Propionate metabolism

Propionate II. Factors

\YARX\jER Ilesearch

Regulating

Adaptation

S. WEGESER,

La’Joralories,

Metabolism’ of Escherichia

co/i to Propionate2

HE?\;R1- C. REEVES,”

Department of Biochemi.str!g, Philadelphia, Pennsylcaniu Received

June

Albert

rZNI)

SAMUEL

h’inslein

Medical

J. AJL f,‘sn(er,

19141

8, 1967

Factors regulating adaptation of Escherichiu coli t,o propionate have t1een esamined. Evidence is presented that growth on propicmate, in contrast t,o butgrate and valerate, involves adaptatio11 rather t,han ml1t,atic,rl-selec.tiotl. The long lag preceding adaptation to this substrate is significantly reduced by the addition of limiting concentrations of CA acids to the medi11m. This C, acid cffcct is partially met by addition of HCO; or vitamin Ba Lo the medium, and s11ggcs1s that this organism may possess a deficiency in tl1e functioning of the propionyl-Co.1 carboxylase pathway. Evidcncc is presented that growth per se it1 the presence of propionate results in inducLio11 of enzymes which enable the cell to 11tilize propiotutte as the sole source of carbon. The metabolic pathway indr1ced 11nder sr1cl1 condit ic111s appears t,o involve ositlation of propionate to acetate and CO?.

;Ilthough the metabolism of propionic acid hns been extensively studied in a varietJ~ of systems (l-.5), little is linow~ regnrding the nwch:u~isms by \\-hich ~:‘s&rict~ ia coli gro\vs awobically using propionatc :w the sole sourw of carbon. I’ropicmlte may be oxidized to :icetntc by several different p:rthways. These include oxidation via nmlonic semialdehydc (6, 7)) oxidation via malonic seminldrl~\-dc-(‘o~~ (S), and osidation \-ia lactate (O-1 1). In aevcral systems, I)ropimate aplwars to be metabolized prirn;tril>* via cwbos\-lation Of ~)rO~)iOll!-l-(‘O.~ tcJ mcth~lmalc m?,l-c’oX (l%lJ) . The ht ter may be isomerized to succirlyl-(.‘oA md oxidized 1)~ the tricnrboxylic acid c\~le. Aliot’hcr pkibility for the mctaboliwl of propionnte is c~cmdensntionof l,ropion!l-(‘c)-~ 1 Paper I in this series, “Propio11:ttc Osidal ion in EschericlGu coli,” appeared it1 this jour11a1, Vol. 121, 440 (1967). z This work was supported by NSF (;raut. (;B 4680 and :iI-03806-07 from the Katic111al Institutes of Allergy a11tl Infectious I>iseases. 3 Ilesearch Career 1)evclopment Awardce of the IYational Institutes of IIenlth n’5-I<:%-11.6928). 55

with glyox>-late lo form ol-ti2.drox?.glrlt3~~te (15) ; t’he latter may be oxidized to succirtic serninldehydc arid hence to succin:~tc (l(i). iI seriesof investigations have been undcrtaken to elucidate t hc factors regulating grO\vth

of

I<.

di

011

~~rO~~iO~l:Lt~~.

l’!lC

first

paper iii this scrics described the (JxidatiOll of propionntc~ to acetate via l:tct:ltc (17). The present, paper considers factors UJntrdling initi:it,ion of growth during ad:tpt:ition to this substrate. Escherichiu coli, strain E-26, was employed in t,hese st udics and was maintained on trypticase soy agar (TS.-1). (:rowth studies were performed as previously described (18) in 500-1111 sidearm culture flasks containing 70 ml of medirun; t,hese cultures were aerat,ed by shaking at 37”. Unless otherwise indicated, growt,h cultures were inoc11latcd to an i11itial turbidity of O-l klett units with washed suspensions of try-pticase soy t1roth (TSR). grown cells. (irowth was mcas11red as trubidity at 660 rnp using a Klett-Summerson calorimeter. ;\ll growth substrates were of comn1ercial 311alyrical grade. Sodium propior1ate (bIatheso11, Colemn11 n11d Bell) ~1s t~mploycd nit haul purification.

