j a x m m n m m m m of m m m m m m w m w m m . cus sa

Jeaaie Muriel Boyd

a

w i $

Submitted to the School for Advanced Graduate Studies
of Michigan Stete University of Agriculture end
Applied Science in partial fulfillment of
the requirements for the degree of
DOCTOR OF BmoSGFHT
Deportment of Chemistry

1958

ProQuest Number: 10008553

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ACKNOWLEDCMBWT
the author wishes to express her sincere gratitude
to Dr. Barnes L. Fairley for the encouragement sod stimu­
lating guidance throughout the course of this work.
2he author also wishes to express her appreciation
to other members of the Chemistry Department whose sug­
gestions were helpful in these investigations.

Finally the author wishes to express her thanks to
the Atomic Energy Commission for financial assistance in
this work*
KKttttXKXKKKWKKX

ii

the author was b o m in Billingsley, Alabama and received her
secondary and high school education in the public schools of Alabama.
She graduated from Sidney Lanier High School, Montgomery, Alabama in
June, 19h7 • The author graduated from Huntingdon College, Montgomery,
Alabama, in 1952 with the degree of Bachelor of Arts with majors in
Chemistry and Mathematics.
She entered Graduate School at Emory University in September,
1952 and held the positions of Teaching Assistant and Besearch
Assistant in the Biochemistry Department of the School of Medicine
for two years. After receiving the Master of Science Degree in
March of 1955* the author was employed as a biochemist at the
University of Michigan.
In the winter of 1956 the author entered the Graduate School at
Michigan State University. In the course of her graduate training
the author served two quarters as a Graduate Teaching Assistant in
Chemistry and two years as a Special Graduate Besearch Assistant
under an Atomic Energy Commission Grant.

Ill

m m m m m m m orm m m m » M H W i

tm
of Michigan Stofco M m i t y of Agrleslliin and
ApptXiod So Swbwni In fty%lii,i ^?^iiflM,<fffii% of

m

m

o? imosofHsr

WKfpxtemmfc of Chwa&otxy
IfSS

Apfnfovod

ABSmCf
It** felloeing imstlcfttioti* * r » eerried out to etni^r the note*
belt# rent* tr
M&eatlde* free eliphAtic eeldei

1) * eetreh for eoapousade which

O 'V

a*fta*^SUt ■**■»» » mjM;A !h .
'*t'V i *1 —1L. -..It -».A j . -■ ■—~j* . j- Jk —. a a I u I A
A
« / tfewiikdMlrtM
U rV u w e
iv ie a e e w e e e e ro cliii^
e it&
J i jjK H s& eo f i w ^ B l.W
%© 8ro M ? IiiiR

t o > y li g |

* > iy t * * f

ecidOj sod dSt^ydbt^lirilSiilL* Itiffffrll^fift yiftiffi

js e ih y iie O L O f iic

doiieffffit?*y soSds# oonpottmlf

fetiett to Ini jrtrorviTeoTe to ptyri&lbln&js t* othsx* opgsnicsi by Kofdwfg)
w n * found to be inectlee* Stadia of the tin*»eoiar** of growth of
the
gee*

ffl vyyimi

eww itf1
-eh^re# *byt ppopdloMBte end ea&nbbetyrete

reffiKHT.e##-r Bete^iiretdopQ^o^Loi^te # M dJJ^ydrcerecil

retired laager period* of adaptation before growth begun.
pareseooe of

ppopioiElo iffiid or gfltbedMgelA eef** in

the growth neditim depreeeed eignlficmti^ the specific aet&rltiee of
the pyristdiBee foreed by the &old to e ftediee also containing uxigU*
JM?***

}^opicmftte~2~ew m e found to be incorporated relatfrely

specifically into the pyrimidine# of the aoid*

e

the purine* of the

mam mpu&umtB

ooxKtoixxid much low#

mmm%*

of ©artou-li*.

prtnsei

of arglnixui, ufe&oh inhibit** growth of the ooldi on certain procoriowi
*m*1* in ja*opionate, boaotariae, airinotatyrate and uracil, vai found to

%md

to on increase in the epecific activity of the pgrria&dlne baae*

foitted by tin aold in a medium also containing WNMriMM***
OtuditOa ftftKtt* tb* ja*W8t©i Of aotlVitlOi

affectin g u ra c il, anlBbbutymt* and botiH ^ljo^«pttoti^eiiso.
On ttae
$ynisddi<is

o f im jjeiw&ie a yvf* j&tbKey fo r tbo flyrffihftifl v o f
ig jOttjggosted, a p5it$osay vfolftfo 3^ 4^ froa propionyl**

l#®®n®jJPBW Jm wMwrilWiyBi vJ0W vCWHwM^yWw A wMK&mwmiw3BW^IhP QT ar®*®***®AoKoAi
Hw JBWB 0®®®**

wifi to

acid w** finally to uildi!i6*>5f**

vi

tkBLB Of CONTESTS
P«g9
INTBOOTCTIDN

X

K X F E W M m m AMD HE3JLTS...................................
Effsct. of Various Compounds on the Growth of Nauroapora
g»gj» ffiP-.......................... ..............
Materials......*...*..................................
Organism............
*......
*....
Growth Procedure
....
Results..... *....... ..............................
Stimulation by Uridine**................................
Arginine Inhibition
...... .......
Growth Curves....... ................ .............
The Utilization of Arainobutyric Acid in the Presence of
Uracil-^-C14 for the Biosynthesis of Pyrimidines in
Heurospora craasa 1298.... ............... ...........
Sterials.1. ....... .... ............ ......
Growth Procedure
.... ...... .
isolation of the Ribonucleotides..... .................
Hydrolysis of Ribonucleotides
... ......... ......
One Dimensional Paper Chromatography.........*.......
Isotope Measurement
....... .................
Results
...... ................... ...............
The Utilisation of Propionate for pyrimidine Biosynthesis in
Neurospora craasa ...... ............................
MaterialsT••"•T.*T .... .... ............ .......... .
Growth Procedure.
....... ......... .
Purification of the Purine and Pyrimidine Bases.
...
Radioautography of Paper Chromatograms.................
Results • ... . ........ ........ . ....................
Utilization of Propionate in the Presence of Uracil-2-C14 for
Biosynthesis of Pyrimidine in Meurospora omasa......
Materials.......... ............ ............... .....
Growth Procedure
... ........... .
Results....
..... .............. ..... ......... .
The Utilization of Umcil-2-C14 in the Presence of Arginine
for the Biosynthesis of Pyrimidines in Benrospom crassa...
Materials.........
..... .777777...
Growth Procedure......................................
Results........ .............. .......... ...........

vii

10

10
10
10
11
11
15
16
IS
id
18

20
20
22

23

2k
25
25
25
26
27
29
30
30
30
30
31
31
31
32
32

M E

OP CONTENTS - Continued
Page

Isolation of Enzymatic Activity Related to Pyrimidine
Metabolism...... *........
Materials.
....
Methods...............
Xnoubation
....
Enzyme Preparations.
......
Remilte.
DISCUSSKB

BIBLIOGRAPHY
APPENDIX...

IS

.....

SUMM^HX.

....

35
35
37
38
39
lib

.....
.......

57
59

.....

62

viii

LIST OF TABLES

nMM

Page

I* Growth Response of Neurospora crass* 1298 to Various
Compounds
.T^77?:77.r.77.~':.77^7.*. ......
XI* Stimulatory Effect of tJridine on Various Supplements.......

12
16

XXX* Arginine Inhibition of Certain Growth Promoting Compounds.. 17
XV* Radioactivity of Ribonucleic Acid Bases from Naurospora
crasaa 1298 after Growth in the Presence of Vai^ous Radio**
active 5ompbunds...... *******...........
*....

ix

33

LIST 0F FIGBTRES
Page

31®®

^TrfBldlne biosynthesis................

$

2* Pyopowd p$t$xivi^r of jyriicidlns d®grada%iosi*•«•«••*«•**#***«

7

1 * Proposed pftthi^

3 * I’
yoploiiate m

t

a

b

o

l

i

a

m

.

b. Rftlative m%«* of gj*o#th.*..

.

l

b

1?

5 . Soggosrted pathway of pyyiadd&ao bioayaihasia in tha

a^nwBom eras** aataat, 1298..........

X

$3

1

INTRODUCTION
The discovery of nucleic acids was made in some investigations
carried out cm the nuclear material of pus cells by Friedrich Miescher
in 169? • Later nucleic acids were shown to be normal constituents of
all cells and tissues studied.

On hydrolysis nucleic acids were found

to yield purine and pyrimidine bases, as well as a pentose sugar, and
phosphoric acid.

Two types of nucleic acid have been found.

One of

these contained the purine bases, adenine and guanine1 the pyrimidine
bases, cytosine and thymine; and a sugar D(-)-d©oxyribose.

The second

type contained adenine and guanine; the pyrimidine bases, cytosine and
uracil; and the sugar, D(-)-ribose.

Both contained phosphoric acid.

Although It is clear that nucleic acids play a fundamental part in
cellular metabolism, the exact nature is difficult to determine.
Deoxyribonucleic acids have been indicated by many indirect lines of
evidence to be the basic genetic material of cells (1).

Ribonucleic

acid seems to be connected with the process of protein synthesis in the
cell.
Host organisms are able to synthesize nucleic acids from simple
metabolites*

The purine and pyrimidine bases are thought to be

incorporated Into the nucleic acids in the form of their nucleotides.
The pathway of purine biosynthesis has been worked out to a large extent
both in mammals and microorganisms. Comparatively little evidence,

2

however, has been found for a pathway by which pyrimidines are
synthesised•
The biosynthesis of pyrimidines from simple precursors was demon­
strated in an experiment by Barnes and Schoanheimer (2) In which N15
labeled ammonium citrate administered to rats was incorporated into
the pyrimidines of nucleic acids*

The investigations of Heinrich and

Wilson (3) showed that position two of the nucleic acid pyrimidines is
derived from C0 a in the rat.

This finding was confirmed by Lagerkvist (U).

In searching for simple metabolic precursors of the pyrimidine
nucleus, Mitchell and Houlahan (5) introduced important information in
studies of the mutants of Neuroapora erases that require uridine for
growth*

In these mutants orotic acid was found to accumulate in the

medium during growth*

For several of these mutants uridine could be

replaced by oxaloacetic acid (6), boring and Fierce (?) found orotic
acid would replace pyrimidines as growth factors for certain pyrimidineless mutants of N* crassa* Orotic acid is also a growth factor for
Lactobacillus bulgguicua ggj if C 14 labeled orotic acid is provided in
the medium, the isotope appears in uridine-5 *-phosphate and cytidine-5 1phosphat© but not in adenine or guanine (8). Eeich&rd (?) had obtained
results earlier in the rat.

Orotic acid is used by animal

tissues, since the administration of N 15 (9 ) or C 14 (10) labeled orotic
acid to rats leads to appearance of the isotopes in the pyrimidines but
not in the purines*
Wright et al. (8 ) demonstrated in L. bulaaricus Op that DL-ureidosuccinic acid was as effective a precursor for the pyrimidines of the

3

nucleic acids as was orotic acid * Reichard (11) showed ansymatically
with rat liver mitochondria that aspartic acid in the presence of
carhasQrl phosphate, adenosine triphosphate and magnesium ion could be
converted to ureidosuccinic acid by mitochondrial fractions and later
he showed labeled aspartic acid to be incorporated into the pyrimidines
(12).

The synthesis of ureidosuccinic acid required several steps,

the first being the production of caxfeasyl phosphate.

The method of

formation of this compound was worked out by Jones, Specter and Liproann
(13)*

In the second part of the reaction, the carbainyl phosphate trans­

ferred its carbamyl group to aspartic acid forming carbamyl^aspartic
acid, often called ureidosuccinic acid.

These reactions were demon­

strated to take place in the mitochondrial fraction* Experiments by
Woods, Ravel and Shlve (lU) with L* arabinosus 17-5, which is an aspartic
acid recpiiring mutant, showed that pyrimidines, as well as threonine
and lysine, could spare the aspartic acid requirement, indicating the
use of aspartic acid for pyrimidine formation in this organism.

The

precursor relationship of carbamyl aspartic acid to orotic acid was
established in nutritional experiments in microorganisms and isotopic
experiments in microorganisms and mammals (3, 11, 12).

