Vol. 18, No. 2, 1965
BIOCHEMICAL AND BlOPHYSlCAt RESEARCHCOMMUNICATIONS
THE EFFECT OF HEMIN ON THE SYNTHESIS OF GLOBIN Gail
P. Bruns
Department of Medicine, and Bronx Municipal
ReceivedNovember
studies
rabbit
reticulocytes
1958),
rabbit
of
two
of
synthetic
cobalt
or purine (Morel1
could
of
a mechanism
synthesis
of
the
(Karibian
may serve
to
which
the
are
summarized of
approximately
marked
and London) coordinate for (1)
briefly:
inhibition
10
-4
in
al,
1958
are
or by the
or by
incubation
rates
and Kassenaar the
in et
normal
agents,
but
the
coordination
and in
evidence
the
proposed
for
of
the
glycine
the
accompanyin
a mechanism
of
The
regulation
hemin
reticulocytes of
question
processes.
mechanism
addition
1957).
of
here
two biosynthetic
al,
a
parallelism
reported
236
the
addition
to be explored.
utilization
al,
influences,
1951)
the
on the
in
remained
provide
et
and dog reticu-
by various
that
M to rabbit the
1958)
physiologic
globin
and Morel1
in
The two mechanisms,
by various
which
basis
et
al,
normal
heme and of
al,
vitro.
indicated
be disturbed
The studies paper
et
heme and globin
1956
ribosides
effects
for
of
affected
(Richmond
Such differential rates
College of Medicine New York, New York
a parallelism
in
be differentially
medium
in
et
indicated
processes
or lead
hypotonic
synthesis
(Morel1
have
by irradiation
e.g.,
Einstein Center,
and Borsook,
marrow
1957)
could
however,
on the (Kruh
bone
(Nizet,
the
Albert Hospital
M. London
18, 1964
Prior
locytes
and Irving
in
which findings may be
a concentration
in
vitro,
there
for
the
synthesis
is
a of
8lOCHEMlCAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS
Vol. 18, No. 2, 1965
the
heme;
principal
are
concerned
(2)
under
site
with
these
an increase
the
the
of
(Nutritional
water
and were
repeatedly
ml of media 1
acids,
L-Valine-Cl4
flasks
flasks
the
and
which
acid
(ALA);
results
in
newly
formed
can increase
of
the
flushed
incubated
at
the
at
with
1500
The cells
were
lysed
twice
x g for
1 volume
punc-
plasma cells
was in
supplemental
plasma,
glucose
84 mg%, and NaHC03 0.01 isotopic
substrate.
M.
Incu-
5 minutes,
stop-
shaking.
15 minutes,
45 minutes
packed
95% O2 - 5% CO2 for
with
20-25%
by cardiac
with
20% rabbit
of hemoglobin,
x g for
with
ml of
iron
distilled of
and the
medium
was the
37O C without
isolation
washed
20,000
0.15
84 mg%, streptomycin
were
obtained
15 minutes
medium,
on "low
a hematocrit
40% Eagle's
labeled)
maintained and deionized
Blood
x g for
40% NC1 culture
were
at
reactions
hemin
into
were
to maintain
contained
consisting
cells
fuged
-valine
own formation
15-20%.
1500
(uniformly
For
the
of
at
Incubation
centrifuged
14
of
Corporation)
bled
count
was centrifuged
and
addition
rabbits
Biochemical
100 mg%, penicillin
pered
of C
New Zealand
and a reticulocyte
bation
the
deltaaminolevulinic
the
its
among
globin.
diet"
removed.
is
of
incorporation
Methods:
aminr,
formation
Heme can inhibit
synthesis
3.4
inhibition
same conditions,
in
hemoglobin.
ture
of
the the
4 volumes of
distilled
at
4O C.
cell
media of
were
isotonic water
were
removed
and
sodium
chloride
and were
centri-
The supernatant
TL alanine, .0007M; arginine HCl, .00016M; aspartic histidine HCl, .0009M; lysine, .0008M; methionine, phenylalanine, .00052M; proline, .00053M; serine, threonine, .0005M; tryptophane, .OOOlW; tyrosine, leucine, .OOlM; cysteine, .00015M 237
suspensions
acid, .OOlM; .OOOlW; .0006lM;. .00026~:
Vol. 18, No. 2,1965
solution
BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNlCATlONS
was applied
Verona1
buffer
optical
density
to
starch
pH 8.6.
of
The hemoglobin
spot
were
Hemoglobin
formation
added
hemin
I 5Xl65M
is
I
was eluted
0.05
M
and the
as a function in
1
I x10-4
in
determined.
shown
Figure
1.
I
2xX)-4
CONCENTRATION
1.
electrophoresis
and radioactivity
Results: concentration
block
of At
the
a concen-
I
3x10-w
4x10-h
OF HEMIN
The effect of various concentrations of added hemin on the formation of globin. The cell suspension was incubated for 30 minutes at 37O C with added hemin; the control was incubated without hemin. At 30 minutes, 1-Valine-C14, 12 mumoles, 8.4 x lo7 cpm/umole was added and the incubation was continued for 2 hours. Results are expressed as the radioactivity of the globin of the experimental samples relative to the control which is assigned a value of 100.
tration
as low
as 5 x low5
M there
is
a considerable
Cl4 -valine
incorporation.
