BIOCHEMICAL
Vol. 175, No. 2, 1991
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages
500-506
PROTAMINE KINASE
FROM
March 15, 1991
CHARACTERIZATION
OF A CAMP-INDEPENDENT
Ca'+-INHIBITED
CANDIDA LIPOLYTICA* M. Rahmatullah',
Received
D.L.
Brenner',
M.W. Wooten',
Departments
ofiBotany
Wildlife,
Alabama
Agricultural
Auburn
University,
February
4,
and Microbiology
and
J.D.
Weetel,+
and 2Zoology
Experiment
and
Station,
AL 36849
1991
A CAMP-independent protamine kinase has been purified from extracts of the yeast Candida lipolvtica by ion-exchange and affinity chromatography. Two subunits with apparent Ml's of 52,000 and 36,000 were resolved by SDS-PAGE. The purified kinase exhibited about 20% activity with casein and histone Type VII-S as substrates relative to protamine. The enzyme was inactive against other protein substrates tested, and was essentially insensitive to AMP, CAMP, cGMP up to 0.2 mM, the polyamines spermine and spermidine up to 1 mM, N-ethylmaleimide (5 mM), 2-mercaptoethanol (20 mM), or dithiothreitol (2 mM), and several cations like Zn2+, N1+, or Co2+ at 0.1 mM each. Ca2+ at 3 mM inhibited protamine kinase activity by 50%, which was reversed by EGTA. 0 1991 Academic Press, Inc.
Phosphorylation-dephosphorylation covalent
modification
transduction of
properties
is and
limited.
For
filamentous
available
specific
fungi
CAMP-independent
and yeast
In fungi, morphogenesis,
kinases
i.e.
dimorphism
emersonii was
have
discoedeum
studied species
CAMP-dependent part
should
0 1991 by Academic Press, Inc. of reproduction in any form reserved.
occuring
function,
signal
Although
awealth
knowledge
of
lower
is (6-8),
are mostly
of the However,
substrates.
as a substrate
the
eukaryotes
cerevisiae
(11,12)
casein
a widely
from
Candida
described. in connection (14)
and zoospore kinases
be addressed.
500
with
protein
by Sandoz No. 00773.
DDD6-291X/91 $1.50 Copyright All rights
from
preferring been
our
Saccharomvces as preferred
Switzerland) and AAES project No. 6-90258OP. 'To whom correspondence
(S),
kinases
from
in Mucor
supportedin
(l-4).
or histone
have been
(15).
protein
systems
of protein kinases
as
organisms
mammalian
kinases (7)
protein
Blastocladiella work
for
casein
protein
(13)
regulating
and Dictvostelium
with
recognized
in eukaryotic
protein
(9,lO) type
for
functions
example,
CAMP-dependent
*This
mechanism
and gene expression
information
albicans
is
CAMP-mediated production
in
and the ras
gene
Ltd. (Easel, AAES publication
Vol.
BIOCHEMICAL
175, No. 2, 1991
product
have been also
kinases
have
involved
in
been the
identified cell
a tyrosine
cycle
regulation
auxotrophic
for
In this kinase [Yarrowia] inhibited
from
S,
sterol
is
activated
CDC28 and More
with
yeast
preferentially
(16).
cdc2+
Saccharomvces pp60v-Jrc
genes it
may play
enzyme
Other
cerevisiae
recently,
a Mg2+-dependent,
ascomycetous
RESEARCH COMMUNICATIONS
of yeasts
This (7). by ergosterol
we characterized that
of (18).
homology
cereviseae
dimorphic,
linolytica)
control of
respectively exhibiting
of
study, the
regulation
m, kinase
in growth as products
cycle
Schizosaccharomvces that
implicated
AND BIOPHYSICAL
from
protein
which
are
(17)
has been
and shown
a role
in cell
a yeast
mutant
(19). CAMP-independent
C_ liuolvtica phosphorylates
protein
(Saccharomvcooosis protamine
and
is
by Cazf. MATERIALS
AND METHODS
Materials: Protamine sulfate (from salmon andherring), dephosphorylated casein, histones (II-A, V-S andVII-S), ovalbumin, bovine serum albumin (BSA), phosvitin, leupeptin, aprotinin, phenylmethylsulfonyl fluoride (PMSF), benzamidine, DEAESephacel, protamine agarose, N-ethyl maleimide, spermine, spermidine, 2mercaptoethanol, Nonidet P-40 (NP-40) and molecular weight standards for polyacrylamide gel electrophoresis were from Sigma Chemical Co., St. Louis, MO. [x3'P]-ATP was purchased from ICN Biomedicals, Inc., Costa Mesa, CA. Reagents for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and dithiothreitolwere from Boehringer Mannheim, Indianapolis, IN. All reagents were of analytical grade and highest purity available. Culture conditions: C_ lioolvtica (NRRLY 1094), obtained from Professor Morris Kates (Department of Biochemistry, University of Ottawa) was grown at 25OC on a rotary shaker in 1% Bacto Yeast Extract (Difco) and 2% glucose. For experiments, 200 ml of the above medium were typically inoculated from an overnight stock culture (10 ml), and harvested during exponential growth at 4.5h following inoculation. Cells from twenty cultures were collected by centrifugation (3000 rpm), pooled, and used for enzyme