The conversions

were shown by Lieberman and Komberg (1$) to require two enzymes, one
which effects the ring closure of carbamyl aspartic acid to dihydroorotic acid and the other, a diphosphopyridine nucleotide requiring
enzyme, which removed two hydrogens from dihydroorotic acid to produce
orotic acid.

k

The mechanism for the formation of uridins-5*^phosphate from
©rotate hag been clarified by Xoraberg and co-irorkere (16, 17, 18).
far the first atop of the conversion of orotic acid to uridine-S*pho*phate, Liebermann end Kornberg (16) showed that the formation of
^pfec«i^oriboeyl*-l*'|37rciph©s|hato from adenosine triphosphate ami riboae»
S-j&osphate mas requiredprior to the ffetsaatiem of ©rotidine<-51-^phosphate
iron ©rotate*

The eaayae, orotidylic pyrophosiiKnylase, which catalyses

this reaction, mas purified from yeast autolysates and mas found to he .
specific for erotic acid. A second emym, oroti^ylic decarboxylase,
catalysed the decarboxylation of ©rottd±ne-5 1^phosphate to fern uridine5 1-phosphate (1?).

ta the absence of 1he orolidylie decarboxylase, '

oretidli^^^^ihesphate accumulated.
be irreversible.

The decarboxylation mas. found to

The conversion of uridine-51-phosphate to

phosphate mas shown by Eernbwg (1®) to take place at the triphosphate
level aed to rs<plp8 adenosine triphosphate and
The sequence of ructions suggests by the above experiments is
the generally accepted pathupr of synthesis of pyrimidine nucleotides.
These reaction* are shown in schematic fashion in Figure 1*

5

HDQC

8D0CL

\

HOOC

WBL

CHa
i

f*

01
O^CGQH
Cdealoaeetic acid

„C

0'

HaH ^ n 000H
Aspartic eel-**

. OH

HHr

nCOOH

tfreidowueeiaie acid

0

^CL

hgffTTi
CH fcyrophoephate
fl
,^ ii»"*|,»'»>'i

Htf
|

^

C

c

.0

0 <-

> 0OOS

QrotidinaH?1-phosphate

diphospho**
GH
jyridlne-

0

,c.

of

f
,0
Wt'

Hs

f

c

^<smn

Orotic acid

N300H
Bihydroorotic mold

V

fa
.0

W
a
ov

0.

CK
II
^-oa
V

1 ^
JL

T

triphosphate ^.C

^,GH

m x

ribose-£*-^phosphate
lJridtna-5*-phosphate

rib©#e-£«-triphosphate
Uridine triphosphate

rifecHWMffc*
triphosphate

Cytidln© triphosphate

Figure 1* Proposed pathway of pyrimidine biosynthesis

Other |*tiw*ya of pyidinidine biosynthesis may assume greater im­
portance under different conditions.

In mutant organisms* where the

normal pathway is blocked, an alternate pathway may be utilised for
growth of the organism. Antimetabolitee such as asauracil, 6~uraeil
methyl «&fone and ethers (19) are known to prevent growth in tuner
sells for a period of time, after which the cells start growing again.

6

This resistance to the antimetabolite may well arise from the adaptive
utilisation of an alternate pathway of uracil production.

Investi­

gations of apparently minor, alternate pathways of biogenesis of
necessary compounds may, therefore, have great importance in certain
cases*
One possible occurrence of such an alternate route was Indicated
whm Fairley (20) reported the growth of several pyrtnddinelese N. erases
mutants on the amino acids, alpha-amlnobutyrate and threonine.

In other

experiments Fairley and Herrmann (21) demonstrated the utilisation of
alpha-amlnobutyrate by the mutant 1298 of N. crassa for synthesis of
pyrimidines. When amlnobutyrate-3*-C14 m s administered to the mold
the labeling of the pyrimidines formed ranged from one-tenth to onefifteenth of the original concentration of radioactivity of the aminobutyrate supplied, fhe pyrimidines formed were labeled to a considerably
greater extent than other mycelial constituents* about four times that
of the ribonucleic acid purines and about ten times that of various
amino acids.

Reisolation of aminobutyr&te also showed a dilution to

about one-twentieth of the original concentration of radioactivity of
&minobutyrat©-3-C14 administered. therefore synthesis of the amino
acid during growth of the mold could explain the dilution when
Incorporated into the pyrimidines.
the aminofoutyrat© was apparently utilised by a route differing from
that shown in Figure 1, in that aspartate and ureidosuccinate were in­
capable of supporting growth.

7

Another alternate .pathway for lyHntdlne synthesis are the reactions described fey FSrfc (ft), m t a s n (23), Oanellakis <aU), Qrisolla
(25), and Rairaan (26). When uraell was administered to rate, Wixk et al.
(St) isolated f r m urine b«teHM«to)pro|3iottic sold end feeia-earlwwjyleadiuspropionio eoid.
ean&siir^^

to eiitdlar experiments with labeled thymine, beta*
sold and bete^Bdj^lei^tjrlc acid were

isolated* Fritasn (23)* CaneHekie (tl*)# Grieolia (2$) end ftetman (26),
bare shown that uraeil-2-C1* ie rapidly degraded by a enayne system of
rat Hirer to form the end prodoote, CG*, H%, and feeta-alanine. She
rechictlon of uracil to ^

deoends uoon

imoleotide, end ie the rate Uniting step (21*). W m eerapononis of the
©sTfoanyl group ere released ansyirottcfiXLy after opening of the ring to
fore <»Jd&a^l-be1»'^alaRine, often sailed feeta-ursidojarcpionfe acid (2U)«

t% ie <|*ii« possible that a reversal of this pathway oool* afford a
pathway of biosynthesis independent of orotic acid • The s$<gnence or
reactions OS a degradatire pathway ie shown in Figure 2. She reversal
of this pathway has been suggested as a Keane of synthesising pyrls&dines.

trxnoH*
Ifcymtaet

m

« n $ n' « h

*'• 0% , B» - H

Orotic, soldi

R « H, R* * 000H

Figtzre 2. Proposed pathway of pyrimidine degradation (22)

8

Although the pyrimidine bases have been shown to be extensively
degraded to carbon dioxide and the beta-amino acid in mamoals,
Caneakkis (27) has shown that in rat liver slices a considerable
portion of uracil-2-C14 was converted to nucleotide pyrimidines and
nucleic acid pyrimidines and that a smaller proportion was degraded to
carbon dioxide. Lagerkvist and Reichard (28) demonstrated that in the
mouse, uracil was efficiently utilised for synthesis of nucleic acid
pyrimidines in both visceral tissues and Ehrlich ascites cells.
In mammals the pyrimidine bases have been demonstrated to be con­
verted to nucleotides by a nucleoside phosphorylase isolated by KaXckar

(29)• Hhcleoside kinases (30, 31, 32, 33) and phosphotransferases (31*)
could convert pyrimidine bases, ribose-phosphate and adenosine triphos­
phate to the nucleotides. Through the use of enzymes isolated from
L. delbrueckii and Thermobacterium acidophilus B-26, synthesis of
certain decacribosides were shown by McKutt (35) to take place by the
transfer of the deoxyribosyl group from on© purine or pyrimidine to
another*

These are methods by which uracil may be efficiently used for

synthesis of nucleic acid pyrimidines.
With the Indication that N. craaaa 1298 is capable of synthesizing
pyrimidine nucleotides by a new metabolic route involving certain
aliphatic acids, it became of interest to determine the precise nature
of this new mechanism.

The present work describes the results of

several different approaches to this problem.

These approaches involved!

1 ) a search for compounds which supported growth to a similar extent as

9

did wainolmtyTic «&£d* teooalna and h m & m r S m , t) traes* studies with
autooa*!!* labelled ompouuad* to ostablish th* utilisation of various
m w & m m Is as Krets&ciiae precursors, mmI 3} attempts to demonstrate
m«wtfai4»

ra(a®tiOIiS*

10

m m m m m * and results
Effects of Various Compounds oa the Growth of
'

Materials
All compounds, with the exception of beta-ureidoproplcnic acid,
l-amiiio-l^carboxy-cyclopEropftne, alplm-hydreaypropioni^ acid and methyl­
malonic acid, were obtained from nutritional Biochemicals Corporation,
the beta-ureidopropionic and ureidosuccinic acids were synthesised by
the procedure described by Lengfeld and StiegHts (36) . two grams of
beta-alanine and 1.85 g. of potassium cyanate were added to an evaporat­
ing dish and enough water added to dissolve both substances,
tion was evaporated on a steam bath to a syrup,

the solu­

the residue was placed

in a small beaker and approximately' 1 ml. of 0.1 M hydrochloric acid
was added.

The beta-ureidopropionic acid precipitated out on standing

in the refrigerator,

the melting point m s l6£-17©° 0.

She 1-amino-

l-carbccsy«cyclop*opane was a gift of Dr. Bans Lau. The beta-hydroxypropionie and methylmalonic acids were gifts of Dr. Minor J. Coon.
Organism
Strain 12?8 of Heurospora crass*. which was produced by Beadle and
Tatum (37) using X-ray and ultraviolet radiation treatment and later
described by boring and Pierce (?), will not grow on a simple media of
salts, sugar and biotin (appendix). This mutant m s assumed to be

n
unable to synthesize pyrimidines since addition of a pyrimidine
compound to the basal media permlts growth of the organism.

The mold

was maintained on culture slant* consisting of taro per cent agar in
basal medium with 1 mg. of uracil per ml. (appendix).
STvifw ilTiKEWttllHI
The wold was grown in 12$ ml. irlsmgsiyer flasks to which 2$ ml. of
a basal nutrient haring, the composition sheen in the appendix mas added.
Bash flash mas stoppered with a cotton ping and autoelaved at 110° C.
far 1$ minutes,
the

rh* heat stable compounds were sterilised awmg with

by autoclavlng while the heat sensitive compounds vers added

are|&ieally after filtration through a Belts bacteriological filter.
After seeling# each flask was inoculated with 0.2 ml. of a spore sue*
pension of the mold made by dispersing tec loopful* of spore* in 10 ml.
Of aterile, distilled water.

The cultures, run in triplicate, were

incubated at 25 0. far four days* At the end of this time the mycelial
pad* were removed, rinaed with distilled water, s<jue©aod to remove
exces* water, and dried overnight in an even at $Q& C.

The amount of

growth id* determined by weighing the dried mycelium.

The weight* were

compared with uracil as a standard. Other controls consisted of basal
medium with no repj&smoat.
Results
Xm Table 1 a H o t of the compounds tasted for growth I* given.
They hare bean placed in three groups indicating their relationship to

12

nm& x

mmm mmmm

or

nmmmom. m m

to

vmms m m m m

*®*>S8®eS8£BS8SiSSSKS5SSSS^

8Bp£L«MBtaxy
Coqxnnds

Waight at
Compound
(««*)

DryW algit of
MyoeliuB
(ag^—U daya)

5
5

57.0
n.x

UraciX QK»$
ffnwll
......... u

opropfcmic acid

XbamtiM
ffcyaddiaaa
IMSM
Aapartic Aeid Group
Aspartic acid
Tfyfeirittffiii*P»^ f» aCid
Orotic aeid
aa&d
Homoserino
A,It

10
5

2

*

x

2.7

2
5

0
0

5
8*55

5
T
5

0
0
0
6
0

6
a

IB

*
8

io.o
0
0
30
0

0

anludnobutyric Acid Group
A# o-Aialnobutyric Aeid
%#eto butyric Aeid
<^!^drosy butyric aeid
f5j^Aoaiue
tr’olaucine
’
Cyclop£ro|»neajoadino'Hsarooixyjlxc ae&ci
I* IToplonie Aeid
Aexylie aeid
p*I^uIi^|il?OplOlwXE WSIm
Kethyl-iaalonio aeid
Succinic aeld
jyrutrie aeid
Aeetle aeid
n-f^Bplandne
Glycerol
Glycine

a
10

5
5
a
a
a*a
10
5
S .1

5
5
6#3

0
2?

5
15
lb
0
0
0
0
0
0
0

13

tit* fMVlttKllir sent!omd pathways of biosynthesis.

