The migration of deuterium during aryl hydroxylation

ARCHIVES

OF

BIOCHEMISTRY

The

.4ND

Migration

BIOPHYSICS

131, 238-244 (1969)

of Deuterium

During

ILEffect of Induction of Microsomal or Polycyclic JOHN DALY,

Laboratory

Aryl

Hydroxylases

Aromatic

Hydroxylation with Phenobarbital

Hydrocarbons

DONALD JERINA, JOYCE FARNSWORTH, AND GORDON GUROFF

of Chemistry, National Institute of Arthritis and Metabolic Biomedical Sciences, National Institute of Child Health and National Institutes of Health, Bethesda, Maryland Received

January

Diseases,

and

Laboratory

of

Human Development, ZOO14

14, 1969

Acetanilide4-2H is converted to 4-hydroxyacetanilide-3-2H during incubation with liver microsomal preparations. The degree of migration and retention of deuterium in this reaction, previously shown to depend on the pH of the media, is also dependent upon the species, strain, and sex of the animal used as the source of microsomal preparations. Pretreatment of animals with phenobarbital causes an increase in the retention of deuterium in 4-hydroxyacetanilide produced from 4-deuteroacetanilide while pretreatment with benzpyrene or 3-methylcholanthrene causes a decrease in the retention. The effect of induction on the migration and retention of deuterium and of tritium is unique to acetanilide4-QH, and is not observed during hydroxylation of anisole-4-zH, chlorobenzene4-2H, or 4-fluorobipheny14’-2H. It is proposed that two “acetanilide hydroxylases” exist in varying amounts in different species, and that one, characterized by exhibiting high retentions of deuterium in the hydroxylation of acetanilide4-2H, is induced by phenobarbital, while the other, characterized by exhibiting low retentions of deuterium in the same reaction, is induced by 3-methylcholanthrene and benzpsyrene.

The induction of drug metabolizing enzymes with phenobarbital, benzpyrene, 3methylcholanthrene, and other agents has received much attention in recent years because of implications in the fields of enzyme induction and protein synthesis, drug therapy, and biological oxidation mechanisms. The types of change in enzyme activities caused by phenobarbital on the one hand, and by the polycyclic aromatic hydrocarbons on the other, have been found to be qualitatively and quantitatively different (1). In addition, while both classesof inducing agents causean increase in a hemoprotein functional in oxidative reactions, evidence has been obtained that two spectrally distinct hemoproteins exist in liver microsomes and that one of these is induced by phenobarbital treatment and the other by poly238

cyclic aromatic hydrocarbons (24). Phenobarbital induction results in the formation of a hemoprotein with an absorption maximum at 418 mp while methylcholanthrene induction causes the formation of a hemoprotein with an absorption maximum at 394 ml*. These two spectrally distinct hemoproteins are, however, spectrally interconvertible upon interaction with certain classes of compounds and the suggestion has been made that two interconvertible forms of a single hemoprotein exist (4). Our own interest in induction of microsomal enzymes stems from investigations on the mechanism of migration of substituents during aryl hydroxylation (“The NIH Shift”) (5). An example of this hydroxylation-induced migration is the conversion of acetanilide-4-3H to 4-hydroxyacctanilide-

MIGR.ATIOK

DURING

ARYL

HYDROXYLATION.

9

II

239

0 NH-&CH3

NH-C-CH, Microsomes

D

02

D FIG.

1. 1\Zigration

of deuterium

during

microsomal

hydroxylation

of acetanilide-4.21%.

