A comparative study of the regulation of pyridine nucleotide formation

BIOCHIMICA ET BIOPHYSICA ACTA

445

BBA 4251

A C O M P A R A T I V E S T U D Y O F T H E R E G U L A T I O N OF PYRIDINE

NUCLEOTIDE FORMATION JOHN IMSANDE*

Group in Biochemical Sciences, Department of Biology, Princeton University, Princeton (U.S.A.) (Received June 2Ist, I963)

SUMMARY

The activity and regulation of nicotinic acid mononucleotide pyrophosphorylase (nicotinic acid mononucleotide:pyrophosphate phosphoribosyltransferase) the ratelimiting enzyme in pyridine nucleotide biosynthesis, was compared in seven species. The enzyme from all species exhibited a uniform requirement for ATP. That pyridine nucleotide biosynthesis in Escherichia coli is regulated, at least in part, by a repression-derepression mechanism was substantiated. In contrast, pyridine nucleotide formation from nicotinic acid in Serratia marcescens, Bacillus subtilis, Torula cmmoris, Tetrahymena pyriformis, and the rat was regulated neither by a repression-derepression mechanism nor by feedback inhibition by NAD. It is concluded that the repression-derepression control mechanism may not be widespread in nature.

INTRODUCTION

Regulation of cellular metabolism has received wide attention in recent years 1, ~. In fact, the phenomena of feedback inhibition3, 4 and repression~lerepressionS, 6 have now been established unequivocally as physiologically useful mechanisms in the regulation of cellular metabolism. Unfortunately, most of the definitive studies on metabolic regulation have been confined to bacteria, particularly Escherichia coli and related species 7. Reports concerning metabolic regulation of various biosynthetic pathways in amphibia, the chick embryo, and the rat, etc., have appeared, but the significance of such studies has not been assessed ~. Hence one wonders how widespread in nature these two types of control mechanisms might be. It appeared that the pyridine nucleotide pathway might be very useful for studies on the comparative biochemistry of metabolic control mechanisms. It has long been known that pyridine nucleotides are vital to all living systems, most of which are capable of forming the pyridine nucleotides that they require. Many of these organisms, or mutants thereof, require nicotinic acid for growth and hence can be made deficient in pyridine nucleotides even though they possess the enzymes necessary for the formation of pyridine nucleotides from nicotinic acid. Abbreviation: NaMN, nicotinic acid mononucleotide. * Present address: Dpartment of Biology, Western Reserve University, Cleveland, Ohio, U.S.A.

Biochim. Biophys. Aeta, 82 (1964) 445-453

446

j. IMSANDE

Recently it was established that pyridine nucleotide biosynthesis in E. coli proceeds from nicotinic acid via nicotinic acid nuclei)tide intermediates, and that NaMN pyrophosphorylase (nicotinic acid mononucleotide:pyrophosphate phosphoribosyltransferase), the first enzyme of the pathway, is rate limiting in pyridine nucleotide formation6, s. Furthermore, it was shown that pyridine nucleotide formation in this organism is regulated, at least in part, by the mechanism of repression and derepression ~. Regulation by feedback inhibition, on the other hand, could not be demonstrated in vitro G. This report will present further evidence that pyridine nucleotide biosynthesis in E. coli and Salmonella is regulated, at least in part, by a repression-derepression mechanism and not by a feedback inhibition mechanism. In contrast, h(~wever, pyridine nucleotide formation in Serratia marcescens, Torula cremoris, Tetrahymena pyriformis and the rat appears to be regulated neither by a repression-derepression mechanism nor by a feedback inhibition mechanism.