Bio uracil grasp

include* the compounds shown by ^various workers to comprise 1sbo pathway
Suggested in Figure 2*

fhese compounds were beto^reidop^ioole teli,

and dibpdrouraoil, leading to the synthesis of uracil, m l dihydrothymina
leading to the synthesis of thymine. Uracil bad boon Shown previously
tO .|SNSSiies .gPCWth# Xa these studies dihylrcmraoil, botaHJUroldoproplcaio
eoldj,'tbjyo&T*» and dlJayd^tbyiBiAS were ffoowrt to replace the yy«*^«rtriigMn
**K|afre««at to a H
thymidine, w

m

degree than uracil itself.

She nucleoside,

not able to satisfy the pyrlitttdine'retirement for growth*'

supld |aftQ bean shf***** to 'bo a degi
PM^ttsift joodict of uncil
through biitltdti gold (36)# but It did not replace uracil for gvcwtb*
the compound* of tho aspartic acid group were those shown in
Figure X* Aspartic acid,

acid, #ad dfhydrooroti© a d d

did not show any fflitseity to satisfy the-iwrlraldine isotiiiiBdt

Us

growth response to orotie sold indicates its ass for pyrimidine bio*
synthesis. (Eutamic aeid has b m m Shown to be readily converted to
aspartic aeid by

reactions*

Since a mutant of H. erases

nMdi laefte glntamie dehydrogenase has been reported by Fineham (39),
the re$iAr«B*t of m al|ha asto© group was visualised.

However, again

no growth response resulted* fhreenine hat been shown previously by
Ifcirley (SO) to be need in place of jyriiaidines. the wain pathway for
the biosynthesis of threonine has bean shown to be eonrsrsion of
aspartic acid to homoserine (bo) which in tarn gives rise to threonine
(111)* Horaoaevine was shown to give a significant amount of growth*

1U

Allothreonine, which differs from the naturally occurring L-threonine
by sin inversion at the second asymmetric carbon atom, was not suitable
for growth.
In the process of looking for compounds which were related to
aminobutyric acid and which also gave significant growth responses,
propionic acid was shown to give very interesting results.

For this

reason the arainobutyric acid group was divided into two sections, one
which contained compounds related to amlnobutyric acid and the other
which contained compounds shown by Stumpf (U2) and Stadtnsan (1*3) to be
related more directly to the metabolism of propionic acid itself.

CHaCH2C00H

Adenosine
triphosphate
coen8yKeA «£►

CHaGHgCO^Coanayme A

Propionic Acid

Propionyl-coenayme A

CHaOH^O-Coensyiae A

J/
V*
fOOH
CH^CBGO-Coenzyme A CHgCHGO-Coenzyme A

HH*
beta~alonyl--coen2iym© A

aciylyl-joenzyme A

methylmlonyl-cpenzyme A

pHgCHgGO-Coenayme A

BDOC-CHaCH2CO-Coonayme A

OH
hydroxypropionyl-

° ° T \

GHgCH^OOOH \
OH

V

HOOC-CH^CH^gCOOH

GH3COOH

hydroxy* acetic
propionic acid
acid
Figure 3*

succinyl-'coenzyme A

succinic acid
p
Krebs Cycle

Propionate Metabolism.

15

Aralnobatyrate, ichick haul already been shown to give a high growth
response in It* erases 1298 (20), gives on deamination the keto &cldf
slpha^kstobatjrric acid, in some organisms ikk)• Although the reaction
has been shewn to he reversible, the keto acid did not support growth.
Heither did the al$l*a-hydroxy?mtyric acid.

fhreoniae which is readily

converted by mat liver to Alpba-**tfdm<^tyric sold (k5 ), was indicated
hgr Ib&rXey to he a precursor. fsoleueima has also been demonstrated
to yield alptm-ketofcutyrio acid (U6)* bat evidently did not give rise
to aminobutyrie acid since no growth resalted •
Of the compounds give® in Figure

propionic, acrylic, Igrdraaty-

propionie and methylmalonic acids gave varying growth responses*
Succinic, pyruvic, and acetic acids and alpha««alanine have been Shown
to be related to the oxidative pathway of propionate metabolism through
the Krebs cycle (U?) * these failed to replace the propionic acid
retirement*

Other three eaibon compounds failing to give growth were

n-propyl amine and glycerol.

Olycine which has such an important role

in purine biosynthesis was found to be inactive here.

ISble II shews the results of adding uridine to sewn of the
previously reported supplements. Lysine showed a slight stimulation by
uridine while uracil and dlMrouraoil give a great response.
X&hydroorotie acid seems to inhibit growth in the presence of uridine.

16

s t m b u jobx snrae* car ukxbhb on tabxqus somotMsam

^pplm m ba

ftfiMiimtiMiMflia of
SupfOaawnt

1. 9*1 mg> Uridine
SlNiShWiiliiBt
aspartic acid
jfld l IbuaaUllM^maASLX Jl
d
3Joiytir©o3?otie

j|lfr i) ■■bia s 1,1 ' I
C■1fl4iis*s
u^yiXJ
rC/QUTC03JL

6.2
5 mg.
5.1
5
5
2
2
2
2

B. 0.5 mg. Uridine
lvalue
aspartic

Wei#* of
Bycelis

5.5
12.6
6.1
0
61.?
6.9

10.2
tiS.S
27.5

5
5.1
5

30.?
31.6
2?.0

Arginine has bean found to hero an Inhibitory effect on the
utilisation of certain of the jyrlmidine precursors (Fairley, unpiblished). Bradl, alpiaMuainobutyric acid, propionic acid, and homo•ariaa have been found to be Inhibited by very email quantities of
arginine.

Xhe remits are Shram la table XXX. Ornithine Shewed the

same effect at about 100 times the concentretionB used for arginine.
Citrullina wui not ao clear-cut.

17

ARGININE INHIBITION OF CERTAIN GROWTH PROMOTING COMPOUNDS

Supplements

Concentration of
Arginine
(mg.)

A. Uracil (3 mg*}
I^arglnine

58
0.01
0.1
1.0
10.0

Uracil (5 mg.)
Dr&rginine
Uracil (10 mg.)
L~arginine

10.0

10.0

z

5

k.l
17
30

0 *001
0.01
0.1
0.5

0.01
0.1
1.0
10.0

20
0
0
0

27
0
0
0
0
18

B. Homosarine (6 mg.)
L*arginine

17

32

C. Propionic acid (6 mg.)
L~arginine

3k

22

B. Amino butyric acid (5 mg.)
L-arginine

Weight of
Mycelium
(rag.— days)

0.01
0.1
1.0

0
0
0

18

Qrowth Curves
Growth curves irere plotted for the time of incubation response of
the mold to selected compounds. Uracil, dihydrouracil, beta-ureidopropionic acid, propionic acid, and alpha-aminobutyric acid were given
in equiiaolar amounts to the mold.

Bach set was run in triplicate.

Five sets of flasks were run for each compound.

The results of these

experiments are shown in Figure i*. Of the various compounds tested,
growth began first on uracil, followed in order by arainobutyric acid,
propionic acid, dlhydrouracil, and finally beta-ureldopropionic acid.
Once growth began the growth rates were

e s s e n tia lly

the same.

Compared

with other compounds aminobutymte and propionate gave lower maximum of
growth.
Utilisation of

Acid in

of

mm
Materials
Uraeil~2-C14 was obtained from the Isotopes Specialties Company.
The specific activity of the sample was given as 0.6 millicuries per
millimole.

This sample m s used in all the experiments involving

administration of uracil to the mutant.

The amlnobutyric acid was

obtained from nutritional Biochemicals.

The organism used was the

N. crassa mutant, 12985 described before.

\

GROWTH

C\J

O

OF

CD

LO
UJ

H
UJ
o C L iT U J
H
< O
cc Q
<
:
3 a
■t;
3
o O 00 5
(T
a o o a. o
3 <1
5 a: 5F CL’ Of
O 3 <a 3
X ■ • o □

>: ui?

J___

o
o

o
00

o

O

<D

lA inn30AlAl

o
00

*9(AJ

ro

4.

o

C\J

FIGURE

-J

RELATIVE

CD

DAYS

RATES

00

20

Growth Procedure
The mold m s grown in 125 ml* Erlenmeyer flasks, to each of which
was added 25 ml. of basal nutrient medium having the composition Shown
in the appendix.

2n each experiment two sets of ten flasks were used,

to each of the first ten flasks were added Q.U mg. of the labeled uracil
and 9*6 mg. of unlabeled uracil.

In addition to the 10 mg. of uracil

the second set of ten flasks contained 10 mg. of arainobatyric acid in
each flask.

The first set served as a control, giving the incorporation

of uracil-2-C14 into pyrimidines, while the second set showed the
influence of aminobuiyric acid on the amount of incorporation of
uracil-2-C14 into pyrimidines.

Tan milligrams of uracil was found from

a study of concentration effects on growth to be the most suitable
concentration for optimum growth,

the autoelaving, inoculation, and

incubation were carried out as described in the previous section
(page 11). At the end of three days of growth the contents of the flask
were filtered on a sintered glass funnel with suction.

The mycelial

pads were washed with a mall amount of water after which they were
placed in 100 ml. of acetone.

The acetone was removed and the uycslia

were placed in the deasicator for 2it hours. At the and of this time
the pads were weighed.
isolation of the Ribonucleotides
The dried, acetone-extracted, mycelial pads were ground to a fine
powder using a mortar and pestle with 120 mesh carborundum powder.

21

This mixture of powders was extracted three times with $ ml. of ice
cold 10 percent trichloroacetic acid
once with l*il ethanoltether.

(wA) •

The residue was washed

The first extraction was carried out for

thirty minute® and the successive extractions for five minutes.
solid was then washed twice with ether.

The

The mixture of carborundum and

extracted mycelial powder was dried, ill of the extractions were carried
out in a 12 ml. centrifuge tube. By using this procedure, the solution
was easily separated from the residue by centrifugation, so that the
residue could be reextracted.
five milliliters of 1 N potassium hydroxide was added to the solid
contents of the tube and allowed to stand for 2U hours, stirring occasion­
ally* After centrifuging, the basic solution was adjusted to a pH of
3.0 by using perchloric acid. During this process and a ten minute
period of standing the solution was kept cold in an ice bath.

The solu­

tion was centrifuged and filtered through a sintered glass funnel.
The pH was taken up to 11 and the precipitate of potassium perchlorate
was filtered off.
This acidic solution was added to a column of Dcwex-l-Cl"', 50-100
mesh, 12X.

The column had b&m previously treated by washing with 200

ml* of 2 H hydrochloric acid, followed by 200 ml. of 5 per cent sodium
hydroxide, and again washing with 200 ml. of 2 H hydrochloric acid.
The final wash was with water until the elnat© was shown to be neutral
to pH paper. When all of the solution containing the ribonucleotides
had filtered onto the column, the column was developed first with 200

22

m l. w f w ater.
a c id .

then tit* tm Xw otM e* war* e lu t*d w ith % K hythfoeblorie

The o p tic *! density o f th * e la a t* was detemdaed a t 26o as*.

Ik * eeaMned fra ctio n # containing tit* nucleotides was evaporated to
dryaeaa oft a fla tis evaporator. Water i* « added eeveral tin * * to old
in m o tte g t il* hgdieoomorto ac id .

mamma#
l&m raAxture of ribcmeleQiddaa m * transferred to * 2 «1 . voXu~
notrlo flaalc tiding small amount# of O.055 X hydrochloric acid.

This

solution m s evaporated to dimness using a (dwurcoal^filtered air stream.
The flask stood overnight in, a vacuum desiccator containing Driwits.
After careful addition of 0*5 ral. oonoaatimtod perchloric m M , tl**
flask' was heated on a steam bath for forty minute# behind a glee*
shield. When the fl&^s

mm

cooled, the contents

mm

transferred to a

12 al* centrifuge tube using snail quantities of eater, the carbon
na* centrifuged down* washed with eater and the wash combined with the
first supernatant,

fhl* solution of purines and pyriioidines was allowed

to filter Into a ectas of Rwwe-50, H %

U X, So x lflO mah.

This

M l boon jrerloasly trotted in tes m m wmamr w the D o m x -1
Mian.

Hwtioa m u tegim wli* utter (201*300 al.) «ad m u

by 6 M S X bj^braebXorle Acid,
of wter.

