Enzymatic hydroxylation of aromatic compounds

ARCHIVES

OF

BIOCHEMISTRY

Enzymatic

AND

BIOPHYSICS

61, 431-441 (19%)

Hydroxylation Compounds’

of Aromatic

Chozo Mitoma, Herbert S. Posner, Henry C. Reitz and Sidney Udenfriend From the Laboratory of Chemical Pharmacoloyfy, National Heart Institute, National Institutes oj Health, Public Health Sewice, C. S. Department of Health, Kdrrcation, and Welfare, Bethesda, Maryland; and Departmen o,f (Themistr{y, Purdae zjniversit!y, West Lafailelfr, Indiana Received

Oct’ober

31, 1955

It has long been recognized that. many aromatic* cwnq~~unds are oxidized in the body t,o form phenolic compounds. The ext,ent of hydroxylation and the orientation of the substituting group have been the subjects of a vast number of studies with intact animals (Z-J), but little information is available concerning the bioc4~emic~al mechanisms invalved . This paper describes an enzyme system in the liver which hydroxylates numerous aromat,ic compounds. It will be shown that t)he hydroxylating enzyme syst,em is located in the microsomes and requires both oxygen a,nd redwed t’riphosphopyridine nucleotide. METHODS Materials DP?i” and TPN of approximately 100% purity was obtained from Pabst Laboratories. TPNH was prepared by t,he method of Kaplan et nl. (5). ATP of 90% purity ’ A preliminary report of this work was presented at the meeting of the American Societ,y for Pharmacology and Experimental Therapeutics at Charlottesville, Va., September, 1954 (1). Part of this paper was taken from a t,hesis submitted by Herbert S. Posner to the Faculty of Purdue University, in partial fulfillment of the requirement for the degree of Master of Science in Chemistry. 2 The following abbreviations are used: DPN and DPNH, unreduced and reduced diphosphopyridine nucleotide; TPN and TPXH, unreduced and reduced 1riphosphop~ridirle nucleotide; ATP. adenosine triphosphate; Tris, tris(hydroxymethyl)aminomethane.

432

MITOMA,

POSNER,

REITZ

AND

UDENFRIEND

was obtained from Sigma Chemical Company. Glucose dehydrogenase was prepared from beef liver by the method of Strecker and Korkes (6). All the substrates and hydroxylated compounds were commercially available with the exception of the 3-, 5-, 6., and 7-hydroxyquinolines which were kindly supplied by Dr. R. T. Williams.

Preparation of Subcellular Fractions Adult male New Zealand white rabbits were stunned and exsanguinated, and the livers3 were removed immediately and placed in ice. All subsequent steps were carried out at O-3”. The liver was homogenized in 9 vol. of cold 1.15’% KC1 using a Potter-Elvehjem type glass homogenizer. The homogenate was filtered through cheesecloth, and unbroken cells, nuclei, and mitochondria were separated by centrifuging for 10 min. at 9000 X g (low speed) in a refrigerated Servall angle centrifuge. To prepare the microsomal fraction, the supernatant was centrifuged at 100,000 X g (high speed) for half an hour in a refrigerated Spinco model L ultracentrifuge. The sedimented microsomes were washed thoroughly with 1.15% KCl, recentrifuged, and taken up in 2-3 vol. of KC1 solution for each gram of the original liver. The suspension w-as lyophilized or was frozen in 5-lo-ml. portions and stored for periods up to 1 week.

Enzyme Assay Incubations were carried out in air at 37” using a Dubnoff metabolic incubator. The incubation mixture contained in general 0.5 ml. of 0.5 M Tris-phosphate buffer of pH 8.5, 10 pmoles of nicotinamide, 0.25 pmole of TPN, 4-30 pmoles* of substrate, and 1 ml. of tissue preparation in a 3.5.ml. final volume. Hydroxylating activity was determined by measuring the amount of p-acetylaminophenol formed by hydroxylation of acetanilide.

