Communication
J. Nutr.
NUCLEOTIDE
PRECURSOR
Sci.
IN
Vitaminol.,
22,
477-480,1976
RIBOFLAVIN
BIOSYNTHESIS1
Hisateru
MITSUDA,
Kenji
Laboratory
NAKAJIMA,
of Nutritional Kyoto
Chemistry,
University,
(Received
Although cursors
it
of
is
riboflavin
precursor. may
purines
and
organism
it
depend
on
that by
an
sp.
present
phosphate
being
labelled
(11).
After
mycelia
soluble (HCOO-)
was as
labelled
described
Liquid
Counter
The
cal
Society
of cell
contents of
Japan
Abrreviations: diphosphate
mannose;
at
this
(TRI-CARB
Kyoto
(April
GA;
guanine
UDP-Gal,
the
presented
BACHER precursor
acid
linear
tri
experiments
with
nutrient
medium
reported guanine
previ for
and on
the
acid 1•~2
incorporation (Cl-)
elution
counted
12 hr,
the
Dowex
1•~2
gradient was
above.
guanosine
solution
Dowex
and
described
analysis
by
of
aerogenes
a natural
labelled
(9,10)
column with
with
0.1
and
end at
radioactivity
of
the
incubation
the
annual
meeting
was
conducted
period of the
(12
Agricultural
as hr)
was Chemi
1976).
uridine
N
a Packard
3320).
content at
8-azaguanine,
and
Furthermore,
nucleotides
isolated
through
by
with
micro purines,
of
experiments
also
of
were
on
of
same
various
results
cell
(7).
a
(8)
riboflavin
chromatographic
by
the
immediate
our
flavino
riboflavin
possibility
perchloric
confirmed
riboflavin
paper
grown
developed
suspension of
were
report
was
Radioactivity
ashbyii.
with
a previous
was
of
E.
incubation
column
GTP
which
Scintillation
1 The
in into
Determination follows.
to
with
non-growing
extracted
subjected
guanine
solution.
the cell
were
chromatography, HCl
which
to
non-growing
obtained
fluid
cells
of
an
pre
immediate for
Aerobacter
the
precursor
an
analogue,
is
compatible
in
that
mutants,
are
interconversion
into
BAUGH
derivative
cells
also
guanine 7).
investigated
nucleotide
submitted
a (6,
is purines
the
possibility
using
are
non-growing ashbyii
were
and and
with
further
direct
in
Eremothecium hr
results
we
of
incorporated
ashbyii guanine
The
a
guanine
24
experiments
purines
purine
uptake
the are
experiments
paper,
which
that
efficiency
purine
discovered
unidentified
to
the
(1-4)
microorganisms
Eremothecium
radioisotope
Corynebacterium the
of
606
reports
as
exogenously by
cells
riboflavin
of
we
of Agriculture,
Kyoto
many
of
NADAMOTO2
11,1976)
that
different
added
triphosphate
suggested
the
activities
Faculty
September
obscure
the
However,
in non-growing
still reported
in
xanthine,
guanosine
ously
is
Tomonori
Sakyo-ku,
through
we
nucleotides
(5).
especially
for
known
Previously,
genesis
In
well
and
ribonucleotidyl-(3•Œ-5•Œ)-adenosine; diphosphate
2 満 田久輝 ,中 島 謙 二,灘 本知 憲 477
galactose.
GDP-M;
guanosine
478
H. MITSUDA,
Fig.
1.
Dowex
1•~2
non-growing Guanine
to
0.5M
heated
in
applied
3M
a
to
a
height
of
2 cm,
riboflavin by
height
same
The
circles
in with
ADP,
incorporated GTP
indicates
on that
reductase cells
into
only
the
active
the
(EC of
the
mold
Fig.
1 show
labelled
(13).
cell
filtered, The
by
the
Dowex
1•~2
distilled
water.
450nm
and
systems:
acid+0.18M (150ml)
order.
