Yonago Acta medica 1998;41:31–44

Studies on a Ca2+- and Cyclic Nucleotide-Independent H1 Histone Kinase
Purified from Rabbit Skeletal Muscle
Norika Kubota, Toshie Akagi, Kazuhiro Kueda, Sawako Saito, Eiji Nanba*, Barry
S. Brewster†, Peter N. Strong† and Eikichi Hashimoto
Department of Pathological Biochemistry, Faculty of Medicine and *Gene Research Center,
Tottori University, Yonago 683-0826, Japan and †Neuromuscular Unit, Royal Postgraduate
Medical School, Hammersmith Hospital, London W12 ONN, United Kingdom
In an attempt to elucidate the regulatory mechanism of microsomal function by protein
phosphorylation, one of the major protein kinases obtained during the preparation of
the microsomal fraction of rabbit skeletal muscle was partially purified and characterized.
This enzyme was a protein serine/threonine kinase and showed similar, but not completely same properties as those of Ca2+-phospholipid-dependent protein kinase (protein
kinase C), judging from its elution profile from an anion-exchange column, molecularmass, responses to protein kinase activators or inhibitors and the substrate specificity.
These results suggest a possible implication of this Ca2+- and cyclic nucleotideindependent H1 histone kinase in protein phosphorylation of microsomal protein(s),although the exact role and the mechanism of regulation of this enzyme are not clear at
this time.
Key words: H1 histone kinase; microsome; protein kinase C; protein phosphorylation; skeletal
muscle (rabbit)

fraction (Edelman et al., 1987). On the other
hand, Ca 2+-phospholipid-dependent protein
kinase (protein kinase C) has been shown to be
implicated in various cellular phenomenareflecting multiple subspecies of this class of
enzyme (Nishizuka, 1995). In order to elucidate the protein kinase involved in the protein
phosphorylation in microsomal fraction, a protein kinase havs been isolated during the preparation of this organelle from rabbit skeletal
muscle in this report. After partial purificationand characterization, this enzyme has beenshown to possess similar properties as those of
the catalytic fragment of protein kinase C (Takai
et al., 1977; Hashimoto and Yamamura 1989).
These results suggest that the protein kinase C
or its related enzyme may have some target in

It has been well established that protein serine/
threonine kinases play important roles in various cellular functions such as cell growth anddifferentiation or various metabolic and cellstructural regulations (Edelman et al., 1987).
For each protein kinase, a specific or multiple
substrate proteins have been assigned according
to its manner of substrate recognition as well as
the topological arrangement of substrate andenzyme (Edelman et al., 1987). Judging fromthe universality of cellular regulation by protein
phosphorylation and dephosphorylation reactions, this type of reactions must be detected insubcellar fractions such as microsome andmitochondria.
Previous reports indicate that a few protein
kinase activities were detected in microsomal

Abbreviations: BSA, bovine serum albumin; cdc2, cell division cycle 2; cAMP, cyclic AMP; CKI, casein
kinase I; CKII, casein kinase II; CKI-7, N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide; CNBr,
cyanogen bromide; MBP, myelin basic protein; Mr, relative molecular mass; protein kinase A, cyclic AMPdependent protein kinase; protein kinase C, Ca2+-phospholipid-dependent protein kinase; SDS-PAGE, SDSpolyacrylamide gel electrophoresis; TLC, thin layer chromatography; TPA, 12-O-tetradecanoylphorbol 13acetate; TPCK, L-1-tosylamido-2-phenylethyl chloromethyl ketone31