E X P E R I M E N T A L AND RESULTS

Materials E7-14CjNicotinic acid (specific activity 9.19 mC/mmole) was obtained from New England Nuclear Corporation; ATP, 5-phosphoribosyl-I-pyrophosphate, NMN, and NAD from Pabst Laboratories; a special niacin-tryptophan-deficient experimental diet and a vitamin B complex control diet for the rat from Nutritional Biochemical Corporation; and 7-week-old CFE male rats from Carworth Farms, Inc. Nicotinic acidrequiring m u t a n t s of Bacillus subtilis were provided by Dr. C. YANOFSK¥ ; the nicotinic acid m u t a n t of Salmonella b y Dr. M. DEMEREC ; Tetrahymena pyriformis strain E by Dr. R. D. ALLEN. Nicotinic acid-requiring E. coli strain 15, No. 9723b, and Torula cremoris, No. 2512, are mutants from the American Type Culture Collection. Methods Cell growth was followed with a Klett-Summerson Colorimeter (filter No. 54). The molar concentration of growth supplements listed for each experiment indicates the initial concentration at the outset of growth. Solutions of NAD were sterilized by millipore filtration. Centrifugation of all cell-free extracts was done in the cold. Protein was determined by the Folin procedure 9, and pyridine nucleotide formation was measured as previously described a. All incubations in vitro were terminated by heating in a boiling-water bath for I rain. The specific activity of the enzyme under study is expressed as m/~moles of NaMN formed per mg of protein per h. Growth of E. coli and enzyme preparation Nicotinic acid-requiring m u t a n t 9723 b, of E. coli strain 15, was grown in a saltsglycerol medium 1° supplemented with either 5" lO-7, I . lO 4, 5" lO-6, or I. lO -4 M NAD, NMN, or nicotinic acid. Cultures, usually I 1, were grown overnight with vigorous shaking at 37 °. The bacteria were collected by centrifugation, washed, and disrupted by alumina grinding 6. The broken-cell preparation was suspended in phosphate buffer s, centrifuged, and the slightly turbid supernatant solution was examined for protein content and its ability to catalyze the formation of pyridine nucleotides s. As shown in Table I, the specific activity of NaMN pyrophosphorylase, the enzyme that Biochim. Biophys. ~-tcta, 82 (19~)4) 4 4 5 453

COMPARATIVE CONTROL OF

NAD

447

FORMATION

catalyzes the formation of NaMN from nicotinic acid and 5-phosphoribosyl-I-pyrophosphate, varied as much as Ioo-fold and was related to the concentration of the growth supplement in the medium. TABLE Na~N

I

PYROPHOSPHORYLASE ACTIVITY IN E . c o l i STRAIN I 5

R e a c t i o n m i x t u r e s c o n t a i n e d 2 0 / z m o t e s M g S O a, 3 / t m o l e s A T P , i /~mole 5 - p h o s p h o r i b o s y l - I pyrophosphate, 5o/,moles Tris-phosphate (I : I ) ( p H 8.5), 4 o # m o l e s N a F , o . i / , m o l e [7-14C~ n i c o t i n i c a c i d , a n d o . I m l e n z y m e i n a t o t a l v o l u m e of I.O ml. I n c u b a t i o n w a s c o n d u c t e d f o r 3 ° m i n a t 37 °. Molar concentration of growth supplement Growth supplement

5" lo ~

1 • to -6

~" Io-6

5" ro *

t • zo 4

Specific activity of enzyme preparation

Nicotinic acid NMN NAD

-44 48

46 41 5°

25 18 34

-0. 4 --

1.5 ---

Growth of Salmonella typhimurium and enzyme preparation The bacterium S. typhimurium is genetically very similar to E. coll. The nicotinic acid mutant of S. typhimurium used for these studies was grown on a glucose-salts medium n which was supplemented with nicotinic acid, or NAD, or both. Conditions for cell growth, harvest, and preparation of cell-free extracts were those described for E. coll. The specific activities of NaMN pyrophosphorylase in the various extracts prepared by this procedure varied approx. 2o-fold (Table II) and was related to concentration of growth factor in the medium. TABLE

I1

N a M N PYROPHOSPHORYLASE ACTIVITY IN S a l m o n e l l a

typhimurium

C o n d i t i o n s f o r i n c u b a t i o n w e r e t h o s e d e s c r i b e d i n T a b l e I, e x c e p t f o r t h e u s e of 5 ° # m o l e s of p h o s p h a t e ( p H 7.5) a s b u f f e r a n d f o r t h e r e d u c t i o n of t h e t i m e f o r i n c u b a t i o n t o 2 0 m i n . Growth supplement Compound