The
Dowex
ex
applied
formate
this
centrifuged.
with
were
formic
and and
solvent
2.5M
in
medium
extracts
ammonium
(100ml)
with
from
washed
indicated
III)
with
the
clear
50W•~4 (Cl-,
fluid
(H+,
200-400 The
was
200-400
mesh)
to
absorbance
radioactivity
nucleotide
GDP
other
or
GTP
was
guanosine
it was
of
a the
determined
which
cells converts
Furthermore,
as of
E,
shown
should
as by
ashbyii to
were
interest
are IMP be
GpA,
closed like in noted
AMP, not
AMP,
clearly
in
a mutant contrast
Fig. lacking
to the
in
[2-14C]guanine GDP-M,
circles
be GA
observed
see,
GMP,
that
mycelia
and could
NAD,
to
the
NAD
UTP of
GTP
such
GMP it
probably
great
of
of
fractions
and
nucleotides,
flavinogenesis,
and
of
pattern
separation
the ATP
hand,
elution
Clear
UDP-Gal,
the
were
(100ml),
and
However,
non-growing
1.6.6.8)
min
guanine.
and
GDP-M, On
non-growing
acid•{0.31M
packed
at
chromatogram.
amounts.
acid
nucleotides
above.
UDP-Gal
the
then eluted
determined
12 hr
GMP,
various
and
the
soluble
incubation.
nucleotides.
formate
10
of
mycelia
developed
formic
first
described
for
on
3M
for
then
was
ADP,
80•Ž
8 cm was
method
and
achieved
and
of and
open
incubated UMP
at
and formic
acid
hr
to
soluble
ammonium
(1•~10cm)
fraction
the
(12),
bath
12th
The
acid
2M
IV)
acid+2.2M
column
mesh)
II)
(100ml),
formic
of
the
shaker.
column
(100ml),
at
(6.42•~106cpm)
give
(HCOO-)
acid
a water
to
10-4M
to
and T. NADAMOTO
chromatography ashbyii
a reciprocal acid
formate
V)
at
on
1•~2
formic
ammonium and
hr
perchloric
a Dowex
I)
column
added
12
with
NAKAJIMA,
of Eremathecium
was for
tracted
(HCOO-)
cells
incubated
K.
the total
GDP 1.
This GMP
growing radioac
COMMUNICATION
Table
1.
Incorporation
Eremothecium 1 and
a Values
tivity
of
GTP).
the
GTP
the
results
matography
from
added
shows
which
[2-14C]guanine
The
results
of initially
These
of
ashbyii.
values
into
were
another
479
nucleotides
calculated
Dowex
and
from
1•~2
(Cl-)
the
riboflavin
results
in
of
Fig.
chromatography.
guanine.
highest
were
value
also
indicated
of
guanosine
ascertained
more
nucleotides
by
clearly
the
Dowex
(GMP,
1•~2
(C1-)
GDP
and
column
incorporation
of
[2-14CJguanine
radioactivity
of
GMP,
chro into
GTP. The
amounts,
and
GDP-M,
from
the
results
graphy. were
The
and
their
The
values
of
are the
guanine
that
activity
of of
exception In
the
present
and
sine
the
1, and
the
amounts
of
of
results into
each
indicate
Under
of
of
of of
at
order a
the
the
of
large
12 hr
GMP, amount
incubation
This
initially
was
added
conditions,
guanosine
GTP
GDP-M, nucleotide.
the
stage.
experimental
value
amounts
that
riboflavin
activity
chromato
guanosine in
exponential
specific
highest
the
GTP
calculated
(C1-)
exception
successively
the
the
1•~2
the
GDP, were
that
with
The
reached
Dowex
indicate
increased
incorporated
experiments incorporated
high
showing for
with
non-growing
only
into
the
GDP-M.