N. Kubota et al.

(Trimmer et al., 1989), KKLGSKKPQK [Na+channel peptide], was synthesized by Tana Labratories, L.C. (Houston, TX). The preparationof H1 and H2B histones from calf thymus was
performed as described earlier (Hashimoto etal., 1976). N-(2-Aminoethyl)-5-chloroisoquinoline-8-sulfonamide (CKI-7), a selective casein
kinase I inhibitor, and cell division cycle 2 (cdc2)
kinase were purchased from Seikagaku Kogyo,
Tokyo, Japan. [γ-32P]ATP (> 185 TBq/mmol)
was obtained from Amersham (Amersham,Bucks, United Kingdom). L-1-Tosylamido-2phenylethyl chloromethyl ketone (TPCK)treated trypsin, bovine brain calmodulin, bovine serum albumin (BSA; fatty acid-free), myelin basic protein (MBP), phosphoserine, phosphothreonine, phosphotyrosine and α-casein
were obtained from Sigma (St. Louis, MO) .
Phosphatidylserine (bovine brain) and diolein
were purchased from Serdary Research Laboratories (London, ON, Canada). N-Bromosuccinimide and leupeptin were obtained from
Wako Pure Chemical and the Peptide Institute,
Osaka, Japan, respectively. P-81 Phosphocellulose paper and 3MM filter paper were purchased from Whatman (Kent, United Kingdom).
Membrane filters (pore size, 0.45 µm) and PVDF
membranes were obtained from Advantec Toyo
(Tokyo) and Millipore (Bedford, MA), respectively.

microsome although the physiologicalsignificance of the existence of this Ca2+- andcyclic nucleotide-independent histone kinase is
not well-established at this time.

Materials and Methods

Materials and chemicals
Rabbits, weighing 3 to 4 kg, were purchasedfrom Kajitani Rabbit Farm (Shimane, Japan).
Excised rabbit skeletal muscle was stored at–80˚C until use. DEAE-Sephacel, cyanogenbromide (CNBr)-activated Sepharose 4B,
Sephacryl S-100, Sephacryl S-200, molecularmass markers for SDS-polyacrylamide gelelectrophoresis (SDS-PAGE) and gel filtration,
Blue Dextran and CleanGel electrode strips (No.
18-1035-33) were obtained from Pharmacia
Biotech (Uppsala, Sweden). H1 histoneSepharose was prepared according to the manufacturer’s instructions (Pharmacia Biotech).
Hydroxyapatite (Bio-Gel HTP) was purchasedfrom Bio-Rad Laboratories (Richmond, CA).
Silica gel 60 thin layer chromatography (TLC)
plates (No. 5626) and cellulose TLC plates (No.
5716) were obtained from E. Merck (Darmstad,
Germany). Cyclic AMP (cAMP)-dependent
protein kinase (protein kinase A) was purified
from bovine cardiac muscle as described
previously (Rubin et al., 1974). Protein kinase
C type I and type II was purified from rat brain
as described previously (Sekiguchi et al.,1988). Casein kinase I (CKI) purified as
described earlier (Liu et al., 1996) was a kind
gift from Mrs. Y. Liu and Dr. H. Kohara in ourlaboratory. Casein kinase II (CKII) was
purified as described previously (Hashimoto etal., 1995). Synthetic peptides, TTYADFIASGRTGRRNAIHD [protein kinase A inhibitor
peptide (Cheng et al., 1986)], RFARKGALRQKNV [protein kinase C inhibitor peptide (House
and Kemp, 1987)], RRLSSL RASTSKA [S6peptide analogue (Wettenhall et al., 1984)], and
LRR ASLG [Kemptide] were prepared as
described previously (Hashimoto et al., 1990a,
1995). The peptide which contains a phosphorylation site in the Na+ channel α subunit

Purification of H1 histone kinase
The H1 histone kinase was purified from rabbit
skeletal muscle as described below. The majority of manipulations were carried out at 4˚C oron ice.
Step 1
Frozen rabbit skeletal muscle, a mixture ofmuscles isolated from the back, buttocks andlimbs, (160 g) was cut into small blocks andground in a meat grinder. The ground muscle
was subsequently homogenized in 800 mL ofbuffer containing 5 mM imidazole-HCl (pH7.4), 0.3 M sucrose, 1 mM phenylmethylsulfonyl fluoride and 5 µg/mL leupeptin using a
Polytron homogenizer (Model K) at speed 6 for
20 × 30 s bursts with 30 s intervals between32

