Eur. J. Biochem.

164,461 -467 (1987)

0FEBS 1987

Rapid purification of protein kinase C from rat brain

A novel method employing protamine-agarose affinity column chromatography
Marie W. WOOTEN Michel VANDENPLAS' and Andrt E. NEL
Department of Medicine, Division of Hematology/Oncology, University of Alabama, Birmingham
Department of Biochemistry, Stellenbosch Medical School, Tygerberg
Department of Internal Medicine, Stellenbosch Medical School, Tygerberg

(Received September 22/December 11, 1986) - EJB 86 1008

We describe a rapid purification of protein kinase C from rat brain cytosol employing a specific substrate,
protamine-coupled to agarose. Sequential chromatography on DEAE-Sephacel, phenyl-Sepharose CL-4B, and
protamine-agarose columns resulted in a 1500-fold purification of protein kinase C. SDS-PAGE analysis of the
purified enzyme resolved a doublet protein of 77 - 80 kDa. This doublet was recognized by a polyclonal antiserum
against protein kinase C. Proteolytic digestion of each protein band generated similar peptide fragments.
The underlying principle of the protamine sulfate purification method was also clarified. Protamine can serve
as a Ca2+/phospholipid-independent substrate. We demonstrate phosphorylation of protamine on the column;
phosphorylated protamine did not bind the enzyme with the same affinity and this covalent modification was
most probably responsible for releasing the bound enzyme from the column after addition of Mg2+ and ATP.
The C kinase inhibitor, H7, inhibits protamine phosphorylation in a dose-dependent fashion but does not prevent
binding of the enzyme to a protamine-agarose column. We therefore conclude that protamine interacts with the
active center of the enzyme enabling it to be phosphorylated, upon which it loses its binding affinity for C kinase.

Protein kinase C has been shown to play a fundamental
role in mediating the actions of hormones, neurotransmitters,
and growth factors [l - 31. This enzyme has received consider-
able attention since its activation is coupled to ligand-re-
ceptor-mediated phospholipid turnover [l]. Protein-kinase C
is predominantly cytosolic in a variety of cell types [l, 21 and
has a ubiquitous tissue distribution, with brain being one of
the richest sources [4]. Increases in intracellular calcium ion
partially activate the enzyme and promote translocation to the
plasma membrane [5, 61. Diacylglycerol, which is transiently
generated from hydrolysis of inositol 4,5-bisphosphate, fully
activates the enzyme and together with phosphatidylserine
provides an anchor to the membrane [l, 71. Since the initial
purification of protein kinase C from rat brain [8] and bovine
heart [9], several schemes have been employed to obtain
homogeneous enzyme [lo - 141, the most commonly used
method being that of Uchida [l11 utilizing phosphatidylserine
column chromatography. However, owing to either degrada-
tion of the enzyme or activation by copurifying Ca2+-depen-
dent proteases, these lengthy procedures have yielded low
quantities of enzyme. Although protamine sulfate has pre-
viously been described as a protein kinase C substrate, which is
phosphorylated in a Ca2+/phospholipid-independent manner
[15], the specific mechanism for this event has remained an
enigma. We were specifically interested to see whether pro-
tamine could be used as a step in developing an affinity purifi-
Correspondence to M. W. Wooten, Division of Hematology/
Oncology, Department of Medicine, University of Alabama at Bir-
mingham, University Station, Birmingham, Alabama, USA 35294
Abbreviations. H7, 1-(5-isoquinolinylsuIfonyl)-2-methylpipera-
zine dihidrochloride; PtdSer, phosphatidylserine; PhMeS02F, Preparation of crude extract
phenylmethylsulfonyl fluoride.
Enzymes. Protein kinase C (EC 2.7.1.37); V8 protease (EC
3.4.21.19).
cation scheme and, if so, provide a molecular explanation
for the event. We demonstrated the following: (a) protamine
coupled to agarose could be used as an adjuvant step during
purification; (b) protamine phosphorylation was Ca2+/
phospholipid-independent but addition of Ca2+/phospho-
lipid could further increase phosphorylation; (c) interaction
with the substrate probably involved the active center in such
a way that protamine was phosphorylated, upon which the
enzyme was released; (d) Ca2+/phospholipid-independent
phosphorylation of protamine could be inhibited by H7,
confirming an inhibitory effect at the level of the active center;
and (e) previously described protamine kinase activity is hom-
ologous to protein kinase C activity.
EXPERIMENTAL PROCEDURE
Materials
Histone (type 111-S), phosphatidylserine, diolein, pro-
tamine sulfate and protamine-agarose were obtained from
Sigma. [y-32P]ATPwas purchased from Amersham. Phenyl-
Sepharose CL-4B was obtained from Pharmacia. DEAE-
Sephacel and Whatman 3MM paper were obtained from
Whatman. Polyacrylamide gel electrophoresis, protein deter-
mination reagents and standard molecular mass markers were
from Bio-Rad. H7 was obtained from Seikagaku Kogyo Co.
and silver-stain reagents were from Accurate Chemical and
Scientific Co. All other reagents were of the highest purity
available.
25 male Sprague Dawley rats (200-250 g) were deca-
pitated and cerebra rapidly removed. The tissue (40g, wet
462

