SHORT COMMUNICATIONS 199

s c 2187
Biosynthesls of 7-chmoro-6-clernethy|tetracyc|ine |n the
presence o f a m | n o p t e r i n and e t h i o n i n e
I t ha~ b e e n d e m o n s t r a t e d t h a t [Me-~4C]methionine a n d [2-~*C~-glycine are efficiently
i n c o r p o r a t e d i n t o 7-szhlorotetracycline~, a n d t h a t t h e t h r e e m e t h y l g r o u p s of 5 - h y d r o -
x y t e t r a c y c l i n e arc d e r i v e d from methionine-",?~. T h e s e findings h a v e led s e v e r a l in-
v e s t i g a t o r s t o s t u d y t h e effects on t e t r a c y c l i n e b i o s y n t h e s i s of various a n t i m e t a -
bolites of c o m p o u n d s f u n c t i o n i n g in biological m e t h y l a t i o n . It h,~ : b e e n s h o w n t h a t
sulfa d r u g s ~, "~a n d e t h i o n i n e n cause t h e p r o d u c t i o n of 7-chloro-6-deme t h y l t e t r a c y c l i n e
by strains which d o not normally synthesize this substance. A methionine-reqairing
s t r a i n of S t r e p t o m y c e s v i r i d i f a c i e n s , p r o d u c i n g 7 - c h l o r o - 6 - d e m e t h y l t e t r a c y c l i n e , g a v e
i n c r e a s e d yields of t h i s a n t i b i o t i c w i t h e t h i o n i n e 6.

TABLE I
7=CHLORO-6-DEMETHYLTETRACYCLINE PRODUCTION XVITH
AMINOPTERIN AND VARIOUS CGMPOUNDG
Diameter of inhibition zone
Concur. Rcversi.~gcompound ConoL on bioautogram(ram)
Inhi6"tor (raM] (raM)

Aminopterin i. x None o 32 27 2z
Aminopterin z. z L-Methionine 6.7 30 tr* I6
Aminopterin z. I ~-methyl-DL-Methionine 6.2 33 tr 2i
Aminopterin i. t DL-Homocysteine 5.6 z8 15 tr
Aminopterin [. t Cyanocobalamin o.37 3° 22 IS
Aminopterin i. l p-Aminobenzoic acid 7-4 30 2o 20
Aminopterin i.i Folic acid 2.2 32 2- i8
None o None o 3" o 30

* tr = trace; CTC = 7-chlorotetracycline; DCTC = 7-chloro-6-demethyltetracycline; TC =

tetracycline.
TABLE II
EFFECT OF AMINOPTERIN AND VARIOUS COMPOUNDS ON TETRACYCLINE POTENCY4

Conch. Telragyclinc pote~tcy,(I~g/ml)

Compound added (raM) + am;ttopterit;~ --aminopteri~t

None o 854 123°

L-Methionine 6.7 517 4 z2
~-methyl-DL-Mcthionine 6.2 z zoo 12 Io
DL-Homocysteine 5.6 29o 805
Folic acid 2.2 84° 829

" Final concentration aminopterin, z.I raM.

We have e x t e n d e d these studies with ethionine and, further, we have shown

t h a t S t r e p t o m y c e s a u r e o f a c i e n s ATCC 13 90o w h i c h p r o d u c e s 7 - c h l o r o t e t r a c y c l i n e a n d
t e t r a e y e f i n e , b u t n o t 7 - c h l o r o - 6 - d e m e t h y l t e t r a c y e l i n e , will s y n t h e s i z e t h i s l a t t e r
s u b s t a n c e w h e n g r o w n in t h e p r e s e n c e of a m i n o p t e r i n .
T h e o r g a n i s m w a s g r o w n in a u t o c l a v e d m e d i u m c o n t a i n i n g e x t r a c t i o n p r o c e s s
s o y b e a n meal, glucose, NaCI, a n d p o w d e r e d CaCOn w i t h sterile a d d i t i v e s . A f t e r a