56

WEGEN’ER,

REEVES,

AND

AJL

RESULTS

Growlh ~4 15. coli OQ j’uUl/ acids. Table I sho\vs t.he ahilit~ of E. co/i, JG%i, to initi:\tc gro\\-th on acetate, propionnte, butyratc, and valerate mineral salts media. It is seen that I+-Z(j

grows

od?-

with

:I

short

lag

Ivhen

transferred from TSH to :1 minernl salts medium containing acc~t:~te :I.S t,hc sole source of carbon. In rontr:M, \vh(w transferred from TSIa to :L propionate medium, l+:-%i exhibits :I lag. of approxim:ltel>~ 00 hours t&ore exponential grou%h lag reflects xi adaptation

is initi:ltcd. process siricc

~~hich have kwl pregro\vn on ~ro\v quickly \vhen trausferrrd frc!sh propionate media. I”urther, propion:LtC

does

I -1.TSB I/ TSB

SHORT-CH.\IX

Inoculum’L

E-20

directly grolvth

population lose the

TABLE Escherichiu

OF

this suhstrate to on

riot appear to involve the

sclcction of a mutant I)ionate-gro\vrl cells

GROWTII

This cells

Primary growth conditions”

since c:~pacit~

proto

I coli

ON

F.\TTY

.I

~!IDS

SERIES

nv

-

econdary growt h conditionsb

Lag (hours)c

8-12

Acetate

E-26 E-26 E-26

(Prop) (Prop)

Propionate TSB

Propionate Propionate Propionate

E-26 E-26 E-26

(But) (But)

TSB Butyrate TSB

Butyrate Butyrate Butyrate

200-300 G-10 S-12

E-26 E-26 E-26

(Val) (Val)

TSB Valerate TSB

Valerate Valerate Valerate

120-150 6-10 8-12

-

8@90 G-10 80-90

-

0 E-26 refers to wild-type cells maintained on trypticase soy agar; E-26 (Prop) refers to cells grown in propionate media and maintained on propionate agar; E-26 (But) refers to cells grown in butyrate media and maint,ained on butyrate agar; E-26 (Val) refers to cells grown in valerate media and maintained on valerate agar. * Cells were cultured as described in tubes of trypticase soy broth (TSB) or in flasks of mineral salt,s medium containing the appropriate fatty acids at a concentration of 0.20%. c Time required to effect an increase in turbidity to 10 klett units when transferred to the secondary growth medium.

FIG. 1. Effect of succinate addition on adaptation to propionate. Washed suspensions of trypticase soy broth-grown E-26 were inoculated into 0.20% propionate media containing varying concentrations of succinate. Duration of lag expresses time required to effect an increase in turbidity to 10 klett units. initiate rapid gro\vth on this substrate after subculture in complex media. In contrast to propionate, the 1011~ lag preceding growth of E-26 on but’yrate and valerate is not the result, of an adaptjive process, but rather involves the selection of a mutant population. It) should he not’ed that both butyrate and valerate grown cells retain the capacit’y t,o init’iatc growth on the respective fatty acids after subculture in complex media. k~ffect of C4 acids on adaptation to puyionate. Adaptive enzyme format)ion is dependent on protein and nucleic acid synthesis. Since such processesrequire an intjracellulur pool of mctabolit’es, the possibility was considered that, adaptation of E. coli to propionate might he limited by the rate of formation of (J4 acids from propionate. To t)est

this hypothesis, the effect of addition of exogenous C, acids to propionate media \vas detcrmined. ;2s Fig. 1 shows, t,he lag preceding gro\vth

initiation

was

considerably

reduced

by the addition of limiting concentjrations of succinate tant to succinate

t,o propionate media. It is imporn&e that) the concentrations of employed were sufficient to effect

ADAPTATION TABLE ABILITI-

Addition .-.-~-

OF

II

V.~RIOUS

SUBSTR.ITES

GROWTH

ON

to mediumU ___-

Sane Siuccinste

=2cetnte Glycolate Glutamate TLictnte a-IIydrosyglutarate Lactose Glucose

TO

TO

STIMUL.ITE

PROPIONATE Lag (hours)*

90 30 45 45 35 35 45 24 24

‘1 0.20y0 propionate-mineral salts; other substrates were added at concentrations of 0.50 pmole/ml. A washed trypticase soy broth-grown suspension of E-26 was employed as the inoculum. b Time required to effect an increase in turbidity to 10 klett units.