It
easily guanine
the
nucleotides
radioactivity
[2-14C]guanine
nucleotide
of was
Accordingly, precursor
well in
GDP-M
was
actively
of
specific with
incorporation known cells
the
(14).
under into that
GTP
GDP-M Thus,
ashbyii,
at the
into
that
obtained
concluded
Eremothecium
nucleotides
incorporated
it is of
cells
guanosine
highest is
mannose-l-phosphate
triphosphate.
immediate
the
nucleotides,
produced.
flavinogenesis,
from
which
chromatogram,
GDP-M.
except
in
Table
with
the
the
from
riboflavin.
exhibited
nucleotides
that
in
on
results
guanosine
comparison
was
GTP
given
already
riboflavin
[2-14C]guanine of
the
and
by
GTP of
1 and
biosynthesis
also
specific
radioactivity
was
riboflavin
and
nucleotides
increased
specific GDP-M
determined
stage
results among
of
added
with
Fig.
radioactivity
GTP,
when
activity
radioactive
of
highest
GDP,
total
being
it
is is
exponential of
guanosine
directly
quite
physiological riboflavin
guanosine
formed
conceivable conditions
through
guano
triphosphate
is
riboflavin.
REFERENCES
1) MCLAREN,J. A., J. Bacteriol., 63, 232 (1952). 2) MCNUTT, W. S., J. Biol. Chem., 210, 511 (1954). 3) BROWN,E. G., GOODWIN,T. W., and JONES, O.T. G., Biochem. J., 68, 40 (1958).
an
480
H. MITSUDA,
K. NAKAJIMA,
and
T. NADAMOTO
4) OSMAN,H. G, and SOLIMAN,M. H., Biochem. J., 333, 351 (1960). 5) MITSUDA,H. and NAKAJIMA,K., J. Vitaminol., 18, 137 (1972). 6) MITSUDA,H. and NAKAJIMA,K., J. Nutr. Sci. Vitaminol.,19, 215 (1973). 7) MITSUDA,H. and NAKAJIMA,K., J. Nutr. Sci. VitaminoL, 21, 331 (1975). 8) BAUGH, C. M. and KRUMDIEK,C. L., J. Bacteriol., 98,1114 (1969). 9) BACHER,A, and MAILANDER,B., J. Biol. Chem., 248, 6227 (1973). 10) MAILANDER,B. and BACHER,A., J. Biol. Chem., 251, 3623 (1976). 11) MITSUDA,H. and NAKAJIMA,K., J. Vitaminol., 18, 131 (1972). 12) MITSUDA,H., NISHIKAWA,Y., and NAKAJIMA,K., J. Nutr. Sci. Vitaminol., 22,115 (1976). 13) AUDLEY,B. G. and GOODWIN,T. W., Biochem. J., 84, 587 (1962). 14) HASSID,W. Z. in D. M. Greenberg (editor), Metabolic Pathways, Academic Press, New York and London, Vol. 1, p. 346 (1967).
J. Nutr.
NUCLEOTIDE
PRECURSOR
Sci.
IN
Vitaminol.,
22,
477-480,1976
RIBOFLAVIN
BIOSYNTHESIS1
Hisateru
MITSUDA,
Kenji
Laboratory
NAKAJIMA,
of Nutritional Kyoto
Chemistry,
University,
(Received
Although cursors
it
of
is
riboflavin
precursor. may
purines
and
organism
it
depend
on
that by
an
sp.
present
phosphate
being
labelled
(11).
After
mycelia
soluble (HCOO-)
was as
labelled
described
Liquid
Counter
The
cal
Society
of cell
contents of
Japan
Abrreviations: diphosphate
mannose;
at
this
(TRI-CARB
Kyoto
(April
GA;
guanine
UDP-Gal,
the
presented
BACHER precursor
acid
linear
tri
experiments
with
nutrient
medium
reported guanine
previ for
and on
the
acid 1•~2
incorporation (Cl-)
elution
counted
12 hr,
the
Dowex
1•~2
gradient was
above.
guanosine
solution
Dowex
and
described
analysis
by
of
aerogenes
a natural
labelled
(9,10)
column with
with
0.1
and
end at
radioactivity
of
the
incubation
the
annual
meeting
was
conducted
period of the
(12
Agricultural
as hr)
was Chemi
1976).