I
10

Fraction number

106

1:
Froctian Number

I E
14oL

Fig. 1 F r o ~ t i o n number
 463

weight) was homogenized in a Waring blender for 10 s with 8
volumes of buffer A (20 mM Tris/Cl, pH 7.4; 2 mM EDTA;
10 mM EGTA; 250 mM sucrose; 1 mg/ml PhMeS02F;
50 mM 2-mercaptoethanol; 250 U/ml aprotinin). The homog-
enate was centrifuged for 60 min at 100000 xg, after which
the supernatant was collected, filtered through glass wool,
and the pH adjusted to 7.6 with 1 M Tris/CI, pH 8.0.
DEAE-Sephacel fractionation
The crude extract (320 ml) was applied at a flow rate of
60 ml/h to a DEAE-Sephacel column (2.5 cm x 20 cm)
equilibrated with buffer B (20 mM Tris/Cl, pH 7.4; 5 mM
EGTA; 2 mM EDTA; 50 mM 2-mercaptoethanol). The
column was washed with 200 ml of the same buffer and then
with 500 ml buffer C (20 mM Tris/Cl, pH 7.4; 1 mM EGTA;
1 mM EDTA; 50 mM 2-mercaptoethanol) at a flow rate of
125 ml/h. The enzyme was eluted from the column by a linear
gradient of 0 - 0.3 M NaCl in buffer C (600 ml) at a flow rate
of 60 ml/h. Fractions of 6 ml each were collected. Every fifth
fraction was assayed for protein kinase C activity, con-
ductivity and absorbance at 280 nm. The eluted protein profile
was analyzed by SDS-PAGE. Fractions containing protein
kinase C activity (Ca2 /PtdSer stimulated above basal) were
+ applied directly to the protamine-agarose column at a flow
pooled in this manner.
Phenyl-Sepharose CL-4B column chromatography
The pooled DEAE-Sephacel fractions were adjusted to the
conductivity of buffer D (20 mM Tris/Cl, pH 7.4; 0.5 mM
EDTA; 0.5 mM EGTA; 10 mM 2-mercaptoethanol (contain-
ing 1.5 M NaCl and applied directly to a 5-ml washed and
packed phenyl-Sepharose CL-4B column at a flow rate of
60 ml/h. The column was washed with 50 ml buffer D 1.5 M +
NaCl at a flow rate of 125 ml/h followed by a 50-ml wash of
buffer D + 0.6 M NaCl at a flow rate of 50 ml/h. The column
was developed with a 30-ml linear gradient of 600-0 mM
NaCl in buffer D at a flow rate of 25 ml/h, followed by an
additional 30-ml wash of the buffer D alone. Fractions of
1 ml each were collected and assayed for protein kinase C
activity and the proteins eluted from the resin were analyzed
by SDS-PAGE.
Protamine-agarose column chromatography
Protamine-agarose (1 ml) was pre-equilibrated in buffer E
(20 mM Tris/CI, pH 7.4; 0.5 mM EDTA; 10 mM 2-mer-
captoethanol) containing 0.6 M NaCI. The fractions contain-
ing kinase activity were pooled, adjusted to the conductivity
of buffer E + 0.6 M NaCl with buffer E + 4.5 M NaCl and
rate of 25 mlih. The column was washed with 30 ml buffer

