The behavior of various antivitamin B6 in Saccharomyces carlsbergensis

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The Behavior of Various Antivitamin BBin Succharomyces ~~Zs~erge~sis’ Taketami Sakuragi and Fred A. Kummerow

Received October 13, 1958

A variety of compounds are currently known as antivitamin B6 , They may be classified into two groups with respect to their mechanism of inhibition: (a) a compound which seems to compete wit,h vitamin B, for a site of absorption or for a site of the enzyme (1, 2), and (b) a compound which seems to block the functional formyl group of pyridoxal or pyridoxal 5phosphate through chemical reactions (3, 4). Although isonicotinoyl hydrazide (5) probably belongs t,o the latter group, it is st,ill uncez~ai~l whether the ability of isonicotinoyl hydrazide to inactivate the vitamin would account for all phases of its toxicity. Attempts have been made t’o different&e the modes of action of vitamin Bg antagonists in rats (6) and in microorganisms (7). The number of ai~tagonists previously tested, however, is rather limited. During the course of our investigation on vitamin B, , it became desirable to differentiate, with regard to the inhibition mechanisms, some of the antagonists such as 4-deoxypyridoxine (2), 5-deoxypyridoxine” (7), 4,5-bisdeoxypy~doxine (8), 4,5-dit.hiopyridoxine~, 4 pyramin (9), 5-deoxypyramin3 (IO), 5-ethoxJrpyraminz* 3 (IO), 2-methylthiopyramin (1 l), isonicotinoyl hydrazide (la), and n-4-amino-3-isoxnzolidone2 (13). A study was therefore undertaken using a, strain of yeast (~ucc~~r~~~c~~ cur~~be~ge~s~~~),which requires vitamin B8 for its growt,h. 1 This work was supported by research grant ?;o. A-257 from the National Institutes of Health, U. S. Public Health Service, Department of Health, Education, and Wetfare. 2 The preparations used in the present study were kindly supplied by Merck & Co., Rahway, N. J., through the courtesy of Drs. S. A. Harris and il. N. Wilson. 3 The following names are tentatively used to designat,e the test compounds; 4,5dithiol~~ridoxine for 2-meth~l-3-hgtfroxy-1,5-l~ismercaptomethylp~ridine, 5-deoxypyramin for 2,5-dimet;tlyl-~-amir~op~~rimidir~e, and 5-ethosypyramin for 2.methgl-iamino-5-ethoxymethylpyrimidine. 4 Sakuragi, T., and Kummerow, I?. A., unpublished. 89

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SAKURAQI AND KU~~EROW

In the present ~~estigation, yeast cells were trained to grow in the presence of various vitamin Be antagonists in order to gain an insight into the mechanism of inhibition through a study of cross-resistant patterns. EXPERIMENTAL

Assay Organism The strain of yeast used in the present study was Saccharomyces carlsbergensis (A.T.C.C. 4228), which requires vitamin A* as an essential growth factor.

Assay procedures The assay procedures and the compositions of the basal medium were essentially the same as that previously employed (9). Throughout this study, the inoculum was prepared as follows: A freshly harvested 24-m. culture of t,he yeast was suspended in sterilized distilled water containing 0.9% sodium chloride to a level so as to give an optical density of 0.05 at 655 rnp against distilled water using a Coleman model 9 Nepho-Calorimeter. This suspension was further diluted with three times its volume of the saline solution. One milliliter of the final suspension was added as an inoculum to each 9 ml. of the culture medium. After incubation at 30°C. for 18 hr., the amount of yeast growth was measured turbidimetrically at 655 rnp against distilled water, and expressed in terms of optical density.