H1 histone kinase purified from skeletal muscle

bursts. The homogenate was centrifuged at
7,000 × g for 10 min in an RF2A rotor (Himac
CR21, Hitachi, Tokyo). The resulting pellets
were re-homogenized in 800 mL of the same
homogenizing buffer containing 0.5 mM Mg2+ATP at the same speed for 10 × 30 s bursts with
30 s intervals between bursts and re-centrifuged
at 7,000 × g for 20 min. The supernatant was
filtered though 4 layers of cheesecloth andcentrifuged at 19,000 × g for 2 h in a JA-20
rotor (J2-21 M/E, Beckman, Palo Alto, CA).
The resultant supernatant was re-filtered thoughanother 4 layers of cheesecloth. This final
supernatant was named Sup 3 and used for further purification. The precipitate obtained bythis centrifugation corresponded to the microsome fraction reported by Inui and coworkers(1987). It is not clear at this time whether theproteins in Sup 3 were originally located on thesurface of the microsome or released from thisorganelle.

was 25–35 mL/h and 5 mL fractions were collected. One major peak of enzyme activity wasrecovered in the fractions eluted with buffer
containing 100 mM NaCl. The active fractions(41.3 mL) were pooled and dialyzed overnight
against 5 L of Buffer B [5 mM potassium phosphate buffer (pH 7.5), 0.3 M sucrose and 10
mM 2-mercaptoethanol].
Step 4The dialyzed one was applied to a hydroxyapatitecolumn (0.9 × 1.3 cm) equilibrated with Buffer
B. After washing the column with the same
buffer, the enzyme was eluted stepwise with 20
mL each of Buffer B containing 100 mM and
300 mM potassium phosphate buffer (pH 7.5)
in place of the 5 mM buffer. The flow rate was
17 mL/h and 1 mL fractions were collected.
The active fractions (8.6 mL) were pooled andused for the present studies as a partiallypurified enzyme preparation.

Step 2
Sup 3 (670 mL) was adjusted to pH 7.5 with 0.5
M Tris-HCl (pH 7.5) and was loaded onto a
DEAE-Sephacel column (2.5 × 4.5 cm) equilibrated with Buffer A [20 mM Tris-HCl (pH 7.5),
0.3 M sucrose and 10 mM 2-mercaptoethanol]
containing 20 mM NaCl. The column waswashed with the same buffer until the absorbance at 280 nm fell to zero. Proteins were
eluted with 320 mL of Buffer A containing 100
mM NaCl at a flow rate of 45 mL/h, and 5 mLof each fraction was collected. When an aliquotof each fraction (20 µL) was assayed for H1
histone kinase activity, 2 active peaks weredetected. The 2nd peak (120 mL) was dialyzed
against 2 changes of Buffer A (5 L) containing
20 mM NaCl for 8 h. The 1st peak was notanalyzed further.

Molecular mass analysis by gel filtration
An aliquot (0.5 mL, 100 µg protein) of the enzyme obtained from the hydroxyapatite column
was applied to a Sephacryl S-200 column (0.8 ×
45 cm) equilibrated with buffer A containing
400 mM NaCl. Proteins were eluted with thesame buffer at a flow rate of 1 mL/h, and 0.5 mL
fractions were collected into tubes in which
ovalbumin had been added as a stabilizer at a
final concentration of 0.1 mg/mL. After
dialysis against Buffer A containing 20 mM
NaCl, each fraction was assayed for the kinase
activity. The elution positions of marker proteins were checked by SDS-PAGE. Molecularmass standards were as follows; Blue Dextran,
void volume; human γ-globulin, 130,000 Da;
BSA, 67,000 Da; ovalbumin, 43,000 Da; cytochrome c, 13,500 Da.