Table 1. Purification ofprotein kinase C

Enzyme activity was determined with histone type 111-S as substrate under reaction conditions as described under Materials and Methods.
Activity was calculated as the difference between activities obtained in the presence of Ca2+/PtdSer/diolein or basal (with EGTA present).
All other conditions are as described in the text

Fraction Volume Protein Specific activity Activity Purification Yield

ml mg nmolrnin-’ mg-’ nmoI min-’ -fold %

Crude pool 250 1500 3.3 5000 1 100
DEAE-Sephacel pool 150 256 11.87 3038 4 61
Phenyl-Sepharose pool 30 18 127.78 2300 40 46
Protamine-agarose pool 6 0.2 4950 990 1500 20

Fig. 1. Chromatographicpurificationsteps together with SDS-PAGE. (A) DEAE-Sephacel column chromatography of rat brain soluble proteins.
The 100000 x g supernatant of rat brain extract was applied to a DEAE-cellulose column equilibrated with buffer B. The column was washcd
extensively with buffers B and C. The proteins were eluted with a 0-0.3 M gradient NaCl in buffer C (gradient increases from left to right).
An aliquot from every fifth fraction was assayed in the presence (0--0) or absence (0-0) of Ca2+/PtdSer/diolein.A peak of C a 2 + /
PtdSer/diolein-stimulated activity was found in fractions 24 - 45. (B) SDS-PAGE of DEAE-Sephacel column chromatography. Every fifth
fraction (30 pl) from the DEAE-Sephacel column was analyzed on a 12.5% SDS/polyacrylamide gel. The Coomassie blue-stained gel is shown
with molecular mass markers on left. Fractions 25 -45 correspond to those with the major peak of C kinase activity. (C) Phenyl-Sepharose
CL-4B column chromatography of protein kinase C. DEAE-Sephacel fractions (25 -45) were pooled and applied to a phenyl-Sepharose CL-
4B column equilibrated in buffer D + 1.5 M NaCI. The bound enzyme was eluted from the matrix with a linear gradient of 600-0 mM NaCl
in buffer D, followed by an additional 30 ml buffer D. Every fifth fraction was assayed for C kinase activity in the presence (0-0) or
absence (0-0) of CaZf/PtdSer/diolein.A sharp peak of activity eluted from fractions 35 - 65. Note: the additional wash effectively
removed all bound enzymatic activity. (D) SDS-PAGE of phenyl-Sepharose CL-4B column chromatography. Every fifth fraction (30 pl) from
the phenyl-Sepharose C L 4 B column was analyzed on a 12.5% polyacrylamide gel. The Coomassie-blue-stained gel is shown with molecular
mass markers on left. Fractions 35 -65 correspond to those with the major peak of C kinase activity (pkc). A doublet protein of approximate
molecular mass 77-80 kDa is readily visible at this point. (E) Protamine-agarose column chromatography of protein kinase C. Phenyl-
Sepharose CL-4B fractions 35-65 were pooled and applied to a I-ml protamine-agarose column pre-equilibrated in buffer E. Every third
fraction was assayed for C kinase activity in the presence (0-0) or absence (0-0) of Caz+/PtdSer. Arrow 1 : flow through; arrow
2: buffer E + 600 mM NaCl; arrow 3 : buffer E + 500 mM NaCl, Mg2+/ATP;arrow 4: buffer E + 600 mM NaCI, MgZ+/ATP;arrow 5 : buffer
E + 700 mM NaCI, Mg2+/ATP;arrow 6: buffer E + 1.5 mM NaCI, MgZ+/ATP.A sharp peak of Ca’+/phosphoIipid-dependent activity
eluted immediately after addition of 600 mM NaCl containing MgZ+/ATP.(F) SDS-PAGE of phenyl-Sephdrose CL-4B column chromatog-
raphy. Every third fraction (30 pl) from the protamine-agarose column was analyzed on a 12.5% polyacrylamide gel. The Coomassie-blue-
stained gel is shown with molecular mass markers on left. A doublet 77/80-kDa protein is visualized in fractions containig C kinase activity
(fractions 31 -42)
464