Concentration added to medium (M)

Spceit~c activity of enzyme preparation

Nicotinic acid Nicotinic acid Nicotinic acid Nicotinic acid Nicotinic acid plus NAD

2.5" lO -7 5.o" I o -7 2 . 0 - IO -~ I.O. lO -4 each at I.O. lO -4

86 65 20 6 4

Growth of Serratia marcescens and enzyme preparation Serratia marcescens, like S. typhimurium and E. coli, is a member of the coliform group of bacteria. The minimal medium employed for the growth of Serratia marcescens was identical to that used for E. coll. Since the particular organism used in these studies does not require nicotinic acid for growth, it could not easily be made deficient Biochim.

Biophys.

Acta,

82 (1964) 4 4 5 - 4 5 3

448

j. IMSANDE

in p y r i d i n e nucleotides. However, a r e l a t i v e l y high concentration of nicotinic acid and N A D (each a t I . IO -4 M) was a d d e d to the m e d i u m of several cultures. In all other respects cultures of Serratia marcescens were grown, harvested, washed, d i s r u p t e d a n d cell-free e x t r a c t s p r e p a r e d as p r e v i o u s l y described for E. coli. The e x t r a c t s were then a s s a y e d for protein a n d N a M N p y r o p h o s p h o r y l a s e activity. As shown in Table I I I , g r o w t h conditions did n o t alter the specific a c t i v i t y of the e n z y m e in the e x t r a c t s examined. TABLE

NaMN

t'YROPHOSPHORYLASE

I 1I

A C T I V I T Y IN ~ g l ' ! / a l i g g ~ q q a Y c e s c e ~ l s

C o n d i t i o n s for i n c u b a t i o n w e r e t h o s e d e s c r i b e d in T a b l e I, e x c e p t for t h e use of 5 ° / ~ m o l e s p h o s p h a t e ( p H 7.o) as b u f f e r . Growth supplement (M)

Specific activity o/ enzvme preparation

Not]e

NAI) (I, lo '4) plus n i c o t i n i c a c i d (~ • 10- 4)

7

Growth of B. subtilis and enzyme preparation Two nicotinic acid-requiring m u t a n t s of the M a r b u r g strain of B. subtilis, No. 114 a n d No. 122, a n d a p r o t o t r o p h , S]3 19, were used in these studies. Some cultures were grown on a m i n i m a l s a l t s - g l y c e r o l m e d i u m 12 w i t h o u t s u p p l e m e n t a t i o n , some were s u p p l e m e n t e d w i t h i . 10 .4 M nicotinic acid or N A D . F o r rich cultures a m i n i m a l salts-glucose m e d i u m was s u p p l e m e n t e d w i t h 1 % y e a s t e x t r a c t . In all cases the cells were grown a n d h a r v e s t e d u n d e r the same condition as described for E. coll. The cellfree extracts, also p r e p a r e d in a like m a n n e r , were a s s a y e d for p r o t e i n c o n t e n t a n d N a M N p y r o p h o s p h o r y l a s e activity. These results (Table IV) s t r o n g l y suggest t h a t the g r o w t h conditions e x a m i n e d do not influence the specific a c t i v i t y of the e n z y m e in question.