Biochim. B i o p k y s . Acta, 71 (~g53) z99-2ox

200 SHORT COMMUNICATIONS

T A B L E III
.-CHLORO-6-DEMETHYLTETRAC~I'CLINE PRODUCTION WITH ETHIONINE AND VARIOUS COMPOUNDS

Diameter of inhibition zone

Inhibitor _. Concn.(mM) Reversing compoutld Cotwn.(mM) on bioautogram(ram)

L-Ethionine O.12 None o 3° 20 tr*

L-Ethionine o.3x None o IO o tr
L-Ethionine 0.62 None o o o o
~-Ethionine o.62 L-Methionine 20. l 28 17 tr
L-Ethionine o.62 L-Methionine 3.3 27 o o
L-Ethionine o.62 ~-m ethyl-DL-Methionine 6.2 tr o tr
L-Ethionine 0.62 DL-Methionine sulfoxide 6.1 26 o 15
L.Ethionine 0.62 DL-Methoxinine 0.75 35 23 14
L-Ethionine o.31 Glycine I3.3 25 o tr
L-Ethionine o.31 DL-Serine 9.5 22 o tr
L-Ethionine 0.31 DL-Threonine 8. 4 3° o tr
D-Ethionine 0.62 None o 24 zo lO
DL-Ethlonine 0.62 None o i8 tr tr
DL-Ethionine o.6z DL-Homocysteine 2.8 25 tr tr
DL-Ethionine o.62 Cyanocobalamin 0.o074 3° o 17
oL-Ethionine 0.62 Co l+ (as Co(NO3) 2) o.17 25 17 18
None o None o 34 o 22

* See Table I.

T A B L E IV
EFFECT OF ETHIONINE AND VARIOUS COMPOUNDS ON TETRACYCLINE POTENCY

Tetracycline potency (pglral)

Compound added Conch. (raM) -
+ ethionine* --ethionilw

None o 3o2 IO2O

DL-Methionine 6.7 664 795
oL-Methoxinine o. 75 994 I x50
DL-Methionine sulfoxide 6.I f~2o x I3O
DL-Homocysteine 2.8 5!2 555
DL-Threonine 8.4 ~64 880
p-Aminobenzoic acid 0.74 356 926
Folic acid o.22 338 880
Cyanocobalamin o.oo74 736 894
Co s+ (as Co(NOa)2) o.ox7 750 750

" Final concentration DL-ethionine, o.62 raM.

7 - d a y i n c u b a t i o n a t 25 ° o n a r o t a r y shak~.r, s a m p l e s w e r e a c i d i f i e d t o p H 2.5 w i t h
H2SO 4 and analyzed for tetracycline antibiotics by filter-paper chromatography and
b i o a u t o g r a p h y u s i n g S t a p h y l o c o c c u s a u r e u s 2 0 9 P (refs. 5, 7, 8). S e m i q u a n t i t a t i o n w a s
achieved by measurement of the diameters of the zones of inhibition in the bio-
autograms. In addition, quantitative data for the total tetracycline potency in the
acidified fermentation samples were obtained using a method based on the inhibition
o f C O 2 e v o l u t i o n b y E s c h ~ r i c h i a coil °.
The data summarized in Tables I and II show that the production of 7-chloro-
6-demethyltetracycfine by S. aureofaciens ATCC x3900 grown in media containing
a m i n o p t e r i n is r e v e r s i b l e b y a d d i t i o n o f L - m e t h i o n i n e a n d ~ - m e t h y i - D L - m e t h i o n i n e .

Biochim. B i o p h y s . Acta, 7~ (I963) I 9 9 - 2 o l

 SHORT COMMUNICATIONS :2OI

I t is to be f u r t h e r n o t e d t h a t L-methionine alone a n d a m i n o p t e r i n only in c o m b i n a t i o n

