STUDIES ON THE METABOLISM OF SOME ACTINOMYCETES
ASSOCIATED WITH POTATO SCAB

by
ROBERT JOHN DOUGLAS

A THESIS
S u b m itte d t o th e S c h o o l of Advanced G ra d u ate S t u d i e s of
M ich ig an S t a t e U n i v e r s i t y of A g r i c u l t u r e and A p p lied S c ien c e
i n p a r t i a l f u l f i l l m e n t of th e r e q u i r e m e n t s
f o r th e d eg ree of
DOCTOR OF PHILOSOPHY

D e p a rtm e n t of M ic r o b io lo g y and P u b l i c H e a l t h
195?

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ACKNOWLEDGMENTS
The a u t h o r

is

p leased

t o P r o f e s s o r E . H. G a r r a r d
B acterio lo g y ,

work*

thanks

of th e D epartm ent of

O ntario A g ric u ltu r a l C o lle g e,

m aking a v a i l a b l e
in th is

to e x ten d h is

for

t h e m a t e r i a l s and e q u ip m e n t u s e d

The s u s t a i n e d

in te re st

o f Dr* C. L*

S anC lem ente, D ep artm en t of M ic ro b io lo g y and P u b lic
H e a l t h , M ichigan S t a te
fu lly

U n iv ersity ,

a lso g ra te ­

acknow ledged*
S incere

H. E l l i s

and J .

thanks are exten d ed to M essrs. R.
J . H o o shley, P h o to g ra p h ic D i v i s i o n ,

O ntario A g ric u ltu ra l C o lleg e,
of the

is

photographs.

for

the

p rep aratio n

TABLE OF CONTENTS
INTRODUCTION

1

LITERATURE REVIEW

2

SOURCE AND IDENTITY OF CULTURES

lU

GENERAL CONDITIONS FOR MAINTENANCE AND GROWTH

lb

RESPIRATION OF MYCELIAL SUSPENSIONS
G en eral T echniques
Age o f C u l t u r e

16

a n d Amount o f S p o r e

A d d i t io n of G lucose t o th e

Inoculum

G r o w t h Medium

17
22

A d ap tatio n vs P e rm e a b ility E f fe c ts

26

O x id a tio n of O rganic A cids

28

E ffect

30

o f S t a r v a t i o n o f t h e M ycelium

The O x i d a t i o n o f Amino A c i d s

35

E n d o g en o u s R e s p i r a t i o n and t h e R e s p i r a t o r y Q u o t i e n t

38

E ffect

M+

of D in itro p h e n o l

The O x i d a t i o n o f

cC -k eto g lu tarate

Pigm ent P r o d u c tio n and T y ro sin e
D iscu ssio n

O x id atio n

^9
56
63

CELL EXTRACTS AND DYE REDUCTION
E x p e r i m e n t a l M ethods and R e s u l t s

67

D iscu ssio n

72

TRANSAMINATION
In tro d u ctio n

77

M ethods

77

R esu lts
SUMMARY
REFERENCES

and D i s c u s s i o n

79
98

STUDIES ON THE METABOLISM OF SOME ACTINOMYCETES
ASSOCIATED WITH POTATO SCAB

by
ROBERT JOHN DOUGLAS

AN ABSTRACT
S u b m itte d t o t h e S c h o o l o f Advanced G ra d u a te S t u d i e s of
M ichigan S t a te U n i v e r s i t y of A g r i c u l t u r e and A p p lie d S c ien c e
i n p a r t i a l f u l f i l l m e n t of the r e q u ir e m e n ts
f o r the degree of
DOCTOR OF PHILOSOPHY

D e p a rtm e n t of M ic ro b io lo g y and P u b li c H e a lth
1955
Approved

ABSTRACT OF THESIS
A number of e x p e r i m e n t s w ere c a r r i e d
o b t a i n a m o re c o m p l e t e
p o ta to scab organism ,

know ledge

of th e m etab o lism

S trep to m y ces

t e c h n i q u e s were d e s c r i b e d f o r

out in order to

the

s c a b i e s ,.

E xperim ental

p re p a ra tio n of m y celial

hom ogenates f o r m anom etric i n v e s t i g a t i o n s ;
such v a r ia b le s
the

in flu en ce

w ere

as the

o f s t a r v a t i o n was d e t e r m i n e d .

o x id atio n ,
oxid ativ e

in

of o x i d iz i n g

a number of m e t a b o l i -

the d e m o n s tra tio n of

su ccin ate,

ty ro sin e,

The R . Q .

p h en y lalan in e).

of the

to the

as

in d icatin g

at lea st

The o x i d a t i o n s
s h o w n t o be

was c o n s i d e r a b l y
resu lt

of th e

in

p art,

P athogenic

of

was d i s c u s s e d .
sto red re se rv e s

coupled w ith th e

obser­

d u r i n g a u t o r e s p i r a t i o n , was

by n i t r o g e n o u s r e s e r v e s . .

of g lu co se,

g lu tam ate,

By t h e

hom ogenates th e
in creased .

in h ib itio n

(e.g *

t h a t t h e e n d o g e n o u s a c t i v i t y was

in co m p lete.

starv ed

added s u b s t r a t e

T his v a lu e ,

v a t i o n t h a t NH^ w a s l i b e r a t e d
in terp reted

t h e maximum

The p o s s i b i l i t y

hom ogenates m e t a b o l i z i n g

w a s f o u n d t o be ~ 0 . 8 6 .

phenol to

o C -k eto g lu tarate

was a t t a i n e d w i t h s e v e r a l compounds

m em b ran e i m p e r m e a b i l i t y

sup p o rted ,

D ifficu lty

and p ro n o u n ce d l a g s were n o te d b e f o r e
rate

of

Hom ogenates

i m p o r t a n t o r g a n i c a c i d s and am ino a c i d s .

was e n c o u n t e r e d

w ere

the e f f e c t

amount of in o c u lu m , m y c e l i a l a g e , and

s h o w n t o be c a p a b l e

c ally

of th e

and s u c c i n a t e

ad d itio n

of d i n i t r o -

a m o u n t ox g l u c o s e

T h i s was b e l i e v e d

o xidized

t o be t h e

of a s s i m i l a t i o n by d i n i t r o p h e n o l .

c u l t u r e s were

shown t o c a u s e

pronounced

p i g m e n t f o r m a t i o n i n a p e p t o n i z e d m i l k medium.
cataly zed

the

w ith the

o x id a tio n of ty r o s in e

and d i h y d r o x y p h e n y l a l a n i n e

f o r m a t i o n of m e la n in .

E x tra cts

o b t a i n e d fro m m y ce liu m d i s r u p t e d by g r i n d i n g

w i t h a lu m in a were u n a b le
uptake

Such c u l t u r e s

t o b r in g a b o u t an exogenous oxygen

i n m anom etric e x p e r im e n ts .

These e x t r a c t s

co n tain ed

d e h y d r o g e n a s e s w h i c h c o u l d be d e m o n s t r a t e d u s i n g t h e d y e
2 ,6 -d ic h lo ro p h e n o l in d o p h en o l.
to stim u late

The a d d i t i o n

dye r e d u c t i o n i n t h e

presence

o f DPN w a s s h o w n

of g lu ta m a te ,

m a la te , and f u r n a r a te .
M y c e l i a l h o m o g en a te s and e x t r a c t s were a b le
glu tam ate
acid

in the

(asp artic,

presence
alan in e,

of

c < -k eto g lu tarate

leu c in e ,

or v a l i n e ) .

g lu tam ic

tran sam in ase

served.

No e v i d e n c e w a s o b t a i n e d f o r a d i r e c t

b etw een

a c t i v i t y was th e

© C -k eto g lu tarate

and t h e

cause

of h ig h

am ino a c i d

in the

glutam ate

fo rm atio n .

and a n amino
The a s p a r t i c -

h ig h e s t of th o se

ob ­

tran sam in atio n

am ides a s p a r a g in e

amine a l t h o u g h b o t h com pounds w ere d e a m i n a t e d .
were e n c o u n t e r e d

t o form

and g l u t ­

D iffic u ltie s

d e m o n s tr a ti o n of t r a n s a m i n a t i o n b e ­
" c o n t a m i n a t i o n ’1 a n d n o n - s p e c i f i c

1

-

-

INTRODUCTION
The s c a b d i s e a s e

on p o t a t o e s h a s c a u s e d c o n s i d e r a b l e

e c o n o m ic l o s s by v i r t u e
extrem e

in stan ces

re n d e rin g the

tem pts to c o n tr o l the
p ractices

of lo w e rin g th e

d isease

the

serio u sly

o t h e r members o f t h e m i c r o b i a l p o p u l a t i o n ,

come a p p a r e n t .

stru c tu re

In occurrence,

although th ere
sev erity ;

on s o i l

is

ev id en ce

certain

so ils

A t­

the

to the

considered

Con­

grow th of

in te rfere

w ith

i n w hich case

and f e r t i l i t y

the d is e a s e

organ­

in h ab itan t.

may b e ­

i s w idespread

of r e g i o n a l v a r i a t i o n

are

sale.

probably because

s o i l unfavourable

scab organism g e n e r a lly a ls o

d e le te rio u s e ffe c ts

or i n

by s o i l am endm ents o r c u l t u r a l

a p p e a r s t o be a n a t u r a l s o i l

d i t i o n s w hich r e n d e r th e

grade

tu b ers u n f it fo r

have b e e n d i s a p p o i n t i n g

ism r e s p o n s i b l e

p o tato

in i t s

t o have a h ig h scab

p o ten tial.
Over t h e

past f if ty

search has been d ire c te d

years an ap p re cia b le
to the

s t u d i e s have d e a l t w ith :
p o ssib le

organism s concerned

(102).

(1),

and t h e

The p r e s e n t w o r k i s

rep o rted

o n some o f t h e m e t a b o l i c a c t i v i t i e s

fo llo w d escrib e
g en ates,
reactio n s

of th e

to carry

of the

concerned

of sc ab -p ro d u c ­

The i n d i v i d u a l s e c t i o n s w h i c h
a c tiv itie s

pigm ent f o r m a tio n of th e

p rep aratio n s

(83),

problem and o b s e r v a t i o n s a re

the r e s p i r a t o r y

of c e l l e x t r a c t s ,

such

physiology

la tter

iso lates.

In d iv id u al

p ro cess as

w ith the

ing actin o m y c ete

aspect

scab problem .

the d is e a s e

m ethods of p r e v e n t i o n

amount of r e ­

cu ltu res,

and t h e a b i l i t y

o f m y c e l i a l homo­
dehydrogenase
of actinom ycete

out tra n sa m in a tio n r e a c tio n s .

-

2 -

LITERATURE REVIEW
In the

study

of the r e s p i r a t i o n

o f "both a c t i n o m y c e t e s

and f i l a m e n t o u s f u n g i m ost w o rk e rs have u sed c e l l u l a r m a t e r i a l
harv ested

from shake c u l t u r e s

(79)•

type

g r o w m uch m ore r a p i d l y

tio n

of a e r i a l m ycelium i s r e t a r d e d .

A gitated c u ltu re s

(23) and,

in a d d itio n ,

hyphae

m ix tu re

p ictu re

b o lism of the

t w o may g i v e a c o n f u s i n g

I t has been n o ted ,

(79)

so t h a t a

of th e m eta­

how ever,

t h a t the

r e d u c i n g c o n d i t i o n s w hich o c c u r i n subm erged c u l t u r e

can give

rise

organism .

the form a­

A e r i a l m y c e l i u m may be

m e t a b o l i c a l l y d i f f e r e n t from v e g e t a t i v e
of the

of t h i s

t o an a c c u m u la tio n of i n t e r m e d i a t e m e t a b o l i t e s w hich

w ould n o t a p p e a r

on s u r f a c e

c u ltu re

(79)-

C o n s i d e r a b l e w ork h a s b e e n done on t h e
strep to m y cetes, e sp e c ia lly
p ro d u ctio n

(79)•

c a s e i n were
monium s a l t s

those

good n i t r o g e n s o u r c e s f o r

b u t no a n t i b i o t i c

p ep tic d ig e s ts

these

organism s.

of

Am­

grow th of S tre p to m y ce s f r a d i a e ,

was f o r m e d .

com plex n i t r o g e n o u s
good a n t i b i o t i c

the

of the

concerned w ith a n t i b i o t i c

Waksman ( 1 0 3 ) n o t e d t h a t

supported

n u tritio n

B ennett

(9) r e p o r t e d

compounds were a l s o r e q u i r e d

y i e l d s w ith S treptom yces g r i s e u s .

th at

to produce
N itrate

was u s e d a s a n i t r o g e n s o u r c e by S tr e p to m y c e s c o e l i c o l o r .
b u t g ood g r o w t h was o b t a i n e d
a g ain st

the

o n l y when p r e c a u t i o n s were t a k e n

developm ent of e x c e s s a c i d i t y

(2^).

of y e a s t e x t r a c t to n itra te -m e d iu m , how ever,
dticea t h e
tlie b

tim e r e q u i r e d

(^+9) c o n c l u d e d

to

th at

The a d d i t i o n

co n sid erab ly r e -

o b t a i n maximum c e l l y i e l d s .
the

actin o m y cetes

G ot­

co uld u t i l i z e

various carb o h y d rates,

alc o h o ls,

compounds a s c a r b o n s o u r c e s ,
in th e ir
of the

a b ility

lack

to u t i l i z e

of s p e c i f i c

a n d Mohan ( 7 5 )

fatty

The d a t a

t h a t w hile

t o t h e m edium r e s u l t e d

i m a t e l y two f o l d ) .

d iffered

d is a c c h a rid e s a p p a re n tly because
of N ickerson

S. f r a d ia e

b o t h c a r b o n and n i t r o g e n fro m g l u t a m a t e ,
glucose

and p r o t e i n

a l t h o u g h some s t r a i n s

h y d ro lases.

in d icated

acid s,

could

the a d d iti o n

i n much g r e a t e r

C o c h r a n e a n d D im m ick ( 2 3 )

obtain
of

grow th (a p p ro x ­
p o in ted

out th a t

a " b a l a n c e " b e t w e e n c a r b o n and n i t r o g e n s u p p l y was a prim e
requirem ent fo r

the

s u c c e s s f u l c u l t i v a t i o n of S.

The w o r k o f U m b a r g e r

(99)

in d icated

appeared to e x e r t a c o n sid e ra b le
p o ten tial

t h a t t h e n u t r i e n t m edium

in flu en ce

on t h e e n z y m i c

of E s c h e ric h ia c o l i .

The p h a s e s

of grow th o b serv ed i n a b a c t e r i a l c u l t u r e

u s u a lly not seen w ith actin o m y cetes alth o u g h i f
ta r d e d by r e d u c in g th e

tem perature

g r o w t h may b eco m e a p p a r e n t
ed t h a t the

grow th r a t e

of a c u ltu r e
cate

d iscrete

(^9,

(^9)*

50, 79), but l i t t l e

p h a ses of g ro w th .

lo g

phase

in u n ic e llu la r

grow th o c c u r r e d ,

and t h a t

to

root

of dry w eight a g a in s t

sh ip .

for

a tim e

organism s.

sh o w e d t h a t

For such a p e rio d

com parable t o

A ssum ing t h a t a p i c a l

p lan es,

at lea st,

tim e

(up t o

in d ic at­

h a s b e e n done t o d em ar­

i n subm erged shake c u l t u r e s

ab le

of

d u rin g the developm ent

Em erson (36)

s p h e r i c a l m a s s e s w o u ld grow i n t h r e e
show t h a t ,

phases

A number o f a u t h o r s

was v a r i a b l e

are

grow th i s r e ­

th en d i s t i n c t

w ith N eu ro sp o ra c r a s s a a grow th phase e x i s t e d
the

co elic o lo r.

t h i s w o r k e r was

a p lo t

of th e cube

gave a s t r a i g h t l i n e
50 h o u r s f o r

the

th is

rela tio n ­

organism )

- 4 a phase a n a lo g o u s t o th e e x p o n e n t ia l phase
A number o f i n v e s t i g a t o r s
m a t u r e m y c e l i u m may d i f f e r
R o lin so n

(84)

cu ltu re
degree

have i n d i c a t e d

and th e

Q0 2 v a l u e s

on t h e n a t u r e

of grow th e x e r t e d

of th e r e s p i r a t o r y

enzymes fo rm ed .

G o ttlieb

w ith th a t

of the

f u n g i e m p h asizin g th e f a c t t h a t the

m ateria l,

hyphal in n a tu re ,

(49 ) com pared a c t in o m y c e te

may be a c t i v e l y

a p i c a l p o r t i o n s and y e t be q u i t e
p arts

of th e m ycelium .

did n o t p a r a l l e l
in d ic atin g
l u l a r m ass.

grow ing a t th e

or even dead in o ld e r

T his a u th o r n o ted

t h a t O2 c o n s u m p t i o n
th is

o b serv atio n as

d eclin ed a f t e r

the

in itia l

a n d t h a t Qq2 v a l u e s f e l l
(50).

to

They s u g g e s t e d

i n r e s p i r a t i o n ” o c cu rred as the

su g g estio n th a t o ld er p o rtio n s
or dead

oxygen u p ta k e ,

(4 9 ), w hile

still

of th e

12 t o 1 5 h o u r

th a t an "in h e r-

c ells

aged.
in p a rt,

by

n o t c o n t r i b u t i n g m axim ally to

in flu en ced

the n i tr o g e n

of o p in io n ap p ears to fav o r

w here h ig h r e s p i r a t o r y

T his

m y c e l i u m , be t h e y

or d ry w e ig h t

b a s i s upon w hich th e r e s p i r a t i o n d a ta w ere b a s e d .
sensus

cel­

o n ly 25 p e r c e n t

o b s e r v a t i o n c o u l d p r o b a b l y be e x p l a i n e d ,

in ac tiv e

c e llu la r

G o t t l i e b and A n d e r s o n showed t h a t w i t h S . g r i s e u s

o f maximum a t 4 8 h o u r s

th e

grow th

a n o n -u n ifo rm ity in p h y sio lo g y th ro u g h o u t the

in c u b a tio n period

latter

sta tic

g r o w t h , and i n t e r p r e t e d

resp irato ry a c tiv ity

e n t change

79)•

I t was a l s o n o te d t h a t th e

of a e r a t i o n d u rin g the e a r ly s ta g e s
in flu en ce

( 50,

chrysogenum changed m ark e d ly a s th e

a g e d f r o m 24 t o 96 h o u r s .

a co n sid erab le

t h a t young and

c o n sid e ra b ly in physiology

showed t h a t b o t h t h e R .Q .

ob tain ed w ith P e n ic illiu m

probably e x is te d *

a ctiv ity

is

The c o n ­

the use

of young m a t e r i a l

d esired

(49,

50, 79).

Cochrane d e m o n s tra te d

th at

5 -

the r e s p i r a t o r y

a c tiv ity

c e lls

o f S . c o e l i c o l o r was h i g h e r t h a n t h a t

cells

(28).

c ellu la r
crease

He i n t e r p r e t e d

o l d e r c e l l s was r e s p o n s i b l e

in re s p ira tio n .

p a rticu la r

on t h e b a s i s

of

I n some c a s e s

for

the

in ­

the a p p a re n t

ch o ice

of a

c e l l age h a s b e e n d e t e r m i n e d by a com prom ise b e ­

tw een u s a b le
a ctiv ity

fin d in g s

of 2 or 3-day

p e r m e a b i l i t y , and s u g g e s t e d t h a t a p e r m e a b i l i t y

in the

in crease

th ese

of h -d ay

q u a n titie s

o f m ycelium and r e s i d u a l r e s p i r a t o r y

(31)*

E r i k s o n and W ebley ( 3 8 ) and W ebley (10*0 r e p o r t e d
ob serv atio n s

on t h e r e s p i r a t o r y a c t i v i t i e s

a c t in o m y c e te M icrom onospora v u l g a r i s .
th e
to

organism
sep arate

d iffic u lt

in s t i l l
the

cu ltu re

These a u t h o r s grew

2 d a y s , and t h e n a t t e m p t e d

a e r i a l and v e g e t a t i v e

hyphae.

W hile i t

t o compare d a t a from a e r a t e d and s t i l l

in cu b a tio n p erio d s

w o rk , had r e l a t i v e l y

and w h ic h , by a n a l o g y t o

low r e s p i r a t o r y a c t i v i t y .

a lso rep o rted

th at rep licates

and s u g g e s te d

th at

the

on c e l l o p h a n e
in d icate

en co u n tered w ith th ese
resp ira tio n

of the

These a u t h o r s

sh o w ed c o n s i d e r a b l e

for

the

d iffic u ltie s

ex p erim en tal tech n iq u es d e scrib e d

tio n v essels)

other

v a ria tio n ,

com plex o r g a n i z a t i o n of su ch a n o rg a n ­

ism was p a r t l y r e s p o n s i b l e

organism

cu ltu res,

produce c e l l u l a r m a t e r i a l w hich

was p h y s i o l o g i c a l l y m a t u r e ,

Some o f t h e

is

o f *+8 h o u r s f o r t h e r m o p h i l i c f o r m s c o u l d

r e a s o n a b l y be e x p e c t e d t o

the

for

of a th e rm o p h ilic

and w a s h in g t h e

en co u n tered .

(e .g .

grow ing

grow th i n t o r e s p i r a ­

the v e ry r e a l t e c h n i c a l d i f f i c u l t i e s
form s.

W e b l e y (10*+) r e p o r t e d

t h a t the

a e r i a l m y c e l i u m o f M. v u l g a r i s w a s i n h i b i t e d

6

-

by a n a tm o sp h e re

of pure

-

oxygen, and i n d i c a t e d

have b e e n th e re s t i lt of i n a c t i v a t i o n
zymes,

t h a t t h i s may

o f -SH c o n t a i n i n g e n ­

The l a t t e r w e r e s h o w n t o be p r e s e n t o n t h e b a s i s

i n h i b i t i o n by i o d o a c e t a t e
although th is

of

and p - c h l o r o m e r c u r i b e n z o a t e ,

i n h i b i t i o n c o u l d n o t be r e v e r s e d by g l u t a t h i o n e .

N i c k e r s o n and Sherm an o b s e rv e d t h a t th e

grow th o f B a c i l l u s

c e r e u s c o u l d be c o n v e r t e d t o a f i l a m e n t o u s t y p e b y c u l t i v a t i o n
of the

o r g a n i s m i n a m ed ium l o w i n Mg+ + i o n s

ed t h a t th e la c k

o f Mg+ + d i d n o t i n h i b i t g r o w t h

was e q u a l t o t h a t o b s e r v e d
peared to
p a ttern
of the

of th e fila m e n to u s

(th e amount

The r e s p i r a t o r y

t y p e c e l l s was c o m p a r a b l e t o t h a t

f u n g i i n t h a t r e s p i r a t i o n was n o t g r e a t l y e l e v a t e d
fo ld )

above th e l e v e l of en d o g en o u s by th e

a d d i t i o n of s u b s t r a t e , i n n o rm ally d i v id i n g
ogenous r a t e

in t h a t norm al c e l l s

g reater

red u ctio n in r e s p ira tio n

acetate

and from t h e s e

p o r t i o n of the

o x id ativ e

The r e s p i r a t o r y
com parable

in the

presence

to the

showed a
of iodo­

io d o ac etate -se n sitiv e

o f f u n g i and a c tin o m y c e te s

in t h a t the endogenous r e s p i r a t i o n

59, 75,

A

sy stem .

a c tiv itie s

v e r y h i g h and r e l a t i v e l y
91)*

co n stan t

is u su a lly

over a c o n sid e ra b le

p eriod

T h is h ig h endogenous r e s p i r a t i o n

and f a i l u r e

t o d e m o n s tr a te a m arked i n c r e a s e

take

presence

in the

the e x ­

d a ta the a u th o rs concluded t h a t the

two f o r m s d i f f e r e d w i t h r e g a r d

(27,

c e lls

was a b o u t t e n t i m e s t h a t , o f e n d o g e n o u s .

f u r t h e r d i f f e r e n c e was n o t e d

of tim e

T h e y sh o w ­

i n t h e c o m p l e t e medium) b u t a p ­

in h ib it c e ll d iv isio n se le c tiv e ly .

(tw o t o t h r e e

are

(76).

i n oxygen u p ­

of exogenous e n erg y so u rc e s le d

some

- 7 w o rk ers t o conclude
w orking a t

t h a t the

"fu ll-lo ad "

and t h a t th e

i n c r e a s e was due t o t h i s
Goddard

term in al r e s p ira to r y

lim itin g

( 3 1 ) , w orking w ith the

failu re

facto r

to observe an

(77)*

c ellu lo se

s y s t e m wa s

Darby and

decom posing fu n g u s

M yrothecium v e r r u c a r i a . a l s o r e p o r t e d h ig h endogenous r e s p i r a ­
t i o n v a l u e s and n o t e d t h a t e x o g en o u s r e s p i r a t i o n c o u ld o n ly
be d e m o n s t r a t e d by s t a r v i n g

th e m ycelium i n a s a l t s - s o l u t i o n

i n the

so t h a t endogenous r e s p i r a t i o n

absence

was d e p r e s s e d
reserve

of n u tr ie n ts

(presum ably because

m ateria ls).

of a d ecrea se

A nalogous s t a r v a t i o n p r o c e d u r e s have

b e e n f r e q u e n t l y em ployed i n a n e f f o r t t o
of added s u b s tr a t e
(29) sta te d

in sto red

sh o w t h e

( 3 5 , *+*+, 5 5 , 7 7 , 7 8 ) ,

o x id atio n

C ochrane and Peck

t h a t prolonged a e r a t i o n of m y c e lia l su sp en sio n s

reduced the endogenous r e s p i r a t i o n

of S, c o e lic o lo r b u t t h a t

s u c h t r e a t m e n t r e d u c e d e x o g e n o u s u p t a k e by a l i k e
g r e a t e r am ount;
d erm ato p h y tes

a s i m i l a r c o n c l u s i o n was r e p o r t e d w i t h

(10).

t i o n red u ced the

S t o u t and K o f f l e r r e p o r t e d

d ry w e ig h t of P .

such s ta rv e d m y c e lia l su sp en sio n s
led

or even

to a rein crease

chrysogenum
i n the

th a t starv a­

(91);

presence

of g lu co se

i n d r y w e i g h t due t o o x i d a t i v e

By t h e u s e

io d ate

o x i d a t i o n fo llo w e d by tr e a tm e n t w ith d e c o lo r iz e d
th e y were a b le

sa cc h arid e -lik e
of glucose

d isap p eared

reserves

assim ilated

co n sid erab le

to

p o rtio n
from th e

tech n iq u e

assim ila ­

tio n .

fu ch sin )

of a s t a i n i n g

shaking

(prelim in ary

per­

show t h e a c c u m u l a t i o n o f p o l y ­

in the m y c e lia l s t r a n d s .