Step 3The dialyzed sample was applied to an H1
histone-Sepharose column (1.5 × 4.5 cm) equilibrated with Buffer A containing 20 mM NaCl.
The column was washed with 150 mL of thesame buffer and eluted stepwise with 100 mLeach of Buffer A containing 100 mM, 250 mM
and 500 mM NaCl, respectively. The flow rate

In-gel kinase assay
An aliquot of the purified enzyme (1.5 mU, 30µL) from the hydroxyapatite column peak fraction was loaded on an SDS-PAGE slab gel. Either 150 µg/mL H1 histone or 150 µg/mL BSA(control protein) was included in the separation
33

N. Kubota et al.

5801 liquid scintillation counter by Cerenkov
radiation. Control assays were performed inparallel where the substrate protein was omitted
and these values were subtracted from those of
the complete reaction system. One unit (1 U) of
the protein kinase activity was defined as theamount of enzyme that catalyzed the transfer of
1 nmol phosphate into H1 histone under the
standard assay conditions described above.
This definition of enzyme activity unit was also
applied to other protein kinases used in thisstudy.
When the substrate specificity of H1 histonekinase was investigated, as well as the effects of
Mg2+, ionic strength and protein kinase modulators, assays were performed as described above,with individual modifications as indicated foreach experiment. The concentrations of proteins and peptides used as substrates in thisstudy were as follows: H2B histone, 60 µg/mL;
MBP, 53 µg/mL; α-casein, 1 mg/mL; S6 peptide
analogue, 0.1 mg/mL; Kemptide, 0.1 mg/mL; Na+channel peptide, 0.1 mg/mL. The reaction with αcasein was stopped by the addition of 2 mL of
10% (w/v) trichloroacetic acid. Acid-precipitates
were collected on a membrane filter and washedwith the same trichloroacetic acid solution. Radioactivity was measured as described above.
For the determination of kinetic parameterswith H1 histone, the kinase activities were determined as described above except that a) H1
histone concentrations were raised between
0.27 and 2.73 µM; b) 1.0 mU of the enzyme was
employed; c) the incubation was carried out for
15 min.
For comparative experiments, the substratespecificity and the effects of protein kinase
modulators were also examined with other representative protein kinases (protein kinase A,
protein kinase C, cdc2 kinase, CKI and CKII).
Protein kinase A, protein kinase C, CKI andCKII were assayed under the respective conditions described previously (Hashimoto et al.,
1985, 1990b, 1995). cdc2 Kinase was assayed
30 min at 30˚C in a reaction mixture (50 µL)
containing 20 mM Tris-HCl (pH 7.5), 10 mM
magnesium acetate, 10 µM [γ-32P]ATP, 1 mg/
mL H1 histone or other substrates as indicatedabove.

gel just prior to the polymerization. Afterelectrophoresis, the gel was treated as describedby Kameshita and Fujisawa (1989) with slight
modifications as indicated below. After renaturation, the gel was preincubated with 2 mLof incubation buffer [20 mM Tris-HCl (pH 7.5)
containing 10 mM MgCl2] at room temperaturefor 30 min. Phosphorylation of H1 histone
within the gel was carried out by incubation of
the gel at room temperature for 2 h with 2 mL of
the incubation buffer in the presence of 50 µM
non-radioactive ATP and 50 µCi [γ-32P]ATP.
After incubation, the gel was washed with 5%(w/v) trichloroacetic acid containing 1% (w/v)
sodium pyrophosphate until the radioactivity of
the washing solution became negligible. The
washed gel was dried on Whatman 3MM filter
paper and exposed to X ray film (New RX, Fuji
Photo Film Co., Kanagawa, Japan) at –80˚C for
24 h or longer time. After the renaturing treatment, the lane of molecular mass markers was
cut from the gel and stained with Coomassie
Brilliant Blue.

In vitro kinase assay
H1 histone kinase activity was measured in areaction mixture (50 µL) comprising 20 mM
Tris-HCl (pH 7.5), 10 mM MgCl2, 60 µg/mL
H1 histone, 10 mM [γ-32P]ATP (5.0–7.0 × 105
cpm/nmol) and 20 µL of enzyme preparation.
The differences in ionic strength due to sodium
chloride or potassium phosphate at the different
purification steps was not corrected for. In
assays (except large scale incubation mixtures),
40 mM potassium phosphate derived from the
enzyme source was constantly included in thereaction mixture. Phosphorylation reactions
were initiated by the addition of [γ-32P]ATP andincubation was usually performed for 30 min at
30˚C. The reaction was terminated by the addition of 25 µL of 5% (w/v) trichloroacetic acid.
The mixture was centrifuged at 3,000 × g for 10
min and 50 µL of the supernatant was spottedon to P-81 phosphocellulose paper (2 × 2 cm).
These papers were then washed 4 times with 10
mL of 75 mM phosphoric acid per each paperfor 2 min each wash. The incorporated radioactivity was determined with a Beckman-LS34