Wtm. « *

fo llo w e d

lb* tsrocil ca m off 3a tbe first 150 ml.

ultw.violet ateorpfefc® n * negLlgibl., ths

aytetttiM ( M *fette& with 2 X hydrochloric m 34 (1*00-550 ml.). 21m
eytosixw m

fond to e e m off la * velum of 120 al. oftor addition

of tho first 60 al. of Mid.

OoMiBo

mm

dinted with 3 x hydrochloric

23

m M (180*200 al#)* She imdJid m m oft 1» a votae of 1*5 «I. after
addition of

m * k to ml. of acid. ® m mtemSm m * elated with

It H hydrochloric acid (l$0*f©O ml.) In & volume of j$ ml. after to al.
of the acid had b a m added to the ee3& m *
yjCKLet SbSCGPbSJBg

fhe elution of the

namm dOtlMythlHf by

t$MI UiMtelmiiattMra SIMffitiroitotoiMlflfi? Wwlftl EfiL finfe fatn^ri 1*Mdh«8ai of
each fraction wm- j&ated and: c&rnted*

tfher© m e a direct proportion

betwmn abaorbanc* end the amount of mdioactivity of each fmetiaa,
indicating She ladiopurity ofe&ch i^tmviolet absorbing compound.

19$# uracil fraction which m e
i|&0 aii of mber

from She column la the first

ammftmd the perchloric y***** «*e for the

u&vQFQjtymXM* mtiwM WHB POmOveO DOlOrC PUMinQr pKPUlvftwluIl OS uISOlJ.
ooo fet
y ffjed oat* l^iret# the fmctlon m e emperebed bgr mane of the
f l m h ampemter to a volxtm of f el*

fbaa the solution m e made

neutral to pKrmpee1'by addition of 1 u potassium hydroxide • $he
Boteeeiom oerchlGr&t© h i iilteopsd off m d She eolation m e further
evaporated by air to about G .5 el* 'fmther ittfifiottlmi m e carried
ffl»i W
m a te

o fj

ttHO *■*wlw<^^trWinhi
y .:m m * i.pe sg^ m j p ™ •

fbe solutions of the aubsteaoee to bo purified vere spotted on
WtMtma #X pipnr. 2km 200 lenbde apt* « m

jOeeed i* inches f n a the

end oa « 7 x 22 inch sheet. The qmta wsre net *o*e then 0.$ as. in
dimeter.

This could be eeccajiLiahed «r'*nfc*a* smell mounts at «

tine and diylng «ith • heir «8*y*r ebile the pep* wee stretched between

2k

theiMfct* After sm&mh* wp&AmUm the paper mm foiled 1*5 iachee
fMt the origin and again in the opposite direction 3 iiiehee frontthe
erlg&u the flap* **r* placed ixigXaea iroagha ead the paper* paaetd
np int w«r #*i* redi* such that the Bret fell coincided eittithe
gleg* red and ibepaper therefore totg straight dcmm from thie point.
A heet rod "M &6 ^ie peper# in the trough*

(ttttragb* m m held at the

top of »&*&i glass mek, and the entire extern

mm

contained in a

12 tgr 2U inch baiisxy jer. ft# aolrent* m m added to the tran^ in

epp^yriiWeteXy 23fei. foexitltiee. /the jarmm covered eith a glass
ptete end the solvent ellowed to flee' A#wi the paper nniil it had olaaoet
reached the bottom, or ihr* .spprosdBsntstlr1

Shears* Ttio papers ears thee

rewserSii sae sr&sci isi tine «©eau wi>w as axt^iefiiCifri **s»p xne
and

ei^eered ee Afffo bliie ehegi%l.gjg spots on e light

gStnfsd#

guaaing, which fluoresces adi^itlgr# appeared eh' a light

Hal# #$#$ m th* f#p^e

back**

1Kb® spots «#r# out out and dlls*# Into tMll

to Q'SSS * hjrtoo-

■tiaeeie **!«» Aftor amSseiM^tmg to* toHrt«ta*NUn«u totoswtoad titm
to* sbsoitono® «t 250 m. OM hawJrsd larabd* of atoh sample -me plated
Had cwoitod. too todtotottosfctr*** ^oaaadto to aMoetotod with to*
aitoariaaat atowbtog apota. '

One tanndrsd lambda ali<jaot« of the motions from the columnand
fra tto purification pe<m&xnm m jflotod m platinum pianeheta.
to «U m m to* a«l»*at mm tonovwt tgrslew <w*jwmti«a orer an

25

infrared lamp with a gentle stream ©f air blowing over the plates,
toe mdimctAviby was determtoed at infinite thinness to a Nuclear
proportional Geiger emater, with the toelear Ultra Sealer Model 1$2.
Since the concentration of each sample had been determined by finding
the absorbance with the Beckmann spectrophotometer, Model DU, the
specific activity expressed as counts par minute per micromole could
be calculated.
Results
After purification by paper chromatography the cytosine Isolated
from the mold which was given uraoil~2-C** alone, showed a specific
activity of 85 and 9k per seat of the specific activity of the original
wraeiX*2*C**. The uracil was labeled to the extent of 77 per cent.
There was a negligible amount of radioactivity to both adenine and
guantoa. When uraell-2-€14 and amtoobutyrate were given together, the
cytosine isolated contained a cementratton of radioactivity Which was
1*0 and 73 per cent of the concentration administered,

toe uracil was

labeled to the extent of 56 per cent of the concentration of radio­
activity given. Again no significant radioactivity m e found to the
purines,

toe results are shown in Table IV.
for fyrimjdtoe
>ra crassa

toe Ut

Materials
Sodium propd.onate-2-C14 m s obtained from the Volk Radiochemical
Company.

Two samples were used,

toe first sample bad a specific

26

activity given at 1.2 millicuriee per millimole.

The second sample

bad a specific activity given as 2.52 millicaries per millimole.
Two experiments mere carried out using the mutant, 1258,
Qrcrwth procedure
The amid mas again grown in 125 nl, flasks, to each of which was
added 25 ml* ef basal mtrient medium. Sixteen flasks were used in
each Qxperiigent. To each of the flaskswas added 0.88 mg. of radio*
active propionate (0.65 mg* of propionic acid) and 6*2 mg. of unlabeled
propionic acid which had been unified by redistillation.

This gave

a concentration of 6.85 mg. of propionic acid par 25 ml* of media, a
concentration found to be satisfactory for optimum growth,

m estab­

lishing the specific activity of the sample, it was found that the
radioactivity of propionic acid must be determined before dilution with
the medium and that two drops of 1 U sodium hydroxide prevented evapor­
ation of the propionic acid from the plates. Observation of these
precautions gave the calculated specific activity, Autoclaving, inocu­
lation, incubation for sir days, and harvesting of the mycelial pads
were carried cut in the same manner as described on page 11.

The iso­

lation and hydrolysis of the ribonucleotides and the chromatography of
the bases m SJowaw 50 were made in the same maimer as described on
pages 21 and 22 ,

27

Purification of the Purine and pyrimidine Bases
the uracil eluted from the column was again removed from the
parehlorate and chromatographed on paper as previously described.
Since the radioactivity of the eluted fractions did not agree
with the ultraviolet absorbtion data, impurity of the cytosine fraction
m s indicated.

The fact that a high radioactivity m s found in those

fractions that gave no ultraviolet absorbance indicated that this
highly labeled compound m s not a purine or pyrimidine.

This m s shown

to be true by chromatographing the highly radioactive fraction that m s
0luted from the columl JUBt before the

fraction.

The Rf (ratio

of distance that the compound moved to that which the solvent moved)
m s determined by locating the radioactivity on the paper.

This m s

carried out using the Forro Chromatographic Scanner (Ferro Scientific
Company, Evanston, Illinois) coupled with a Nuelear-Chicago Model 1620 A
ratemeter and a Model AW Esterline-Angus graphic ammeter.

The

of the

activity peak m s 0.36.
When the cytosine fraction m s chromatographed on paper four spots
were located using the ultraviolet lamp.

Their Rf values were*

(1) 0.55, (2) 0.65, (3) 0.79, a»d (it) 0.88.
spot, while the others were light absorbing.

Spot (2) was a flourescent
Significant radioactivity

was found to be associated with spot (1) which from the spectrum obtained
using the Beckmann spectrophotometer, Model DK-2 m s shown to be guanine,
and with spot (3) which m s shown also by spectral studies to be
cytosine. Scanning of the paper chromatograph showed a high peak of

28

radioactlvlty where the cytosine migrated, a lower peak where the
gamine migrated and a much lower peak at a

of 0.37 where there was

no ultraviolet absorbing spot* The R^ of this latter spot corresponded
to that found above in chromatography of the radioactive, non-ultraviolet
light absorbing fraction and probably indicates slight contamination of
the cytosine fraction with this material*

Elution of the spots from

the paper chromatograph and counting were carried out as previously
described.
This non-ultraviolet light absorbing material was originally eluted
from Dowex-50 by 0.055 N hydrochloric acidi whereas cytosine required
2 N hydrochloric acid*

However, there was not a complete separation of

the two compounds and thus considerable purification was required for
the cytosine fraction as was described above. Since this contaminating
compound does not absorb light in the ultraviolet region it cannot very
well be a pyrimidine or purine. Although the compound did not give a
positive test with nlnhydrin {0.2 per cent ninhydrin in 1-butanol
saturated with water), the concentration may not have been great enough
to detect an amino acid.

However, calculations based on the amount of

radioactivity and the weight of an average amino acid, assuming no
dilution of isotope, indicated there should have been sufficient
material present to detect had this been of amino acid nature.

Neither

was a positive test for a ureido acid obtained after spraying with
and para-diraethylaminobenzaadehyde, but again there may have
been an insufficient concentration.

n

Adenine was reehromatographed to giro on idea of the parity of the

VUtin* fraction* two ultraviolet absorbing spots were found on the
paper*

the n£ values were (X) 0 *57, and (a) O.??.

The adenine wee

fouad ty the Beckmann 0K-2 to he located la spot (2). Elution front the
peper sad counting of the elntate wee carried out In the sente manner as
the uracil end cytosine*
Ikdioatiiography of Paper Chromatograms
Strips tc be radloeutogrepbad were taped to 3 by ID inch sheets of
Kedah BXue Brand X-ray film. A Spot of radioactive solution was placed
In the upper right hand comer In order to nark the relation of the
chromatogram to the developed film*

The films, with attached papers,

were placed between plywood boards which were clamped tightly together
to Insure close contact of chromatograms and films# After a period of

% weeks the chromatograms were removed and the films were developed for
four minutes with Kodak D-X9 Developer, then placed in a stop bath of
1 per cent (w/v) acetic acid for 10 seconds, and left In a fixer solu­

tion of sodium thiosulfate for at least 10 minutes.

The films ware

then washed in cold tap water for apprrarimtely X hour, and then hung
by clips to dry.
Badioautographs run on the chromatographs of the cytosine fraction
Showed the radioactivity of spot (3), which corresponds to cytosine
itself, to be clearly associated with this spot and not contaminated
with ether substances#

30

Results
After purification by chromtography on paper, the specific
activity of the cytosine was 31 to 33 per cent of the concentration of
radioactivity originally given in the medium.
per cent.

The uracil was 33 to 3it

The guanine erne 8 to 11* per cent and the adenine uas 9 to 12

per cent* A summary of the data is recorded in Table XT.
t yresenee of tlraotl-2-C**
ie jaHeuroapora diEWsaa
Materials
Tiro samples of uracil-2-C*5* were used#

The first sample with a

specific activity given of 0.6 milliouries per millimole was obtained
from the Isotopes Specialties Company and was used in experiments with
the mutant 12p8. One experiment was carried out.

The second sample

with a specific activity given of 1*2 adllicuries per millimole was
obtained from Volk Badioehemicals Company and was used with the wild type
of $. erases. One experiment was carried out.
Orewth TToeedare'
Two sets of ten 125 mi* Erlcmmeyer flasks wears used in the experi­
ment.

Twenty-five milliliters of basal medium was added to each flask.

In both sets* O.lt mg. of the labeled uracil and 9*6 mg. of unlabeled
uracil were added to each of the flasks. The second set of ten flasks
p #92 mg. of propionic acid in addition to the 10 mg. of uracil.