Assay and IdentiJication

of Reaction Products

p-Acetylaminophenol and p-aminophenol were assayed by the methods described by Brodie and Axelrod (7). To determine o- and m-acetylaminophenols, the incubation mixture was saturated with KtHPOa and extracted with ether. The combined ether ext,ract from several incubation beakers was concentrated, extracted with dilute alkali, buffered to pH 9.2, and treated with 2,6-dichloroquinone chloroimide (8). The resulting blue color was measured at 610 mp. To identify the o&o and n&a isomers, the combined ether extract was evaporated to dryness, the residue was taken up in a small amount of alcohol, chromatographed6 3 Preparations obtained from rabbit livers were consistently more active than those from rats, guinea pigs, and dogs. 4 Tween 80 was used to solubilize diphenyl and naphthalene; 12.3 mg. (80 pmoles) of diphenyl or 10.2 mg. (SO pmoles) of naphthalene was dissolved in 200 mg. of w-arm Tween 80 and made up to 4 ml. with water. Of this solution, 0.2 ml., containing 4 pmoles of substrate and 10 mg. of Tween 80, was used for each incuhat,ion. 5 All paper chromatograms were developed on Whatman No. 1 paper using the ascending technique.

AROMATIC

HYDROXYLATION

433

using benzene-glacial acetic acid-water (2:2:1) as a solvent, and sprayed with the chloroimide reagent followed by borate buffer, pH 9.2 (9). The El values for the ortho and meta isomers were 0.53 and 0.15, respectively. Hydroxylated products of nitrobenzene were reduced with stannous chloride and HCl. The resulting m-aminophenol was det,ermined using the Bratton-Marshall reagent (10) ; p-aminophenol was assayed as previously described. In the determination of gentisic acid, the acidified reaction mixture was cxtracted with chloroform to remove salicylic acid. The residual aqueous phase was then saturated Tyith NaCl, and gentisic acid was estracted into ether. An equal volume of heptane was added to the ether, and the gentisic acid was returned to an aqueous phase by shaking with a small volume of 0.1 il1 sodium pyrophosphatc. The gentisic acid was identified spectrophotofluorometrically (11) exhibiting :I fluorescence peak at 436 rnp and being maximally activated at 320-325 mp. It was assayed with the Folin phenol reagent (12). The hydroxydiphenyls were extracted into heptane from the acidified incubiilion mixture and were returned to an aqueous phase by extracting t.he heptanc \vith 0.1 N NaOH. o-Hydroxydiphenyl was assayed by reacting with 2,6-dichloroquinone chloroimide. Since the Folin phenol reagent reacts with both o- and p-hydrosydiphenyl, the para isomer was determined as the difference between the values obtained with the Folin and chloroimide reagents. p-Hydroxydiphenyl was identified chromatographically using the solvent syst,em 0.02 N NaOH iu 20% ethanol. o-Hydroxydiphenyl lvas unstable and could not be chromatographed under similar conditions. The procedure for ext,raction of the naphthols was the same as that for the hydroxydiphengls. a-Xaphthol was assayed wit,h the chloroimidr reagent and also spectrofluorometrically. It had a fluorescence peak at 475 rnp and \yas masimnlly activated at 330 ml. 8.Naphthol had a fluorescence peak at. 415 rnp and W:LS mnsimally activated at 310 mF. In estimating quinoline derivatives, the alkalinized incubation mixture \!-:I,? first extracted with heptane to remove quinoline. The residual mixture was thrn adjusted to pH 6, and the hydroxyquinolines were extracted into ether and then returned to an aqueous phase by extracting the ether \vith :I small volume of 0.1 :V HzSO, . 3-Hydroxyquinoline was determined by it,s absorption at 350 mp arrtl identified fluorometrically. It had a fluorescence peak at 450 rnp ant1 n-as mahimall!: activated at 345 mF. 3-, 6-, and 7-Hydroxyquinolines were identified I)>chromatographing them in benzene-acetic acitl-water (2:2.2:1) :ind viewing the fluorescent spots under an ultraviolet lamp, The Ri v:rlurs were 0.55, 0.1 I, :~rrtl 0.26, respectively. For each type of determination, standards were prepared by- carrying kno\\rr amounts of the hytlrosylated compound through the entire extraction procedure. Comparable recoveries were obtained when the hgdroxylated compounds were added to boiled enzyme. As further controls, each hydrosylatcd compound LVW also incubated with the microsomal system, in an amount equal IO about 5-10% of its unhydroxylated precursor. p-Aminophenol, p-h~drox~:lcrt:Lrlilitfe, anti gentisic acid were quantitatively recovered after incubat.ion. All i he other compounds were metabolized to varying extents, but in all bllt two ~:ISPSisee Tallle II) at least 50% of the compound survived incubat.ion.