only. m:q$nul grodh; however, such cultures grew to high turbidit(y and exhibited a monophasic pattern of growth. The :lhility to stimulate adaptation to propionnte is not, specific for succinate but is n-wt 1,. :L vtlriet>of subskates (Table II). It should be emphasized that glucose and lactose were quite effective in init’iating grcnvth. These sugars often result in cstabolitc rc~pression of enz\.me formation, but unclog the experiment:rl conditions described hcrc provide metaholites required for ellZJYW iriduction. a-Y~droxvglutarat,e also stimuh~tetl growth imtlntion ; this is in accord with the finding that whydroxyglutar:ttch synthase constitutive mutants exhibit a decreased lag during ndnpt:~tion to propit )n:lt(L i22‘. ()11(’ ~;o~sihilii~ for the direct formation of acids from propionate is via the proc’4 pion\-1-(‘(A carboxylnse pathlvay. This enz!xx wcluence is :I major pathway of metabolism in animal tissues propion:itc and wver:d microorganisms. The csrhox,vlntion of propion~l-CoA4 to form methJ.lm:il0n~~l-c‘oA require.5 hiotin 3s 3 cofactor; the subsequent isomerixation of meth>-lm:don~~l-C’o;Z to succinyl-CoA requires Blscocnz~mc as :I cofactor. The foregoing has been review31 (I 1).

5’7

Pl:.Ol’IONATE

there is no apparent requirement for cxogcnous groivth factors. Ncvert(heless, the possibility was considered that, adaptat#ion to propionate might, he limited by a deficienq in the synthesis of one or more cofactors. Of those tested, vitamin BP2 stimulatjed adapt:lCon to propionnte (Table III). It, is of int,crcst that HC’O~ also st imulaked growth ; the latter is not due to :I pH effect since the medium is highly buffered and remains ncutr:d. Aloreover, B12 , HOOF, and succinatcx were :ltlditivc in effect. Thcsc data suggest, that :Ldap)t:~tioll to lxcqkm:~te may be regulated by t,he activit) of the I~ropioll~l-C’oA c:ubox,vlase p:bthwq-. I:act,ors stimulating the operatioii of this pathlvay may facilitntc~ adaptation to propionatc through ;L primary cffcct on the form:ttion of C’i acids presun~ably requiwd for enzyme indllction. l’reliminw-y cxpermerits suggcstS that the activity of prol~ionylC’oh c:arbos\-1:isc in enzyme extracts of prol)ioll:lte-grow\-ii I:‘. cwli is considelabl~lower than that reported (14) for animal tissues. This c~lzymc was assayed (19) b> measuring t hc I)ro~)io~l;\~l-C’o-~ dependent illcorpor:ltiolt of H’4(‘O:i- into :~cid-insr)llll)lr material. That isonic’r;w rract,ion ~3s ]I( )l. assaycY~.

13,~ scquenti:ll

transfer of E-26 from TSB plus HCOU media, :I populntion c;ul be select cd which grr)\\.s in unsupplemc~ltetl l)tqiorlnte media \\-ith :t signific:ultlq

to

propicwlte

TABLE EFFECT

OF

HCOa-

AND

IXITIATION Addition

III \T~~~~.\~~~

0s

BLZ

ON

(>rton-,rrr

PILOPIONATE

to medium”

Lag

(hours?

None

90

HCO-

65 70 65 55 30

BE Succinate IICOJ+ Bl2 HCW + l&

+

Succinatc

a 0.20% propiorlate-mineral salts; inoculated as described in Table II. KHCOr was added at a concent ralioll of 5 rmoles/ml ; crystalline vitamin Blz (California (lorporation for Biochemical Research) at a concent ration of 0.5 pg/ml, and suecinate at a concentration of 0.1 ~mole/ml. b Time required to effect an increase in klrbidit) to 10 klefl lillits.

58

WEGENER,

REEVES,

reduced lag (from approximately 90 hours to 24-30 hours). It is possible that such conditions result in the selection of a mutant which possesses an altered propionyl-CoA carboxylase enzyme with a higher affinity for COz. This would be consistent with the observation that wild-type E. coli E-26 possesses a low level of activity of propionvlCoA carboxylase. Induction oj propionate oxidation. Figure 2 shows the results of an experiment designed to determine whether growth per se, in the presence of propionate, results in adapt’at’ion to this substrate. In this experiment, E-26 was grown first on succinat’e and then incubated in a propionat’e medium containing a limit’ing concentration of succinate. At various intervals, samples of the culture were withdrawn, washed, and inoculated into flasks of unsupplement’ed propionate media (Curve A). Under these conditions, cells become adapted to propionate as evidenced bs the fact t,hat such cult#ures exhibited a re-

)r

FIG. 2. Adaptation to growth on propionate. In system A, E-26 was grown first on succinate and then incubated at a turbidity of 25 klett units in a 0.200/, propionate plus 0.01% succinate medium. At the times indicated, samples of the culture were withdrawn, washed, and inoculatjed at an initial turbidity of cl klett units into unsupplemented propionate media. System B was identical except that the inducing medium contained 0.20% succinate plus 0.01% propiollate. Duration of lag expresses time reqllired, in unsupplemented propionate media, to effect an increase in turbidity to 10 klett lmits.

