the isolation and characterization of a...
TRANSCRIPT
The isolation and characterization of apigment polymer formed by an adenine-
requiring mutant of Saccharomyces cerevisiae
Item Type text; Thesis-Reproduction (electronic)
Authors Gottfried, Richard Joseph, 1939-
Publisher The University of Arizona.
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Link to Item http://hdl.handle.net/10150/318291
THE ISOLATION AND CHARACTERIZATION OF A PIGMENT
POLYMER FORMED BY AN ADENINE-REQUIRING MUTANT
OF SACCHAROMYCES CEREVISIAE
by
R ichard Joseph G o ttf r ie d
A T h esis S u b m itted to th e F acu lty o f th e
DEPARTMENT OF MICROBIOLOGY AND MEDICAL TECHNOLOGY
In P a r t i a l F u lf illm e n t o f th e R eq u irem en ts F o r th e D egree o f
MASTER OF SCIENCE WITH A MAJOR IN MICROBIOLOGY
In th e G raduate College
THE UNIVERSITY OF ARIZONA
1971
STATEMENT BY AUTHOR
T his th e s is has been su b m itte d in p a r t ia l fu lf i l lm e n t o f re q u ire m en ts fo r an advanced degree a t The U n iv e rs ity o f A rizona and is deposited in th e U n iv ers ity L ib ra ry to be m ade availab le to b o rro w ers under ru le s o f th e L ib ra ry .
B r ie f q u o ta tio n s f ro m th is th e s is a re allow able w ith o u t spec ia l p e rm ission , provided t h a t a c c u ra te acknow ledgm ent o f so u rce is m ade. R eq u ests f o r p e rm issio n f o r extended q u o ta tio n f ro m o r rep ro d u c tio n o f th is m an u sc rip t in whole o r in p a r t m ay be g ra n te d by th e head o f th e m ajo r d e p a r tm e n t o r th e Dean o f th e G raduate C ollege when in h is judgm ent th e proposed use o f th e m a te r ia l is in th e in te r e s t s o f scho larsh ip . In a ll o th e r in s tan ces , how ever, p e rm issio n m u s t be ob tained f ro m th e a u th o r.
SIGNED
APPROVAL BY THESIS DIRECTOR
T his th e s is has been approved on th e d a te shown below:
r i n h iJ IRVING YALL ' D a te
P r o fe s s o r o f M icrobiology and M edical Technology
ACKNOWLEDGMENTS
I would like to ex p ress my ap p rec ia tio n to D r. Irv ing Y all, my m ajo r
adv iso r, f o r h is many he lp fu l su g gestions, c o n s tru c tiv e c r i t ic is m s , encour
agem en t and aid during my co u rse o f s tu d y a t The U n iv e rs ity o f A rizona.
I w ish to thank my m o th e r and la te f a th e r f o r encouraging m e to continue
on to g rad u a te s tu d ie s . I especially w an t to thank my w ife and fam ily f o r
th e i r continued encouragem ent and fo r th e many s a c r if ic e s th e y have u n d er
gone to m ake th is possib le .
TABLE OF CONTENTS
LIST OF ILLUSTRATIONS
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
o o o o o o O O O © 0 0 © o
e e o o o o o o e o o ©
o e o o o o o o
Q O O O O 0 G © O o © O ©
p © © o o ©
O O O O O O O O O O O
o o o o o o o o
O rganismG row th m edium e E x tra c tio n p rocedu re Gel f i l t r a t io n Ion exchangeHigh v o ltag e e lec tro p h o re s is B en to n ite a d so rp tio n . . Solvent e x tra c t io n P ig m en t id e n tif ic a tio n K in e tics o f p igm en t fo rm a tio n
o e © o o o o
RESULTS
DISCUSSION
SUMMARY .
REFERENCES
o o o o o o o o o o o o e o o o e
e o e o d o o o o o o
LIST OF ILLUSTRATIONS
F igure P ag e
1. The purine b io sy n th e tic pathw ay, a f t e r Buchanan andH artm an . . . 0 . . . . . . o . . . . . . . . o . . . 3
2. E le c tro p h o re tic sep a ra tio n o f nucleic acid p re c u rso rs ta n d a rd s and n eu tra liz ed p e rch lo ric acid e x t r a c t sa t pH 2, 3j and 4.8 . . . . . . . . . . . . . . . . . . 14
3. High v o ltag e e le c tro p h o re tic an a ly sis o f su p e rn a ta n tand p e lle t f r a c t io n s f ro m a c e to n e -p re c ip ita te dp e rch lo ric acid e x t r a c ts . . . . . . . . . . . . . . . . 17
4. High v o ltag e e le c tro p h o re tic analysis o f s u p e rn a ta n tand p e lle t f r a c t io n s f ro m e th a n o l-p re c ip ita te dp e rch lo ric acid e x t r a c ts . . . . . . . . . . . . . . . . 1 8
5. U ltra v io le t ab so rp tio n s p e c tra ob tained w ithS-adenosylm eth ionine in 6 N su lfu r ic acid b e fo re (------- ) and a f t e r (-------- ) p igm en t rem o v a l bye th an o l p re c ip ita tio n . . . . . . . . . . . . . . . . . 20
6. C om parison o f th e v is ib le s p e c tra o f th e B ra t to n -M arsha ll chrom ophores produced f ro m th e n e u tr a lized p e rch lo ric acid e x t r a c t (--------) and f ro m th ep u rif ied p igm ent ( ) . . . . . . . . . . . . . . . . 22
7. R ela tionsh ip o f p igm ent co n ce n tra tio n to o p tic a ld en sity , m easu red a s th e B ra tto n -M a rsh a llchrom ophore . . . . . . . . . . . . . . . . . . . . . 23
8. C om parison o f th e am ount o f p igm en t fo rm ed in th ece lls (--------) to ce ll g row th , a s m easured bytu rb id ity ̂ ) . . . . . . . . . . . . . @ . . . . . . 25
v
ABSTRACT
A p rocedu re has been developed fo r th e iso la tio n o f th e red p igm ent
f orm ed by an ad en ine-requ iring m u ta n t o f Saccharom yces c e re v is ia e , c o n s is t
ing o f p re c ip ita tio n in 90% ethano l, follow ed by f u r th e r p u r if ic a tio n by high
vo ltage e le c tro p h o re s is and gel f i l t r a t io n . T h is p rocedu re a lso allows th e
se p a ra tio n and iso la tio n o f RNA and nucleic acid p re c u rso rs f ro m th e soluble
pool o f th e se ce lls . The p igm ent c o n s is ts o f polym ers o f random chain leng th ,
p rev en tin g i t s s e p a ra tio n f ro m th e se com ponents by ion exchange c h ro m a to
graphy.
Id e n tif ic a tio n o f th e p igm ent as 5-am inoim idazole r ib o tid e was based
on th e ab so rp tio n sp e c tru m o f i t s B ra tto n -M a rsh a ll d e riv a tiv e , i t s negative
re a c tio n w ith th e Pauley re a g e n t fo r im idazo les, and i t s ab so rp tio n s p e c tra(s~x
in th e u l tr a v io le t and v is ib le ran g es. On th e b a sis o f i t s accum ulation in th e s e
ce lls , th e m u ta n t block in th is o rgan ism has been id e n tif ie d a s th e re a c tio n
co nverting 5-am inoim idazole r ib o tid e to 5 -am ino-4-im idazo lecarboxy lic acid
rib o tid e , an.Ad-2 m u ta tio n .
The B ra tto n -M a rsh a ll chrom ophore was used fo r th e sp ec ific m e a su re
m en t o f p igm ent fo rm a tio n during grow th o f th e m u ta n t in exogenous adenine.
P ig m en t fo rm a tio n w as found to begin during log phase, b e fo re v isib le p igm ent
is fo rm ed in th e ce lls , and i t was found to reach a m axim um during th e
s ta t io n a ry phase o f g row th .
