Cystine and Cysteine Metabolism by Proteus vulgaris and Proteus Mmorganii

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cr& n ss AND CYSTEINE METABOLISM BY PE oiius m m m

m am

w m m

Reino H ail K a llio

A d is s e r ta tio n subm itted in p a r tia l fu lfillm e n t o f th e requirem ents fo r th e degree o f Doctor o f Philosophy in the Department o f B acteriology in th e Graduate C ollege o f th e S tate U n iversity o f lorn dune 1950

ProQuest Number: 10991962

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uest ProQuest 10991962 Published by ProQuest LLC(2018). C opyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C o d e M icroform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 4 8 1 0 6 - 1346

7 ~ I S ■>o Kh C *op &

u

Acknowledgement Dr* J . It* Portia* has given fr e e ly o f h is heavilyburdened tim e in encouraging th e p resent study* I t i s a p o in t o f pride and a p r iv ile g e to be con­ sid ered one o f h is s c ie n t if ic offsp rin g *

ill

table

or

contests

INTRODUCTIOW*, ** *.------------------------------------------------------ 0. . . . . . * ------. . . 1 EOT8BSBHTAL METHODS****........................

* * ................... . . . . . . . . . . ...........12

RESULTS AMD DISCUSSION.................. s r o m T .* . APPENDIX BIBLIOGRAPHY.,

. . ................................. ».............20

........... ............................... .............. . . . . . . . . . . . . ».. 43 ............

44

........... . . . . . . . . . . . . . . . . . . . . ............. 52

iv

TABLE OF FIGURES F igure 1 * th e e f f e c t of gH on. c y ste in e deaulfhydrase a c t i v i t y . . . . . .

22

Figure 2 . C ystine and c y ste in e u t ilis a t io n by Proteus v u lg a r is c e l l s grown in c y s t e i n e *

25

.

Figure 3 . C ystine and c y ste in e u t ilis a t io n by c e lls grown in c y s tin e ................ .........* . .

26

Figure 1 * D issim ila tio n o f cy stein e under aerobic and anaerobic c o n d itio n s.......................* ^ .......,.,.. . . . . . . . . . . . . . . . . . .

51

V

TABLE OF TABLES Table I . C ysteine desulfhydrase a c t iv it y o f P roteus v u lg a r is c e ll s groisn under va rio u s co nd it io n s * • • « • • • « . • • • • • • . . * • * « « * . • • * • • • • » • 23 T able II* Anaerobic u t iliz a t io n o f c y ste in e by Proteus v u lg a r is adapted to c y s t e i n e * * * * * * * * * * * . . 28 Table III* E ffe c ts o f variou s in h ib ito r s on hydrogen s u lfid e pro­ du ction from c y ste in e by Proteus vul gar i s . . . . . . . . 31 Table IV.

Sodium a aide in h ib itio n o f anaerobic pyruvate u t iliz a t io n v u l g a r i s . 33

Table V* Anaerobic c y ste in e d issim ila tio n by a a id e -in h ib ite d r e s tin g c e l l s of Proteus v u lg a r is .* * . . . * . . .............................................. 35 Table VI* Anaerobic d is s im ila tio n o f c y ste in e by to lu en e tr e a ted c e l l s o f P roteus morganil. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 Table VII* E ffe c ts of various in h ib ito r s on th e c y stein e d e su lf­ hydrase a c t iv it y o f tolu en e k ille d c e l l s o f Proteus g iq rg a n ii.. . 39 Table VIII* B ea ctiv a tlo n o f "aged", tolu en e k ille d Proteus m organil c e lls toward c y s t e i n e , . , , . , 4^ Table IX. B iochem ical c h a r a c te r istic s o f Proteus v u lg a r is and Proteus m organii used in th e present s t u d y * * . . . . . , . . , . . . , * . . . . . 47 Table X, Production o f hydrogen s u lfid e from variou s su lfu r compounds by Proteus v u lg a r is and P roteus marganl i *.

48

fa b le X I, Hydrogen s u lfid e production as a fu n ction o f the number Proteus v u lg a r is c e l l s p r e s e n t ... Table XII* Gcopounds te s te d s in g ly and in com bination fo r a c tiv a ­ tio n o f c e l l fr e e e x tr a c ts to®/ard c y s t e i n e , . . . * . . . . , . , * . , . . . . . . 5 0

1

The ob servation th a t a v a r ie ty o f h eterotrop h ic organ ism produce hydrogen s u lfid e and oth er v o la t ile s u lfu r compounds from p ro tein sub­ s tr a te s i s o ld and w e ll e sta b lish e d .

For example, in 1089 Nenckl and

S ieb er noted th e production o f m ethyl mercaptan from p ro tein p u tre­ fa c tio n brought about by a v a r ie ty o f anaerobic organism s.

R ettger

(1 9 0 6 ) and E erier (1 9 0 6 ) co n fim ed th is observation a t le a s t in th e ca se o f the o b lig a te anaerobes C lostridium n o vv i. C lostridium f e s e r l and C lostridium len to o u treso en s.

In la t e r stu d ie s on p ro tein putre­

fa c tio n R ettger (1 9 1 2 ) pointed out th a t p u trefa ctio n was e s s e n tia lly an anaerobic process and was ch a ra cterised by "the ev o lu tio n o f fo u l­ sm ellin g products which are c h a r a c te r istic o f ordinary cadaveric de­ com position*

I t should be noted th a t mercaptan i s o f p a r tic u la r

sig n ific a n c e and th a t indole* sk a to le and hydrogen s u lfid e are o f le s s importance”.

R ett gear demonstrated fu rth er th a t hydrogen s u lfid e was a

'teomcion p ro tein decom position product” and was n o t in d ic a tiv e o f tru e p u trefa ctio n .

3h th e same stud y th e p u trefa ctiv e a b il it ie s o f a number

o f fa c u lta tiv e anaerobes, e s p e c ia lly th e Proteus group, were in v e s ti­ ga ted ,

Under com pletely anaerobic co n d ition s none o f th e members o f

th e genus Proteus were capable o f degrading th e pure p ro tein s used (egg and serum albumin; and © d estin ).

ilhen, however, oxygen was ad­

m itted in to th e growth medium rapid decom position o f th e p ro tein s oc­ curred w ith th e lib e r a tio n o f some hydrogen s u lfid e but no m ethyl morcaptan*

2 Sperry and H ettger (1915) added to th ese o b serv ation s by n o tin g th a t fa c u lta tiv e anaerobes fa ile d to m u ltip ly in th e presence o f pure p rotein s when ammonium s a lt s and oxygen were a b sen t.

I f , however, pep­

tone m s added to th e c u ltu re medium rapid growth and degradation o f p rotein took p la c e , a s evidenced by liq u e fa c tio n o f th e protein* m ethyl mercapt&n was produced even under th ese co n d itio n s*

No

Thus i t was

noted q u ite ea rly th a t th ere seemed to be a fundamental d iffe re n c e in p ro tein breakdown between the o b lig a te anaerobes and th e fa c u lta tiv e groups*

I f th e d iscovery and ch a ra cteriza tio n o f m ethionine had taken

p la ce p r io r to th ese in v e stig a tio n s perhaps a t le a s t p art o f th e ex­ plan ation fb r t h is d ifferen ce m ight have been p a ten t, but m ethionine m s not d iscovered u n til 1 9 2 2 . S in ce c y stin e had been d iscovered in 1 89 9 by Corner and c y ste in e lay Embdon in 1 9 0 1 , i t seemed c le a r to the e a r lie r workers th a t the hydrogen su lfid e and m ethyl mercaptan observed must a r ise from th ese two stalfu r-con tain in g p ro tein components.

Wohlgemuth (1904.), fo r

example, claim ed th e production o f methyl mercaptan and e th y l s u lfid e from c y stin e decomposed by b a c te r ia l a c tio n .

These claim s were v igo r­

ou sly denied by Burger (1914) who found hydrogen s u lfid e but no oth er s u lfid e or mercaptan produced from c y stin e by 2 3 sp e c ie s o f bacteria* Caapek (1920) suggested th e course o f th e rea ctio n was a prelim inary red uction of c y stin e to c y ste in e follow ed by a t^rdrolytic deam inations HSOHgCH (Nil?)GOGH + H20

V

HSCH^CHGHCOOH

+ NH 3

The ^ -th io la c tlc a cid formed was then dacarboxylated and o x id ised to

3 t h lo g ly c o llic acid* H3GH2 GiiGBGG0H + 0 2 ------>

HSCH^COOH ♦ C02 * h 2o

The t h io g ly c o llic a cid m s subsequently decarboxylated to m ethyl mercaptan. Betake (1924) agreed w ith th e above r ea c tio n sequence but was Jinab le apparently to o ff e r mutch experim ental proof fo r h is view*

The d is~

s lm ila tlv e pathway o f c y stin e proposed fey Wohlgemuth and Kotake was stro n g ly questioned fey o th er in v e stig a to r s who were unable to fin d m ethyl mereaptan a s an end product o f c y stin e breakdown*

As a m atter o f f a c t ,

as e a r ly a s 1905 fa o i had disagreed w ith th e proposed scheme inasmuch as h© found th a t hydrogen s u lfid e (but never m ethyl mercapt&n) m s fo m ed from th io la c tic o r th io g ly c o llie a cid by b a c te r ia l a c tio n .