ASD

AJL

I4CO7 evolved from

50 -

zw, m-

u II.

40

1-14C- Proplonate

!

0

i

TIME OF INDUCTION

(hours)

FIG. 3. Adaptation

to oxidize propionate. A succinate-grown suspension of E-26 was incubated at a turbidity of 25 klett units in a 0.20% propionate plus 0.01% succinate medium. At the times indicated, samples of the culture were withdrawn and incubated in mineral salts buffer containing propionate-l-14C. The incubation mixture was aerated at 37”, and respired 14C02 was trapped by bubbling effluent air into tubes containing hyamine hydroxide. To quantitate 14CO? evolved, aliquots of the trapping solution were analyzed with a liquid scintillation spectrometer. Time of incubation in the inducing medium is plotted versus the percentage of initial propionate-l-14C activity recovered as 14C02 after a 20-minute incubation period.

duced lag when t’ransferred to unsupplemented propionate media. Curve B in Fig. 2 sholvs the results obt,ained when succinategrown cells were similarly incubated in a succinate medium containing a limiting concentrat’ion of propionate. As above, samples were &hdrawn at. various intervals, washed, and inoculated into unsupplemcnted propionate media. Under the lat.ter conditions, cells likewise adapt’ed to propionate, although these conditions were lessefficient than those employed for Curve A. Figure 3 showst#hat these condit,ions result in the induction of enzymes catalyzing the oxidation of propionate t,o CO,. As above, succinate-grown cells were incubated in a

propionate medium containing limiting succinute. Samples \\-cre I\-it)hdratn-n at various harvested by centrifugation, int~erval~, lvnshed, and suspended in mineral sslt,s buffer to n t,urhidit!of 300 l&t units. One ml each of the respective cell suspensions IW,S then placed in 50.ml, roundbottom S-neck fl:~sks, and the volun~c WRS adjusted to -2.5 ml xvit.h rnincml salts buffer. The flasks Lverc equilibrated at X7”, 0.5 ml of water containing 3 PC (2 @moles) of propionate-1-14C 1~~1s added. and the incubation mixture wxs aerated at. a conskmt flow rat’e. Respired “C0, ~vas trapped and quant,itnted as described previousI?(17). Figure 3 sho1v-s the Ltbility of cells harvested after varying periods of incubation in the inducing medium to oxidize propionate to CO,. The capacity to oxidize propionate is expressed as a percentage of the initial propionate-l-14C activity recovered as 14COS) _ aft,er a ZO-minut,e incuhntion period. A relatively short time of incuhnt.ion in propionate plus succinate media ~-as sufficient to induce enzymes &ich catalyze propionate oxidation. In

view of these results, it was of int’erest

to determine the respective pattern of utiliz:Lt ion of propionate and succinate during grolyth of E. coli in mineral salts media containing both of these sub&rates. To invcstigate this aspect, tn-o parallel cultures, c:icli containing 0.20 7% propionate plus

0.01 % succinate, were inoculated lvith a \vashcd suspension of succinate growl cells at :111 initial turbidity of O-l klett units. To OIIC culture was added propionate-UJ4C; the ot,her received succinate-c-14C ; the final specific nctivit;v of each n-as 0.01 &Y/mole. At various intervals during growth, samples front each of the cultures were wit.hdratvn,

harvested, and washed. The respective cell pellets n-ere lvsed 113th hot methanol and cooled, :lrd protein n-as precipitated

and hi

addition of cold t,richloroacetic acid. l’rotein ~3s collected by centrifugation, nashed t\vicc \I-ith HCl and soluhilized in HCO:i, i111(1 tllc 14C‘ :Ictivity ~1s then determined. The concentration of protein in the respective solutions W:W determined hv the method of T,owy rt nl. (ZO), and spec’ific :ictivitics IVW(’ cspresscd as cprn *“c incorpornte#mg protein. I’igure 1 sho\\.s the pattern of propionatc~-