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UDESFRIEND

RESULTS Requirements of the Enzyme System in Liver Homogenates

Homogenates of rabbit liver required the addition of both nicotinamide and TPN for activity; DPN was relatively inactive. The addition of glucose 6-phosphate (2 pmolea) also stimulat.ed activity. Hydroxylation did not take place in t.he absence of oxygen. The optimal pH for hydroxylation was found to be 8.2,6 the reaction proceeding linearly up to 30 min. At pH values below 7, the enzyme system was unst.able. Use of the Waring blcndor for homogenization also resulted in preparations 1vit.h diminished activity, indicating the 1abilit.y of t.he system. Intracellular

Distribution

of the Ilydroxylating

Enzyme System

The homogenate was subjected to fractional centrifugation as described under Methods, and each fraction was assayed for hydroxylating activity (Table I). Almost all the activity was in the 9000 X g supernatant fract.ion which contains the soluble protein and microsomes. Further fractionation into microsomcs and soluble fract,ion showed t.hat the activit.y was not demonstrable with either component, alone but that combination of t,he two restored the activity. Pactors Involved in Hydroxylation The requirement of TPN for hydroxylation at first suggested dehydrogenation, with TPN acting as the hydrogen acceptor. However, the enhancement of activity by glucose B-phosphate suggested that TPN might be acting in its reduced form, since glucose 6-phosphate dehydrogenase is a known constituent of the soluble fract,ion of liver cells. To test this hypothesis, washed microsomes were incubated with acetanilide, TPS, and a TPNH-generating system. The latter, 400 /Imoles of glucose and 150 units of glucose dehydrogenasc, was added t.o the incubation mixt,urc for microsomcs described in Table I. I-nder these conditions 70-80% of the original activity of whole liver homogenate could be restored. In addition, t.he reaction was stimulated by ATI (1 ctmole), but the magnit.ude of this efIect was found t.o \Tary with each microsomal preparation. For optimal activity the microsomal system also required inorganic phosphate. Air could not be replaced by nitrogen. No requirement of Mn++ or &rate was observed, even after dialysis, as found in the hydroxylation of aniline by intact mycobacteria (13). Direct evidence 6 Five-tenths milliliter of 1: 1 Tris-phosphate (0.5 :M) buffer at pH 8.5 in 3.5 ml. of incubation mixture gave a find pH of 8.2.

AROMATIC

TABLE

Distribution

Ir&acellrtlar Fractions :lcetanilide,

equivalent as described

435

HYDROXYLATION

I

of Acetanilide-HUtl~oxlllatirLg

Activity

t,o 250 mg. of liver were incubated with 10 rmoles under Methods, and incubated for 30 min.

Celllllar

9.i\cetylamino11i1eno1 formed

fmctions

Kclative”

a Activity

in whole

+ microsomes

homogenate

is arbitrarily

taken

activity r,r

prrlolc

Whole homogenate 9000 x g dupernatant 100,000 x g Microsomes Soluble fraction Soluble fraction

Of

1.14

100

1.14

100

0.01 0.01 I .14

0.9 0.9 loo

a:; 100.