Methyl raer-

captan was produced from c y stin e on ly when a ferm entable carbohydrate was pp esentf f o r © stab le, in th e case o f E sch erich ia c o il m ethyl mercaptan m s formed in th e presence o f g lu c o se, la c to s e , sucrose or d u lclto l* Since in no ca se d id methyl mercaptan appear to be a major m etab olic endproduct i t m s concluded th a t i t s appearance in th e presence o f a f e r ­ mentable sugar was th e r e s u lt o f secondary rea ctio n s* By 1915 the a b ilit y of raany microorganism s to produce hydrogen s u l­ fid e from p ro tein , peptones or c y stin e had been w e ll e sta b lish e d and th e rea ctio n was being u t ilis e d w idely as a p h y sio lo g ic a l a id in id e n tify in g b acteria*

Jordan and V ictorson (1917) used n u trien t agar to which had

been added lea d a c eta te as a means o f p a r tia lly d iffe r e n tia tin g members o f th e sp e cie s o f Salm onella* Tam er (1918) used hydrogen s u lfid e pro­ duction to help d istin g u ish c e r ta in y e a s ts , and more r ec e n tly the re­

4 a c tio n has been a l l i e d to d iffe r e n tia tin g between spec le a in th e genus B ru cella (Huddleson and A b e ll, 1927)*

A grea t many stu d ie s have been

sad© w ith resp e c t t o making hydrogen su lfid e production by b a cteria more e a s ily d iscern ab le—among th ese may be mentioned th ose o f Z obell and Feliham { 1 9 3 4 ) Stekol and Renameie r (1942) , and Hansmoier and S tek o l (1942)*

There i s l i t t l e p oin t in d isc u ssin g or ©3±ending th is

l i s t fo r though th ese in v e stig a tio n s contribu ted valuable adjuncts t o the armamentarium o f th e d ia g n o stic b a c te r io lo g is t th ey are p u rely q u a lita tiv e in nature and con trib u te l i t t l e or nothing to an under­ standing o f th e mechanism o f th e rea ctio n whereby hydrogen s u lfid e i s lib e r a te d from c y stin e or cy stein e* Almy and Jamas (1926) developed r e la tiv e ly s p e c ific and s e n s itiv e methods fo r th e estim atio n o f hydrogen s u lfid e and mercaptans and ca r­ r ie d out q u a n tita tiv e stu d ie s on th e lib e r a tio n o f th ese compounds from various peptone media by E scherich ia c o li, Proteus v u lg a ris and Salmon­ e lla aertrveke*

Apart from the fa c t th at various peptones d iffe r e d in

the amounts o f hydrogen s u lfid e lib e r a te d i t was e sta b lish e d th a t no mercaptan appeared and th a t th e hydrogen su lfid e produced was d ir e c tly proportional to th e c y stin e content o f the peptone being used*

When

c y stin e was added t o a peptone medium the su lfu r could q u a n tita tiv e ly be recovered as hydrogen su lfid e *

I t was a ls o noted th at a la rg e pro­

portion o f the hydrogen s u lfid e lib e r a te d from a given medium was pro­ duced in a com paratively sh o rt tim e in th e e a r ly incub ation period * -6 to 12 hours fo llo w in g Inoculation*

Ho attem pt was made to a sc e r ta in

th e o th er m etabolic products produced fro a cy stin e*

However, i t i s

5 in te r e s tin g th a t Als^r and James sta ted th a t in th e e a se o f Protons v u l­ g a r is th e production o f hydrogen s u lfid e I s enhanced by aerob ic condi­ tio n s*

T his study apparently s e ttle d the q u estion o f m ethyl mercaptan

form ation from c y stin e by t h is group o f organisms* A more d e ta ile d study o f c y stin e m etabolism m s carried out by fa r r (1933)*

lashed suspensions o f Proteus v u lg a r is were suspended in

b u ffe r so lu tio n s con tain in g c y stin e , and hydrogen was bubbled through the suspension and then through appropriate bubblers to absorb v o la t ile end products*

At th e end o f 21 hours an alyses were ca rried out and th e

r e s u lts le d fa r r to advance th e fo llo w in g scheme fo r th e o v e r -a ll re­ a c tio n s o f anaerobic c y stin e breakdowns (sCH2CH(HH2 )aOOH)2

♦ 28

2BSCK2Cm(M2)C©GH +

mg>

VaHSGI^Gff (fSf^COQI! + 2IH3 * SCH^CQQH * 2BC0GH

A ll end products q u a n tita tiv e ly f it t e d t h is scheme excep t formate* However, i f th e carbon d ioxid e lib era ted m s converted to i t s equi­ v a len t a s formate and added to th a t recovered as form ate th e equation m s r e a liz e d .

This conversion seems sound sin ce I t i s w e ll known th a t

th ere i s an enzyme system which m ediates th e o v e r -a ll reactio n s m om

----->•

GOg + h2

The system may a c tu a lly c o n sist o f a number o f enzymes*

(Bakes and

Jollym aa, 1 9 0 1 ; Quaatel and Ihetham* 192 5 j Stephenson and S tick la n d , 19315 Stephenson and S tick lan d , 1932? Waring and '.verkman, 1944; Ordal and HaLvceraen, 1939)*

These enzymes are found in th e EnterobaG teriaceae

and in many ©idler groups.

They appear to be p a r tia lly adaptive and so

6

far a® i s known account fo r tfe® t r m hydr®g©« relea sed during b a c te r ia l carbohydrate ftattM&a&tan* Sserr*® a t t e s t s to is o la te la c t ic a cid or a cid from th e rea c tio n m ixtures a fte r c y stin e had been metab­ o liz e d were uniform ly u n su ccessfu l* An im portant con trib u tion to th e knowledge o f c y stin e am! c y ste in e f®babelism was made by a s e r ie s o f in v e stig a tio n s by F rom geot and e©~ workups covering both b a c te r ia l and mammalian asp ects*

I t i s not in ­

tended to review th e lite r a tu r e o f mammalian su lfu r m etabolism but I t would s e e s o f in te r e s t to touch upon th ose mammalian rea c tio n s which appear to p a r a lle l th e enzymic rea ctio n s in bacteria*

fh® presence o f

an enzyme which lib e r a te s hydrogen s u lfid e from c y stin e and c y stein e in mammalian tis s u e s was f i r s t observed by Fromageot, Wooky and Chaix {1919)*

la subsequent stu d ies th ese authors found dog liv e r to be a

r ic h source o f th e enzyme and th ey were able to prepare sta b le acetone powders from ground liv e r which retain ed th e ir a c t iv it y toward c y ste in e fo r some tim e*

(Fromageoi* m dkp and Chaix, 1910? Froaageot, Chaix

and f hibatit, 194.6).

O nparified acetone p r e c ip ita te s o f dog liv e r incu­

bated w ith c y ste in e le d to r e s u lts which were best expressed by th e equation s 3H30H2CH{®?2}C0QH — V H2S ♦ CH^CBClH^GOGS *

(£CH2GH(M!2)€0GH)2

P a r tia l p u r ific a tio n o f the crude preparation by chloroform treatm ent y ield ed no alanira© but rather la c t ic a c id and ammonia in a d d itio n to c y stin e and hydrogen su lfid e s 3H3CB2 { H ( M 2 )G0CH — V H2 S + m ^ + CH3 GH

H2S +

+ CH3 COGOOH

Pyruvate was iso la te d # ch a ra cterized and q u a n tita tiv e ly estim ated*

the

©nzym© m s p a r tia lly p u r ifie d j i t s optimum j>H was a t 7*2 , i t was power­ f u lly in h ib ite d by HCH, carbonyl reagents and arsen iou s o x id e .

Contrary

to Froraageot, however# Suythe found no in h ib itio n by amino acids*

At­

tem pts t o rev erse th e rea c tio n s ta r tin g w ith pyruvate, ammonia and hy­ drogen s u lfid e were u n su ccessfu l although a sm all amount o f radio­ a c t iv it y could be lo c a te d in c y stein e when the liv e r preparation and c y ste in e were incubated to g eth er w ith S^5 as sodium s u lfid e (Smythe and H o llid ay , 1942),

In a review o f enzyme rea ctio n s o f su lfu r com­

pounds (Smyths, 1944) th e name cysteirm se m s considered m isleading and th e more d e sc r ip tiv e c y stein e d esu lf hydrase m s suggested*

Since

th is la t t e r name has been w idely accepted i t w ill be used in th e re­ mainder o f th is t h e s is . Based on h is fin d in g s fo r th e o v e r -a ll rea ctio n cteythe advanced th e fo llo w in g mechanism fo r th e a c tio n o f cy stein e desulfhydrase on c y s te in e : ch2sh

— h2s

GM 2

r

GGCH

^ —X

,

4 - H23

ch 2

gh^

OmBn — y

G — HH

I

COGH

huh

------->

I

GGOH

m 3

G = y ♦ KHq

1

CQOH

fh© mechanism proposed alm ost n a tu r a lly suggests a oojs^arisen w ith th e format ion o f pyruvic a cid from 2 -p h osp h o-glyccric acids

9 OHgCK

— H20

CHOI^Hg

CHx

^

'm m

+

BOH

CHGPO3 H2

nao

GH3

>

C= 0 ♦ HOPO^Hg

oooh

com

One r a th e r ia^orfcant d if f e r e n t in t h is analogy i s th a t in ih s easo o f phosphoglyceric a c id th e proposed interm ediate i s sta b le enough to be recoverab le whereas la th e cy stein e ca se th e interm ediate suggested* amino a c r y lic a cid i s not s ta b le . According to Charg a ff and Sprinson (19A3) serin e i s attack ed by r e s tin g c e ll s o f B. c o ll to y ie ld pyruvic a cid presumably v ia amino a c r y lic a c id , th u s! GggOB

~

CH2

CH3

CW®2

m m 2

HCGQH ♦ C t^JtO O P C ^ £ t seems reasonable to suppose th a t d esp ite Tarr*s fa ilu r e to is o la te pyruvate from th e r ea c tio n m ixture t h is m a teria l may w e ll be th e a c tu a l precursor o f the a c e ta te and form ate found. The p resen t stud y was undertaken to attem pt a q u a n tita tiv e c l a r i f i ­ ca tio n o f th e products f orjaad by b a c te r ia l d esu lfh yd ration o f c y s te in e , th e c h a r a c te r istic s o f th e enzyme system , and i f p o ssib le to prepare a c e ll- f r e e a c tiv e preparation fo r mere d e ta ile d study* rep ort o f fin d in g s in th ese a rea s.