Isolation of Cells Resistant to ~-Deoxypyr~dox~~e Ten milliliters of the medium, which contained 120 m&g. pyridoxine hydrochloride and 300 pg. 4-deoxypyridoxine hydrochloride, was heavily inoculated with S. carlsbergensis and incubated at 30°C. When growth became vigorous, one loopful of the culture was transferred to a tube containing the same level of pyridoxine and 600 ag. 4-deoxypyridoxine hydrochloride/l0 ml. Incubations and transfers were similarly conducted until the amount of 4-deoxypyridoxine hydrochloride reached 30 mg./lO ml. in the tenth tube. Without changing the levels of either supplement, six transfers were then made in a lo-ml. culture medium containing 40 mpg. pyridoxine hydrochloride and 20 mg. 4-deoxypyridoxine hydrochloride. An agar slant was prepared by the addition of 270 of agar to this medium, and the resistant cells carried on the agar slant were designated as R4DP.

Isolation of Cells Resistant to J,5-~~sdeoxypyridoxine A medium which contained 40 mpg. pyridoxine hydrochloride and 10 mg. 4,5-b&deoxypyridoxine hydrochloride/l0 ml. was inoculated with S. car~sbergens~s. During the course of five transfers the concentration of pyridoxine remained unchanged, while the level of the antagonist was gradually increased so as to reach 50 mg./lO ml. Nine additional transfers were made in a medium supplemented with 40 m&g. pyridoxine hydrochloride and 50 mg. 4,5-bisdeoxypyridoxine hydrochloride/lo ml. An agar slant was prepared by adding 2% agar to this medium, and the resistant cells thus isolated were designated as RBDO.

Isolation of Cells Resistant to 5-Ethoxypyramin In the first tube the medium contained 40 mpg. pyridoxine hydrochloride and 20 mg. 5-ethoxypyramin/lO ml. In five successive transfers, the concentration of the

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latter was increased to 100 mg./lO ml., the level of pyridoxine remaining unchanged. The following ten transfers were made in a medium which possessed the same concentration of the supplements as that found in the last tube, and the cells thus isolated, designated aa RSEP, were carried on an agar slant.

Isolation

of Cells

Resistant to Isonicotirwyl

Hydraxide

The procedures were essentially identical with those described above. The first set of seven transfers was made from a medium containing 120 mpg. pyridoxine hydrochloride and 30 mg. isonicotinoyl hydrazide/lO ml. The last transfer was made into the medium which contained 120 mpg. pyridoxine hydrochloride and 100 mg. isonicotinoyl hydrazide. Four subsequent transfers were made in a medium containing 40 mpg. pyridoxine hydrochloride and 100 mg. isonicotinoyl hydrazide/lO ml. The cells thus isolated were designated as RINH, and carried on an agar slant,. RESULTS In the present

it was confirmed that the previously known 4-deox~~yridoxine (2), 5-deox~yridoxine (7), 4,5-b&antivitamin B, , deoxypyridoxine (8), 4,5-dithiopyridoxine:~ 4 pyramin (9), 5-deoxypyramir? (lo), 5-ethoxypyramin3 (lo), 2-methylthiopyramin (ll), isonicotinoyl hydrazide (12) and D-4-amino-3-isoxazolidone (13) depressed the growth of ~~~harornyces carlsb~ge~s (A.T.C.C. 4228). 2-Methyl-3hydroxy -4 - methoxymethyl -5 - hydroxymethylpyridine has been reported to be an antivitamin Bg in rats (6) and chicks (14). The hydrochloride of this compound at a level of 3 mg./lO ml. of the medium in the presence of 40 mpg. pyridoxine hydrochloride was not able to impair t.he growth of the strain of yeast. At lower concentrations, the 4-methoxymethyl analog of pyridoxine stimulated growth. No growth depression or stimulation was induced with 2-methyl-3-amino-4-methoxymeth;yl-5-aminomethylpyridine in S. carlsbergensis; the structurally similar compound, 2-ethyl-3-amino-4ethoxymethyl - 5 - aminomethylpyridine has previously been reported to serve as a vitamin Be antagonist in a strain of Saccharomyces rerevisiae (15). Although L-penicillamine is known to act as an antivitamin Bs in rats (ES), it was apparently innocuous to the strain of yeast used in the present study. 2-Mercaptoethylamine, which is a compound structurally related to penicilIamine, has been reported to interfere with the growth of yeast (16). The mechanism, however, has been proven to be independent of the metabolism of vitamin Bs (16). Growth of 8. carlsbergensis was determined at various concentrations of an antivitamin Be in the absence and presence of another antagonist in the medium. When upon supplementation with the second antagonist enhanced growth inhibition resulted over the test range, the effect of the two compounds was designated to be additive, although not necessarily on a quantitat,ive basis. In four cases the effects of 4-deoxypyridoxine and isollicoti~oyl hydrazide were additive, whereas 4-deoxypyridoxine and 4,5-bisdeoxyinvestigation,