uridine
N
a Packard
3320).
content at
8-azaguanine,
and
Furthermore,
nucleotides
isolated
through
by
with
micro purines,
of
experiments
also
of
were
on
of
same
various
results
cell
(7).
a
(8)
riboflavin
chromatographic
by
the
immediate
our
flavino
riboflavin
possibility
perchloric
confirmed
riboflavin
paper
grown
developed
suspension of
were
report
was
Radioactivity
ashbyii.
with
a previous
was
of
E.
incubation
column
GTP
which
Scintillation
1 The
in into
Determination follows.
to
with
non-growing
extracted
subjected
guanine
solution.
the cell
were
chromatography, HCl
which
to
non-growing
obtained
fluid
cells
of
an
pre
immediate for
Aerobacter
the
precursor
an
analogue,
is
compatible
in
that
mutants,
are
interconversion
into
BAUGH
derivative
cells
also
guanine 7).
investigated
nucleotide
submitted
a (6,
is purines
the
possibility
using
are
non-growing ashbyii
were
and and
with
further
direct
in
Eremothecium hr
results
we
of
incorporated
ashbyii guanine
The
a
guanine
24
experiments
purines
purine
uptake
the are
experiments
paper,
which
that
efficiency
purine
discovered
unidentified
to
the
(1-4)
microorganisms
Eremothecium
radioisotope
Corynebacterium the
of
606
reports
as
exogenously by
cells
riboflavin
of
we
of Agriculture,
Kyoto
many
of
NADAMOTO2
11,1976)
that
different
added
triphosphate
suggested
the
activities
Faculty
September
obscure
the
However,
in non-growing
still reported
in
xanthine,
guanosine
ously
is
Tomonori
Sakyo-ku,
through
we
nucleotides
(5).
especially
for
known
Previously,
genesis
In
well
and
ribonucleotidyl-(3•Œ-5•Œ)-adenosine; diphosphate
2 満 田久輝 ,中 島 謙 二,灘 本知 憲 477
galactose.
GDP-M;
guanosine
478
H. MITSUDA,
Fig.
1.
Dowex
1•~2
non-growing Guanine
to
0.5M
heated
in
applied
3M
a
to
a
height
of
2 cm,
riboflavin by
height
same
The
circles
in with
ADP,
incorporated GTP
indicates
on that
reductase cells
into
only
the
active
the
(EC of
the
mold
Fig.
1 show
labelled
(13).
cell
filtered, The
by
the
Dowex
1•~2
distilled
water.
450nm
and
systems:
acid+0.18M (150ml)
order.
The
Dowex
ex
applied
formate
this
centrifuged.
with
were
formic
and and
solvent
2.5M
in
medium
extracts
ammonium
(100ml)
with
from
washed
indicated
III)
with
the
clear
50W•~4 (Cl-,
fluid
(H+,
200-400 The
was
200-400
mesh)
to
absorbance
radioactivity
nucleotide
GDP
other
or
GTP
was
guanosine
it was
of
a the
determined
which
cells converts
Furthermore,
as of
E,
shown
should
as by
ashbyii to
were
interest
are IMP be
GpA,
closed like in noted
AMP, not
AMP,
clearly
in
a mutant contrast
Fig. lacking
to the
in
[2-14C]guanine GDP-M,
circles
be GA
observed
see,
GMP,
that
mycelia
and could
NAD,
to
the
NAD
UTP of
GTP
such
GMP it
probably
great
of
of
fractions
and
nucleotides,
flavinogenesis,
and
of
pattern
separation
the ATP
hand,
elution
Clear
UDP-Gal,
the
were
(100ml),
and
However,
non-growing
1.6.6.8)
min
guanine.
and
GDP-M, On
non-growing
acid•{0.31M
packed
at
chromatogram.
amounts.
acid
nucleotides
above.