purification. Preparation of crude extract. Cells (typically 5-6g wet weight) were harvested by centrifugation at 3,000 g for 10 min, washed twice with phosphate buffered saline at pH 7.4 and resuspended in 50 mM imidazole-Cl(pH 7.5) containing O.lmM each of EDTA, NaF, aprotinin, and 20 mM 2leupeptin, PMSF, benzamidine mercaptoethanol. The cells were disrupted in a Bead-Beater (Biospec Products, Bartlesville, OK) in the presence of glass beads (0.45-0.50 mm). The beads were removed by centrifugation at 500 g for 15 min, and the supernatant containing soluble proteins and membranes was treated with 0.1% NP-40 and 10 mM EGTA for lh with gentle inversion. The extract was then cleared by centrifugation at 12,000 g for 15 min. All procedures were performed at 4OC. DEAE-Sephacel fractionation. The crude extract was applied to a DEAE-Sephacel column (1.2 cm X 15 cm) previously equilibrated with 50 mM imidazole-Cl, pH 7.5 containing 2 mM each of EDTA and EGTA, 20 mM 2-mercaptoethanol and 0.1 mM each of PMSF, aprotinin, benzamidine and leupeptin (Buffer A). The column was washed extensively with Buffer A followed by successive washings with one column volume each of Buffer A containing 0.05, 0.1, 0.2 and 0.3M NaCl. Protamine kinase was next eluted with Buffer A + 0.4 M NaCl. Fractions of 5 ml were collected during the last elution step. The fractions containing enzyme activity were pooled and diluted (1:l) with Buffer A. Protamine agarose chromatographY. The kinase-containing fraction from the DEAESephacel column was loaded onto a protamine agarose column (0.67 cm x 7 cm) preequilibrated in Buffer A containing 0.2M NaCl (Buffer B). This column was washed successively (two column volumes each) with increasing concentrations of NaCl (0.3, 0.4 and 0.6M, respectively) in Buffer A. Protamine kinase was then eluted from the column with Buffer A + 0.8M NaCl. Fractions of 1 ml were collected during the last step. The active fractions were pooled, dialysed overnight (10h) 501
Vol.
BIOCHEMICAL
175, No. 2, 1991
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
with Buffer A and concentrated on a mini DEAE-Sephacel column (0.9 cm X 1.5 cm) previously equilibrated in Buffer A. The eluant (in Buffer A + 0.4M NaCl) was stored at 4OC. At this stage, the enzyme retained activity for at least 15 days if not subjected to a freeze-thaw cycle. For kinase activity studies, the enzyme was further dialysed for a 16h period in 50 mM imidazole-Cl, pH 7.5 (with two changes of buffer). Protamine kinase assay. Protamine kinase was assayed by a modified procedure of Fontana et al. (20). To tubes temperature equilibrated at 26'C was added 40 mM PIPES (pH 6.5), 10 mM MgCl,, 0.05 mg protamine sulfate, kinase samples and effecters (see Tables) in a final volume of 100 ml. Following incubation for 6Os, the kinase reaction was initiated by adding [$-32P]ATP (1200 cpm/pmol) to a final concentration of 20 pM. Samples (40 1) were withdrawn at 60s and 12Os, and the reaction terminated by applying them to dry Whatman 3MM paper discs (2.5 cm diameter) previously soaked in 12.5% (w/v) trichloroacetic acid (TCA). The discs were washed three times for 30 min each and then washed for 14h with 12.5% TCA. Protein bound radioactivity was determined as described previously (21,22). One unit of protamine kinase activity is defined as the amount of enzyme that incorporated 1 pmol of 32P into protamine/min. Miscellaneous methods: Protein was determined by the Lowry procedure. SDS-PAGE was performed in 10% slab gels (23); proteins were detected with Coomassie Blue. RESULTS Protein
kinases
kinase
in C. liuolvtica.
activity
order
to
yeast,
identify
the
using
in
crude
unfractionated
crude
and characterize
the
protein
a O-1M NaCl
kinase
activity
unbound
the
column
proteins,
0.4 M NaCl.
preparation
gradient. was
and then
To further containing
protamine
A protein
by elution
(1.2M)
a minor
protamine, 0.8M
it
Table
1.
2. 3. 4.
protamine
detected
had a very
low specific and further
Purification
g (wet
weight)
activity.
kinase
Volume (ml) 60.0
Protein (w)
cells
Candida
for
column. which
was
of NaCl
kinase
linolvticaa activity protein) -976 2569 2748
protein
eluting
DEAE-Sephacel
Specific (units/mg 4.5 0.8 0.5
used
0.3MNaC1,
column
thatphosphorylated
140.0
were
agarose
to the
CAMP-
above
on a smaller from
for
concentrations
The protamine
of protamine
from
the
with
remove 0.05
eluting
from
eluted
concentrated
A to
assayed
a protamine
preparations
this
- 0.4M NaCl.
were
on
At higher
some
20.0 3.0 0.5
of
fractions
activity in
peak
The fractions
at 0.8M NaCl.
