Vol.3, No.3
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Sept. 1960
NUCLEOSIDE TRIPHOSPHATE
DEPENDENT PEPTIDE ACTIVATION BY
A CRUDE SOLUBLE PROTEIN FRACTION OF BAKER'S YEAST:
A NEW TYPE OF ACTIVATING
MECHANISM
A.H.W.hI.
van
Schuurs,
It
Hoff
S.R.
de Kloet
Laboratory,
Utrecht,
and V.V,
Koningsberger
State University
The Netherlands
of Utrecht
Received September 7, 1960
The occurrence
of carboxyl
of baker's
yeast
was reported
laboratory
about
three
b).
activated
by a group
years
1959)
1960)
bound
identified,
tide
material
(s-RNA)
yeast
1960
and particulate
a).
Moreover,
synthesizing
a number
isolated
protein
Hase
et al.,
During
a trial
1960)
by a crude
natant)
protein
formation
preparations
activated
peptides
(e.g.
both
Dirheimer
nucleoit
was
soluble
fractions
(de Kloet
nucleotide
various
and
without
from
of
et al.,
bound
pep-
tissues
and microorganisms
et al.,
1958;
Harris
et al.,
1959).
to study
fraction
of yeast
a new type
was found
peptide
to occur.
activation
soluble
of energy-dependent
In
300
this
paper,
a,
et al.,
isolated
(m-RNA)
of similar
from
been
1957
Furthermore,
ribonucleoprotein
carboxyl
has been
1959;
acid
this
Konings-
(Schuurs
peptides
1960 b).
from
1959;
have
bound
et al.,
ribonucleic
contain
tides
as phosphate
(de Kloet
shown that
peptides
extracts
et al.,
-monophosphate
activated
as well
of workers
(v.d.Grinten,
and cytidine-5'
carboxyl
in
ago (Koningsberger
Adenosine-5'-monophosphate
berger,
peptides
(Tuboi
(100.000
et al.,
g super-
hydroxamate
preliminary
data
Vol.3, No.3
BIOCHEMICAL
are presented
Sept. 1960
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
on the mechanism
involved
in
this
activating
obtained
commercial
baker's
Gist-
en Spiritusfabriek
reaction.
Materials
The yeast
was a freshly
("koningsgist",
Koninklijke
Delft).
protein
The crude
prepared
by repeated
100.000
g yeast
et al'(l956).
dialysed
overnight
containing
in
have
1957 b).
been
and Subbarow
against
Before
with
Norit
described
triphosphates
and/or
Hydroxamate
hydroxsmate
formation
is
at least
90 minutes
that
ATP,
to Chao
0.2
and 0.7
water
and
pH 7,
M buffer
the
g/ml)
dialysed
pro-
in
to re-
order
determine
hydroxamic
(Koningsberger
were products
et al.,
according
to Fiske
of Sumner
from
(1944).
Pabst.
For paper-
experiments
Whatman
the
catalysed
amount
by the
formed
without
the
is
feature
crude
of the
yeast
protein
proportional
addition
to time
of any substrate
for
but
1).
hydroxamate
UTP,
results
A remarkable
Mg++ and NH20H (Fig.
of either
use,
the modification
formation.
Furthermore,
a
was used.
fraction
ATP,
a 0.002
and paperelectrophoresis
1 filtersaper
in
was determined
Experimental
2.
between
(0.2
previously
phosphate
with
from
was
material.
used to locate
(1925)
chromatography
no.
cold
nucleotide
Inorganic
Nucleoside
the
experiments
according
was redissolved
was treated
The methods
our
prepared
collected
Mg++ per ml.
move contaminating
in
yeast
at
of the, protein
extract
saturation
2 ,umol
solution
used
precipitation
The precipitate
sulphate
acids
fraction
supernatant
ammonium
tein
and methods
formation
CTP or GTP (Table
301
requires
I).
the
presence
Vol.3, No.3
BIOCHEMICAL
p mol
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table
Hydroromotr/ml
0.8 -
IS
JO
45
60
75
time
obtained
(Pi)
of the
so far
is released
2.0
1.3
0.7
0.6
Hydroxamate
(Hx) formation
in the presence
of equal
(10 ,umol/ml>
concentrations
of different
nucleoside
triphosphates.