The a m o u n t

by m y c e l i a l s u s p e n s i o n s r e p r e s e n t e d a

( 7 0 t o 79 p e r c e n t )
shake f l a s k s .

of the

On t h e

g lu c o se w hich

basis

of m anom otric

-

d a ta ,
the

8

-

D arb y and Goddard c a l c u l a t e d

glucose

added to

the r e a c t i o n f l a s k s

th u s did n o t c o n trib u te
(31)-

It

is

to a m easu rab le

tem p tin g to t r y

O ginsky e t a l
resp ira tio n ,

(77)

t h a t u p t o 90 p e r c e n t o f
was a s s i m i l a t e d ,
in crease

i n C>2 u p t a k e

to e x p la in the f a i l u r e

to d em o n strate

and

of

a p p re c ia b le exogenous

even over 6 hour p e rio d s ,

on t h i s b a s i s

of r e ­

s e r v e f o r m a t i o n by a s s i m i l a t i o n *
In the

in te rp re ta tio n

of m anom etric d a ta th e d i f f e r e n c e

b e tw e e n ex o g en o u s and endogenous oxygen u p ta k e
a s s u m e d t o be t h e

resu lt

of o x id a tio n of the

S u c h a n a s s u m p t i o n may n o t be
i n w hich endogenous a c t i v i t y
lab e lle d

c e lls,

the r e s p i r e d
presence
the

ju stifie d
is high

is

g en erally

added s u b s t r a t e *

e s p e c i a l l y w ith form s

(13)-

U sing r a d i o c a r b o n

C o c h r a n e and G ib b s showed t h a t r a d i o c a r b o n i n

CO2 o f S . c o e l i c o l o r w a s n o t r e d u c e d b y t h e

of added s u b s t r a t e

(25);

th u s th e y concluded t h a t

s u b t r a c t i o n o f e n d o g en o u s r e s p i r a t i o n was j u s t i f i e d .

S im ilar

tech n iq u es c a rrie d

d icated

t h a t r a d i o c a r b o n i n t h e r e s p i r e d CO2 w a s n o t r e d u c e d

d u rin g g lucose

o x id atio n ,

t i o n of a c e t a t e .

out w ith P . chrysogenum (13)

b u t was r e d u c e d d u r i n g

re sp ira tio n
a range

showed t h a t

from th e

occurred.

(10?)

on t h e

to ta l

of c o n c e n tra tio n s

ed t o a r e l a t i v e

and S t o u t and K o f f l e r

i n the

of added s u b s t r a t e ,

c o n stan cy in the

oxygen u p tak e

(91)*

s u b t r a c t i o n of endogenous

oxygen up tak e

T h i s was i n t e r p r e t e d

of endogenous

oxida­

A d i f f e r e n t e x p e r i m e n t a l a p p r o a c h was d e s ­

c r i b e d b y W i l n e r and C l i f t o n
These w o rk e rs

the

in ­

to

presence

the

d ata

of

p o in t­

percentage

o x i d a t i o n w hich

in d icate

t h a t no d e p r e s s i o n

occurred

in the

presence

of added

- 9 o x id izab le

su b stra te.

t i o n v a r i e s w ith the
in v o lv ed

(10 1) ,

Since

the e f f e c t

su b strate

on e n d o g e n o u s r e s p i r a ­

and p r o b a b l y w i t h th e

i t w ould a p p e a r p r u d e n t t o

endogenous a c t i v i t y
The p r o d u c t s

fo r each tis s u e

of th e

observe

tissu e

and r e c o r d

p rep aratio n stu d ied .

o x id a tio n of c a rb o h y d ra te

by a c t i n o ­

m ycete s a re

t h o u g h t t o be CO2 a n d H2 O a l t h o u g h t h e r e s u l t s

o f C ochrane

in d icated

least

th a t in term ed iate

i n some s p e c i e s

(26).

was fo u n d t o a c c u m u l a t e
of fo rm a tio n of t h i s

W i t h S« c o e l i c o l o r

in co n sid era b le

c o m p o un d i s

t h a t a lth o u g h the key r e a c t i o n s
were

shown t o

d icated

th at

pro d u cts

occur w ith t h is

am ounts.

of c o n s id e ra b le
in d ic ativ e

form

(28),

occurred a t
su ccin ic acid
The mode
in te re st

in

of a K re b 's cy cle

f u r t h e r work i n ­

the a cc u m u latio n of s u c c in a te

resu lte d

from th e

c a r b o x y l a t i o n of p y ru v a te and su b s e q u e n t r e d u c t i o n of the
product to su ccin ate

(26).

T hus, w hile

c y c l e was p o t e n t i a l l y a v a i l a b l e ,
t h e k e y compounds
through t h i s

acid

t h e a c c u m u l a t i o n o f one o f

(nam ely s u c c i n a t e )

cy cle.

a tric arb o x y lic

O ther s t u d i e s

a p p a re n tly did n o t a r is e

in d icated

th a t a conven­

t i o n a l Em bden-M eyerhof-Parnas g l y c o l y t ic

sc h e m e p l u s K r e b f s

cycle

g r i s e u s . G arner and

did n o t o p e r a te ,

K o ffler

(*+3) i n d i c a t e d

and in d eed f o r S.
th at

only the

o x i d i z i n g enzym es were c o n s t i t u t i v e
volved

i n t h e K r e b 's c y c l e were

pyruvate
- the

o n ly w here

in g m edia.
by t h e u se

the

A later

o t h e r enzymes i n ­

c o n s i d e r e d t o be a d a p t i v e .

A c o n v e n tio n a l s u c c in ic dehydrogenase
e x ist

and s u c c i n a te

s y s t e m was s a i d

to

o r g a n i s m w a s g rov m 011 s u c c i n a t e - c o n t a i n ­
r e p o r t by K o f f l e r

of c e l l - f r e e

ex tra cts,

the

( 6^ )

in d icated

th at,

e s se n tia l reactio n s

of

th e K re b 's c y c le

c o u l d be

c o n fir m a tio n of the
has re c e n tly

sh o w n f o r

g riseu s.

o p e r a t io n of a K r e b 's

appeared

( 1 5 , 3 0 , *+6 )*

a n d Mohan ( 7 5 ) w i t h S« f r a d i a e
a c tiv ity

S.

a g a i n s t K re b 's c y c le

cycle

dehydrogenase
ene b lu e

a c tiv ity

sh o w e d t h a t d e h y d r o g e n a s e
i n t e r m e d i a t e s c o u l d be d e t e c t e d

c o u l d n o t be d e m o n s t r a t e d w i t h m e t h y l ­

b u t t h a t by u s i n g a 1 :3 0 0 0 s o l u t i o n o f 2 , 6- d i c h l o r o -

a c tiv itie s

D ehydrogenase

fo r c i t r a t e , °C -k e to g lu tara te , m a la te , pyruvate,

and s u c c i n a t e were fo u n d .

t h a t th e y were u n a b le

It

is

of i n t e r e s t to note

t o d e m o n s t r a t e e x o g e n o u s O2 c o n s u m p t i o n

m a n o m e t r i c a l l y u s i n g whole c e l l s
th e

These w o rk ers

i n d i c a t o r d y e was i m p o r t a n t i n t h a t

p h e n o l i n d o p h e n o l d y e r e d u c t i o n was o b s e r v e d .

la c ta te ,

i n S« g r i s e u s

The w o r k o f N i c k e r s o n

by dye r e d u c t i o n m ethods u s i n g c e l l e x t r a c t s .
found t h a t th e E 0 of the

F u rth er

organism a s s im ila te d

and t h e y p o s t u l a t e d

th e c a r b o n s o u r c e and t h e n m e t a b o l ­

iz e d e n d o g e n o u sly from a m e ta b o lic

pool.

They a l s o r e p o r t e d

t h a t e n d o g e n o u s r e s p i r a t i o n was h i g h a n d r e l a t i v e l y
over long

p erio d s

and y e a s t s .

of tim e

in c o n tra st to th a t

E x p e r i m e n t s were d i s c u s s e d

a c t i n o m y c e t e s were a b l e

th at

to reduce

co n stan t

of b a c t e r i a

in d icatin g

t h a t the

trip h en y ltetrazo liu m

c h lo rid e,

and t h i s

o b s e r v a t i o n i n c o n j u n c t i o n w i t h t h e work

of B rodie

and G ots

(1 ^ ) would i n d i c a t e

f l a v o p r o t e i n H - tr a n s p o r t sy stem ,
Cochrane

and Peck

re sp ira tio n
c e ll-fre e

(28)

stressed

of i n t a c t c e l l s

p rep aratio n s.

at least

in th is

of a

organism .

t h a t the h ig h endogenous

c o u l d be a v o i d e d b y t h e u s e

By t h i s

show e x o g e n o u s r e s p i r a t i o n

the e x is te n c e

a p p r o a c h t h e y were

of c i t r a t e

ab le

of
to

a n d <*- k e t o g l u t a r a t e ,

11

-

-

tw o com pounds f o r w h i c h u n e q u i v o c a l 03 u p t a k e
shown w i t h i n t a c t

c ells..

ed t h a t a p e n to s e

sh u n t system f u n c tio n e d

c e t e s and t h a t t h i s
fo r a co n sid erab le
of the f a t e

c o u l d n o t be

L a t e r work by C ochrane

(29)

in d ic at­

i n some a c t i n o m y ­

pathw ay o f hexose d e g r a d a t i o n a c c o u n te d
p o r tio n of the

of la b e lle d

glucose

ge num p r e v i o u s l y m e n t i o n e d
scheme o f c a r b o h y d r a t e

glucose u t i l i z e d .

during

A nalyses

o x i d a t i o n by P . c h r y s o -

( 6 3 ) were a l s o

consonant w ith t h is

degrad atio n .

P r e p a r a t i o n o f H om ogenates and T e c h n iq u e s
The s t u d y o f f i l a m e n t o u s

organism s i s

d iffic u lt

in th a t

c o n v e n tio n a l m anom atric te c h n iq u e s a re n o t a p p li c a b l e
o u t some m o d i f i c a t i o n

(31, 3 8 ).

V arious a u th o rs

w ith ­

have

"blended"

o r h o m o g e n i z e d m y c e l i u m i n o r d e r t o make t h e

resp irin g

s u s p e n s i o n m o re h o m o g e n e o u s

t o do t h i s
makes t h e
(103).
tio n

resu lts

(31,

72).

F ailu re

i n poor ag reem en t betw een r e p l i c a t e s

in te rp retatio n

Darby and Goddard

o f e x o g e n o u s C>2 u p t a k e
(31)

and

d iffic u lt

sh o w e d t h a t p r o l o n g e d m a c e r a ­

o f t h e m y c e l i u m o f M. v e r r u e a r i a h a d a d e l e t e r i o u s

effect

on t h e

d a ta are
regard

a ctiv ity

su b je c t to c r itic is m ,

to the

g en izer.

resp irato ry

d ata rep o rte d

They r e m a rk e d t h a t

occur from fra g m e n te d
t h e m ycelium a f t e r

from th e u se

o f a g l a s s homo-

on d i s r u p t i o n

m y celial stran d s

in to

"b leed in g "

could

and y e t t h e y w ashed

The d e c r e a s e

could r e a d i l y

of enzymes a n d /o r c o f a c t o r s

T heir

how ever, e s p e c i a l l y w ith

d isru p tio n .

of such p r e p a r a tio n s

of th e m ycelium .

in r e s p ir a tio n

be o c c a s i o n e d b y t h e

th e wash l i q u i d

lo ss

a n d n o t be

- 12 c a u s e d by th e h o m o g e n i z a t i o n p r o c e d u r e .
grounds i t
p rio r

On t h e o r e t i c a l

w o u l d a p p e a r t h a t w a s h i n g s h o u l d be c a r r i e d

out

to c e l l d is ru p tio n .
The e f f e c t

of the

o f t h e a m o u n t o f i n o c u l u m on t h e

grow th o b ta in e d has b een i n v e s t i g a t e d

Goddard

(31).

m ycelium were

p r o d u c e d i n sh a k e c u l t u r e s when l a r g e
in tro d u c e d , w hereas,

of

num bers

a s m a l l e r number of

l a r g e r m y c e l i a l m a sse s r e s u l t e d when th e

sira b le

by D arby and

T h e s e w o r k e r s sh o w e d t h a t s m a l l p e l l e t s

of s p o r e s were

decreased.

ch aracter

spore

i n o c u l u m wa s

They a l s o commented t h a t s m a l l p e l l e t s w ere d e ­

in th at

the r a t e

o f O2 d i f f u s i o n

m a s s e s c o u l d be a l i m i t i n g

facto r

in to larg e

cell

in the r e s p i r a t i o n r a t e s

o b tain ab le.
P relim in ary
and th e r e s u l t s

s t a r v a t i o n p ro c e d u re s have been d i s c u s s e d ,
appear c o n flic tin g .

endogenous r e s p i r a t i o n r a t e

is

C e rta in ly a decreased

d esirab le;

c a n be a c h i e v e d o n l y a t t h e e x p e n s e

how ever,

if

of a p ro p o rtio n a l

th is
or

g re a te r th an p ro p o rtio n a l decrease

in exogenous o x id a tio n

(1 0 , 2 8 ) ,

of q u e stio n a b le

th e n such a procedure

Work m e n t i o n e d b e f o r e

(31,

is

91) in d ic a te d

th at

ox id ativ e

a s s i m i l a t i o n b y f u n g i may a c c o u n t f o r m ore o f t h e
l o s t from th e r e a c t i o n m ix tu re
tio n .

Such a p r e f e r e n t i a l

in agreem ent w ith the

glucose

th an does o x id a tiv e

co n v ersio n to reserv e

c o n c e p t of u t i l i z a t i o n

v a lu e .

degrada­

m aterial is

a d v a n c e d by

N i c k e r s o n a n d Mohan ( 7 5 ) *
The b i o l o g i c a l a c t i v i t y
upon the

pH o f t h e

sy stem ,

of o rg a n ic a c id s

is

a n d S im o n a n d B e e v e r s

dependent
(89)

noted

-

th at

the

d ifferen ce

f o r m s was s u c h t h a t

13

-

in a c tiv ity
statem en ts

of the

a n i o n and u n d i s s o c i a t e d

of a c t i v i t y were r e l a t i v e l y

m e a n i n g l e s s u n l e s s a c c o m p a n ie d by d a t a c o n c e r n i n g t h e

hydro­

g e n i o n c o n c e n t r a t i o n a t w h ic h t h e e f f e c t was o b s e r v e d .
K re b 's c y cle

in term ed iates

co n sid erab le

sig n ifican ce

Peck (28) has in d ic a t e d
c itra te

of c o n s id e r a b le

v alu e

p e r m e a b i l i t y e f f e c t s may be o f
(100);

th e work of C ochrane and

t h i s w ith re g a rd to the

by S . c o e l i c o l o r .

W ith

The u s e

of c e l l f r e e

o x i d a t i o n of
m ethods i s

i n overcom ing p e r m e a b i l i t y d i f f i c u l t i e s

( 2 8 , 82, 100).
The f o r e g o i n g s u m m a r i z e s a n u m b e r o f i n v e s t i g a t i o n s
w h ic h show:
1 ) the

sim ila rity

of f u n g i and a c t i n o m y c e t e s w i t h r e g a r d

to th e ir r e s p ira to r y

a c tiv itie s,

2 ) the h e te ro g e n e o u s n a tu re
v a ria tio n
3)

of m y c e l i a l g ro w th and

i n m etab o lism upon a g in g of the

c ells,

some o f t h e m e t h o d s w h i c h h a v e b e e n u s e d t o g r o w
c e l l u l a r m a t e r i a l and p r e p a r e

it

for re sp ira tio n

w ork,
*+) t h e

p o ssib ility

of d e m o n s tr a tin g d e h y d ro g e n ase

re a c tio n s w ith c e l l e x tr a c ts .

lb

-

SOURCE AND IDENTITY OF CULTURES
The c u l t u r e s u s e d i n t h i s w o r k w e r e
from s c a b l e s i o n s
O n tario .

o b ta in e d from v a r i o u s

iso lated

p arts

of

T h o s e d e s i g n a t e d w i t h a p r e f i x P h a v e b e e n sh o w n

t o be c a p a b l e

of cau sin g

in fec tio n tr ia ls
sitic

on t u b e r s

o rig in a lly

ty p ic a l scab le s io n s

in greenhouse

a g a in s t the v a r i e t y K a tah d in .

These p a r a ­

s t r a i n s r e p r e s e n t a r a t h e r homogeneous gro u p w i t h r e ­

gard to

g ro ss ap p earan ce, biochem ical a c t i v i t y

se ro lo g ic al reactio n s
There
sp ecies

Is

still

( 3 *+)? a n d p h a g e s e n s i t i v i t y

co n sid erab le

or a group of s p e c ie s

p o tato scab
se ttled

(3*0*

(^7 ),

adds to the

c o n f u s i o n a s t o w h e t h e r one

is resp o n sib le

problem s

p o ten tial v a ria b ility
in v o lv ed

(86).

i n many i n s t a n c e s

on c a r b o h y d r a t e u t i l i z a t i o n
(8).

At p r e s e n t ,

th o se

organism s c ap ab le

fo r causing

th ere

is

of th e s e

is un­
organism s

C r i t e r i a used in c l a s s i f i ­
alth o u g h a re c e n t r e p o r t

stu d ies

shows c o n s i d e r a b l e

prom ise

some j u s t i f i c a t i o n f o r c o n s i d e r i n g

of cau sin g

s c a b t o be s t r a i n s

S tre p to m y c e s s c a b i e s as opposed to th o se
cause

( 7*+)*

The t a x o n o m y o f t h e a c t i n o m y c e t e s

and th e

c a t i o n a re vague

(102),

iso lates

of

un ab le

to

the d i s e a s e .
GENERAL CONDITIONS FOR MAINTENANCE AND GROWTH
C u l t u r e s were m a i n t a i n e d

g lu co se-asp arag in e
m edia,

agar

(3^)*

s p o r e s were s c r a p e d

so lu tio n

on s l a n t s

For i n o c u la tio n of l i q u i d

off larg e

o f ’’T w ee n 2 0 ” , a n d w e r e

even su sp en sio n .

of a s y n t h e t i c

sla n ts

i n t o a 1 :5 0 0 0

s h a k e n t o b r i n g a b o u t more

For those ex p erim en ts

i n w hich m asses of

- 15 m ycelium w ere r e q u i r e d
1 t o 2 week o ld
e x h ib ited

spore

i t was f o u n d a d v i s a b l e
p rep aratio n s;

a slo w er grow th r a t e ,

older

to use

spore

only

c u ltu res

p ro b ab ly as the r e s u l t of

reduced g erm in atio n .
Of a n u m b e r o f m e d i a u s e d , D i f c o p e p t o n i z e d m i l k
appeared

to

give

th e m ost r a p i d and p r o f u s e

grow th.

w e r e i n c u b a t e d a t 2 5 t o 3 0 C on a r e c i p r o c a t i n g
cy cles

per m in u te,

2 in ch t r a v e l ) .

(PM)
C u ltu re s

shaker

(100

- 16 RESPIRATION OF MYCELIAL SUSPENSIONS
G en eral T ech n iq u es
R e s p i r a t i o n was m e a s u r e d by c o n v e n t i o n a l m a n o m e tric
tec h n iq u e s
ing r a t e

(101) w ith a b a th te m p e ra tu re

of 110 s t r o k e s

per m inute w ith a i r

C a r b o n d i o x i d e was a b s o r b e d by 0 . 2 m l .
added t o th e
fu g atio n ,
cen t),

of 30 C and a s h a k ­

cen tre w e ll.

as the

gas p h ase.

o f 20 p e r c e n t KOH

M ycelium was h a r v e s t e d by c e n t r i ­

and w ashed w i t h s a l i n e

o r KC1 s o l u t i o n

(0 .0 2 p e r­

b l e n d e d f o r a p p r o x i m a t e l y two m i n u t e s i n a P o t t e r -

E lv e jh e m h om ogenizer and susp en d ed i n b u f f e r

so lu tio n .

less

o therw ise

(pH 5 . 6 ) w a s

used

(28).

n o t e d 0.025M p h o s p h a t e b u f f e r

C e l l s u s p e n s i o n was ad d ed t o t h e m a in c o m p a r tm e n t,

and s u b s t r a t e

(or b u f f e r )

f l a s k s were a llo w e d

was a d d e d t o t h e

to e q u ilib ra te

s t o p c o c k s were c l o s e d

15 m in u te s b e f o r e

d i g e s t i o n m ix tu re

Ammonia w a s d i s t i l l e d

b

arm .

The
the

o f h o m o g en ates were d e te r m in e d by

K je ld a h l a n a ly s e s w ith the

tio n u n it in to

side

and m e a s u re m e n ts b e g u n .

N itrogen c o n te n ts

Hanna ( 1 8 ) .

Un­

percent b o ric

of C am pbell and

f r o m a Markham d i s t i l l a ­

acid ,

and t h i s was t i t r a t e d

w i t h s t a n d a r d H2 S 0i+ t o t h e e n d - p o i n t o f a m e t h y l - r e d b r o m o creso l-g reen in d ic ato r
P relim in ary

(22).

o b serv atio n s

in d icated

t h a t w ide v a r i a t i o n s

e x i s t e d w h e n r e s p i r a t o r y m e a s u r e m e n t s w e r e made i n t h e
presence

or ab sence

of o x id iz a b le

f a c t o r s were t h e n i n v e s t i g a t e d
co n d itio n s necessary
assured re s p ira to ry

f o r the
a c tiv ity .

su b strate.

A number o f

in order to defin e

those

p r e p a r a tio n of su sp en sio n s

of

- 17 Age o f C u l t u r e

a n d Am ount o f S p o r e

Two s e r i e s

of f l a s k s were

s u s p e n s io n of s t r a i n P2^.
oculum ;

the

Inoculum

in o c u la te d w ith a heavy spore

One s e t r e c e i v e d

seco n d r e c e i v e d 0 . 5 m l.

Incu b ated fo r

2 , 3 ? and

and th e r e s p i r a t o r y
b u f f e r and on t h e

b

days,

The c u l t u r e s w e r e

a c t i v i t y was m e a s u r e d i n t h e

a d d i t i o n of g lu co se

m y c e l i u m a p p e a r s t o b e m ore a c t i v e

th ere

is

little

O2 u p t a k e .

differen ce

i n v e s t i g a t o r s who

th a n m ature c e l l s .

T his

s e c t i o n on o r g a n i c

sm a ll inoculum 2 -d ay c u l t u r e s ,
b e tw e e n e x o g e n o u s and e n d o g en o u s

From s u c h d a t a no c o n c l u s i o n s
of the

c o u l d be d r a w n

reg ard in g

the a b i l i t y

stra te s.

O ld e r p r e p a r a t i o n s do n o t e x h i b i t t h i s

to the

2, and 3 .

( 3 1 , 5 2 , 5*0 , y o u n g

g e n e r a l i z a t i o n w i l l appear a g a in in the
W ith th e

of

(5>uM).

sh o w n i n FIGURES 1 ,

of m ic ro b ia l t i s s u e s

o x id atio n .

presence

or s u c c i n a t e

I n a g re e m e n t w i t h th e work of o t h e r

acid

of in ­

t h e m y c e l i u m was h a r v e s t e d ,

O x y g e n u p t a k e s p e r mgm. o f N a r e

used a v a r ie ty

6 .0 m l.

organism to o x id iz e

these

sub­

behaviour

same d e g r e e .

An a d d i t i o n a l e x p e r i m e n t w a s c a r r i e d
e stab lish ed

cu ltu res

2b

hours

old .

g e n a t e s were a llo w e d t o e q u i l i b r a t e
presence

of s u b s tr a te

before

k.

In th is

case,

f o r *+5 m i n u t e s

t h e homo­
i n the

th e m anom eters were c l o s e d ,

t h e r e s p i r a t i o n was r e c o r d e d .
i n FIGURE

out w ith s im ila r ly

D etails
is

experim ent

are

presented

use

o f m ycelium e s t a b l i s h e d w i t h l a r g e

the

d e m o n s t r a t i o n o f e x o g e n o u s 0 2 u p t a k e w h i c h c o u l d n o t be

o b s e r v e d when s m a l l s p o r e

A gain i t

of t h i s

and

observed t h a t the
in o c u la allow s fo r

i n o c u l a were u s e d .

The e n d o g e n o u s

-

18

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- 22 up tak es are

a p p ro x im a te ly the

exogenous u p tak es

are

same i n b o t h c a s e s w h e r e a s t h e

d ecid ed ly d if f e r e n t.

D a ta o b ta in e d a s d e s c r i b e d above a re
lead in g ,
cin a te )

in th at

the l a g w hich o c c u rs

c l e a r - c u t b u t m is­

(e s p e c ia lly w ith suc­

b e f o r e maximum r e s p i r a t o r y a c t i v i t y

is attain e d

is

not ev id en t.
A d d i t io n of G lucose

to the

G r o w t h Medium

The d e c i s i o n t o u s e y o u n g m y c e l i u m p r e s e n t e d d i f f i c u l t i e s
in t h a t adequate

y i e l d s w e r e m ore d i f f i c u l t t o

sh o rt in cu b atio n p e rio d s.
th at

the a d d itio n

o b ta in w ith

(75)

N i c k e r s o n a n d Mohan

of glucose

in creased

the

showed

grow th of S. f r a d i a e

i n a g l u t a m a t e medium .
To d e t e r m i n e
the
were

tim e

one c o n t a i n i n g

glucose

on c e l l

w ith 1 .0 m l.
in cu b ated

o f medium.

of a f i l t e r e d

on t h e

fo llo w in g

Dry w e i g h t .

shaker.

spore

of f l a s k s

second c o n ta in in g

of 1 . 0 m l.

of

5

per­

These were i n o c u l a t e d

su sp en sio n

D u p licate

grow th and

tw o s e t s

o n l y PM, t h e

(PMG) a d d e d a t t h e r a t e

c e n t s o l u t i o n p e r 30 m l.

and th e

of glucose

o f maximum m y c e l i a l d e v e l o p m e n t ,

prepared,

PM p l u s

the e f f e c t

(P29 ) ,

and were

c u l t u r e s were rem o v e d ,

d e t e r m i n a t i o n s were made:
C u l t u r e s were f i l t e r e d

thro u g h ta re d

a n d t h e m y c e l i u m r i n s e d w i t h 20 m l .
P a p e r s and m ycelium were d r i e d
and r e - w e ig h e d .