+
E 0.6 M NaCl. Elution from the protamine-agarose resin
was accomplished by four successive step-washes consisting
+
of 5 ml each of buffer E 10 mM MgC12, 1 mM ATP and (a)
500 mM NaCl; (b) 600 mM NaCl; (c) 700 mM NaCl; and
(d) 1.5 M NaCl. Fractions of 0.5 ml were collected at the
beginning of the Mg2+/ATPsalt elutions. Both protein kinase
C activity and SDS-PAGE profiles were determined across
the column.
For Coomassie blue visualization of proteins, the gels were
stained with 1.5% Coomassie brilliant blue in 45% methanol
and 10% acetic acid, and destained in 12% methanol, 12%
acetic acid. The following marker proteins were used:
200 kDa, myosin; 116 kDa, galactosidase; 92 kDa, phosphor-
ylase b ; 66 kDa, bovine serum albumin; 45 kDa, ovalbumin;
31 kDa, carbonic anhydrase; 21 kDa, soybean trypsin in-
hibitor; 14 kDa, lysozyme.

Protein kinase C assay One-dimensional peptide mapping

Protein kinase C activity was routinely assayed by measur- Purified protein kinase C, which was resolved on a 7-
ing the incorporation of 32Pfrom [y-32P]ATPinto lysine-rich 12.5% gradient gel, was subsequently excised and digested
calf thymus histone [8 - 141. The reaction mixture (100 1-11> with Staphylococcus aureus V8 protease in situ. The peptides
contained 20 mM Pipes, pH 6.5, 20 pg histone, 10mM generated were analyzed on 17.5% SDS/polyacrylamide gel
MgC12, 250 pM EGTA and/or 500 pM CaC12, 5 pg PtdSer containing 1 mM EDTA [18]. Peptides were visualized by
and 1 pg diolein in the presence or absence of H7. In some silver staining of the gel.
reactions histone was substituted by 20 pg protamine sulfate.
Reactions were initiated by addition of 1 nmol [ Y - ~ ~ P I A T P
Immunoblotting
containing (0.5- 1.0) x lo6 cpm, and the plastic vials were
incubated at 30°C for 5 min. The reaction was stopped by Proteins separated by SDS-PAGE were transferred to
spotting onto Whatman 3MM paper and washing extensively nitrocellulose membranes [19] for 90 min at 100 V in a buffer
in 20% trichloroacetic acid, followed by two successivewashes containing 25 mM Tris/Cl, pH 8.3, 20% (v/v) methanol and
in 10% trichloroacetic acid and the radioactivity determined 0.1OO/ SDS. Immunoreactive protein kinase C was detected by
by counting Cerenkov radiation. The incorporation of [y- serial reaction with a polyclonal rabbit antiserum to C kinase,
32P]ATPwas linear during the course of the reaction, and the peroxidase-labelled goat-(anti-rabbit antibody) and 4-chloro-
activity was proportional to the amount of enzyme employed. 1-naphthol as substrate.
Protein kinase C activity was expressed as the stimulated
activity (pmol 32P transferred min-' mg protein in the Protein determination
presence of Ca2+/PtdSer/diolein above basal (EGTA-contain-
ing vials). Protein was determined by the method of Bradford [20]
Phosphorylation of protamine in situ on the agarose using bovine serum albumin as a standard.
column was attempted by addition of enzyme obtained from