The increased
hemoglobin
in
of
the
presence
out
a 24-hour
period
very
rapidly
and is
incubation were
(Fig.
incubated
Cl?-valine;
2b). with
in
other
of
added
incubation
readily In hemin
observed these for
experiments
formation
hemin (Fig.
is
in
238
which
first
the prior hemin
in
labeled through-
The effect the
experiments,
of
demonstrable
2a).
during
30 minutes
increase
hour
occurs of
reticulocytes
to
the
addition
and C14 -valine
of
BlOCHEMlCAL
Vol. 18, No. 2,1965
2
4
6
AND BIOPHYSICAL
6
10
RESEARCH COMMUNICATIONS
I
20
"16
12
24
26
TIME IN HOURS
$400-
b
E” l.200‘E p
~~-
1 P
800-
I” +
600-
& :
400-
a8
200I IO
2.
were
I 20
I I 30 40 TIME IN MIN.
I 50
I 60
The effect of added hemin on globin formation. After incubation for 30 minutes at 37O C with added hemin (L x 10'4 M) or without it (control) l-Valine-C14, 12 mumoles, 8.4 x lo7 cpm/umole was added and the incubation was continued for various periods of time, (a) hours, (b) minutes.
added
observed
formation
of protein
was
'in 15 minutes. Previous
laboratory
enhanced
simultaneously,
(Kassenaar
studies et al,
by Kruh 1957) 239
and Borsook have
(1956)
demonstrated
and from enhanced
this forma.
Vol. 18, No. 2,1965
BIOCHEMICAL
tion
of hemoglobin
iron
and of hemin
effects
on the
was observed question
on the
iron
in
cells
by
diet
from
the
of
hemin
have peptide
of the
newly
(a)
the
if
would
lead
synthesis: 204
(b)
of
enhancing
study.
effects
of hemoglobin
in
The on the
its
rendered
cells
of hemin
are observed
The stimulatory
iron-deficient effects
with
possible
formation
of hemin
normal
or elevated
the
of hemin chains are
subunits
to tetramers less
hemin
would
the
increase
may be a combination
240
of the
rate of
the
than in
conversion of the
the
tetramer,
effects
formatio
exert of
a feedsubunits
subunits
of polypeptide the
poly-
conversion
increased
should
concentration in the
l
the
may
of hemoglobin;
stable
of hemoglobin
synthesis,
there
(1)
may promote
be reflected
of polypeptide diminish
are:
by which
on the biosynthesis
of heme would if
mechanisms
of globin
effect addition
to a secondary (c)
effect
have been
of hemoglobin
effect
tetramer
that
polypeptide
subunits
inhibition
to the
the
formed
of hemoglobin; back
the
(2)
a stabilizing
together.
most marked
formation
or absent
accelerating
chains;
the
The principal
increases
a primary
added
increase
iron.
Discussion: added
were
of
stimulatory
or no further
stimulatory
phlebotomy.
may be minimal
concentrations
maximal
or by virtue
that
animals
and extensive
their
heme is under
be noted
increasing
concentrations
its
independently of
At
little
and iron
exerts
RESEARCH COMMUNICATIONS
iron.
produced
hemin
iron
should
derived
of
of globin,
synthesis
in
iron
singly
when both
It
addition
synthesis
of globin
effect
or of
which
of whether
synthesis
or of
on the
AND BIOPHYSICAL
and chain
of 2(a)
and
BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS
Vol. 18, No. 2, 1965
A major enhancing
question
effect
studies
of
proteins
in
on the
that
addition
globin the
concerned
of hemin
indicate
formation
is
the while
the
reticulocyte
cubated
in protein
in
the
is
of
The inhibitory coupled
with
provides
test.
It
(3)
is
and
(2)
currently
being
the
the
inhibition
stimulation
papers.
If could
by globin, mechanism
the
the
raises
subunits
been
might
in-
of globin of
the
to experimental
speculative
its
system
be more
sensitive
and
(4)
own synthesis.
and considered
processes
formation
possibilities
by globin
described
to a dual
own
of heme on the
of
syn-
as a working
by heme of its
it
and fourth
contribute which
inhibition
or its
third
nuclei
formation
subjected
effect
have
independently
own synthesis
is presented
of heme synthesis
by globin two processes
the
stimulatory
In addition,
of other
a schema of regulation
the
increased
erythrocyte
of heme on its
schema
(1)
in
bulk
observed
avian
increasing
for
are
found
of regulation, than
the The
in
these
to be operative, by heme and
regulation
by either
alone.
This work of the Office
the
Preliminary
results
of the
of
hemin.
This
includes:
of globin.
grant
basis
which
synthesis
they
in
of hemoglobin.
hypothesis
first
effect
a partial
thesis
of
its
of globin.
have
in
effect
specificity
stimulated.
(1964)
added
the
of hemin
not
synthesis
presence
synthesis
synthesis
Hannnel and Bessman an increase
with
was supported by USPHS grant of Naval Research, Nonr-4094. 241
HE+02803
and a
Vol. 18, No. 2, 1965
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
REFERENCES
Hammel, C. and Bessman, S., Fed. Proc. 23, 317 (1964) Karibian, D. and London, 1-M. (acc-mpanying paper) H. and London, I.M., J. Biol. Chem. 22p, 423, Kassenaar, A., Morell, (1957) Kruh, J. and Borsook, H., J. Biol. Chem. 220, 905 (1956) Morell, H., Savoie, J.C. and London, I.M., J. Biol. Chem. 233, 923, (1958) Nizet, A., Bull. sot. chim. biol. 39, 265 (1957) Richmond, J.E., Altman, K.I. and Salomon, K., J. Biol. Chem. 190, 817 (1951)
242