H1 histone kinase purified from skeletal muscle(Beemon and Hunter, 1978) with slight modifications. After freeze-drying, the tryptic peptides were dissolved in electrophoresis buffer(formic acid:acetic acid:H2O = 5:15:80, v/v)
and spotted on Silica gel 60 TLC plates. High
voltage electrophoresis was performed at 1000
V for 50–60 min at 4˚C (Pharmacia Multiphor
II flatbed electrophoresis apparatus) as described by Naka and coworkers (1983). For onedimensional mapping, the dried plate was
exposed to an X ray film for an appropriate timeat –80˚C. For two-dimensional mapping,ascending chromatography was carried out atroom temperature on the TLC plate with thebuffer (n-butanol:acetic acid:pyridine:H2O =32.5:25:5: 20, v/v) as a solvent system. After
chromatography, plates were exposed to X ray
films as before.

Tryptic peptide mapping
H1 histone (36 µg) was phosphorylated by the
purified H1 histone kinase (1.0 mU) or otherprotein kinases; protein kinase A (3.0 mU),
protein kinase C (0.96 mU) or cdc2 kinase (2mU) under the respective reaction conditions
described above except that a) the specific activity of [γ-32P]ATP was increased to 2.4–3.0 ×
10 6 cpm/nmol, b) the reaction mixture wasscaled up by 3-fold (150 µL), c) the incubation
time was 3–4 times longer. The reaction was
stopped by the addition of Laemmli buffer, andthe reaction mixtures were separated by SDSPAGE (Laemmli, 1970). From an estimate of
the radioactivity, 50–125 µmol of phosphate
was incorporated per mol of H1 histone by eachprotein kinase. The radioactive H1 histone was
extracted from the gel and digested overnight at
37˚C with TPCK-treated trypsin (final concentration 50 µg/mL) as described previously

2.0

1.5

1.0

0.5

FFF
FF
FF
FF F
F FFF
FFFF
FF

0.5

0.4

0.3

(mM)
100

60
40

0

20

40

0.1

20
0

0.0

0.2

80

OD at 280 nm

H1 Histone kinase activity

2.5

Phosphorylation of H1 histone by the purified
kinase and the separation by SDS-PAGE wasperformed as described under the previous
section. The labeled H1 histone was extracted
from the gel and precipitated with trichloroacetic acid as described by Beemon and Hunter(1978). The precipitate was washed and hydrolyzed in 6 N HCl for 2 h at 110˚C. The hydrolysate was lyophilized and resuspended in distilled water. An aliquot of this solution was spotted on a cellulose TLC plate together with 3
phosphoamino acids (50 nmol each). Electrophoresis was carried out at 1000 V for 1 h at
16˚C with the buffer (pyridine:acetic acid:H2O= 1:10:189, v/v) on a Pharmacia Multiphor II
flatbed apparatus as described by Mahoney andcolleagues (1996). Positions of phosphoaminoacids were visualized by ninhydrin spray andradioactive phosphoamino acids were detectedby autoradiography.

II

NaCl

I

(× 10–3 cpm)

Phosphoamino acid analysis

0.0

60

Fraction number
Fig. 1. Purification of H1 histone kinase by DEAESephacel chromatography. Sup 3 fraction was
chromatographed on a DEAE-Sephacel column andmeasurements of H1 histone kinase activity were
performed as described under Materials andMethods. The 2 eluting peaks were named DEAE-I
and DEAE-II, respectively. , H1 histone kinase
activity; , absorbance at 280 nm; ---, NaCl concentration.