the phenyl-Sepharose pool to a small volume of beads in
+
buffer E 0.6 M NaCl. After 30 min the beads were washed
and resuspended in buffer E containing 0.5 M NaCl, 10 mM RESULTS
MgClz and 1 mM ATP with 100 Ci/mol [Y-~~PIATP. In- Summary of purification
cubations were carried out for different time periods and
reactions terminated by boiling in Laemmli SDS sample buf- Protein kinase C activity eluted from the DEAE-Sephacel
fer. column at approximately 0.1 M NaCl (Fig. 1A), a similar
salt concentration to that previously described for rat brain
cytosol [8]. The enzyme was maximally stimulated in the pres-
Determination of amount of enzyme hound ence of calcium and phospholipid. Fractions containing the
to protamine agarose peak of protein kinase C activity (Fig. 1 A and B, fractions
utilizing the probe [3H]phorbol 12,13-dibutyrate 25 -45) were pooled and applied to a phenyl-Sepharose CL-
4B column in the presence of 1.5 M NaCl. Upon development
Aliquots of 3 -4 pg sempurified C kinase from the phenyl-
of the resin with decreasing NaCl (0.6-0 M), the bulk of
Sepharose pool were premixed with 0 - 100 pM H7 before protein kinase C activity eluted toward the end of the gradient
addition of 50 ~ 1 5 0 %suspension of protamine-agarose beads (Fig. 1C). An additional 30 ml buffer eluted the remaining
in buffer E plus 0.6 M NaCl. After shaking at 4°C for 1 h, enzyme associated with the resin. SDS-PAGE analysis of
the beads were washed twice in the same buffer and re-
fractions collected from the elution revealed a doublet protein
suspended in 90 pl of a 20 mM Tris buffer, pH 6.8, containing of 77-80 kDa (Fig. 1D), which was coincident with the
100 mM KC1, 0.5 mM CaC12 and 100 &ml PtdSer. To this
enzymatic activity. The peak fractions of protein kinase C
was added lop1 suspension to give a final of 30nM
activity were pooled and applied directly to a protamine
[3H]phorbol 12,13-dibutyrate in the absence or presence of a agarose column. The column was developed with buffer con-
100-fold excess of non-labelled probe [6, 161. After three
taining increasing amounts of NaCl(0.5- 1.5 M) and Mg2+/
washes in the same buffer, the beads were transferred into
ATP. A sharp peak of protein kinase C activity eluted from the
scintillation vials and counted. All determinations were done
resin in the presence of Mg2+/ATP(Fig. 1E). Eluted fractions
in duplicate.
were analyzed by SDS-PAGE and revealed a doublet of I7 -
80 kDa (Fig. l F ) , which coincided with the peak of Ca2+/
One-dimensional gel electrophoresis phospholipid-dependent kinase activity. No contaminating
proteins were visualized by Coomassie staining or by
One-dimensional SDS/polyacrylamide gel electrophoresis autoradiography of an aliquot of the enzyme that was
(SDS-PAGE) was performed either on a 12.5% gel or a 7- autophosphorylated in the presence of [ Y - ~ ~ P I A(results
TP not
12.5% gradient as described by Laemmli 1171. shown}.