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o-o0

40

80

120

0

60

120 1;

5ETHOXYPYRAMIN (MICROGRAMS/IOML.) FIG. 1. The growth inhibition of Saccharomyces cnrtsbergensis (A.T.C.C. 4228) by an antivitamin I30 in the absence and presence of another antivitamin Bg . For each 10 ml. culture medium/tube, 40 mpg. pyridoxine hydrochloride was added. Supplements : A. I. 4-Deoxypyridoxine hydrochloride. II. 4-Deoxypyridoxine hydrochloride plus isonicotinoyl hydrazide (3 mg./lO ml.). A. I. Isonieotinoyl hydrazide. II. Tsonicotinoyl hydrazide plus 4-deoxypyridoxine hydrochloride (5 rg./lO ml). C. I. 4,5-Bisdeoxypyridoxine hydrochloride. II. 4,5-Bisdeoxypyridoxine hydrochloride plus 4-deoxypyridoxine hydrochloride (5 pg./l0 ml.). D. I. 5-Ethoxypyramin. II. 5-Ethoxypyramin plus 4-deoxypyridoxine hydrochloride (5 fig./10 ml.) _

pyridoxine, and 4-deoxypyridoxine and 5-ethoxypyramin were not additive (Fig. 1). The results of similar tests with a variety of antivitamins B6 indicate that, except for isonicotinoyl hydraside and n-4-amino-3-isoxazolidone (Table I), 4-deoxypyridoxine failed to show an additive effect with other antagonists. Isotlicotinoyl hydrazide and D-4”amino-3-isoxazolidone exhibited their additive functions with all the antivitamin Be test~ed in the present investigation (Table I). Additivity was also noted within any pair

of the

following

compounds:

5-deoxypyridoxine,

4, &bisdeoxypyri-

doxine, 4,5-dithiopyridoxine, pyramin, 5-deoxypyramin, 5-ethoxypyramin, and Z-methylthiopyramin (Table I). The results of similar tests by Rabinowitz and Snell indicated that the effect of 4-deox~yridoxine and ~-methyl-

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Bs IN

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YEAST

I

and ~onadd~t~v~ Erects in Growth Inhibition of Saccharomycescarlsbergensis (A.T.C.C. l%98) Within a Pair of Yarious Antivitamin Re - - 2 - -

Z’he Additive

-

.d

8 '2 4 'Z >; R x

A: Additive N: Not additive -: Not tested

.4,5-Bisdeoxypyridoxine ..-__--__ 4,5-Dithiopyridoxine Pyramin -------------5-Deoxypyramin 5-Ethoxypyramin 2-Methylthiopyrsmin Xsonicotinoyl hydrazide