INTRODUCTION
E arly s tu d ie s by Y all (29) and by Y all e t a l. (31) on an adenine-
requ iring , pink, m u ta n t o f th e y e a s t , Saccharom yces ce rev is ia e , have shown
t h a t th e m u ta n t’s adenine re q u ire m e n t can be s a t i s f ie d by supplying th e ce lls
w ith an exogenous source o f adenine, hypoxanthine, o r S -adenosylm eth ionine
(S-AM). Guanine and xan th ine cannot s a t i s f y th e re q u ire m e n t, no r can a c t iv i ty
f ro m "^C -labeled exogenous guanine be found in adenine re is o la te d f ro m th e
ce lls (Yall, unpublished r e s u l ts ) , ind ica ting th a t th is m u ta n t cannot re v e rs e
th e re a c tio n which c o n v e rts adenine to guanine via xan th ine . In addition,
adenosine, adenylic acid (AMP), adenosine d iphosphate (ADP), adenosine t r i
phosphate (ATP), and m ethy lth ioadenosine (MTA) cannot se rv e a s exogenous
purine so u rces f o r th is m u ta n t. A lthough sm all am ounts o f adenosine w ere
found by t r a c e r s tu d ie s to e n te r th e ce lls , i t could n o t su p p o rt th e i r g row th
(29). V ery l i t t l e MTA w as found to e n te r th e ce lls , and th e ce lls w ere t h e r e
fo re considered im perm eable to th is compound (31). S-A denosylhom ocysteine
(S-AH) can e n te r th e ce lls and se rv e a s a purine source only i f th e ce lls a re
t r a n s f e r r e d in to m edium contain ing th is supplem ent during th e log phase o f
g row th (16).
S tud ies by Knudsen (15) and Knudsen, M oore and Y all (16) on th e
m etab o lism o f S-AM and S-AH in th is m u ta n t showed t h a t exogenously supplied,
-labeled adenine, S-AM and S-AH a re in co rp o ra ted in to th e purine pool
and nucleic acids o f th e m u ta n t c e lls . S-AM and S-AH a re broken down by th e
ce lls to supply adenine f o r g row th . M easu rem en t o f th e sp e c if ic a c t iv i t ie s
o f th e se compounds, re is o la te d f ro m ce ll e x t r a c ts , showed each compound to
be d ilu ted by tu rn o v e r o f unlabeled adenine f ro m th e c e l l 's nucleic ac id s. The
e x te n t o f th is d ilu tion , how ever, w as found to be m uch g r e a te r th an could be
explained e n tire ly by th e tu rn o v e r o f unlabeled adenine p re s e n t in th e ce lls .
p r io r to th e add ition o f th e labeled compounds.
F u r th e r s tu d ie s on th e in co rp o ra tio n o f -lab e led fo rm a te and
glycine in to th e se ce lls showed t h a t th e lab e l w as in co rp o ra ted in to b o th
adenine and guanine (13, 15, 30). F o rm a te h as been shown to be in co rp o ra ted
in to th e purine rin g s t r u c tu r e a t tw o p o in ts during i t s b io sy n th es is — s te p s
4 and 10 shown in F ig . 1, Glycine may be in co rp o ra ted a s an in ta c t m olecule
a t s te p 3 (5). The in co rp o ra tio n o f labeled fo rm a te and glycine by th is m u ta n t
in d ic a te s t h a t th e se ce lls may have som e m eans o f overcom ing th e m u ta n t
block in th e purine b io sy n th e tic pathw ay so t h a t de novo sy n th es is can occu r.
A f te r exhaustion o f exogenous adenine supp lem ent, th e m u ta n t c e lls
cease to grow, and th e y fo rm a pale re d p igm en t (31). W ith adenine plus
L -m eth ion ine, th e co lo r becom es deep red ; exogenous S-AM causes th e fo r m
a tio n o f a b r ic k -re d p igm en t. T h is p igm ent is an in te rm e d ia te p roduct o f
purine b io sy n th esis , which accu m u la tes due to th e block in th e purine b io
sy n th e tic pathw ay.
Fig . 1. The purine b io sy n th e tic pathw ay, a f t e r Buchanan and H artm an (5).
The common ab b rev ia tio n s a re l is te d in p a re n th e se s below th e nam e o f th e in te rm e d ia te s .
3
Ribo se-5 -p h o sp h a te
(1> i5 -P hosphoribosy 1-pyrophosphate
(2) 1
Phosphoribosy lam ine(PRA)
(3) r GLYCINE
5 -P hosphog lycinam ide-ribo tide(GAR)
(4) r ■FORMATE
F orm axn ide-g lycinam ide-ribo tide
(5) | <FGAR>
F o rm ylg lycinam ide-r ibo tide(6, | (FGAM)5-A m ino im idazo le-ribo tide(7, | ^
5-A m .ino-4-im idazole-carboxylic acid r ib o tid e
(8) | ^5 -A m ino-4-im idazo le-N -succino-carboxam ide r ib o tid e
(9, | <SAICAR) .
5 -Amino -4 -im idazo le-carboxam ide r ib o tid e(AICAR)
(10) f--------------------------FORMATEV
5-F orm am ide-4~ im idazo le-carboxam ide r ib o tid e
( i d J .(FAICAS)
Inosine m onophosphate
ADENINE GUANINE
F ig . 1. The purine b io sy n th e tic pathw ay.
S im ilar p igm en t fo rm a tio n has been re p o r te d in th e l i t e r a tu r e f o r
aden ine-requ iring m u ta n ts o f y e a s t (6, 8, 11, 21, 23, 27), m olds (3, 11), and
b a c te r ia (11, 20, 22). Rom an (23) p o s tu la te d t h a t tw o m u ta tio n a l blocks in th e
purine pathw ay could lead to th e accum ula tion o f a red -p ig m en ted in te rm e d ia te ,
and he designa ted th e se Ad-1 and Ad-2 m u ta tio n s . T hese r e s u l t e i th e r f ro m
th e m u ta tio n a l block occu rrin g in th e re a c tio n co n v ertin g 5-am in o -4 -im id azo le -
carboxylic acid r ib o tid e (CAIR) to 5 -am m o-4-im idazo le-N -succind -carboxam ide
r ib o tid e (SAICAR) in th e Ad-1 m u ta n ts (F ig. 1, s te p 8), o r f ro m th e block
occu rrin g in th e re a c tio n converting 5-am inoim idazole r ib o tid e (AIR) to CAIR
(Fig. 1, s te p 7), in th e Ad-2 m u ta n ts (21). T he p ro d u c ts which accum ula te in
th e se m u ta n ts a re CAIR (in th e Ad-1 m u ta n ts ) o r AIR (in th e Ad-2 m u ta n ts ) .
C onventional m ethods o f s ep a ra tin g and pu rify in g nucleic acid p re
c u rso rs f ro m ce ll e x t r a c ts o f th e se m u ta n ts , p a r tic u la r ly th e ch ro m ato g rap h
ic re so lu tio n o f th e s e compounds, have been u n su ccess fu l due to th e
biochem ical p ro p e r tie s o f th e se p ig m en ts (26). B oth th e A d-1 and Ad-2
m u ta n ts may accu m u la te AIR. CAIR is an u n s tab le compound which b reaks
down to fo rm AIR (21). In th e p re sen ce o f oxygen, AIR w ill po lym erize to fo rm
th e c h a r a c te r is t ic re d p igm en t (26). T h is p igm ent has been s tu d ied by
Sm irnov e t ah (27) and has been found to c o n s is t o f m ixed po lym ers o f AIR,
which v a ry w idely in chain leng th , and which con tain a num ber o f amino ac ids
bound to th e backbone th rough th e am ide groups.