0.35
A containing
protein
placed
in
of protamine
Buffer
of Buffer
pooled
peak
about with
In
on DEAE-Sephacel
a large
a major
were
kinase
of the kinase
Total Extract DEAE-Sephacel(1) Protamine agarose DEAE-Sephacel( 2)
a 5.2
gradient
the
species
fractionated with
protein
C_ lioolvtica.
kinase
extensively
followed
activity
Step 1.
a step
kinase,
proteinpeakwas
but
washed
significant
from
protein
eluting
(data not shown). kinase activities.
kinase
NaCl was dialysed
major
in the fraction first
the
peak without
followed
at
purify
preparations
trials,
kinase
column at about 0.2M NaCl dependent and independent
to detect
initial
with
The protamine
unable
was initially
In our
was detected
Thereafter,
We were
extraction.
Vol.
175,
No.
BIOCHEMICAL
2, 1991
Table
2.
Substrate
AND
BIOPHYSICAL
RESEARCH
specificity of protamine Candida lbolvtica
kinase
Enzyme actiyjty ( mol of P transferred/assay)
Substratea
COMMUNICATIONS
from
Relativeb activity
Protamine sulfate (Salmon) 4.72 100.0 Protamine sulfate (Herring) 4.58 97.0 Casein 0.93 19.7 Bovine serum albumin NA= NA Ovalbumin 0.39 8.3 Histone Type II-A NA NA Histone Type V-S 0.18 3.8 Histone Type VII-S 1.12 23.7 Phosvitin NA NA a The final concentration of the proteins tested was 0.5 mg/ml. Reactions were performed as described under the Materials and Methods section. Activity of protamine kinase with casein was tested with Tris-Cl buffer, pH 7.5. b Activity is expressed as a percent of the activity observed with protamine sulfate from salmon. 'NA=no activity. column. fold
The specific
by the purification The
kinase
substrate with
BSA,
of the protamine
steps
exhibited
(Table
20% of that
2).
for
ovalbumin,
Molecular
weight kinase
two bands
with
outlined
and
subunit
In some preparations, peptide.
Catalvtic
properties
but
of
required
Mg2+ to
activity
was reduced
the
this
phosphorylate
Co2+ and Zn2+ at 0.1
kinase
(data
about
19%;
not
half-maximal
shown).
At
inhibition
concentrations
with
stimulated
kinase
by about
of exogenous
activity
Ca2+ (Table
CAMP or cGMP (up to 0.2 dithiothreitol
(2 mM) or
inhibition.
Protamine
spermidine
(1 mM each;
observed
3).
Activity
and unchanged
substituted
503
also
for
of NaCl
kinase
Mg'+, (up to
activity
3 mM CaC12.
kinase
insensitive
on by
EGTA at
Ca2+ inhibition
by 2-mercaptoethanol was
shown).
of
of Mg2+ had no effect
(5 mM). AMP at 0.2
activity not
of apparent
C_ linolvtica
at 3 mM level
of the protamine
N-ethylmaleimide
kinase
with
showed
proteolysis
Addition
abolished
50% when present
d-f),
data
presence
Ca2+ completely
band
from
1 mM, Ca2' inhibited was
equimolar
kinase
Mg".
purified
on SDS-PAGE (Fig
a third
The with
mM in the
the
enzyme
due to partial
When Mn2+ was
obtained
the about
activity
of
respectively,
been
kinase.
protamine.
to 25% of that
as
was only
The purified
on storage,
protamine
200 m&i) or Ni2+, activity
homogeneity
and 36,000, may have
3000-
and V-S.
The
and occasionally
protamine VII-S
or no phosphorylation
by electrophoresis.
of 52,000
M, of &O,OOO was observed,
toward
and histone
II-A
composition.
almost
1.
was little
or histones
apparentM,'s
the upper
of casein
and there
was determined
was increased
activity
preferential
phosvitin
kinase
in Table
Phosphorylation
protamine,
protamine 1).
activity
and
in the absence
was unaffected
by
(up to 20 mM), mM caused to
spermine
a 15% or
Vol.
175,
No.
BIOCHEMICAL
2, 1991
A Figure
1.
SDS-polyacrylamide livolvtica. from DEAE-Sephacel
BIOPHYSICAL
C
D
RESEARCH
COMMUNICATIONS
gel electrophoresis of purified protamine left to right: A) 18 g crude extract, column, C) 6 g fraction from protamine and D) 2 g each of molecular weight standards (1) -galactosidase bovine serum albumin (69 kD), (3) ovalbumin (45 kD), (4) -1actalbumin (14.2 kD). (29 kD), and (5)
from Candida fraction columns, kD), (2) anhydrase
B
AND
From
kinase 3) 6 g agarose (116 carbonic
DISCUSSION The distinct
protein
from
protamine is
over
from
other
CAMP-independent.
kinase
from two
from other
proteins It
C_ albicans
SDS-PAGE (13). into
kinase
those
is
C_ linolvtica or slime
(casein,
histones,
also
that
different
was resolved
In contrast,
the protamine
non-identical
characterized
fungal
subunits
in
sources
phosvitin) from
into kinase
during
mold the
three
in
this that
study it
as a substrate,
CAMP-independent non-identical
fromg
and protein
subunits
liuolvticawas
electrophoresis
is
prefers
by
resolved
under
denaturing
conditions.
Protaminekinases and Escherichiad
have beendescribed These
(28).