FIG. 4. Comparison of incorporation of succinate-14C and propionate-‘“C inlo cell material. A washed suspension of succinate-groan E-26 was inoculat.ed into parallel cultures cont.aining either propiorlate-W4C plus succirmtc, or succinateTJJ4C plus propionate. The concentrations of propionate and succinate were 0.20 and O.Ol%, respcatively. The specific aclivit,y of each substrate was 0.01 pC/pmole. At the times indicated, samples were withdraw11 from the cultures and incorporation of 14C into acid-insoluble material was determined as described in the text. The incorporation of succirlate-14C aud propionate-1°C at, various times is expressed as a percentage of the respective maximum incorporation obtained during growth. 0, growth; 0, succitlate-14C incorporation; n propionate-14C illcorporation.

W versus succin:ite-14C incorporat,ion into protein. Incorporation of propionate-‘4C and succinate-14Cduring various phasesof growth was determined from the specific activity of the isolated protein, and is expressed as a percentage of the respective maximum 14C incorporation. It is seen thatt incorporation of succinaW4C was highest in the earl! growth phase and declined wit’h further growth. In contrast, propionate-14C incorporation did not reach a maximum until the supply of succinate in t.hc cult.ure had become limiting. However, at the earliest stage of gro\vth measured significant incorporation

(1’ klett units), of propionute-t4(’

occurred. Under these conditions, grolvth initiation involves concomitant utilizat,ion of succinate and propionatc. The supply of (‘Xo#2Ilolls sacci11:1tc ilt the medium so011

and further gro\vt,h is becomes limit’ing, effected bJ- using propionate as the sole source of carbon.

Bflect of substrate concentt~ationon adaptation to propionate. The observ&on \vas made that the lag during adaptation to propionate It-as markedly influenced bq’ the concentrat,ion of t,his subst,rate. In the cxperimerit sho\vn in Fig. 5, propionate concent,ration was varied from 0.05 to 0.40 ‘%. Under these condit,ions, the lag increased from 30 to 215 hours, respectively (Curve A). This effect is a characteri& only of the adaptacells exhibit t,ion process since adapted lags at all propionate markedly shorter concentrations. Ho\\-ever, cells lvhich had been adapt,ed to 101~ concentrat’ions of propionate (0.05 ‘3%; Curve B), gre\v more slo~vl~ on high concentrations of propionate than

1

did cells 15.hich had been adapted to higher concentrations of this substrate (0.02 or 0.30 %; C’urve V). TVhile t,he concentration of propionate in tho medium dramat’ically influenced the duration of lag during adaptat’ion to propionate, the rate of exponential gro\vth under these conditions \vas not significantl?- altered by substrate concentration. J,ikeuGe, the maximal turbidity obtained in

such cultures n-as proportjional t,o the concentration of propionntc. This phenomenon might

result

from

a

contaminant in t,he commercial preparutions of propionate which inhibits initiation of gro\vth. The observed lag may therefore reflect the time required for cells to convert t,his inhibitor to an inact,ive form. Adapt,ed cells, then, ma!- possesst)hc capacity t,o quicklv metabolize such an inhibitor. To test this possibility, a mineral salts medium containing 0.40 ‘3%propionate \vas inoculated with adapted cells, and the cultures \vcre gro\vn to a turbidity of 15 ltlett units. The culture filtrate bias collected asept~icall~-in a l\Iillipore filter and redispenscd into sterile culture

flasks.

This

medium

was

reinocu-

lated n-it’h unadapted cells and the lag before growth initiation was determined. Under these conditions, a long lag again was apparent. These results appear to rule out the possibilit~y that an inhibit,or present in commercial

preparations

of propionate

is re-

sponsiblefor the effect described above. DISCUSSION

.O

010 PROPIONATE

020

030

CONCENTRATION

040 ( % )

FIG. 5. Effect of propionate concentration on duration of lag in propionate media. Washed suspensions of appropriately grown cultures of E-26 were inoculated at an initial turbidity of O-1 klett units into mineralsaltsmediumconl,ainingvarying concentrations of propionste. Propionatc concentration is plotted versus duration of lag (time required to effect an increase in tllrbidity to 10 klett units). Curve A, cells grown first in trypticase soy broth; Curve B, cells grown first in 0.05% propionate media; Cllrve C, cells grown first in either 0.20 or 0.40% propionate media.