This th e s is i s a

12 M ODS

Approxim ately fo r ty str a in s o f Proteus vu lgarla were screened fo r

th e ir a b ility to produce hydrogen s u lfid e by in o c u la tio n on Kligler*3 iron agar sla n ts*

The tu b es were in sp ected a t frequent in te r r a le fo r

blackening and th e most rapid producer o f hydrogen su lfid e was chosen for fu r th e r study*

Somewhat la t e r in th e course o f th e in v e stig a tio n

i t became evid en t th a t th ere was no connection between hydrogen s u lfid e

production on Kligler* s medium and th e a b ilit y , under proper conditions* to decompose c y ste in e so th e screen ing was discontinued*

The cu ltu re

©f Proteus vulgaris chosen was th e stock str a in o f th is la b o ra to ry ; th e f m t e m m & w il used was a s tr a in d esignated a s M32 and was o r ig in a lly receiv ed from Dr« &* F# Baass (see Hauss, 1936) as h is Ho* ?Q strain * The B sch erich ia c o ll used in a few in sta n ces was a lso th e stock str a in

of t h is laboratory*

The biochem ical c h a r a c te r istic s o f th ese organisms

are lis t e d in Table X o f th e Appendix* B acteria were grown in a medium c o n s istin g o f 6 parts n u trien t broth and 3 p arts fr e sh meat in fu sio n troth* to a jsH o f 7 *6 ,

The medium was adjusted

Immediately p rio r to in o c u la tio n a c y ste in e hydro-

ch lo rid e s o lu tio n was n e u tra lise d to phenol red and s t e r ilis e d by f i l ­ tr a tio n through Corning f r it t e d g la s s f il t e r s (Grade U F ),

The s t e r ile

cy stein e was added to th e cu ltu re medium to a f in a l con cen tration o f 0*G5/S* and in o c u la tio n s were made by washing o ff th e en tir e growth o f a 1 2 hour agar sla n t*

Incubation was ca rried out a t 3 7 ° C in 1 2 l i t e r

scrum b o ttle s s nipped w ith bubblers*

A naerobiosis was m aintained by

13 a cu rren t o f s t e r i l e n itrogen w hich, a fte r p a ssin g through the b roth , was bubbled In to a strong cadmium a ceta te so lu tio n to absorb hydrogen su lfid e #

g a lls were u su a lly harvested a t 1 0 -1 4 hours, having by t h is

tim e a tta in e d maxima l a ct iv it y and growth#

Growth was never as lu x u ri­

ant a s s ig h t be expected on a medium ©f th e type used, probably because o f th e heavy production o f hydrogen s u lfid e r e s u ltin g in enzyme poison­ in g a s w e ll a s p r e c ip ita tio n o f e s s e n tia l tra ce m eta ls.

H arvesting m s

c a rr ie d out w ith a Sharpies Super cen trifu ge* th e c e ll s were th en washed tw ice by suspending in d is t il le d w ater and were recovered each tim e by ordinary c e n tr ifu g a tio n .

$hen la r g er q u a n titie s o f c e lls wore used th e

washing was accom plished by suspending th e c e llu la r mass in 3 l i t e r s o f d is t ille d w ater in a 4 l i t e r serum b o t tle , shaking v ig orou sly and r e h a rv estin g by passage through th e Sharpies c e n tr ifu g e . During th e course o f the study more c e lls were required than would grow a n a ero b ica lly under the co n d itio n s enumerated*

While a e r o b ic a lly

grown c e lls d isp layed seme deoulfhydrase a c tiv ity t h is could be in ­ creased by p rio r adaptation to c y ste in e , out as fo llo w s t

The adaptation was carried

a fte r h arvestin g a e ro b ica lly grown c e lls the b a cteria

were suspended in 5 0 0 m l, o f 0 * 1 M phosphate b u ffer (|p 7 , 4 ) to which had been added c y ste in e t o a f in a l con cen tration of 0.05^*

The sus­

pension was placed in a one l i t e r Erlenmeyer fla s k submerged in a con­ sta n t temperature bath a t 3 7 * C and a vigorous current o f nitrogen was passed through the suspension and in to a cadmium a c eta te absorber. P e r io d ic a lly , th e araount o f hydrogen s u lfid e produced per u n it tim e was measured.

By t h is r e la t iv e ly sim ple method adaptation appeared com plete

u la about an boor*

The c e l l s were then harvested and washed as d escrib ed

above* C onventional Ifeirburg apparatus was used to incubate th e c e l l s in th e presence o f th e v ariou s su b str a te s.

Bnzym preparations (5-3 mg* o f r e s t­

in g c e l l s )» b u ffer and c o fa c to rs or in h ib ito r s were in th e main compart­ ment o f th e v e sse l? su b stra te was tip p ed in from th e sid e bulb fo llo w in g a 1 5 m inute ga ssin g w ith nitrogen and a 5 minute equ ilib r a t io n period* Substrate was p resen t in a f in a l concen tration of 0 * 0 2 M u n less oth er­ w ise noted*

The c e n te r -w e ll contained 0 ,3 m l, o f 2 M cadmium a c eta te t o

abscrb hydrogen s u lfid e a© described by Smyths (1941)*

A fter the rea ctio n

had progressed th e r e q u is ite tim e f if t e e n p er cen t tr ic h lo r o a c e tic a cid from th e sec©nd s id e bulb was tip ped in to step th e r ea c tio n .

Shaking

was continued fo r an a d d itio n a l 1 0 m inutes to in su re com plete absorption o f th e hydrogen su lfid e *

F ollow ing high speed centrifug& t ion o f fla s k

co n ten ts a liq u o ts o f th e c le a r supernatant were used fo r analyses* C ysteine was determ ined e s s e n tia lly by th e method o f Shinohara and P adis (1936; 1936 a)*

The general procedure c o n sisted o f adding t o an

appropriate san^ple 1*0 ml* o f fr e sh ly prepared 1*0 M sodium b is u lf it e , 2 .0 ml* o f 2*0 M sodium a c e ta te , 0*6 ml* o f 2 ,0 M a c e tic acid and f in a lly 1 .0 m l. o f molybdenum-free phosphotungstie aoid prepared according to th e

d ir e c tio n s o f f o lin - (1934) * The volume was made up to IQ m l., th e con­ te n ts o f each tube was w e ll mixed and a fte r standing 2 0 m inutes a t room tem perature the blu e c o lo r was read in a Coleman Model 14 Spectrophoto­ m e te r

a t 680 m/a.

* R esu lts were ca lcu la ted from a standard curve pre­

pared from c y ste in e p u r ifie d according to Shinohara and Padis * The non-

15 c y ste in e reducing m a teria l » a determ ined by carrying ou t a d u p lica te of th e a n a ly sis as described accep t th at c y ste in e c o lo r form ation m s s p e c if­ ic a lly in h ib ite d by th e p rio r a d d ition o f 1 . 0 ml. o f 0 .1 H m ercuric ch lo r­ id e .

These la t t e r tubes were used as blanks to su b tract th e extraneous

color form ation from th a t due to c y ste in e .

There was l i t t l e n on -cystein e

reducing m a terial p resen t. Hydrogen s u lfid e m s determined by tr a n sfe rr in g th e cadmium s u lfid e from th e cen ter w e ll quant it a t iv e ly to an Brleameyer fla s k w ith a syringe and blunt tu b ercu lin n eed le, th e cen ter w e ll was flu sh ed sev era l tim es w ith d is t ille d water* tha washings bein g added t o th e fla s k and f in a lly th e cen ter w e ll was c a r e fu lly wiped w ith absorbent cotton sev e r a l tim es and th e co tto n wads were a lso added to th e s u lfid e suspension in th e fla s k . A fter a c id ific a tio n w ith &? hydrochloric a cid a known ex cess o f 0.002 M iod in e so lu tio n was added and b a c k -titr a tio n w ith fr e s h ly prepared and standardised sodium th io s u lfa te (0.002 H.) was carried o u t.

R ecoveries o f

hydrogen s u lfid e from a c id ifie d sodium s u lfid e in amounts ranging from 2-50 micromoles were $7-89 per cent a s compared w ith th e 95-96 per cent value reported by Smythe. In a few in stan ces elem ental s u lf ur was determined by the method pro­ posed by Guthrie (19 3$ ).

The method in v o lv es th e use o f an ex cess o f s u lf -

hydryl compound and measurement o f the hydrogen s u lfid e produced from th e reaction s 2 RSH * S

V-

RSSR * HgS

Guthrie poin ted out th a t th e method m s not s p e c ific but a la r g e number o f su lfh y d ry l compounds w ill rea ct in t h is manner.

In our hands c y stein e at

16 a f in a l con cen tration o f 2*0 M appeared q u ite e ffe c tiv e *

A liqu ots o f tin-

cen trifu ged Warburg fla s k con ten ts *©r© removed a fte r incub ation and solved in 6*0 K hydrochloric a cid and centrifuged*

d is­

The p r e c ip ita te was

washed once w ith hydrochloric a cid and tw ice w ith d is t ille d water and then extracted w ith 4-5 ml. o f b o ilin g alcohol*

Aliquots® u su a lly 1*5 to 2 .0

ml* o f t h is e x tr a c t were placed in the sid e bulb o f a 50 ml* Marburg fla sk * th e main compartment con tain in g 5 ml* o f th e concentrated c y ste in e so lu tio n ad ju sted to jgp 7 * 0 and th e cen ter w e ll con tain in g cadmium ace­ ta te *

A fter th e system m s clo sed and eq u ilib ra ted the ex tr a ct was tip p ed

in and th e r ea c tio n was allow ed t o go on fo r two hours a t 3 7 * G sad th e hydrogen s u lfid e production determined*

R ecoveries o f added su lfu r were

85 - 9 o per cen t—somewhat low er than recorded by G uthrie,

Ammonia was determ ined by adding 3*0 ml* o f saturated KaGH to a d i­ lu ted a liq u o t o f sample and aera tin g fo r 60 m inutes in to 0 .0 2 & s u lfu r ic acid*

K essler iz a t io n was th en carried out and th e colo r read in the

spectrophotom eter and compared t o a standard curve* Pyruvic a c id m s Iso la te d as th e 2 s 4 -dinitrophenyXhydra^onc f o l­ low ing th e incubation o f 2 0 mg. amounts o f c e lls w ith 15 mg. o f cy stein e in 1 * 0 M p h o sp h a te b u ffer a t pH 7*4 fo r one hour in the presence o f sodium a sid e which in h ib its pyruvate u t iliz a t io n .