Analysis of the phosphorylated domainof H1 histone by N -bromosuccinimide
cleavageF

The phosphorylation and subsequent purification of H1 histone were performed as indicatedin the previous section. The extracted histone35

N. Kubota et al.

H1 Histone kinase activity

0.4

1.5

1.0

0.5

0.0

F
F
F
F
F F F FFF
FF
FF
0

20

40

400
0.3
300

0.2

200

OD at 280 nm

2.0

NaCl

H1 Histone kinase activity

(mM)
500

0.1
100

0

16.0

8.0

0.0

0.0

Fraction number(mM)

F
F FF FFFFFF F

0

60

0.16

FF
F

12.0

4.0

0.20

10

20

30

300

0.12

200

0.08

100

0

OD at 280 nm

F

2.5

F
F

(× 10–3 cpm)
20.0

0.5

KPB

(× 10–3 cpm)
3.0

0.04

0.00

40

Fraction number

Fig. 2. Purification of H1 histone kinase by H1
histone-Sepharose chromatography. Purification of the DEAE-II fraction on a H1 histoneSepharose column and measurements of H1 histone kinase activity were performed as described under Materials and Methods. , H1 histonekinase activity; , absorbance at 280 nm; ---,
NaCl concentration.

F

Fig. 3. Purification of H1 histone kinase by hydroxyapatite chromatography. Purification on a hydroxyapatite column of the active enzyme fraction obtained
from the previous step (Fig. 2) and measurements of H1
histone kinase activity were performed as describedunder Materials and Methods.
, H1 histone kinase
activity; , absorbance at 280 nm; ---, concentration ofpotassium phosphate buffer (KPB).

F

was freeze-dried and resuspended in 0.8 mL of 1
N acetic acid with 2 mg of non-phosphorylated
H1 histone as a carrier. N-Bromosuccinimide
cleavage of the H1 histone was performed as described by Bustin and Cole (1969). The cleaved
fragments were applied to a Sephacryl S-100
column (1.5 × 115 cm) equilibrated with 0.02 N
HCl and eluted with the same solution at room
temperature. The flow rate was 8.5 mL/h and
2 mL fractions were collected. The radioactivity of each fraction was monitored using a
Beckman liquid scintillation counter in the
Cerenkov mode and the absorbance at 230 nmof each fraction was measured with Hitachi 181
UV-VIS spectrophotometer. The amino-terminal and the carboxyl-terminal domains of H1
histone were confirmed by SDS-PAGE (15%polyacrylamide separating gel) and protein
staining.

0.1 cm) with 12.5% separating gel and 4.5%
stacking gel by the method of Laemmli (1970).
Electrophoresis was usually performed at 30
mA for 1 h. Protein was determined according
to the method of Bradford (1976) using Bio-Rad
reagent with BSA as a standard. Each experiment had been repeated at least 2 or 3 times anda representative result was presented.

Results

Purification of H1 histone kinase activity
from rabbit skeletal muscle
Sup 3 fraction (670 mL, 502.5 mg protein) wasused as the starting point for purification. It wasadsorbed onto a DEAE-Sephacel column andproteins were eluted as broad 2 peaks with buffer containing 100 mM NaCl as shown in Fig. 1.
They were named DEAE-I (fractions 8–25,
91.5 mL, 23.4 mg protein) and DEAE-II (fractions 26–48, 120.0 mL, 7.6 mg protein), respectively. The specific activity of the H1 histone

Other procedures and determinations
Unless otherwise indicated SDS-PAGE wasperformed in a mini-slab gel (8.5 cm × 6.5 cm ×36

H1 histone kinase purified from skeletal muscle

Table 1. Purification of H1 histone kinase from rabbit skeletal muscle
Step

Volume(mL)

Protein Total activity
(mg)
(mU)

Specific activity(mU/mg)Yield(%)

Purification(-fold)1. Sup 3 fraction
670
502.5
367
0.73
100.0
1.00
2. DEAE-Sephacel
I
91.5
23.4
624
26.5
185
36
II
120.0
7.6
734
96.9
200
133
I + II
211.5
31.0
1358
43.9
385
60
3. H1 histone-Sepharose
41.3
1.95
440
225.5
120
309
4. Hydroxyapatite
8.6
0.75
297
396.6
81
543
Purification of H1 histone kinase and determination of enzyme activity and protein concentration were
performed as described under Materials and Methods. Purification was repeated on several occasions andtypical data are presented.