Table 3. protamine
fromvarious
kinases
Effects kinase
of
exhibit
Relative
EGTA,
3 mM + CaCl,,
a
Materials
See
of Activity (8) 100.0 81.6 153.5 43.8
1 mM 3 mM mM
mixture.
tissues
as well
CaCl on the activity from &ndida livolvticaa
Additive None CaC12, CaCl EGTAf'3
mammalian
overlapping
and
3 mM Methods
91.4 for
504
composition
of
the
assay
(24-27)
as distinct
Vol.
175,
No.
2, 1991
properties. cells
(25)
differ
and the cytosol
of bovine
of 45,000
shares
described
Generally,
enzyme Our protamine
testis
protamine
ionic
several.
For
protamine
kinase
the
of
preliminary
may play
two
a catalytic
leukemia
kinase
cation
a role
in
of
regulatory
composed
studies described
about
subunits and
cells
for
(data here
the
is
the
example,
kinase
both
of
the C_ lipolvtica by Ca2+ but
C_ lipolvtica
protamine
composition,
not
indicate increasing
of cell
growth.
C_
described,
and the latter
with
to
ion exchange
previously
inhibited
polypeptide
regulation
from
50% inhibition
shown)
but
and response
protamine
of a single
higher
CAMP-independent
eluted
kinases were
leukemia
On the other hand, a by CAMP, and had an M,
were The
cell
3mM of the
are
respectively),
kinases
strengths,
with
and leukemia
(27)
COMMUNICATIONS
promyelocytic
was activated
characteristics
Also,
from
cells
vs 43,000,
to any of the protamine
(26).
suggestive
fromboth
is not identical
required
0.5mM
high
RESEARCH
respectively).
trout these
relatively
here
former
kidney
(110,000
rainbow
BIOPHYSICAL
kinases
non-inhibitory,
from
(24). at
lipolvtica
weight vs
kinase
columns
kinase
the protamine
(inhibitory
protamine
AND
For example, in molecular
Ca"
but
BIOCHEMICAL
unlike
the
the only
kinase
is
110,000
M,
(25). that
the
growth.
activity If
so,
of this
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
Hunter, T. (1987) Cell, 50, 823-29. Blackshear, P.J., Nairn, A.C. and Kuo, J.F. (1988) FASEB J. 2, 2957-69. Taylor, S.S., Buechler, J.A. and Yonemoto, W. (1990) Ann. Rev. Biochem. 59, 971-1005. Nishizuka, Y. (1988) Nature (London), 334, 661-65. Edelman, A.M., Blumenthal, D.K. and Krebs, E.G. (1987) Ann. Rev. Biochem. 56, 567-613. Biochemistry 26:4207-4212.14. Sternbach, H. and Kuntzel, H. (1987). Dahl, C., Biemann, H-P. and Dahl, J. (1987) Proc. Natl. Acad. Sci. USA, 84, 4012-16.14. J. Biol. Chem. Kolarov, J., Kulpa, J., Baijot, M. Goffeau, A. (1988). Biophys. 199:321-330. S. and Passeron, S. (1980). Arch. Biochem. Moreno, 263:10613-10619.14, Judewicz, R.D., Glikein, G.C., and Torres, H.N. (1981) Arch. Biochem. Biophys. 206:87-92. Jimenez, B., Pestana, A. and Fernandez-Renart, M. (1989) Biochem. J. 260:557-561. Ospina, B. and Fernandez-Renart, M. (1990) Biochem. Biophys. Act. 1052:483488. Gupta, B.R. and Datta, A. (1986) Biochem. J. 234, 543-6. P.R. and Rogers, P.J. (1983) In: Fungal Differentiation (Smith, Steward, J.E., ed.), Marcel Dekker, Inc., New York and Basel, pp 267-313. Silverman, P.M. (1978) J. Bacterial. 135, 976-80. Matsumoto, K., Uno, I., Oshima, Y. and Ishikawa, T. (1985) Yeast, 1, 15-24. Reed, S.I., Hadwiger, J.A. and Lorincz, A.T. (1985) Proc. Natl. Acad. Sci. USA, 82, 4055-59. Simanis, V. and Nurse, P. (1986) Cell, 45, 261-8. Dahl, J.S. and Dahl, C.E. (1985) Biochem. Biophys. Res. Commun., 133, 84450. Fontana, J.A., Emler, D., Ku, K., McClung, J.K., Butcher, F.R. and Durham, J.P. (1984) J. Cell. Phys. 120, 49-60. Rahmatullah, M. and Roche, T.E. (1985) J. Biol. Ghem. 260, 10146-52. 505
Vol.
22. 23. 24. 25. 26. 27. 28.
175, No. 2, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Hucho, F., Randall, D.D., Roche, T.E., Burgett, M.W., Pelley, J.W. andReed, L.J. (1972) Arch. Biochem. Biophys. 151, 328-40. Laemmli, U.K. (1970) Nature (London), 227, 680-5. Jergil, B. and Dixon, G.H. (1970) J. Biol. Chem., 245, 425-34. Durham, J.P., Butcher, F.R. and Fontana, J.A. (1982) Biochem. Biophys. Res. Commun. 108, 840-5. Damuni, 2. and Reed, L.J. (1988) Arch. Biochem. Biophys. 262, 574-84. Damuni, Z., Amick, G.D. and Sneed, T.R. (1989) J. Biol. Chem. 264, 6412-6. Kuo, J.F. and Greengard, P. (1970). J. Biol. Chem. 245~4067-4073.