3-3H with 44-50 % retention of t’he tritium 4-chlorophenol (ll), and 4-hydroxyanisole (8). was isolated by originally present in the 4-position (6). 4-Fluoro-4’-hydroxybiphenyl thin-layer chromatography (silica gel GF, benStudying the same reaction, but using liver acetate, 95:5, 12~ 0.42) and the deutemicrosomsl preparations from rabbits pre- zene-ethyl rirlm content determined by mass spectrometry. treated with phenobarbital, Tanabe et al. (7) Young adult animals were used throughout. report’ed much higher retentions of tritium The rats weighed from 200 to 300 g. Animals were (72 %) in t’he isolat’ed 4-hydroxyacetanilide. pretreated with eit.her sodium phenobarbital (80 The only apparent difference between the mg/kg, one injectiolr daily) for 4 days and killed two sets of experiments was the use of on day 5 or with benapyrene (20 mg/kg in cott,onmicrosomesfrom phenobarbital-induced rab- seed oil, one injection daily) or 3-methylcholanoil, one injection bits in the latter experimenk, suggesting threne (40 mg/kg in cottonseed t’hat phenobarbital caused an increased nc- daily) for 2 days and killed on day 3 unless otheracetanilide-@H tivit? of an “acetanilide hydroxylase” char- wise noted. For in viva studies, was administered 24 hr after acterized by high migration and retention of or chlorbenzene-4JH the last injection of indllcing agent. tritium. The results reported here on the hydroxylation of acetanilide-4-2H (Fig. 1) RESULTS and acet’anilide-4-3H support this hypotheThe retention of deuterium in the 4-hysis and, further, indicate that induction with polycyclic aromatic hydrocarbons such as droxyacetanilide formed from acetanilide3-methylcholanthrene causes the opposite 4-2H was found to be quite dependent on effect’, namely, a lower ret’ention of deute- the species, strain, and sex of the animal used as the source of liver microsomes. riunl or tritium in 4-hydroxyacetanilide. These data are presented in Table I along with the effect of pretreatment of animals with phenobarbital or benxpyrene. ConDeuterated and tritiated acetanilides n-ere prepared as previously described (6). Other deutsist’ently lower deuterium retention values crated substrates were prepared by standard were obtained using microsomes from animethods. Microsomal hydroxylations were carried mals pretreated with benzpyrenc than with ollt for 15 min with either crude microsomal prepmicrosomes from untreated animals. Conarations or resuspended microsomal pellets as versely, higher deuterium retention values described previously (8). Substrates were added were observed using microsomes from anias ethanol solutions. mals which had been pretreated with phenoAcctanilide-4-2H (50 mg/kg) or chlorobenzenebarbital. 4-*H (400 mg/kg) was administered intraperitoneTable II presents a comparison of the ally to male Sprague-Dawley rats and the urine effect of pretreatment of animals with was collected and treated with @-glucuronidase as previously described (9). phenobarbital or 3-methylcholanthrene on Hydrosylated products from in viva or in vilro the retentions of either deuterium or tritium stltdies were extracted into et,hyl acetate. The in 4-hydroxyacet’anilide obtained following extract was dried with anhydrous sodillm sldfate incubation of acetanilide-4-2H or acetanilideand concentrated to a small volume. The phenolic 4-3H with rat liver microsomes. The retenproducts were separat,ed and isolated by paper or tion of both deuterium and tritium in 4thirl-layer chromatography and t,he denterium or hydroxyacetanilide was increased by phenotritium content of the products was determined. barbital pretreatment and decreased by The isolation and determination of deuterium or methylcholanthrene pretreatment. tritium in variolls hydroxylated products has The effect of pretreatment’ of mice, male been described for l-hydroxyacetanilide (6,9, 10).

240

DALY, TABLE

RETENTION

OF

TANILIDE

AFTER OF

Species

JERINA,

AND

I

DEUTERIUM

IN

MICROSOMAL ACETANILIDE-4-'Ha Percent No. of--experimats* Normal

4-HYDROXYACEHYDROXYLATION

retention

of deuteriumC Induced phenob&ita

Induced benzPYrene

Rat Sprague-Dawley (Male) Sprague-Dawley (Female) Wistar (Male) Osborne-Mendel (Male) Fisher 344/N (Male) NIH Black (Male) Mouse Hamster Cat Guinea Pig Rabbit (New Zeland White)

4

43 f

1

34 f

150

f

1

4

38 f

1

32 =t 146

f

1

1 1

45 45

-

-

1

40

-

-

2

50

-

-

6 2 1 2 4

46 f 2 37 36 30 31 f 1

36 h 28 28 f

152

f: 2 36 1 37 f 2d

a Incubations were carried out at 37” for 15 min and contained 15 ml of microsomal suspension corresponding to 5 g of liver, 3.5 ml of 0.5 M Tris buffer, pH 8, 15 rmoles of NADP, 50 pmoles of glucose-6-phosphate, 10 units of glucose-6-phosphate dehydrogenase, and 30 pmoles of acetanilide4-2H added in 0.5 ml of ethanol in a final volume of 20 ml. 4-Hydroxyacetanilide was isolated by extraction into ethyl acetate and subsequent acidchromatography-[Benzene-acetic paper water (2: 2: l)] as previously described (9). * Each experiment used pooled microsomes from either three rats, five mice, two hamsters, one cat, two guinea pigs, or one rabbit. Animals were given daily injections of 20 mg/kg of benzpyrene in cottonseed oil or 80 mg/kg of sodium phenobarbital for 4 days and killed on day 5. Microsomes were prepared as previously described (8). c Percentage retention of deuterium was determined from the mass spectra of the isolated 4hydroxyacetanilide. Deviations from the mean are presented for experiments which were repeatedt a least four times. d This retention was observed 24 hr after the first injection of phenobarbital and remained constant during 5 days of induction (see also Fig. 2). In one experiment retention of 48y0 was obtained.