Growth of Torula cremoris and enzyme preparation T h e yeast, Torula cremoris ATCC 2512, requires nicotinic acid for g r o w t h and was c u l t u r e d on a highly s u p p l e m e n t e d m e d i u m ~3. However, to produce a chemically TABLE

NaMN

PYROPHOSPHORYLASE

IX:

A C T I V I T Y IN ]Y~acill~lS

subtdis

C o n d i t i o n s for i n c u b a t i o n w e r e t h o s e d e s c r i b e d in T a b l e I, e x c e p t f o r t h e use of o n e - h a l f t h e a m o u n t of e n z y m e a n d for t h e r e d u c t i o n of t h e t i m e of i n c u b a t i o n t o 2o r a i n . Growth supplement Organism

None

Nicotinic acid

NA D

~ "to 4 M

z .to 4 M

Rich*

Specific activity o/enzyme preparation S B ~9 It4 122

93 --

85 9°

83

10 5

" too ~5

* C o m p o s i t i o n of rich m e d i u m is d e s c r i b e d in t h e t e x t . B i o c h i m . B i o p h y s . A c t a , 82 ( i 9 0 4 ) 4 4 5 - 4 5 3

COMPARATIVE CONTROL OF N A D

FORMATION

449

defined medium, casamino acids plus tryptophan were substituted for the charcoaltreated peptone. Cultures grown on this medium were supplemented with either 5" I0 -7 or 1.10 -4 M NAD or nicotinic acid, the lower concentration being growth limiting TM. When a rich medium was desired the casamino acids plus tryptophan were replaced by 1% yeast extract and 1% bactopeptone. In all cases the cultures were grown overnight with vigorous shaking. Cells were collected by centrifugation, washed, suspended in 0.05 M phosphate (pH 7.0) (5 ml buffer per g wet weight of cells), and disrupted by passage through a French press (6000 lb/in2). The disrupted cell suspension was centrifuged, the slightly turbid supernatant solution was retained and examined for protein content and NaMN pyrophosphorylase activity. As shown in Table V, the specific activity of the enzyme was not altered significantly by the growth conditions examined. TABLE V N a M N PYROPHOSPHORYLASE ACTIVITY IN T o r u l a c r e m o r i s C o n d i t i o n s for i n c u b a t i o n were those described in Tabl e I, e x c e p t for t h e use of 5 ° /*moles o f p h o s p h a t e (pH 7-5) as buffer, i n c r e a s i n g t h e a m o u n t of A T P to 5/*moles, a n d for t h e r e d u c t i o n of t h e t i m e of i n c u b a t i o n to 20 min. Molar concentration of growth supplement Growth supplement

5" ~o 7

I • xo *

Rich* + 5" to s

Specific activity of enzyme preparation

N i c o t i n i c acid NAD

52 4°

29 3°

-32

* C o m p o s i t i o n of rich m e d i u m is t h e described in t h e t e x t . TABLE VI N a M N PYROPHOSPHORYLASE ACTIVITY IN T e t r a h y m e n a p y r i f o r m i s R e a c t i o n m i x t u r e s c o n t a i n e d 15 /*moles MgSO4, i o / * m o l e s A T P I.O/*mole 5 - p h o s p h o r i b o s y l - I p y r o p h o s p h a t e , 5 ° / , m o l e s p h o s p h a t e (pH 7.o), 4 ° / * m o l e s N a F , o. I / * m o l e [7-1iC2nicotinic acid a n d 0. 5 ml of e n z y m e in a final v o l u m e of i.o ml. I n c u b a t i o n w a s for 60 m i n a t 37 °. Growth supplement

N i c o t i n i c acid ( i . i o -~ M) Nicotinic acid (I. IO - e M ) N i c o t i n i c acid (5" lO-5 M) Rich medium

Specific activity of enzyme preparation

18 io

IO io

Growth of Tetrahymena pyriformis and enzyme preparation Stock cultures of nicotinic acid-requiring Tetrahymena pyriformis, strain E, were maintained on a medium composed of 2 % proteose peptone plus 0.2 % yeast extract. Cells for experimentation were grown on a chemically defined medium 14, supplemented with I . io -7, I. lO 4, or I. io -5 M nicotinic acid or on the rich medium designated for the stock cultures. The growth rate is approx, one-half maximal on I . io -7 M nicotinic acid 15. All experimental cultures were grown in the dark at 25 o. Aeration was accomplished by limited shaking. The cells were collected by centrifugation at 4000 × g for B i o c h i m . B i o p h y s . A c t a , 82 (1964) 445-453

450

J. IMSANDE

IO min, washed in 0.05 M phosphate (pH 7.o), centrifuged, resuspended in cold phosphate buffer (I g wet weight of cells per 9 ml buffer) and then disrupted with a Potter-Elvehjem homogenizer (2-3 min). The slightly turbid supernatant solution, which was obtained from the broken-cell suspension by centrifugation at I2 ooo i~: g for IO min, was assayed for protein and NaMN pyrophosphorylase activity. Results from such determinations suggest that growth conditions do not influence significantly the specific activity of the enzyme (Table VI).