506
Vol. 175, No. 2, 1991
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages
500-506
PROTAMINE KINASE
FROM
March 15, 1991
CHARACTERIZATION
OF A CAMP-INDEPENDENT
Ca'+-INHIBITED
CANDIDA LIPOLYTICA* M. Rahmatullah',
Received
D.L.
Brenner',
M.W. Wooten',
Departments
ofiBotany
Wildlife,
Alabama
Agricultural
Auburn
University,
February
4,
and Microbiology
and
J.D.
Weetel,+
and 2Zoology
Experiment
and
Station,
AL 36849
1991
A CAMP-independent protamine kinase has been purified from extracts of the yeast Candida lipolvtica by ion-exchange and affinity chromatography. Two subunits with apparent Ml's of 52,000 and 36,000 were resolved by SDS-PAGE. The purified kinase exhibited about 20% activity with casein and histone Type VII-S as substrates relative to protamine. The enzyme was inactive against other protein substrates tested, and was essentially insensitive to AMP, CAMP, cGMP up to 0.2 mM, the polyamines spermine and spermidine up to 1 mM, N-ethylmaleimide (5 mM), 2-mercaptoethanol (20 mM), or dithiothreitol (2 mM), and several cations like Zn2+, N1+, or Co2+ at 0.1 mM each. Ca2+ at 3 mM inhibited protamine kinase activity by 50%, which was reversed by EGTA. 0 1991 Academic Press, Inc.
Phosphorylation-dephosphorylation covalent
modification
transduction of
properties
is and
limited.
For
filamentous
available
specific
fungi
CAMP-independent
and yeast
In fungi, morphogenesis,
kinases
i.e.
dimorphism
emersonii was
have
discoedeum
studied species
CAMP-dependent part
should
0 1991 by Academic Press, Inc. of reproduction in any form reserved.
occuring
function,
signal
Although
awealth
knowledge
of
lower
is (6-8),
are mostly
of the However,
substrates.
as a substrate
the
eukaryotes
cerevisiae
(11,12)
casein
a widely
from
Candida
described. in connection (14)
and zoospore kinases
be addressed.
500
with
protein
by Sandoz No. 00773.
DDD6-291X/91 $1.50 Copyright All rights
from
preferring been
our
Saccharomvces as preferred
Switzerland) and AAES project No. 6-90258OP. 'To whom correspondence
(S),
kinases
from
in Mucor
supportedin
(l-4).
or histone
have been
(15).
protein
systems
of protein kinases
as
organisms
mammalian
kinases (7)
protein
Blastocladiella work
for
casein
protein
(13)
regulating
and Dictvostelium
with
recognized
in eukaryotic
protein
(9,lO) type
for
functions
example,
CAMP-dependent
*This
mechanism
and gene expression
information
albicans
is
CAMP-mediated production
in
and the ras
gene
Ltd. (Easel, AAES publication
Vol.
BIOCHEMICAL
175, No. 2, 1991
product
have been also
kinases
have
involved
in
been the
identified cell
a tyrosine
cycle
regulation
auxotrophic
for
In this kinase [Yarrowia] inhibited
from
S,
sterol
is
activated
CDC28 and More
with
yeast
preferentially
(16).
cdc2+
Saccharomvces pp60v-Jrc
genes it
may play
enzyme
Other
cerevisiae
recently,
a Mg2+-dependent,
ascomycetous
RESEARCH COMMUNICATIONS
of yeasts
This (7). by ergosterol
we characterized that
of (18).
homology
cereviseae
dimorphic,
linolytica)
control of
respectively exhibiting
of
study, the
regulation
m, kinase
in growth as products
cycle
Schizosaccharomvces that
implicated
AND BIOPHYSICAL
from
protein
which
are
(17)
has been
and shown
a role
in cell
a yeast
mutant
(19). CAMP-independent
C_ liuolvtica phosphorylates
protein
(Saccharomvcooosis protamine
and
is
by Cazf. MATERIALS
AND METHODS
Materials: Protamine sulfate (from salmon andherring), dephosphorylated casein, histones (II-A, V-S andVII-S), ovalbumin, bovine serum albumin (BSA), phosvitin, leupeptin, aprotinin, phenylmethylsulfonyl fluoride (PMSF), benzamidine, DEAESephacel, protamine agarose, N-ethyl maleimide, spermine, spermidine, 2mercaptoethanol, Nonidet P-40 (NP-40) and molecular weight standards for polyacrylamide gel electrophoresis were from Sigma Chemical Co., St. Louis, MO. [x3'P]-ATP was purchased from ICN Biomedicals, Inc., Costa Mesa, CA. Reagents for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and dithiothreitolwere from Boehringer Mannheim, Indianapolis, IN. All reagents were of analytical grade and highest purity available. Culture conditions: C_ lioolvtica (NRRLY 1094), obtained from Professor Morris Kates (Department of Biochemistry, University of Ottawa) was grown at 25OC on a rotary shaker in 1% Bacto Yeast Extract (Difco) and 2% glucose. For experiments, 200 ml of the above medium were typically inoculated from an overnight stock culture (10 ml), and harvested during exponential growth at 4.5h following inoculation. Cells from twenty cultures were collected by centrifugation (3000 rpm), pooled, and used for enzyme