5
Ion exchange ch rom atography h as been used by m any in v e s t ig a to rs
to iso la te th e p igm ent fo rm ed by such m u ta n ts (3, 11, 18, 20, 22). The o th e r
com ponents o f th e nucleic acid and soluble pools, how ever, -could n o t be
iso la ted by th e se a u th o rs . S tud ies on th e purine b io sy n th e tic pathw ay o f th e
m u ta n t re q u ire t h a t com ponents o f th e nucleic acid and soluble pools be
iso la te d in pu re fo rm and in q u a n tita t iv e am oun ts . I t is p a r tic u la r ly im p o r ta n t
to rem ove th e p igm ent po lym ers f ro m th e s e com ponents, a s -^C -labeled
fo rm a te and glycine a re in co rp o ra ted in to th e p igm ent a t s te p s 3 and 4 ,
shown in F ig . 1.
T h e re a re th re e approaches to th is problem . Knudsen e t a l . (16)
iso la te d com ponents o f th e nucleic acid and soluble pools f ro m e x t r a c ts o f
ce lls h a rv e s te d during th e log phase o f g row th , b e fo re th e exogenous purine
so u rces w ere exhausted and p igm ent fo rm a tio n occu rred . Iso la tio n p ro ced u res
under th e se conditions a re u n su itab le f o r studying th e de novo purine b io
sy n th e s is o f th e m u ta n t, a s th e purine pathw ay may be p a r t ia l ly o r to ta l ly
sh u t down a t th is s ta g e o f ce ll g row th . B urns (6) found t h a t th e p re sen ce o f
exogenous adenine in h ib its th e e n tire purine b io sy n th e tic pathw ay in a s im ila r
m u ta n t o f Saccharom yces c e rev is ia e .
S ilver and E a to n (26) iso la ted p igm en t f o r c h a ra c te r iz a tio n by grow
ing th e i r ce lls under anaerobic cond itions, th u s p rev en tin g th e oxygen-
ca ta liz e d po ly m eriza tio n o f th e AIR m onom er u n its . T h is m ethod does n o t,
how ever, p re v e n t AIR f ro m polym erizing when exposed to oxygen during
subsequent se p a ra tio n and p u r if ic a tio n p ro ced u res .
6
The th ird approach is to s e p a ra te th e p igm ent, e x tra c te d f ro m th e
m u ta n t c e lls h a rv e s te d f ro m s ta t io n a ry phase c u ltu re s a f t e r exhaustion o f
th e exogenous purine supplem ent, f ro m th e o th e r com ponents. The m ajo r
ob jective o f th is study w as to develop a m ethod f o r th e co m p le te sep a ra tio n
o f a ll com ponents o f th e nucleic acid and soluble p re c u rso r pools o f th e se
c e lls , and th e i r p u r if ic a tio n f o r subsequen t q u a n tita t iv e s tu d ie s on de novo
purine b io sy n th esis . In addition , th e id e n tif ic a tio n o f th e m u ta n t block in th e
b io sy n th e tic pathw ay has been d e te rm in ed by th e id e n tif ic a tio n o f th e p igm en t,
and th e k in e tic s o f p igm en t fo rm a tio n has been in v e s tig a te d .
MATERIALS AND METHODS
O rganism
Saccharom yces c e rey is iae SC -10-80-3-5 , an ad en in e-requ iring
s tr a in , o rig inally supplied by F. P„ H ungate (G eneral E le c tr ic Co., R ichland,
W ashington), w as m ain ta ined on ag ar s la n ts (m a lt e x t r a c t , 0.6%; y e a s t
e x tr a c t , 0.6%; pep tone, 1%; glucose, 2%; ag ar, 2%) by incubation a t 25 C
f o r 48 hours and a t 4 C u n ti l u se . S ubcu ltu res w ere m ade a t app rox im ate ly
m onthly in te rv a ls .
G row th Medium
The sy n th e tic m edium described by Rom an (23), supplem ented w ith
6^iM adenine and 400 jiM m ethionine p e r 100 m l, w as used f o r m u ta n t grow th
and p igm ent fo rm a tio n . C ells w ere grown in 100 m l volum es, contained in
500 m l, co tton -p lugged , E rlen m ey er f la s k s , incubated a t 25 C, w ith c o n s ta n t
shaking.
E x tra c tio n P ro ced u re
C u ltu re s fo r p igm ent fo rm a tio n w ere h a rv e s te d a f t e r approx im ate ly
36 h r g row th in Rom an’s m edium , cen tr ifu g e d , and w ashed w ith saline . T he
ce lls w ere e x tra c te d w ith th re e volum es o f 1.5 N p e rch lo ric acid by th e
p rocedu re described by Knudsen e t a l. (16). A f te r th r e e e x tra c tio n s , th e
7
su p e rn a ta n t flu id s w ere pooled, n e u tra liz ed w ith 10% KOH, and allowed to
p re c ip i ta te overn igh t in th e cold. The p o ta ss iu m p e rc h lo ra te p e rc ip i ta te w as
rem oved by c e n tr ifu g a tio n a t 5000 X g, and th e n e u tra liz ed e x t r a c ts w ere
fro z e n u n til u se .
Gel F i l t r a t io n
M olecular seive ch rom atography w as ru n on B io-G el P -2 and P -1 0
gels (Bio-Rad L a b o ra to r ie s , Richmond, C a lifo rn ia ) and on Sephadex G-10 gel
(P harm acia F ine C hem icals, U ppsala, Sweden). The gel beds w ere p rep ared
according to th e m a n u fa c tu re rs ’ recom m endations, and w as e lu ted w ith
e i th e r d is tille d w a te r o r 0.5 M phosphate b u f fe r , pH 7. In a ll column ch rom
a tog raphy p ro ced u res , th e f r a c t io n s w ere m o n ito red w ith an LKB Uvicord
a b so rp tio n m e te r and co lle c ted w ith th e aid o f a f r a c t io n c o lle c to r .
Ion Exchange
Dow ex-50 (N af) (Bio-Rad L a b o ra to rie s ) was used f o r th e iso la tio n
o f S-AM by th e m ethod ou tlined by Shapiro and Ehninger (25). D E A E -cellu lose
(Cellex-D , B io-R ad L ab o ra to ries .) w as p rep a red f o r p igm en t iso la tio n by th e
m ethod o f Sm irnov e t a l. (27). H ydroxyapatite (B io-G el HT, B io-R ad
L a b o ra to rie s ) was p rep a red according to m a n u fa c tu re r’s recom m endations,
and was e lu ted by a s tep w ise , in c reasin g ionic co n cen tra tio n , g rad ien t o f
phosphate b u f fe r , pH 6.8.
High V oltage E le c tro p h o re s is
A n a ly tica l e le c tro p h o re tic sep a ra tio n s w ere ru n on S ch leicher and •
Schuell 3043-B paper, 20 x 40 cm, in th e Cam ag high v o ltag e e le c tro p h o re s is
a p p a ra tu s . In it ia l in v e s tig a tio n s ru n under th e conditions suggested by
S tran sk y (28) f o r th e se p a ra tio n o f adenine n uc leo tides, showed t h a t th e
com ponents o f th e n e u tra liz ed p e rch lo ric acid e x t r a c ts could b e s t be s e p a r a t
ed in 0.01 M c i t r ic acid -sod ium c i t r a t e b u f fe r , pH 3.0.
Known nucleic acid p re c u rso r s tan d a rd s w ere applied as sp o ts to th e
c e n te r o f th e b u ffe r-so a k e d paper. The p e rch lo ric acid e x t r a c t could n o t be
applied in th is m anner, as th e individual com ponents w ere to o low in concen
t r a t io n to be d e te c te d a f t e r e le c tro p h o re tic sep a ra tio n . T hese sam ples w ere
d ried onto th e pap er, using sev e ra l ap p lica tio n s p e r sp o t. The b u f fe r w as
applied to th e se p ap ers f ro m b o th ends, allowing th e b u f f e r f r o n ts to reach
th e sam ple sp o ts sim ultaneously .
S ep ara tio n s w ere ru n f o r 30-45 m in a t 3000 v o lts . A f te r a i r drying,
th e com ponents w ere lo ca ted a s dark, absorb ing sp o ts a t w avelengths o f
app rox im ate ly 260 nm.