ii 4 N

2 ‘W 4 ‘E B 2 2 _I_ -& _---

_---A -:u .- -- _--N -- _-N .- ---N A -- _-.-

s .3 a2 ‘C 2 3 a2 .9 m II; -J; -

-i-

i

2 .m w 2 ‘E 2 .,o &I .z R d -G

/

.* i ! -a” I-

I-

N

N --

A _--A

-AC -- --

A -- _- -- _A A -- _- -- -A A A __.- -- _A A -_- -..- -I- -A A A A -- _-- _- -A -

_-

/_

pyridoxine was additive, whereas that of 4-deoxypyridoxine and 5-deoxypyridoxal was not (7). The additivity does not necessarily mean that the inhibition mechanisms of the two antivitamin Be are similar. However, it seems conceivable that if their modes of inhibition are identical and a direct chemical reaction between the two antagonists does not take place, additivity should result. The order of effectiveness as an antivitamin B6 is shown with approximate molar potencies, in which the activity of 4-deoxypyridoxine is expressed by an arbitrary number of 1000, as follows: 4-deox~y~doxine (1000) > 5-deox~yridoxine (500) > 5deox~~amin (2~) > 4,5-bisdeoxypyridoxine (75) > pyramin (50), 5-ethoxypyramill (50) > 4,5dithiopyridoxine (40) > 2 -methylthiopyramin (7.3) > I) -4 -amino - 3isoxasolidone (1.5) > isonicot,inoyl hydrazide (1.2). 4-Nitrosalicylaldehyde is reported to replace pyridoxal in non-enzymic transamination (17) and competitively inhibits the biochemical reaction which requires pyridoxal 5-phosphate (18). This has been explained by the structure of 4-nitrosalicylaldehyde which is functionally identical with that of pyridoxal (5, 17). 4-Nitrosalicylaldehyde was found to be a growth

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1 ,

0

4-NITROS~ICYLAL~HY~ (MlCRO~AMS/ IOML.) FIG. 2. The growth inhibition of Saccharomyces carlsbergensis (A.T.C.C. 4228) by 4-nitrosalicylaldehyde in the absence and presence of an antivitamin Rs , For each 10 ml. culture medium/tube, 40 mpg. pyridoxine hydrochloride was added. Supplements: I. 4-Nitrosalicylaldehyde. II. 4-Nitrosalicylaldehyde plus 4-deoxypyridoxine hydrochloride (5 pg./l0 ml.). III. 4-Nitrosali~ylaldehyde plus 4,5-bisdeoxypyridoxine hydrochloride (60 pg./l0 ml.). A pattern essentially identical with III was also obtained with 4-nitrosalicylaldehyde plus 5-ethoxypyramin (90 fig./10 ml.). IV. 4-Nitrosalicylaldehyde plus isonicotinoyl hydrazide (4 mg./lO ml.). A pattern essentially identical with IV was also obtained with 4-nitrosalicylaldehyde plus n-4-amino-3-isoxazolidone (2.5 mg. as its calcium salt/l0 ml.).

inhibitor for 8. curEsbergensis,b and the inhibition was enhanced in the presence of 4,5-bisdeoxypyridoxine or 5-ethoxypyramin (Fig. -,2>.Nonadditivity, however, was noted between the aldehyde and 4-deoxypyridoxine (Fig. 2). In the presence of 4-nitrosalicylaldehyde and isonicotindyl hydrazide or n-4-amino-3-isoxazolidone, the effects observed in the growth of yeast were that of isonicot~oyl hydrazide or n-4-amino-3-isoxazolidone alone, the toxicity of 4-nitrosalicylaldehyde being completely masked (Fig. 2). This is probably due to chemical condensation between the two growth inhibitors so as to inactivate each other. However, the molar concentrations of the isonicotinoyl hydraElide (29 bmoles/lO ml.) and the n-4-amino3-isoxazo~done (21 ~moles/lO ml.) were considerably higher than that of 4-nitrosalicylaldehyde (up to 1.1 pmoles/lO ml.). The loss of the first two compounds through chemical condensation might therefore not have been reflected in the growth of the microorganism. 5 The toxicity of 4-nitrosalicylaldehyde to S. carlsbergensis could not be reversed by vitamin Bg . Similar observation has previously been reported with other microorganisms (17, 18).