purification. Preparation of crude extract. Cells (typically 5-6g wet weight) were harvested by centrifugation at 3,000 g for 10 min, washed twice with phosphate buffered saline at pH 7.4 and resuspended in 50 mM imidazole-Cl(pH 7.5) containing O.lmM each of EDTA, NaF, aprotinin, and 20 mM 2leupeptin, PMSF, benzamidine mercaptoethanol. The cells were disrupted in a Bead-Beater (Biospec Products, Bartlesville, OK) in the presence of glass beads (0.45-0.50 mm). The beads were removed by centrifugation at 500 g for 15 min, and the supernatant containing soluble proteins and membranes was treated with 0.1% NP-40 and 10 mM EGTA for lh with gentle inversion. The extract was then cleared by centrifugation at 12,000 g for 15 min. All procedures were performed at 4OC. DEAE-Sephacel fractionation. The crude extract was applied to a DEAE-Sephacel column (1.2 cm X 15 cm) previously equilibrated with 50 mM imidazole-Cl, pH 7.5 containing 2 mM each of EDTA and EGTA, 20 mM 2-mercaptoethanol and 0.1 mM each of PMSF, aprotinin, benzamidine and leupeptin (Buffer A). The column was washed extensively with Buffer A followed by successive washings with one column volume each of Buffer A containing 0.05, 0.1, 0.2 and 0.3M NaCl. Protamine kinase was next eluted with Buffer A + 0.4 M NaCl. Fractions of 5 ml were collected during the last elution step. The fractions containing enzyme activity were pooled and diluted (1:l) with Buffer A. Protamine agarose chromatographY. The kinase-containing fraction from the DEAESephacel column was loaded onto a protamine agarose column (0.67 cm x 7 cm) preequilibrated in Buffer A containing 0.2M NaCl (Buffer B). This column was washed successively (two column volumes each) with increasing concentrations of NaCl (0.3, 0.4 and 0.6M, respectively) in Buffer A. Protamine kinase was then eluted from the column with Buffer A + 0.8M NaCl. Fractions of 1 ml were collected during the last step. The active fractions were pooled, dialysed overnight (10h) 501
Vol.
BIOCHEMICAL
175, No. 2, 1991
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
with Buffer A and concentrated on a mini DEAE-Sephacel column (0.9 cm X 1.5 cm) previously equilibrated in Buffer A. The eluant (in Buffer A + 0.4M NaCl) was stored at 4OC. At this stage, the enzyme retained activity for at least 15 days if not subjected to a freeze-thaw cycle. For kinase activity studies, the enzyme was further dialysed for a 16h period in 50 mM imidazole-Cl, pH 7.5 (with two changes of buffer). Protamine kinase assay. Protamine kinase was assayed by a modified procedure of Fontana et al. (20). To tubes temperature equilibrated at 26'C was added 40 mM PIPES (pH 6.5), 10 mM MgCl,, 0.05 mg protamine sulfate, kinase samples and effecters (see Tables) in a final volume of 100 ml. Following incubation for 6Os, the kinase reaction was initiated by adding [$-32P]ATP (1200 cpm/pmol) to a final concentration of 20 pM. Samples (40 1) were withdrawn at 60s and 12Os, and the reaction terminated by applying them to dry Whatman 3MM paper discs (2.5 cm diameter) previously soaked in 12.5% (w/v) trichloroacetic acid (TCA). The discs were washed three times for 30 min each and then washed for 14h with 12.5% TCA. Protein bound radioactivity was determined as described previously (21,22). One unit of protamine kinase activity is defined as the amount of enzyme that incorporated 1 pmol of 32P into protamine/min. Miscellaneous methods: Protein was determined by the Lowry procedure. SDS-PAGE was performed in 10% slab gels (23); proteins were detected with Coomassie Blue. RESULTS Protein
kinases
kinase
in C. liuolvtica.
activity
order
to
yeast,
identify
the
using
in
crude
unfractionated
crude
and characterize
the
protein
a O-1M NaCl
kinase
activity
unbound
the
column
proteins,
0.4 M NaCl.
preparation
gradient. was
and then
To further containing
protamine
A protein
by elution
(1.2M)
a minor
protamine, 0.8M
it
Table
1.
2. 3. 4.
protamine
detected
had a very
low specific and further
Purification
g (wet
weight)
activity.
kinase
Volume (ml) 60.0
Protein (w)
cells
Candida
for
column. which
was
of NaCl
kinase
linolvticaa activity protein) -976 2569 2748
protein
eluting
DEAE-Sephacel
Specific (units/mg 4.5 0.8 0.5
used
0.3MNaC1,
column
thatphosphorylated
140.0
were
agarose
to the
CAMP-
above
on a smaller from
for
concentrations
The protamine
of protamine
from
the
with
remove 0.05
eluting
from
eluted
concentrated
A to
assayed
a protamine
preparations
this
- 0.4M NaCl.
were
on
At higher
some
20.0 3.0 0.5
of
fractions
activity in
peak
The fractions
at 0.8M NaCl.