The is o la tio n m s ac­

complished by p o olin g th e con ten ts o f sev era l fla s k s , cen trifu g in g to re­ move c e ll s and adding tw ice th e th e o r e tic a l amount o f 2 , 4 -d in itro p h en y lfeydrasine in 2 .0 II hydrochloric a c id .

The m ixture was allow ed to stand

overnight a t 5 ° -C and was then ex tra cted th ree tim es w ith e th y l a c eta te and th e la t t e r was In turn ex tra cted three tin e s w ith 1 0 per cen t sodium

17 carbonate.

The f in a l p r e c ip ita tio n m s made by a c id ify in g th e sodium

carbonate s o lu tio n .

The p r e c ip ita te m s washed w ith 0 .1 If hydrochloric

a c id and d ried in vacuo over Pa0B. The m eltin g p o in t was 21li°C (un­ c o r r e c te d .)

The mixed m eltin g p o in t w ith th e 2 , 1|-din itrophenylhydra zone

made from p u r ifie d lith iu m pyruvate was a lso 2lU° C {u n corrected .) Pyruvate was q u a n tita tiv e ly determ ined by the 2 ,It-d in itrop h en ylhydrazone method o f Freidman and Haugen (19U3) •

In a few in sta n ces to

check the p o s s ib ilit y o f in terferen ce by sodium a sid e the s a lic y la ld e hyde method o f Straub (1936) was a lso used. A number o f methods were used in attem pts to produce a n on -viab le a c tiv e system .

The f i r s t was the standard method fo r grinding w ith Py-

rex g la s s (K aln itsk y, U tter and Werkman, 19U5).

Proteus v u lg a r is was

grown a n a ero b ica lly in the presence o f 0 .0 5 per cen t c y stein e and the c e lls were harvested a t 1$ hours.

From 20 l i t e r s approxim ately 15 grams

wet w eight o f c e lls were obtained.

Per gram o f w et c e lls two o f fin e ly

ground Pyrex g la s s were added to the c e ll paste and mixed to form a firm «b at te r ” which was ground by fo rcin g through Pyrex g la ss cones as di­ rected by K alnitsky and co-workers (19US)•

C entrifugation a t speeds of. r

1 0 ,0 0 0 RPM or over fo r periods from 1 0 to 1 5 minutes s u ffic e d to c lea r

th e f lu id o f d eb ris.

The c le a r supernatant liq u id was used as c e l l

fr e e e x tr a c t- on seme occasion s the lig h t,, f lu f f y second la y er which appeared to c o n s is t o f c e llu la r debris was a ls o used.

V iable organisms

in the various preparations were determined by standard p la te count methods. The second method used fo r non -viable preparations was one o f

16 chem ical treatm ent.

Acetone* e th e r , chloroform , cyclohexanol and tolu en e

were a l l used to tr e a t c e l l suspensions whose subsequent c y ste in e d e s u lfhydrase a c t iv it ie s were then determ ined.

Toluene was chosen fo r more

d e ta ile d in v e s tig a tio n a fte r prelim inary survey showed th a t under the proper experim ental co n d itio n s c e lls trea ted with tolu en e were nonv ia b le and had considerab le c y stein e desulfhydrase a c t iv it y .

These

p reparations were in a c tiv e a g a in st pyruvate thus d e lin ea tin g the system under stud y.

The procedure as f in a lly adopted was sim ple in Hie extrem e.

A heavy suspension o f c e ll s was d isp ersed in approxim ately 2$ m l. o f phosphate b u ffer in a $0 m l. cen trifu g e tube, 1 0 ml. o f r e d is t ille d tolu en e was added, th e tube was tig h tly stoppered and shaken v ig o ro u sly fo r approxim ately f iv e m inutes.

A fter standing a few m inutes the tube

was cen trifu g ed fo r 10 m inutes a t 2$O0 EPM.

Both the tolu en e and super­

natant b u ffer were removed by means o f a p ip e tte attach ed to an a sp ir a to r . The s id e s o f the tube were c a r e fu lly wiped w ith absorbent co tto n swabs and the packed c e ll s were then resuspended in an appropriate volume o f b u ffer fo r use in the Warburg apparatus.

I t was found th a t fu rth er

washing o f the c e ll s could be ca rried out to remove more toluene but there appeared to be no enhancement o f a c t iv it y fo llo w in g a d d itio n a l washings from which i t seems ev id en t th a t tolu en e does n ot in te r fe r e w ith desulfhydrase a c tio n .

The c y ste in e desulfhydras© system was n ot

e x tr a c tib le from the c e l l s .

C e lls trea ted in th is manner were alm ost

com pletely n o n - v ia b le as determined by p la te counts. A f in a l method fo r the production o f n o n -liv in g system s was the drying o f c e ll s over P20s .

A heavy paste o f a e r o b ic a lly grown c e lls

19

was adapted to c y ste in e and was then cen trifu ged and washed tw ice w ith d is t il le d w ater.

The b a c te r ia l c e ll paste was spread on a P e tr i d ish

l i d and p laced in a d esicca to r over P2 0s . room tem perature.

Drying was carried out a t

I f the drying was rap id ly accom plished by use o f a

vacuum pump to evacuate the d esicca to r the c e l l mass flu ffe d up in to a gray spongy mass which was com pletely dry in from two to th ree hours. While the suspension was a c tiv e the supernatant follo w in g cen trifu g a tio n was very s lig h t ly o p alescen t and in a c tiv e .

P la te counts in d ica ted the

c e ll s p o ssessed a high su rv iv a l ra te to th is treatm ent. _ On the oth er hand, c e l l p a stes d ried slo w ly over P2 06 using a w ater pump to evacuate the d e sicc a to r on ly to the p o in t where sm all bubbles appeared in the mass req uired tw elve to six te e n hours fo r complete drying and y ield ed a hard, g la s sy , dark brown preparation.

These slow -dried preparations

when ground and ex tra cted w ith w ater, sa lin e or b u ffer y ie ld e d an a c tiv e suspension.

C en trifu gation removed debris but l e f t the a c t iv it y in the

lig h t brown, o p a lescen t su p em ate.

A ll ex tr a ctio n s and cen trifu g a tio n s „o o f the d ried preparations were carried out a t > C.

20

RESULTS AKD DXSCUSSIOW

Tfe© primary o b je c tiv e o f t h is stu d y was t o determ ine the products formed from th e b a c te r ia l d issim ila tio n o f c y stin e and cystein e*

C ystine

m etabolism seemed a lo g ic a l sta r tin g p o in t In view o f Anderson* s (1917) d e n ia l o f th e claim o f DesnueUe (1939) th a t c y stin e i s an aerob ically reduced to c y ste in e and then d esulfh ydrated.

Anderson reported no hydro­

gen s u lfid e production from c y stin e by Proteus v u lg a ris Jn th e absence o f a reducing agen t although hydrogen su lfid e was produced from c y stein e when no reducing m a teria l was present*

However, ammonia m s produced

from both amino a c id s under th ese c o n d itio n s. The optim al jjH a t which th e rea c tio n s might proceed was f i r s t de­ termined*

Bach Warburg v e s s e l contained 50 micrograms o f c y stin e , 5

to 8 mg. dry w eight o f washed Proteus v u lg a ris c e l l s grown a n a erob ica lly in th e presence o f c y s tin e , and b u ffer to a f in a l volume o f 2*8 ml* cen ter w e ll contained cadmium a ceta te as p rev io u sly described*

The

A fter

g a ssin g and e q u ilib r a tin g su b strate was tip p ed in to the main compart­ ment a s a n eu tra l su sp en sion .

Incubation was c a rr ie d out fo r one hour

a n a ero b ica lly a t 3 7 * C and hydrogen s u lfid e product io n was determ ined. Measurable amounts o f hydrogen su lfid e were produced a t a l l jgBs from 5 *0 t o 8*5 w ith a p la tea u o f maximal production between 7 * 2 and 7*5*

From th ese r e s u lts th e con clu sion was drawn th a t hydrogen s u lfid e pro­ duction apparently tak es place w ithout an extern al hydrogen donor in th e ca se of cy stin e*

These data do n o t, o f cou rse, r u le out the a c tio n

o f endogenous hydrogen donators*

C ysteine sim ila r ly te s te d showed an

21 I d e n tic a l

optimum a s measured by hydrogen su lfid e produ ction.

r e s u lt i s shewn g r a p h ic a lly in F igure I .

This

The s im ila r ity in pH optima

does n o t n e c e ssa r ily im ply th a t th e red u ction o f c y stin e and the d o su lfh y d ra tio n o f c y ste in e are in th e same r e a c tio n sequence.

Accord­

in gly* oth er experim ents were design ed to determ ine whether the mecha­ nism o f c y stin e m etabolism involved c y ste in e . S ta n ier *s (1947) concept o f sim ultaneous adaptation seemed a p p li­ ca b le fo r th e reasons which fo llo w .

Almy and James (1926) b eliev ed th a t

aerated c u ltu re s produced more hydrogen s u lfid e than a n a ero b ica lly grom c e ll s in th e case o f Proteus v u lg a r is.

In attem pting to check t h is con­

c lu sio n we cams to q u ite th e op p osite view , namely th a t anaerobJbsis en­ hances c y ste in e de s u lf hydras© a c t iv it y o f Proteus v u lg a r is.