T o tal carb o h y d rate.

of s a l i n e .

o v e rn ig h t a t 10? C

A d d itio n al f i l t e r

e a c h day t o c o r r e c t f o r

d i s c s were d r i e d

the w e ig h t l o s s

F iltrates

papers

on d r y i n g .

were a n a l y s e d f o r

to ta l

carbohydrate

using

23 the

anthrone re a g e n t

(73).

Pre­

l i m i n a r y a n a l y s e s made w i t h t h i s r e a g e n t r e v e a l e d
th at

o p tic a l d e n s ity d e te rm in a tio n s a t

5 ^ 0 m>u.

w ould s u c c e s s f u l l y m easure an i n t e r n a l g lu c o s e
s t a n d a r d a d d e d t o PM.
pH*.

H y d r o g e n i o n c o n c e n t r a t i o n was d e t e r m i n e d by means
of the

R esu lts
It

is

g lass e le c tro d e .

of th e se d e te rm in a tio n s are

p resented

i n TABLE 1 .

e v i d e n t t h a t m y c e l i a l g r o w t h was maximum a t t h e

sa m p lin g , and t h a t th e

a d d i t i o n of g lu co se

hanced m y celial y ie ld s .
in te r v a l stu died

No d r a s t i c

although l y s is

C arbohydrate d isa p p ea ran c e
3 days and c u l t u r e s
to cause

ly sis

occurred

over the

maximum v a l u e a t

s u p p lie d w ith a d d i t i o n a l glucose

a very ra p id disappearance

of the

a c t i o n b e c a m e p r o g r e s s i v e l y more a l k a l i n e

It

co n sid erab ly en­

does occur in o ld e r c u l t u r e s .

approached the

t i o n p e r io d presum ably because
s h o u l d be n o t e d

2-d a y

sugar.

t h a t the

filte re d

The r e ­

d u rin g the

of the l i b e r a t i o n
spore

appeared

incuba­

o f NH^

( 7 2 ),

su sp en sio n

u s e d a s i n o c u l u m i n t h i s e x p e r i m e n t d i d n o t c o n t a i n a s many
sp o re s as th e m assive
the r e s p i r a t i o n
w ere
to

5

in o c u la used to e s t a b l i s h c u ltu r e s fo r

tria ls.

W ith l a r g e r

i n o c u l a maximum y i e l d s

o b t a i n e d m o re q u i c k l y a n d some l y s i s w a s e v i d e n t a t H
days.

increase

F u rth er ad d itio n s

the

of g lu c o se d id n o t a p p r e c i a b l y

grow th o b ta in e d .

FIGURE 5 sh o w s t h e r e s p i r a t o r y
h a rv e ste d

a c tiv ity

o f m y c e li u m

f r o m PM m edium w i t h a n d w i t h o u t a d d e d g l u c o s e .

in c o rp o ra tio n

of g lu c o se

in to

th e

c u ltu re

The

m edium a p p e a r s t o

-

2k

-

TABLE 1
MYCELIAL WEIGHT, RESIDUAL CARBOHYDRATE, AMD
pH DURING ACTINOMYCETE GROWTH

Time
Me dium

C a rb o h y d ra te
days

PM

pH

mgms. *

0

-

100

6 .7

1

-

100

7 .0

2

9 3 .5

92*3

8 .5

3

100

81

8 .7

V

9*+

77

•
00

PMG

4-*

D ry w e i g h t

5

92

7 5 .5

8 .8 5

0

-

1

-

2
3

5

100

,

6 .7

9 2 .5

6 .9

171

53

8 .0

1^7

i*i+

8 .5

1H5

kh

8 .5

m5

3 7 .5

8 .7 5

.......... ..........1
* m y c e l i a l w e i g h t s n o t d e t e r m i n e d on t h e f i r s t
l i t t l e g ro w th w i t h th e s m a l l in o cu lu m *

day - v e ry

* * c a r b o h y d r a t e e x p r e s s e d a s p e r c e n t a g e o f t h a t i n a s a m p le
o f t h e u n i n o c u l a t e d m edium a n a l y s e d e a c h d a y *

1

i

- 25 -

03
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stim u la te

resp irato ry

26 -

a c tiv ity

as w e ll as in c re a s in g

grow th.

A d a p ta tio n vs P e rm e a b ility E f fe c ts
D a t a i n FIGURE 5 i n d i c a t e d

th a t a lag

a t t a i n m e n t o f maximum r e s p i r a t o r y r a t e
added t o h o m o g e n a te s, and s u g g e s te d
m ig h t be i n d u c t i v e

in n atu re.

o c c u rred i n the

when s u c c i n a t e was

th at th is

To t e s t t h i s

type

p o s s i b i l i t y my­

c e l i u m w a s g r o w n i n PMG a n d i n PMG p l u s s u c c i n a t e
5 p e r c e n t sodium s u c c i n a t e

p e r 30 m l. m edium );

m a t e r i a l was h a r v e s t e d and t h e r e s p i r a t i o n
glucose

or s u c c in a te

perim en ts are

as the

su b strate.

rate

to elim in a te

of su ccin ate

R esu lts

the

in to the

W i t h PMG c e l l s

i n low c o n c e n t r a t i o n

su ccin ate

T his r e d u c tio n

t h e maximum
is

observed

i s added t o

(5>cM p e r f l a s k ) .

co n cen tratio n is

is

a c h i e v e d more

increased

(2 5//.M

i n l a g by i n c r e a s i n g c o n c e n t r a ­

s h o w n i n TABEE 2 .

These d a ta

in d icate

t h a t the

presence

t h e m e d iu m i n some way " c o n d i t i o n e d "
effect

of th ese ex­

T his resp o n se

t h e maximum r e s p i r a t i o n r a t e

r a p i d l y when th e

is

c e llu la r

g r o w t h medium

la g encountered b efo re

o x id atio n b eg in s.

th e W arburg v e s s e l s

tio n

the

observed w ith

c o n s i s t e n t l y a n d i s m ore e v i d e n t \7h e n s u c c i n a t e

per f l a s k ) .

( 1 . 0 m l.

g i v e n i n FIGURE 6 .

The i n c o r p o r a t i o n o f s u c c i n a t e
appears

of resp o n se

of in c re a s in g

the

su b strate

the

of su c c in a te

c ells

in

sc t h a t the

c o n c e n t r a t i o n i s no l o n g e r

ap p aren t.
On t h e

surface

of an in d u ctiv e

th is

response;

appears

t o be a c l e a r

how ever,

d em o n stratio n

p e r m e a b i l i t y e f f e c t s have

- 27

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us
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- 28
b e e n shown t o c a u s e
D e fin itiv e

s im ila r r e s u l t s w ith A zo to b acter

reso lu tio n

of t h i s

p o i n t c o u ld be a c h i e v e d by

show ing a n im m ed iate a c t i v i t y
trac ts

(see

sectio n

(82)*

on s u c c i n a t e

in m y celial ex­

on c e l l e x t r a c t s ) .

TABLE 2
EFFECT OF THE ADDITION" OF SUCCINATE TO THE MEDIUM ON THE
OXIDATION OF SUCCINATE BY MYCELIAL HOMOGENATES

R e sp iratio n
C o n cen tratio n

for

p e rio d 20-120 m in u tes

f o r m ycelium grown i n

of

Exp. 1

>6c M / f l a s h

*

PMG + s u c c i n a t e

PMG

su ccin ate

Exp.

2

Exp. 1

Exp. 2

5

135

187

208

■ 183

2?

226

250

200

176

expressed as

percentage

of endogenous.

O x id a tio n of O rg an ic A cids
The r e s p i r a t i o n
in the
acid s

presence
is

of young and o ld m y c e l i a l h o m o g en ates

of s e v e ra l m e ta b o lic a lly

sh o w n i n TABLE 3«

su sp en sio n s can ox id ize
a c e ta te .

S u ccin ate

very v ig o ro u sly .

It

These d a ta i n d i c a t e

su c cin a te ,

and f u m a r a t e
is

im p o rta n t organic

fu m arate, c i t r a t e ,

in p a rtic u la r

in te re stin g

th at c e ll

to note

are

and

o x id ized

t h a t the

presence

- 29 -

TABLE 3
RESPIRATION OF YOUNG AND OLD
MYCELIAL HOMOGENATES
1 7 - h o u r hom ogenate
- 1

R esp iratio n fo r

p eriod

*

S u b strate
0 -6 0

6 0 -1 2 0

1 2 0 -1 8 0

0 -1 8 0

endogenous

383

195

182

760

glucose

^80

39^

382

1256

su ccin ate

1+58

520

8 O5

1783

fum arate

1+08

682

1+85

1575

c itrate

551

5 i5

238

1105

ace ta te

1+95

375

318

1187
■

1

9 6 - h o u r hom ogenate
R e s p ira tio n f o r p erio d *
S ubstrate
0 -6 0

60-120

0-180

120-180

endogenous

lk 2

122

99

363

glucose

22?

2k3

312

780

su ccin ate

171

25 9

27 b

70*+

fum arate

180

232

301

71 3

c itrate

117

130

139

386

a ce ta te

195

227

171

02

u p t a k e / m g m . N.

Time p e r i o d s a r e

j
!
A

1
1

59 3
.................................-

g iv e n i n m inutes*

!

- 30 of c i t r a t e
c ells;

appears

to e x e rt a d ep ressin g e f f e c t

in ad d itio n ,

before

the

th ere

resp irato ry

is

rate

a lag

on h - d a y

of a p p ro x im ately 2 hours

b eg in s to exceed th a t of endo­

genous c o n tr o ls *
A lso e v i d e n t from the
m y c e l i u m i s m o re a c t i v e
a p p e a r s t o be t h e
ig atio n s*

T his

grow th d a ta
c ellu la r

above d a t a

is

the

f a c t t h a t young

t h a n o l d e r m y c e liu m , and t h e r e f o r e

p referred m aterial for re s p ira to ry

in v est­

o b s e r v a t i o n m ight have b e e n a n t i c i p a t e d from

p rev io u sly given.

These d a ta

s y n t h e s i s had c e a se d a f t e r

s h o u l d be p o i n t e d

o u t, how ever,

o b tain ed u sin g a f i l t e r e d

spore

in d icated

th at net

2 days incu b atio n *

It

t h a t t h e g r o w t h d a t a was
s u s p e n s io n a s inoculum w here­

a s t h e m y c e l i u m f o r r e s p i r a t i o n was d e r i v e d b y u s i n g m a s s i v e
spore

in o cu la*

in c re a se d ,
e a rlie r
E ffect

L arg er in o c u la cause

and n e t s y n t h e s i s

tim e

i n the

these

genous r e s p i r a t o r y

experim ents
a ctiv ity

o u t i n w hich a c e l l

i n th e W arburg v e s s e l s .

th is

^ r e p a r a t i o n a n d o f a sam ple

dure

p relim in ary

on t h e

h o m ogenate.

shaker before

of the

i t was

a ctiv ity

of

same s u s p e n s i o n n o t

starv atio n

i n t e r f e r e d w ith the

An

s u s p e n s i o n was

The r e s p i r a t o r y

o b s e r v a t i o n would i n d i c a t e

has s e r io u s ly

of the

h i g h and p e r s i s t e n t e n d o ­

of hom ogenates i s e v i d e n t .

used

F irst

p ro b a b ly com pleted a t an

the

•’s t a r v e d ” i n b u f f e r f o r 3 h o u r s

to th is

t o be

o f th e M ycelium

e x p e r i m e n t was c a r r i e d

subjected

grow th r a t e

in cu b a tio n p erio d .

of S ta r v a tio n

In a l l

is

the

is

th at

g i v e n i n FIGURE 7 .
the

shaking

resp irato ry

C lo s e r e x a m in a tio n of the d a t a ,

proce­

a ctiv ity
how ever,

- 31
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- 32 shows 't h a t th e
m ycelium i s ,
ence
th e

in itia l

for

the m ost p a r t ,

in a c tiv ity
slo p es

n o ted .

of th e

m ycelium th e
d u rin g the

resp irato ry

a ctiv ity

resp o n sib le

C>2 u p t a k e

curves

the d i f f e r ­
from

show t h a t f o r n o n - s t a r v e d

in itia lly

37?, f a l l s

second h o ur of r e s p i r a t i o n .
is

for

The Qq^CU) v a l u e s c a l c u l a t e d

Qo2 * w h i c h i s

i n i t i a l v alu e

o f t h e n o n —s t a r v e d

200, and d u rin g the

t o a b o u t 200

For starv ed

c ells

second hour d e c re a s e s

173 ~ a d e c r e a s e

o f o n l y 1 2 . ? p e r c e n t c o m p a r e d w i t h a *+6.7

percen t decrease

f o r the u n s ta rv e d

show t h a t a f t e r

c o n stan t fo r ^ to
2 hours

su sp en sio n .

a p p ro x im a te ly 1 hour the r a t e

re s p ira tio n decreases,

and f o llo w in g

? hours

(th at

is,

th is

k

t o show t h a t t h i s
are

in te rv als

fo r about 3 hours;

th e m anom eters were

s h o w n i n FIGURE 8 .

decreases

sig n ific an tly

lin e

observed.

in itia l

co llected

It

r e s p i r a t i o n and s u b ­

observed.

The t r e n d

An e x p e r i m e n t w a s

opened, r e s e t ,

is

c lo sed ,

of t h i s

experim ent

1 hour,

after

c o n s t a n t over the

w hich th e
tim e

th at a short

slope

period
th is

decrease.
in d ic ate

and

e v id e n t t h a t endogenous a c t i v i t y

Exogenous r e s p i r a t i o n does n o t e x h i b i t

These d a ta

is

a t app ro x im ately 1 hour

The r e s u l t s

after

is re la tiv e ly

sig n ific a n t rate

in

endogenous and exogenous r e s p i r a t i o n

r e s p i r a t i o n a g a in m easured.

of the

of endogenous

3 hours s t a r v a t i o n plus

i n young c u l t u r e s .

o u t i n w hich th e

w ere f o l lo w e d

rap id

co n sisten tly

e s p e c ia lly n o ticeab le

are

These d a t a

i n t h e r e s p i r o m e t e r )*

sequent d eclin e

c arried

to

rem ains r e l a t i v e l y

D a ta fro m a number o f e x p e r i m e n t s a r e
TABLE

the

(1 t o 1 . ? h o u r )

- 33 sta rv a tio n

p eriod

p iratio n ra te ,

s i g n i f i c a n t l y reduced th e endogenous r e s ­

a n d i n m u ch o f t h e

t e c h n i q u e was u s e d

s u b s e q u e n t work t h i s

i n o rd er to low er th e

c o n tr o l uptake

valu e s .

TABLE

k

THE DECREASE IN ENDOGENOUS RESPIRATION
OF MYCELIAL HOMOGENATES
------------ ----------------— — ------------- ------------------------M y c e lia l age

R e sp iratio n

for

the

period

1

(hours)

3 0 -6 0

0-30

6 0 -9 0

9 0 -1 2 0

65

62

6l

109

105

85

83

17

177

140

118

55

22

Ilk

100

76

—

72

102

100

85

—

96

77

65

66

57

1 5 .5

100

16

.

|
i_ .

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- 35 The O x i d a t i o n o f Amino A c i d s
E x p e r im e n ts were c a r r i e d
of c e l l

su sp en sio n s

M ycelium h a r v e s t e d
washed i n s a l i n e ,
(pH 5 . 6 ,

to

out to d eterm ine

o x id ize

b y c e n t r i f u g a t i o n f r o m PMG m edium w a s
d isru p ted ,

suspended
on th e

i n phosphate b u f f e r
shaker fo r 1 hour.

s u b s t r a t e s w e r e n e u t r a l i z e d w i t h NaOH a n d a d d e d

t o W arburg v e s s e l s

a t l e v e l o f 5Q/*M p e r f l a s k .

a c i d s u se d were L form s e x c e p t f o r v a l i n e ,
and t h r e o n i n e w h ich were r a c e m ic m i x t u r e s .
the r e s u l t s

of these

flask s

pooled,

trifu g a tio n .
for

acid

(TCA) s o l u t i o n ,

and th e
Sam ples

(uptake

su b tracted )

are

The d a t a

of th e

before

c o llected

are b u t slow ly

w hereas

the

a sp artic,

the

of d u p l ic a t e

The r e s u l t s

of th ese

the 3 hour r e s p i r a ­

a d d it i o n of s u b s tr a te

o x id ized

v a lin e,

has been

p articu larly

and p h e n y l­
stu d ied

g l u t a m i c and

Thus, fo r

cells

amino a c i d s a p p e a r t o r e p r e s e n t

p o t e n t i a l energy source

o x i d a t i o n of a s p a r t i c

g ly cin e,

over the 3 hour p e rio d

o x id ized v ery v ig o ro u s ly .

grown i n a c a s e i n d i g e s t th e
a co n sid erab le

co n ten ts

i n TABLE 5 .

o t h e r am ino a c i d s ,

are

o f 20 p e r c e n t

s u p e r n a t a n t f l u i d were a n a ly s e d

show t h a t l e u c i n e ,

alan in e

in te n se

the

a l o n g w i t h t o t a l O2 u p t a k e s f o r

tio n period

FIGURE 9 s h o w s

p r e c i p i t a t e d m ycelium removed by c e n ­

by d i r e c t n e s s l e r i z a t i o n .

an aly ses

p h en y lalan in e,

t h e f l a s k s were re m o v e d , th e

s to p p e d by th e a d d i t i o n of 0 . 5 m l.

tric h lo ro a ce tic

The a m i n o

tria ls.

Follo\*7ing r e s p i r a t i o n ,
reactio n

a b ility

a number o f am ino a c i d s .

0 . 0 2 5 M ), and s t a r v e d

Amino a c i d

the

fo r the

organism .

and g l u t a m i c a c i d s ,

co upled

The

- 36 -

-P

a
cS

O E-t
C\J

H

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o w ap-i
O

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- 37

TABLE ?
OXYGEN UPTAKE AND AMMONIA RECOVERY DURING
THE OXIDATION OF AMINO ACIDS

Oxygen
S u b strate

uptake

#

**
Exogenous

^CM MI3

Endogenous

recovered

endogenous

17?

100

7 .2

g lu tam ate

820

468

14 .4

a sp arta te

579

330

1 5 .8

t h r e onine

4-27

244

1 3 .2

11

phe n y l a l a n i n e

349

199

5 .0

;

v alin e

251

143

6 .6

alan in e

5 40

308

14.1+

leu cin e

250

143

5 .9

glycine

321

183

1 2 .8

* ^ 1 .

o? consumed f o r p e r i o d I 5 ~ l 8 0 m i n u t e s ,

^expressed

as percentage

of e n d o g en o u s c o n t r o l .

- 38 w ith
the

th eir

a c tiv ity

p iv o ta l ro le

in tran sam in atin g

sy stem s,

em phasizes

of th e se

compounds i n am ino a c i d and e n e r g y

o f NH3 a r e

f a r b e lo w w h a t w o u ld be e x p e c t e d

m etabolism .
The y i e l d s
if

sim ple

take

o x idativ e

p lace,

d e a m in a t io n w ere

the

only r e a c t i o n t o

v iz:

H
R - C - COOH

0 .5 02

R - C - COOH 4 II

nh2

J

0

I n th e above c ase
be e x p e c t e d t o

a n O2 c o n s u m p t i o n o f 11.2>uJL. w o u l d

o c c u r f o r each/*M of

O2 u p t a k e s f a r e x c e e d e d t h i s

form ed.

fig u re,

The a c t u a l

s o t h a t i t m u st be

assum ed t h a t th e r e s i d u a l c a rb o n s k e l e t o n of th e
is

am enable

to f u r th e r

o x id atio n .

s u p p o r t a g r e a t e r O2 u p t a k e
sim ple d e a m in a tio n to th e
FIGURE 9 a l s o

lag ,

the

sim ila rity

on a l a n i n e
are

in stru c tu re

fairly
is

rap id .

in te restin g

of th ese

F o l­

The s l o w
i n view of

acid s.
Q uotient

endogenous r e s p i r a t o r y a c t i v i t y

m y c e l i a l h o m o g e n a te s and th e
(see

occurs p rio r

and p h e n y l a l a n i n e .

E n d o g e n o u s R e s p i r a t i o n and th e R e s p i r a t o r y

been d iscu ssed

and v a l i n e

t h a n c a n be a c c o u n t e d f o r b y

and l e u c i n e

The c h a r a c t e r i s t i c

amino a c i d

co rresp o n d in g k eto a c id .

the r a t e s

o x id a tio n of v a lin e

Even le u c in e

shows t h a t a s i g n i f i c a n t l a g

t o t h e maximum a c t i v i t y
low ing t h i s

NHo

step s

tak en to reduce

p a g e s 32 a n d 3 3 )*

n atu re

it
ol

of

have
the

- 39 endogenous s u b s tr a te

is

lo ss

a f t e r r e s p i r a t i o n by s t a i n i n g

of c a rb o h y d ra te

n o t known, and a t t e m p t s

(91) w ere n o t s u c c e s s f u l .
source

In an e f f o r t

of t h i s endogenous a c t i v i t y

to d e te c t a
m ethods

to d eterm in e

the r e s p i r a t o r y

the

q u o tien t

( R . Q . ) was d e t e r m i n e d b o t h fro m e n d o g e n o u s r e s e r v e s and up o n
the

a d d itio n

sim p le st case

of s u c c i n a t e ,

o x id ized .

d escrib ed ,

or g lu ta m a te .

In the

t h e R . Q . w o u l d be e x p e c t e d t o be u n a l t e r e d b y

the a d d i t i o n of s u b s t r a t e
b ein g

pyruvate,

sim ilar

in n atu re

to th a t alre ad y

O x y g e n u p t a k e was m e a s u r e d a s p r e v i o u s l y

a n d CO2 e v o l u t i o n w a s m e a s u r e d b y W a r b u r g ' s " d i r e c t

m e t h o d " w i t h a c o r r e c t i o n b e i n g made f o r C02 r e t e n t i o n i n t h e
buffer

(101).

FIGURES 1 0 a n d 1 1 sh o w d a t a
t i o n and C02 e v o l u t i o n .
maximum a c t i v i t y
p y ru v ate•

are

For th is

obtained fo r

The t i m e l a g s w h i c h o c c u r r e d b e f o r e

ag ain ev id en t e s p e c ia lly
su b strate

the

phases,

p rev io u sly been rep o rte d

by B a r r e t t e t a l

of c i t r a t e

suggested

in the

case

of

o x i d a t i o n a p p e a r s t o be

c o m p o s e d o f tw o d i s t i n c t

tio n

oxygen consump­

a s i t u a t i o n w hich h a s
(6) f o r the

by a Pseudomonas s p e c i e s .

oxida­

These w o rk e rs

t h a t a n a c t u a l a d a p t a t i o n was o c c u r r i n g d u r i n g

w hich p r o c e s s a n in d uced s y n t h e s i s

of " c a r r i e r m o le c u le s"

w h ic h were c o n c e rn e d w i t h s u b s t r a t e

t r a n s p o r t a c ro s s the

cell

m em b ran e w a s b r o u g h t a b o u t .
TABLE 6 i n d i c a t e s R . Q . v a l u e s
in te rv als

d u rin g the

been c a lc u la te d

course

from th e

maximum a c t i v i t y .

of r e s p i r a t i o n .

slopes

A ll th ree

tab u lated

over th re e

tim e

V alues have a l s o

of the c u rv e s a t p o i n t s

compounds c a u s e d a l a r g e

of

in itia l

•H

O ^
CM

O

i—
I

©jfBQ.dn S0 s«ieq.*?Toao-pM

Oxygen uptake by l5*5-hour homogenate (0.10 mgm. N/flask)#
Endogenous •
•
su ccin a tes
s, glutamate *
*, pyruvate n
a# Substrates at 2J>>UM/flask
A-endogenous not subtracted, B-endogenous subtracted#

vO

Fig# 10

- l|0 -

o

rH

o

p© A IO A 9

O

2qq

sa .Q % J X O J -°T U

Carbon dioxide evolution by
l5«5*hour homogenate* Endogenous • ----- • ,
succinate a
a , glutamate *--------- x, pyruvate □
d *Substrates at
25 A M /flask
A-endogenous not subtracted, B-endogenous subtracted.

CVJ

Fig* 11

- ill -

-

kz

-

CO
CVJ

-ch O co
O'

CVJ

CO

rH

O

IA

O

o

00

O

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O
•
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rH

rH

8
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p\

iH
rH

C*-

O
1A

r~i

r-i

r-i

rH
O '

CVJ
CVJ

o

rH

w
Eh

TABLE 6

-P

CtJ
>H
PH#N

•H

CO

rH

vO
CO

Eh

•S*

3C5
fe

O
5s;
o
M
Eh

rH

iH

ft

CO

M

x;

rH

o

o
rH

-P

■P

CO

-P

-P

- i*3 in crease

i n R .Q . v a l u e s w h ic h d e c r e a s e d

gressed.
ever,

D ata c a l c u l a t e d

from th e

may he m o re s i g n i f i c a n t

p re v io u s ly m entioned.
sig n ific an t rise

slopes

of the l i n e s ,

i n view of th e

lag

The a d d i t i o n o f p y r u v a t e

in R .Q .,

d e c a r b o x y l a t i o n were

as r e s a i r a t io n pro*

a situ atio n

as the r e s u l t

d ata

ob tain ed

i n the

th at

sig n ific a n t q u a n titie s
the

in p a rt a t le a s t,

in d ic ate d
present
could

serve

p iratio n

of p r o t e i n d e g r a d a tio n .

C hrom atographic a n a ly s e s

be e x p e c t e d

p ro te in c o n trib u ted

A p r o m in e n t am ino a c i d c o n s t i t u e n t

the

It

is

resu lt

the a d d i t i o n

suggested

th at

of am ino a c i d

q u a n titie s

of g lu tam ic

su fficie n t

to

valu e

for

cause

These

su b stan ces

if

(N P N ), a c o n d i t i o n t o

to the endogenous a c t i v i t y .
o f c e l l e x t r a c t s wa s

of w hich e l e v a t e d

th e R .Q .

o b tain ­

t h e e n d o g e n o u s r e s p i r a t i o n were

o x i d a t i o n th e n the

a c i d w h ich were

th e R .Q .