0.35
A containing
protein
placed
in
of protamine
Buffer
of Buffer
pooled
peak
about with
In
on DEAE-Sephacel
a large
a major
were
kinase
of the kinase
Total Extract DEAE-Sephacel(1) Protamine agarose DEAE-Sephacel( 2)
a 5.2
gradient
the
species
fractionated with
protein
C_ lioolvtica.
kinase
extensively
followed
activity
Step 1.
a step
kinase,
proteinpeakwas
but
washed
significant
from
protein
eluting
(data not shown). kinase activities.
kinase
NaCl was dialysed
major
in the fraction first
the
peak without
followed
at
purify
preparations
trials,
kinase
column at about 0.2M NaCl dependent and independent
to detect
initial
with
The protamine
unable
was initially
In our
was detected
Thereafter,
We were
extraction.
Vol.
175,
No.
BIOCHEMICAL
2, 1991
Table
2.
Substrate
AND
BIOPHYSICAL
RESEARCH
specificity of protamine Candida lbolvtica
kinase
Enzyme actiyjty ( mol of P transferred/assay)
Substratea
COMMUNICATIONS
from
Relativeb activity
Protamine sulfate (Salmon) 4.72 100.0 Protamine sulfate (Herring) 4.58 97.0 Casein 0.93 19.7 Bovine serum albumin NA= NA Ovalbumin 0.39 8.3 Histone Type II-A NA NA Histone Type V-S 0.18 3.8 Histone Type VII-S 1.12 23.7 Phosvitin NA NA a The final concentration of the proteins tested was 0.5 mg/ml. Reactions were performed as described under the Materials and Methods section. Activity of protamine kinase with casein was tested with Tris-Cl buffer, pH 7.5. b Activity is expressed as a percent of the activity observed with protamine sulfate from salmon. 'NA=no activity. column. fold
The specific
by the purification The
kinase
substrate with
BSA,
of the protamine
steps
exhibited
(Table
20% of that
2).
for
ovalbumin,
Molecular
weight kinase
two bands
with
outlined
and
subunit
In some preparations, peptide.
Catalvtic
properties
but
of
required
Mg2+ to
activity
was reduced
the
this
phosphorylate
Co2+ and Zn2+ at 0.1
kinase
(data
about
19%;
not
half-maximal
shown).
At
inhibition
concentrations
with
stimulated
kinase
by about
of exogenous
activity
Ca2+ (Table
CAMP or cGMP (up to 0.2 dithiothreitol
(2 mM) or
inhibition.
Protamine
spermidine
(1 mM each;
observed
3).
Activity
and unchanged
substituted
503
also
for
of NaCl
kinase
Mg'+, (up to
activity
3 mM CaC12.
kinase
insensitive
on by
EGTA at
Ca2+ inhibition
by 2-mercaptoethanol was
shown).
of
of Mg2+ had no effect
(5 mM). AMP at 0.2
activity not
of apparent
C_ linolvtica
at 3 mM level
of the protamine
N-ethylmaleimide
kinase
with
showed
proteolysis
Addition
abolished
50% when present
d-f),
data
presence
Ca2+ completely
band
from
1 mM, Ca2' inhibited was
equimolar
kinase
Mg".
purified
on SDS-PAGE (Fig
a third
The with
mM in the
the
enzyme
due to partial
When Mn2+ was
obtained
the about
activity
of
respectively,
been
kinase.
protamine.
to 25% of that
as
was only
The purified
on storage,
protamine
200 m&i) or Ni2+, activity
homogeneity
and 36,000, may have
3000-
and V-S.
The
and occasionally
protamine VII-S
or no phosphorylation
by electrophoresis.
of 52,000
M, of &O,OOO was observed,
toward
and histone
II-A
composition.
almost
1.
was little
or histones
apparentM,'s
the upper
of casein
and there
was determined
was increased
activity
preferential
phosvitin
kinase
in Table
Phosphorylation
protamine,
protamine 1).
activity
and
in the absence
was unaffected
by
(up to 20 mM), mM caused to
spermine
a 15% or
Vol.
175,
No.
BIOCHEMICAL
2, 1991
A Figure
1.
SDS-polyacrylamide livolvtica. from DEAE-Sephacel
BIOPHYSICAL
C
D
RESEARCH
COMMUNICATIONS
gel electrophoresis of purified protamine left to right: A) 18 g crude extract, column, C) 6 g fraction from protamine and D) 2 g each of molecular weight standards (1) -galactosidase bovine serum albumin (69 kD), (3) ovalbumin (45 kD), (4) -1actalbumin (14.2 kD). (29 kD), and (5)
from Candida fraction columns, kD), (2) anhydrase
B
AND
From
kinase 3) 6 g agarose (116 carbonic
DISCUSSION The distinct
protein
from
protamine is
over
from
other
CAMP-independent.
kinase
from two
from other
proteins It
C_ albicans
SDS-PAGE (13). into
kinase
those
is
C_ linolvtica or slime
(casein,
histones,
also
that
different
was resolved
In contrast,
the protamine
non-identical
characterized
fungal
subunits
in
sources
phosvitin) from
into kinase
during
mold the
three
in
this that
study it
as a substrate,
CAMP-independent non-identical
fromg
and protein
subunits
liuolvticawas
electrophoresis
is
prefers
by
resolved
under
denaturing
conditions.
Protaminekinases and Escherichiad
have beendescribed These
(28).