GUROFF

Sprague-Dawley rats, and rabbits with various compounds on the retention of deuterium in 4-hydroxyacetanilide is presented in Table III. Pretreatment with barbiturates, diphenylhydantoin, or acetanilide resulted in increased retention of deuterium while benzpyrene, 1,2-benzanthracene and 3-methylcholanthrene pretreatment resulted in decreased deuterium retention. Pretreatment of mice with a variety of other compounds including various steroids, bile acids, aminopyrine, aniline, nicotinamide, pyridine, naphthalene, phenanthrene, caffeine, 4-hydroxyacetanilide, and barbituric acid had no significant or consistent effect at the dose (50 mg/kg) studied on the retention of deuterium in the 4-hydroxyacetanilide. Maintaining male Sprague-Dawley rats under conditions which cause induction of aryl hydroxylases (12) (5” for 4 days) did not result in a change in the retention of deuterium. Preincubation of liver microsomes from untreated rats with either phenobarbital (1O-4 M) or benzpyrene (lop4 M) for 5 min before the 15-min incubation with acetanilide-4-2H (1O-3 M) did not change the retention of deuterium in 4-hydroxyacetanilide. The retention values obtained for control, phenobarbital, and benzpyrene incubations TBBLE COMPARISON TRITIUM

II

OF RETENTION OF DEUTERIUM IN hHYDROXYACETANILIDE

MICROSOMALHYDROXYLATION 4-2H OR ACETAN1LIDE-‘k-3H' Species

Rat, SpragueDawley (Male) Rat, SpragueDawley (Male) Rat, SpragueDawley (Male)

Induction

AND AFTER

OF ACETANILIDE-

Percentage Deuterium

retention Tritium

None

44 It

1

45 f

3

3.Methylcholanthrene Phenobarbital

34 +

1

24 rt

3

50 f

1

63 f

3

a Incubations and isolation of 4-hydroxyacetanilide were carried out as described in Table I. Deuterium contents were determined by mass spectrometry and tritium contents by assay and liquid scintillation counting as previously described (6).

MIGRATION TABLE ~~ETENTION

OF

MENT

WITH

~HYDROXYACE-

day Mice

P;’

EFFECT

HYDROXYLATION OF PRETREAT-

VARIOUS

Dose, Percentage Compound

HYDROXYLATION.

retention

of deuterium

SpragueDa$yeyleyts a

Rabbits

Species

75 75

46f2 53 zt 52

75 100

52 54

50

50

50 20 30

51 f 36 f 36 f

50

42

1

43f131fl 50 f 1 -

37 f -

2 Rabbit

47 1 1 1

-

1

IV

Pretreatment

X-Methylcholananthrene Phenobarbital None Phenobarbital

HYDK~XYLATIOS

PercentPercentage retention of age increase deuterium in hydroxylation with Kormal Acetone acetone

170

36

43

330 140 180

50 31 3G

+.;; 46

a Incubations and isolation as described in Table I. Incubations with acetone were 0.45 Y in this substance.

38

49 34 f 34

241

OF ACETOXE ON THE OF ACETANILIDE”

COMPOUNDW

Rat n’one Phenobarbital Secobarbital Hexobarbital Diethylbarbituric acid Diphenylhydantoin Acetanilide Benzpyrene S-blethylcholanthrene 1, a-Benzanthracene

II TABLE

IN

MICR~SOMAL EFFECT

OF ANIMALS

ARYL

III

DEUTERIUM

T~NILIDE AFTER OF ACETANILIDE-4-‘H:

DURING

38 28 f: 26

1

-

a Incubations and isolation of C-hydroxyacetanilide were carried out as described in Table I. Five mice were induced for 2 days and killed on day 3. Three rats or one rabbit were induced for 4 days and killed on day 5 and microsomes were prepared as described (8). Deviations from the mean are reported for experiments which were repeated at least 4 times.