Experimental
conditions
were the same as
described
in Fig. 1.
hydroxamate
indicate
that
per pmol
formation.
one /urn01
hydroxamic
Analytical
of inorganic
acid
(Hx)
(Table
Table
II
This
'{I
fact
formed
II).
finding
that
protein
I
3.3
I
3.3
Release
at 250 C of inorganic
hosphate (Pi) and hydroxamate
(Hx P
formation
by a mixture
containing
20 mg protein,
10 ,umol
ATP,
10 )rnOl
Mgc12, 10 pmol
NaF and
1 mm01 NH20H. As a blank
for Pi
formation
the ssme mixture
was used
with the exception
of NH20H.
302
1
data
phosphate
seems
be confirmed
90
hr
(min)
,“~~~o;~;“,e,‘~r~;~~
Stoichiometry
Hx/ml/2
no
ATP
UTP
CTP
GTP
yeast protein
fraction.
The
reaction
mixture
contained
15 e protein,
10 ,umol
ATP,
1Op mol Ifig++, 100,umol
TRIS
buffer
pH 7 and 1 mm01 NH20H
per ml. Tyrosine
hydroxamate
was used as a standard
for
the determination
of
hydroxamate
(v.d. Ven, 1958).
2.
mol
90
hcubotton
.E&+
I
nucleoside
-A TP
1960
Sept.
by the
the
same
fraction
catalyses
the
exchange
of P32 labelled
organic
ATP,
to
phosphate
UTP,
inwith
CTP and GT?:
Vol. 3, No. 3
BIOCHEMICAL
this
could
gram
of
taining
be shown by radioautography
the
nucleotides
isolated
32 mg protein,
(20 /UC>
Na2HP3204
was incubated
(Fig.
7.5 ,umol
a total
of a paperelectrophero-
from
69 @
It
ml for
may be assumed
phosphates
action
+
30,umol
which
45 min.
at
25'
C
formed
we could
chromatography
the
products
in
the
separate
diwith
in yeast
of the
reaction
them
with
quite
well
separations,
were
showing
6 N HCl,
one half
of
re-chromatograyw
hydroxamates
products
septide
amino
have
a ferric
been
eluted
each eluate
being
of the
blanks
and spraying
with
formed
in
the
reaction
hydroxamates,
acids.
This
which
(Dixon
and
finding
suggests
303
9 distinct
colour.
and hydrolysed
kept
So
with
as a blank.
From
HCl-hydrolysed
ninhydrin,
each peptide
to stick
(4 : 1 : 5) as
hydroxamate
and the
with
reaction.
by repeated
2 chromatographic
spots
P32 by the
ATP tend
solvent.kfter
4 of these
and mono-
activation
acid-water
obtained,
amounts
phosphokinases,
n-butanol-acetic
spots
with
small
1958).
of the
The hydroxamates
together;
were labelled
are known to occur
Identification
that
nucleoside
of nucleoside
Webb,
ent
NaF,
triphosphate,
of 1.3
of contaminating
were
con-
Fig, 2 Radioautogram
showing the incorporation of P32 labelled
inorganic
phosphate
in
ATP, UTP, CTP and GTP. Experimental
conditions
are mentioned
in the text.
The circled
areas represent
U.V. quenching,
the shaded
areas radioactivity.
Electrophoresis
at
5 V.cm-1 for 16 hrs in 0.02 1 citrate
buffer,
pH 3.9.
u c 4__"_
--_.
far,
10 pmol
nucleoside
volume
mixture
2).