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The strains of S. carlsbergensis (A.T.C.C. 4228), R4DP, RBDO, R5EP, and RINH were morphologically indisting~shable. Unlike the original strain, the last four grew with ease on a medium devoid of vitamin 3, , although supplementation with pyridoxine further stimulated their growth. 4-Deoxypyridoxine, 4,5-bisdeoxypyridoxine, and 5-ethoxypyramin were still capable of impairing the growth of R4DP, RBDO, and R5EP (Fig. 3). However, it, was often noted that as the concentration of the antimetabohte in the medium was increased, the degree of ixlhibition reached more or less a plateau (Fig, 3). The results indicated that the culture consisted of cells which were highly resistant to the inhibitor as well as those which were not. This is not surprising, since, throughout the course of isolation, no attempt had been made to select a single cell from the culture. In the medium which contained neither vitamin B, nor antivitamin B6 , the growth of the original strain of cells was slight so as to give an optical density of approximately 0.04 against distilled water under the experimental conditions. In the presence of 160 mpg. pyridoxine hydrochloride/l0 ml. of the assay medium, the growth of the original strain of S. carlsbergensis was completely prevented upon supplementation with 0.1 mg. 4-deoxypyridoxine hydrochloride, 0.5 mg. 4,5-bisdeoxypyridoxine hydrochloride, or 1 mg. 5-ethoxypy~~n~in. The results thus indicate t8hat R4DP possessed marked resistance to 4-deoxypyridoxine as well as 4,5-bisdeoxypyridoxine or 5-ethoxypyramin. R4DP, however, was as sensitive as the original strain toward the toxicity of isonicotinoyl hydrazide (Fig. 3). Similarly, RBDO and R5EP were much less sensitive to 4-deoxypyridoxine, 4,5-bisdeoxypyridosine, and 5-ethoxypyramin than t#he original strain, whereas the toxicity of isonieot,inoyl hydrazide to RBDO and R5EP remained as high as that to the original strain (Fig. 3). It is of interest to note that RINH could tolerate well 4-deoxypyridoxine, 4,5-bisdeoxypyridoxine, and 5ethoxypyramin, although RINH seems to have attained only a slightly improved resistance to isonicotinoyl hydrazide (Fig. 3). The original strain of S. car~sberg~r~~swas also transferred 15 times in a medium containing neither pyridoxine6 nor an antivitamin Bs , and in a medium supplemented with 40 mpg. pyridoxine hydrochloride/l0 ml. without an antimetabolite. The two types of cells which had thus been treated showed no improved resistance to 4-deoxypyridoxine, 4,5-bisdeoxypyridoxine, 5-ethoxypyramin, and isonicotinoyl hydra&de over the original strain. DISCUSSION

Isonicot,inoyl hydrazide is believed to antagonize vitamin B6 through formation of a hydrazone with pyridoxal or pyridoxal 5-phosphate (5). 4 Sinee “vitsmirr-free easein hydrolyzete” had been used as an ingredient, the basal medium was not absolut~ely free from vitamin Bs .

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1

?

r 0.6 m 2 0.4 J sO.2 F !3 IO

RBDO 1

--------

k lx (3

CONCENTRATIONS OF ANTIVITAMIN Bs (PER10 ML.) FIG. 3. The effect of antivitamin BF, on growth of the normal strain of Saccharonzyees C~Tlsb~r~~n~is (A.T.C.C. 4228), the 4-deoxypyridoxine-resistant cells (R4DP), the 4,5-bisdeoxypyridoxine-resistant cells (RBDO), the 5-ethoxypyramin-resistant cells (RSEP), and the isonicotinoyl hydrazide-resistant cells (RINH) in the presence of 160 mpg. pyridoxine hydrochloride/l0 ml. of the medium. The broken lines represent the level of growth in the presence of neither pyridoxine nor antivitamin Bg . Concentrations of antivitamin Be : I. 4-Deoxypyridoxine hydrochloride: (A) 7.5 mg., (B) 15 mg., and (C) 30 mg. II. 4,5-Bisdeoxypyridoxine hydrochloride: (A) 20 mg., (B) 40 mg., and (C) 80 mg. III. 5-Ethoxypyramin: (A) 40 mg., (B) 80 mg., and (C) 160 mg. IV. Isonicotinoyl hydraaide: (A) 10 mg., (B) 20 mg., and (C) 40 mg.