/A MOLES TPNH FIG. 1. Dependence of hydroxylation of acetanilido on Tl’NH. Each flask cont ained 50 mg. of I)-ophilized microsomes (equivalent to 750 mg. of liver), 12 Mmoles of acetanilide, 5 pmoles of nicotintmide, 1 pmole of ATP, 0.5 ml. of 0.5 4f Trisphosphate buffer at pH 8.5, and water to make a final volume of 3.5 ml. Incubation was carried out in air at 37” for 30 min.

that TPNH was involved in hydroxylation was obtained when chemically prepared TPNH added alone to washed microsomes was found to effect the formation of p-acetylaminophenol (Fig. 1). In the presence of chemically prepared TPNH, ATP and inorganic phosphate no longer stimulated hydroxylation. I)istribhon,

of Hyh-ozylating

&~stem in Rabbit

Tiswes

Studies with 9000 X g supernatant fractions of homogenates of brain, kidney, liver, lung, and muscle showed that only the liver contained

436

MITOMA,

Substrate Rabbit incubated

POSNER,

Specificity

REITZ

TABLE and Relative

AND

UDENFRIEND

II Amounts

of Product Formed

liver microsomes were fortified as reported under with 10 pmoles of substrate for 30 min. at 37”.

Methods

and were

Hydroxylated products= pmole/g./hr.

Acetanilide Aniline Nitrobenzene Salicylic acid Naphthalene Quinoline

Diphenyl

4 2 and 3 4 3 2, 5 1 2 3 5 6 7 8 2 4

4-8 (2-3)” 0.03 1-2 0 Trace 0.1 0.04-0.08 oa 1.5 Ob Trace Trace Ob 2.3” 0.4”

a Incubated with 4 pmoles of substrate and 10 mg. of Tween 80 per flask. b Four-tenths pmole of 5- and %hydroxyquinolines were almost completely destroyed under the incubation conditions. c Number refers to position of hydroxyl group.

this enzyme system. Using 30 pmoles of substrate, the rate of hydroxylation of acetanilide in the liver supernatant was 4-8 pmoles/hr./g. liver. Substrate Specificity and Identification

of Products

In Table II are listed some of the compounds which were tested as substrates of the microsomal hydroxylating system. Conclusions as to the relative extent of hydroxylation of the various substrates listed may not be justified since, with the exception of the aminophenols, acetylaminophenols, and gentisic acid, the hydroxylated compounds were also destroyed to some extent during incubation with microsomes. One of thestriking features of this system is that none of a number of normally occurring substrates known to be hydroxylated in vivo was oxidized (Table III). In addition, two other compounds, antipyrine and theophylline, which are hydroxylated in vivo, were not affected by the microsomal system. When acetanilide was incubated with rabbit liver microsomes, p-acetylaminophenol was practically the only product formed, but trace

AROMATIC

HYDROXYLATION

TABLE ligdws!glatiolL Substrate

Reactions

r,-Phen~lnlanine l,-'rryptopll;iIl

Iiynurenine Anthranilic Phenylacetic

Tryptamine

acid acid

437

III

Xot Catalyzed 6~ Liver Microsames Hydroxylated product

1,.Tyrouine (Z, 3., or 4hydroq phenylalanine) 5-OI-I-L-tr~l,toph:rlll 3-Hydrorykynureninc 3-Hydrosyant,hranilic acid 2-Hydrosyphenylacetic acid 3-Hydrosyphenyl:Lcet ic ;icitl 4Hydroxyphenylacetic :rcid .i-OH-tryptsminr (srrotonin)

amounts of both o- and m-acetylaminophenols wuld be detected on paper chromatograms after spraying n-it,h t,he chloroimide reagent,. Aniline gave rise t#o only the p-hydroxy derivatix-e. So e~idewe for acetylation or deavetylation w.s observed with awtanilicle and aniline, acetanilide giving rise to only acet,ylamiaophc~lol, and aniline to only free p-amiilopheuol. A trace amount of p-nitrophenol was dctecked \vhen nitrobenzene. was incubat~ed in oxygen to minimize its reduction to p-aminophenol No hydroxylation product ot’her than gentisk acid was formtl aft,er incubating salicylic acid. Napht’hol formed by the microsomes was exclusively t,he wform on the basis of the fluorescence spectrum. However, the major product from naphthalene was a dihytlrodiol-like compound which upon acid hydrolysis yielded mainly oc-naphtzhol (I-l). This may be a clue as to t,he mechanism of microsomal hydroxylation since it, m:ly be an intermediate in the enzymatic formntioir of oc-uapt,hol from napht~halene. Incubat’ion of quinoline wit’11 rabbit liver microsomcs yielded 3-hy _ droxyquinoline as the major product,, nit,h only traces of Ci- and 7-hydroxyquinolines. No evidence was obtained for the formation of the 5- and 8-isomers, although the chloroimide reagent was sufficientzly sensitive t’o detect 0.007 pmole of either compound. With diphenyl the microsome system yielded at least two products. One of them was p-hydroxydiphenyl which could be identified by color test and by paper chromatography. The sewnd gave a color t,est, similar to o-hydrosydiphenyl but was not further idtwt~ified. Inhibitors