Growth of the rat and enzyme preparation Eleven 7-week-old rats were divided arbitrarily into two groups. The control group (consisting of four rats) was fed a standard vitamin B complex rat diet. The experimental group (consisting of seven rats) was maintained on a special nicotinic Time (days) 250

0

5

10

j

15

~

j

20 ~

25

30

~

,

35 ,

40

200

~00 ~D

o Cn

"~

~

150

o _c

o 5.0 m ~ 4.0 2

~)

E s.o ~-~

,oo

I l

~3

45

I 6

78

910

I

Io

II

Individual r a t Fig. I. G r o w t h c u r v e s a n d t h e specific: a c t i v i t i e s of r a t - l i v e r N a M N p y r o p h o s p h o r y l a s e . T h e a v e r a g e g r o w t h r a t e of a c o n t r o l r a t is s h o w n b y t h e u p p e r c u r v e ( O - - O ) w h e r e a s t h e a v e r a g e g r o w t h r a t e of a n e x p e r i m e n t a l r a t is i l l u s t r a t e d b y t h e lower c u r v e ( [ ] - - kl ). I n d i v i d u a l r a t s were sacrificed on t h e d a y s i n d i c t e d b y t h e arrows. The specific a c t i v i t i e s of t h e e n z y m e from i n d i v i d u a l c o n t r o l r a t s is i n d i c a t e d b y t h e open b a r s a n d t h o s e from i n d i v i d u a l e x p e r i m e n t a l r a t s are repr e s e n t e d b y t h e s h a d e d bars. C o n d i t i o n s for e n z y m e i n c u b a t i o n in vitro were t hos e d e s c r i b e d in T a b l e I, e x c e p t t h a t 5 ° / * m o l e s of p h o s p h a t e (pH 7.o) were used as buffer, t h e a m o u n t of MgSO 4 was r e d u c e d to 15/*moles, t h e level of ATP w a s i n c r e a s e d to 5/*moles, a nd t h e t i m e of i n c u b a t i o n was e x t e n d e d to 6o min.

acid-free, tryptophan-free vitamin B complex rat diet, which was supplemented with 4 mg of L-tryptophan per IO g of diet. After 3o days on this diet the level of L-tryptophan was increased to IO mg per Io g of diet. Daily rations provided were 15 g for each control rat and Io g for each experimental rat (experimental rats seldom consumed the entire ration). Rats from both groups were weighed every other day and the Biochim. Bioph2/s. Acta, 82 (i q04) 445-453

COMPARATIVE CONTROL OF

NAD

FORMATION

451

average growth curve for each group is shown in Fig. I. A rat from the corresponding group was sacrificed on the days indicated by the arrows, the liver excised immediately and weighed. A portion of each liver (1.5 g) was minced and then homogenized for TABLE

VII

COMPARISON OF THE SPECIFIC ACTIVITIES OF N a M N PYROPHOSPHORYLASE FROM VARIOUS SPECIES

Source

Specific activity

Rat liver

3 12 37 92

Tetrahymena pyriformis Yeast

B. subtilis Serratia marcescens S. t y p h i m u r i u m E. coli K - I 2 w i l d t y p e * E. coli s t r a i n 15 m u t a n t E. coli K - I 2 m u t a n t *

9 4-86 1. 5 o.4-46 o.o5-4 o

* T h e s e v a l u e s a r e t a k e n f r o m a p r e v i o u s p u b l i c a t i o n °.