kinase in DEAE-II was 3.5 times higher thanthat of DEAE-I, and was therefore used for the
next step in the purification. After decreasing
the NaCl concentration to approximately 20
mM by dialysis, DEAE-II was applied to a H1
histone-Sepharose column. H1 histone kinase
activity was eluted as a single peak with thebuffer containing 250 mM NaCl as depicted inFig. 2. The active peak fractions (fractions 23–
30; 41.3 mL, 1.95 mg protein) were dialyzed
and loaded to a hydroxyapatite column. The
kinase activity this time was eluted with a buffer containing 100 mM potassium phosphate as
indicated in Fig. 3. Further purification was
attempted by several methods (gel filtration andcation exchange chromatography, affinity

H1 Histone kinase activity(× 10–3 cpm)
3.0

a c
Vo b

2.5

d

F
F
F
F
F
FFFFFFFFFFF FFFFFFFFF FF

2.0

1.5

1.0

0.5

0.0

0

20

40

60

80

Fraction number
Fig. 4. Molecular mass analysis of H1 histone kinase by
Sephacryl S-200 gel filtration chromatography. Gel filtration of the enzyme was performed and H1 histonekinase activity ( ) was assayed as described under
Materials and Methods. Black bar indicates the peakfractions. The elution position of each molecular mass
standard is pointed by an arrow. Vo, void volume determined using Blue Dextran; a, human γ-globulin; b, BSA;c, ovalbumin; d, cytochrome c.

F

37

Fig. 5. Detection of H1 histone kinase activity
by an in-gel assay method. The H1 histonekinase preparation was separated on SDS-PAGE
containing either H1 histone (A) or BSA, as a control (B) and then the H1 histone kinase activity
was detected as described under Materials andMethods. The position of the radioactive bandwith M r 45,000 is indicated by arrow head.
Numbers on the left side of the figure show thepositions of molecular mass markers: phosphorylase b (94,000 Da), BSA (67,000 Da), ovalbumin (43,000 Da), carbonic anhydrase (30,100Da) and soybean trypsin inhibitor (20,000 Da).

N. Kubota et al.

–3

1/Kinase activity

H1 Histone kinase activity(× 10 cpm)
chromatography on a heparin8.0
Sepharose or a polylysine-agarose),AB
6.0
but did not result in a significant in6.0
crease in the specific activity of the
4.0
enzyme (data not shown). Although
4.0
the enzyme preparation was still contaminated with other proteins, the
2.0
2.0
active fractions recovered from thehydroxyapatite column (fractions 4–
0.0
0.0
0
10
20
30 40
50
60
0
50
100
150
200 250
12; 8.6 mL, 0.75 mg protein) was
(mM)
(mM)
2+
Mg
NaCl
used for further analyses of this H1
histone kinase. A summary of the puriFig. 6. Optimum concentration of Mg2+ and the effect of ionicfication procedure is given in Table
strength on H1 histone kinase activity. Protein kinase activities were measured using 0.25 mU of H1 histone kinase as de1, indicating an approximately 540scribed under Materials and Methods except that the concenfold purification from the Sup 3 fractrations of Mg2+ (A) or NaCl (B) were changed as indicated.
tion with an 81% overall recovery.
Although the reason is not clear atpresent, the high recovery of enzyme activity around Mr 50,000 after staining with Coomassie
may be due to the presence of an endogenous Brilliant Blue (data not shown). However,inhibitor in the starting Sup 3 fraction. The re- radioactive H1 histone bands were detected onsult shown in Fig. 4 depicts the elution profile the autoradiogram of the gel after the activeof kinase activity from a Sephacryl S-200 gel enzyme fractions were incubated with H1
filtration column. The molecular mass of the histone and [γ-32P]ATP and the reaction mixenzyme was estimated to be approximate tures subsequently separated by SDS-PAGE
50,000 Da by comparison with the eluting posi- (data not shown). In an attempt to obtain further
tions of marker proteins. An SDS-PAGE analyses information on the molecular mass of the H1of the active fractions (fractions 29–35; black histone kinase, an in-gel assay method wasbar, Fig. 4) showed very faint protein band(s) employed, and Fig. 5 shows the existence of aradioactive protein band Mr ~45,000 in the gel
containing H1 histone; this radioactive protein(pmol phosphate/min)–1
band does not appear to be due to autophos-–2