The resu lts

o f hydro gen s u lfid e production from cy stein e by r e stin g c e l l s cultu red under variou s co n d ition s are g iv en in Table I .

E vid en tly, an aerob iosis

in crea ses th e a c t iv it y and t h is a c tiv ity can be fu rth er increased by the in c lu sio n o f c y ste in e or c y stin e in th e growth medium.

The observation

o f in creased hydrogen su lfid e production under anaerobic co n d itio n s i s in lin e w ith a rep o rt by Skerman (1949) in which s u lfid e su lfu r was de­ term ined p o la ro m etrica lly in cu ltu res o f Proteus v u lg a r is. were n ot fom ed w hile oxygen was present in the medium.

S u lfid e ions

S in ce, under

anaerobic c o n d itio n s, th e ad d ition o f c y stin e enhances c y ste in e d e s u lfhydrase a c t iv it y th en c e l l s which have been adapted (by p rior growth) t o c y stin e should m etab olize both c y stin e and cy stein e a t a rapid ra te in th e even t th a t cystin© i s f i r s t reduced to c y s te in e .

On the oth er

hand c e l l s adapted to c y ste in e might not be adapted to c y stin e u n less

0 RESTING CELLS Proteus vulgaris 0 TOLUENE TREATED

H 2 S PRODUCTION MOLES/mf CELLS/HOUR

3.0

2.5-

2. 0 -

-0 -

OS-

8.0

Figure X Th© e f f e c t o f j>H or cystein© desulfhydrase a c tiv ity .

23 TABLE 1 GXSTEXHB BESULFHTDEA3E ACTIVITT4* OF PHOTEUS m&ARIS CELLS GROSS' UNDER VARIOUS conditions H2S Production Growth C onditions Moles x 10 "^/lag/hr Agar Surface (A erobic)

0.13

Agar Surface (Anaerobic)

1 .0 3

H u trlen t Broth (A erated)

0,80

N u trien t Broth (Anaerobic)

1 .4 5

N u trien t Broth ♦ 0.05$ C ysteine (Anaerobic)

2 .2 0

N u trient Broth ♦ 0 .1 0 $ C ysteine (Anaerobic)

2 .0 0

N u trien t Broth + 0 ,0 5 $ C ystine (Anaerobic)

2 .2 2

^Determined by HjsS production in Warburg v e s s e ls . Bach v e s s e l contained 8 -1 0 lag. c e l l s , 0 .0 2 M c y ste in e , 0 .0 5 H phosphate b u ffer jgH 1*1*} cen ter w e ll contained cadmium a c e ta te . Temperature 37® C. Time = 1 hour, a n a ero b ica lly .

24 fchap® i s a strong ravers® rea ctio n and cystein e-ad a p ted c e l l s would, a t­ tack c y stin e on ly a fte r a la g period o f adjustment*

As w ill be shown

la t e r th ere is no evidence fo r an ap p reciab le reversal o f th e rea ctio n s c y stin e —V

2 cy stein e*

th e r e s u lts of c y ste in e and c y stin e d is s im ila ­

tio n by c e l l s grown in c y ste in e are shown in F igure II*

The r a te s a t

which th e two amino a c id s were attack ed were determ ined by a n a ly sis o f * th e amount o f hydrogen s u lfid e lib e r a te d over th e course o f 9 0 m inutes a t 15 minute in ter v a ls*

I t i s r e a d ily apparent th a t c y stin e 13 metabo­

lis e d but only fo llo w in g a la g period* itiic h i s sh ort but d e fin ite and rep rod u cib le.

Once th e la g p eriod i s over th e r a te o f c y stin e d issim i­

la tio n equals o r a c tu a lly exceeds th a t o f c y ste in e breakdown*

ih en

c e ll s were grown in c y stin e -c o n ta in in g media th e y m etabolize both c y stin e and c y stein e a t alm ost equal r a te s w ithout passing through a la g phase in e ith e r e a se .

These r e s u lt s are shewn in Figure I H .

It

should be p oin ted ou t th a t in th ese experim ents c y stin e was present in h a lf th e con cen tration o f c y ste in e , i . e . , the p o te n tia l c y stein e con­ te n ts o f each fla s k in th e two s e r ie s were eq u al.

The r e s u lts obtained

in d ic a te th a t c y stin e forms c y stein e and the la t t e r then undergoes d es u lf hydration. The use o f the Shinohara method fo r c y ste in e may be made to in ­ clude a determ ination o f both c y stin e and c y stein e sin ce the method measures c y ste in e fo llo w in g reduction w ith sodium b is u lf it e .

I f the

b is u lf it e ad d ition i s om itted in one a liq u o t and included in another id e n tic a l a liq u o t both amino a cid s may be ca lcu la ted by th e equations c y st in s * t o t a l o f cy stein e + c y stin e - 2 x cy stein e presen t (valu es

a5i

_i

_i

LlJ

LlJ

_I CD

CD

g

CD

to-

CYSTEINE UTILIZATION BY CYSTEINE-ADAPTED CELLS

QC Q_ OO rJ

05-

0

m

15

30

CYSTINE UTILIZATION BY CYSTEINE-ADAPTED CELLS

60

75

TIME IN MINUTES Figure 2 Cystine and c y stein e u t iliz a t io n by Proteus v u lg a r is c e l l s grown in c y ste in e .

90

H z s PRODUCED MOLES x l O '6/m j. C ELLS

3.5 1

3.0-

2.0

CYSTEINE UTILIZATION BY CY5TINE -ADAPTED CELLS CYSTINE UTILIZATION BY CYSTINE-ADAPTED CELLS

0.5

45

T I M E IN M IN U T E S

Figure 3 C ystine and cy stein e u t iliz a t io n by c e l l s grown in cystine*

v

27 are expressed in m illigram s.)

The use o f t h is m od ification in d icated

th a t th ere was alm ost no accumulation o f c y stein e from c y stin e metabo­ lis m .

Apparently c y stein e was d esu lf hydra ted very rapidly by the c e l l s

under th e experim ental con d itio n s used.

In c y ste in e experiments there

appeared to be no formation o f c y stin e as measured by th is technique. A ll o f th ese data are in accord w ith D asnuelie’ s views regarding c y stin e metabolism in E scherichia c o l i and do not agree w ith the observations o f Anderson. L o g ic a lly , the next step was to attempt an elu c id a tio n o f the pro­ ducts formed from c y ste in e under the conditions being employed in these experim ents.

Following the incubation of Proteus v u lg a ris c e l l s with

c y stein e an a n a ly sis of fla s k contents was made fo r ammonia, hydrogen s u lf id e , fr e e su lfu r and pyruvic a cid .

Pyruvic a cid determ inations in

experiments o f t h is type were so low as to be in c o n c lu siv e .

A ty p ic a l

s e t o f experim ental valu es i s recorded in Table I I 5 pyruvate values are not included.

These experiments were performed in t r ip lic a t e } fo r each

three experim ental fla s k s co n tro ls were provided as fo llo w s: ( 1 ) a v e s s e l which had no su b stra te, but was otherw ise id e n tic a l w ith the ex­ perim ental f la s k s , to check fo r endogenous hydrogen s u lfid e production} ( 2 )a fla s k id e n tic a l w ith experim ental v e s s e ls except the c e l l s had been b o ile d fo r 1 0 minutes as a check fo r non-enzyraatic hydrogen s u lfid e production, and} ( 3 ) a reactio n v e s s e l id e n tic a l w ith the experim ental— but stopped a t zero tim e.

Since c y stein e was made up and n eu tra l­

iz e d p rio r to each run th is zero time a n a ly sis was r e la t iv e ly important.

28

TABLE I I

ANAEBQBIC UTILIZATION OF CYSTEINE BY frotbus vuloaris adapted to cysteine

Bfcles x 1 0 - 6 /mg/hour H2 S Produced

nh3 Produced

3 Produced

3.5

2 .0

1 .6

0 .1

3 .5

2 .1

4 .0

0 .1

3 .2

1 .5

0 .6

0 .0

3 .0

1 .6

3 .8

0 .0

3 .6

2 .0

0 .2

0 .3

3 .7

2 ,1

2 .8

0 .1

3 .9

2 .1

3 .1

0 .1

3 .0

2*0

3 .6

0 .2

2 .0

1 .7

2 .5

0 ,0

4*1

2 ,9

0 .9

0 .0

3 .0

2 ,3

3 .6

0 .1

3 .2

2 ,2

3 .6

0 .1

Averages 3 .0

2 .1

2 .5

0 .1

C ysteine Disappearing

Each experim ental v e s s e l contained c e l l s , cy stein e 0*02 M, and phosphate b u ffer 0 . 1 M jdH 7*4-* Center w e ll contained cadmium a c e ta te . T o ta l volume = 2 .8 ml* 1 hour incubation a t 37® C,

29

Under th e co n d itio n s of th ese experiments endogenous or non-en?ymat ic hydrogen s u lfid e was newer produced*

Ammonia and elem ental s u lfu r de­

term inations were performed in d u p lic a te , f t i s evid en t from Table I I th a t hydrogen s u lfid e recovered amounted to about 70 per cen t o f th e c y ste in e lo s t* a fig u re higher than Smythe (1941) found in mammalian liv e r (6 6 per c e n t) and o f the same order as fisted by Tarr (1933) in th e case o f Proteus v u lg a ris (75 per cent)* Am­ monia values w hile e r r a tic in d ic a te ammonia i s produced and on a molar b a sis poin t to a r a t io o f one mole o f ammonia produced per mole o f cys­ te in e metabolized*

Somewhat variable r e s u lt s were noted in many o f th e

experiments but in no case were the data such th at th e r ea c tio n could be in terp reted as y ie ld in g no ammonia* Elemental s u lfu r fig u r e s were c o n s is te n tly low and in a number o f in stan ces n o n -e x isten t • two ways,

These, re s u its may I® in terp reted In a t le a s t

lb i s p o s s ib le th a t th e production o f elem ental su lfu r i s an

unimportant s id e r ea c tio n , s l ig h t a s compared to c y ste in e desulfhydras© and a c tu a lly having no r e la t io n to i t .