This mechanism of inactivation has been supported by the present results which showed that the toxicity of ~-nitrosali~ylaldehyde to Saccharomyces car~sberg~~~i~ (A.T.C.C. 4228) was completely masked in the presence of isonicotinoyl hydrazide (Fig. 2). Such a possibility would explain the enhancement of the inhibition by a vitamin BE antagonist in the presence of the hydrazide (Table I). Because of the comparable distribution of the functional groups in the molecule, n-4-amino-3-isoxazolidone may also behave in a manner similar to isonicotinoyl hydrazide (19, 20). If 5-deoxypyridoxine is converted to 5-deoxypyridoxal in tivo, as pyridoxine is oxidized to pyridoxal, it seems reasonable to expect alleviation of the toxicity in the presence of isonicot*inoyl hydraeide. It was, however, noted that the effects of ii-deoxypyridoxine and isonicotinoyl hydrazide were additive (Table I). The results thus appear to suggest that the conversion of the 5-deoxy compound to the corresponding 5-deox~yridoxal in oiuo is absent or occurs to a ~egli~ble extent.

ANTIVITAMIN

B,j IN YEAST

97

The antivitamin Bs activity in pyramin has been explained by it.s structural similarity to the vitamin (21). Pyramin &phosphate is an antagonist of pyridoxal 5-phosphate in a tyrosine decarboxylase system as is 4-deoxypyridoxine &phosphate (1, 22). It was therefore implied that pyramin 5phosphate might be the active form of the antagonist in Go, and pyramin and 4-deoxypyridoxine served as an antivitamin Bs through a comparable mechanism. In the present study, however, it became evident that because of their nonadditivity, the modes of il~hibitio~ of the two compounds may differ (Table I), The direct involvement of the 5-hydroxymethyl group of the pyramin molecule in the inhibition mechanism might also be questionable at least in the strain of yeast employed, since pyramin belongs to a group which includes 5-deoxypyridoxine, 4, Sbisdeoxypyridoxine, and 5deoxypyramin. Furthermore, the 5-deoxy analog of pyramin was approximately four times more active than the mother compound as an antivitamin Bs on a molar basis. This is in contrast, to the fact that the 5-deoxy analog of 4-deoxypyridoxine, namely, 4,5-bisdeoxypyridoxine, was about 413 as active as 4-deoxypyridoxine, and that 4,5-bisdeoxypyridoxine and 4-deoxypyridoxine showed nonnddit’ivity in inhibition (Table I) _ The findings, however, do not necessarily exclude the possibility that pyramin may undergo phosphorylation in &IO through the 5-hydrox~~~ethyl group (23). Through a series of transfers in a medium containing an antivitamin Bc , it has been possible to isolate cells of S. carlsbergensis which are capable of surviving in the presence of antivitamin Bs . During the course of isolation, a lag period of S-5 days often elapsed before the growth of yeast became noticeable, especially in the early stages of the transfers. This might suggest that the cells so select,ed were the ones which had adapted themselves so as to grow in the presence of antivitamin B, rather than those which had originally possessed such resistance. The cells of S. carlsbergensis which were able to tolerate either 4-deoxypyridoxine, 4,5-bisdeoxypyridoxine, or 5ethoxypyramil~ simult,aneousl,~ exhibited resistance to the renlai~ling two antimetabolites (Fig. 3). If the three compounds are antagonists specific to vitamin B6 , the results may readily be understood. On the other hand, R4DP, RBDO, and R5EP were as sensitive as the original strain to isonicotinoyl hydrazide, and RINII was significantly insensitive toward 4deoxypyridoxine, 4,5-bisdeoxypyridoxine, and 5-ethoxypyrarnin (Fig. 3). The results imply that isol~icotinoyl hydrazide possesses toxic functions which cannot be accounted solely by its ability to inact,ivate vitamin Bs . SUMMARY