w i t h I)L-homocysteine are i n h i b i t o r y to tetracycline biosynthesis.
I n Tables I I ! a n d IV, it can be seen t h a t L- a n d DL-ethionine are quite i n h i b i t o r y
to t e t r a c y c l i n e synthesis, in p a r t due to g r o w t h inhibition, b u t t h a t these c o m p o u n d s
as well as l~-ethionine cause production of 7-chloro-6-demethyltetracycline. Certain
c h e m i c a l agents can increase or eliminate biosynthesis of this substance a n d o v e r c o m e
t h e general inhibition of t e t r a c y c l i n e production in t h e presence of ethionine. These
agents include methionine, m e t h i o n i n e sulfoxide, m e t h o x i n i n e , g!ycine, serine,
threonine, h o m o c y s t e i n e , c y a n o c o b a l a m i n , a n d Co 2+ ions.
T h e inhibition of the 6 - m e t h y l a t i o n of 7-chlorotetracycline b y a m i n o p t e r i n
s u p p o r t s t h e hypothesis offered in an earlier report 4 t h a t t h e insertion of this m e t h y l
g r o u p is a folic a c i d - d e p e n d e n t reaction• The effects of ethionine m a y be ascribed to
e i t h e r a direct c o m p e t i t i o n w i t h m e t h i o n i n e or an e t h y l a t e d folic acid deriwttive
a c t i n g as an a ntagor/ist of t h e n o r m a l m e t h y l a t e d coenzyme.

Squibb Institute for M d i c a l Research, S. L. NEIDLEMAN

N e w Brunswick, N . J . (U.S.A.) E. BIENSTOCK
R. E. BENNETT

t p. A. MILLER, J. R. D. MCCORSnCKAnD A. P. DOERSCHUK,Science, tz 3 (I956) to3o.

J. F. SNELL, A. J. BIRCH AND P. J. THOMSON,J. Am. Chem. See., 82 (I960) 2402.
a A. J. BIRCH, J. F. SNELL AND P. J. THOMSON,J. Chem. See., (*.962) 425.
t j. j. GOODMANAND M. M•TRISHIN, J. Bacteviol., 82 (I96I) 615.
5 D. PERLMAN,L. J. HEUSIgR, J. B. SESIAR, W. R. FRAZlER.~ND J. A. 13OSKA,J. Am. Chem. See.,
83 (I96I) 448I.
6 D. HENDLIN, E. L. DULANEY, D. DRE-2.ItER,T, COOK AND L. CHAIET, Biochim. Biophys. Acta,
58 (x962) 635.
J. H. MARTIY, A. J. SHAY, L M. PzVEss, J. N. POR'rLR, J. H. MOWAT .~ND N. BOHONOS,
Antibiot. Ann., (t954-t955) to-o.
8 G. A. SELZER ANt) W. W. WRIGHT, Antibiot. Chemotherapy, 7 (1957) z9z.
9 j. R. GERKE, T. A. HANEY, j. F. PAGANOAnD A. FERRARI,Ann. N.Y..dcad. Sci., 87 (r96o) 78-'.
~o j. R. D. McCoRMICK, U. I'IIRSCH, E. R. JE~SE~ AND x-Nl'.O. ~JOLANDER, U.S. Patent ,878280
(1959).

R e c e i v e d S e p t e m b e r 7th, 1962
Biochim. Biophys..4cta, 71 (I963) I99-2oi

s c 2203
Unavailability of chromaffin granule adenosine triphosphate
for metabolic reactions
T h e a d r e n a l m e d u l l a c o n t a i n s c o n s i d e r a b l y m o r e A T P t h a n a n y o t h e r tissue thus far
s t u d i e d z. A l m o s t all of this A T P is held w i t h i n t h e chromaffin granules 2, which store
t h e a d r e n a l i n e a n d n o r a d r e n a l i n O . T h e adrenaline a n d noradrenaline are known to
be h e l d w i t h i n t h e chromaffin granules in a biologically i n a c t i v e form. Thus, when
a p r e p a r a t i o n of isolated a d r e n a l m e d u l l a " l a r g e ~ a n u l e s " suspended in isotonic
sucrose is i n j e c t e d i n t r a v e n o u s l y i n t o a cat o n l y a small fraction of t h e presser a c t i v i t y
of t h e c o n t a i n e d amines is i m m e d i a t e l y a p p a r e n t . On t h e o t h e r hand, if t h e granules
a r e first " l y s e d " in distilled w a t e r a n d t h e n i n j e c t e d t h e full presser a c t i v i t y of t h e
c o n t a i n e d amines is i m m e d i a t e l y observed*.

Biochim. Biophys. Acta, 7 z (z963) 2Ol-2O3