The r e s ­

s o l u t i o n was a c c o m p a n i e d

in n o n -p ro tein n itro g en

ed.

amino a c i d s were

f o r endogenous a c t i v i t y .

by an in c r e a s e

acid ,

of n i t r o ­

of c e l l e x t r a c t s

am ounts of f r e e

of hom ogenates i n b u f f e r

g lu tam ic

o x id atio n in d icated

on a t t h e e x p e n s e

s e c t i o n on t r a n s a m i n a t i o n ) .

if

O ther

o f NH^ w e r e p r o d u c e d , a n d t h i s

c arried

as s u b s tra te s

co n stan t

concept t h a t endogenous a c t i v i t y

th a t co n sid erab le

(see

if

p a r t of the r e s p i r a t i o n

s t u d y o f amino a c i d

o b s e rv a tio n supported

genous r e s e r v e s .

caused a

t h e mode o f a t t a c k .

f e i O .8 6 ) w h i c h s u g g e s t e d t h a t a t l e a s t

w as,

periods

t o be e x p e c t e d

The e n d o g e n o u s R . Q . v a l u e w a s r e l a t i v e l y

probably arose

how­

p r e s e n t may h a v e b e e n

t o be i n e x c e s s

p ro tein deg rad atio n .

sig n ifican t

of th e e x p e c te d

A n o t h e r p o i n t e v i d e n t f r o m FIGURE 1 0 i s
to ry ra te s
(Qo2 W

on s u c c i n a t e

v a lu e s are

not su b tracted ).

t h a t the r e s p ir a '

and g l u t a m a t e a r e v e r y s i m i l a r .

su c cin a te

5^ 7 * g l u t a m a t e

609, endogenous

A p o ssib le

ex p lan atio n is

t h a t g lutam ate

o x id a tio n proceeds as fo llo w s:
A
glu tam ate — —
and t h a t

the

s t e p s A and B a r e

b e i n g t h e more r a p i d .
a ll-o v er rate

end p ro d u c ts

d issim ila r

in r a te ,

T h is c o n d i t i o n w ould r e s u l t

s t e p A.
in the

b e in g d e te r m in e d by B, th e r a t e - l i m i t i n g

The Qq ^ v a l u e s w o u l d b e e x p e c t e d
were t r u e .

B

succinate

F u rth er

d eg rad atio n is

support fo r

developed

in the

t o be t h e
th is

same i f

step .

th e above

pathw ay of g lu ta m a te

s e c t i o n on <*-ket o g l u t a r a t e

o x id atio n .
E ffect

of D in itr o p h e n o l

(DNP)

The d a t a p r e v i o u s l y d i s c u s s e d
latio n

s u b s t r a t e was n o t a s s t r i k i n g

w i t h some o t h e r m i c r o o r g a n i s m s
the f a c t t h a t

the

added s u b s t r a t e
f o r com plete

e x tra

stim u­

in h ib its

(7 6 ,

91)•

as th a t

obtained

A l s o e v i d e n t was

oxygen ta k e n up i n th e

presence

of

w a s much b e l o w t h a t w h i c h w o u l d be e x p e c t e d

o xidation;

t i o n was o c c u r r i n g .

th is

f a c t suggested t h a t a s s im ila ­

V a r i o u s w o r k e r s h a v e s h o w n t h a t DNP

a s s i m i l a t i o n a n d t h u s a l l o w s f o r t h e mor e c o m p l e t e

o x i d a t i o n o f added s u b s t r a t e
7,

t h a t the

of r e s p i r a t i o n c a u s e d by th e a d d i t i o n of a n exogenous

o x id iz ab le

(2,

in d icated

51).

The p r o c e s s

t h a n w ould o c c u r i n i t s

absence

of p h o s p h o r y l a ti o n w hich accom panies

o x id atio n ,

and w h ich s u p p l i e s

a ssim ila tio n ,
( 2 , H8) •

is

the en erg y f o r

t h o u g h t t o be i n h i b i t e d

W ith m ost t i s s u e s ,

the

process

by t h i s

compound

low c o n c e n t r a t i o n s c o n s i d e r a b l y

increase

r e s p i r a t i o n w hereas h ig h e r c o n c e n tra tio n s are

h ib ito ry

(88).

The a c t i o n o f DNP on r e s p i r a t i o n i s

pH s e n s i t i v e

a s shown by t h e e x t e n s i v e

Sim on ( 8 8 ) .

D a ta o b ta in e d by t h i s w orker a re

v alu e

in e stab lish in g

the

w hich m ust be em ployed.
d eterm in e w hether t h i s
d ifferen ce

of

stro n g ly

in v estig atio n s

c o n cen tratio n s

in­

of

of c o n s id e r a b le

of the

in h ib ito r

An e x p e r i m e n t w a s c a r r i e d

out to

com p o un d c o u l d b e u s e d t o i n c r e a s e

the

b e tw e e n t h e e x o g e n o u s and e n d o g e n o u s r e s p i r a t i o n

of actin o m y c ete

hom ogenates.

A 1 0 - 3 M s t o c k s o l u t i o n o f 2 , * + - d i n i t r o p h e n o l was p r e ­
pared;

and from t h i s ,

of 0 . 5 m l.
10- \
these

d i l u t i o n s w e r e made s o t h a t t h e a d d i t i o n

of s o l u t io n e s ta b lis h e d

co n cen tratio n s

a n d 2 . 5 x 1 0 " “** M i n t h e W a r b u r g v e s s e l s .
co n ce n tra tio n s

of 5x10“ ,

The e f f e c t o f

on e n d o g e n o u s a n d g l u c o s e r e s p i r a t i o n

was t h e n o b s e r v e d .
FIGURE 1 2 p r e s e n t s

the r e s u l t s

o f one e x p e r i m e n t .

a c o n c e n t r a t i o n o f 5 x 1 0 “ ^ M, DNP s t i m u l a t e d

At

endogenous

r e s p i r a t i o n w h e r e a s a t 2.5x10""** M b o t h e x o g e n o u s a n d e n d o ­
g e n o u s r e s p i r a t i o n were d e p r e s s e d .
w e r e m o re d i f f i c u l t

to

in te rp re t.

The r e s u l t s
In fiv e

10"*** M c o n c e n t r a t i o n g a v e a n i n c r e a s e
t i o n w hereas
ap p reciab ly
ed t h a t

th is

a t 10*“ * M

e x p erim en ts the

i n endogenous r e s p i r a ­

i n a n o t h e r e x p e r i m e n t t h e 0 2 u p t a k e was n o t
g reater

th an th a t

of the

co n cen tratio n rep resen ts

c o n tro l.

It

a c ritic a l

is

su ggest­

lev el at

-

k6

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CM

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01

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A

- V? w hich th e

opposing r e a c t i o n s

are r a th e r
The
by a l l

d e li c a t e l y b alan ced .

o x i d a t i o n of added g lu c o se

co n cen tratio n s

2,5 x 1 0 ^ M t h i s
tio n

o f s t i m u l a t i o n and i n h i b i t i o n

o f DNP t e s t e d .

t o be d e p r e s s e d

At a c o n c e n t r a t i o n of

e f f e c t was p r o b a b l y t h e r e s u l t

of p h o s p h o ry la tio n ,

quired fo r

appeared

glucose

a sy n th etic

ox id atio n .

h o w e v e r, were e x p e c t e d
o x id a tio n over th a t

to

in h ib i­

p ro c e s s presum ably r e ­

The l o w e r DNP c o n c e n t r a t i o n s ,

show a n i n c r e a s e d

of the

of the

rate

of glucose

c o n tro ls.

T h ese a p p a r e n t l y a n o m a lo u s r e s u l t s w ere c l a r i f i e d when
i t was f o u n d t h a t t h e
presence
tio n

p a tt e r n of glucose

o f DNP w a s s t r i k i n g l y

c h a n g e d by p r e l i m i n a r y s t a r v a ­

of th e m y c e li a l ho m og en ates.

above were r e p e a t e d w i t h

o x i d a t i o n i n the

The e x p e r i m e n t s o u t l i n e d

5x10"5 and 10"^ c o n c e n t r a t i o n s of

DNP u s i n g c e l l s w h i c h h a d b e e n s t a r v e d f o r 1 h o u r i n b u f f e r
so lu tio n .

R e su lts

of th ese

Here a g a i n th e
DNP i s

o x i d a t i o n b y l o w DNP c o n c e n t r a t i o n s

latter

duced t o

experim ents

of the

2 hour

to

lev el

to y ield

is ev id e n t.

In

g l u c o s e c o n c e n t r a t i o n was r e ­

a n O2 u p t a k e

o f ^=267><.1«

o x id atio n
By t h e e n d

o b s e r v a tio n p e rio d the u p ta k e r a t e s had f a l l e n
of th o se

upon s u b tr a c tio n
ob served t h a t the
age

the

s t i m u l a t i o n of

2 y /_ M /f la s k , a n amount w h ic h on c o m p l e t e

was e x p e c t e d

the

sh ow n i n FIGURE 13*

s t i m u l a t i o n o f e n d o g e n o u s r e s p i r a t i o n by

o b serv ed b u t i n a d d i t i o n the e x p e c te d

glucose
these

experim ents are

in the

flask s

c o n t a i n i n g no g l u c o s e ;

of endogenous from exogenous u p ta k e s
a d d i t i o n o f DNP h a d i n c r e a s e d

o x i d a t i o n t o 62 p e r c e n t

the

( 5 x 1 0 - 5 M) f r o m a v a l u e

i t \^as

percen t—
of only

- 1+.8 -

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-

33 p e r c e n t i n t h e
T h is
strik in g

absence

o f DNP.

s t im u l a tio n of glucose
w ith o ld er

d em o n strated
serv atio n s

c e lls,

only a f t e r

o x i d a t i o n by DNP w a s more

a n d a s m e n t i o n e d above c o u l d be

p relim in ary

lev el

c o n s i s t e n t \tfith t h e

i n some way d e p e n ­

c o n c e p t t h a t when

i s h i g h some n a t u r a l l y p r e s e n t r e g u l a t o r y

m echanism c o n t r o l l i n g t h e r e l a t i v e
and o x id iz e d

and p r e v e n t s th e

is

T hese ob­

o f e n d o g e n o u s r e s e r v e s w i t h i n t h e homo-

The d a t a a r e

th e r e s e r v e

u lated

sta rv atio n .

s u g g e s t t h a t t h e DNP e f f e c t

d e n t upon the l e v e l
gen ate.

1*9 ~

o p erates

am ounts of g lu c o s e

in favor

of th e

latter

d e m o n s t r a t i o n o f t h e DNP e f f e c t .

w o r k w i l l b e n e c e s s a r y t o d e f i n e mor e p r e c i s e l y

assim -

process
F u rth er

the v a r ia b le s

in v o lv ed ..
The O x i d a t i o n o f

-k eto g lu ta rate

C o n s i d e r a b l e d i f f i c u l t y was e n c o u n t e r e d i n a t t e m p t i n g
to d em o n strate
hom ogenates.

the

o x i d a t i o n of

I n m ost c a s e s ,

c a u s e d no s i g n i f i c a n t
c o n tro l.

Since

K re b 's c y c le

the

presence

in the

its

or i n d i r e c t l y .

of a n o th e r k e to a c i d ,

t o b r in g a b o u t the

pyruvic a c id .

u s u a l l a g was n o t o d b u t f o l l o w i n g
of r e s p i r a t o r y

of the

i n amino a c i d

was t h o u g h t i m p o r t a n t t o d e m o n s t r a t e

d irec tly

com pound

a key i n t e r m e d i a t e

plays a p iv o ta l ro le

C e l l h o m o g en a te s were a b le

tio n

of t h i s

in r e s p i r a t i o n over t h a t

com p o un d i s

and a l s o

m etabolism i t
u tiliz a tio n

th is

change

< K - k e t o g l u t a r a t e by c e l l

a c tiv ity

In th is

th is

occurred.

o x id atio n

o x id a tio n the

a d e fin ite
T erner

stim u la­

(93? 9^0?

w o rk in g w i t h hom ogenates
DNP d e p r e s s e d t h e
p rep aratio n s

50 -

o f mammary g l a n d t i s s u e ,

endogenous r e s p i r a t o r y

and a llo w e d f o r th e

ous r e s p i r a t i o n w h ic h o t h e r w i s e
d ata

of T e rn e r

in d icated

o x id a tio n of keto a c id s
The e f f e c t

b rate

for

of h is

d e m o n s tra tio n of an exogen­
c o u l d n o t be d e t e c t e d .

The

or t h e ir d ir e c t p re c u rso rs.

o f DNP on t h e

o x i d a t i o n of p y ru v a te and
C e l l h o m o g en a te s w ere com bined

( ? 0 > u . M / f l a s k ) a n d DNP, a n d a l l o w e d t o e q u i l i ­

20 m i n u t e s

w ere t h e n c l o s e d ,
R esu lts

a ctiv ity

t h a t t h e a c t i o n o f DNP w a s u p o n t h e

« C - k e t o g l u t a r a t e was e x a m in e d .
w ith s u b s tra te

sho w ed t h a t

i n th e W arburg v e s s e l s .

The m a n o m e t e r s

and 0^ u p t a k e s r e c o r d e d .

of t h i s e x p e rim e n t a re

sh ow n i n TABLE

TABLE 7
EFFECT OF DINITROPHSNOL ON
KETO ACID OXIDATION

S u b strate

R e s p i r a t i o n w i t h DNP c o n c e n t r a t i o n o f
0

1 0 -lt M

5 x 1 0 " 5 {,[

j

endogenous

302

398

328

©C- k e t o g l u t a r a t e

303

397

3 “+9

pyruvate

ifl8

bk8

1+29

julI . O2 u p t a k e b y a h o m o g e n a t e f r o m a n 1 8 - h o u r c u l t u r e o f
O b s e rv a tio n p e rio d 1?0 m in u te s.
Hom ogenate n o t s t a r v e d so
endogenous valu e i s h ig h .
1 . 0 m l . o f h o m o g e n a t e ( 0 . 5 ^ mgm.
N /m l.) per f l a s h ;
t o t a l volume 2 . 2 m l.

.

The d a t a

51 -

sh ow t h a t DNP a f f e c t e d

r e s p i r a t i o n to about the

same d e g r e e ;

can t s tim u la tio n of k e to acid
the

presence

o f DNP.

b o t h en d o g en o u s and e x o g en o u s
c e r t a i n l y no s i g n i f i ­

o x id a tio n occurred because

The r a t i o

of exogenous/endogenous

o x y g e n u p t a k e w i t h p y r u v a t e was s l i g h t l y
presence

o f DNP a t b o t h c o n c e n t r a t i o n s

c e n t r a t i o n s were n o t u se d s i n c e
DNP s i g n i f i c a n t l y

H igher con­

i t was f o u n d t h a t 2 . 5 x 1 0 “ ^ M

reduced b o th endogenous a c t i v i t y

o f DNP w a s n o t d o n e b e c a u s e

g lu ta rate

d e p re ss e d by the

teste d .

exogenous a c t i v i t y w ith added g lu c o s e .
effect

and a l s o

F u r t h e r work on th e

i t was f o u n d t h a t

noted t h a t

o b serv atio n s

on a m i n o a c i d

o x i d a t i o n i t was

oxygen u p t a k e s were s i g n i f i c a n t l y

w ould be e x p e c t e d f o r
NH^ r e c o v e r i e s ) .

sim p le d e a m in a tio n

W ith g lu ta m a te

as the

h ig h er th an

(a s c a l c u l a t e d from
su b strate

t h e Op

u p t a k e was c o n s i d e r a b l y g r e a t e r t h a n c a l c u l a t e d f o r
d ehydrogenation,
im ated t h a t

and th e

rate

to

of added s u c c i n a t e .

Such a c o n d i t i o n w ould o ccu r

rates

p rev io u sly

p rev a ile d ).

th en an in h ib ito r
an i n h ib it io n
of e v e n ts

<<-ket o g lu t a r a t e , the l a t t e r

11s u c c i n a t e ” a n d t h i s

(p ro v id ed the

If

in tu r n f u r t h e r degraded

su g g ested c o n d itio n w ith r e s p e c t to

glutam ate

of s u c c in a te

of g lu tam ate

is

o x i d a t i o n p ro v id e d the

The c l a s s i c a l c o m p e t i t i v e
first

o x id iz ed thro u g h su c c in a te

o x id a tio n should a ls o y ie ld

s u g g e s te d above a c t u a l l y

g e n a s e by m a lo n a te

glutam ic

of r e s p i r a t i o n c lo s e ly approx­

g l u t a m a t e were d e a m in a te d t o

oxid ized

©C-keto-

o x i d a t i o n c o u l d be d e m o n s t r a t e d d i r e c t l y .

D uring th e

if

of

tak es

in h ib itio n

sequence

p lace.
of s u c c i n i c d e h y d ro ­

d i s c o v e r e d by Q u a s t e l and c o - w o r k e r s

h as b een r e p e a t e d ly confirm ed
su g g e stio n as to

52 (62,

t h e mode o f g l u t a m a t e

g e n a t e s were a llo w e d

to re s p ire

bo th in the

and a b se n c e

tio n
gives

presence

over a 2 hour p erio d

is

and t h e

lin e s

degree

in the

To t e s t

of the

the

o x id atio n ,

added s u c c i n a te

presence

c e l l homo­

in h ib ito r*

R esp ira­
TABLE 8

from th e s lo p e s

and ab sen c e

of i n h i b i t i o n caused by the

above

or g l u ta m a t e

s h o w n i n FIGURE l H .

t h e Qq2 <N) v a l u e s c a l c u l a t e d

C>2 u p t a k e

96 ) .

of the

of the

in h ib ito r

presence

of t h i s

compound.
TABLE 8
INHIBITION OF RESPIRATION BY MALONATE

Qo 2 W

Percent

S u b strate

*

in h ib itio n

no m a lo n a te

m alonate

endogenous

100

82

18

su ccin ate

191

118

38

g lu tam ate

282

l*+5

1+9

*

a s com pared t o th e c o r r e s p o n d in g t r e a t m e n t I n th e
absence of m alonate.

T he i n h i b i t i o n o f e n d o g e n o u s r e s p i r a t i o n c a u s e d by m a l o n ­
a t e w ould

in d ic ate

resp irato ry

a c t i v i t y was p a s s i n g t h r o u g h s u c c i n a t e .

l e v e l of m alonate
to b lo ck

t h a t a t l e a s t a p o r t i o n of the endogenous

su ccin ate

T his

(20Q /C -M /flask) was e v i d e n t l y n o t s u f f i c i e n t
o x i d a t i o n c o m p le te ly thou g h th e

in crease

- 53 -

CM
rH

•H

o
o

CM

o
O

rH

G3fBq.dn 2 q Scr©q.fx°^TM

bO

in in h ib itio n
tended t o
was t h e

confirm

in ten se

presence
tiv e

over th a t
the

step s

of m alonate a c tio n *

in h ib itio n

in th e

-

o b s e r v e d i n thG e n d o g e n o u s c o n t r o l

site

of m alonate*

ft

of g lu tam ate

More s t r i k i n g

o x i d a t i o n in the

These d a t a s u g g e s te d t h a t the

pathw ay from g lu ta m a te

oxida­

t o s u c c i n a t e were

very ra p id

and d ep ended upon th e r a p i d rem o v a l of s u c c i n a t e

by f u r t h e r

o x id atio n .

remove
the

su ccin ate

in itia l

o v erall

in the

step s

p ictu re

is

suggested t h a t the f a i l u r e

presence

in the

of i n h i b i t o r

oxidative

of an in c re a se d

In the absence
re sp ira tio n rate

It

of m alonate

in h ib itio n .
a t th e end of 3 h o u rs th e

w ith added s u c c in a te

added a t

A sim ila r d eclin e
the

(°2 u p ta k e

120M 1 .

5/x.M l e v e l .

g lu tam ate

( 5yO^M) d e c r e a s e d a n d
co n tro l

The d i f f e r e n c e

i n oxygen consumed

s u c c i n a t e ) was a p p r o x i m a t e l y

w h e r e a s 1 1 2 / / . 1 . was th e c a l c u l a t e d

c o n v e r s i o n o f 5/*.M g l u t a m a t e

c e n t of th e
w ith added

calcu lated
su ccin ate

(endogenous)

i n r a t e was n o t e d w i t h g l u t a m a t e

- 02 uptake

t i o n o f s u c c i n a t e was f a r

s l o w e d down

p ro cess th u s g iv in g the

was a p p r o x i m a t e l y e q u a l t o t h a t i n t h e
v essels.

to

to

d ifferen ce

5/*M s u c c i n a t e *

from c o m p le te ;

f o r the

The o x i d a ­

o n ly a b o u t 25 p e r ­

o x y g en had b e e n consumed when t h e r a t e

d eclin ed

to the

lev el

of t h a t

in the

co n tro l v e sse ls.
The d a t a
p erm eab ility
(28)*
c e lls
w hich

of Cochrane

a n d P e c k sh o w ed t h a t v a r i a t i o n s

in

o c c u r r e d a s t h e m ycelium of S. c o e l i c o l o r aged

O th e r work w i t h w hole c e l l s
were a b le

to c a rry

-k eto g lu tarate

in d icated

t h a t 1 and 2 - d a y

out tra n s a m in a tio n re a c tio n s

was one o f t h e

re a cta n ts,

in

a f a c t w hich

- 55 stro n g ly
in to

sug g ested

t h a t the

o ld er c e lls *

In the

m y celiu m was u s e d

it

o x id a tio n co u ld ,
tio n

com p o un d w a s a b l 9 bo g a i n e n t r a n c e

course

o f o t h e r wort; w h e r e

became e v i d e n t t h a t

in f a c t,

^ -k eto g lu ta rate

be d e m o n s t r a t e d d i r e c t l y

on p ig m e n t p r o d u c t i o n ) .

older

(see se c ­

TABLE 9 c o n t a i n s d a t a d e r i v e d

f r o m a n u m b e r o f e x p e r i m e n t s w h i c h sh o w v e r y c l e a r l y t h e
effect

of m y c e l i a l age

on

^ -k eto g lu ta rate

ox id atio n .

TABLE 9
INFLUENCE OF THE AGE OF THE MYCELIAL HOMOGENATE ON
THE DEMONSTRATION OF °(-KETOGLUTARATE OXIDATION

r—

.......
......
R e s p ira tio n w ith
1 —
—
18
1 ? .?
17

S u b strate
endogenous
o (-k eto g lu tarate
* y 6 c l . O2 u t p a k e
in hours.

the

o u t by th e s e
d iffic u ltie s,

’

22

22

>+90

*+80

384

3 64

334

U 6l

*+92

H86

558

490

485

o v e r 1 2 0 m i n u t e s/m g m . N.

of th e

Homogenate age

above d a t a

o x id a tio n of © C -ketoglutarate
organism s a lth o u g h , because
the

is

as

c a n be c a r r i e d

of p e rm e a b ility

d i r e c t d em o n stratio n of t h is r e a c tio n is

n e t alw ays s u c c e s s f u l .
su p p lied w ith g lu tam ate,

H ow ever, when t h e
a p o stu lated

g lu tarate

a n d a c o m pound w h i c h r e a d i l y

c e ll,

organism

the

21

¥+9

A sugg ested i n t e r p r e t a t i o n
fo llo w s:

♦if

hom ogenate o f age

o x id izes

the

organism s are

precursor

ofoC -keto-

p en etrates

glu tam ate

i n t o the

and b e h a v e s to w a rd s

m alonate

in h ib itio n as

56 -

one w o u l d p r e d i c t a s s u m i n g t h a t t h e

o x id a tio n occurred as fo llo w s:
g lu tam ate
It

is

k eto g lu tarate —

in te restin g

tio n rate

is

of the

w hich e x i s t s

the

su ccin ate.

p o t e n t i a l f o r © C -ketoglutarate

of V aisey
probably

(92) a

biochem ical

a ctin o m y cetes w ith p a th o g e n ic ity .

(102) in d ic a te d

t h a t th e brown r i n g t e s t

The w o r k
( 9 2 ) wa s

o nly a m a n i f e s t a t i o n of m elan in p r o d u c tio n a r i s i n g -

as the r e s u l t

of t y r o s i n a s e

a c tiv ity .

An a t t e m p t w a s made t o m e a s u r e t h e
by th e

th an th a t

O x id atio n

o r i g i n a l w ork of T a y l o r and D eck er

of the

o x idation

o r d e r of m agnitude

n u m b e r o f a t t e m p t s h a v e b e e n made t o c o r r e l a t e
p ro p erties

These

su ccin ate.

P igm ent P r o d u c t io n and T y ro sin e
Since

oxida­

p o i n t of com plete g lu ta m ic d eh y d ro ­

same o r a g r e a t e r

for

end p r o d u c ts

glu tam ate

j u s t as h ig h as t h a t f o r

d a ta su g g e st t h a t the
m u st be

—

to note th e f a c t t h a t the

(even beyond the

genase a c t i v i t y )

su ccin ate

m ethod o f H o l l i s

fo rm a tio n of m elanin

(56) w hich in v o lv e s th e

e s t i m a t i o n of FeC l^ p r e c i p i t a t a b l e

su b stan ces.

g rav im etric
Under th e

e x p e r i m e n t a l c o n d i t i o n s u s e d , h o w e v e r, b l a n k v a l u e s were h i g h
and r e p l i c a t e s
m ea su re d the
(57?
tio n s

90).

showed p o o r a g r e e m e n t .

ty ro sin ase

r e a c t i o n by c o l o r i m e t r i c m e th o d s

To i n v e s t i g a t e

o f a PK c u l t u r e

i n g w e r e made

O th e r w o r k e r s have

the

filtra te

p o ssib ility

of t h i s

m ethod d i l u ­

w h i c h sh ow ed i n t e n s e b l a c k e n ­

( w i t h u n i n o c u l a t e d PM a s t h e

d ilu e n t),

and th e

- 57 o p tic al d e n sitie s
series

resu ltin g

of w av elen g th s u sin g

The r e s u l t s
These
lin e

of the

of th e s e

s o lu tio n s determ in ed a t a

t h e Colem an s p e c t r o p h o t o m e t e r #

tria ls

are

g i v e n i n FIGURE 1 5 .

d a t a show t h a t t h e r e

was a s a t i s f a c t o r y

stra ig h t-

r e l a t i o n s h i p b e tw e e n o p t i c a l d e n s i t y and p ig m e n t c o n ­

c e n t r a t i o n a t a n u m b e r o f wave l e n g t h s #

The r e s u l t s

at

s h o w e d g o o d s e n s i t i v i t y a n d a g r e e m e n t w i t h B e e r * s Law.
wave l e n g t h w a s u s e d

5^ 0 mjju
T his

t o com pare p ig m e n t f o r m a t i o n by a number

of c u l t u r e s .
A series

of p a th o g e n ic

( p r e f i x S) a c t i n o m y c e t e
medium , t h e

( p r e f i x P) and n o n -p a th o g e n ic

c u l t u r e s w e r e g r o w n f o r 6 d a y s i n PM

g ro w th rem oved, and the

w i t h u n i n o c u l a t e d PM m e d i u m , a n d t h e
d eterm in ed .
FIGURE 1 6 ,

The r e s u l t s

filtra te

betw een th e s e
filtra te s

form s.

d e te rm in a tio n s are

5^0

mjju

sh o w n i n

in physio­

C hrom atographic a n a ly s e s

however r e v e a l e d t h a t t y r o s i n e

e d f r o m t h e m edium i n t h e

d ilu ted

o p tic a l d en sity a t

and s u g g e s t t h a t a v e ry r e a l d i f f e r e n c e

logy e x i s t s
of c u l t u r e

of th ese

cu ltu re

absence

of d e te c ta b le

d isappear­

pigm ent

p ro d u ctio n .
L erner has in d ic ate d
fo r ty ro sin e

o x id atio n

t h a t a number of p a th w a y s e x i s t

(65,

66).