B en to n ite A dso rp tion
B en to n ite w as p rep a red by th e m ethod outlined by F ra e n k e l-C o n ra t,
Sanger, and T su g ita (10) and resuspended to a c o n ce n tra tio n o f 1.5% in 0.01.K
sodium a c e ta te b u f f e r o f th e d esired pH. To one m l o f th e n eu tra liz ed
p e rch lo ric acid e x t r a c t w as added 100 mg b e n to n ite , plus 0.04 m l o f 0.1 M
. 1 0
EDTA. This m ix tu re was s t i r r e d v igerously f o r 30 m in a t room te m p e ra tu re ,
and th e b en to n ite was rem oved by c e n tr ifu g a tio n a t 120,000 X g in th e B eckm an
Model L u ltr a c e n tr ifu g e . The su p e rn a ta n t flu id was exam ined f o r lo ss o f
u ltra v io le t-a b so rb in g m a te r ia l a t 260 nm in th e Beckm an DU sp ec tro p h o to
m e te r , using one cm q u a r tz c u v e tte s . The individual com ponents w ere
exam ined by high vo ltag e e le c tro p h o re s is .
Solvent E x tra c tio n
N eu tra lized p e rch lo ric acid e x t r a c ts w ere p re c ip ita te d w ith ace to n e
o r e thano l in an ice b a th f o r 30 m in. The p re c ip i ta te w as rem oved by
c e n tr ifu g a tio n a t 27,000 X g fo r 30 m in a t 4 C in th e S erva ll RC-2 r e f r i g e r a t
ed c e n tr ifu g e . The so lv en t was ev ap o ra ted f ro m th e s u p e rn a ta n t f r a c t io n
in th e cold by a sp ira tin g a ir th rough i t . The su p e rn a ta n t and p re c ip ita te
f r a c t io n s w ere resuspended in sm all volum es o f d is til le d w a te r . The com pon
e n ts o f bo th f r a c t io n s w ere exam ined by high vo ltage e le c tro p h o re s is .
F ig m en t Id e n tif ic a tio nv
P u r if ie d p igm ent, iso la ted f ro m th e n eu tra liz ed p e rch lo ric acid
e x tr a c t , w as id e n tif ie d by th e exam ination o f th e v is ib le ab so rp tio n sp e c tru m
o f i t s B ra tto n -M a rsh a ll d e riv a tiv e , fo rm ed by th e m ethod o f F laks and
Lukens (9). The Pauley re a c tio n fo r th e .id e n tif ic a t io n o f im idazoles w as
p e rfo rm ed as described by Ames and M itch e ll (1). The v is ib le and u l tr a v io le t
ab so rp tio n s p e c tra o f th e p igm ent be tw een 200 and 600 nm w ere d e te rm in ed
using th e Beckm an DU sp e c tro p h o to m e te r .
K in e tic s o f P ig m en t F o rm atio n
M u tan t ce lls w ere grown in te n l i t e r s o f Rom an1 s m edium supp lem en t
ed w ith adenine and L -m eth ion ine, in a 12 l i t e r V ir t i s f e rm e n te r , a t 25 C,
a e ra te d a t 10 p s i w ith a flow r a t e o f 40%, and a g ita te d by s t i r r in g a t a r a t e
o f 3500 rp m e T u rb id ity m easu rem en ts w ere m ade on th e B ausch and Lomb
S pectron ic-20 sp e c tro p h o to m e te r a t 540 nm. During th e la te log and s ta t io n
a ry phases o f grow th , 200 m l sam ples w ere ta k en f o r p igm ent as say. The
ce lls w ere cen trifu g ed , washed w ith saline, and lyophilized to d ryness.
P ig m en t was e x tra c te d f ro m weighed sam ples o f d ried cells w ith
1.5 N p e rch lo ric acid, as a lready described . The p igm ent w as converted to
th e B ra tto n -M a rsh a ll chrom ophore and m easu red sp e c tro p h o to m e tr ic a lly
in th e Beckm an DU at: 505 nm.
RESULTS
P re lim in a ry in v e s tig a tio n s showed t h a t an a r t i f i c i a l m ix tu re o f
adenine, S-AM and S-AH could be co m p le te ly s e p a ra te d on a Sephadex G-10
column by e lu tio n w ith phosphate b u f f e r , Sephadex G-10 excludes m olecules
having a m olecu lar w eigh t g r e a te r th a n 700 da lto n s f ro m th e gel m a tr ix
(Sephadex tech n ica l l i te r a tu r e ) , allowing th e m to p ass th rough th e gel
u n re ta rd e d , w hereas m olecules which have m olecu lar w e ig h ts below 700
da lto n s a re r e ta rd e d to vary ing deg rees, depending on th e i r m olecu lar s ize .
The la rg e m o lecu lar w eight o f th e p igm en t po lym ers su g g ested th a t th ey
could be rem oved f ro m th e p e rch lo ric acid e x t r a c t in th is m anner. No s e p a r a t
ion occu rred on Sephadex G-10, no r did i t occur on B io-G el P -2 o r B io-G el
P -1 0 , which exclude m olecules having m olecu lar w eigh ts in excess o f 1800 and
20,000 d a lto n s , re sp e c tiv e ly (Bio-Rad te ch n ica l l i t e r a tu r e ) .
Gel f i l t r a t io n was unable to give sharp se p a ra tio n o f any of th e
com ponents o r groups o f com ponents because o f th e la rg e num ber o f m o lecu lar
sp ec ies, closely r e la te d in m o lecu lar w e ig h t, B raun (4) found th a t Sephadex
G-10 is u n su itab le f o r sep a ra tio n o f com plex m ix tu re s . T he la rg e r th e
num ber o f com ponents, th e p o o re r th e re so lu tio n o f th e individual peaks
becom es.
12
13
High v o ltage e le c tro p h o re s is o f known nucleo tide, nucleoside, and
o th e r nucleic acid p re c u rso rs gave good sep a ra tio n . F ig . 2 shows th e
re so lu tio n ob tained a t th r e e pH lev els.
A t pH 2, m o s t o f th e com ponents m ig ra te d to w ard th e cathode, o r
rem ained n ea r th e orig in on th e anodal side. E xam ination o f th e p e rch lo ric
acid e x t r a c t showed t h a t th e p igm en t m ig ra te d s lig h tly to w ard th e cathode,
occupying th e sam e p o s itio n a s AMP, A DP, and A TP. A t pH 4.8, th e se th re e
com ponents w ere s e p a ra te d f ro m th e p igm en t, b u t th e p igm en t m ig ra te d to
th e sam e p o s itio n a s did adenine, adenosine, S-AM and S-AH.
A t pH 3, th e p igm en t rem ained a t th e origin, w h ereas th e o th e r
com ponents m ig ra te d away fro m i t , to w ard e i th e r th e anode o r th e cathode.
H ypoxanthine, inosine and AMP w ere lo ca ted very c lose to th e p o sitio n
occupied by th e p igm ent, and th is m ay m ake th e i r s e p a ra tio n d if f ic u l t by
th is m ethod.
B a tch se p a ra tio n o f th e com ponents o f th e p e rch lo ric acid e x t r a c t
was ob tained by applying th e sam ple to a s t r ip o f f i l t e r p aper, 2 x 150 mm ,
which w as laid on th e o rig in o f th e b u ffe r-so a k e d p aper. T h is m ethod o f
app lication p rev en ted uneven m ig ra tio n caused by th e ap p lica tio n o f too
c o n ce n tra te d a sam ple in a single sp o t. E xam ination o f th e com ponents
e lu ted f ro m th e pap er in d is tille d w a te r ind ica ted th a t m any o f th e bands
could be re so lv ed in to tw o o r m ore com ponents by gel f i l t r a t i o n on Sephadex
G-10.