1-2 min in 13.5 ml of cold o.25 M sucrose with a Potter-Elvehjem homogenizer. The broken cell preparation was centrifuged at 8000 × g for IO rain and the slightly turbid supernatant solution was collected and examined for protein content and NaMN pyrophosphorylase activity. As shown in Fig. I, the specific activity of the enzyme appeared to be independent of the growth conditions examined. DISCUSSION

That pyridine nucleotid.e biosynthesis in E. coli is regulated, at least in part, by a repression-derepression mechanism has been demonstrated with another strain of that organism (Table 1). This control mechanism is also important in the regulation of pyridine nucleotide formation in S. typhimurium (Table II). However, the evidence presented for other organisms strongly suggests that regulation of pyridine nucleotide formation by this mechanism is not widespread in nature. This conclusion is based on the fact that the growth conditions (i.e. nicotinic acid starvation or high levels of NAD or nicotinic acid), which are known to initiate more than a Ioo-fold repression-derepression in E. coli, alter the level of NaMN pyrophosphorylase in all other organisms studied, except S. typhimurium, by less than two-fold (Tables II and V1 and Fig. I). The culture of Serratia marcescens used in these studies did not require nicotinic acid for growth, hence a critical test for derepression could not be made. It will be noted, however, that the level of NaMN pyrophosphorylase in Serratia marcescens could not be repressed (Table 111). This finding suggests that Serratia marcescens, an organism closely related to E. coli, does not possess regulatory mechanisms identical to those found in E. coli. The lack of repressibility of NaMN pyrophosphorylase in B. subtilis and yeast is even more striking since the amount of enzyme activity in cell-free extracts is extremely high regardless of growth conditions (Tables III and IV). Indeed, the specific activity of NaMN pyrophosphorylase in B. subtitis is approx. 20 % of that for orotidBiochirn. Biophys. Acta, 82 (1964) 4 4 5 - 4 5 3

452

j. IMSANDE

ylic pyrophosphorylase even though orotidylic acid is formed 300 times faster in viva than is pyridine nucleotides 16. The ciliate protozoan, Tetrahymena pyriforrnis, also appears to have a fixed level of NaMN pyrophosphorylase (Table V). Studies on the level of NaMN pyrophosphorylase in rat liver revealed that derepression of the enzyme in this organ is unlikely. All rats fed the low-tryptoi)han , nicotinic acid-free diet for a period of 2 weeks or longer developed the symptonls of nicotinic acid deficiency, yet the specific activity of the hepatic enzyme did not change (Fig. I). It should be noted that after 3o days the amount of L-tryptophan in the experimental diet was increased 2.5-fold, the higher level being slightly in excess of the minimal daily requirement. During the succeeding 4 days the weight of an average liver doubled, a strong indication that protein synthesis is taking place. As will be seen in Fig. I, the specific activity of the enzyme did not increase during this period, a suggestion that derepression of NaMN pyrophosphorylase in rat liver is not likely. The significance of the recently discovered liver enzyme which catalyzes the formation of NaMN from quinolinic acid and 5-phosphoribosyl-I-pyrophosphate~7,~s was not assessed. In all of the incubations conducted i~t vitro with the various cell-free extracts, NaMN was the first pyridine nucleotide formed. However, during such incubations, the amount of NaMN in the reaction vessels did not increase greatly with time. Instead, a low level of nicotinic acid adenine dinucleotide soon appeared and immediately thereafter NAD accumulated at a rapid rate. Thus it appears that, in all the cellfree extracts examined, NaMN pyrophosphorylase is the rate-limiting enzwne in the formation of NAD from nicotinic acid. It has previously been established that this is the rate-limiting enzyme in NAD biosynthesis by cell-free extracts of E. co/i", s. Cell-free extracts from all organisms studied, including yeast which is reported to contain NADase and nicotinamide deamidase ~", were examined for inhibition (}f NaMN pyrophosphorylase activity by 5"IO-a M NAD. In no case was inhibition detected. Since the concentration of NAD in the intact cell is approximately equal to that used above 6,8, one can conclude that inhibition of this enzyme bv NAD feedback is not important in the regulation of pyridine nucleotide synthesis in the organisms studied. From a comparison of the specific activity of the enzvme from the various sources studied it will be seen that, with the exception of yeast and B. subtilis, the level is uniformly low (Table VI). Indeed this result is to be expected since, as coenzymes, pyridine nucleotides are required and produced only in catalytic amounts. Perhaps the rate of production of the enzyme in some species is regulated bv the constitutive control process recently advanced by PARDEE AND BE(;KWlTN"°. An important observation in these studies is the absolute and unifl~rm requirement of ATP for NaMN pyrophosphorylase activity. ATP dependence and stimulation of this enzyme from other sources has been previously reported 2L'2. The role played by nucleoside triphosphates in the biosvnthesis of NaMN is currently under investigation. ACKNOWLEDGEMENTS