In anim al t i s s u e s

ty ro sin e

may u n d e r g o t r a n s a m i n a t i o n w i t h o C - k e t o g l u t a r a t e , a n d t h e
acid

p r o d u c e d may be f u r t h e r

and fu m a r ic

acid s

ty ro sin e

o x id ized

is

to an in d o le - lik e
m elanin

(66).

(66).

degraded t o y i e l d

In the

tru e

ty ro sin ase

ace to a ce tic
reactio n ,

through d ih y d ro x y p h en y lalan in e

in te rm e d iate

T his l a t t e r

and f i n a l l y

k eto

(dopa)

polym erized to

r e a c t i o n has been s tu d ie d

i n some

- 58 -

♦8

Optical

d en sity

•6

2

1

2

R e la tiv e pigment co n cen tra tio n
Fig#

R e la tio n sh ip between o p t ic a l d e n sity and pigment
co n ce n tra tio n a t a number of w avelengths.

8

si

S22 S23 s26 s 27 S28 p3

p10 pl 4 P21 p23 P2lj. P29

Actinom ycete cu ltu re
F ig # l 6

Pigment form ation by a group o f non-pathogenic ( S)
and p ath ogen ic (P) actinom ycete c u ltu r e s .

- 60 d e ta il,

and the

d a ta r e v e a l t h a t the

reactio n

( t y r o s i n e — >—d o p a )

how ever,

once c a t a l y t i c

the

is

in itia l

s t e p i n the

s l o w a n d may be r a t e —l i m i t i n g 5

q u an tities

of dopa have a c c u m u la te d

o x i d a t i o n p r o c e e d s a t a much i n c r e a s e d r a t e .
The o x i d a t i o n o f t y r o s i n e ,

of o ( - k e t o g l u t a r a t e

alo n e

or c a t a l y t i c

and w i t h th e a d d i t i o n

q u a n titie s

( 5/ c M / f l a s k )

of

d o p a , was f o l l o w e d u s i n g h o m o g e n a te s fr o m a p ig m e n t p r o d u c i n g
s tr a in P29*

T y r o s i n e was a d d ed a s a s u s p e n s i o n of c r y s t a l s

(0 . 2^ m l./fla s k )
of

a n d ° C - k e t o g l u t a r a t e wa s a d d e d a t t h e r a t e

^0 m*M / f l a s k .

The r e s u l t s

obtained are

sh o w n i n FIGURES

17 and 1 8 .
These d a ta
c(-k eto g lu tarate
the u p tak e

in d icate

ap p aren tly

observed

i n the

c lo s e ly approxim ated
v a lu e s.

the

long the

in d u ctio n

t h a t the

very s im ila r
ex p ected

ty ro sin e

if

since

in d iv id u a lly

observed

th e a d d i t i o n of dopa d id
tended to p ro­

A m o re s i g n i f i c a n t p o i n t ,

how ever,

o x i d a t i o n o f t y r o s i n e -h d o p a p r o c e e d e d a t a r a t e
to

th at

ty ro sin e

served w ith dopa.

the

sum o f t h e

and

of b o th s u b s t r a t e s v e ry

o x id atio n , but ra th e r

of t y r o s i n e

L erner

L form .

a l o n e - a c o n d i t i o n t o be

o x i d a t i o n o c c u r r e d th r o tig h dopa and th e
fo rm e r were n o t l i m i t i n g .

t i o n c a n be a d v a n c e d f o r

as

presence

period .

c o n c e n t r a t i o n of th e

m ix tu re.

o x i d a t i o n of t y r o s i n e

proceeded in d e p e n d e n tly ,

C ontrary to e x p e c ta tio n s

not stim u late

is

t h a t the

the

prolonged

No e x p l a n a ­

in d u c tio n period

The p r e p a r a t i o n u s e d was a r a c e m i c
(6 6 ) r e p o r te d

th at th is

C hrom atographic a n a ly s e s

th e W arburg v e s s e l s

rev ealed n e g lig ib le

ob­

(DL)

may n o t be a s a c t i v e
of the

co n ten ts

g lu tam ate

of

fo rm atio n

- 6l -

200

M icro liters

0£

uptake

300

100

120

Time (minut e s )
P ig . 17

Oxygen uptake by 22-hour homogenate (O.63 mgm.
N /f la s k ) . Dopa#—— • , t y r o s in e s
ty r o sin e
p lu s dopa x
x. C alcu lated valu e fo r the
sim ultaneous o x id a tio n o f ty r o sin e p lu s dopav
v-.
Endogenous uptake (209>m.1* a t 120 m in .) su b tracted .

- 62 -

300

<D

cs

■p

Q*
3

oN
CQ 200
©

-

•P

*rl
r|

O
S«
o

100-

30

120

Tim© (m inutes)
Pig* 18

Oxygen uptake by 2 2 -hour homogenate (0*70 mgm*
N /fla sk )# <<-ket oglu tar ate •
t y r o s in e s ----- A ,
< *-k etoglu tarate p lu s ty r o sin e x----- * , and
glutam ate o
o .
C alcu lated value for the
sim ultaneous o x id a tio n o f ty r o sin e p lu s oC-ketog lu ta r a te v
Endogenous uptake (2f?5?^l. at
120 min#) subtracted#

- 63 i n t h e e C - k e t o g l u t a r a t e H- t y r o s i n e
d a t a were
tio n s

the

o b tained w ith c e l l e x t r a c t s .

alan in e

g lu tam ate

flask s*

and v a li n e

fo rm atio n .

Under s i m i l a r c o n d i­

c o u l d be shown t o c o n t r i b u t e

The d a t a s u p p o r t t h e

o x id a tio n of ty ro sin e

fo rm atio n .

C onsiderable

S im ilar n eg ativ e

to

conclusion th a t

o c c u r r e d c h i e f l y b y way o f m e l a n i n
pigm ent f o r m a tio n o c c u rre d i n th e s e

v e sse ls co n tain in g ty ro sin e

o r d o p a , b u t was m o st a d v a n c e d

i n th e f l a s k s w hich a l s o c o n ta in e d added « C -k e to g lu ta ra te *
A s i m i l a r e x p e r i m e n t was c a r r i e d
p roducing

stra in

out w ith a non-pigm ent

T h is organism a l s o o x id iz e d t y r o s i n e ,

b u t n o o x i d a t i o n o f d o p a c o u l d b e sh o w n o v e r a 2 h o u r o b s e r ­
v a tio n perio d .

No m e l a n i n f o r m a t i o n was o b s e r v e d i n a n y o f

th e W arburg v e s s e l s .
w hether t h i s

F u r t h e r w ork i s

failu re

to

o x id ize

planned to d e te rm in e

dopa i s

ch aracteristic

of a l l

th e n o n -p ig m en t form ing s t r a i n s .
D isc u ssio n
Recent re p o rts
S. g r i s e u s
th ese

on m an o m e tric

(15", *+6) a n d S . n i t r i f i c a n s

to th a t

sh o w n i n t h e

The p r e s e n t d a t a a r e
in the

d a ta alo n e
proof

term in al

c o n stitu te

of t h i s

p r e s e n t work w i t h S . s c a b i e s .

consonant w ith the
oxidative

o p e r a t i o n of a K r e b 's

pathw ay a lth o x ig h m an o m etric

i n s u f f i c i e n t evidence

to advance as

(28).

Endogenous r e s p i r a t o r y
ing

(72) r e v e a l t h a t

o rg a n ism s p o s s e s s a p a t t e r n of r e s p i r a t o r y a c t i v i t y

sim ilar

cycle

in v e s tig a tio n s using

a c t i v i t y wa s q u i t e

i n agreem ent w ith the r e s u l t s

hig h ,

of o t h e r s w orking

a fin d ­
^ ith

-

actin o m y cetes

(15* 2 8 , 7 2 ) ,

6k

-

and was a c c o m p a n ie d by a n i n c r e a s e

i n NPN a s d e t e r m i n e d b y p a r t i t i o n w i t h TCA.

T h is l a t t e r

o b s e r v a t i o n s u p p o r t e d th e c o n t e n t i o n drawn from R .Q . d a t a
th a t n itro g en o u s re se rv e s c o n s titu te d
endogenous s u b s tr a t e .
co n sisten tly

part

of the

The p a t t e r n o f e n d o g e n o u s a c t i v i t y

observed

(i.e .

by a l o w e r , r e l a t i v e l y
genous r e s e r v e

at least

the r a p id

co n stan t r a te )

in itia l rate

follow ed

su g g e sts t h a t the endo­

m a t e r i a l may be h e t e r o g e n o u s

in n atu re.

A t­

te m p ts t o r e v e a l a change i n t o t a l c a r b o h y d ra te a f t e r r e s ­
p ira tio n

in b u ffe r,

how ever, f a i l e d

t o show a n y c o n s i s t e n t

decrease.
The s h o r t s t a r v a t i o n p e r i o d e m p l o y e d i n much o f t h i s
work a s a n a i d

in red u cin g a u to r e s p i r a t i o n has a ls o been

found v a lu a b le w ith S. n i t r i f i c a n s
co u pled w ith th e use
resu lte d

in the

of l a r g e

T his tec h n iq u e

i n o c u l a and young m ycelium

s a t i s f a c t o r y d e m o n s tr a ti o n of exogenous

r e s p i r a t i o n w ith m ost s u b s t r a t e s
the l a t t e r

(72).

co n d itio n s,

teste d .

how ever, r e s u l t e d

F ailu re
i n the

to observe

grow th of

m y c e liu m w i t h w h i c h e x o g e n o u s r e s p i r a t i o n was d i f f i c u l t
im possible

to d em o n strate.

In those
term in ated
the

experim ents

d u rin g the

o x i d a t i o n s were

amino a c i d
in te rp re te d

or

i n w hich exogenous r e s p i r a t i o n

o b se rv atio n period

in co m p lete.

i t was e v i d e n t t h a t

Low NH3 r e c o v e r i e s

o x i d a t i o n o b s e rv e d by o t h e r w o rk e rs
as in d ic a tin g

oxidative

( 1 0 , 1 1 ) were

assim ilatio n .

p o i n t wa s o f t e n n o t a v a i l a b l e

during

ev id en ce

on t h i s

long la g

p e r io d s e n c o u n te re d and th e f a c t t h a t th e

D e fin itiv e

because

of the

o b serv atio n

- 65 p e r i o d was n e c e s s a r i l y r e s t r i c t e d
ig atio n s.

The u s e

in creased
on t h i s

in " re s tin g

of h ig h s u b s t r a t e

o x id atio n ra te s

c ell"

in v est­

c o n c e n tra tio n s to o b tain

a ls o precluded f u r th e r

observations

p o in t.

C e llu lar

im p e rm e a b ility to added s u b s t r a t e s

i s a source

of d i f f i c u l t y

i n much r e s p i r a t i o n w o r k ( 2 8 , H5 , 6 2 , 8 2 ) .

Timbre i t

in d ic ate d

(100)

th at c itr a te ,

iso c itra te ,

o x alo su c-

c i n a t e , and © C - k e t o g l u t a r a t e were a p p a r e n t l y n o t m e t a b o l i z e d
by I n t a c t c e l l s

of E .

c o l i w h e re a s compounds of th e K reb*s

c y c l e b e lo w <= C -ketoglutarate d id
t o be o x i d i z e d b y c e l l
observed
m alate

the

failu re

su sp en sio n s.

of S.

or fum arate u n le s s

very high

pen etrate

g riseu s
the

su b stra te

ficu lt

to d em o n strate.

of b o th the

em ployed

99).

(58,

m alonate

tu la ren sis

su b stra te.

p erm eab ility

c u ltu r a l co n d itio n s

f o r e x a m p le , were a b l e t o

o x i d a t i o n , b u t were u n a b le

in h ib i t io n w ith in ta c t c e l l s

t o d em on­

of P a s t e u r e l l a

(62).

The l a g
is

p eriod s

on t h e

o c c u r r i n g b e f o r e maximum o x i d a t i v e

re a c h e d have b e e n th e

v e stig a tio n s.

B ack and M i t c h e l l
b asis

in d ic ate d

req u irin g

c ellu la r

o r g a n i s m and t h e

Kann and M i l l s ,

show o C - k e t o g l u t a r a t e

w orkers

c o n c e n t r a t i o n was

o x id a tio n has been d i f ­

U ndoubtedly,

is a fu n ctio n

tio n s

su ccin ate,

o r t h e m y ce liu m was p r e i n c u b a t e d w i t h t h e

p r e s e n t w ork <> C -ketoglutarate

a c tiv ity

G i l m o u r e t a l . (H6 )
to o x id ize

In the

stra te

a n d c o u l d b e sh ow n

s u b j e c t of num erous i n ­
(5) e x p la in e d

th eir

observa­

of "tem p o rary im p e rm e a b ility " w h ile
th at

"ad ap tatio n "

the
(3)»

process

other

i n v o l v e d was a n e n e r g y -

G erhardt e t a l.

(^+5) s u g g e s t e d

- 66 s e v e ra l e x p e rim e n ta l approaches to
pH, e s t e r i f i c a t i o n

of s u b s t r a t e s ,

p e r m e a b i l i t y and th e use

is

problem

a lte ra tio n

of e x t r a c t s ) .

c e l l e x t r a c t s w i l l be r e p o r t e d
it

the

of c e l l u l a r

D ata o b ta in e d u s in g

in a la te r

s e c tio n alth o u g h

e v i d e n t t h a t s u c h o b s e r v a t i o n s do l i t t l e

or deny th e

c o n cep t of "a d a p tiv e

( v a r i a ti o n of

to confirm

t r a n s p o r t 11 p r o p o s e d b y

W illia m s and W ils o n (1 0 5 , 1 0 6 ) .
D uring the m alonate
w e r e made t o d e m o n s t r a t e

i n h ib i t io n experim ents a ttem p ts
su ccin ate

a c c u m u l a t i o n by p a p e r

chro m ato g rap h y

( 3 2 ) when c e l l h o m o g en a te s w ere

w ith g lu tam ate

and m a lo n a te .

su c ce ssfu l.
a l.

S im ilar

T hese e x p e r i m e n t s were n o t

o b s e r v a t i o n s w ere r e p o r t e d by M arr e t

( 6 8 ) who c o n c l u d e d t h a t

o x i d a t i o n was v e r y

se n sitiv e

the

in itia l

to the

c e l l h om ogenates p r e s e n t s

or s u c c i n a t e .
P.

su ccin ate

a n a p p a r e n t anom aly i n t h a t th e

(62) a lth o u g h w ith t h i s

are

of e i t h e r

It

organism the r a t e
is

probable

sim p ly a f u n c t i o n of p e r m e a b i l i t y

G ilm our e t a l .

(*+6 ) h a v e s h o w n t h a t p r o g r e s s i v e l y

c o n cen tratio n s

of o C -k eto g lu tarate r e s u l t e d

in creased

rates

g lu tam ate

o b s e r v a t i o n s h a v e b e e n made w i t h

o x i d a t i o n was a l s o l o w .

o b serv atio n s

of i n h i b i t o r s .

o b tain ed w ith o ld er

w a s much l o w e r t h a n t h a t

S im ilar

tu la re n sis

s te p in g lu tam ate

presence

The o x i d a t i o n o f < - k e t o g l u t a r a t e

re sp ira tio n rate

in cu b ated

o f o x i d a t i o n b y S* g r i s e u s

of

th a t these
sin ce
in creasin g

in correspondingly
su sp en sio n s.

- 67 CELL EXTRACTS AND DYE REDUCTION
The t y p i c a l l a g s
stra te s

in the

o x i d a t i o n o f a number of s u b ­

by c e l l h o m o g e n a te s and th e

w ith c e r t a i n t y betw een in d u c tiv e
prom pted th e

g lass

av ailab le

(58) and of th e s e

and p e r m e a b i l it y e f f e c t s

for

the

the

g rin d in g w ith an a b ra s iv e

(sand,

I t was

o u t s e t t h a t n e g a tiv e r e s u l t s w ith such

p r e p a r a t i o n s m i g h t be o f l i m i t e d v a l u e
g en erally

A number

p r e p a r a t i o n of c e l l

o r a l u m i n a ) a p p e a r e d t o be m o st p r o m i s i n g .

recognized a t

is

to d istin g u is h

i n v e s t i g a t i o n of m y c e lia l e x t r a c t s .

of te c h n iq u e s are
e x tra cts

in a b ility

o b ta in e d only a f t e r

s i n c e maximum a c t i v i t y

some r e c o n s t r u c t i o n

(58,

82,

97)*
E x p e r i m e n t a l M ethods an d R e s u l t s
G r i n d i n g i n a m o r ta r w i t h sand and i n a g l a s s homogeni z e r w i t h a lu m i n a was t r i e d ;
m ost r e l i a b l e ,

latter

a p p e a r e d t o be t h e

an d was u s e d t h r o u g h o u t t h i s w o r k .

iu m w a s h a r v e s t e d
(0 .0 2 p e rc e n t)

the

and washed w i t h s a l i n e

in the

c en trifu g e,

The m y c e l ­

o r KC1 s o l u t i o n

t h e n groiind f o r 10 m i n u t e s

w i t h a n e q u a l volume o f a lu m i n a i n a g l a s s h o m o g e n iz e r c h i l l ­
ed i n a n i c e b a t h .
phosphate b u f f e r

(pH 7 * 0 , 0 . 0 2 5 M) a n d c e n t r i f u g e d

i m a t e l y 1 6 0 0 XG f o r
m ain tain ed

The d i s r u p t a t e was e x t r a c t e d w i t h

5 t o 10 m i n u t e s .

a t approx­

The e x t r a c t s w e r e

in an ice w ater b a th u n t i l u se d .

A n u m b e r o f a t t e m p t s were made t o o b t a i n c e l l e x t r a c t s

c a p a b le o f b r i n g i n g about oxygen uptake i n W arburg v e s s e l s .
P r e p a r a t i o n s o b t a i n e d w i t h g l a s s p o w d e r or a l u m i n a d o show

- 68 a sm a ll endogenous uptake bu t l i t t l e
observed

in the

ad en o sin e

presence

trip h o sp h ate

tiv e

sy stem

of added s u b s t r a t e .

of
had

M e th y le n e b l u e was ad d ed i n a n e f f o r t
lim itin g

facto r

in the

te rm in a l oxida­

( 7 1 ) , b u t n o s i g n i f i c a n t s t i m u l a t i o n was o b s e r v e d .

The a d d i t i o n o f M g i o n s

was a l s o w i t h o u t e f f e c t .

was o b s e rv e d o n ly w i t h e x t r a c t s
and u s u a l l y

A d d itio n s

and d ip h o s p h o p y rid in e n u c le o ti d e

no s t i m u l a t o r y e f f e c t .
to by-pass a p o ssib le

o r n o s t i m u l a t i o n was

from young

A c tiv ity

( 1 , 2- d a y ) c e l l s ,

d i d n o t p e r s i s t beyond 30 m in u te s a f t e r

the

e q u ilib ra tio n p erio d .
The f a i l u r e
accep to r

of c e l l e x t r a c t s

prom pted th e u se

F ollow ing

the

(2 0 ) t h e dye 2 , 6 - d i c h l o r o p h e n o l

( p h e n o l - i n d o - 2 : 6 d i c h l o r o p h e n o l , B .D .H .) was c h o s e n .

of c a r r y i n g

(Thunberg t e c h n iq u e )
of the e x t r a c t s
f u l use

of the

dye i s

C olem an j u n i o r

n eg lig ib le

out the re a c tio n s
is

av o id ed .

in evacuated

The r e l a t i v e l y

tubes

low a b i l i t y

o u t i n 1 2 x 7 5 mm c u v e t t e s w i t h a

spectrophotom eter.

Phosphate b u f f e r

of th e m ix tu re

was t h e n a d d e d , m ixed b y i n v e r s i o n ,
o b se rv e d by fo llo w in g
600)

(pH 7 # 0 ,

and e x t r a c t were a d d e d , m ixed b y i n v e r s i o n ,

o p tic a l d en sity

m/JL (&0 . D .

so t h a t th e

dye more l i k e l y .

0 . 0 2 5 M), s u b s t r a t e

600

(5 3 )

t o u s e O2 a s a t e r m i n a l a c c e p t o r made s u c c e s s ­

R e a c ti o n s w ere c a r r i e d

and th e

( 3 3 , 7 1 )*

s u g g e s t i o n o f t h e w o r k o f N i c k e r s o n a n d Mohan

A u to - o x id a tio n of t h i s
n ecessity

oxygen a s an e l e c t r o n

of an a r t i f i c i a l c a r r i e r

(7 5 ) end C a s id a and K n ig h t
indophenol

to use

the

decrease

o v e r a Pe r i o d

s e t to

and th e

zero.

The d y e

d e co lo riz atio n

in o p tic a l d en sity a t

of tim Q *

ThG d y e c o n c e n t r a ­

- 69 tio n

in itia lly

u s e d was 0 . 0 0 2 p e r c e n t

Some r e a c t i o n s w e r e c a r r i e d
of 0 .0 1 3

( f in a l co n cen tratio n )#

o u t w i t h a f i n a l dye c o n c e n t r a t i o n

p e r c e n t s o t h a t a more f a v o r a b l e

sp ectro p h o to m eter

p o r tio n of the

s c a l e was e m p lo y e d .

As i n t h e m a n o m e t r i c w o r k i t w a s a p p a r e n t t h a t y o u n g c e l l
m a t e r i a l w ould pro d u ce th e m ost a c t i v e e x t r a c t .
c e lls

gave a c t i v e

p r e p a r a t i o n s h u t no a c t i v i t y

d e m o n s tra te d w ith e x t r a c t s from

of a number of t r i a l s

f u m a r a t e , and c i t r a t e
genous r a t e .

a r e s h o w n i n FIGURE 1 9 .
in d ic ated

in ra te

11s p a r k ” a c e t a t e

(su c cin a te ,

f u m a r a t e ) were n o t s u c c e s s f u l . .

change

and L e v y ) .
lin e

of

acid s

o f e x t r a c t s may be c o m p a r e d b y m e a s u r i n g

(T his i s

e sse n tia lly

the

in itia l

stag es

of

t h e method of E n g l e s b e r g

TABLE 1 0 s h o w s t h a t a n a p p r o x i m a t e l y s t r a i g h t -

r e l a t i o n s h i p e x i s t s b e t w e e n t h i s m e a s u re m e n t and t h e

amount o f e x t r a c t u se d
h o w ever, and
cause

o x i d a t i o n by th e use

in o p tic a l d e n s ity d uring

the r e a c t i o n .

su ccin ate,

c o u l d be s h o w n f o r a c e t a t e .

A ttem pts to

the

th at

s t i m u l a t e d dye r e d u c t i o n o v e r t h e e n d o ­

No i n c r e a s e

The a c t i v i t y

c o u l d be

day c e l l s .

T y p i c a l dye r e d u c t i o n r e s u l t s
The r e s u l t s

One o r 2 - d a y

in the r e a c t i o n .

A maximum i s r e a c h e d

i n c r e a s i n g am ounts o f e x t r a c t

p ro p o rtio n al in creases
A number of a t t e m p t s t o

th e n no lo n g e r

in o p tic a l d e n sity

change.

sh o w d e h y d r o g e n a s e a c t i v i t y

w i t h o ^ - k e t o g l u t a r a t e were u n s u c c e s s f u l .

The i n t e r p r e t a t i o n

of o th er d a ta

hinged upon s u c c e s s ­

(e .g .

glu tam ate

f u l d e m o n s tr a tio n of a c t i v i t y
t r a c t s were f o r t i f i e d

o x id atio n )

tow ards t h i s

w i t h DPN i n a n e f f o r t

su b strate

so e x ­

to b ring about

• 70 1 . 0 *-

0.8

o-5

1

2

k
Time (m inutes)

P ig . 19

R eduction o f 2 ,6 -d ic h lo r o p h e n o l indophenol (0 .0 2 $ )
by 0#5 and 0 .7 ml# o f e x tr a c t from l|Ij.-hour mycelium.
Endogenous •
su c c in a te 10>tM a
a.

- 71 -

TABLE 1 0
RELATIONSHIP BETWEEN THE AMOUNT OF EXTRACT
AND THE RATE OF DYE REDUCTION

E xpt#

AO.D. 1 - 2 *
6 00

E x tra ct
ml*

1

2

3

observed

calcu lated

**

0 .5

120

-

0 .6

136

144

0 .7

164

168

0 .6

170

192

0 .3

139

•

0.1*.

187

184

j

o .5

208

230

j

0 .3

108

i

1

135

144

0 .5

185

CO

0 .5

39

;

O
H

. 14.

0

1

.(

*

j

[

0*7

49

46

)

0 .9

56

59

j

72

i

|
1 .1
____
—
%

:

73
..............

change in o p t i c a l d e n s i ty
1 and 2 m in u te s. (x l0 "3 )

i

... -ia t 600

-------------1

m/4. b e t w e e n

** c a l c u l a t e d from t h e o b se rv e d v a lu e w i th th e
l e a s t am ount o f e x t r a c t used*

a c tiv ity

a g a in st °6 -k eto g lu tarate.

ex p erim en t are

The r e s u l t s

s h o w n i n FIGURE 2 0 , a n d i n d i c a t e

a d d i t i o n o f DPN w i l l a l l o w f o r t h e

as

A d d i t i o n o f DPN a l s o

f u m a r a te , m a l a t e , and g lu ta m a te

s h o w n i n TABLE 11*

shown a g a i n s t t h e s e

t h a t the

d e m o n s tra tio n of dehydro­

genase a c t i v i t y w ith o C -k e to g lu ta ra te .
stim u la te s

of such an

dehydrogenation

W i t h some e x t r a c t s a c t i v i t y

latter

c a n n o t be

compounds w i t h o u t th e a d d i t i o n o f

pyr id in e nuc l e ot id e .
D iscu ssio n
The f a i l u r e

to d em o n strate

ap p reciab le

02 uptake w ith

c e l l e x t r a c t s was n o t a l t o g e t h e r u n e x p e c t e d ;
ig ato rs
is

have r e p o r t e d

b eliev ed

or the
v a lu e .

sim ilar d if f ic u ltie s

ad d itio n

of p r o t e c t i v e

p ro tein

O th er e x p e r i m e n t a l w ork h a s

in v est­

(5*+, 7 2 , 7 5 ) •

t h a t f u r t h e r m ethods of r e c o n s t r u c t i o n

d e r i v a t i v e s may b e m o re e f f e c t i v e
b lue

other

It

(58, 81,9 6 )

( 6 7 , 8 1 ) may p r o v e

of

sh ow n t h a t p h e n a z i n

carriers

th an is

m ethylene

(28, 3 3 ).
T hroughout th e

endogenous a c t i v i t y

i n v e s t i g a t i o n i t was a p p a r e n t t h a t t h e
o f e x t r a c t s was v e r y h i g h .

p r o b a b l y due

in p a rt to the

acid s

e x tra cts.

i n the

by d i a l y s i s

sig n ific an t q u a n titie s

O th e rs have r e d u c e d

(82) a lth o u g h t h i s

the r e c o n s tr u c tio n

T h i s wa s

procedure

of amino

such a c t i v i t y

g e n e ra lly adds to

p r o b l e m s m e n t i o n e d a b o v e ( 2 0 , 29).