were respectively, 42, 44, and 44. Similar results were obtained with rabbit liver microsomes. Incubations of acetanilide-4JH were carried out in the presence of such competitive inhibitors as the isomeric chloroacetanilides, methylacetanilides, and fluoroacetanilides, or with SKF-525 or carbon monoxide (CO/O2 ratio for 50% inhibition = 2.6-2.8 for microsomes from either phenobarbital or methylcholanthrene-induced rats). No significant effect on the degree of retention of deuterium in the 4-hydroxyacetanilide was observed. When acetanilide-4JH was incubated with microsomal preparations containing 0.45 M acetone, conversion to 4-hydroxyacetanilide was greatly enhanced (13). The migration and retention of deuterium was also increased as shown in Table IV. Hydroxylation of anisole-4-2H and 4-fluoro-

biphenyl-4’-2H was also enhanced by acetone but no effect on migration and retent,ion of deuterium was observed. In vivo experiments in which male Sprague-Dawley were given acetanilide4JH (50 mg/kg) afforded 4-hydroxyacetanilide with retention of 34, 40 and 30 % deuterium for control animals, animals pretreated with phenobarbital, and animals pretreated with benzpyrene, respectively. These values may be compared to corresponding retention of 37, 46, and 32% obtained at pH 9 in vitro. The pH of the incubation medium affects the retention of deuterium in 4-hydroxyacetanilide using rat or rabbit microsomes from control animals and animals prekeated with either phenobarbital or 3-methylcholanthrene as shown in Fig. 2. The retentions obtained with rabbit microsomes are much more dependent on pH than are the retentions found using rat microsomes. The effect of administration of phenobarbital or 3-methylcholanthrene on “:lcetanilide hydroxylase” activity and on the retention of deuterium in 4-hydroxyacetanilide over the initial 24-hr period after injection is shown in Fig. 3. The chaugc in percent retention of deuterium preceeds the increase in “acetanilide hydroxylase” activity. When deuterated substrates other than acetanilide were examined either in zlif~o with anisole-4-2H and 4-fluorobiphenylA’2H or in vivo with chlorobenzene-4-“H using

242

DALY,

JERINA,

RABBIT

AND

I

60 -

RAT

403 ‘“t_ 7

9

8 PH

FIG. 2. Dependence of migration and retention of deut,erium in 4-hydroxyacetanilide on the pH of the incubation medium using microsomes from rats and rabbits. (a), control; (A), pretreated with phenobarbital; and (O), pretreated with methylcholanthrene.

GUROFF

control animals or animals pretreated with phenobarbital or 3-methylcholanthrene, no signijicant cliflerencesin the degree of retention of deuterium in the 4-hydroxylated product were observed. Anisole-4JH was converted at pH 8 to 4-hydroxyanisole which retained 63 II= 4% of the deuterium. This retention was independent of the source of microsomes (rat, mouse, or rabbit) and was not affected by pretreatment of animals with phenobarbital or benzpyrene. 4-Fluorobiphenyl-4-2H was converted at pH 8 to 4-fluoro-4’-hydroxybiphenyl with retention of 63 % deuterium by microsomes from control male Sprague-Dawley rats and those pretreatedwithphenobarbitalorbenzpyrene. For comparison, biphenyL4JH is converted with rabbit liver microsomes to 4-hydroxybiphenyl with a migration and retention of 64 % of the deuterium (9). When chlorobenzene-4-2H was administered to control male Sprague-Dawley rats and to animals pretreatedwithphenobarbital orbenzpyrene, the retention of deuterium in 4-hydroxychlorobenzene was, respectively, 54, 55, and 54%. These differences are not significant and the percentage retention obtained with the rat is the same as previously observed with rabbits (14).

i I a b 2 5 B 0

0.7

-70

_ l -------------.