A lte r n a tiv e ly i t may be argued

th a t elemeribal s u lfu r I s produced from c y stein e and i s then ra p id ly re­ duced to hydrogen su lfid e *

Indeed, Tarr has demonstrated (Tarr, 1933 a;

1934) th a t Proteus v u lg a r is i s capable o f reducing both su lfu r and t h io s u lfa te to hydrogen su lfid e*

The f i r s t explanation seems most lo g ic a l

inasmuch as l i t t l e o r no hydrogen s u lfid e was produced when su lfu r was added t o v e s s e ls under th e conditions used here although t h is may have been a fu n ctio n o f th e low order o f w ettin g o f su lfu r p a r tic le s by th e

30

aqueous medium*

However* s u lfu r v a lu es were even lower In la t e r exp eri­

ments u sin g non -viab le system s. R estin g c e l l s o f Proteus morganli and E scherichia c o l i y ie ld e d sim i­ la r r e s u lt s on incubation w ith c y ste in e although th ese values were some'what lower than th o se obtained w ith Proteus v u lg a r is, gg& m t. m m M

the f a c t that

I s able to attack c y ste in e a t a r e la t iv e ly rapid r a te

in th e same manner as Proteus v u lg a r is i s surprisin g in view o f th e observation (Meyers and Porter* 1945) th a t Proteus mcrganli cannot u t i l i z e c y ste in e as a source of su lfu r f o r growth* in a medium other­ wise devoid o f organic s u lfu r compounds.

Cystine* however* under these

circum stances i s w e ll u t iliz e d fo r growth. In order t o lea rn more o f the nature of the system th e e f f e c t s o f various in h ib ito r s were in v e s tig a te d ,

th e in h ib ito r s ineluded sev era l

well-known substances which block enzyme reaction s* a s w e ll as a few substances th at are c lo s e ly r e la te d in c er ta in r e sp e c ts to c y stein e • AH o f th e experiments were performed in t r ip l ic a t e using Warburg fla s k s as p reviou sly d escrib ed . Three v e s s e ls had no in h ib ito r present and the average of hydrogen s u lfid e production in th ese v e s s e ls was taken* in each experim ent, a s 1 0 0 per c e n t. carried out at le a s t in tr ip lic a t e *

In h ib itio n experiments were

The c e l l s , bu ffer and in h ib ito r

were incubated fo r 15 minutes prior t o the tip p in g in o f th e su b stra te. R esults o f these experiments are tabulated in Table I I I .

Of in t e r e s t

are the r e s u lt s obtained w ith hydroxylamino and semicarfoa zide •

The

powerful in h ib itio n by th ese reagents p o in ts up the importance o f ca r­ bonyl groups in t h i s system and agrees w ith the fin d in g s o f D esnueile

31

3&BI3 m e f f e c t s of m a w s b h jb it o b s oh hyerqgen SULFKB PSOKJCTiai m M CYST3BIE B? m m tw m M

in h ib ito r

Concentration

% In h ib itio n

lo d oaoetic Acid

.010 M

100

lo d o a cetic Acid

.005 H

90

lod oacetlc Acid

.0 0 1 11

75

Sodium A rsenite

.005 K

100

Sodium A rsenite

.002 M

100

Sodium A rsenite

.0005 M

91

Fluoroacetaie

.015 M

5

Fluoroacetate

.005 M

0

Sodium Azide

.015 M

21

Sodium Aside

.0 0 6 M

15

Sodium Azids

.001 M

9

Serine

.015 M

10

Serine

.0 2 0 M

10

Alanine

*021 M

9

Homocysteine

.020 M

20

Hydroxylamine

.001 II

90

Semiearbazide

.0 0 1 12

65

■gach experimental v e s s e l contained in h ib ito r , c e l l s , c y stein e 0,02 M, phosphate b u ffer 0*1 M, pH 7+1* Cadmium a ceta te in cen ter ttoII. T otal volume * 2*3 ml* 1 hour incubation a t 3 7 * C.

32

(1939) in th e case o f E scherichia c o i l and with Sm yths?s (1941) fin d in g s In mammalian l i v e r c y ste in e ds s e l f hydras©*

Contrary to D esnuelle and in

agreement with Siaythe no in h ib itio n by amino a c id s was observed even though the concentration was r a ise d to equal th a t o f the substrate*

The

r e s u lt s w ith iodoacetate are adm ittedly o f dubious value as i t i s very p o ssib le th a t th e

SH groups blocked may be those o f c y ste in e j on the

other hand th e very considerable in h ib itio n observed may r e s u lt fro/a blocking segue portion o f the a c tu a l enzyme or some m aterial a ctin g us an endogenous c o -fa c t op.

The most s ig n if icant fin d in g from th ese ex­

periments was th e fa c t th a t a s id e , in r e la t iv e ly high concentrations* did n ot seem t o in h ib it markedly cy stein e desulfhydras© a c t iv it y bet appeared to block th e a b i l i t y o f r e s tin g c e l l s to u t i l i s e pyruvate* T h is fa c t was discovered in a ro u tin e a n a ly sis o f f la sic contents fo r py­ ruvate fo llo w in g experiments w ith in h ib ito r s . In order t o check more c lo s e ly th e a ctio n o f azide on anaerobic pyruvate u t iliz a t io n an experiment was sot up in which pyruvate, b u ffer, c e l l s and varying concentrations of asid e were incubated fo r one hour and pyruvate was determined in th e fla s k contents* tabulated in Table IV.

These r e s u lts are

Four fla s k s were s©t up fo r each concentration o f

azide employed, one b ein g stopped at zero tim e.

Pyruvate determinations

were carried out in duplicate follow in g tr ic h lo r o a c e tic a cid p r e cip ita ­ tio n and c en tr ifu g a tio n by the Freidman-Haugen (1943) method and In some ca ses by th e procedure o f Straub (1936 ) .

These values were averaged.

Thus, each fig u re in Table IV represents at le a s t th ree experiments* From th e r e s u lts i t i s evident that under the con d ition s here employed

33

t u u rr 3 0 0 m AZIDE INHIBITION OF AHMBGBIG by frotmb m m a x s

fm m xm m m z m m Aside A dditions

Pyruvate U tilis e d (pyruvate added—Pyruvate recovered; Moles x 1 0 "^/iag/hr

m Aside

4*7

Ho Aside

4*6

Ho Aside

4*0

•001 M Aside

4 .6

•001 M Aside

4*6

•001 M Aside

4*0

•004 £ Asid e

4*5

.004 U Aside

4*3

*004 & Aside

4 .0

.008 M Aside

2 .5

•008 U Aside

2 .4

,008 M Aside

3 .0

•010 M Aside

1*5

.010 M Aside

1*4

.010 U Aside

1 .2

•015 M Aside

1 .1

.015 U Aside

1 .2

.015 H Aside

0 .9

Each experimental v e s s e l contained 60 moles x 1 0 - 6 pyruvate, 8 tag* c e l l s , a s id e a s noted and phosphate bu ffer nH 7*4* Total volume ® 2 . 8 m l. 1 hour a t 37° C.

n t o study c y ste in e d©sulfhydras© action the addition o f sodium a sid e pre­ vents th e d is s im ila tio n of pyruvate*

The usefu ln ess o f t h i s fa c t i s

r e a d ily evid en t in in te r p r e tin g the mechanism o f c y stein e breakdown. There appears t o be nothing in th e lit e r a tu r e concerning the e f f e c t s o f a sid e on pyruvate d issim ila tio n but a note by Loomis and Lipm&rm (194.9) in d ic a te s th at a sid e may act by blocking phosphorylation.

The system

used by Loomis and Lipmann involved y east hexokinase fr u c to se as phos­ phate acceptor and ferrlcyan id e as a hydrogen acceptor*

I t may be danger­

ous to gen eralize from t h e ir data on th e a ctio n o f a sid e on pyruvate meta­ bolism b at inasmuch as pyruvate i s d issim ila ted v ia a phosphorclastic s p l i t th e blocking o f phosphorylation may be involved in th e in h ib itio n o f pyruvate in Proteus v u lga ris c e l l s . T ypical cy stein e desulfhydras© experiments w ith r e s tin g c e l l s were again s e t up but sodium azid e was added to a f i n a l concentration o f *008 M* Follow ing Incubation analyses were again made fo r metabolic products in clu d in g pyruvic a c id ,

the experiments were performed in t r ip lic a t e and

pyruvate „ c y s te in e , and ammonia determinations were carried out in dupli­ c a te .

The r e s u lt s of th ese experiments are lis t e d in Table V,

The re­

s u lt s are in terp reted to mean th a t th e major rea ctio n involved I s cysteln© — >■ H2S +

+ pyruvate

Again, only a very sm all amount of elem ental s u lfu r appears*

Proteus

morganii and B scherichia c o li g iv e e s s e n t ia lly the same type of r e s u lt s but th ese have not been tabulated her© because only a r e la t iv e ly few de­ term inations were made to cheek t h i s p o in t. Simple* non-viable systems are ideal fo r enzyme stu d io s and i t was

35 TABLE V ANAEROBIC G*5ffE2K& DISSIMILATION BT AZIDE-HKHIBITMi* RESTING CELLS OF PROTEUS TOGARIS M oles x lO ^ /n ig /h o o r

C ysteine D isappearlog

Has Produced

2 .A

1 .3

2 .0

2 .0

—

2 .0

1 .0

0 .2

2*5

—

2*2

1 .8

0 .8

2.5

—

2*1

1 .0

0 .8

1 .6

—

2 .0

1 .2

1 .0

1 .3

—

3 .0

1 .7

1 .0

1*3

0 .0

2 .6

1 .5

wariftv

2 .8

0 .0

2 .6

1 .3

1 .0

2 .9

0 .1

2*5

1 .6

0 .2

2 .0

0 .1

1 .6

1 .3

0 .8

1 .0

—

1 .?

