G 3359 from the National Science Foundation and grant E - i 2 o i from the National Institute of Health. REFERENCES 1 C. CHIANG, C. J. SIH AND S. G. KNIGHT, Biochim. Biophys. Acta, 29 (1958) 664. 2 C. CHIANG AND S. G. KNIGHT, Biochim. Biophys. Acta, 35 (1959) 454. 3 B. L. HORECKER, P. Z. SMYRNIOTIS AND H. KLENOW, J. Biol. Chem., 2o 5 (1953) 661. 4 Z. DlSCHE AND E. BORENFREUND,J. Biol. Chem., 192 (1951) 583. 5 C. D. WEST AND S. RAPOPORT, Proc, Soc. Exptl. Biol. Med., 7° (1949) 141. e O. H. LOWRY, N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265. 7 L. P. ZILL, J. Z. KHYN AND G. M. CHANIAE, J. Am. Chem. Soc., 75 (1953) 1339. 8 A. BEVENUE AND K. T. WILLIAMS, Arch. Biochem. Biophys., 34 (1951) 225. 9 R. M. HOCHSTER, Can. J. Microbiol., I (1954) 346. 10 O. T. SCHMIDT AND R. TREIBER, Bet., 66 (1933) 1765. 11 T. REICHSTEIN, Helv. Chim. Acta, 17 (1934) 996. 12 p. A. LEVENE AND F. B. LAFORGE, J. Biol. Chem., 18 (1914) 319. la I. GREENWALD, J. Biol. Chem., 88 (i93 c) i. 14 R. L. ANDERSON, Federation Proc., 19 (196o) 82. 15 S. HOLLMANN AND O. TOUSTER, J. Biol. Chem., 225 (1957) 87. le D. B. McCoRMICK AND O. TOUSTER, J. Biol. Chem., 229 (1957) 451.

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