A4ninvestigation was undertaken to assess the factors regulating growth of E. coli strain E-26 on propionate. The question considered first was-why is there a long lag before growth is initiated on this substrate? In this regard it should be noted that growth on propionate, unlike but,yrate or valcrate, is the result of an adaptive process rather than a mutation-selection phenomenon. This is evidenced by the fact, t’hat, cells which have been pregrown on propionate initiate rapid growth when transferred directly t,o fresh propionate media, but lose this capacit)- n-hen subcultured in complex media. In contrast, gro\vth on but\-rate or valerate (1s) results in selection of a mutant popuhttion, since the cnp:\cit!- of such cells IO grow on thcsc fatty acids is not repressed.

JwoJ)iowte

1nct:~holisni

is

considerc~d

in

the

follo\~-ing arlicle (2). REFERENCES

X11 olism

import;mt relates

f:rct,or to

the

in

J)ropion:1tc

mcch:u1ism

111&J,1)~

\vhich

f5.

cd’ forms Cd acids recluired for the synthesis of :d:q)tivt: enzymes. One possibility for the tlircct forn1:rlio11 of C’4 acids from propion:ite is

vi:1

the

J~roJ~ior1~~l-(lo=\

c:trhox~~lase

J):Lth-

therefore of interest that, both vit:\min B1, and HCK& partially replace tlw C’, :tcid rcxpircmeiit~ during daptation to propionate. In addition, HI.‘, HUX, and succiiiate :rrecuniulativein stimlll:ltillgncl~.ptation to this substrate. It is suggested that gr.(nvth initiation during :&pt:Ltion to proJknatcb is limited J)y the :ictivity of the JJr,,J,iorl~l-(‘OX\ cnrlwqd:w pathw:L)-. The f~J1CYXtiOll of this path\\-ay ma!- lx restricted by :I lvw affinity of the curhuxylase enzyme for CO2 or by :I deficialcy in the enzyme catalyzing the isomcrization of methylmnlonyl-Co8 to succiny1LC’oA. Alternatively, since E. fdi classicwlly dots riot synt,hesize By., this reaction might, hc limiting during gro\vth on propion:~te. The presence of the isomcrasc :111d the co-factor requircment~ arc currently under invest~igxtioii. In :&ptd cells. Jwopio1l:ttc is oxidized via the lxctatc pathrvay (17). The operation of this pathn-:r> provides :m ndditiord mechanism for (I, ncid form:ition via carboxylation of pyruvate of J’hosphoeriolp~ruvatt~. The significance of cnrhox~l:~tion rcnctioiis to growth init,i:Ltion on propiom~te is considered in :mothcr paper of’ this series (23). AI alternate mcchnnism for formation of Cd :wids diwctl~- from propionate is vi:1 the a-h!.tlrox~alut:~rnte path IV:I~ (21). The aig~ific:mcc~ of this pathw:~~~ to \\x!-

(l-1).

It

is

1. S~hI)i'hi.\N, 5:. I:., Bull. Sot. (‘him. Biol. 37, 931 (1955). 2. STADTMAN, B. I:., ANI) V.LGELO~, 1’. R., Proc. In&ml. &mp. ihzy?t/c Chem., I[‘okyo, Kyoto, 1957, p. 86 (1958). I’. R., &a~, J. bl., AND STADTMAN, 3. VAGELOS, E. R., J. Hiol. (‘hem. 234, 7F5 (1959). 4. ?VI.L*ILER, ANV ~IUENXEKENS, F. &I., I-I. IL, Biochim. Hiophys. ilcta 11, 575 (1953). 5. LEAVER, F. W., Woon, II. ct., .na STJERNHOLY, IX., J. Bactcriol. 70, 521 (1955). 6. GIOV.\SELLI, J., .\ND STUMPB, P. IL, ,r. Biol. Chem. 231, 411 (1958). 7. RENDIN.\, G., ASL, COON, 31. J., .r. Biol. (Them. 226, 523 (1957). 8. V.\GEI,OS, 1’. R., J. &ok Chem. 235, 34ti (1960). 9. CARDON, B. P., AND B.LRKER, Ii. .I., .lrch. Biochem. 12, 165 (1947). 10. BALDKIN, R. L., WOOD, W. .4., MU I
23. KOLOI)ZIEJ, S. J., (19(Z).

13. *J., WEC:ENER,

.,l rch.

Biochem.

W.

P.,

Biophjla.

.1x1)

.-\JL,

123, fifi