-
2.
a reaction
YgC12,
and 3 pmol
in
Sept. 1960
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
it
appeared
ATP as an energy
containing
that
the
that
source
7-9
activating
differ-
the
Vol.3,No.3
BIOCHEMICAL
reaction
proceeds
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
with the products
cess, as no peptide
dialysed
Sept. 1960
of some proteolytic
or amino acid substrate
pro-
was added to the
protein.
Discussion
Recently,
nucleotide-bound
as a product
occur
albumin by liverthis
finding
of peptide
coupling
synthetic
and brain
and with regard
nucleotide
mitochondria.
of serum
As a consequence of
to the rather
general
occurrence
compounds, Penn (1960) has suggested a
between certain
energy-dependent
breakdown and bio-
processes.
mechanism of the peptide
paper or about its
reaction
have been shown to
of an energy-dependentbreakdown
It is not yet possible
synthesis.
peptides
But it
to draw any conclusion
activating
possible
is hardly
role
reaction
(Hoagland,
an energy-dependent
speculative
to conclude
and -synthetic
1955) end that its
coupling
in this
bio-
that this
for amino acid
occurrence
between some protein
indicates
breakdown
pathways as suggested by Penn (1960).
There may also be a reiation
(1960 a, b) who synthesized
with the finding
peptides
the aid of the amino acid incorporation
of nucleoside
described
in the process of prote.in
is not the same as that described
activation
about the
of Beljanski
from amino acids with
enzyme in the presence
triphosphates.
References
Beljanski,
M. (1960 a) Biochim.Biophys.Acta
41, 111.
(1960 b) Faraday Society, Informal
discussion
on cytoplasmic particles
and their
role in protein
synthesis (Reading, March 28th and 29th).
Chao, F.-C. and Schachman, H.K. (1956) Arch.Biochem.Biophys.
& 220.
Compt.rend.
Dirheimer,
G., Weil, J.H. and Abel, J.P. (1958)
246,
3384.
Dixon, M. and */{ebb, E.C. (1958) &z
es (Longmans,
co. ) London, New York, Toronto 3".
304
Green
and
Vol.3, No.3
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Sept. 1960
Fiske,
C.H. and Subbarow,
Y. (1925) J.Biol.Chem.
66, 375.
Chr.0.
van
der
(1959)
Thesis,
Utrecht.
Grinten,
Harris,
G. and Davies,
J.W. (1959) Nature I&,
788.
Hase, E., Mihara,
S., Otsuka,
H. and Tamiya,
H. (1959) Arch.
Biochem.Biophys.
&,
170.
Iioagland,
M.B. (1955) Biochim.Biophys.Acta
l6, 288.
Kloet,
S.R. de, Schuurs
A. H.Z.M.,
Koningsberger,
V.V. and
Overbeek,
J.Th.G.
[1960 a) Koninkl.Ned.Akad.Wetenschap.
Proc.,
--3 63, 374.
Kloet,
S.R. de, Xeene, J.G.C.
van der
and Koningsberger,
V.V.
(1960 b) to be published.
Koningsberger,
V.V.,
Grinten,
Chr.O.van
der
and Overbeek,
J. Th.G. (1957 a) Koninkl.Ned.Akad.Wetenschap.Proc.
60 144.
-B -9
and
(1957 b)
Biochim.&ophys.Acta
26, 483.
Koningsberger,
V.V. (1959) lO.Coll.d.Gesellschaft
f.physiol.
Chemie,
Mosbach,
50 (Springer
Vex-lag, Berlin).
Penn, N.W. (1960) Biochim.Biophys.Acta
&
55.
Schuurs,
A.H.Yi.M.
and Koningsberger,
V.V. (1960) Biochim.
Biophys.Acta,
in press.
Sumner, J.B.
(1944) Science 100, 413.
Tuboi,
S. and Huzino,
A. (1960) Arch.Biochem.Biophys.
86, 309.
Ven, A.W.van de, Koningsberger,
V.V. and Overbeek,
J.Th.G.
(1958) Biochim.Biophys.Acta
28, 134.
305