0.5 =*=--.----

L&E 3. g-o.3

-I-----

-A----

4

8

5 E

------------a’

-

1

12

16

20

24

HOURS

FIG. 3. Relationship between induction of acetanilide hydroxylase activity barbital or methylcholanthrene and the change in migration and retention Curves represent pmoles of 4-hydroxyacetanilide per incubation (see legend, injection of phenobarbital (0-O) or methylcholanthrene (A-A) and tion of deuterium in 4-hydroxyacetanilide aft&r injection of phenobarbital methylcholanthrene A-----A. Points are averages of two experiments with point. No further change in percentage deuterium retention was found during days of phenobarbital administration.

with phenoof deuterium. Table I) after percent reten(O-----O) or five mice per 4 additional

MIGRATION

DURING

ARYL

DISCUSSION

The migration and retention of deuterium, tritium, and various other substituents has been found to be a characteristic of enzymatic hydroxylation (5) and appeared to be strongly dependent on the nature of the substrate (9) rather than on the enzyme. Thus, phenylalanine-4JH with either phenylalanine hydroxylase from liver or from Pseudomonas (15) or wit*h adrenal tyrosine hydroxylase (16) yielded tyrosine-3-3H with approximately 90 % retention of tritium. In addition, a similar substrate, amphetamine-4-3H formed 4-hydroxyamphetamine3-3H with a microsomal hydroxylase and again approximately 90 % of the label was retained (10). It was, therefore, surprising to discover that migration and retention during hydroxylation of acetanilide-4-2H was dependent on the source of the microsomes and on pretreatment of animals with either phenobarbital or polycyclic aromatic hydrocarbons. Induction of “aryl hydroxylases” with phenobarbital or benzpyrene did not, however, affect the migration and retention of deuterium with other aromatic substrates such as anisole-4-2H, chlorobenzene-4-2H and 4-fluorobiphenyl-4’-2H. Only acetanilide-4-2H showed this dependence. The migration and retention of deuterium during the hydroxylation of acetanilide-4-2H and closely related compounds such as benzanilide-4-2H were also unique among aromatic substrates in showing a dependence on the pH of the incubation media (9). Lower retentions were observed at higher pH values as is shown in Fig. 2. The dependence on pH was more pronounced with liver microsomes from rabbits than with those from rats. It now seems that the effect of pH on migration of deuterium in the microsomal hydroxylation of acetanilide-4-2H is more complicated than it first appeared (9). The explanation based on alternate stabilization of cationoid intermediates by ionization of the amide in a pa-dependent equilibrium still seems satisfactory but other factors are also affecting the observed apparently retentions during the hydroxylation of acetanilideWH. The present study delineates two of these factors namely, t’he species employed :LS a source of liver micro-

HYDROXYLATION.

II

243

somes and the exposure of the animal to either barbiturates or polycyclic aromatic hydrocarbons. It is possible to interpret the data obtained in the following manner: There may be two hydroxylase” which types of “acetanilide occur in varying amounts in the species studied. One of these “acetanilide hydroxylases” would then be characterized by a high migration and retention of deuterium. This enzyme would be the major functioning “acetanilide hydroxylase” in microsomes from mice or rats. It would be preferentially increased by phenobarbital treatment, and would yield a retention of 50-55% deuterium. The retention with this enzyme would be slightly dependent on pH if at all. The other “acetanilide hydroxylase” would be characterized by low migration and retention of deuterium. This enzyme would be the major functioning “acetanilide hydroxylase” in microsomes from rabbits and guinea pigs. It would be preferentially increased by 3-methylcholanthrene or benzpyrene treatments, and would yield a retention of 26-29% deuterium. The retention with this “acetanilide hydroxylase” would be strongly pH dependent. i\Iechanistically, it seems likely that the unique differences (pH dependence, source of microsomes) in retention with acetanilide-4-2H hydroxylation are due to the possibility of stabilizing charged intermediates by ionization of the amide grouping (9). In the postulated high retention “acetanilide hydroxylase” this ionization and resulting stabilization of charge might be prevented by binding of the acetanilide at a hydrophobic site on the enzyme while in the low retention “acetanilide hydroxylase” the amide group may be in a hydrophilic region so that ionization is possible and may be effected by the pH of the medium. These observations certainly must be considered in relation to the already large body of evidence that indicates t’hat the induction of “microsomal hydroxylases” with phenobarbital or polycyclic aromatic hydrocarbons are quite different (1). Qualitatively, the apparent levels of the two “acet’anilide hydroxylases” estimated from deuterium retentions appear to correlate with published data on the ratio of the two

244

DALY.