14
A
pH 2
B
+ CDRNA
<£UJZZZ2>
CDAMP A DP ATP
CD CDAd SAMAdsSAH
A
pH 3
B
pH 4.8
B
ATPCDA DP
CD CD CDHx Ad,Ads SAMIs
RNAAMPSAH
C 7cz>o a w zsm
+ C D CZ> OATP A DP AMP
C=RNA
C
CD C7 2D
CD Ad Ads
Is SAH Hx SAM
<2^> C ?
tO rigin
F ig . 2. E le c tro p h o re tic sep a ra tio n o f nucleic acid p re c u rso r s tan d a rd s and n e u tra liz ed p e rch lo ric acid e x t r a c ts a t pH 2 ,3 , and 4.8.
A. S tandardsB. N eu tra lized p e rch lo ric acid e x t r a c t s . H atched a re a ind ica tes
p igm en t.
A b b rev ia tio n s: Ad AdenineAds AdenosineAMP Adenylic acidADP Adenosine d iphosphateATP A denosine tr ip h o sp h a teSAH S-adenosy Ihom ocysteineSAM S -adenosy Im eth ionineHx HypoxanthineIs Inosine
15
High vo ltage e lec tro p h o re s is tu rn e d o u t to be to o cum bersom e a
p rocedure f o r th e b a tch sep a ra tio n o f th e individual com ponents o f th e
p e rch lo ric acid e x t r a c ts . I t w as, how ever, a valuable techn ique f o r analyzing
th e e ffe c t iv e n e s s o f o th e r m ethods. Sm all am ounts o f p igm en t a re concen
t r a t e d a t th e o rig in and can be easily lo ca ted by th e i r orange o r red d ish -
orange appearance under u l tr a v io le t l ig h t.
B en to n ite ad so rp tio n has been used f o r th e p u r if ic a tio n o f nucleic
acid p re p a ra tio n s by sp ec ifica lly adsorb ing p ro te in s , such a s ribonuclease (10).
The amino acid c o n te n t o f th e p igm ent, shown by Sm irnov e t a l. (27) su g g ested
th a t b e n to n ite ad so rp tio n could be used a s a m ethod f o r th e rem o v a l o f th e
p igm ent f ro m crude p e rch lo ric acid e x t r a c t s .
B en to n ite w as found to rem ove m uch o f th e p igm en t f ro m th e crude
e x t r a c ts , b u t high v o ltage e le c tro p h o re tic an aly sis showed t h a t th e p igm ent
was n o t e n tire ly rem oved a t pH 6.8 R ecom m ended by F rae n k e l-C o n ra t e t a l.
(10)_/, no r a t any pH betw een 5.0 and 8.1. A t pH 8.1, v e ry l i t t l e p igm ent was
rem oved. E xam ination o f known nucleic acid p re c u rso r s ta n d a rd s showed t h a t
app rox im ate ly 30% o f th e adenine s ta n d a rd w as adsorbed to th e b en to n ite . T h is
m ethod w as abandoned fo r th e se re a so n s .
Hydroxy a p a t i te has been used to f r a c t io n a te nucleic acids on th e
b a s is o f th e i r lin ea r s ize and n e t charge (2). D ilu te b u f f e r e lu te s low
m olecu lar w eight com ponents, w hereas th e longer po lym ers a re re ta in e d ,
being e lu ted by h igher ionic c o n ce n tra tio n s o f phosphate b u f f e r . S ep ara tio n
o f th e crude p e rch lo ric acid e x t r a c t on h y d ro x y ap a tite rem oved th e sm a lle r
16
nu c leo tid es , nucleosides, and re la te d com ponents, f ro m th e re d p igm en t.
However, th e sm a lle r p igm en t po lym ers w ere a lso e lu ted a t th is ionic
co n cen tra tio n . T hese w ere yellow in co lo r, and w ere found to ag g reg a te to
fo rm th e c h a r a c te r is t ic o ran g e -red p igm en t sp o t a f t e r se p a ra tio n by high
vo ltage e le c tro p h o re s is .
Iso la tio n o f th e p igm ent on D E A E -cellu lose by th e m ethod d escribed
by Sm irnov e t a l . (27) could n o t be dup lica ted , a s th e p igm en t w as n o t
adsorbed to th e colum n under th e conditions th ey d esc rib ed . The p igm ent
was e lu ted w ith th e sm a lle r com ponents o f th e p e rch lo ric acid e x t r a c t .
The n e u tra liz ed p e rch lo ric acid e x t r a c ts w ere p re c ip ita te d w ith
e th an o l o r ace to n e a t co n ce n tra tio n s betw een 50 and 90% so lv en t c o n c e n tra t
ions. F ig . 3 shows th e r e s u l t s o f e le c tro p h o re tic analy sis o f th e ace to n e -
' p re c ip ita te d f r a c t io n s , and F ig . 4 shows th e e le c tro p h o re tic an aly sis o f th e
e th a n o l-p re c ip ita te d f r a c t io n s .
A cetone p re c ip ita tio n rem oved m o s t o f th e sm a lle r com ponents f ro m
th e e x t r a c t a t th e 90% co n ce n tra tio n . N e ith e r so lv en t rem oved any d e te c ta b le
m a te r ia ls below 70% so lv en t c o n ce n tra tio n . A lm o st a ll th e p igm ent was
rem oved by p re c ip ita tio n in 90% e thano l, which se p a ra te d bo th p igm ent and
RNA fro m th e sm a lle r com ponents. Since th e ace to n e p re c ip ita tio n rem oved
many o f th e sm a lle r m olecu lar sp ec ies , 90% ethano l p re c ip ita tio n was chosen
fo r f u r th e r in v es tig a tio n . The p igm ent rem ain ing co n sis ted o f sm a lle r m o le
cu la r w eight spec ies which could n o t be p re c ip ita te d by e i th e r so lven t.
17
SUPERNATANT FRACTION
90% + t ^ o -
80% c o - < c s
70% § c o < 3
WHOLE EXTRACT
c------ — ' -_) tz z ' ■'x s-— x z-— -xV— ----——— '~c.
PE L L E T FRACTIONS
90% ----- "X ^r ----> %
80% C -J g 9 C )
70% , c _ : y s 9 = _
tO rigin
Fig. 3. High v o ltag e e le c tro p h o re tic an a ly sis o f su p e rn a ta n t and p e lle t f r a c t io n s f ro m a c e to n e -p re c ip ita te d pe rch lo ric acid e x tr a c ts .
The p e rc e n t ace to n e in re la tio n to e x tr a c t is in d ica ted a t l e f t . P ig m en t is in d ica ted by th e ha tch ed a rea .
18
SUPERNATANT FRACTIONS
90% +
80% Q> CD CD
70% c C CD CD>
WHOLE EXTRACT
PE L L E T FRACTIONS
90% C_
80%
70% +
IO rigin
Fig . 4. High vo ltage e le c tro p h o re tic analy sis o f su p e rn a ta n t and p e lle t f r a c t io n s f ro m e th a n o l-p re c ip ita te d pe rch lo ric acid e x tr a c ts .
The p e rc e n t e thano l in re la tio n to e x t r a c t is ind ica ted a t l e f t . P ig m en t is ind ica ted by th e ha tch ed a re a .
19
A f te r p re c ip ita tin g th e n e u tra liz ed p e rch lo ric acid e x t r a c t in 90%
ethano l to rem ove th e p igm en t, S-AM w as iso la ted f ro m th e su p e rn a ta n t
f r a c t io n by chrom atography on Dow ex-50 (Na+ ) ion exchange re s in . The u l t r a
v io le t ab so rp tio n o f th is compound in 6 N su lfu r ic acid w as com pared to t h a t
o f S-AM iso la ted f ro m th e n e u tra liz ed p e rch lo ric acid e x t r a c t w ith o u t p r io r
p re c ip ita tio n . The d if fe re n c e shown be tw een th e tw o ab so rp tio n s p e c tra
(Fig. 5) is due to th e p re sen ce o f p igm ent in th e u n p re c ip ita te d sam ple. T he
S-AM iso la ted a f t e r e th an o l p re c ip ita tio n has an ab so rp tio n m axim um a t
256 nm w avelength , which ag rees w ith th e c h a r a c te r is t ic s o f th e S-AM
iso la ted by Schlenk and D ePalm a (24).