0 .8

1 .2

1 .2

—

2 .0

1 .2

2 .1

2 .5

—

1.3

0 .9

1 .9

0.05

hh3 Produced

Pyruvate Produced

3 Produced

Averages 2 .4

.

36 f e l t th a t such a system might he o f value in th e present study.

By methods

alread y d iscu ssed a non-viable preparation was made from washed, c y ste in e adapted Proteus morganii c e l l s which showed considerable a c t iv it y a g a in st c y s t e in e ,

In these experim ents i t ras noted th a t while Proteus marm a ll

would stand tolu en e treatment up to 20 minutes and s t i l l d isp la y consider­ able c y ste in e desulfhydrase action Proteus v u lg a r is was com pletely in a c t i­ vated by only a few minutes exposure to to lu en e.

A number o f experiments

were performed in th e cold (5* C) but even under these co n d itio n s i t m s im possible to produce an a c tiv e preparation o f Proteus v u lg a r is .

Accord­

ingly* Proteus morganii was used f o r th is phase o f the study* The toluene trea ted c e l l preparation ex h ib ited i t s optim al a c t iv it y a g a in st c y ste in e a t j>H 7 .4 - 7 . 6 * (Figure I)* and experiment showed i t to be com pletely in a c tiv e a g a in st pyruvate.

I t is almost superfluous t o

note th e q u a n tita tiv e aspects o f c y ste in e d issim ila tio n # sin ce th ese para l l e l c lo s e ly th e r e s u lts obtained w ith a sid e in h ib ited Proteus vu lg a r is. VI*

However, a few rep resen tative experiments are tabulated in Table

These were performed in t r ip lic a t e as in the case o f r e s t in g c e lls *

U nfortunately, a ll e ffo r ts to ex tra ct th e enzyme from th e toluene treated c e l l s were u n su ccessf u l.

Extract ions were t r i a l w ith d i s t i l l e d w ater,

s a lin e , b u ffers in various concentrations and in 4 . 5 to pH 8 *5 .

0 .5

u n it step s from

dH

The e f f e c t s of various temperatures on th e extraction

process were stu d ied , as w e ll a s various lengths of ex tra ctio n time but uniformly the a c t iv it y remained in th e c e l l m a ter ia l. P la te counts in d icated th a t th e c e l l s were not v ia b le and i t seemed lik e ly th at some information regarding the c y stein e desulfhydrase system

37 tm s vi MAEROBIC nmmmTMM m Gf&mm m TOLtJMs rmktm mus of froesib m m x x Moles x 10^/iag/hour

Cysteine Disappearing

Has

M3

Pyruvate

Produced

Produced

Produced

1.5

0*8

1*0

1*7

1*3

0*8

1*0

1.5

1*0

0 .5

0*2

0 .6

1*0

0*3

0*8

1.5

1*1

0*5

1 .6

0 .3

1 .6

0*9

1*6

1 .1

1*7

1*1

0*5

1*0

0*7

0*9

1*0

Average 1*3

38 m ight be gained through th e use o f s p e c ific in h ib ito r s , th e fa c to r of perm eab ility having been removed* in part*

For example, Binkley had

claimed th a t magnesium, raangams© or zin c ions are necessary t o th e a c tio n o f c y stein e desulfhydras®.

In the event t h is were true the ad­

d itio n o f ntrapping reagen ts” such as 8 -hydroxyqu in o lin e might in d ic a te th e n e c e s s ity o f m e ta llic ions by in h ib itin g the r e a c tio n .

Various in ­

h ib ito r s were te s te d fear t h e ir a c tio n on the tolu en e k ille d c e l l s and the r e s u lts are given in Table VII*

The importance o f th e carbonyl group

i s again apparent from these experiments because o f the powerful in h i­ b itio n exerted by carbonyl rea g en ts.

Noteworthy i s the lack o f in h ib itio n

by 8 -hydroxyquincline and °t" ^ —dip yridyl in d ic a tin g th a t d iv a len t ion s may not p la y as important a r o le as has been previou sly suggested.

Some

fu rth er evidence in t h is d ir e c tio n may be afforded by th e f a c t th a t to lu ­ e n e -k ille d c e l l s held fo r on® hour (aged) a t £>H 4 . 5 in phosphate b u ffer lo s e much of th e ir a c t iv it y toward c y s te in e .

Ttiile r e a c tiv a tio n may be

brought about by th e add ition o f b o iled c ell, ex tra ct no aestivation oecurs when magnesium, manganese, or zinc ion s are added to a f i n a l concentration o f 0 .0 0 1 M, Wood and Guns a l us (1949) in t h e ir in v e stig a tio n of serin e and threo­ nine dehydrases found th at th ese systems were a c tiv a te d by g lu ta th io n e and adenosine-5-*phosphoric a c id .

In view o f the s im ila r ity o f t h e r e ­

a ctio n s involved attempts were made to r e a c tiv a te th e c y stein e d e s u lfhydrase system by the ad d ition o f th ese compounds.

No r e a c tiv a tio n oc­

curred w ith th ese m aterials alone, in combination or in combination w ith divalen t io n s .

In addition to th e compounds l i s t e d in Table ¥111 th e

J8&FKQTS GF tmicm 3>IKI8iT0RS CB THE em&xm mmmmmsE activity of m vmk kilusd cells* of

m x em rnm m xi

In h ib ito r

Concentration of In h ib ito r

Per B en t’ In h ib itio n o f HjgS Production

~ B ipyridyl

.0 0 1 M

0

~ D ipyridyl

.005 M

10

8 -hydroxyquinoline

.oca M

5

Semicarba sid e

.0 0 1 M

75

Rydroxylaedn©

.001 M

m

Phenylhydrasine

* 001 u

88

Phenylhydra sin e

* 002 K

%

*S&sed on hydrogen su lfid e production, Each fla sk contained cystein e 0.02 M* buffer jaH 7*6* in h ib ito r, and to lu en e-k illed c e lls 5 sag* T otal volume = 2 .8 ml*, one hour anaerobically a t 37° S .

40

« a s ls n u m m tm m im o f

m otm s m m m xi

cells

m b m m -x m g ® «FQ;+

1 *2

Aged c e l l s ♦ A dencslae- 3 **f10 / * G lutathione * Mg**

1 .4

Aged c e l l s + AdenosineHHPOA * G lutatiiione ♦ Mg**

1*4

Aged c e l l s * b o ile d c e l l ex tra ct

3 .9

Aged c e i l s ♦ b o iled c e i l ex tra ct

3*7

u the fo llo w in g were u n su ccassfu lly tr ie d f o r r e a c tiv a tio n alone and in com­ b in atio n s adenine, guanylic a c id , c y tid y lic acid* ATP, manganese, cobalt* iro n (ferrous and f e r r i c ) , z in c , pantothenate, adenosine, ascorbic acid., TPN, QPN, pyridoxal and pyidoxamine*

I t would appear that some c o -fa c to r

was b ein g destroyed on standing a t pH 4*5*

However, t h is fa c to r has not

been determined t o date. One further p oin t i s o f in te r e st w ith respect to toluene treatment e f Proteus morraftii c e lls *

While the cy stein e deaulf hydrase system appears

t o be r e la t iv e ly unharmed by the action o f to lu en e, cystin e i s no longer attacked by c e l l s k il le d in t h i s maimer*

Obviously, th e system respon sib le

fo r reducing c y stin e to c y ste in e i s no longer present*

That t h is enzyme

system i t s e l f i s destroyed and not m erely in a ctiv a ted by th e removal o f a to lu en e-so lu b le c o -fa c to r i s in d icated by the fa c t th at the m aterial remaining a ft e r toluene removal under vacuum w ill not a c tiv a te to lu en ek il le d c e l l s toward cystine* Successf u l preparation of a c e ll- f r e e system was accomplished r e la ­ t i v e l y la t e in th e study*

C e ll pastes dried slow ly as described in the

s e c tio n on Experimental Methods—were extracted w ith d i s t i l l e d water and the c e llu la r d eb ris was removed by high speed cen trifugation*

The

supernatant flu id appeared fr e e o f c e l l s and exh ib ited considerable ac­ t i v i t y as evidenced by hydrogen s u lfid e production.

Furthermore, the

dried c e l l paste was qu ite sta b le and could be held fo r seme time under vacuum w ithout appreciable lo s s of a c t i v i t y .

The c e ll - f r e e e x tra ct was

in a c tiv e a g a in st pyruvate and analyses follow in g incubation of the c e l l Juice and cy stein e m erely confirmed th e previous fin d in g that ammonia*

A2

hydrogen s u lfid e and pyruvate are the major products o f the reaction* C ysteine desulfhydrase a c t iv it y was maximal in the f i r s t e x tr a c t.

One

fl£L. o f the f i r s t water extra ct yield ed 1 4 .3 0 micromoles of hydrogen su l­ f id e from 1 5 mg* c y ste in e in phosphate buffer In a one hour anaerobic in cu b ation .

The second and third ex tra ct uhder id e n tic a l conditions

produced 1 0 .0 0 and 3 .1 0 micromoles# resp ectiv ely *

In a number o f ex­

periments conducted on t h is crude extract no appreciable lo s s in ac­ t i v i t y could be noted fo llo w in g d ia ly s is o f one* two or three hours. I t i s p o s s ib le , however, th at th e d ia ly s is of a more high ly p u rified system migM. rev e a l a dlalyzab le component.

The ad d ition o f b o iled

c e l l s t o the ex tr a cts uniformly resu lted in a small but reproducible in crease in a c t iv it y .

This increment amounted t o about 10-15 per cent

above th e a ctio n o f the ex tr a cts alone*

h3

mmmx Cystine and c y stein e metabolism by Proteus v u lg a r is and Proteus morg a n ii has been in vestigated *

A fter anaerobic growth in c y stin e (or c y s t­

e in e ) containing media r e s tin g c e l l s o f both sp e c ie s in phosphate b u ffer £B 7 . 1* a n a erob ically reduce c y stin e to c y ste in e j each mole o f the la t t e r y ie ld s , v ia desulfh ydration, one mole each o f hydrogen s u lfid e , ammonia and pyruvate.