for the l&action

The data iu Table IV show t,hat hydroxylation of awtanilide strongly inhibited by LY,a’-dipyridyl and p-chloromerc.uriherlzoate,

was sug-

438

MITOMA,

POSNER,

EJect of Inhibitors

REITZ

AND

UDENFRIEND

TABLE IV on Hydroxylation

of Acetanilide

Each flask contained 0.5 ml. of 0.5 M Tris-phosphate microsomes equivalent to 500 mg. of liver, 10 amoles each of TPNH and ATP, and inhibitor in 3.5 ml.

Final Percent concentration inhibition

Inhibitors

1.4 1.7 8.5 1 1 2.8 2.8 2.8 2.8

a, a’-Dipyridyl p-Chloromercuribenzoate 2,4-Dichlorophenol Ascorbic acid SKF 525a” Versene Sodium arsenate Sodium fluoride Sodium pyruvate 0 Diethylaminoethyl

buffer pH 8.5, lyophilized of nicotinamide, 1 pmole

x x X x x X X X X

10-s lo-’ lo-’ 10-S 10-a 10-a 10-s 10-S 10-s

87 83 67 40 20 0 0 0 0

diphenylpropylacetate’HC1.

gesting that Fe* and a sulfhydryl group are involved in t,he enzyme system. The mode of inhibition by dichlorophenol, a catalase inhibitor (15), is not clear.

DISCUSSION One of the striking properties of the microsomal system is the variety of aromatic compounds that it can hydroxylate. Since aromatic hydroxylation can also be produced non-enzymatically, utilizing Hz02 and Fe++ (16), or ascorbic acid, 02, and Fe++ (17, IS), the possibility was considered that microsomal hydroxylation was non-enzymatic. However, the evidence, reviewed below, points to a direct enzymatic involvement. Experiments on intact guinea pigs have indicated that in scorbutic animals t,he extent of hydroxylation of various aromatic compounds, including acetanilide, is depressed (19). This effect is probably indirect, since liver microsomes normally contain at most traces of ascorbic acid? Furthermore, the addition of ascorbic acid to microsomes obtained from both normal and scorbutic guinea pigs was without effect; in fact, at concentrations of 1O-3 M, the addition produced an inhibition. The requirement of TPNH and oxygen in the hydroxylation of aromatic compounds suggests the involvement of a peroxide. In fact Gillette et al. (20) have reported that TPNH is oxidized by liver microsomes even 7 J.

J. Burns,

personal

communication.