JERINA,

spectral forms of the hemoprotein, cytochrome P-450, and the effect of induction with phenobarbital and polycyclic aromatic hydrocarbons on this ratio. The time course for the change in observed retention and the increase in “acetanilide hydroxylase” activity in mice after treatment with either phenobarbital or S-methylcholanthrene must also be considered. In both cases the change in retention values is marked within 7 hr while at this time no apparent increase in “acetanilide hydroxylase” activity is yet apparent. Similarly in rabbits the change in deuterium retention for phenobarbital induction is complete within the first 24 hr while 3-5 days are necessary for maximal induction of enzyme activity (Table I, footnote d). The hypothesis of two “acetanilide hydroxylase” enzymes must, of course, be subjected to further experimentationl. The enhancement of “acetanilide hydroxylase” activity with acetone in vitro reported previously by Anders (13) and the concommitant increase in deuterium retention is surprising. Anders (13) has interpreted the enhancement of “aniline hydroxylase” activity with acetone and the concommitant changes in K, for aniline hydroxylation as tentative evidence for the occurrence of two “aniline hydroxylases.” Other kinetic studies have also been reported as evidence for two “aniline hydroxylases” (17). The “aniline hydroxylase” with the larger K, value appeared to be selectively induced by phenobarbital. These and other reports (18-20) indicate a multiplicity of drug metabolizing enzymes which tends to support our interpretation of the variation of deuterium retentions with acetanilide-4-2H hydroxylation as due to the functioning of at least two “acetanilide hydroxylases.” 1 Inhibition of hydroxylation of acetanilide-42H with S-trifluoroacetylaniline results in deuterium retentions that are G - 7% lower than those obtained without inhibitor using rat liver microsomes.

AND

GUROFF REFERENCES

1.

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Rev., 19,317 (1967). E., AND MANNERING, G. J., Biothem. Biophys. Res. Commun., 24,668 (1966). 3. ALVAHES, A. P., SHILLISG, G., LEVIN, W., AND KUNTZMAN, B., Biochem. Biophys. Res. Commun., 29, 521 (1967). 4. HILDEBRANDT, il., REMMER, II., AND EST.\BROOK, R. W., Biochem. Biophys. Res. Commun., 30, 607 (1968). 5. GUROFF, G., DALY, J., JERINA, D., RENSON, J., UDENFRIEND, S., .~ND WITICOP, B., Science 167, 1524 (1967). 6. UDENFRIEKD, S., ZALTZM:~N-NIRENBERG, P., DSLY, J. W., GUROFF, G., CHIDSEY, C., AND WITKOP, B., Arch. Biochem. Biophys. 120, 413 (1967). 7. TAN~BE, M., YASUDA, D., TAGG, J., .~XD MITOM.~, C., Biochem. Pharmacol. 16, 2230 (1967). 8. JERINA, D., GUROFF, G., AND DALY, J., Arch. Biochem. Biophys. 124, 612 (1968). 9. DALY, J., JERINA, D., AND WITKOP, B., Arch. Biochem. Biophys. 128, 517 (1968). 10. DALY, J., GUROFF, G., UDENFRIEND, S., AND WITKOP, B., Arch. Biochem. Biophys. 122, 218 (1967). 11. KRISCH, K., AND STAUDINGER, H., Biochem. 2. 334, 312 (1961). 12. INSCOS, J. K., AND AXELROD, J., J. Pharmacol. Exptl. Therap. 129, 128 (1900). 13. ANDERS, R/I. W., Arch. Biochem. Biophys. 126, 269 (1968). 14. JERINS, D. M., DALY, J. W., AND WITKOP, B., J. Am. Chem. Sot. 89, 5488 (1967). 15. GUROFF, G., LEVITT, M., DALY, J. W., AND UDENFRIIEND, S., Biochem. Biophys. Res. Commun. 25, 253 (1966). 16. D)SLY, J. W., LEVITT, M., GUROFF, G., .QYD UDENFRIEND, S., Arch. Biochem. Biophys. 126, 593 (1968). 17. Wa~.4, F., SHIMAKA~.~, H., TAKASUGI, M., KOTAKE, T., AND S.~K.~MOTO, Y., J. Biochem. (Japan) 64, 109 (1968). 18. KUXTZM.~N, R., LEVIN, W., JA4COBSON, RI., END CONNEY, A. II., Life Sci. 7, 215 (1968). 19. ALVBRES, A. P., SCHILLING, G. R., .~ND KUNTZMAN, R., Biochem. Biophys. Res. Commun. 30, 588 (1968). 2. SLADEK,

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GURTOO, H. I,., AND PLOWMAN,

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CAMPBELL, K. >I.,

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Biochem. 31, 588 (1968).

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