Sodium asside in r e la t iv e ly high concentrations has l i t t l e

or no a c tio n on c y ste in e desulfhydrase a c tio n but prevents pyruvate u t i­ l i s a t io n - the formation and accumulation o f pyruvate can thus be demon­ s tr a te d ,

Toluene treatment o f Proteus morganii y ie ld s a non-viable

preparation which i s a c tiv e a g a in st cy stein e but not pyruvate or c y stin e , ib i s non -viable system i s in h ib ite d by carbonyl reagents but not by a sid e , 8 -hydroxyquinoline, OL-dl-dipyridyl, or amino a c id s,

preparation i s g r e a tly diminished by holding a t

The a c t i v it y o f the i* .5 fo r one hour.

This lo s s i s restored by the ad d ition o f b o iled c e l l s but not by gluta­ th io n e, adenosine- 2-phosphoric a cid , adenosine- 5 -phosphoric a c id , di­ v a le n t m e ta llic ions or combinations o f these fa c to r s, thus in d ica tin g th a t ser in e dehydrase i s n o t id e n tic a l m th cy stein e desulfhydrase-—the former being a c tiv a te d by glutathione and adenosine- 5-phosphorie a cid . Ho in h ib itio n was noted wl th reagents capable of forming complexes with d iv a len t io n s .

C ell fr e e ex tra cts of dried, adapted c e l l s do n ot appear

to have a dialyaable component nor are these ex tr a cts a c tiv a te d by di­ v a le n t m e ta llic io n s .

The c y stein e desulfhydrase system i s sim ila r in

both sp e cie s o f Proteus studied and may be condderably enhanced by p rio r growth o f the c e l l s anaerobically in the presence o f c y stin e or c y ste in e .

44 APPENDIX

The biochem ical c h a r a c te r is tic s o f the Proteus v u lg a ris and the Proteus morganii are recorded in Table ±X. The Proteus v u lg a ris was o f the “spreader” typ e, growing in a th in gray film on an agar su rfa ce. The s p e c if ic i t y o f both organisms toward various su lfu r compounds under the con d ition s employed in t h is study are given in Table X. G enerally, the r e s u lts .a r e in e x c e lle n t accord w ith the r e s u lts of Tarr (1933 * )•

Of some in t e r e s t i s the fa c t th a t r e stin g c e l l s o f Proteus

morganii produce some hydrogen s u lfid e from homocysteine w hile Proteus v u lg a r is produced none from th is compound.

Because o f the c lo se s im ila r i­

t y between methionine and homocysteine the a ctio n o f Proteus morganii on methionine was c u r so r ily in v e stig a te d .

No hydrogen s u lfid e was produced

which in view o f the structure o f methionine i s not su rp risin g .

Methyl

mereap tan, on the other hand, might be an expected end product.

A few

an alyses fo r methyl a®reap tan were carried out and the r e s u lts in d icated l i t t l e or no roercaptan was being produced.

A nalysis was carried out by

p lacin g 0 . 1* ml o f 3 N MaOH in the cen ter w e ll, the a lk a li was removed a t the end o f the experiment, a c id ifie d and the so lu tio n was tit r a t e d w ith io d in e .

These r e s u lts do not exclude the p o s s ib ilit y th a t under conditions

more optimal fo r methionine u t iliz a t io n methyl mercaptan might be produced and the remainder of the molecule might w e ll be handled in the same way as the portion o f the homocysteine molecule remaining a ft e r hydrogen s u lfid e is s p lit o ff.

Studies are proceeding toward is o la tin g and id e n tify in g 4

the products of homocysteine breakdown and toward e sta b lish in g conditions

under which the mechanism o f methionine d issim ila tio n may be in vestigated * While attem pting to make a c e ll - f r e e preparation by grinding with g la s s a s described in ISxperimental Methods i t was f e l t th a t a q u a n tita tiv e r e la tio n s h ip between v ia b le c e l l s and hydrogen s u lfid e production w>uld be o f some v a lu e .

These r e la tio n sh ip s were carried out w ith Proteus v u lg a ris

c e l l s in the same manner as already in d ica ted fo r c y stein e desulfhydrase a c t iv it y .

Various d ilu tio n s o f c e l l suspensions were used and follow in g

the incubation period p la te counts were made o f the Warburg v e s s e l contents by standard p la te counting techniques.

The r e s u lts are given in Table XI.

Attempts to separate c y stein e desulfhydrase a ctio n and whole v ia b le c e l l s were never s u c c e s sfu l.

The a c t iv it y present was always referab le to

the v ia b le c e l l count o f the preparations.

In attempts to a c tiv a te these

preparations numerous ad d ition s to the systems were made, s in g ly and in various combinations. f e c t iv e .

None of th ese m aterials or combinations were e f­

They are l i s t e d in Table XII.

A study was a ls o made o f hydrogen s u lfid e production under aerobic and anaerobic conditions because o f divergent statem ents in the lite r a tu r e regarding hydrogen s u lfid e production from aerated c e l l suspensions and suspensions through which nitrogen or hydrogen was being bubbled (Anderson, 19ii7, Tarr, 1933# James and Almy, 1926). in Figure IV.

The r e s u lts are shown g ra p h ica lly

Obviously, due to the p ecu lia r nature o f the substrate when

high e e l l concentrations are present (or high cy stein e concentrations) re­ ducing conditions obtain regard less o f the gas phase above the suspending medium.

This i s shown in th a t portion of the curves involvin g compara-

46 l i v e l y large amounts of c e l l m aterial.

In th ese in stan ces the hydrogen

s u lfid e production i s id e n tic a l under anaerobic and aerobic conditions# On the other hand when the concentration o f c e llu la r m aterial i s lowered the hydrogen s u lfid e production in the aerobic experiments rap id ly f a l l s to zero w hile the anaerobic hydrogen s u lfid e production remains r e la t iv e ly high*

From t h i s , and other evidence c ite d in th is study, i t i s concluded

th a t cy stein e desulfhydrase i s e s s e n t ia lly an anaerobic reaction*

47 TABLE IX

BIOCHEMICAL CHAEACTEHISTICS OF PROTEUS VULGARIS AND PROTEUS MORGANII USED IN THE PRESENT STUDY C haracteris t i c

Proteus v u lg a ris

Proteus morganii

M o tility

+

+

A cetylm ethylearbinol

-

-

Glucose

AG

AG

Fructose

AG

AG

G alactose

AG

AG

Maltose

AG

-

Sucrose

AG

-

Dextrin

-

-

Lactose

-

-

Mannitol

-

D u lc ito l

♦

Indol

■¥

♦

HaS

♦

-

C itrate U tiliz a tio n

♦

-

G elatin L iquefaction

+

-

Agar Colony

* K lig le r 5s Iron Agar

Gray-Blue, Spreading

+ -

Gray-Blue, E n tire, no spreading

48 TABLE X PRODUCTION OF HYDROGEN SULFIDE FROM VARIOUS SULFUR COMPOUNDS BY PROTEUS VULGARIS AND PROTEUS MORGANII

Compound

H2S Production P. v u lg a ris

P. morganii

Cysteine

100 #

100 #

Cystine

10

%$9656

Homocysteine

Trace

2

%$Homocystine

0

0

C ysteic Acid

0

0

Taurine

0

0

Methionine

0

0

C ysteine S u lfin ic Acid

0

0

C ysteine D isu lfoxid e

0

0

T h io la c tic Acid

0

0

T h io g ly c o llic Acid

0

0

^Determined in Warburg v e s s e ls a? described fo r cy stein e desulfhydrase a c t iv it y

49

TABLE XI HYDROGEN SULFIDE PRODUCTION AS A FUNCTION OF THE NUMBER OF PROTEUS VULGARIS CELLS PRESENT

Number o f C e lls (M illio n s) / Warburg V essel 1 7 , 0 0 0 - 1 8 ,0 0 0

~6

Moles x 10

/h r

13.3

7 ,000-9,000

7.7

5,00 0 -7 ,0 0 0

5 .2

3 ,itOO-lt,UOO

h .2

1 , 6 0 0 - 2 ,0 0 0

3 .3

1 8 0 -2 2 0

l.k

1 2 0 -1 8 0

1 .0

50-80

0 .6

1 0 -1 0

0 .3

3 -1 0

0 .3

1-3

0 .1

Determined in Warburg Vessels as described for cysteine desfulfhydrase activity#

50 TABLE XII COMPOUNDS TESTED SINGLY AND IN COMBINATION FOR ACTIVATION OF CELL FREE EXTRACTS TOWARD CYSTEINE

Adenosine triphosphate

Zinc ion s

Diphosphopyidine n u cleotid e

Cobalt ions

Pantothenic a c id

Ferrous ions

F o lic a c id

Glutathione

B io tin

Guanylic Acid

Pyridoxin©

C ytid ylic a cid

Pyridoxamine

Adenine

Pyridoxal

Adenosine

Glucose-1-phosphate

Adenosine-3-phosphoric acid

Glucos e - 6 -phosphate

Adenosine-5-phosphoric a cid

Fructose-1-6-diphosphate

Thiamin

Pyruvic a c id

N ic o tin ic a cid

Su ccinic a cid

Nicotinamide

Fumaric a cid

Yeast e x tr a c t

Magnesium io n s

B oiled Proteus c e l l ex tr a ct

Manganese ion s

H 2 S PRODUCED MOLES x lO ^ /ra j C E L L S/H O U R

51

5.0 -

4.0 -

2.0 -

1. 0

-

18

16

14

12

10

8

DRY W E IG H T OF CELLS IN MILLIGRAMS

Figure 4 D issim ila tio n o f cy stein e onder aerobic and anaerobic co n d itio n s. CD Aerobic © Anaerobic

6

2

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