AROMATIC

HYDROXYLATIOS

439

in the absence of substrate, producing “peroxide.” However, hydroxylation cannot be due to peroxide alone. Thus kidney microsomes were found to oxidize TPNH in a similar manner, but are unable to hydroxylate aromatic compounds. Furthermore, DPNH, although just as readily oxidized by liver and kidney microsomes, cannot substitute for TPNH. Attempts to demonstrate a direct participation of Hz02 in this react’ion were also unsuccessful. Thus addition of catalase did not inhibit, and Hz02 generated by glucose oxidase (notatin) or by n-amino acid oxidase csould not replace TPNH in this system. In addit’ion t,o an oxidizing system for TPNH, hydroxylation requires a labile component in liver microsomes. Microsomes t,hat were aged, or solubilized by deoxycholate, lipase, and sonic disintegration, or which had been excessively aerated at pH 7 no longer catalyzed hydroxylation but still retained TPNH oxidase activity. Consistent with an enzymatic mechanism for rnicrosomal hydroxylation is the finding that acetanilide yielded almost’ exclusively p-hydroxyacetanilide (Table IV). Non-enzymatic hydroxylation, whether by Hz02 and metals (16), or by ascorbic acid, I’e 1 ++, and Versene (18)) produces almost equal amounts of the ortlzo and para isomers. On t’he basis of available informat’ion, the recluirement for TPNH and 02 may best be explained by the oxidation of TPNH t,o form a peroxide-like compound which a&s as the hydroxylating agent in t’he presence of a peroxidase-like enzyme in the microsomes. Similar requirements for a reduced pyridine nucleotide and O? have hern shown in other oxidative reactions. Thus, DPNH and O2 have also been found to be required by phenylalanine hydroxylase (21). The microsomal system Ilot only resembles phenylalanine hydroxylase in the requirement, for rcdutrcd pyridinc nucleotide and O? , hut in a number of other aspc>ctsas ~~11. In neither (ease can there be demonstrated a direct participation of II& . Roth syst’ems are inhibited by the ferrous ion c~omplexing agent, LY,a’-dipyridyl. 111the case of phenylalanine hydroxylaso, direct evidence for ferrous ion involvement has actually beelk obtained. Iiowcver, unlikr the microsomal syst’em, phenylalanine hydroxylase is highly spcGfic. and au yet no substrate other than L-phenylalanine has been found. DPNH and O2 have also been shown to he involved in the cleavage of the imidazole ring (22). The 11-p hydroxylasc of steroids in adrenal extracts is reported to require TPN, 0, , and fumarat,c (23). Onr might, speculatt that TPNH is also active in this systenl.

440

MITOMA,

POSNER,

REITZ

AND

UDENFRIEND

Previous studies have shown that microsomes contain other enzyme systems which oxidatively metabolize drugs (24). Thus, side-chain oxidation of barbiturates, ether cleavage of codeine, deamination of amphetamine, and dealkylation of aminopyrine are all catalyzed by microsomes and require TPNH and 02 , Although these reactions have similar requirements to the hydroxylating system, the instability of the present system and its relative refractoriness to the inhibitor SKF 525-A (diethylaminoethyl diphenylpropylacetate), as contrasted to some of the other microsomal enzyme systems, indicate that it is an entity distinct from all the others. Some differences exist between the present findings and previous in viva studies concerning the relative abundance of hydroxylated isomers. Naphthalene has been found to be metabolized to both CX-and P-naphthols in viva (14), whereas only the a-form was detected in the microsomal systems. Furthermore, only the para isomer of hydroxydiphenyl has been found in the urine of rabbits after feeding diphenyl (a), while the microsomal system yielded at least two isomers. The main product after feeding quinoline to dogs has been found to be the 3-hydroxy isomer (25). 3-Hydroxyquinoline is also the major product obtained on incubation of quinoline with rabbit liver microsomes. HOWever, although large amounts of other hydroxylated isomers of quinoline have been found after its oral administration (26), only traces of 6- and 7-hydroxyquinoline are formed by rabbit liver microsomes. Some of these observed differences may be due to varying stabilities of the hydroxylated products, either in the intact animal or in the microsomal system. However, it is also likely that catalytic sites for other types of aromatic hydroxylation may exist other than in liver microsomes. It is apparent from the data presented above that, in general, the extent of hydroxylation is related to the polarity of the substrate; the less polar compounds are more readily attacked by the microsomal system. This may be because polar compounds cannot penetrate the microsomes and may explain why the normal aromatic metabolites investigated, all of which are highly polar compounds, are not acted upon. The present studies do not, therefore, exclude the possibility that there exist normally occurring substrates of this enzyme system in the living animal. It may be, however, that the primary function of the microsomal hydroxylating system is to “detoxify” nonpolar aromatic compounds (25).