E le c tro p h o re tic analy sis o f th e p re c ip i ta te f r a c t io n f ro m 90% e th an o l
(Fig. 4) showed only tw o com ponents, RNA and p igm en t, which a re widely
se p a ra te d a t pH 3. A la rg e b a tch o f p re c ip ita te d p igm ent w as sep a ra te d f ro m
th e RNA by e le c tro p h o re s is , e lu ted f ro m th e paper in d is tille d w a te r , and
p u rif ied by gel f i l t r a t io n o f Sephadex G-10. A f te r e lu tio n o f th e p igm ent f ro m
th e column, an add itiona l peak o f u l tr a v io le t-a b sorbing m a te r ia l was e lu ted
fro m th e column. T his l a t t e r peak could be e i th e r a sm a ll m olecu lar w eigh t
com ponent which had th e sam e e le c tro p h o re tic p ro p e r tie s a s th e p igm ent, and
which c o -p re c ip ita te d w ith i t , o r a n o n -sp ec if ic su b stan ce e lu ted fro m th e
paper i t s e l f . The p igm en t peak w as th e n c o n ce n tra te d by lyophilization .
The ab so rp tio n s p e c tra o f th e p u rif ied p igm ent in th e u l tr a v io le t and
v isib le ranges showed no ab so rp tio n peaks betw een w aveleng ths o f 200 and
600 nm . The B ra tto n -M a rsh a ll chrom ophore derived f ro m th e p u rif ied
r
Opt
ical
de
nsit
y
20
0.400
0.300
2560.200
2520.100
0.000280 300240 260220
W avelength (nm)
Fig. 5. U ltra v io le t ab so rp tio n s p e c tra obtained w ith S-adenosylm eth ioninein 6 N su lfu ric acid, b e fo re (--------) and a f t e r p igm ent rem oval (------- )by e thano l p re c ip ita tio n .
21
pigm ent showed a single ab so rp tio n peak in th e v isib le range, w ith i t s m axim um
ab so rp tio n a t 505 nm (Fig. 6). T h is in d ica te s t h a t th e p igm en t is com posed o f
5 -am inoim idazole r ib o tid e (AIR), r a th e r th a n 5 -am ino -4 -im idazo le carboxylic
acid r ib o tid e (CAIR), which has an ab so rp tio n m axim um a t 420 nm (26).
The Pauley re a c tio n f o r im idazo les, which gives a b lue-co lo red
compound when re a c te d w ith CAIR (1) w as negative when r e a c te d w ith th e
p u rif ied p igm en t f ro m th is m u ta n t.
The n eu tra liz ed p e rch lo ric acid e x t r a c t w as r e a c te d w ith th e
B ra tto n -M a rsh a ll re a g e n ts and th e v isib le sp e c tru m ob tained w as com pared
w ith t h a t o f th e p u rif ied p igm en t (F ig. 6). The ab so rp tio n m axim um w as
found to be a t 506 nm, r a th e r th a n 504 nm ob tained w ith th e p u rif ied p ig m en t.
The v isib le ab so rp tio n sp e c tru m o f th e crude p e rch lo ric acid e x t r a c t showed
no ab so rp tio n peaks, being q u ite s im ila r to th e ab so rp tio n sp e c tru m ob tained
fro m th e p u rif ied p igm ent.
D ilu tions o f th e p e rch lo ric acid e x t r a c t w ere p rep a red , and th e
B ra tto n -M a rsh a ll chrom ophore w as fo rm ed in each. F ig . 7 shows th a t a
lin ea r re la tio n sh ip e x is ts be tw een th e p igm en t co n ce n tra tio n and o p tica l
d en sity a t 505 nm. E quivalen t d ilu tions o f th e p e rch lo ric acid e x t r a c t did n o t
give any appreciab le ab so rp tio n a t th is w avelength . The s p e c if ic ity o f th e
B ra tto n -M a rsh a ll re a c tio n can be used f o r th e sp e c tro p h o to m e tr ic m e asu re
m en t o f th e am ount o f p igm en t sy n th esized in th e ce lls during grow th .
P ig m en t fo rm a tio n during c e ll g row th was exam ined in ce lls growing
in R om an 's m edium , supplem ented w ith adenine and L -m eth io n in e . F ig . 8
22
0.900
504
0.700
5060.500
0.300
0.100
400 450 500 550 600
W avelength (nm)
F ig . 6. C om parison o f th e v isib le s p e c tra o f th e B ra tto n -M a rsh a ll chrom o-phores produced f ro m th e n e u tra liz ed p e rch lo ric acid e x t r a c t (-------- )and f ro m th e p u rif ied p igm ent (------ _).
Opt
ical
de
nsit
y
23
0.500
0.400
0.300
0.200
0.100
0.00041 2 3 65 7 8 9 10
R ela tiv e am ount o f p igm ent
Fig. 7. R elationsh ip o f p igm ent c o n ce n tra tio n to o p tic a l d en sity , m easured a s th e B ra tto n -M a rsh a ll chrom ophore.
24
show s th e am ount o f p igm en t produced by th e m u ta n t during th e la te log and
s ta t io n a ry phases o f g row th . V isib le p igm en t is n o t ev iden t u n ti l 29 ho u rs o f
grow th , corresponding to th e end o f th e exponential g row th s ta g e . AIR, as
in d ica ted by th e p resen ce o f th e B ra tto n -M a rsh a ll chrom ophore, is produced
much e a r l ie r in th e ce lls , during th e log g row th s ta g e . M axim um levels o f
p igm ent a re reached when th e ce lls re ach th e s ta t io n a ry phase.
Cul
ture
tu
rbid
ity
(O.D
. 54
0 ru
n)
25
0.600
0.500
1.00.400 V isible
p igm en t / 0.80.300
0.6
0.2000.4
0.1000.2
0.00.0002520 30 35
T im e in hours
Fig . 8. C om parison o f th e am ount o f p igm ent fo rm ed in th e ce lls (--------)to c e ll g row th , as m easured by tu rb id ity (-------- ).
Rel
ativ
e am
ount
of
pigm
ent
DISCUSSION
Ion exchange chrom atography is generally used f o r th e sep a ra tio n o f
com plex m ix tu res o f nuc leo tid es , nucleosides, and o th e r com ponents o f th e
soluble pool (4, 12, 17). T h is m ethod o f s ep a ra tio n is based on th e a d so rp tio n
o f th e m olecules to th e re s in , and th e i r d isp lacem en t by a m ore s tro n g ly
adsorbed ion (17). The heterogenous n a tu re o f th e p igm ent causes i t to
e lu te f ro m an ion exchange re s in w ith any and a ll com ponents. F o r th is
reaso n , Dow ex-50 (Na+ ) re s in could n o t s e p a ra te pure S-AM f ro m e x t r a c ts
contain ing th e p igm ent (F ig. 5).
H ydroxyapatite s e p a ra te s po lynucleotides on th e b a s is o f th e e le c t r o
s t a t i c in te ra c t io n betw een th e negatively -charged , phosphate groups o f th e
nucleic acid and th e p o s itiv e charge o f th e calcium ions o f th e h y d ro x y ap a tite
c ry s ta ls (7). Increasing th e m o lar c o n ce n tra tio n o f phosphate b u f fe r used f o r
e lu tio n p ro g ress iv e ly red u ces th is in te ra c t io n to ze ro . The polynucleotide
m olecules a re th e n e lu ted on th e b a s is o f th e i r lin ea r charge d ensity , depend
e n t on th e num ber o f phosphate groups along th e len g th o f th e m olecule. T he
sm a lle r com ponents w ill be e lu ted f i r s t , w ith th e longer m olecules being
e lu ted a t h igher c o n ce n tra tio n s o f phosphate b u f f e r .