D ata

o b t a i n e d w i t h c e l l h o m o g e n a t e s sh o w ed t h a t m y c e l i u m h a r v e s t e d
b efo re e x ten siv e

reserve

f o r m a tio n had o c c u rre d e x h i b i t e d

low er endogenous a c t i v i t y .

The p r e p a r a c i o n o f e x t r a c t s

from

- 73

0 .6

o. d

o
o
vO
«

0 .3

_u.

1

2

3

J

5

Tim© ( m i n u t e s )
F ig.

20

R e d u c tio n o f 2 , 6 d i c h i o r oph e n d in d o p h e n o l (0 .0 1 3 $ )
b y 0 * 5 ml*
e x t r a c t from 1 8 -h o u r m ycelium and
t h e s t i m u l a t i o n b y DPN (500>c<gm./3 m l . r e a c t i o n
m ix tu re.
Endogenous*
•, endogenous+DPN °
o,
g l u t a m a t e * —— *, g l u t a m a t e + D P N ^
v,o(-keto+D PN *
x.

- 7if- -

CD
-P

aj
t—
i
cd

03
aj

4

XA

O

O '

QD

O

h

ai

1A

c\J

O

S

rH

S

is

CD

-P

aj

-P

oo
rQ

<D
-P

3

00

a*
•P

3

§

E-h

OA

O

XX\

rH

rH

rH

h

o in

rH

bO

€
r-i
rH

CA

O

co

1A
I

us O
• c
O sC

g

PQ

<

Eh

a

CM

O
cd
Jh

-P

*

o

rH
0s
O

CM
C\J
rH

CO
CO
O

CO
vQ
rH

<3
* O EH
(54

35

P-t

o
09
a

o

*H
-P

•H
■d
d

3;
Ph
Q

01

03

O

O

3

CD
-P

CD

3

CD

cri

otjO

U

-P

-p

d

X>

m

m

3

3

oW

d

3

CD

d

3

CD

oo

3

CD
-P

a
u

C
O

3

75

-

-

v e r y young m ycelium r e p r e s e n t s a n o t h e r a p p ro a c h t o th e
problem .
C e l l e x t r a c t s were a b le
r e d u c t i o n i n the

presence

t o b r i n g a b o u t a n i m m e d i a t e d ye

of* s u c c i n a t e ;

o b s e r v e d w i t h h o m o g e n a te s c a n n o t be
ing a d a p ta tio n ,
genase

is

at least

i n the

form ed d u r in g th e

the la g p e rio d s

in te rp reted

as in d ic a t­

sen se t h a t s u c c in ic dehydro­

lag p e rio d .

As d i s c u s s e d

pre­

v io u sly ,

how ever,

ad ap tiv e

p ro c e ss concerned w ith th e t r a n s p o r t of s u b s tr a t e

across

the

these

th u s,

o b s e r v a t i o n s do n o t c o n t r a d i c t a n

c e l l mem brane.

The f a c t t h a t d e h y d r o g e n a s e a c t i v i t y
c o n sid erab ly g re a te r
suggested

the

of the l a t t e r
th is

2 .0 m l.

alo n e

th an t h a t

p o ssib ility
during

observed w ith c C -k eto g lu ta rate

of d em o n stratin g a n et fo rm atio n

g lu tam ate d e h y d ro g e n atio n .

sa m p le s of c e l l e x t r a c t were

and w ith added g lu ta m ic a c id

m ix tu res

To b o t h r e a c t i o n

A f t e r 2 h o u r s a t room

s u p e r n a t e s exam ined f o r k e t o

a c i d s b y t h e m ethod o f C a v a l l i n i and F r o n t a l i
to g ra p h y of th e

phenylhydrazone

b utanol

in d icated

tra tio n

i n the m ix tu re

an in c re a s e

reactio n

d e riv a tiv e s

t o w hich g lu ta m a te

glu tam ate

(21).

Chroma­

in w ater s a tu r a te d

in the o f - k e to g lu ta r a te

T h i s e x p e r i m e n t sh o w e d t h a t t h e

the

in b u ffer

t h e r e a c t i o n was s t o p p e d and th e m i x t u r e s d e -

p r o t e i n i z e d w i t h TCA, a n d t h e

o u t the

To t e s t

in cu b ated

( 1 0 0 /< .M ) .

2 . 5 mgm*. o f DPN w e r e a d d e d .

tem p eratu re

on g l u t a m a t e was

concen­

had b e e n ad d ed .

o rg a n is m s were a b le

to carry

©£—k e t o g l u t a r a t e , a n d s u p p o r t s

o x i d a t i o n scheme p r e v i o u s l y a d v a n c e d .
The s t i m u l a t i o n

in the

a ctiv ity

on f u m a r a t e

c a u s e d by

the

76 -

a d d i t i o n o f DPN w a s p r o b a b l y a r e f l e c t i o n

of the

co facto r

r e q u i r e m e n t o f t h e m a l i c d e h y d r o g e n a s e e n zy m e i n t h e e x t r a c t .
A s i m i l a r DPN s t i m u l a t i o n h a s b e e n r e p o r t e d
a phycom ycete

(19);

how ever,

for e x tra c ts

these e x tr a c ts a ls o

of

showed

m alic dehydrogenase a c t i v i t y .
The d i s r u p t i o n a n d e x t r a c t i o n p r o c e d u r e s u s e d w e r e
ad m itted ly

in efficien t.

h a v e b e e n shown t o r e s u l t
p e rc e n t of the

of th e
strom

in reco v eries

of o n ly 10 t o 2?

o r i g in a l N p re s e n t in A zo to b acter c e l l s

These a u th o r s a l s o
stan d ard ized

S i m i l a r a l u m i n a - g r i n d i n g m ethods

(52).

o b s e r v e d t h a t t h e m e t h o d c o u l d n o t be

and t h a t no d e f i n i t e

frac tio n

i n t a c t c e l l c o u l d be a s s u r r e d

of the a c t i v i t y

in the e x t r a c t .

L ind-

( 6 7 ) n o t e d t h a t d u r i n g a l u m i n a g r i n d i n g much p r o t e i n

w as l o s t by a d s o r p t i o n .
e ffic ie n t
p roducible

E x tracts

p r e p a r e d b y t h e more

s o n i c v i b r a t i o n m ethod c o n t a i n e d a l a r g e r , r e ­
amount of th e

shown t o r e q u i r e

o r i g i n a l c e l l n i t r o g e n and w ere

l e s s r e c o n s t i t u t i o n f o r maximum a c t i v i t y

( 52).

- 77 -

TRAN SAMINATION
In tro d u ctio n
The a c t i n o m y c e t e

stra in s

used

i n t h i s w ork grew r a p i d l y

on a g l u e o s e - a s p a r a g i n e - s a l t s medium.
g ested
ab le
is

t h a t the

am ino a c i d

b e liev e d

to

o r g a n is m s were a b l e
sy n th esis,

c u ltu res

to cause

o b se rv a tio n sug­

to b rin g about co n sid er­

an a c t i v i t y

p lay an im p o rtan t r o le

e x p e r i m e n t s were c a r r i e d
these

T his

i n w hich t r a n s a m i n a t i o n
(69, 70).

out to d em o n strate

A number of

the a b i l i t y

of

tran sam in atio n .

M ethods
I n m o s t o f t h e w o r k t o be r e p o r t e d
used.

These w ere

c e l l e x t r a c t s were

o b t a i n e d by g r i n d i n g t h e m y celiu m w i t h

a lu m in a i n a g l a s s hom ogenizer as d e s c r ib e d
on dye r e d u c t i o n .

In itia lly

i n e v a c u a te d T hunberg t u b e s ,
were o b t a i n e d by u s i n g
of th e ease
th e w ork.

the cap -

W ith th e

m ixed w i t h t h e

test

the

tu b es.

t h e y were u s e d

Because
i n m ost of

of Thunberg tu b e s i s
d istilla tio n

occurs in to

s m a ll volum es em ployed c o n s i d e r a b l e
th is

condensate
co n ten ts

care

i s w a s h e d down a n d

of the

tube

d isto rtio n

i n the

c o n c e n t r a t i o n r e l a t i o n s when

carried

out

same r e s u l t s

rest

a n a ly se s are

i n o rd e r t o avoid

out.

E x t r a c t s were added t o
acid

latter

in cu b a tio n co n sid erab le

m ust be t a k e n t o e n su re

p o ssib le

but e ss e n tia lly

A c o m p lic a tio n i n th e use

th a t d u rin g

sectio n

t h e r e a c t i o n s were c a r r i e d

sm all sto p p e red

of h a n d lin g th e

in the

and p h o s p h a t e b u f f e r

tubes

c o n tain in g

( 0 . 0 2 ? K, pH 7 - 0 ) .

N H ^-donor, k e t o
N eu tralized

so lu tio n s

78 -

o f am ino and k e t o a c i d s were added t o e s t a b l i s h

f i n a l co n cen tratio n s
of 0 .2 5 m l./m l.

of jO /iM /m l.

E x t r a c t was a d d e d a t r a t e

of r e a c t i o n m ix tu re u n le s s

T ubes w ere in c u b a te d

otherw ise n o te d .

i n a w a t e r b a t h a t 37 C.

S u itable

c o n t r o l s w ere i n c l u d e d a s shown i n t h e f o l l o w i n g
TUBE

n h 2 - d on g r

KETO ACID

ch art:

BUFFER

EXTRACT

1

4

+-

4

-4

2

—

H-

4

+-

—

*4

4-

+

+-

3

k

—

—

5

b o iled

HTube 1 r e p r e s e n t s

the

com plete r e a c t i o n m ix tu re ;

sam ple was rem oved fro m t h i s m ix t u r e a t
d esig n ated
ed b o ile d

(5 m in u te s)

th e r e a c t i o n was e n z y m a tic
w hich th e r e a c t a n t s
were

included

p ro d u c t form ed
in cu b atio n

z e ro tim e and i s

as tube 1° in sub seq u en t a n a ly s e s .
ex tra ct

p eriod

to ensure

th at
in

were s i n g l y and s i m u l t a n e o u s l y o m i t t e d ,

later

d iscu ssio n ).

the

source

of the

At th e end of t h e

t h e r e a c t i o n was s t o p p e d e i t h e r by p l a c i n g

the

tubes

in a b o ilin g w ater b a th fo r

the

t u b e s f o r 10 m in u te s a f t e r w h ic h th e

and s e t a s i d e

5 co n tain ­

T u b e s 2 , 3? a n d 4 ,

to e s t a b l is h w ith c e r ta in ty
(see

Tub e

and was i n c l u d e d

in n atu re.

a

5 m in u tes

or steam ing

c o n t e n t s were c o o l e d

fo r ex am in atio n .

R e a c t i o n m ix t u r e s were a n a ly s e d by p a p e r c h ro m a to g ra p h y
w i t h W h atm a n No.
solvent

(12).

k

p a p e r and w a t e r - s a t u r a t e d

D e v e lo p m e n t was c a r r i e d

phenol as the

o u t by th e d e s c e n d i n g

- 79 m ethod;

the

c a b i n e t was h e l d

in an in c u b a to r a t ^ 2 6

F o llo w in g c h ro m a to g ra p h ic developm ent the
pended i n f r o n t
tu re

(42).

h eld

p a p e r s were s u s ­

o f a f a n a nd a l l o w e d t o d r y a t room t e m p e r a ­

P a p e rs w ere sp ra y e d w it h 0 .1 p e r c e n t e t h a n o l i c

n i n h y d r i n s o l u t i o n and th e
24 h o u r s .

C.

c o l o r was a l l o w e d t o d e v e l o p f o r

When r a p i d r e s u l t s w e r e r e q u i r e d

the

p a p e r s were

i n f l o w i n g s t e a m f o r a b o u t 1 m i n u t e t o a l l o w f o r mor e

rap id

c o lo r developm ent.
Where s e m i - q u a n t i t a t i v e

size

sp ots

(co n tain in g

pap er and th e
(v/v)

r e s u l t s were d e s i r e d , e q u a l

t h e amino a c i d ) were c u t o u t o f t h e

co lo r e x tra c te d

aqueous e th a n o l.

b y s h a k i n g w i t h ?0 p e r c e n t

The o p t i c a l d e n s i t y

of th e r e s u l t i n g

s o l u t i o n was d e t e r m i n e d w i t h a C olem an s p e c t r o p h o t o m e t e r a t
a wave l e n g t h
e x tra c ts
sh eet.

o f ? 7 0 m>U.

B la n k v a l u e s were

obtained w ith

from a p a p e r s p o t of e q u a l a r e a c u t from th e

same

S u c h a m e t h o d h a s b e e n sh o w n t o h a v e a n a c c u r a c y o f

it 7 p ercen t

(60).

I n t h i s w ork i t was fo u n d t h a t w i t h i n a c h r o m a to g r a p h ic
a n a l y s i s e x c e l l e n t a g r e e m e n t was o b t a i n e d b e t w e e n t h e
d e n s ity v alu es
the

of re p lic a te d

sp o ts.

o p t i c a l d e n s i t y was d i r e c t l y

I t was a l s o fo u n d t h a t

rela te d

to co n cen tratio n

f o r a m o u n ts o f g l u t a m i c a c i d u p t o 13y*M /m l.
how ever,
tio n

The v a l u e s ,

c a n n o t be com pared fro m day t o day b e c a u s e

i n the

o p tic al

of v a r i a ­

d e p th of c o lo r form ed.

R e s u lts and D isc u ssio n
The a b i l i t y
PMG m e d iu m t o

of e x t r a c t s

tran sfer

the

fro m 1 and 2 - d a y c e l l s

grown i n

NH2- g r o u p t o <*-ke t o g l u t a r a t e

from

a la n in e ,

g ly cin e,

a n d 22*

T hese e x t r a c t s were

ity ;

the

80 -

and a s p a r t i c

acid

is

sh o w n i n FIGURES 21

of a p p ro x im a te ly the

s l i g h t d if f e r e n c e s w ith ala n in e

not b e liev e d

t o be s i g n i f i c a n t .

a nd g l y c i n e w e r e

Some q u a n t i t a t i v e

was u s u a l l y n o t e d e v e n when s i m i l a r l y

same a c t i v ­

v a ria tio n

p r e p a r e d e x t r a c t s were

used.
In m ost e x p e rim e n ts
tubes

s u b s t a n t i a l am o u n ts of amino a c i d s ,

g lu tam ic

a c i d were

m arginal a c t i v i t y
requirem ent fo r
In the

produced.
d iffic u lt

a ll

g lu tam ate

the

alon e

p a rticu larly

and e s t a b l i s h e d a n a b s o l u t e
p rev io u sly d e sc rib e d .

i t was n o t e d t h a t

In the

suggested

a ctiv ity ,

th at

t h e r e was g e n e r a l l y a g r e a t e r f i n a l

some m a t e r i a l i n t h e e x t r a c t was s u p p l y i n g

3*5 m l.

of phosphate b u f f e r

sy n th esis.

In an e f f o r t to reduce

o f e x t r a c t w e r e d i a l y z e d a g a i n s t 50 m l .

( 0 . 0 2 5 M, pH 7 . 0 )

i n the

co ld f o r 2 hours

w ith c o n sta n t s t i r r i n g .

T his d ia ly z e d

d ialy zed

same e x t r a c t w e r e t e s t e d

p o r tio n of the

m ate f o r m a t i o n w i t h a s p a r t a t e
R e su lts

of th is

p r e p a r a t i o n and an u n -

and a l a n i n e

ex p erim ent are

T h e s e d a t a sh o w t h a t t h e

apparent lo ss

m

a c tiv ity

a sp artate-g lu tam ate

R e s u l t s w ith the

fo r g lu ta ­

as N ^ -d o n o rs.

sh o w n i n FIGURE 2 3 .
g lutam ate

lev e ls

w e r e l o w e r e d c o n s i d e r a b l y by t h e d i a l y s i s

the

presence

c o n c e n tr a ti o n th a n i n the e x t r a c t p lu s b u f f e r c o n t r o l .

NH2“ Srcru P s f o r g l u t a m a t e
th is

co n tro l

T h i s made t h e e v a l u a t i o n o f

c o n tro ls

c o n tro l tu b es

of ^C -k eto g lu tarate

T his

i t was f o u n d t h a t i n t h e

also

in the

co n tro ls

treatm en t but th a t

occurred.

The r e s u l t s

w ith

s y s t e m w e r e r e n d e r e d more d e c i s i v e .

alan in e-^C -k e t o g l u t a r a t e

s y s te m were n o t

( OAJ . q . qj u oT q m iq u sou oo sq.mirBq.uxo

Fig# 21

Formation of glutamate by extract from 1-day mycelium (1#10 mgm. N/ml
Reaction mixtures contained oC-ketoglutarate and A-glycine, B -alanine,
or C-aspartate# For tube contents see “Methods".

- 81

22

-d-

Cr\

CM

H

O
pH

U\

-d*

CO

&

(0 ^ £ * q * q ) u o x q e a q u e o u o o
<D

OJ

0

q.T3 wBq.nxo

Formation of glutam ate by e x tr a c t from 2-day mycelium (0.99 mgm. N / m l .)
Reaction m ixtures contained o(-keto glut ar ate and A -g ly c in e , B -a la n in e or
C -a s p a r ta te .
For tube co n ten ts see "M ethods11.

v\

F ig.

~ 82 -

concentration

(O.D

- 83 -

1° 1

2

3

4

5

1°

1

2

3

4

5

i°

1

2

3

4

5

Glutamate

Tube

0 1
*

"

0 .0 5

i°

1

2

3

4

5
Tube

Fig* 23

Formation o f glutam ate from -k e to g lu ta r a te p lu s
a sp a r ta te (A, B) or alan in e (X, Y)* Charts A, X
show r e a c tio n w ith o r ig in a l e x tr a c t ( 1.1 5 mgm. N/m l*)
B, Y r e a c tio n w ith d ia ly z e d e x tr a c t (0*70 mgm. N /m l.)*
For tube co n ten ts se e "Methods11*

8k

-

sig n ific an tly

changed.

The l a t t e r

c o n s id e r a b ly low er a c t i v i t y
T his d i a l y s i s

h o w e v e r, was of

tre a tm e n t reduced the n itr o g e n c o n te n t of

o f a p p r o x i m a t e l y Wo p e r c e n t #
p ro tein

system ,

even before d ia ly s is .

t h e e x t r a c t f r o m 1.1 5" m g m . / m l .

the

-

t o 0*70 m g m ./m l., a r e d u c t i o n
In s e v e r a l o th e r experim ents

( p r e c i p i t a t a b l e ) and n o n - p r o t e i n

(so lu b le)

n i t r o g e n w ere d e te r m in e d by f r a c t i o n a t i o n w i t h an e q u a l
volum e

o f 2 0 p e r c e n t TCA a n d s u b s e q u e n t n i t r o g e n d e t e r m i n a ­

t i o n s by th e K j e ld a h l m ethod.

The NPN o f s e v e r a l e x t r a c t s

was f o u n d t o v a r y b e t w e e n 3 5 and 60 p e r c e n t of t h e
n itro g en .

To d e t e r m i n e

the n a tu re

of th e

com ponents of th e

NPN f r a c t i o n ,

s a m p le s w ere c h ro m a to g ra p h e d

p h e n o l, a lo n e

and w i t h a d d i t i o n s

R esu lts

of th ese

sig n ific an t

q u an tities

the

and l e u c i n e .

ex tra cts

of o C -k eto g lu tarate
the

absence

o f know n a m i n o a c i d s o l u t i o n s .

of n in h y d r in - p o s itiv e
to those

m a t e r i a l s whose

of g lu ta m a te , g ly c in e ,

O ther n i n h y d r i n - p o s i t i v e

g iv in g le s s w all d efin ed
th en ,

in w ater s a tu ra te d

e x p e r i m e n t s showed t h a t t h e e x t r a c t c o n t a i n e d

Rp v a l u e s w e r e c o m p a r a b l e
a la n in e ,

co n tain ed
a lo n e ,

su b s ta n c e s w hich,

c o u ld give r i s e

h y d rin -p o sitiv e

the

E v id en tly

in the

presence

to g lu tam ic a c id

in

o f a n e x o g e n o u s NH2 - d o n o r .

g r a p h i c a n a l y s e s was t h a t n o n s p e c i f i c

to

m ateria ls

s p o t s were a l s o p r e s e n t .

A n o th e r f a c t w h ic h became e v i d e n t fro m th e

p erio d .

to ta l

m aterials

In p a rtic u la r
p o sitio n s

o b serv atio n is

based

p ro d u c tio n of n in ­

o c c u rre d d u rin g the

i t was n o t e d t h a t

of a la n in e

ch ro m ato­

in cu b atio n

sp o ts corresponding

and l e u c i n e w ere

on c o m p a r i s o n s “w i t h t h e

form ed.

(T his

an alyses

of

- 8? -

b o ile d c o n tr o l and zero-tim e c o n tr o l tu b e s ).
w a s made t o d e m o n s t r a t e

the r e a c tio n :

g l u t a m a t e -h p y r u v a t e

a l a n i n e 4- ° c - k e t o g l u t a r a t e

b u t no s i g n i f i c a n t a la n i n e
suggested t h a t t h i s
d u rin g

ase a c t i v i t y
titra tio n s
buffer

sy n th esis

n o n -sp ecific

in c u b a tio n arose

appearance

i n p a r t by th e

o u t d u rin g the

showed a n i n c r e a s e

v a t i o n w hich s u p p o rte d th e

in

th e

a b ility

It

is

of "am ino a c i d s "

sim ultaneous p r o t e i n ­
in cu b atio n #

Form ol

in c u b a tio n of e x t r a c t in
groups o c c u rre d ,

an obser­

c o n cep t of p r o t e o l y t i c

a c tiv ity .

I n dye r e d u c t i o n e x p e r i m e n t s
cases

c o u l d be s h o w n .

of the e x t r a c t d u rin g th e

carried

An attem pt

i t was f o u n d t h a t

of the e x t r a c t to reduce

i n some

t h e dye was l o s t

q u i c k l y when t h e e x t r a c t was n o t k e p t i n a n i c e w a t e r b a t h
u n til used.

P r e v i o u s d a t a s h o w e d t h a t i n some e x p e r i m e n t s

on t r a n s a m i n a t i o n t h e
sy n th esis
a ctiv ity

( s e e FIGURE 2 3 ) ,

tim e

heated

to

scrib ed .

in creased ,

W ith t h i s

co n stan t

of th e r e a c t i o n b etw een a s p a r ­

and a t

in terv als

sa m p le s were rem oved,

and a n a ly s e d as p r e v i o u s l y d e ­

of such an experim ent are

sho w n i n FIGURE

p a r t i c u l a r e x t r a c t t h e a c t i v i t y was e s s e n t i a l l y

over the

showed t h a t

the

ph asize

fact

the

out

The a m o u n t s o f t h e a d d i t i o n s t o

s to p the r e a c t i o n ,
R esu lts

An e x p e r i m e n t was c a r r i e d

course

and c C - k e t o g l u t a r a t e .

tu b e 1 were

2*+.

the

g lutam ate

and s u g g e s t e d t h a t t r a n s a m in a s e

m i g h t be v e r y r a p i d .

to in v estig ate
tate

z e ro tim e c o n t r o l i n d i c a t e d

2-hour in c u b a tio n p e rio d .

a c t i v i t y may d i m i n i s h a f t e r
th a t co n sid erab le

O ther e x p e rim e n ts
1 hour,

q u a n titativ e

a n d em­

v a ria tio n

-

86 -

o
V\

O
•r4
-P

0 .3 ~

0.2

-

0.1
-p

A

B

0

E

D

F

2

3

Tube
F ig . 2l|.

Time course o f the g lu ta m ic -a sp a r tic
transam inase r e a c tio n . Tubes A to F
r e p r e se n t glutam ate co n ce n tra tio n s in
th e com plete r e a c tio n m ixture sampled
at 0, 15, 3 0 , J
4.5 , 6 0 , and 120 minutes
r e s p e c t iv e ly . Bars 2 , 3 , k-» anc* 5
re p r e se n t glutam ate in th e c o n tr o l
tu b es (s e e "Methods” ) sampled a t 120
m in u tes. E xtract from 1-day mycelium
( 1 . 19 mgm. N / m l .

)»

-

o c c u rs b etw een e x t r a c t s .

87 -

PLATE 1 sh o w s a c h r o m a t o g r a m o f a

s i m i l a r e x p e r i m e n t i n w h i c h g l u t a m a t e f o r m a t i o n was d e t e r m i n ­
ed a t

i n t e r v a l s u p t o 90 m i n u t e s .
A ll ex perim en ts d iscu ssed

w ith 0 .0 2 5 M b u f f e r ,
on enzyme a c t i v i t y ,
and t h e b u f f e r
o b tain f in a l

pH 7 * 0 .

To d e t e r m i n e

of 7 .0 ,

p a re d and t e s t e d f o r

1 —tu b e

amino a c i d

is

su ggested th a t

for

b)

g reater

3 —t u b e *+)J .

are

T h r e o n in e and l y s i n e

d ata

i n d i c a t e d where

sum o f t h e a m o u n t s

added s i n g l y

From t h e s e

amino a c i d s

com plete r e a c t i o n m ix tu re

th a n the

a la n in e , leu cin e,

system .

a s NH2 d o n o r s w i t h c C - k e t o -

fo rm a tio n i n the
is

th is

s o l u t i o n s were p r e ­

T ran sam in atio n is

fo rm e d when t h e r e a c t a n t s

donors.