UDP-Gal
the
then eluted
determined
12 hr
GMP,
various
and
the
soluble
incubation.
nucleotides.
formate
10
of
mycelia
developed
formic
first
described
for
on
3M
for
then
was
ADP,
80•Ž
8 cm was
method
and
achieved
and
of and
open
incubated UMP
at
and formic
acid
hr
to
soluble
ammonium
(1•~10cm)
fraction
the
(12),
bath
12th
The
acid
2M
IV)
acid+2.2M
column
mesh)
II)
(100ml),
formic
of
the
shaker.
column
(100ml),
at
(6.42•~106cpm)
give
(HCOO-)
acid
a water
to
10-4M
to
and T. NADAMOTO
chromatography ashbyii
a reciprocal acid
formate
V)
at
on
1•~2
formic
ammonium and
hr
perchloric
a Dowex
I)
column
added
12
with
NAKAJIMA,
of Eremathecium
was for
tracted
(HCOO-)
cells
incubated
K.
the total
GDP 1.
This GMP
growing radioac
COMMUNICATION
Table
1.
Incorporation
Eremothecium 1 and
a Values
tivity
of
GTP).
the
GTP
the
results
matography
from
added
shows
which
[2-14C]guanine
The
results
of initially
These
of
ashbyii.
values
into
were
another
479
nucleotides
calculated
Dowex
and
from
1•~2
(Cl-)
the
riboflavin
results
in
of
Fig.
chromatography.
guanine.
highest
were
value
also
indicated
of
guanosine
ascertained
more
nucleotides
by
clearly
the
Dowex
(GMP,
1•~2
(C1-)
GDP
and
column
incorporation
of
[2-14CJguanine
radioactivity
of
GMP,
chro into
GTP. The
amounts,
and
GDP-M,
from
the
results
graphy. were
The
and
their
The
values
of
are the
guanine
that
activity
of of
exception In
the
present
and
sine
the
1, and
the
amounts
of
of
results into
each
indicate
Under
of
of
of of
at
order a
the
the
of
large
12 hr
GMP, amount
incubation
This
initially
was
added
conditions,
guanosine
GTP
GDP-M, nucleotide.
the
stage.
experimental
value
amounts
that
riboflavin
activity
chromato
guanosine in
exponential
specific
highest
the
GTP
calculated
(C1-)
exception
successively
the
the
1•~2
the
GDP, were
that
with
The
reached
Dowex
indicate
increased
incorporated
experiments incorporated
high
showing for
with
non-growing
only
into
the
GDP-M.
It
easily guanine
the
nucleotides
radioactivity
[2-14C]guanine
nucleotide
of was
Accordingly, precursor
well in
GDP-M
was
actively
of
specific with
incorporation known cells
the
(14).
under into that
GTP
GDP-M Thus,
ashbyii,
at the
into
that
obtained
concluded
Eremothecium
nucleotides
incorporated
it is of
cells
guanosine
highest is
mannose-l-phosphate
triphosphate.
immediate
the
nucleotides,
produced.
flavinogenesis,
from
which
chromatogram,
GDP-M.
except
in
Table
with
the
the
from
riboflavin.
exhibited
nucleotides
that
in
on
results
guanosine
comparison
was
GTP
given
already
riboflavin
[2-14C]guanine of
the
and
by
GTP of
1 and
biosynthesis
also
specific
radioactivity
was
riboflavin
and
nucleotides
increased
specific GDP-M
determined
stage
results among
of
added
with
Fig.
radioactivity
GTP,
when
activity
radioactive
of
highest
GDP,
total
being
it
is is
exponential of
guanosine
directly
quite
physiological riboflavin
guanosine
formed
conceivable conditions
through
guano
triphosphate
is
riboflavin.
REFERENCES
1) MCLAREN,J. A., J. Bacteriol., 63, 232 (1952). 2) MCNUTT, W. S., J. Biol. Chem., 210, 511 (1954). 3) BROWN,E. G., GOODWIN,T. W., and JONES, O.T. G., Biochem. J., 68, 40 (1958).
an
480
H. MITSUDA,
K. NAKAJIMA,
and
T. NADAMOTO
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