Fig. 2. Silver-stained gel after Cleveland peptide mapping of protein
kinase C doublet. A sample of protein kinase C ( 5 pg) was subjected
to 7- 12% SDS-PAGE. After staining and destaining, the visible
upper and lower protein bands were excised individually from the gel
and incubated with several changes in 62.0 mM Tris/HCl buffer,
pH 6.8, containing 1% SDS, 1 mM EDTA and 10% (v/v) glycerol
(equilibration buffer) at room temperature for 60 min. Gel slices were
then transferred to the wells of a second SDS/polyacrylamide gel
(17.5%) with a 10-cm stacking gel and overlayed with 20 pl equilibra-
tion buffer containing 20% glycerol. This, in turn, was overlayed with
10 p1 equilibration buffer containing 10% glycerol and 100 ng S.
aureus V8 protease. Following electrophoresis, the gel was silver-
stained and the peptide fragments were visualized. Lane A: upper
band fragments resolved; lane B: lower band fragments resolved
Table 1 summarizes the results obtained following this
purification scheme. The yield from 40 g rat brain was
typically 200 pg. The homogeneous protein has a specific ac-
tivity of 4950 nmol min-' mg-'. This represents a purifica-
tion of 1500-fold relative to the initial DEAE-Sephacel pool,
with a 20% yield. The enzyme was unstable if frozen in buffer
E and lyophilized but remained fully active for several months
at - 80 "C is stored in buffer E containing 50% glycerol.
Characterization ofprotein kinase C
To investigate the nature of the doublet the homogeneous
proteins were subjected to SDS-PAGE utilizing a 7 - 12.5%
gradient. In this manner the bands were slightly separated.
The individual proteins were excised from the gel and treated
with S. aureus V8 protease in situ following the method of
Cleveland et al. [18]. Analysis of the peptide fragments by
SDS-PAGE and silver staining revealed similar peptide
fragments generated from either the 80-kDa (upper band)
or the 77-kDa (lower band) species (Fig. 2). Moreover, the
465
Table 2. Effect of protamine phosphorylation upon binding of protein
kinase C
An aliquot (100 pl) of protein kinase C , obtained from the phenyl-
Sepharose pool was equilibrated with protamine-agarose (50 p1 of a
50% slurry equilibrated with buffer E + 0.6 M NaC1) in a microfuge
tube at 4°C. After 30min the bound enzyme was eluted from the
protamine-agarose by a 15-min incubation of the resin with buffer E
+ 0.5 mM NaCl containing 10 mM Mg CI2/1 mM ATP (500 pl).
The resin was washed and spun in an Eppendorf microfuge. The
phosphorylated protamine-agarose was then re-used as a means to
determine whether phosphorylation of the substrate had an effect
upon enzyme binding. Equal aliquots of phosphorylated and non-
phosphorylated protamine agarose (50 p1 of a 50% slurry in buffer E
+ 0.6 M NaC1) were incubated with 100 pl phenyl-Sepharose pooled
enzyme. The enzyme was eluted as described above and 10-pl aliquots
were used to assay for protein kinase C activity
Resin Enzyme Decrease
bound in binding
pmol min-' %
mg-
Non-phosphorylated protamine-agarose 3548 f 144 -
Phosphorylated protamine-agarose 367 137 90
Fig. 3. Autoradiogram of in situ protarnine substrate phosphorylation.
Protein kinase C (PKC) obtained from the phenyl-Sepharose pool,
was mixed with protamine-agarose in a microfuge tube. After 30 min
the agarose beads were washed with buffer E + 0.6 M NaC1. To the
beads (containing bound C kinase) were added 10 mM MgClz and
1 mM ATP containing 100 Ci/mol [Y -~~PIA TP. Incubations were
carried out for 0 (lane I), 1 (lane 2), 3 (lane 3), 5 (lane 4), 10 (lane 5 )
and 20 (lane 6) min respectively followed by addition of Laemmli SDS
sample buffer, boiling, and SDS-PAGE/autoradiography. A standard
protamine sulfate (non-immobilized) phosphorylation reaction in the
presence (lane 7) or absence (lane 8) of enzymes was terminated by
boiling in SDS sample buffer for comparison. Note: the increased
phosphorylation of protamine in situ (triplet) compared to the
phosphorylated protamine sulfate standard
doublet was recognized by the same polyclonal antiserum
against protein kinase C (data not shown).
Analysis of protein kinase Clprotamine interaction
We next undertook experiments to elucidate the mecha-
nism of protein kinase C binding to protamine. When pro-
tamine sulfate was utilized as a substrate to measure protein
kinase C activity, its phosphorylation was independent of
added cofactors (Table 2), compared to lysine-rich histone.
However, upon addition of CaZt /PtdSer/diolein the phos-
466