It

The g l u t a m a t e f o r m a t i o n w i t h t h e s e

the n e t glu tam ate

a sp arta te ,

G lutam ate

s y s te m was o b s e r v e d , b u t

of 7 .0 - 8 .0 e x is ts

a ctiv ity

s h o w n i n FIGURE 25*

(tube

t h e e f f e c t o f pH

and 8 . 0 .

i n a c t i v i t y were n o t e d .

A number o f n e u t r a l i z e d

(tube

7 .5 ,

asp arta te -g lu ta m a te

a r a t h e r b r o a d pH r a n g e

Is

out

s o l u t i o n in the r e a c t i o n m ix tu res v a rie d to

fo rm a tio n in the

g lu ta rate .

p o i n t were c a r r i e d

g ro u n d m y celiu m was e x t r a c t e d w i t h s a l i n e ,

pH v a l u e s

no d i f f e r e n c e s

to th is

it

and v a l i n e

is

( tube 2 - tube

b) +

apparent th a t

c a n be u s e d a s am ino

have n e g l i g i b l e

a c tiv ity .

T r a n s a m i n a t i n g a c t i v i t y was r e a d i l y d e m o n s t r a b l e w i t h
m y c e l i a l hom ogenates i n l i e u

of c e l l e x t r a c t s .

phic a n a ly s e s

of th e

r e a c ti o n m ix tu res

not as a ru le

a s c o n f u s e d by n o n - s p e c i f i c

m ation a lth o u g h g lu tam ate
tro l

tu b es.

c o n sisten tly

in th ese

C hrom atogra­
s y s te m s were

"am ino a c i d " f o r ­

appeared

in the

con­

E x t e n s i v e work was n o t done w i t h w hole c e l l s

-

88

-

PLATE I .
Chroma to"!* a n s h o w i n g t h e t i m e c o u r s e o f s s o a r t i c g l u t a m i c t r a n s a n i n a s e a c t i v i t y by a m y c e l i a l h o r n o g e n a t e . The
f i r s t 6 s p o ts r e p r e s e n t a n a ly s e s o f th e com plete r e a c t i o n
m i x t u r e s a m p l e d a t 0 , 1 $ s 3 0 , I l % 9o O , a n d oQ m i n u t e s r e s p e c t ­
iv ely .
2 , 3 , If a n d 5 r e p r e s e n t t h e c o n t r o l t u b e s ( s e e "I.’e t h o d s ” )
sam pled a t °0 m in u te s .
upper s n o ts are a s o a r t i c a c id , low er
sp o ts glu tam ic ac id .

- 89 -

o

ru\
•
Q
o

o.k--

a
o

*rt
P
aS
-p

S3
©
o
s
o
o

0.2

©

-p

as
%
p
3
rH
e
m
B

F ig .

25

D

E
F
Tubes

ELE
it.

5

The f o r m a t i o n o f g l u t a m a t e f r o m < < - k e t o g l u t a r a t e
and A - a s p a r tic , B -a la n in e , C -th reo n in e,
D - l e u c i n e , E - l y s i n e , and F - v a l i n e .
B ars 2 t o 5
r e p r e s e n t g lu tam ate c o n c e n tra tio n s in the c o n tro l
t u b e s o f t h e a s p a r t a t e - g l u t a m a t e sy stem .
Incuba­
t i o n tim e 120 m in u t e s .
E x t r a c t from 1 -d a y
m y c e liu m ( 1 . 2 0 mgm.N /m l. ) .
F o r t u b e c o n t e n t s s e e MM e t h o d s " *

-

sin ce

it

was b e l i e v e d

to c ritic is m

in th a t

im p erm eab ility to
The a b i l i t y

90 “

th a t neg ativ e

r e s u l t s w o u l d be s u b j e c t

th ey could a r i s e

because

of c e l lu l a r

one o f t h e r e a c t a n t s .
of th e am ides,

a s p a r a g i n e , and g l u t a m i n e ,

t o a c t a s N E ^ - d o n o r s t o ° C - k e t o g l u t a r a t e was i n v e s t i g a t e d .
R e su lts

of c o lo rim e tric

in d icated

th at

g lu ta rate

d i d n o t y i e l d m o re g l u t a m a t e

form ed i n th e
sep arately
absence
the

the

a n a ly s e s of g lutam ate c o n c e n tr a tio n

com bined p r e s e n c e

o m itted .

W ith a s p a r a g i n e

of k eto a c id

deam inated to a s p a r t i c

sum o f t h a t

co m ponents were

c o n tr o l tube

i n the

su g g est th a t asp arag in e

is

probably

a c id w hich i n t u r n t r a n s a m in a t e s w ith

t o form g lu ta m a te

m ix tu re.

is

i n the

com plete r e a c t i o n

no good e v i d e n c e f o r a d i r e c t t r a n s a m i n a ­

tio n r e a c tio n w ith e ith e r

glutam ine

or a s p a r a g in e

and oC -keto-

PLATE 2 s h o w s a c h r o m a t o g r a m f r o m a s i m i l a r

experim ent c a r r ie d
c e ll e x tra ct.

o u t w i t h a m y c e l i a l hom ogenate

The r e a c t i o n i n w h i c h a s p a r t a t e

in stead

of

h o m ogenate.
I t was p r e v i o u s l y

n o t be d e m o n s t r a t e d
t r a c t s were
th e

of

s u p p lie s the

1^ 2 “ g r o u p h a s b e e n i n c l u d e d a s a c o n t r o l o n t h e a c t i v i t y
th e

but

i n a m o u n t w h e n ° c - k e t o g l u t a r a t e wa s

o C -k eto g lu tarate
There

the

k eto —

showed t h e f o r m a t i o n o f a s p a r t a t e ,

These r e s u l t s

g lu ta ra te .

th a n the

c o n t r o l tu b e s i n w hich th e s e

l a t t e r was r e d u c e d

added.

o f g lu ta m in e and

sta te d

th a t ala n in e

in r e a c t i o n m ix tu res

in c u b a te d w ith g lu tam ate

o pposite re a c tio n

(glutam ate

s y n t h e s i s could

i n w hich c e l l ex ­

and p y r u v a t e ,

alth o u g h

f o r m a t i o n from a l a n i n e

o C - k e t o g l u t a r a t e ) c o x ild b e d e m o n s t r a t e d .

and

W i t h t h e mor e a c t i v e

-

M

I

91

-

A

PLATE 2 .
F o rm a tio n o f g lu ta m a te from
®C- k e t o g l u t a r a t e a n d
a s p a r t a t e , a s p a r a g i n e , o r g l u t a m i n e by a m y c e l i a l h o m o g e n a te .
Tube c o n t e n t s a r e d e s c r i b e d i n " M e t h o d s " .
I n c u b a t i o n tim e
90 m i n u t e s .

- 92 -

g l u t a m a t e —a s p a r t a t e
d em o n strate
these

sy ste m , how ever,

the r e a c t i o n

a c tiv itie s

in both d ir e c tio n s .

sodium e t h y l

itse lf,

the

presence

b u t the e s t e r

gave c o n f l i c t i n g r e s u l t s .

o f o t h e r am ino a c i d s

c o n s i s t e n t glu tam ate

sim u ltan eo u sly

o b tained

ib ility

i n the

o x id ized

(80,

of the d a ta d i f f i c u l t *

o b se rv in g the r e s p i r a t i o n

87)*

s y s te m w ere s i n g l y and

To i n v e s t i g a t e

the f e a s ­

o f s t a r v e d h o m o g e n a te s was o b s e r v e d u p o n t h e
or o C -k e to g lu ta ra te

am ino and k e t o a c i d w ere b o t h p r e s e n t .
g l u t a m a t e was a l s o
on t h e

proposed r e a c tio n p ro d u ct.

slow ly o x id iz e d ,

and th e

an a p p re c ia b le
because the

as rap id

as th a t

experim ents are

and when t h e

The o x i d a t i o n o f

observed in order to e s t a b l i s h an a c t i v i t y

am ino a c i d s were c h o s e n b e c a u s e th e

ig ated

th at

i n d i r e c t ev id en ce f o r t r a n s ­

po stu lated

a d d it i o n of a la n in e , v a l i n e ,

a fter

i t was f e l t

o f t h i s m ethod o f t r a n s a m i n a s e d e m o n s t r a t i o n th e

re sp ira tio n

lev el

NH2 ~ a c c e p t o r

" c o n ta m in a tio n " of the e x t r a c t s

a m i n a t i o n by m an o m etric m e th o d s,
when th e r e a c t a n t s

a s the

sin ce

w o u l d make u n e q u i v o c a l i n t e r p r e t a t i o n
O ther w o rk e rs have

Tn some

o x a l a c e t a t e was u s e d i n s t e a d

No w o r k w a s a t t e m p t e d w i t h o x a l a c e t a t e
in the

to

PLATE 3 sh o w s

w i t h an e x t r a c t from s t r a i n

p rev io u s experim ents
of o x a la c e ta te

i t was p o s s i b l e

lag

other

one

( v a l i n e ) was o n l y

( a l a n i n e ) was o x i d i z e d

p eriod*

only

A s p a r t a t e wa s n o t i n v e s t ­

o x id a tio n of t h i s

of glu tam ate

These p a r t i c u l a r

itse lf.

amino a c i d

is

The r e s u l t s

alm ost
of th ese

s h o w n i n FIGURE 2 6 .

In the v a lin e -g lu tam ate
o b se rv a tio n p eriod th e re

sy stem tow ard th e end of th e

w a s some i n d i c a t i o n o f t r a n s a m i n a s e

PLATE 3*
C hrom atogram show ing g l u t a m i c - a s p a r t i c t r a n s a m i n a s e
a c t i v i t y by a m y c e lia l e x tr a c t .
The r e a c t i o n b e t w e e n a s p a r ­
t a t e a n d o C - k e t o g l u t a r a t e i s sh ow n o n t h e l e f t ; t h e r e v e r s e
r e a c t i o n b e t w e e n g l u t a m a t e a n d o x a l a c e t a t e i s show n o n t h e
r i g h t . Time 1 2 0 m i n u t e s . A r r o w s A i n d i c a t e a m in o a c i d s r e ­
duced i n th e c o m p le te r e a c t i o n m i x t u r e ; h i n d i c a t e s th e d i s ­
a p p ea ra n c e o f endogenous g lu ta m a te i n th e p re se n c e of
o x a la ce ta te .

_ 9*+ -

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- 95 -

a c tiv ity

a l t h o u g h th e r e s u l t s were n o t c o n c l u s i v e .

alan in e-g lu tam ate

sy stem , how ever,

re v e a l any tran sam in ase
rep licate

a ctiv ity .

t h i s m ethod f a i l e d

W arb u rg v e s s e l s w ere p o o le d and t h e

i n b o th system s *

reactio n s
sitiv e

It

is

su p ern atan ts

f o r m a t i o n was

a p p a re n t, a t l e a s t fo r the

a b o v e , t h a t t h e m a n o m e tric m ethod i s a l e s s

d ev ice

fo r fo llo w in g

to

When t h e c o n t e n t s o f

a n a ly s e d by p a p er ch rom atography g lu ta m ate
ev id en t

W ith th e

tran sam in ase

a ctiv ity

sen­

th an is

th e m ethod of d e t e r m i n i n g e n d - p r o d u c t f o r m a t i o n .
I t was o b s e r v e d t h a t

some e x t r a c t s w e r e a b l e

to carry

o u t t r a n s a m i n a t i o n r e a c t i o n s , b u t p o s s e s s e d no d e h y d r o g e n a s e
a c tiv ity

as

refle ctio n

ju d g e d b y dye r e d u c t i o n .
of the

T h i s was p r o b a b l y a

a v i d i t y w i t h w h ic h c o f a c t o r s were h e l d by

d i f f e r e n t enzyme s y s t e m s .
p y rid o x a l phosphate,

the

O t h e r w o r k e r s h a v e sho w n t h a t
co facto r

i n t r a n s a m i n a t i o n , may

n o t be r e s o l v e d fro m t h e apoenzyme e v e n by e x t e n s i v e
(H i,

70,

85).

A d d itio n s

a n d ATP ( 5 m g m . ) t o t h e
no s t i m u l a t i o n ,

of p y rid o x al h y d ro ch lo rid e
a sp artate-g lu tam ate

l e v e l was n o t l i m i t i n g .

a d d itio n

of p y rid in e

showed t h a t

in th ese

enzym es was p r e s e n t
In the
carry

ex tra cts

oth er hand,

the

i n dehydrogenase e x p erim e n ts

a co facto r fo r re s p ira to ry

i n s u b —o p t i m a l a m o u n t s .

stu d ies

or to a s s e s s

by o x i d a t i o n p r o c e s s e s .

d e m o n strate

t h a t the

p r e c e d i n g e x p e r i m e n t s no a t t e m p t was made t o

out b alan ce

rea cta n ts

n u cleo tid e

On t h e

(3^gm *)

system caused

an o b s e r v a t i o n w hich s u g g e s te d

co facto r

d ialy sis

the

tran sfer

the

p o ssib le

lo ss

I n view of th e f a i l u r e

of e le c t r o n s

t o oxy g en and th e

of
to

- 96 -

apparent d eficien cy
e x tra cts

o f r e s p i r a t o r y e n zy m e c o f a c t o r s

i t was f e l t

p r o c e s s e s w o u ld be

th a t loss

of r e a c t a n t s by o x i d a t i o n

of o n ly m inor

The r e l a t i v e l y h i g h l e v e l
am ination is
of o th er
have

of g l u ta m a t e - a s p a r ta te

(Ho, 6 l ,

80),

o b serv atio n s

sup p lies

tran sam in ase

a c tiv ity

of th e

as the r e a c t i o n
N eisseria

t o have an a c t i v e

glu tam ic-

b u t n o a l a n i n e - g l u t a m i c enzym e

has a ls o been noted

p l o y e d may l i b e r a t e

on a number

g lu tam ate-

t h e NH2 g r o u p ( 1 6 ) .

gonorrhoeae has been re p o rte d
asp artic

the

system i s ab o u t 6 tim es as a c tiv e

in w hich a la n in e

tran s­

H o w ever, Camm arata and Cohen

shown t h a t w i t h p i g k i d n e y e x t r a c t s

asp arta te

It

im portance.

a t v a ria n c e w ith the

system s

i n the

(98).

t h a t t h e e x t r a c t i o n p r o c e d u r e s em­

only a p o r t i o n of th e

o rig in a l tissu e

(17)*

It

tran sam in ase
is

q u ite

p o ssib le

t h a t s u p e r i o r d i s r u p t i o n and e x t r a c t i o n p r o c e d u r e s would
in d ic ate

a sim ilar

s i t u a t i o n f o r the

strep to m y cetes.

S e v e r a l a t t e m p t s w e r e made t o d e m o n s t r a t e

g lutam ate

f o r m a t i o n f r o m NHi,.'*' a n d c £ - k e t o g l u t a r a t e , b u t t h e s e w e r e n o t
su c ce ssfu l.

T h is

is

perhaps n o t s u r p r is in g

i n view of th e

r e q u i r e m e n t o f g l u t a m i c d e h y d r o g e n a s e f o r DPN p r e v i o u s l y
d iscu ssed .
asp arta te

C e l l e x t r a c t s were a l s o u n a b le
sy n th esis

to brin g about

f r o m f u m a r a t e a n d NH^ i o n s .

D uring

g r o w t h i n p e p t o n i z e d m i l k i t w a s f o u n d t h a t t h e medium
co n tain ed

in creasin g

a m o u n t s o f NH^ a s g r o w t h p r o g r e s s e d .

I t may bo t h a t u n d e r t h e
c e lls

c u l t u r a l c o n d i t i o n s employed th e

do n o t d e v e l o p a n y a p p r e c i a b l e

u tiliz a tio n .

c a p a c i t y f o r NH^

-9 7 -

T he “ c o n t a m i n a t i o n "
endogenous g lu tam ate
tissu e s

(H, 1 6 , 3 9 ,

been suggested
trated

of e x t r a c t s w ith g lu ta m ate

f o r m a t i o n have b e en o b se rv ed w ith o th e r
95)*

To a v o i d t h e s e d i f f i c u l t i e s

t h a t e n z y m e p r e p a r a t i o n s be d i a l y z e d ,

by l y o p h i l i z a t i o n ,

and e x c e s s

added to th e r e a c t i o n m ix tu re s
co m bination of th ese

(16).

tech n iq u es

r u p t i o n p r o c e d u r e w ould i n d i c a t e
ing a c t i v i t y

and h i g h

it

has

concen­

p y rid o x a l phosphate
It

is

p o ssib le

th at a

p l u s a more e f f i c i e n t d i s ­
m ore e x t e n s i v e

th a n has been observed in the

tran sam in at-

p r e s e n t w ork.

-

98 -

SUMMARY
T e c h n iq u e s were d e s c r i b e d

for

the

p r e p a r a t i o n o f hom o-

g e n a t e s o f fL tre.ptomyces s c a b i e s w i t h a s s u r e d r e s p i r a t o r y
a c tiv ity .

D a ta were

p resen ted to

t i o n c o u l d be c o n s i s t e n t l y
e s ta b lis h e d w ith larg e
tio n

d e m o n s t r a t e d w h en y o u n g c u l t u r e s

i n o c u l a were u s e d .

c ally

im p o rtan t o rganic

in d icated

the

Pronounced la g

t o o x i d i z e a number o f m e t a b o l i -

a c i d s and amino a c i d s .

t h a t both ty p es

energy sources fo r

e x tra c ts

in d icated

(su ccin ate,

p e r i o d s were c o n s i s t e n t l y

fu m arate, c i t r a t e ,

p ro b ab ly the r e s u l t

incom plete,

in d icated

im p o rtan t

organism s *

su b stra tes

and g lu ta m a te )

of low c e l l

of g lu co se,

observed d u r­

D ata o b ta in e d w ith c e l l

t h a t f o r a number o f t h e

The o x i d a t i o n s

The d a t a

o f compounds r e p r e s e n t e d

i n g much o f t h e r e s p i r a t i o n w o r h .

were

A short starv a­

p e r i o d was recommended t o r e d u c e e n d o g en o u s a c t i v i t y .

Such h o m o g en a te s were a b le

were

show t h a t e x o g e n o u s o x i d a ­

th ese

lags

p erm eab ility .

su c cin a te,

and g l u t a m a t e

and o b s e r v a t i o n s were d e s c r i b e d w h ic h

th a t co n sid erab le

a s s i m i l a ti o n probably

occurred.

A m eth o d was d e s c r i b e d e m p l o y i n g d i n i t r o p h e n o l by w h ic h a
decrease

i n a s s i m i l a t i o n d u rin g glucose

o x i d a t i o n c o u l d be

observed•
On t h e b a s i s
the

lib e ratio n

tio n
in

of th e

of s o lu b le

i t was c o n c l u d e d

p art,

o b s e r v e d r e s p i r a t o r y q u o t i e n t and
n itr o g e n d u rin g endogenous r e s p i r a ­

t h a t the

l a t t e r was s u p p o r t e d , a t l e a s t

by n itro g e n o u s r e s e r v e s .

C e l l e x t r a c t s w e r e show n

- 99 -

to c o n ta in co n sid erab le
am ino a c i d s ,

p articu larly

M alonate
g lu tam ate

in h ib itio n

g lu tam ic a c id .
of endogenous,

s u c c i n a te , and

s u p p o r t i n g a pathw ay of g lu ta m a te

cC -k eto g lu tarate

O b s e r v a tio n s on th e
hom ogenates i n d i c a t e d

stra in s.

o x id atio n

and s u c c i n a t e were d i s c u s s e d *
o x id a tio n of ty ro s in e

th at

the

th ro u g h d ih y d ro x y p h en y lalan in e
ing

of r e a d i l y e x t r a c t a b l e

o x i d a t i o n b y c e l l h o m o g e n a t e s wa s d e m o n s t r a t e d .

O b serv atio n s
through

q u a n titie s

by c e l l

o x id a tio n p ro b ab ly proceeded
to m elanin i n pigm ent-form ­

No e v i d e n c e w a s o b t a i n e d f o r a p r e l i m i n a r y

tra n sa m in a tio n w ith

cC -k eto g lu tarate

as is

o b s e r v e d i n some

an im al t i s s u e s .
C ell e x tra c ts
w ere u n a b le

p r e p a r e d from m ycelium ground w i t h a lu m in a

to cause a p p re c ia b le

ex p erim en ts.

C>2 u p t a k e

i n m anom etric

A t e c h n i q u e was d e s c r i b e d u s i n g t h e dye 2 , 6 -

d i c h l o r o p h e n o l in d o p h e n o l by w h ic h d e h y d ro g e n a se a c t i v i t y
c o u l d be d e m o n s t r a t e d w i t h a number o f s u b s t r a t e s .
w ere d e f i c i e n t i n p y r i d i n e
stim u la tio n

n u cleo tid e

a s e v id e n c e d by th e

o f d y e r e d u c t i o n c a u s e d b y DPN i n t h e

of g lu ta m ate

hom ogenates and e x t r a c t s
a c tiv ity .

f o r m a t i o n was a l s o
ed w ith

a c tiv ity

possessed

th at c ell

po ten t g lu ta m ic -a sp a rtic

A sm all b u t s i g n i f i c a n t

g lutam ate

o b s e r v e d when c e l l e x t r a c t s w ere

o C -k eto g lu tarate

N eg lig ib le

presence

and m a l a t e •

Paper ch ro m ato g rap h ic a n a ly se s re v e a le d

tran sam in ase

E x tracts

and a l a n i n e ,

v a lin e,

in cu b at­

or l e u c i n e .

was o b s e r v e d w i t h t h r e o n i n e ,

ly sin e,

- 100 -

g ly cin e,

and t y r o s i n e

a s N E ^-donors.

ence f o r d i r e c t t r a n s a m i n a t i o n betw een
the

am ides, a s p a ra g in e ,

D iffic u ltie s

encountered

No c o n c l u s i v e e v i d ­
o C - k e t o g l u t a r a t e and

and g l u t a m i n e c o u ld be o b t a i n e d .
in the d e m o n stra tio n of tran sa m in ­

a se a c t i v i t y w ere d i s c u s s e d .

- 101 REFERENCES
1# A f a n a s i e v ,

M.M# ^ C o m p a r a t i v e p h y s i o l o g y o f A c t i n o m y c e s i n
r e l a ti o n to p o tato scab.
N ebraska A g n c . E x p t.
S ta . B u l l. 92: 1 -6 3 , 1937.

2. A llen , P .J .

T o x in s and t i s s u e
43: 221-229, 1953.

resp ira tio n .

P hytopath.

3 . A tk i n s o n , D .E .
The b i o c h e m i s t r y o f H y d r o g e n o m o n a s .
II.
The a d a p t i v e o x i d a t i o n o f o r g a n i c s u b s t r a t e s .
J . B a c t. 69: 310-315, 1955.
4 . A w apara,

J.

and B. S e a le .
in r a t organs.
1952.

D i s t r i b u t i o n of tran sam in a se s
J . B i o l . Chem. 1 9 4 : *+97-502,
’

5. B a c k , K . J . C . and R . M i t c h e l l .
The e f f e c t o f g l u c o s e i n
t h e g r o w t h m edium on t h e s u c c i n i c a c i d o x i d ­
i z i n g system of E s c h e r i c h i a c o l i . A u st. J .
S c i . R e s . (B ) 5: " 2 5 6 - 2 6 3 , 1 9 5 2 .
6. B a rre tt,

J . T . and R .E . K a l l i o .
T erm inal r e s p i r a t i o n in
Pseudom onas f l u o r e s c e n s ; com ponent enzymes of
the t r i c a r b o x y l i c a cid c y c le .
J . B a c t. 66:
517-525, 1953.

7 . B e e v e r s , H.

2 , 4 - d i n i t r o p h e n o l and p l a n t r e s p i r a t i o n .
A m e r. J . B o t . *+0: 9 1 - 9 6 , 1 9 5 3 .

8. B e n e d ic t, R .G ., P ridham , T .G ., L in d e n f e l s e r , L .A ., H a ll,
H . H . a n d R . W. J a c k s o n .
F u rth er stu d ies in
th e e v a lu a tio n of carb o h y d rate u t i l i z a t i o n
t e s t s as a i d s i n th e d i f f e r e n t i a t i o n of s p e c ie s
of S tr e p to m y c e s .
App. M i c r o b i o l . 3 : 1 - 6 , 1 9 5 5 .
9. B e n n e tt, R .E .
N u t r i t i o n of S tre p to m y ce s g r i s e u s i n r e l a ­
t i o n to strep to m y cin p ro d u ctio n .
J . B a c t . 5 3:
254, 1947. (A b st.)
1 0 . B e n t l e y , M .L.
Enzymes o f p a t h o g e n i c f u n g i .
M ic r o b io l. 8: 36 5 -3 7 7 , 1953-

J.

Gen.

1 1 . B e r n h e i m , F . a n d W .E. Do T u r k .
The e f f e c t o f c h l o r a m ­
p h e n i c o l a n d c e r t a i n o t h e r d r u g s on t h e o x i d a ­
t i o n o f a r o m a t i c a m i n o a c i d s by a s t r a i n o f
Pseudomonas a e r u g i n o s a .
J . Pharm . and E x p t.
T h e r a p . 105": 2 4 6 -2 5 1 ', '" " 1 9 5 2 .
1 2 . B l o c k , R . J . , Le S t r a n g e , R . a n d G. Z w e i g .
P a p e r Chroma­
to g rap h y .
A L a b o r a to r y M anual.
Academ ic
P r e s s I n c . , N .Y . 1 9 5 2 .

- 102

13* B l u m e n t h a l , H . J . , K o f f l e r , H. a n d E . P , G o l d s c h m i d t *
The
r a t e o f e n d o g e n o u s r e s p i r a t i o n a s a f f e c t e d by
th e o x i d a t i o n of exogenous s u b s t r a t e s *
S ci.
116 : L+75~l+77, 1 9 5 2 .
1*+. B r o d i e , A .F * a n d J . S . G o t s *
E f f e c ts of an is o la te d
d e h y d r o g e n a s e e n z y m e a n d f l a v o p r o t e i n on t h e
r e d u c tio n of tr ip h e n y lt e tr a z o l i u m c h lo r id e .
S c i . i l k * tfO-Hl, 1 9 5 1 .
1 5 . B u t t e r w o r t h , E . M . , G i l m o u r , C.M. a n d C . H . Wang.
S tu d ies
on t h e b i o c h e m i s t r y o f th e S t r e p to m y c e s .
II*
F i x a t i o n o f C O2 b y i n t a c t c e l l s o f S t r e p t o ­
myces g r i s e u s .
J . B a c t. 6 9 : 725-727, 1955.
1 6 . C am m arata, P . S . and P . P . C ohen.
The s c o p e o f t h e t r a n s ­
a m in a tio n r e a c t i o n i n anim al t i s s u e s .
J. B io l.
Chem. 1 8 7 : ^ 3 9 - ^ 5 2 , 1 9 5 0 .
1 7 . C am m arata, P .S . and P .P . Cohen.
S p ectro p h o to m etric
m easurem ent of t r a n s a m in a tio n r e a c t i o n s .
B i o l . Chem. 1 9 3 : *+5-52, 1 9 5 1 .