SUMMARY Au aromatic hydroxylatiug system has beer1 found in liver microsomes which requires TPNH and oxygen for its activity. Evidence for the cnzymatic nat,ure of this reactiou is presented. A uumber of suMrates fol this system have been investigated and their pheuolic m&Mites hays, been identified. REFEREWES 1. XIITOK\, (1.. .\sD UDESFRIEND, S., .J. Phamacd. Krptl. Therap. 113, -IO (1955~. 2. SUI,~II. J. N., Riochem. Sot. S~~~~posia(Cambridge, I*:n~l.) No. 6, 1.5 (19.50). 3. 1-or-se:, I,., Riochenr. Sot. Synzposin (Cambridge, Kngl.) No. 5, 27 (1950). 4. WILLIAMS, Ii. T., A&tracts, 1,. 30. Third national medicinal chemistry sympsium of the Americxn Chemical Society, Ch:trlottesvillc, Vx.. 1952. 5. IihPLAS, s. o., CoLowrcK, s. l',, ASD NEITmLD, 1,:. F.. -1. ISiol. t'hcul. 195, 107 i1951). 6. SYRECKER, H. J., .~ND KORKES, S., J. Riol. ('hem. 196,i69 (19.52). 7. BRODIE, B. I<., AKD AXELROD, J., J. Phamacol. B.rptZ. Therap. 94, %2 (19481. 8. ~'IMROS, \V. I<., Hiochenz. ./. 38, 399 il!M). 9. BK-\Y, II. O., .WD TIWRPE, W. V., in “&IN hods of I%iochemic:tl Analysis” (D. Glicli. ed.), Vol. I, p. 873. Interscience l’ut~lishers, Sew \-ark, 1951. 10. RRATTOS, A. C., .~ND MARSH.YLL, 1:. li.,J. Rid. f'hem. 128, 53i (1039). 11. ~owv.li~~s~ 11. I,., (:A~-LFIEI,I), l'., -\sI) I'DENFRIEXD, S., ,%irnw 122.31% (1055i. 12. ~Jor,~~, 0.. .\SD CIOC.4LTEV, r., ./. uiol. f'hem. 73, 627 (1027). 13. S~oasx, N. H., S.~MKEI.S, &I., ~\ND MAYER, R. I,.! -1. Viol. ('hem. 206, 731 (195-t).

18. ~~RODIE,

f'hm. I!).

B.

,~XE:r,ROD , J.,

mcrnp. 20.

I<.,

AXELROD,

208,541

<;ll,l,~TE:

Thcrnp.,

J., 8110~~.

I'.

.\..

:\11)

\‘I)ESFRIEND.

s..

J. Jjio/.

(1954).

UDESFME~D,

S.,

z~sr) RRODIE,

13. 11.. .I.

I'hrrmcml.

Ki-p(l.

111, 176 (1954,. J. Ii.,

I,.4 DE,

u.

s.,

ASD

I~ROD~E.

I$. I$., .J. I'~/n,t/lrrr~!.

Elptl.

in press, or Abstracts c‘., Arch. Biochcrn. ad

Am. Chcm. SOV. 128th meeting 1BC (19551. Hioph,ys., in prcsp. .~ND HAY~ISH~, T.. .I. .tt~t. (‘hem. SW. 76, 5570

21. MITOM\, 22. H.~Y~ISIII, O., TABOR, H., (1054). 2.1. HAY.\SO, >\I., AND DORFMO, R. I., J-. Riol. f’hem. 211, 227 (195-1). 21. RIGIDIF:. 13. B., I~XELROD, ,J., COOPER, J. R.. G ~UI>ETTK, I,.. T, \ Ill-, blrron~.t, ('., ?YSD ~:DESFRIEND, S., Science 121, 60X (1955). 2.5. ~'OKWK, I,., .\sD I~RODIS, B. I<., .J. Biol. C’hrttl. 187, 78; (1950). 26. $~111'lr, .J. x.. AND ~r~~~~,~~4~~~, 11. T., J3iochenl. .J. 60, 284 (1!)55j.

13. S..