31

As in the experiment utilising uracil-2-G14 and ejainobutyrete, the first
set of flasks served as a ©octroi and gars the incorporation of uracil
into the jyrijtd4inea. the second sot shewed the influence of propionic
acid m the amount of incorporation of uracil-2-C*4 into syrimidines of
the imitant, 1298.

The autoclarlng, inoculation, incubation, and harvest­

ing sere carried out as described on page H *

the isolation and hydroly­

sis of the ribonucleotides and the ohromtography of the bases vers
carried out in the manner already described,
coluaaa m m listed end counted.

the fractions £rm the

Hie radioactivity was in direct

proportion to the ultraviolet absorbance.
Results
the cytosine from the mold which was fed uracil-2-C1* and propionate
was found to contain a concentration of radioactivity *dyi.«h ass 53 per
cent of the initial concentration of the radioactivity furnished the
mold as uracil-2-C14. there m s essentially no labeling of the purines,
admins and guanine. Again the cytosine from the mold fed only uracil2-C*4 m ad basal constituents showed a dilution of specific activity of
83 per cent of the original specific activity.

fhe fftilimtim of Umcil-2-CX 4 in the Presence of Arginine for
:T,n$SSjSlpi^^
Kmroapors Crassa
Materials
Uraeil-2-Ci4 m s obtained from the isotopes Specialties Company,
the specific activity of the sample m s given as 0.6 raillicruries per

3a

nU lim olA . lh$ L-arginine mui obtained £rc® P fta .tle h l Ghsnlcal
Cowpanjr. The argaalea nm& m i t t e l . <mam aataBt. 1196, dewrlbad
before.

■

«*wwi jrrcesiuurs

As. wold was grown In 125 nl. Brlenmeyer flasks, to each of which
was added 25 nOL. of basal asdlust* two sets of ton flasks were used,
A both sets, Q.li stge of the labeled uracil and 9.6 ng. of unlabeled
uracil were added to each of A t flasks.

The second set of ton flasks

contained 10 luge of arginine In addition to the 10 stg* of uracil per
flask* Again tho first set sowed as the control, while the second eet
Showed whet effect arg*'filTt<a hod on the inoorporatIon of

i into

nucleic sold ppdaildiaas* A s antoelawiag, inoculation, incubation, .end hawestlng wise carried out m

previously daecrlhod.

The isolation

end hydrolysis of the ribomcleotldea and A s chroratography of the
bases w o w cabled set es jjreviously described.
colmm were plated and counted.

The fractions free* the

The radioactivity wee in direct

proportion to the ultraviolet absorbance.
Eesolts
A e cytosine fraction f m a the mold which was fed uraeH~2~C**
end arginine wee found to contain a concentration of radioactivity
which wee 100 per cent of the initial concentration of the radioactivity
ihralehed the wold as iiracll*^^.

Acre wee essentially no labeling

33

TABLE 17
KADX0A.CTI7ITX OF RIBONUCLEIC ACID BASES FROM KEgROSPORA CRASSA 1298
A F « R GROWTH IN THE PRESENCE OF VARIOUS RADKAC’TIVff*COMPOUNDS
Compound
Isolated
From Mold

Supplement

Specific
Activity of

Specific
Activity of
Compound
Isolated

Ratio

2.60 x 104

cytosine
uracil

2.J*it x 104

adenine
UracH-2-*C ^
«■ aminobutyrate

•9U

76
125

.003

1.89 x 1C4

.72

2.60 x 104

cytosine
uracil

.OOOU
.001

lu
23
2.51 x 10*

.85
.77
0
0

2.13 x ID4
uracil

1.9k x 104

adenine

0
0

Uracil-2-C14
* Aodnobu'tyrate

2.51 x 1G4
1.21 x 104
1.1*0 x 104
8

uracil
guanine
adenine
Experfjwnt 3
>piomie-2-C14

91

.0003

•ooit

l.Ui x 10®
cytosine
uracil
guanine
adenine

k

.1*8
.56

14

104
104
104
104

.31
.33
.11
.13

X 1G4
X IQ4
X 104
X 104

.33
•3U
.08
.09

i*.1*3 X
it.72 X
2.05 X
1.80 X

1.2it x 10®
cytosine
uracil
guanine
adenine

It .10
it .19
0.96
1.03

Continued

3U

TiBLE XV «. Continued

Supplement

Compound
Isolated
From Hold

Specific
Activity of
Supplement

Specific
Activity of
Compound
Isolated

Ratio

Experiment 5
2.50 x 10*
cytosine
uracil
guanine
adenine
Utfacil-24*CX*
♦ propionate

2.21 x 10*

.08

0.2 x 10*
0.23 x 10*

.09

1 .1*5 x 10*

.58

0 .51* x 10*
0.63 x 10*

.21
.21

I.I4I x 10*

.7U

2.50 * X04

cytosine
uracil
guanine
adenine
Experiment 6
OmoiX-S^14

mm

1.90 x H )4
cytosine
uracil
guanine
adenine

tJw.cil-2-C14
+ Arginine

•88

mm

0
0
1.90 x 104

cytosine
uracil

/

i.n

x
0
0

io *

1.01

35

m d gaaatna. Again th© eyiosln© ttm the wold

#£■ the pariaee*
£*d «aOy

«&4 basAL eaasUtamfcs shoved « dilatloa ©f

epeoifie activity of 7U g«p e«ai ©f the original specific activity,

Hal^rielsi
Sodium gvejfaiwtfN^M^ was obtained from the Volk Radl©che»&c*XB
Company*

tym specific activity was gfcrattte 1-2 milliettries ]ser

m lm . Dihydrooracil and eoantfyaft. A (product of ftebei Laboratories)
%uMMiUik

i
l
k
A
m - Iff
diwfrmanflms Jr Jl A th
ttklfcfliiiMtoria iMIuhtMi
WWFW w
P
T
^
M
S
I
W
I
J
£
3
P
G
*
!
w
!
tW
O
P
M
R
v
t
i
i
.
T
O
y
i
w
p Ivw
lEIS^m iW f
l
l
w
B
W
B
l
lwA
*
®
8
H
P
9
e
@
1
8
i
l
*
CMLi^. wMMMMdMI

JCBfl

JSIjjnG^

%YfA the baainja salt ©f

ASc.Mi

MfcJ f ^tokab iMmtiMa
dkUM
am
MkjLvLa
mrn-rnb^mfai^mdm
iS^OpSJw
v)%|^
8ffiKw5
PKBjroW
^ftWU&lfipv

was obtained fpom BuiritA©Bal

IMJI ml
tiitti arinin'i© AM I Mb.'
H ill.
MnBnMAJMttdS
A.M<SbJ jS.Mn. i«.^iiJ AmUm MatkM iilHilkM iM IW M id M A ittM
o
lOS
lflBil(ftiiS* Tw®
QJSXpmfmiLn©MnS'jtopbJtuniAaBfc0
pQnP3s,5jQS IfilCiSOMQ®
8BQ wX|^3pPO|^l M3316

naeleotlde were obtained iws Sigma Chemical Corporation. Bsta-aJUnixw
n u a product o£ Katheson, Colasan and Bell, obtained Srtm the Metro
Industries. Tbs bota-alanyl-Faatetbalne, profdce^jwatefchelne, and
beta-awddoproislonto sold were sgprtfcwiead by tbe rather* Jha ivqatc
tlon of the latter has bam described m page 10.
A * paptm^pmtetbslniS and bsta-alaoyl-pentethiBlae were prepared
tqr tbs thio^imol pnoeedure used br VicOaad CM) bo synthesise thio*
eaters. She propionyl chloride required la tele procedure was synthe­
sised according to the proeedare of Broun (10). Banacyl ohloride
(0.375 aolaa) and redistilled jaajdml* aaid (0.25 »°l«e) ware placed
la a round batten flask and ware distilled through a Synder-Shrinar

36

reflux condenser in such a manner that the temperature at the top of
the column did not go above 76.5° C.

The propionyl chloride distillate

{0.12 moles) was dissolved in ether.

Thiophenol (o.lh moles) m s added

slowly, and the solution m s left standing under nitrogen at 5° C. to
form propionyl-thiophenol. Pantethen®, obtained from the Mann Research
Laboratories, m s reduced with a $ per cent solution of sodium cyanide
in 1 If sodium hydroxide,

the redaction m s followed by the appearance

and the depth of the purple color obtained in the presence of a 5 per
cent solution of sodium nitroprusside.
of free aulfbydiyl groups.

This is a teat for the presence

To the pantetheine (0.08 moles) m s added

approximately SO mg. of sodium bicarbonate, and nitrogen m s bubbled
through. A slight excess of the propicmyl-ihiophenol (0.10 moles) m a
added.

The reaction proceeds for a few minutes during which the solu­

tion changed to a bright yeHcw color and the test reaction with sodium
nitroprusside for free sulfhydryl groups disappeared.

The mixture now

gave a purple color with metbanolic hydraxylamine and ferric chloride,
a test for an ester group. After adjusting the solution with hydro­
chloric acid to a blue color with Congo red, it was extracted with
ether.

This removed excess thiophenol.

The aqueous solution m s

lyophillzed, producing a yellow powder. The beta-alanyl-pantetheine
m s prepared in the same manner with the exception that the acid chloride
m s prepared using thionyl chloride. These thiophenol procedures were
not found to be satisfactory since the investigator developed an extremely
painful rash from contact with even veiy small amounts of thiophenol.

37

Amm

cafciofcctory procctec for nicking proj&on^-pimteiaieijaa

mto imxuI ift

use of propionic safaydriri© (O.X mdea) end puitethclae

(0-12 mclcc) * The di^pp&ft3nu%60 of fro* st&fl^drya gr<mpc

md

app«u»ne# of the eater gimp teak place £& aboat 10 minetea.

the
the

chloridesef bote-alanine made with an aacce** of thloegrl chloride (o.lk
moles*) reacted with the reduced paatetfcene (0*1 melee) In 3 z&« of
triethacioljunine buffer (0*1 H, pH 7*W to give ^tiMOacar^f^

of theee oompouaade in cm .@0 pear cant l^propanel eelTemt
®0r 'ttw^WSi^Ai
apfll ^wW«BEw!i^^w JPWr
epet

i|©l|5$w

gave e xy^eitfy**

41X1.^(*vroBEy3WwWv JWSHSNsWWeyiB^
jiitropruaaide teet before and

after hydrolysis with 0*1 if r ^ l w 1^^dxtsssddtf
Kk dMMli^K MK^iMBdAMKMlJUi
a
anp^rw^A group*

the presence of

Mjai -At itffA la IfkiC-wlTa|kt III Ir iill ril*! f
dfi .ilk
.wflaC ^ dN.M&. &
tdfe. S&r%la^h.
CilMt il
jJt oak
#enx#xjie>wse
xvocx* xa
revccisxaec
w nee oxauxixcie

fojssi ifyiyjy^g

ifiny eiyt doee not give the teat with nitrepecsside

cmleee reduced again:*
Methods

Both alpha and hat* amino aoida ware detected after paper chroma­
tography fay reaction With nlnhyarir!. Brneil M l determined both In
eolation team lta absorbance using the Beetaisim spectrophotometer,
Modal

W,and after chwaatograiiiir on paper fay looating tbs spots with

an ultrwriolet lamp. Attempts at chromatographing propionate ware wa»
successful baaaasa such small amounts ware not detectable by present
methods.