The author is greatly indebted to Dr. A. B. PARDEE, in whose laboratory these experiments were conducted, for helpful suggestions throughout the course of this work, and Biochim. Bioph/s..4cia, ,% (19~)4) 445 4523

COMPARATIVE CONTROL OF

NAD

FORMATION

453

to Miss L. S. PRESTIDGE for invaluable technical assistance during a portion of this work. These studies were supported in part by U. S. Public Health Service Grant No. AI-o44o 9. The author was a Postdoctoral Fellow of the National Cancer Institute, National Institutes of Health. REFERENCES 1 A. B. PARDEE, in P. D. BOYER, H. LARDY AND K. MYRBACK, The Enzymes, Vol. I, Academic Press, New York, 1958, p. 681. Cold Spring Harbor Syrup. Quant. Biol., Vol. 26, Long Island Biological Assoc., Cold Spring H a r b o r , New York, 1961. a j. C. GERHART AND A. B. PARDEE, J. Biol. Chem., 237 (I962) 891. a H. S. MOYED, J. Biol. Chem., 236 (1961) 2261. 5 R. A. YATES AND A. B, PARDEE, J. Biol. Chem., 227 (1957) 677. 6 j . IMSANDE AND A. B. PARDEE, J. Biol. Chem., 237 (1962) 13o5. 7 A. C. WILSON AND A. B. PARDEE, in M. FLORKIN AND H. S. MASON, Comparative Biochemistry, Vol. 6, Academic Press, New York, p. 73. s j. IMSANDE, J. Biol. Chem., 236 (1961) 1494. 9 0 . H. LowRY, N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265. 10 R. A. YATES AND A. B, PARDEI~, j . Biol. Chem., 221 (1956) 743. ll B. N. AMES, B. GARRY AND L. A. HERZENBERG, J. Gen. Microbiol., 22 (196o) 369. 12 j. SPIZlZEN, Proe. Natl. Aead. Sci. U.S., 44 (1958) lO72. 13 W. L. WILLIAMS, J. Biol. Chem., 166 (1946) 397. 14 A. M. ELLIOT AND R. E. HAYES, Biol. Bull., lO 5 (1953) 269. ls A. M. ELLIOT, Physiol. Zool., 23 (195o) 85. 16 j. IMSANDE, unpublished results. 17 y . NISHIZUKA AND O. HAYAISHI, J . Biol. Chem., 238 (1963) PC 483 . 18 R. K. GHOLSON, I. UEDA AND L. M. HENDERSON, Federation Proe., 22 (1963) 651. 19 j. G. JOSHI AND P. HANDLER, J. Biol. Chem., 237 (1962) 929. ~0 A. B. PARDEE AND J. BECKWlTn, Biochim. Biophys. Acta, 60 (1962) 452. 2i j. IMSANDE AND P. HANDLER, J. Biol. Chem., 236 (1961) 525. ~z j . PREISS AND P. HANDLER,J. Biol. Chem., 233 (1958) 493.

Biochim. Biophys. Acta, 82 (1964) 445-453