The p igm ent m olecules show a high a f f in i ty f o r th e calcium ions o f
h y d ro x y ap a tite . They a re e lu ted w ith th e RNA com ponents o f th e p e rch lo ric
26
27
acid e x t r a c t , ind ica ting a polym er o f ex ten s iv e lin ea r d im ensions. The
re a g g reg a tio n o f th e yellow p igm ent m olecu les, e lu ted w ith th e sm a lle r
m olecu lar w eight com ponents, in to re d p igm en t, has been observed by o th e r
in v e s t ig a to rs . H un ter and Hlynka (14) found th a t 5-am inoim idazole undergoes
spontaneous d iazo tiza tio n betw een tw o m olecules o f th e sam e compound,
passing th rough shades o f red , and even tually tu rn in g a d a rk blue. F riedm an
and M oat (11) su g g es t t h a t s ta b iliz a tio n a t th e pink o r re d s ta g e may be due
to th e r ib o tid e fo rm o f th is compound in th e pink, aden in e-req u irin g m u ta n ts .
B a tch sep a ra tio n by high v o ltag e paper e le c tro p h o re s is was d iscarded
a s an in it ia l s ep a ra tio n m ethod , due to th e e r r a t ic r e s u l t s ob tained in th e
iso la tio n o f th e individual com ponents. T h e ir iso la tio n by th is m ethod re q u ire s
f u r th e r f r a c t io n a tio n o f each e le c tro p h o re tic band by e i th e r ion exchange
ch rom atography o r by gel f i l t r a t io n . T his com plication m akes th e p rocedu re
to o cum bersom e f o r th e se p a ra tio n o f th e com ponents o f m u ltip le sam ples.
The sep a ra tio n p ro to c o l b e s t su ite d f o r th e p ig m en t-con ta in ing ,
p e rch lo ric acid e x t r a c ts c o n s is ts o f p re c ip ita tio n o f th e p ig m en t in 90%
eth an o l as an in i t ia l s te p . The p igm en t is th e n easily s e p a ra te d f ro m th e RNA,
which c o -p re c ip ita te s , by high vo ltage e le c tro p h o re s is . T he o th e r com ponents
o f th e soluble nucleo tide pool can be iso la te d f ro m th e s u p e rn a ta n t f r a c t io n
by ch rom atography on Dow ex-50 (Na+ ) and Dow ex-50 (H+ ), a s described by
Shapiro and Ehninger (25).\
The p igm ent fo rm ed by th is m u ta n t has been id e n tif ie d a s 5-am ino
im idazole r ib o tid e (AIR) by th e follow ing c r i te r ia . The B ra tto n -M a rsh a ll
28
d e riv a tiv e has an a b so rp tio n m axim um a t 504-506 nm„ The ab so rp tio n
m axim um o£ th e B ra tto n -M a rsh a ll chrom ophore derived f ro m CAIR is found
a t 520 nm (21, 26). The P au ley re a c tio n , which tu rn s blue in th e p resen ce o f
CAIR (1) fo rm ed no v is ib le co lor w ith th e p igm en t iso la te d f ro m th is m u ta n t./
Levenberg and Buchanan (18) found t h a t AIR produces an orange co lo r which
fad es a lm o s t im m ed ia te ly . The re d co lo r o f th e p igm ent probably m asked
th is co lor re a c tio n .
No ab so rp tio n peaks a re found in e i th e r th e v is ib le o r u ltr a v io le t
, ran g es o f the ' sp e c tru m w ith th is p igm en t. Levenberg and Buchanan (18)
found th a t p u rif ied sam ples o f AIR gave no ab so rp tio n m axim um in th e u l t r a
v io le t above 210 nm , a lthough m o d e ra te ly s tro n g , g en era l a b so rp tio n w as
ev iden t in th e low er w avelengths. CAIR, on th e o th e r hand, has tw o a b so rp t-
, c ■ -ion peaks, a t 234 nm and a t 264 nm (18, 21).
The r e s u l t s ob tained by Sm irnov e t a l . (27) a re confusing, as th ey
find t h a t th e p igm ent iso la te d f ro m th e i r o rganism , which th e y id e n tif ie d as
AIR, h as tw o ab so rp tio n m axim a in th e v is ib le range, a t 490 nm and a t 540 nm .
F u r th e rm o re , th e i r p igm en t fo rm ed a B ra tto n -M a rsh a ll p ro d u c t which a b so rb
ed m axim ally a t 515 nm .
S evera l d iscrep an c ies a re found in th e l i t e r a tu r e concerning th e
ab so rp tio n m axim um o f th e B ra tto n -M a rsh a ll chrom ophore. I t has been found
t h a t th e shape o f th e ab so rp tio n curve o f th e B ra tto n -M a rsh a ll chrom ophore
produced f ro m CAIR is n o t s t r i c t l y rep roducib le , due to th e in s ta b il i ty o f
CAIR, which is decarboxy la ted to AIR under th e acidic cond itions o f th e
29
B ra tto n -M a rsh a ll re a c tio n (7, 19, 21, 26). T h is decarboxy la tion s h i f t s th e
ab so rp tio n m axim um to w ard th a t o f AIR. I t is m o s t p robable t h a t th e
p igm ent iso la ted by Sm irnov e t a l. (27) is CAIR. T his would accoun t f o r th e
in ab ility o f th e polym erized AIR p igm en t to be re ta in e d on D EA E -cellu lose
under th e conditions desc rib ed by th e se a u th o rs , and would a lso explain th e
d if fe re n c e s no ted in th e v is ib le ab so rp tio n s p e c tra o f th e p ig m en ts .
On th e b a sis o f th e c h a ra c te r iz a tio n o f th e p igm ent produced by th is
m u ta n t as AIR, th is s t r a in m ay now be d esigna ted as an Ad-2 m u ta n t o f
Sac ch ar om yces c e re v is ia e .
In th e p re sen ce o f exogenously supplied adenine, v is ib le p igm ent
fo rm a tio n is n o t ev iden t u n ti l th e ce lls e n te r th e s ta t io n a ry phase o f g row th .
However, inc reasin g am ounts o f p igm ent m onom er, AIR, can be d e te c te d in
log phase ce lls as e a r ly as 10 hours b e fo re th e ce lls e n te r th e s ta t io n a ry
phase. B urns (6) fin d s t h a t th e fo rm a tio n o f AIR in an adenine -u ra c il-
h is tid in e -re q u irin g , t r ip le m u ta n t o f Saccharom yces c e rev is ia e is dependent
solely upon th e co n ce n tra tio n o f adenine supplied exogenously. As l i t t l e as
1.5 _pg p e r m l adenine w as found to reduce th e fo rm a tio n o f AIR appreciably .
The ra p id ity w ith which adenine reduced th e fo rm a tio n o f AIR in th is m u ta n t
ind ica ted th a t th is e f f e c t w as due to an a l lo s te r ic inh ib ition o f th e b io
sy n th e tic pathw ay by adenine.
A f te r th e ce lls re ach th e s ta t io n a ry phase o f g row th , th e am ount o f
AIR p e r c e ll reach es a m axim um level, and rem a in s a t t h a t level w ith o u t being
degraded by th e ce lls . T his ag rees w ith B u rn 's finding (6) t h a t AIR n e ith e r
in h ib its i t s own fo rm a tio n , nor is i t degraded by th e ce lls . F u r th e r p ro d u c t
ion o f AIR by th e ce lls is ap p aren tly stopped by th e exhaustion o f endogenous
and exogenous purine so u rces , leading to th e c e s sa tio n o f th e m etabo lic
a c t iv i ty of th e ce lls .
SUMMARY
The p igm ent produced by a pink, aden in e-req u irin g m u ta n t o f
Saccharom yces ce rev is iae has been shown to be com posed o f a polym eric
fo rm o f 5 -am inoim idazole ribotide* On th is b asis , th e m u ta n t has been
designa ted an Ad-2 m u ta n t. T h is p igm en t is fo rm ed during fh e la te exponent
ia l phase o f g row th , and po lym erizes to fo rm th e v isib le p igm en t when th e
ce lls e n te r s ta t io n a ry phase a f t e r exhaustion o f exogenously supplied adenine.