Dihydrouracil and beta-ureidoproplonic sold were detected on

chromatograms (with or without previous nimhydrin treatment) by spraying

SB

ilitfa 0*5 X a©dia» faydrtarfde, | ^ M i | after drying for thirty minutes,
fay spraying with a station containing 100 ml. of ethanol, 10 ml. of
concentrated hydrochloric acid, oid 1 gnu of para*»dlmetfay:i^^
defayde. thlo loot spray destroyed guy color produced fay ninhydrin and
produced a yellow color with ureMo acids (formed by the sodium bytaadLde
ipXtySxig or

m m d^i^ied m

tyr tuo

** ffe® fiap»fe *tea&

of chromatograms with a k K faydroacyiamim isolation {pX 6-5*7 *0) followed fay aprwyiaag with ferric
t t . d E . *■‘•Muifc.
r'l tMl■>'
Wfca&!tlkiMiMiMM
k,JW ‘JiE?
T Uu%iM
JifcJMMhd* X^JlkfWln^kd: A
^fcW**
cixiori»e %& MmxMmtiFQ
orw$mz
i wewMMNi
of
per cent
f e m e cntorxQ©,
jus
Jkhfafc^t iiK■*&.*&.jtfh Jr

ttf tfl‘■Vllit^^rtfli i^ Vl*t I*i'.tiHKlf XiL. acag/•
Mfcait if litf 1
ifTl *4 HIMl»h<14^2
per cent iLmaJ .Ikfafc^fa hii^r*» l i ilih iSftl Jt j^. jite-.rih'&.ddt'
acm, and. 3ffa w
i!0^»rociy.oric
lie secons
tkhJMibfe

Tsrocedare iarolTiKi faydroiywle of the tfaieaitar with strong
of sodium hydroxide im

100 i&* of 95 per cent

(3 esu

methanol), followed fay

spraying with Sodium nltropameside reagent (1.5 gn. of sodium nitroprusflide aired la $ ml* of 2 X sulfuric acid, nod adding 95 »&• methanol
and 10 el* of 26 pen* cent ammonia.

M i

solution was filtered to remove

aalte).

Us* enaymatie reaction was allowed to proceed while kept at constant
teaperatare is • w t « r tw«b. All wcpMflnmito iwre cuwisd out «t 35°

C. fop wajying pwioda of tiiM .

39

A number of d i H M t procedures were us©d for preparing sdations of the ncrooULal constituents to bo spoMiineci for ©osyisatic activity,
the prteipl© mim in ail of the procedures was comj&et© destruction of
tb» J^Slial nails to free tbs inner constituents.

In cats of the first

attempts, jayoelta grown for four days were harvested by filtering on &Buchner funnel, and the pads wars stored in tbs deep frees© for two
days* After slight thawing the pads ware ground with carborundum powder
in 0.1 M phosphate buffer. During grinding tbs mixture was kept sold
by dipping in an ethanol-dry is© bath,

tbs

was

frosen

and upon rethawing and. allowing insoluble particles to settie, tb©
supernatant was used la tests for ©nasnsaiie activity uoon arainobutyric
acid* (Ms milliliter of th® solution was used with various oonoesstrations
of aaaiEiolmtyric sold in phosphate buffer (j® ?.?).

fhe total vote© was

§ si* and. the reaction time was for 30 and 60 minute periods. .After
addition of 1 *&« of £ per ©wot trichloroacetic acid and filtering,
50 lambda spots were placed on Chateau #3 paper,

the developing aolveat

was l«fcute&olfglacial acetic aoldidlstilled water in a ratio of 80*30*80
by vote®. After derelopiag for IT hours, drying, and spraying with
ninhydrin, no change in tee oonoaatration of andnobu^ric acid was
cbserved. When this expertent was repeated the m m results were ob­
tained. Wtm freshly harvested mycelia, ground with carborundum and
incubated with aminebutyrlc acid showed no activity over periods of 30
laimtes, 80 minutes, 120 minutes, and k hours incubation time.
*

kO

Another treatment of the mycelial pads in order to study enzyme
activity involved preparation of an acetone dried ponder*

The freshly

harvested mycelia were homogenized in the growth medium followed by
extraction of the suspension with 6 volumes of acetone at -20° C.

The

acetone was filtered off and the powder was placed in a dessicator.
One hundred milligrams of the powder were allowed to stand in 1 ml. of
water for 6 hours*

Three substrates were used*

propionic acid (3.7 mg.),

aminobutyrlc a d d (5*15 mg.), aspartic acid (U.US mg.) and aminobutyrate
plus 1 mg. of jyridaxd-hydrochloride. Incubation was carried out for
50 minutes followed by filtration through a sintered glass filter.
Aliipiots were placed on Whatman #1 paper aiad the development of the
paper was carried out for 16 hours with a solvent consisting of equal
volumes of 0.1 N sodium acetate and ethanol. Again no change was
observed except for a small decrease in concentration of the aminobutyrate
with the {yddoxal«-hydrochloride. This experiment was tried again using
acetone dried powder of the residue left after centrifuging the homo*
genized nycelial pads, but again no activity was observed. All of these
preparations showed succinic dehydrogenase activity as determined by the
common methylene-blue procedure.
A third preparation was very similar to the first one described
in that the freshly harvested mycelia were frozen in the deep freeze
for I*.# hours after which they were ground in phosphate buffer (0.1 N
pH 7*9) using a tea Broeek hand grinder.

The homogenate was centrifuged

in a Servall refrigerated centrifuge for 10 minutes at 11,000 revolutions

ia

per minute at h° C.
1 mg* of uracil.

One milliliter of the supernatant urns used with

The controls were 1 mg. of uracil to which 1 ml. of

enzyme was added at the end of the experiment.

One mi31.iliter of the

ens^me served as a blank. At the end of 12 hours each of the solutions
was diluted to 100 ml. and the concentration of uracil determined on
the Beckmann spectrophotometer, Model 00. Ho change in uracil eoncentration was observed.
A fourth method of preparation, which was more successful than the
previous ones, involved lyophilization of the harvested myeelia.

This

removes the water under vacuum and at low temperatures* leaving a brittle
dried myoelia which can be ground to a powder.

One hundred milligrams

of this freshly prepared powder, when incubated in phosphate buffer in
the presence of uracil showed a slight activity as was detected by a
decrease of the uracil concentration determined by the absorbance.
This experiment was repeated with similar results for uracil, and at the
same time aminobutyrate decreased in concentration as was shown by
chromatography on paper. Ho activity was observed with respect to action
on dihydrcuraeil. Ho activity could be found 1» the powder that had
been standing at room temperature in a dessicator for several days.
The fifth and most successful preparation consisted of freezing
the freshly harvested mycelial pads on blocks of dry ice and homogenizing
in trie (tris-hydroxymethylaminomethane) buffer (pH 7*9). A modification
of this procedure involved homogenizing the frozen mycelia in a Waring
blender with the dry ice and diluting afterwards with tris buffer.
One milliliter of the homogenate m s used with 0.05 mg. uracil, 0.07 mg.

1*2

tf ribot* t o 0.01 «ig* ttootto

t o total volnmo

m»

3 Mil* 3DaoabatlGM uaa carried oat fo r 12 hour*, ffee eszymatic a c tiv ity
*** fealiod by tailing t o solutions ,i» a watar bath for %$ minutes.
Ali<$iot« (i$© Itoda) tot- t o t o t o g x m i M ©n ^tptr t o t o uxtol »pot#

lo e a to by u#a of «a a lto v lo ltt lamp* U racil 4& t o ionmmo# o f t o ’
a y G tlitl preparation o ta to a groat to x to t to. e v e n tra tio n . Wltb
ftoat t o AW t o a g to l eonctototon decreased ta t not to t o tan*

d&gro* a s v ith a m o il alca®* $ to t o t o o f prsptJEwblott uas trie d using
m rlotts substrates. A to g to ta t o t dAbydrouracil (0 .1 » g .), amia©~
ta % to acid (0*1 Mg*)# tatA to astto (0.05 mg*)* beta-^roidopropim ic
to d (0*1 mg*)* propionic to ld (0*1 m g.), u r to l (0 .1 'Mg-*)f prop&onAs

m M f t o ritaat^**|ta8$m t© ( I mg*), tata*nmdd©p^pi©nie acid plus
(2 »g*5, t o p3E^io^l*»pant«th«l3ae (1 mg.) plus rS ta to
**jiiifeospbsta (2 Mg*-}* $$$&' fixtures Ad a final tMXaws of j? ml* hhwps

ineubstod fo r ilHrtKi bourns. offcor * M

tte s c tlrib jr * * * dootrpyod by

hosting. On. hundred lajfcds *3_l<*aoia * * r * spotted on p *j» r. The paper
was deraioped fo r 2tt iK un tn * satttmted phaaoHvuter solvent. A fter
-drying flw arnl#tt the phenol s t ill prevented detection o f any e ltrs rio le t
absorbing spots. Sp«gri«j5sith niiihyteia ahsiwd *

in a ria o -

butyric asltd but no eherago la b et*-*la ain » . Spraying a ith aCUcali,

followed by spraying s ith p a*w *d lM tl$te*d »fi^

so change

is ecraeenfcratiea ot dlhydrawacJi or bote-woidopropioaie a d d . Ib is
lsfctsr sywKUal p w i***i±o n « u found to giro interesting results in *
study « f its action on propionate ted bete-alanyi-paHtethelne.

1*3

H e incubation mixture eoaaisbcd <xt the following substituents to which
was added .2 aa# of the supernatant frem the centrifuged homogenstet

ppO|4oaot««^^S^ {0.1 uM, 1.1& x 10® counts per minute per micromole),
H*wtfhate buffer (10 W , & 7.9), potassium chloride (50 uH), adenosine
triphosphate (1 uH), coeneyme A (0.3 uH), dbLphosphoKjrtridime nucleotide
(0.2 tali), iriphosj^pp&d^ nucleotide (0.1 uH), glutathione (5 UH),
awssmiiim chloride (1*00 uH), ribose^-phoejhate (0*1 mg.).
volarne was 2.7 si*

H e total

H i t eas e comhimtlm of the experiments carried

cut by Stampf (1*2) and Stadtman (1*3). After incubation for 3 hours
w*i3MjptW wF OX ttw k 0O *O l vi»CBElJI W03P® ^XXAQCKi CBS pfltpflfiP 0JSm CW|pV0iOpOCl 321 fittt 0 0

yOiP OOHv JL*^^p9rOIpOftOX 0033rW0v* SftQDi OwiP03®XX^^3WtlItt U3H8 Xlitt 3H wJtXpl a(SJtvO 3Xt
order that several types of expounds could be located.

One chromato**

graph wee need for ecanning purposes, however, the propionate evaporated
during clnroxm^graphy * H i e seats chromatograph was need to locate
ultraviolet absolving spots. In the control experiment which ocntained
all components but was heated to boiling at the beginning of the axperi~
ment, Six ultraviolet absorbing spots were noted, two of which were
associated with the components added to the mixture. One of these was
aa overlapping area of eoensytas A, dis^sj^opjrridine nucleotide and trijhosphoiyrldine meleotide and the other was adenosine trt:$x»ephate*
la H e case of H e active preparations, the ultraviolet light absorbing
spot that was associated with Cosnsyme A, di^osphopgrridin© nucleotide
and trti&osj&opyridine nuelsotM© was not present, and where ribose-5phosphate and beta*al«j^l*pant6thelne were added adenosine triphosphate

i*U

m m not p m l

nor wee m m of the fcrnr spots froa the heeaogenmte Itself.

0»e of the ehromtogr&sfos m s treated with niabandrin, then was sprayed
with

#a*d MttjmtaafHawfetatylmnHwrihdMwal»3M*nrrij»„ Hn yuan »4nlwi{ly<ii»

positive spots m

m

found*

He bel^weid© acids eon34 be detected.

Sparaying another chromatograph with al&ali end then with the sodium
i&ir©pmsai4e reagent Viewed a new tbio*estsr in the aetivo prejwrailoa

eontelniiig

flm*

M
M
f
c
f
l
l|k
f
i
4

trith *
t
f
r
»
a
a

t ^ ^ sters were

- this spot
^
D
w
a
i
w
f
c
j
D
't
f
o
w
t
w
aiis
jR
Illf* ■
lift

C&foMBP

-

dR
va
bm
um
n iwIELw^llW
tkbawiMtM^^assiki^aMs- m
asg
tat
nM.
b'n
seQ
^E
Mif
eSf
itff^n4a.4%stfW!^fc4fr^unKp '
SSjaenwS'^n e
^tejAd
Mtfawt^MMBK
tt
HS
ist
s
Ell
M^F^4
mkJ
wfw
mel
^.m
meW y t
fpf
Ee'
m^a
twt
ya^
wa r
WWwt^^pURlW*
WMwKw
w»3b
fH
fi
HM^pWr
aaJ
IaW
Ii
ei
tw
dm. CD^lod 011% overnight* At ths place nis*T8 the thio*6stw >pot should
have been a n**r ultrsvlalat abasibiBg spot appeared xhiah mgr be

£eeK&hs&3&0 fraa

of the satar#
SasuXts

She preparation found be be m e t sueoeeaful was that uslas a Waring
blaaiar hoaogenate of the fremt M i d aad dry ios dilated Dith tris
buffer. Being thia preparaiioB diaappoamnae of aaiadfeatyxate sad
uracil n a n obeereed. Also » the jawwaoe of beta-alaayX-jaatatheinB,
rlboae-S-ndMepbate, jwopdanie acid, adanoaine triptoajtoate, aad
eoauqnm A, fe*»atiaa of a smr thio-eatar sa# * W M i .