Table 3. Effect of H7 on protein kinase C activity Table 4. Effectof H7 on protein kinase C binding to protamine agarose
Protein kinase C activity was assayed with either protamine or histone as assessed by the probe (3H]phorbol 12,lS-dibutyrate
as substrate in the presence or absence of calcium, phosphatidylserine Aliquots of semipurified enzyme were premixed with 0- 100 pM H7
or diolein as described under Materials and Methods. Results are before addition of protamine-agarose beads in suspension in buffer
reported as nmol min-' mg enzyme protein-' and represent the mean E + 0.6 M NaCl. After washing to get rid of non-bound enzyme, the
f 1 standard deviation (SD) of triplicate experiments beads were resuspended in 20 mM Tris, pH 6.8,lOO mM KCl, 0.5 mM
CaClz and 100 pg/ml PtdSer. Reaction volumes were made up to
Incubation Activity Inhibition 100 pl by the addition of 30 mM [3H]phorbol 12,13-dibutyrate
+
- SD (12.5 Ci/mmol) in the presence or absence of a 100-fold excess of non-
labelled phorbol dibutyrate. After 30 min at 30°C the beads were
nmol min-' % washed, transferred to scintillation vials and counted. Radioactivity
mg-' in the presence of unlabelled phorbol dibutyrate was regarded as
6.34T0.830 background and subtracted from estimations obtained in the presence
Protamine -
of label only. All estimates were done in duplicate and are reported
Protamine + C a 2 + ,PtdSer/diolein 11.60 f 2.65 -
asmeans 1 SD
Protamine + H7, 1 pM 5.14+0.791 19
Protamine + H7, 10 pM 3.89 f 0.953 39 Amount of H7 [3H]phorbol
Protamine + H7, 50 pM 2.04 f 0.125 68 12,13-dibutyrate bound
Protamine + H7, 100 pM 2.05 f 0.406 68
Protamine + Ca", PtdSer/diolein PM dPm
+ H 7 , l pM 1.29 & 0.544 37 0 16 769 f 5072
Protamine + C a 2 + ,PtdSer/diolein 1 15711 f 6500
+ H7, 10pM 5.92 f 1.03 49 10 17462 f 4551
Protamine + Ca2+,PtdSer/diolein 100 17921 f 2741
+ H7, 50 pM 3.49 T 0.956 70
Protamine + C a Z f ,PtdSer/diolein
+ H7,lOO p M 2.70 f 0.792 77
Histone 1.53 f0.311 - amount of enzyme bound to the column in the presence or
Histone + C a 2 + ,PtdSer/diolein 13.25f 1.80 - absence of H7 (Table 4).
Histone + C a 2 + ,PtdSer/diolein
+ H 7 , l pM 9.85 0.778 26 DISCUSSION
Histone + C a 2 + ,PtdSer/diolein
+ H7,lO pM 6.60f 1.95 50 Protamine, a highly basic protein, which has been de-
Histone + C a 2 + ,PtdSer/diolein scribed as a specific substrate for protein kinase C [15], was
+ H7, 50 pM 3.03 f 0.40 77 employed for the purification of the enzyme. The three-step
Histone + C a 2 + ,PtdSer/diolein procedure described, provides a comparatively simple method
+ H7,lOO pM 2.50 f 0.665 81
for the preparation of homogeneous protein kinase C. This
method results in adequate yields and permits the use of
purified protein kinase C as a biochemical reagent. Recently
phorylation of protamine was stimulated twofold. These re- described purification procedures [ l l , 121 are costly and time-
sults suggest that protamine may interact with the enzyme in consuming, yielding very low quantities of purified enzyme.
the absence of Ca2+ of phospholipid in such a way that A direct comparison of our purification scheme with other
the active center is triggered to phosphorylate the binding published procedures to obtain homogeneous protein kinase
substrate in the presence of Mg2+ and ATP. Phosphorylation C is presented in Table 5.
of protamine sulfate may in turn be responsible for its release Our purification further substantiates the previous obser-
from the column. To test this hypothesis further, protein vation that protamine kinase and C kinase are one and the
kinase C was bound to protamine agarose in buffer E + 0.6 M same activity [23].Apart from copurification, they had similar
NaCl followed by washing away the unbound protein and peptide maps and similar antigenic properties. The reason for
T Pthe presence of Mg2+.Analysis of appearing as a doublet is uncertain at this stage but could
addition of [ Y - ~ ~ P ] Ain
the phosphorylated products by SDS-PAGE/auto- involve posttranslational modification. Furthermore, this ob-
radiography (Fig. 3) revealed a time-dependent increase in servation was not restricted to brain tissue but applied also
phosphorylation of protamine in situ on the resin (triplet of to spleen (M. Wooten, unpublished results). Moreover, the
bands) as well as slight autophosphorylation on the enzyme properties of our homogeneous preparation are quite similar
itself (top arrow). Moreover, if protamine was phosphorylated to those described for protein kinase C utilizing other tissue
it no longer bound protein kinase C (Table 2). This suggests sources or purification methods [ l l , 121. Likewise, the specific
that the resin may not be used again unless treated with a activity was roughly the same as that obtained by other meth-
phosphatase prior to binding of protein kinase C. ods (4619 or 4135 nmol min-' mg-' respectively) [ll, 121.
H7 has recently been described as a potent and specific Moreover, our data shed light on the manner in which C
inhibitor of protein kinase C [21, 221 and could, therefore, be kinase interacts with protamine. In the absence of Mg2+ and
used to further define the interaction between kinase C and ATP the enzyme is able to bind to immobilized protamine
protamine. Both protamine sulfate and histone type 111-S under the buffer conditions described. When MgZ and ATP +