The antagonistic functions of ten representative ant,ivitamin B, have been studied with the aid of ~~~~ur~~~e~s earlsbergensis (A.T.C.C. 4228),

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which requires vitamin I36 for its growth. The results seemed to indicate that 4-deoxypyridoxine competed with vitamin Bs in a manner which was different from that of 5-deoxypyridoxine, 4,5- bi~eoxypyridoxine, 4,5dit.hiopyridoxine, pyramin, 5-deoxypyramin, 5 -ethoxypyramin, and 2methylthiopyramin. Indirect evidence implies that the direct involvement of the 5-hydroxymethyl group of the pyramin molecule in its inhibition mechanism is questionable. The cells which were resistant to 4-deoxypyridoxine, 4,5-bisdeoxypyridoxine, 5-ethoxypyramin, and isonicotinoyl hydrazide were isolated. The cross-resistant patterns appeared to indicate that the first three compounds were probably the antagonists specific to vitamin Be , and the toxicity of isoni~otinoyl hydrazide t,o S. ~~r~~~~rg~~~~ was partly due to its ability to block the function of vitamin E$,and partly due to an undefined function. The behavior of n-4-amino-3-isoxazolidone seems to be comparable to that of isonicotinoyl hydrazide. REFERENCES UMBREIT, W. W., AND WADDELL, J. G., Proc. Sot. Exptl. Biol. Med. 70,293 (1949). RABINOWITZ, J. C., AND SNELL, E. E., Arch. Biochem. Biophys. 43,399 (1953). KUCHINSKAS, E. J., AND DU VIGAEACD, V., Arch. Biochem. B~op~~s. 66,1 (1957). UMBREIT, W. W., Am. J. C&n. Nutrition 3, 291 (1955). MAURON, J., AND BUJARD, E., Bull. sot. chim. Beiges 66, 140 (1956). PORTER, C. C., CLARK, I., AND SILBER, R. H., J. BioE. Chem. 167,573 (1947). RABINOWITZ, J. C., AND SNELL, E. E., Arch. Biochem. Biophys. 43, 408 (1953). SAKURAGI, T., J. Org. Chem. 23, 129 (1958). SAKURAGI, T., AND KUMMEROW, F. A., Arch. Biochem. Biophys. 71, 303 (1957). SHINTANI, S., J. Pharm. Sot. Japan 77, 746 (1957). SAKTJRAGI, T., Arch. Biochem. Biophys. 74, 362 (1958). BOONE, I. U., ANR WOODWARD, K. T., Proe. Sot. ExplE. Biot. Med. 64,292 (1953). (Osaka) 3, 68 (1957). YAMADA, K., SAWAKI, S., AND HAYAMI, S., J. Vitaminol. OTT, W. H., Proc. Sot. Ezptl. Biol. Med. 66, 215 (1947). MARTIN, G. J., AVAKIAN, S., AND Moss, J., J. Biol. Chem. 1’74,495 (1948). SAKURAGI, T., AND KUMMEROW, F. A., Arch. Biochem. Biophys. 79, 284 (1959). IKA~A, M., ANX) SNELL, E, E., J. Am. Chem. Sot. 76,653 (1954). OLIVARD, J., AND SNILL, E. E., J. BioE. Chem. 213,203 (1955). CYMERMAN-CRAIG, J., RUBBO, S. D., WILLIS, D., AND EDGAR, J., Nature 176, 34 (1955). 20. JONES, L. R., Anal. Chem. 28,39 (1956). 21. MAKINO, K., KINOSHITA, T., SASAKI, T., AND SRIOI, T., Nature 17% 34 (1954). 22. MAIIINO, K., AND KOIKE, M., Enzymologia 17, 157 (1954). 23. HARRIS, D. L., AND YAVIT, J., Federation hoc. 16, 192 (1957).

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