U5

DISCUSSION
From the results presented In the previous section, various de­
grees of growth of the mold were observed in the presence of certain
compounds in the basal medium.

Other compounds when added to the basal

media failed to promote growth of the mold* Some of these latter find­
ings were expected since the idea that the mutant utilises a pathway
different from the normal organism has already been introduced, Thus,
finding that ureidosucclnic acid and aspartic acid would not support
growth of the mold was not surprising, although these compounds have
been described by Korriberg (IB) as playing an important role in pyrimi­
dine biosynthesis In other organisms. Another compound, orotic acid,
shown to be an Intermediate in this same sequence of reactions studied
by Komberg, has Already bean shown by boring (7) to be used by the
imitant for growth.

The small amount of growth, in comparison to that

found for alpha-aminobtttyric acid and some of the other compounds
shcnm in the results in Table I, probably indicates that orotic acid
does not give rise to orotidine-S1-phosphate and then to uridine-#* phosphate as was suggested by Korafeerg for ih© normal pathway. A rela­
tively slow decarboxylation to form uracil followed by conversion of
uracil to uridine-#1-phosphate, seems to be another possibility.
failure ©f the mutant to grow either on alpha-ketobutyric acid or
on alpha-hydroxybutyrie acid indicates that these compounds are not
intermediates in the use of the amino acids by the mutant.

1*6

Both horaoaerin© and threonine hare bean ehoim to give rise to alphaketobutyrlc acid (1*1). Klmcay et &1. (14*) have traced the catabolism
ef Cx*-lab©l©d axdnobutyric acid in rat liver homogeaates and found
that the amino acid is converted to the koto acid followed by oxidative
decasbcocylation to the next loser monoe&rboxylic acid, propionic acid.
Fairley (20) has demonstrat$d that aminobutyric acid, homoterlne, and
threonine were utilized by the mutant to about the earn* extant for
growth.

Homoserine and threonine are interconvertible and this inter**

conversion has been suggested to take place through the hydration of a
vinylgiycin® intermediate (hi).

Hydrogenation of this intermediate has

been suggested to occur giving alpha-aminobutyric acid (5Q) • Allothreonine
did not replace threonine as an additive to the basal media supporting
growth.

Threonine raeemase, tharefore, would not seem to be present in

the stttant, although it has been found in Escherichia coll (£1).
Since isoleucine can be derived from the alpba-ketobutyrie acid formed
from either threonine or homoserine (52), it was expected to act in a
maimer similar to these two compounds.

However, the failure of isoleu­

cine to support growth seems to further indicate that the Iceto acid is
not the common intermediate between threonine, homoserln© and aminobutyric acid.

Failure of cyclopropane aminocarboxylic acid to support

growth suggests that the mold cannot open th© cyclopropane ring to form
either homoserine, threonine or aminobutyric acid.
Since threonine is known to give rise to butyric acid and propionic
acid (55), both of these compounds were tested as possible intermediates

hi

im the utilization of the aliphatic amino acids.

In higher animals

threonine can he cleaved to yield glycine and acetate (5U). The results
show that of the four compounds, propionic acid was the only' one used
by the mold for growth,

these results were comparable to those obtained

when aminobutyric acid was added to the medium.

This finding Is strongly

indicative of the use of propionic acid for pyrimidine formation in the
mutant by & pathway closely related to that by which the amino acids
are utilised.
The metabolism of propionate has received considerable attention.
St&dtman (1*3) has shewn that dried cells of Clostridium propjonlcum
metabolize propionic acid in the presence of ammonium salts to form
beta^alanine.

These reactions tales place through Coenzyme A derivatives.

Traplonyl-Coensyme A is formed from propionic acid, adenosine triphos­
phate and Coenssy&e A . Propionyl-Ooenzyme A or acrylyl-Coonzyme A in
the presence of ammonium ions is converted to beta-alanyl-Cosnzyme A.
An early step in the utilisation of propionate by pig heart involves the
addition of carbon dioxide to the three carbon compound (55)*

This can

occur only after the propionic acid is converted to propionyl-Coenzyme A.
The product of the reaction is methylmalonyl-Coenzyme A and the reaction
requires adenosine triphosphate.

This product is then converted to

succinic acid by an isomerization reaction.
Other pathways of propionate metabolism exist.

In cow udder

propionic acid-l-C14 Is converted to acetic aeid-l-C14* & reaction
which can not take place by way of the above mentioned mechanism (56).

Ia peanut mitochondria, propionate appears to be acidised to beta*
hydroooosropionic acid,

pmfamp byway

of

the

Coms&Mrii derivatives

propionic acid* acrylic acid, and beta4$rdrc«ypr^

of

acid (M)*

Ift Trie* of the atAebence of these jntbirays, Bam of the inter*
modim m of these reaction* m m tested as growth js*Gmoters for the
mutant. 0g the compounds tried acrylic acid, beta*lydroaypiropi©nic
acid and met^lmadcwsic acid aapported growth, suggesting that boas or ^
all of these Gfmpmi&et may be iatemediatee in the nee of propionate
for pyrimidine formation*
fhe finding that propionate end related compounds supported growth
of the syriaddineleas atmin, led to the suggestion that these expounds

m m used for pyrimidine foimtion by omrerslon to beia-el&nine, beta*
ureidopropionie acid, and dthydrouraoil* When the compounds were tested
^ iU
iMkfcdt UUii?l:
llb^UlkjA
fw h *ih
At dSa*tokfa'Mfcd& Jb.rfk
a l r 4 m |-ft
VXtfu<(ltMJMltiM**aK4^fc
as
growth supplements^
osta^aianins
was
xcmm to me *S*tftUflk
inactive*
However^

the remaining two compounds dtd permit growth to occur*

beta*

alanine itself Is not utilised for growth, the pathway by which propionic
acid is used might require formation of the activated form, bei*«w&anylCoanzyme A.

fhs failure of the m M to use beta-elanine indicates the

absence of the wrnym needed for the direct activation*
fhe results obtained from experiments In Which the growth of the
mold was deteradned as a function of time for these various growth*
supporting ccropownds, indicate, however, that neither propionate nor
aminobutyrate can be used by a pathway which involves free beta-ureidopropionate or dihydrouracil as intermediates,

these conclusions were

readied on the basis of the results shown in figure k, showing

149

amino-butyrate and propionate to give & lower maximum of growth as
well as beginning growth at a tin© far before dihydrouracil or ureidopropionate.

If propionate and aidnobutyrate were utilised by conversion

to ureidopropionic add, dihydrouracil and then to uracil, the lag in
initiation of growth, which probably indicates adaptation to the substrate,
should hare been of the sane order or greater than with dlhydrouraeil
and ureidopropionio acid*

Since the maximum reached in the curves when

the latter substances were used as growth substituents, is similar to
that reached by uracil, these substances probably are utilised after
conversion to uracil by the reversal of the degradative pathway shown
in Figure 2. Once growth begin, the growth rates as measured by the
slopes of the curves were essentially the sane, indicating no great
destruction of any of the compounds occurred during the adaptive period*
Compared with the other compounds, aminobutyrate and propionate were
found to give a lower maximum of growth,

this is presumably related to

the use daring growth of substantial amounts of these compounds for
metabolic reactions other than pyrimidine formation.
Since the absence of uridine derivatives for coensyme reactions
has been suspected as the major deficiency in the mold as it begins
growth, selected compounds were chosen to see whether the addition of a
small amount of uridine would cause any significant growth effect.

The

results in Table II showed that only dihydrouracil gave a more than
additive response in the presence of uridine* A similar effect has been
shown (21) for aminobutyric acid and threonine.

These results are con­

sistent with the use of an initial supply of a trace of uridine to

So

jMrittft * e m p i i»»wii«upy In th# utilisation of t&ee® cmpmatei
tonWW|| tfeli 8©a« not rt&ft out t&© po»«tbiIity tfeai uridine siatply
allawit a worn rapid adaptation by m m k m m pcmmm*

m * reaulta In Table 17* Bhawing tba uiilimttc® bgr 1&e sold of
fe®t& B&dieaeiiim uraull and «ttiudbu%mte supplied together in tbs basal
awdtem# jawida laxlton* support fear ths utiligsatiosi of aaincAmtyrate in
ipdadd&*is biosynthesis* Sfc the Gonirol sa^&idwaats with

mmam ljnfo*rtd*9

AltiK!&» tbs
8S to W

par o m t of tbs initial oous«nti*mtim at jmdioaeiirtty aippLisd

In tbs nmoll#
WHO

* vmtsr M <*h *gttSBt*

afeMiftMkjfeat ^4 ----.'at

If the ^ntlnidln«a for»ad had a# their sol® pp9o$t*n*s?

i,Jftmi^t,

€5* M ®

lUibh^MfaErtb

9fcfeA Atj^ii

w R fl^ m U m Jm fllw w ' 0 9 933 4 .9 0 9 X 9 0

vO i n

Idflmttiaal sactent as tbs praeureQi*# fjfyft dilution of the isotope which
nan aolnally fw iwt
at the

that a^ntkssls of ths pyirt&idi&&$ occurred
the

s©tup©es of the

ymeteS't'ty»_> fRIffh on fffitftftftdfiftfotsci tantnat@j $$ WOll && fPQBi the

8UP|>3JLOd#

Whsn wijMtoatyrie m i d m s *Ma& «3m g with tbs uracil-2-C1*, utilisation
of

d m docresood to on ermn groator octant, and produced

EPrliddinea which n » labeled to <afly 5S to 72 per cant of the concen­
tration of radioactivity capriied a* uiMi!*®"®1*.

®»mo eKpopteaot#

t t w that whan « d » M p i t e and «**«41 w w a present at tbs m u m time
the sold m o d both for syattisols of KWlaJ4ta98.

Ttrnra «xpn<tMgst8 did

not iaMoots «Nrihar ooinob«%»4o ooid evasod dilution of tbs csdio>
sattoi^r bgr ojmtbasis at xamlX itsolf or wbstbsr it m s atilissd bp•Bother mitts.

Bsmmr* tits duts pressntsd jsmiousljr in tbs grcarth

51

curves (page 19) suggests tbs two substances were used by different
routes*
If propionate and aminobutyrate are used by the wold for pyrimidine
biosynthesis by tbs same pathway, then the concentration of radioactivity
found in the pyrimidines Isolated from the mold would be expected to be
of the same order as that found for aminobutyrate*

Indeed, the results

shewn in liable IV do shew considerable dilution, 31 to 33 per cant of
the initial concentration of radioactivity, to have taken place in
formation of the pyrimidines by the mold when propionate-2-C14 was added
to the medium*

However, aminobutyrate-3-C14 was found to be utilised

to the extent of li* pen* cent of the initial concentration of radio*
activity*

Thus, although the dilution Is to a much greater degree than

uracil itself, it is to a lesser degree than aminobutyrate*

The rela­

tively low concentration of activity found in purines, ID per cent of
the original radioactivity, indicates that there is a specific incorp­
oration of propionic acid into the pyrimidines.
Further evidence for utilization of propionate for pyrimidine bio­
synthesis by the mold is found in the results expressed in Table IV.
Although in the above experiment the dilution of the initial radioactivity
m s great enough to section the specificity of propionate utilisation
for pyrimidine biosynthesis, this experiment shorn that propionate is
used to a considerable extent* Again in the control experiment using
uracil-2-C14 alone, a slight dilution of the isotopic carbon was noted
as the compound m s used for nucleic acid pyrimidine formation. When

52

ppoptonlo m U mm m m u tth the mwmlMMfl* to ths b siftl swdius^ th *
flutt* mood both of tfa© coapcmnd*

torpyriiaiaia# syathesi*.