J.

1 8 . C a m p b e l l , W.R. a n d M . I . H a n n a .
The d e t e r m i n a t i o n o f
n i t r o g e n by m o d i f i e d K j e l d a h l m e th o d s .
J.
B i o l . Chem. 1 1 9 : 1 - 7 * 1 9 3 7 .
1 9 . C a n t i n o , E . C . a n d M.T . H y a t t .
F u r th e r ev id en ce f o r the
r o l e of th e t r i c a r b o x y l i c a c i d c y c le i n m orphog en sis in B la s to c la d ie lla em erso n ii.
J. B act.
6 6 : 712- 7 2 0 , .19^3.
20. C a sid a,

L .E . J r . and S.G . K n i g h t .
The o x i d a t i o n o f
t r i c a r b o x y l i c a c i d c y c l e c o m p o u n d s by P e n i c i l liu m chrysogenum .
J . B a c t . 6 7 : 6 5 8 - 6 6 1 , 195*+•

2 1 . C a v a l l i n i , D. a n d N. F r o n t a l i .
Q u a n titativ e d eterm in atio n
o f k e t o a c i d s by p a p e r p a r t i t i o n c h r o m a to g r a p h y .
B i o c h . a n d B i o p h y . A c t a . 1 3 : Lt'39"1+^5? 1 9 5 ^ .
22. C o le,J . 0.

and
C .R . P a r k s .
S em im icro -K jeld ah l procedure
fo r co n tro l la b o ra to rie s .
I n d . a n d E n g . Chem.
A nal. Ed. 18: 61-62, 19^6.

2 3 . C o c h r a n e , V.W. a n d I . Dimmick*
The m e t a b o l i s m o f s p e c i e s
of S tre p to m y ce s.
I . The f o r m a t i o n o f s u c c i n i c
a c i d and o t h e r a c i d s .
J . B a c t . 56: 723*730?
19*+9.

2k •

C o c h r a n e , V.W. a n d J e a n E . C o n n .
The m e t a b o l i s m o f s p e c i e s
of S trep to m y ces.
I I . The n i t r a t e m e t a b o l i s m o f
S . c o e l i c o l o r • B u l l . T o rre y B o t. Club 77: 1 0 1 8 , 19 $0~.

- 103 2 5 . C o c h r a n e , V.W. a n d M. G i b b s .
The m e t a b o l i s m o f s p e c i e s
of S tr e p to m y c e s .
I V . The e f f e c t o f s u b s t r a t e
on t h e e n d o g e n o u s r e s p i r a t i o n of S tre p to m y c e s
c o elic o lo r.
J . B a c t. 6 l: 305-3077 1951.
2 6 . C o c h r a n e , V.W.
The m e t a b o l i s m o f s p e c i e s o f S t r e p t o m y c e s .
V. The r o l e a n d p a t h w a y o f s y n t h e s i s o f o r g a n i c
a c id s i n S treptom yces c o e l i c o l o r .
J . B a c t. 63:
k59-k71 9 1 9 ^ 2 7
2 7 • C o c h r a n e , V.W. a n d H . D . P e c k .
The r e s p i r a t o r y m e c h a n i s m
of S. s c a b i e s .
P h y t o p a t h . *+2; 5 , 1 9 5 2 . ( A b s t . )
2 8 . C o c h r a n e , V.W. a n d H .D . P e c k .
The m e t a b o l i s m o f s p e c i e s
of S trep to m y ces.
V I. T r i c a r b o x y l i c a c id c y c le
r e a c t i o n s i n S treptom yces c o e l i c o l o r .
J . B act..
6 5 : 3 7 - ^ 4 , 19531“ ^
2 9 . C o c h r a n e , V . W . , P e c k , H .D . a n d Anne H a r r i s o n .
The m e t a b o l ­
ism o f s p e c i e s o f S t r e p t o m y c e s .
V I I . The h e x o s e
m on o p h o sp h ate s h u n t and a s s o c i a t e d r e a c t i o n s .
J . B a c t. 6 6 : 1 7-23, 1953.
30. C oty, V .F .,

G a r n e r , H . R . a n d H. K o f f l e r .
T ricarb o x y lic
a c id c y c le r e a c t i o n s i n S treptom yces g r i s e u s .
B a c t . P r o c . 1 9 5 3 : 7km

3 1 . D a r b y , R .T . and D .R . G o d dard.
S t u d i e s on t h e r e s p i r a t i o n
of th e m ycelium of th e fu n g u s M yrothecium v e r ru caria.
A m er. J . B o t . 3 7 : 3 7 9 - 3 8 7 , 1 9 ? 0 7
3 2 . D e n i s o n , F.W . J r . and E . F . P h a r e s .
R a p id m ethod f o r p a p e r
c h ro m a to g ra p h y of o r g a n ic a c i d s .
A n a l . Chem.
2 kt 1 6 2 8 - 1 6 2 9 , 1 9 5 2 .
33. D ickens, F .

a n d H. M c l l w a i n .
P h e n a z i n e compounds a s
c a r r i e r s i n th e hexose m onophosphate s y s te m .
B iochem . J . 32: 1615-1625, 1938.

3*+. D o u g l a s , R . J . a n d E . I f . G a r r a r d .
S e ro lo g ic a l o b servations
on t h e a c t i n o m y c e t e s a s s o c i a t e d w i t h p o t a t o
scab.
C a n . J . B o t . 3 2 : 1+8 0 - 14 8 5 , 1 9 5 ^ .
3 5 . E i m h j e l l e n , K . E . a n d H. L a r s e n .
The m e c h a n i s m o f i t a c o n i c
a c i d f o r m a t i o n by A s p e r g i l l u s t e r r e u s .
2 . The
e f f e c t of s u b s t r a t e s and i n h i b i t o r s .
Biochem .
J . 60: 139-1^7, 1955.
3 6 . Em erson,

S.

The g r o w t h p h a s e i n N e u r o s p o p a c o r r e s p o n d i n g
t o th e l o g a r i t h m i c phase i n u n i c e l l u l a r o rg a n ­
ism s.
J . B a c t. 60:^221-223, 1950.

-

10k

-

37* E n g l e s b e r g

E.
and J .B . Levy.
Induced s y n t h e s i s of t r i ­
c a r b o x y l i c a c i d c y c le enzymes as c o r r e l a t e d
w i t h t h e o x i d a t i o n o f a c e t a t e and g lu c o s e by
P a ste u rella p e s tis .
J . B a c t . 6 9 : Hl8-*+31, 1 9 5 5 .

38. E rik so n , D

a n d D. W e b l e y .
The r e s p i r a t i o n o f a t h e r m o ­
p h i l i c a e tin o m y c e te M icrom onospora v u l g a r i s .
J . G e n . M i c r o b i o l . 8V *+55-*+63, 1 9 5 3 •

39* F e l d m a n , L I . a n d I . C . G u n s a l u s .
The o c c u r r e n c e o f a
w ide v a r i e t y of t r a n s a m i n a s e s i n b a c t e r i a .
B i o l . Chem. 1 8 7 : 8 2 1 - 8 3 0 , 1 9 5 0 .

ko.

J.

F i n e ham, J R . S .
E f f e c t s o f a gene m u t a t i o n i n N e u r o s p o r a
c r a s s a r e l a t i n g t o g lu ta m ic dehydrogenase form a­
tio n .
J . G e n . M i c r o b i o l . 1 1 : 2 3 6 - 2^ 6 , 195*+*

*+1. F o w d e n , L . a n d J . D o n e .
A new t r a n s a m i n a t i o n r e a c t i o n .
N a t. 1 7 1 : 1 0 6 8 , 1953*
*+2. F o w d e n , L .

The q u a n t i t a t i v e r e c o v e r y a n d c o l o r i m e t r i c
e s t i m a t i o n o f amino a c i d s s e p a r a t e d by chrom a­
to g rap h y .
B i o c h e m . J . *+8 : 3 2 7 - 3 3 3 ? 1 9 5 1 •

**3* G a r n e r , H.3

a n d H* K o f f l e r .
P r e lim in a r y evidence a g a i n s t
th e e x is te n c e of K re b 's c y c le i n S treptom yces
g r i s e u s . B a c t . P r o c . 1 9 5 1 : 139*

kk.

G en tile,

C a r b o h y d ra te m e ta b o lis m and o x a l i c a c i d
A ,C.
s y n t h e s i s by B o t r y t i s c i n e r e a . P la n t P h y s i o l .
2 9 : 2 5 7 - 2 6 1 , I9 W :

*+5* G e r h a r d t , :

M acG rego r, D . H ., M a rr, A .G ., O l s e n , C.B .
and J .B . W ilso n .
The m e t a b o l i s m o f B r u c e l l a :
The r o l e o f c e l l u l a r p e r m e a b i l i t y .
J. B act.
6 5 : 5 8 1 - 5 8 6 , 1 95 3 *

k6 .

G i l m o u r , C . M . , B u t t e r w o r t h , B .M ., N o b le , E . P . and C.H.
W ang.
S t u d i e s on t h e b i o c h e m i s t r y of t h e
S trep to m y ces.
I . T erm inal o x id a tiv e m etab o l­
ism i n S tr e p to m y c e s g r i s e u s .
J. B act. 6 9 :
719-72*+,' 1 9 ^ .

k7*

P roposed group of c h a r a c t e r s
G o r d o n , R.: S. a n d M.M. S m i t h .
f o r t h e s e p a r a t i o n of S t r e p t o m y c e s and N o c a r d i a .
J . B a c t. 69: 1^7-150, 1 9 5 ? * ’

*+8 . G o t h o s k a r ,

S . S . , S c h e f f e r , B . P . , S t a h m a n n , M.A. a n d J . C .
W alker.
F u r t h e r s t u d i e s on t h e n a t u r e o f
F u sariu m r e s i s t a n c e i n tom ato.
P h y to p ath .
ktfz 3 0 3 - 3 0 7 , 1 9 5 5 *

- 105 *+9* G o t t l i e b , D .
The p h y s i o l o g y o f t h e a c t i n o m y c e t e s .
Symposium ( A c t i n o m y c e t a l e s ) V I. I n t . Cong.
M i c r o b i o l . Rome, 1953*
50.

G o t t l i e b , D. a n d H.W. A n d e r s o n .
The r e s p i r a t i o n o f S t r e p t o myces, g r i s e u s .
S c i . 1 0 7 : 1 7 2 - 1 7 3 , 19*+8.

51 • G o u c h e r , C . R . , W o o d s i d e , E . E . and W.. K o c h o l a t y .
The i n ­
f l u e n c e o f 2 , ^ - d i n i t r o p h e n o l on t h e o x i d a t i o n
o f a c e t a t e a n d s u c c i n a t e by E s c h e r i c h i a c o l l .
J . B a c t . 6 7 : 5 9 3 - 5 9 6 , 1 9 5 1*.
52* G r e e n , M. a n d P.W . W i l s o n .
H y d ro g e n ase and n i t r o g e n a s e
A zo to b acter.
J . B a c t. 65: 511-517, 1953.
53. H aas, E .

5^+. H a r r i s ,

in

A c o lo rim e tric d e te rm in a tio n fo r s tu d ie s in v o lv ­
ing coenzym es.
J . B i o l . Chem. 1 5 5 : 3 3 3 - 3 3 5 ,
1 9 1^ .
J.O .
A study of a d a p ta tio n in A z o to b a c te r.
S c i . S o c . A m e r. P r o c . 1 8 : 1 5 ^ - 1 5 6 , 195*+•

S o il

5 5 . H o c k e n h u l l , D . J . D . , W a l k e r , A . D . , W i l k i n , G.D. a n d F . G .
W inder.
O x i d a t i o n o f p h e n y l a c e t i c a c i d by
P e n i c i l l i u m c h r y s o g e n u m . B i o c h e m . J . 50 :
6o5-609, 1952.
56. H o l l i s ,

J.P .
S t u d i e s on S t r e p t o m y c e s s c a b i e s .
II.
S t i m u l a t i o n o f m e l a n i n p r o d u c t i o n by am ino
a cid s.
B u l l . T o rrey B o t. C lub.
81: 96-103,
195^.

57. H o ro w itz ,

N .H . a n d S . C . S h e n .
J . B i o l . Chem. 1 9 7 :

5 8 . H u g o , W .B•

N eurospora t y r o s i n a s e . .
513-520, 1952.

The p r e p a r a t i o n o f c e l l - f r e e e n z y m e s f r o m m i c r o ­
organism s.
B a c t. R evs. 18: 87-105, 195^.

59.

I s e n b e r g , I i . D . , S c h a t z , A . , A n g r i s t , A . A . , S c h a t z , V. a n d
G .S. T re la w n y .
M i c r o b ia l m e ta b o lism of c a r ­
b a m a t e s . I I . N i t r i f i c a t i o n o f u r e t h a n b y S t r e . p t omvces n i t r i f i c a n s .
J . B a c t . 6 8 : 5 - 9 , 195^#

60.

Jones,

61.

J o r d a n , D .C .
T r a n s a m i n a t i o n i n c e l l - f r e e e x t r a c t s of
e f f e c t i v e and p a r a t i s t i c R h i z o b i a .
J. B act.
65: 220-221, 1953•

N .R.

The f r e e a m i n o a c i d s o f f i s h .
1-m ethyl
h i s t i d i n e and & - a l a n i n e l i b e r a t i o n by s k e l e t a l
m uscle a n s e r i n a s e of c o d li n g ( G a d u s c a l l a r i a s ) .
B i o c h e m . J . 6 0 : 8 1 - 8 7 , 1955*

- 106

62* K a n n , E*E* a n d R .C * M i l l s .
O x id a tio n of g lu ta m ic a c id
by P a s t e u r e l l a t u l a r e n s i s .
J. B a ct. 6 9 : 659o o 4 ^ 3-95^5^#
6 3 . K o f f l e r , H.

(convenor)
S y m p os iu m on p h y s i o l o g y
C o rn e ll U n iv e rs ity , Ith a c a , 1952.

6 k., K o f f l e r , H

P r o b l e m s o f o x i d a t i v e m o ld m e t a b o l i s m .
Symposium ( M i c r o b i a l M e t a b o l is m ) V I. I n t .
M i c r o b i o l . Rom e, 1 9 5 3 .

of f u n g i .

Cong.

6 5 . L e r n e r , A* B . a n d T . B . F i t z p a t r i c k .
B io c h e m is try of m e la n in
fo rm atio n .
P h y s io l. R evs. 30: 91-126, 1950.
6 6 . L e r n e r , A* B.
M e ta b o lis m of p h e n y l a l a n i n e and t y r o s i n e .
A d v . i n E n z . I 1*: 7 3 - 1 2 8 , 1 9 5 3 .
67. L in d stro m , E .S .
The oi - k e t o g l u t a r a t e
A zo to b acter.
J . B a ct. 65:
68.

o x id ase system of
565-570, 1953.

M a r r , A.G . , O l s e n , C . B . , U n g o r , H . S . a n d J . B . W i l s o n .
The o x i d a t i o n o f g l u t a m i c a c i d b y B r u c e l l a
ab o rtu s.
J . B a c t. 66: 606-610, 1953.

6 9 . M e i s t e r , A•

i n Amino A c i d M e t a b o l i s m ,
e d . b y W. D.
M cElroy and B. G l a s s .
J o h n H o p k i n s P r e s s , 1 9 55*

7 0 . M e i s t e r , A•

T ran sam in atio n .
1955.

71. M illm an,

Adv. i n E n z . 1 6 : 185-2*46,

The c h a r a c t e r i z a t i o n o f
I . a n d G . P . Y o u m a n s.
t h e t e r m i n a l r e s p i r a t o r y e n z y m e s o f t h e H37Ra
s t r a i n of M ycobacterium t u b e r c u l o s i s .
J. B act.
6 9 320 3 2
19

:

- ^,

#.

M icro b ial
7 2 . M o h a n , R . R • , T r e l a w n y , G . S . a n d A. S c h a t z .
m etabolism of c arb am a te s.
I I I . P rep aratio n
and r e s p i r a t o r y a c t i v i t y of n o n p r o l i f e r a t i n g
c e l l s of S treptom yces n i t r i f i c a n s .
J. B act.
69: 387-392, 1955.
73* N e i s h , A.C .

A n a l y t i c a l m ethods f o r b a c t e r i a l f e r m e n ta ­
tio n s.
N a t . R e s . C o u n c i l o f C a n . R e p o r t *-+68-3 R, 1950.

7k.

S t u d i e s on a c t i n o p h a g e
N ewbould, F .H .S . and E .H . G a r r a r d .
f o r S trep to m y ces s c a b i e s .
( T h a x t . ) Waksman a n d
H e n ric i.
C a n . J . B o t . 3 2 : 3 8 6 - 3 9 1 , 195*+»

75.

S t u d i e s on t h e n u t r i t i o n
N i c k e r s o n , W . J . a n d R . R . M oh an .
and m e ta b o lism of S tr e p to m y c e s .
Sym posiu m
( A c t i n o m y c e t a l e s ) V I. I n t . Cong. M i c r o b io l .
Rome, 1953*

- 107 -

76.

N i c k e r s o n , W .J. and F .G . Sherm an.
M etab o lic a s p e c ts of
b a c t e r i a l grow th i n th e absen ce of c e l l d i v i —
sio n .
I I . R e s p i r a t i o n of n o rm a l and f i l a m e n ­
tous c e l l s of B a c illu s c ereu s.
J . B a c t . 6k •
6 6 7 - 6 7 8 , 1 9 5 2 . ---------------

77* O g i n s k y , E L . , S m i t h , P . H . a n d W.W. U m b r e i t . . The a c t i o n
o f s t r e p t o m y c i n on t h e r e s p i r a t i o n o f S t r e p t o myces g r i s e u s .
J . B a c t . 6 l : 639-6*42, 1 9 5 1 7
78. P erlm an, D

a n d G. Wagman.
S t u d i e s on t h e u t i l i z a t i o n o f
l i p i d s by S trep to m y ces g r i s e u s .
J . B a c t. 63:
253- 2 6 2 , 19^27

79* P e r l m a n , D

P h y s i o l o g i c a l s t u d i e s on t h e a c t i n o m y c e t e s .
B o t . R e v . 1 9 : *+6-97, 19 53*

80. P o lia k ,

a n d D. F a i r b a i r n .
The m e t a b o l i s m o f A s c a r i s
lu m b rico id es o v a rie s.
I I . Amino a c i d m e t a b o l ­
ism .
Gan. J . B iochem . and P h y s i o l . 33: 3 0 7 -

J.]

316 , 1955 .
8 1 . P r i c e , C.A

82.

and K .V . T h im an n .
The e s t i m a t i o n o f d e h y d r o ­
genases in p la n t tis s u e .
P l a n t P h y s io l. 29:
1 1 3 - 1 2 ^ , 1951*.

R e p a s k e , I :* a n d P.W . W i l s o n .
O x id a tio n of i n t e r m e d i a t e s
o f t h e t r i c a r b o x y l i c a c i d c y c l e by e x t r a c t s o f
A zo to b acter a g i l e .
P ro c . N at. Acad. S c i. 39:
2 2 ^ - 2 3 2 , 1 9 ^3 .

83* R i c h a r d s o n

8*+. R o l i n s o n ,

J.K .
The i n f l u e n c e o f t u b e r d e v e l o p m e n t on
scab i n f e c ti o n in K atahdin p o ta to e s .
P hyto­
p a th . k2 : 297-298, 1952.
( r.N* R e s p i r a t i o n o f P e n i c i l l i u m c h r y s o g e n u m
in p e n ic illin ferm en tatio n s.
J . Gen. M ic ro ­
b i o l . 6 : 336-3^3* 1952.

8 5 . R o w s e l l , E ,V.

T r a n s a m i n a t i o n t o p y r u v a t e a n d some o t h e r
acid s.
N at. 168: 10*
4 , 1951*

oC - k e t o

86.

S ch aal,

V a r i a t i o n and p h y s i o l o g i c a l s p e c i a l i z a t i o n
L.< l.
i n t h e common s c a b f u n g u s . (A. s c a b i e s . ) J .
A g r i c . R e s . 6 9 : 1 6 9 - 1 8 6 , 19*+*+"*

T r a n s a m in a tio n as a s t e p i n t y r o s i n e m eta­
8 7 . S c h e p a r t z , B.
b o lism .
J . B i o l . Chem. 1 9 3 : 2 9 3 - 2 9 8 , 1951*
88.

S i m o n , E.W

The a c t i o n o f n i t r o p h e n o l s on r e s p i r a t i o n
a n d on g l u c o s e a s s i m i l a t i o n i n y e a s t .
J. E x p t.
B o t . ki 3 7 7 - 3 9 2 , 1 9 5 3 .

- 108 -

89.

S i m o n , E.W

90.

S p e n c e r, R .P . and J .B . F i e l d .
Q u alitativ e c o lo rim e tric
a s s a y o f t y r o s i n a s e s u b s t r a t e s and i n h i b i t o r s .
P .S .E .B .M . 88: 576-578, 1955.

91.

S to u t,

and H. B e e v e r s .
The q u a n t i t a t i v e r e l a t i o n ­
s h i p b e tv/e e n pH a n d t h e a c t i v i t y o f w e ak a c i d s
• and b a s e s i n b i o l o g i c a l e x p e r i m e n t s .
S ci.
11*4; 1 2 * 4-1 26 , 1 9 5 1 .

H.A . a n d ^H . K o f f l e r .
B io c h e m istry of fila m e n to u s
fu n g i.
I . O x i d a t i v e m e ta b o lis m of g l u c o s e by
P e n i c i l l i u m chrysogenum .
J . B a c t. 62: 253-

268 , 1951 .

9 2 . T a y l o r , C. F . and P . D e c k e r.
A c o r r e l a t i o n betw een p a th o ­
g e n i c i t y and c u l t u r a l c h a r a c t e r i s t i c s i n th e
genus A ctinom yces.
P h y t o p a t h . 3 7 : *49- 5 8 , 1 9 *47.
93* T e r n e r , C.

The m e t a b o l i s m o f c i t r i c a c i d i n t h e mammary
gland.
I . The e f f e c t o f p - n i t r o p h e n o l o n t h e
s y n t h e s i s and o x i d a t i o n of c i t r i c a c i d by
h o m o g e n a t e s o f mammary g l a n d .
Biochem . J .
6 0 : 88-95, 1955.

9*+* T e r n e r , C.

The m e t a b o l i s m o f c i t r i c a c i d i n t h e mammary
g l a n d . I I . The e f f e c t o f p - n i t r o p h e n o l a n d o f
f l u o r i d e on t h e s y n t h e s i s o f c i t r i c a c i d i n
f l u o r o a c e t a t e - b l o c k e d hom ogenates.
B iochem .
J . 6 0 : 9 5 - 1 0 1 , 1955*

T rans­
95* T h o r n e , C. B . , Gomez, C . G . a n d R . D . H o u s e w r i g h t .
a m i n a t i o n o f D -a m in o a c i d s by B a c i l l u s s u b tilis.
J . B a c t . 6 9 : 3 5 7 - 3 6 2 , 1955*
I n h i b i t i o n by m a lo n a te of s u c c i n i c d e h y d ro ­
9 6 . T h o r n e , K. B .
genase i n h e a rt-m u sc le p r e p a r a tio n s .
Biochem .
J . 5*+: 5 ^ 0 - 5 ^ 7 , 1 9 5 3 .
97# T i s s i e r e s ,

98. Tonhazy,

A . , M i t c h e l l , H . K . a n d F . A. H a s k i n s .
Studies
on t h e r e s p i r a t o r y sy s te m o f th e poky s t r a i n
of N eurospora.
J . B i o l . Chem. 2 0 5 : *423-*433,
1 9 5 T.

O x i d a t i o n of amino
Nr. E . a n d M . J . P e l c z a r J r .
a c i d s a nd c o m p o u n d s a s s o c i a t e d w i t h t h e t r i ­
c a r b o x y l i c a c i d c y c l e by N e i s s e r i a gon o r r h o e a e .
J . B a c t. 65: 368-377, 1953.

The i n f l u e n c e o f t h e e n v i r o n m e n t o n
99 . U m barger, H .E .
a c e ta te m etabolism in E s c h e ric h ia c o l i .
J*
B a c t . 6 8 : 1*40-1*45, 195*4 •

- 109 -

1 0 0 . . U m b r e i t , W.W. R e s p i r a t o r y c y c l e s .
J . C e l l , a n d Comp.
P h y s i o l . Ul (Supp. 1 ): 3 9 -6 6 , 1953.
1 0 1 . U m b r e i t , W.W. , B u r r i s , R.IT. a n d J . F . S t a u f f e r .
Manom e t r i c t e c h n i q u e s and t i s s u e m e t a b o l i s m .
B u r g e s s P u b . C o . , M i n n . 19*49.
1 02. V a is e y , E .B .
An i n v e s t i g a t i o n o f a c t i n o m y c e t e s i s o l a t e d
fro m t h e s o i l and t h e i r a b i l i t y t o c a u s e
p o tato scab.
M .S.A . T h e s i s , U n i v e r s i t y o f
T o r o n t o , 19 5 3#
1 0 3 . W a k sm a n , S . A .

N e o m y c i n . R u t g e r s U n i v e r s i t y P r e s s , 1 953*

1 0 H . W e b l e y , D.M.
The e f f e c t o f o x y g e n on g r o w t h a n d m e t a b o l ­
ism of th e a e r o b i c t h e r m o p h ilic a c tin o m y c e te
M icrom onospora v u l g a r i s .
J . G e n. M i c r o b i o l .
1 1 ": 114—1 2 2 , 195*4.
1 0 5 . W i l l i a m s , A.M. a n d P .W . W i l s o n .
E q u i l i b r a t i o n of su c ­
c i n a t e s o l u t i o n s w i t h a d a p te d and u n a d a p te d
A zo to b acter c e l l s .
Can. J . M i c r o b i o l . 1 :
3 6 - 4 !+, 1 9 5 ^ .
1 0 6 . W i l l i a m s , A.M. a n d P .W . W i l s o n .
A d a p ta tio n of A z o to b a c te r
c e l l s to tr ic a r b o x y lic acid s u b s tr a te s .
J.
B a c t. 67: 353-360, 195^.
1 0 7 . W i l n e r , B. and C .E . C l i f t o n .

B a c illu s s u b t i l i s .

O x i d a t i v e a s s i m i l a t i o n by
J . B a ct. 6 7 : 5 7 1 - 5 7 5 *