A m ethod has been developed f o r th e iso la tio n o f th e p igm ent f ro m
ce ll e x t r a c ts and f o r th e p u r if ic a tio n o f th e o th e r com ponents o f th e nucleic
acid and soluble nucleo tide pools o f th e s e ce lls fo r f u r th e r s tu d ie s on th e
de novo sy n th es is o f p u rines in th is o rgan ism .
31
REFERENCES
1, A m es, N. B ., and H« K0 M itchell. 1952. The paper chrom atography o fim idazo les. J. Am . Chem. Soc. 74:252-253.
2. B ernard !, G. 1965. C hrom atography o f nucleic acids in h y d ro x y ap a tite .N a tu re . 206:779-783,
3. B e rn s te in , H. 1961. Im idazole compounds accum ulated by purine m u ta n tso f N eurospora c ra s s a . J. Gen. M icrobiol. 25:41-46.
4. B raun, R. 1967. S ep ara tio n o f b a se s , nucleosides and nucleo tides onSephadex G-10. B iochem . Biophys. A c ta . 142:267-270.
5. Buchanan, J. M., and S. C. H artm an . 1959. E nzym atic re a c tio n s in th esy n th es is o f th e p u rin es . Adv. in Enzymology. 21:200-261. I n te r science P u b lish e rs , New Y ork.
6. B urns, W0 V. 1964. R egulation and coord ination o f pu rine and pyrim idineb io sy n th esis in y e a s t . I. R egu lation o f purine b io sy n th esis and i t s re la tio n to t r a n s ie n t changes in in tra c e llu la r nuc leo tide levels. B iophys. J. 4:151-166.
7. D orfm an, B. 1963. In trag en ic co m plem en ta tion and p re c u rso r accum ula tio n a t s ev e ra l lo c i co n tro llin g adenine b io sy n th esis in y e a s t . G en etics . 48:887-888.
8. F ish e r, C. R. 1969. Enzymology o f th e p igm ented aden ine-requ iringm u ta n ts o f Saccharom yces and S ch izosaccharom yces. B iochem . Biophys. A c ta . 34:306-310.
9. F lak s , J. G., and L. N. Lukens. 1963. The enzym es o f purine nucleo tidesy n th es is de novo. A ssay f o r d iazo tizab le am ines, in Colowick, S. P . , and N. D. Kaplan. M ethods in Enzymology. j6:55. A cadem ic P r e s s . New Y ork.
10. F rae n k e l-C o n ra t, H., B. Sanger, and A. T su g ita . 1961. P u r if ic a t io n o f v ira l RNA by m eans o f b en to n ite . V irology. 14:54-58.
32
33
11. F riedm an , H., and A. G„ M oat. 1958. A com parison o f n u tr i t io n a l andgen etic blocks in th e sy n th es is o f pu rines by y e a s ts , m olds, and b a c te r ia . A rch . Biochem . Biophys. 78:146-156.
12. G ilb e rt, D. A ., and E. W. Yemm. 1958. Soluble nuc leo tides and n u c leo tid e-amino acid com ponents o f y e a s t . N a tu re . 182:1745-1746.
13. H offm an, M. 1969. F a c to r s a f fe c t in g endogenous p roduction o f purinein an aden ineless m u ta n t o f Saccharom yces ce rev is ia e . M a s te rs th e s is , Univ. o f A rizona.
14. H u n te r, G ., and P . Hlynka. 1941. On 4 - (or 5-) am ino - glyoxaline (im idazole).Can. J. o f R esearch . 319:296-304.
15. Knudsen, R. C. 1969. The u p take and u ti l iz a t io n o f S -adenosylm eth ionineand S -adenosylhom ocysteine in an adenine m u ta n t o f Saccharom yces c e re v is ia e . M a s te rs th e s is , Univ. o f A rizona.
16. Knudsen, R. C„, K„ M oore, and I. Y all. 1969. U ptake and u ti l iz a tio n o fS -adenosy l-L ^m eth ion ine and S- adenosy l-L -hom ocyste ine in an adenine m u ta n t o f Saccharom yces c e rev is ia e . J. B a c te r io l. 98:629-636.
17. L ed e re r , E ., and M. L e d e re r. 1957. C hrom atography, a review o f p rin c ip le sand ap p lica tio n s . 2nd ed. E lse v ie r P ubl. Co., New Y ork.
18. Levenberg, B ., and J. M. Buchanan. 1957. B io sy n th es is o f th e pu rin es .XII. S tru c tu re , enzym atic sy n th es is and m e tab o lism o f 5-am ino- im idazole r ib o tid e . J. B iol. Chem . 224:1005-1018.
19. L even thal, M ., J. Fogel, and D. D. H u rs t . 1962. G enetic and b iochem icalan a lysis o f pu rine b io sy n th e tic pathw ays in S accharom yces. G en e tic s . • 47:467.
20. Love, S. H0, and B. Levenberg. 1959. F o rm a tio n o f 5-am inoim idazolerib oside by E sch erich ia c o l i . Evidence fo r i t s s t r u c tu r e and m e ta bolic re la tio n sh ip to th e p u rin es . Biochem . B iophys. A c ta . 35:367- 373.
21. Lukens, L. N„, and J. M. Buchanan. 1959. B io sy n th esis o f th e pu rin es .XXIV. The enzym atic sy n th es is o f 5 -a m in o -l-r ib o sy l-4 - im id a z o le - carboxylic acid 5 '-phosphate f ro m 3-am ino-1 -rib osy lim idazo le 5*- phosphate and carbon dioxide. J. B iol. Chem. 234:1799-1805.
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22. R abinow itz, J. C. 1956. P u rin e fe rm e n ta tio n by C lo strid iu m cy lindrosporum .J. B iol. Chem . 218:175-187.
23. Roman, H. L. 1956. A sy s te m se le c tiv e f o r m u ta tio n s a f fe c t in g th esy n th es is o f adenine in y e a s t . C om pt. Rend. Lab. C a rlsb e rg . S er. P hysio l. 26:299-314.
24. Schlenk, P ., and R. E. D ePalm a. 1957. The fo rm a tio n o f S -adenosyl-m eth ionine in y e a s t . J. B iol. Chem . 229:1037-1050.
25. Shapiro, W. K., and D. J. Ehninger. 1966. M ethods f o r th e analysis andp re p a ra tio n o f adenosylm ethionine and adenosylhom ocysteine.Anal. B iochem . 15:323-333.
26. S ilver, J. M., and N. R« E ato n . 1969. F u nctiona l blocks o f th e Ad-1 andAd-2 m u ta n ts o f Saccharom yces ce rev is iae . B iochem . Biophys. R es . Comm. 34:301-305.
27. Sm irnov, M. N., V. N„ Sm irnov, E. I. Budosky, S. G„ Inge-V echtom ov, andM, G. Serebrjakov . 1967. Red p igm ent o f adenine-dependent y e a s t Saccharom yces cerev isiae ,, B iochem , B iophys. R es . Comm. 34:299-304.
28. S transky , Z. 1963. D e te rm in a tio n o f adenine nuc leo tid es by paper e le c t r o p h o res is . J. C h ro m ato g r. 10:456-462.
29. Y all, I. 1962. B io sy n th esis o f S -adenosylm eth ionine by Saccharom ycesc e re v is ia e . I. Adenine and M ethionine R eq u irem en ts . J. B a c te r io l. 83:1336-1340.
30. Y all, I., M. H offm an, and R. C. Knudsen. 1969. Endogenous purine fo rm a tio n in an aden ine-requ iring s t r a in o f Saccharom yces c e re v is ia e . B a c te r io l. P ro c . 133.
31. Y all, I ., S. A. N o rre ll, R . Joseph, and R. C. Knud sen. 1967. E f f e c t o fL -m eth ion ine and S - ad eno sylm eth ionine on g row th o f an adenine m u ta n t o f Saccharom yces c e rev is ia e 0 J. B a c te r io l. 93:1551-1558.