phosphorylation were inhibited by H7, whether reactions were are supplied, the substrate is phosphorylated and the enzyme
run in the presence or absence of Ca'+/phospholipid (Table released. Because this occurs in a Ca2+/phospholipid-inde-
3 ) . Since H7 was shown to compete with ATP but not substrate pendent manner, it is likely that upon initial binding a
binding to the active center [21, 221, it may be expected not conformational change ensues, which activates the enzyme.
to interfere with C kinase binding to the protamine-agarose This implies at least some interaction with the active center of
column. This notion was confirmed by determining the actual the enzyme, either directly or via a closely associated allosteric
 467

Table 5. Compurison of protein kinuse C purification schemes

Tissue protein Number Quantity Specific activity Appearance on Reference

of columns purified SDS-PAGE

(mg) Pg nmoI min-' mg-' kDa

Bovine brain (12000) 5 24-76 4735 79.5 [I 21
Rat brain (672) 5 43 1090 82.0 181
Bovine heart (99970) 4 70 1743 83.5 PI
Pig spleen (64800) 4 540 55 68.0 [I 31
Rat brain (990) 4 (+ HPLC) 200 820 80.0 (doublet) ~ 4 1
Rabbit kidney (946) 2 2 4619 80.0 [I 11
Rat brain (1 500) 3 200 4950 80 - 77 (doublet) as described

effector site. It remains to be proven whether the same pro-
tamine molecule can compensate for putative closely associ-
ated PtdSer and Ca2+-bindingsites or whether more complex
stoichiometric alterations are brought about at more distal
sites. C a 2 + , PtdSer and diolein could enhance phos-
phorylation even further, implying that at least some of these
allosteric binding sites remained unoccupied.
Thus far, most inhibitors of protein kinase C interact
directly with the phospholipid-binding site [24, 251. We, how-
ever, demonstrated that H7 inhibits the Ca2+/phospholipid-
independent phosphorylation of protamine. This confirms
that H7 interacts with the active center and the protamine
phosphorylation assay could provide an easy way of
determining the site of action of putative C kinase inhibitors
[21, 221.
Considerable attention has been focused on protein kinase
C in the last few years owing to its regulatory role in diverse
cellular processes. With the aid of H7, a specific C kinase
inhibitor, and studies regarding specific substrate phosphory-
lation we may begin to unravel the complex regulatory role
that this kinase system plays in modulating biological
functions. Protamine phosphorylation may be used to screen
possible inhibitors of C kinase operating on its active center
and protamine-agarose chromatography can be used to purify
the enzyme in relatively large amounts. Woodgett and Hunter
[26] have recently described the existance of two forms of
protein kinase C (78 - SO-kDa species). They have provided
immunological and physiochemical data supporting the no-
tion that isoforms of this protein exist in several tissues. In
further support for the existance of isoforms of the enzyme,
analysis of rat brain protein kinase C cDNA clones have
shown that the heterogeneity may be due to alternative
splicing [27]. Our data would be in line with these findings. It
will be of interest to determine whether the two forms are
preferentially expressed and the possible physiological signifi-
cance of this expression.
This work was supported by grants from the South African Medi-
cal Research Council to A. E. Nel and National Cancer Institute (CA-
08038-01) to M. W. Wooten. This work was completed in partial
fulfillment of a doctoral thesis (M. V.). We would like to thank Mrs
W. Visser for skilful secretarial assistance.
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