Table 3. protamine
fromvarious
kinases
Effects kinase
of
exhibit
Relative
EGTA,
3 mM + CaCl,,
a
Materials
See
of Activity (8) 100.0 81.6 153.5 43.8
1 mM 3 mM mM
mixture.
tissues
as well
CaCl on the activity from &ndida livolvticaa
Additive None CaC12, CaCl EGTAf'3
mammalian
overlapping
and
3 mM Methods
91.4 for
504
composition
of
the
assay
(24-27)
as distinct
Vol.
175,
No.
2, 1991
properties. cells
(25)
differ
and the cytosol
of bovine
of 45,000
shares
described
Generally,
enzyme Our protamine
testis
protamine
ionic
several.
For
protamine
kinase
the
of
preliminary
may play
two
a catalytic
leukemia
kinase
cation
a role
in
of
regulatory
composed
studies described
about
subunits and
cells
for
(data here
the
is
the
example,
kinase
both
of
the C_ lipolvtica by Ca2+ but
C_ lipolvtica
protamine
composition,
not
indicate increasing
of cell
growth.
C_
described,
and the latter
with
to
ion exchange
previously
inhibited
polypeptide
regulation
from
50% inhibition
shown)
but
and response
protamine
of a single
higher
CAMP-independent
eluted
kinases were
leukemia
On the other hand, a by CAMP, and had an M,
were The
cell
3mM of the
are
respectively),
kinases
strengths,
with
and leukemia
(27)
COMMUNICATIONS
promyelocytic
was activated
characteristics
Also,
from
cells
vs 43,000,
to any of the protamine
(26).
suggestive
fromboth
is not identical
required
0.5mM
high
RESEARCH
respectively).
trout these
relatively
here
former
kidney
(110,000
rainbow
BIOPHYSICAL
kinases
non-inhibitory,
from
(24). at
lipolvtica
weight vs
kinase
columns
kinase
the protamine
(inhibitory
protamine
AND
For example, in molecular
Ca"
but
BIOCHEMICAL
unlike
the
the only
kinase
is
110,000
M,
(25). that
the
growth.
activity If
so,
of this
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
Hunter, T. (1987) Cell, 50, 823-29. Blackshear, P.J., Nairn, A.C. and Kuo, J.F. (1988) FASEB J. 2, 2957-69. Taylor, S.S., Buechler, J.A. and Yonemoto, W. (1990) Ann. Rev. Biochem. 59, 971-1005. Nishizuka, Y. (1988) Nature (London), 334, 661-65. Edelman, A.M., Blumenthal, D.K. and Krebs, E.G. (1987) Ann. Rev. Biochem. 56, 567-613. Biochemistry 26:4207-4212.14. Sternbach, H. and Kuntzel, H. (1987). Dahl, C., Biemann, H-P. and Dahl, J. (1987) Proc. Natl. Acad. Sci. USA, 84, 4012-16.14. J. Biol. Chem. Kolarov, J., Kulpa, J., Baijot, M. Goffeau, A. (1988). Biophys. 199:321-330. S. and Passeron, S. (1980). Arch. Biochem. Moreno, 263:10613-10619.14, Judewicz, R.D., Glikein, G.C., and Torres, H.N. (1981) Arch. Biochem. Biophys. 206:87-92. Jimenez, B., Pestana, A. and Fernandez-Renart, M. (1989) Biochem. J. 260:557-561. Ospina, B. and Fernandez-Renart, M. (1990) Biochem. Biophys. Act. 1052:483488. Gupta, B.R. and Datta, A. (1986) Biochem. J. 234, 543-6. P.R. and Rogers, P.J. (1983) In: Fungal Differentiation (Smith, Steward, J.E., ed.), Marcel Dekker, Inc., New York and Basel, pp 267-313. Silverman, P.M. (1978) J. Bacterial. 135, 976-80. Matsumoto, K., Uno, I., Oshima, Y. and Ishikawa, T. (1985) Yeast, 1, 15-24. Reed, S.I., Hadwiger, J.A. and Lorincz, A.T. (1985) Proc. Natl. Acad. Sci. USA, 82, 4055-59. Simanis, V. and Nurse, P. (1986) Cell, 45, 261-8. Dahl, J.S. and Dahl, C.E. (1985) Biochem. Biophys. Res. Commun., 133, 84450. Fontana, J.A., Emler, D., Ku, K., McClung, J.K., Butcher, F.R. and Durham, J.P. (1984) J. Cell. Phys. 120, 49-60. Rahmatullah, M. and Roche, T.E. (1985) J. Biol. Ghem. 260, 10146-52. 505
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AND BIOPHYSICAL
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Hucho, F., Randall, D.D., Roche, T.E., Burgett, M.W., Pelley, J.W. andReed, L.J. (1972) Arch. Biochem. Biophys. 151, 328-40. Laemmli, U.K. (1970) Nature (London), 227, 680-5. Jergil, B. and Dixon, G.H. (1970) J. Biol. Chem., 245, 425-34. Durham, J.P., Butcher, F.R. and Fontana, J.A. (1982) Biochem. Biophys. Res. Commun. 108, 840-5. Damuni, 2. and Reed, L.J. (1988) Arch. Biochem. Biophys. 262, 574-84. Damuni, Z., Amick, G.D. and Sneed, T.R. (1989) J. Biol. Chem. 264, 6412-6. Kuo, J.F. and Greengard, P. (1970). J. Biol. Chem. 245~4067-4073.
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