fho urm cil-

u&§ fcworpormted to th * octant o f 53 |w r cent of the in itia l
concentration o f
u tiliis tio a fo3p jairineSa

iftfco th * isnri«M*ae* end w w tie slla r a *
It

mpptmraolmr,%hemtore,th at propionic

acid mi»t bo- added bo the H o t of krnam jtf&jMMttie precursors.
W ith th is demonstration of the moo of tW

acids* ostd&&*

butyrle tod propiento eeide, fo r jgrrfaddiae biogadaeeie toe problem o f
to r J*to fcy toiash theyere u tilis e d jeraeanto lt w if . S m w m m of to r
Of tho &VX$g$$P ©f growth, it. #»QW
bfttk compounds are mtilisoi by the
lioosoBtof by tho sxpopisiBiits

to ftOflOfitO that

pathway >■ With the 5jifo:naaiian
this thesis* a ^**ffeaT. potiomy

oo21 8 W bo suggested*
.^totototot ki^i
©:
MS idMflWkik f
ir APl^Mb .
^toS tofc
The pftipesea
pathway <in
giTea «Sin^h. *OFigoo
5*
po-tho 1^*.^.
basis
of peo»-

'OMktkldlL

p4

gut oadbiobutyTio acid being closely related* the suggestion '

io wed** that they go

a

intermediate

i© betft^alAnyl**

cosnsyis© A« Ib is la tte r s tita ftw * has ib N r boom shew* to be resdfay
derived

pa^i^oogrl^oOBSiyiao A. Hot© should bo sod© th at th is is so

activated fopi: of bots^oissiso which might

the fa ilu re of the

to tte r eubetence to euEpert ®fcwto. B » eddition of rlboee-piwepbete
to bttlawed to tokr piece « t to la etoj> v lth tha £nrm1A.m of th« bate-

tlMc&X-Qemusr** i-rib o tld e . 5Eh« iuact step wwOd b« the foroitlc® of
the

To^'*ax*iAoT^$£my%MtomtfmA-^rSbotide. Treatth is ooaqpennd,

dl^<draim ell.-nhotiiSe oonld be fomed end then reAieed to nrl<llae^>‘ phoephato. A etaalljr* toe tofo«cto3tl«4 o f toe «UM>ee«tooa|tokto could

HO— C H 2

CH*
PROPIONIC

H Y DR OXY PRO PIO Nld1

ACID

ACID
a d e n o s in e

CDEn Z Y M E

t r ip h o s p h a t e

a

CO- C oenz ^

CO-COENZYME A

\

CH,
/ 2
CH,

\

P R O P IO N Y L -C O E N Z Y M E

_

CH

-----

//
CH„

A

1

ACRYLY L - COENZYH

HN—

C

/

\

0=C

\
N—

»
CH

(—

//
CH

1

R t B OTIDE

URIDINE-5 - P

FIGURE

5-SUGGESTED

P A T H W A Y OF PYRIM
CR ASSA

Ml

c

o

2h

H ,N — C H

\

C H a <r

H _N —
2

—

/

/

\
c h

CH

C H

HO

C OoH
C H
2

3

AMI NO B U T Y R I C

H OMOSERINE

A C I D
N»
C O - C

/

c h

o e n z y m e

2‘

C O - C O E N Z Y M E

a

R t b '’o t i d e

2

'-ALANY

l

COENZ Y M

^3 - A L A N

E

A

/
o=c

A-

CO-COENZYMEI

\

\N

/ C H 2

C H„

R IS O T I D E

R I B O TID E

/?-

H Y D R O U R A CIL-

[129 8

YL - COEN Z YM E

H ,N

I

UR E I D O P R O P I O N Y L -

C O E N Z Y M E -A -R IB O T IO E

e

pO S Y N T H E S I S

CH2

R IB O T ID E

p
HN—-C
\
/
C
CH
/
\
ch2
N

Ri b o t i d

^ C H 2

HN

CHo

h2 n

\

A

IN

THE

N E U R O SPQR A

Sk

occur after tbs formation of b«ta«weid©propia^

A or the

role*** of Coenaya© A wight also ocour nitb tbs foraation of beta*
alassyl*rlfeebideo At any rate, it should bo noted that this route differs

turntbs

simple reversal of the reactions described by Fink (22), which

indicate that eid^tii90^*«|hoa^to is degraded to beta-elanine by ngr
Of free betaMireidoi^ptodc «sM, sod dil^riireniraoil, in that activated
compounds la which the glyeoaidic linkage already cadets are meed*
T w & i M m w evidence has already been presented by arisolia (57) shosN*
ing that beta*«*F«Sdoi^

and dihydrimtraeil^rlbotide are

eaayiratloally conrertcd to uridine*#•-phosphate by rat liver.
little or ho evidence is available for the remainder of the proposed

pathway*

that part e^gges^iftg the route by

eet4 m y be

a&Lnobutyrio

to enter the lyrimidiae stracture# As ***** beau stated

l»eriou«3y it i s known that aminobutyrate can be deandnatsd to fora the
koto

»w^ tbefr decarboxjrlatad to fora cror&caaic acid* However. the

koto «Sl4 4o«S not support growth, unking this pathway saw unlikely.
Aacthor suggestion Is that ths fowoatlm of henossrlms from aadnobutyrato
fcyssy of the vinylgiyoine interaedlate gives rise to frrdrrayproptonlc
•014 hy • nochanlim of dsawtnatian and dooarbeoylation. ^ytfrraqrproploalc
•014 is wOated to projAanis aelA, sa|*oss«ly through aoxylis sold or
bstetsCLsisiBO'
I n n tfaoo# little disroot ssSAsaeo Is awafUKlo, this pattsmy Is
seseeUrtsm* with the available cvidanee sal is offered as a guide for
Author study. HArlsy (20) has reported the inhibitory action of

55

atfglai&s m tbs use of aminobtttyri© acid and uracil for grartb of ths
aatanbe

Tbssa studlas wars axiaadad In the peasant wc*te allowing tho

asms lahiMtory effect of arginIn# oa sropteat© acid and honiossriiie
aiiMaatfcsn.
of

The experiments demonstrated that o»2y a ▼aiy swell saoant

(0*01 ag.) ia needed to completely iiflilblt growth la tho

presence of 5 rog* of «inin<^tyrie acid, inropieaio add, or hemoseriiie.
fhio Is a further Indication that these compomido are used by tho same
pth*

fhe growth of the mold In the presence of uracil, is inhibited

hf arginine, bat only at snob Mghsr e<»*ceatrations.

This indicates .

that uracil probably does not follow the same pathway as propionate and
adnbbutyrate*

Further evldeaiee of this point was cfotained in the

^BCBCJ^B^P3LMB0fiKlCkJ^i 3J9l iBBw@jBk H ' 3 H | M 5 ■

Wiftfll jWaBBU9;t

L

vO

ISOXCl 3Jft tlJMJ

prasestee of arginine* With the uracil-2**C*■'* alone, a snail amount of
dilution of the isotopic carbon use again noted. However, with the
arginine this dilation was not p m t *

This seems to indicate that the

dilution, canned by the formation *€ |ff^£o^|i*Coni]iM’A fSroa ths edmgiLo
carbon source* cnee motabolio pathmye a m undermy, la Inhibited and
• U of law pyrimidines oro feraed ixm tho unwil-i-0**.
tho enuyaatio experiments were undertaken to obtain avidonc# an
to whether or m b m m of tho reaction* postulated for the use of
propionate for pyrimidine synthesis actually da occur In tho mold.
Considerable effort m e expanded trying methods by which tho tough
nyeelial w i le oould ho raptured, at tho earn time giving active enzyme
prepexwUeno.

Bost rosulte mere obtained with the frozen aycolia

56

bomoganisad In <liy le«* Activity v n abonn on uracil, aainobntypate,
and

13*© I&tfes? wKptmA t m triad instead of

lt® €©Oii*ys» A derivative for m m m & m t I reasons.

!ton w m oil

preliaiinary eapariaaaia end, on ouch, ekm that onaymiie activity
tovard certain substrata# 1® present.

However, no eoncIasAons can bo

drawn on tbo ralation. of these activities to the proposed mechanism.

57

m m n
!• Wartime evidence for the utilisation of aminobutyrate for synthesis
ef nucleic m M jyrimldincs by the mutant K. crassa 1290 ma* obtained
ia experiments carried cut with the addition of ttnell~2*€*4 end
andnobutyrate to the bneal medium.

The control experiments with

nreoil-*2*C14 aims showed a Smell auction of the radioactivity of
the uracil adidjalstered, m

it nee incorporated into the pyrimidines

of the nucleic acids. With uracil~2-<ii* #ad aminobutyrate both in
the medium the mold Shewed the ability to use both of the substances
at the sane tim*
activity* in the

This m b ehown by the great dilution of the radio*
jyrlmidiRSM>.

2* A search for further grcsifth^^pportin compounds for If* jgasssiai
added the following compounds*

propionic, acrylic, beta-hydrcay-

praj&onic, beta-uraidoptropiGnie, ffiSthylnalonic acids and dibyxhreuraeil.
Various intermediates of the Koraberg scheme were not need for growth.
Studies of the time-cours© of growth of the mold on various supple*
meats shewed that propionate and mdnobatyrate gave similar responses.
Beta^oreidoproplon&te and dihydrouraeil required longer periods of
adaptation before growth began*
3* Broplnate*2-C14 m e found to be incorporated, relatively specifically
into the pyrimidines of the mold*

The purines of the same expert*

manta contained much loser amounts of carbon-lU*

58

It. She { M M B N of nnlalwHed propionic acid la the growth wMam
depreeeed significantly tits specific aetlTitt.es of the pyrimidines
fexnad by the sold la a madia® also containing ttr*cH-2*Cw .
$. Arginine m

foam to inhibit strongly tits growth of tits mold with

propionate or heaoaerine a» mpg&emHits. A weak inMbttico of tits
use of uracil for grcarth m

noted, tits jmssnss of arginine in

heml wedfww containing araoll-a-C1* led to aa lacrosse in the
specific aetiwities of tits pyrlMidines fonaed.
6. Preliminary experlzRenbs shewed that ewsymas wars present In the mold,
l
u
u
J
&
aSIXQOIm a
r
a
l
B
O
D
t
t
t
y
i
U
t
i
O
r t
&
f
S
&
i
XA
X
U
Z

?. Ob tha basis of tbs results a slur pathway far the synthesis of
pyriiflidt&e compounds is suggested, a pathway which leads fro®
p^pl<»iyl*^3oenay«e & throu# the Coomym I derivatives of bate*
alanine and beta^ureidopropionic acid to dihydrouridylic acid and

finally to uridine-*#*-phosphate.

59

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1. Hotchkiss, R. D.j Brachet, J., The Suclelo Adds, Vol. XI, Chargaff,
E. mod Davidson, J. H., eds., Chapters 27 and 28, Academia Press,
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60

17. Koxnberg, A., liebaiwan, I., and Sirama, E. S., J. Biol. Chat.,
215, 389, 1(03, 1(17 (1955).
IB. tiebermn, I., J. Biol. Cham., 222. 7 ® (1956).
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62

X* Basal M

dh

Calcium chloride
l ga.
Ammonium tartrate
go gn.
Ammoniumnitrate
XO gm.
Potassium d%drogeaa phosphate
10 gm.
l&gneeium sulfate heptahydrate
g gm.
Sodium chloride
1 gm.
Sucrose
101 gnu
Blotla
26
Trace element solution
10 ml.
Distilled water
to 10 1.

2, Trace element solution!
Sodium tetraborate
Ammonium molybdate
Fmric chloride
sulfate h&it&hydiftte
Cupric chloride
IrfirtiTrri initfi’lfra ii*ih» iW 4W-lfk^l 1
1wol mltm '
m
n &mcnia onionme
Distilled water
3* Guitar® slantst

8*6 gm*
6.i* gen.
' 50.0 &*.
200.0 gm.
27.0 gm.
1. £f
a*>
gm*
to 500*0 mi.

Th© mold is iiaintained on culture slants eoasisting

of Vtaaai vftariium containing 2 per cent agar a*** 1.0 rag, of uracil per
ml*

ttm agar and uracil are dissolved in basal medium by heating

and 10 ml* fractions of the resulting culture median are transferred
to fytm tost tubes.

Hie tubes are stoppered with cotton plugs and

are sterilized by autocXaving. The tabes are placed m a slant while
Still hot to provide maximum surface area and the contents allowed
to gel.

The mold is transferred from tube to tube at two^week

intervals using standard sterile technique.