2

Table 2. Effects of protein kinase activatorsand inhibitor on H1 histone kinase activity
1

Addition02

Relative kinase
activity (%)

None
100
cAMP
(0.4 mM)
115
CaCl2 (0.2 mM)+ Calmodulin (10 µg/mL)
90
Heparin(0.25 µg/mL)
107
cAMP, cyclic AMP.
H1 histone kinase activities were measured as described under Materials and Methods except that 0.3
mU of the enzyme was employed and protein kinase
activators or inhibitor were added as indicatedbelow. Results are presented as % activities taking
that obtained in the absence of the modulator as 100.

4 (µM)–1

1/[H1 Histone]
Fig. 7. Double-reciprocal plot of phosphorylation of
H1 histone by H1 histone kinase. Protein kinase
activities were measured using 1.0 mU of H1 histonekinase as described under Materials and Methods except that the incubation was performed under various concentrations (from 0.27 to 2.73 µM) of H1 histone for 15 min. The results are presented as a
double-reciprocal plot.

38

H1 histone kinase purified from skeletal muscle

Table 3. Ca2+ and phospholipid requirementof H1 histone kinase and protein kinase C
activities

phorylation. In contrast, no radioactive band
was detected in the control gel. These results
suggest that the purified H1 histone kinase isprobably a monomeric protein with a molecularmass of 45,000–50,000 Da.

Effector

Enzymatic properties of the purified H1
histone kinase+ Ca2+ + Phospholipid
100
100+ Ca2+ – Phospholipid
73
9+ EGTA + Phospholipid
108
71+ EGTA – Phospholipid
97
11
2+
Protein kinase C, Ca -phospholipid-dependent
protein kinase.
Protein kinase activities were measured as described under Materials and Methods except that i)
0.25 mU of the H1 histone kinase and 0.96 mU ofprotein kinase C were incubated for 30 and 10 min,respectively; ii) the final concentrations of Ca2+,
EGTA and phospholipid are 0.4 mM, 0.5 mM and 8µg/mL phosphatidylserine plus 0.8 µg/mL diolein,respectively. Results are presented as % activities taking that obtained in the presence of Ca2+ and phospholipid as 100 for the respective protein kinases.

The effect of Mg2+ on kinase activity was examined and the data in Fig. 6A indicate that the
optimum concentration of Mg 2+ was approximate 10 mM. Figure 6B depicts the effect ofionic strength on kinase activity. A small effectwas observed at NaCl concentrations below 75
mM, but enzyme activity decreased rapidly
above 100 mM NaCl.
A kinetic analysis of H1 histone phosphorylation was shown in Fig. 7. From the doublereciprocal plot, Km and apparent Vmax values
were calculated to be 0.51 µM and 1.36 pmol/
min, respectively.
125

(%)

A

Kinase activity

Relative activity (%)
H1Protein
histonekinasekinase
C

B

C

100
75
50
25
0
0

10–8 10–7 10–6 10–5 10–4(M)Inhibitor concentration0

10–8 10–7 10–6 10–5 10–4(M)Inhibitor concentration

0

10–6

5 × 10–5 10–4 (M)CKI-7 concentration

Fig. 8. Effects of various protein kinase inhibitors on H1 histone kinase activity. A: Effect of protein kinase
A inhibitor peptide. H1 histone (60 mg/mL) was phosphorylated by H1 histone kinase (0.25 mU) or protein
kinase A (1.2 mU) under the respective standard assay conditions described under Materials and Methods
except that the inhibitor peptide was added at the indicated concentrations and 0.4 mM cAMP was included asindicated below. Results are presented as % activities taking that obtained in the absence of the inhibitor as
100. and , activities of H1 histone kinase in the presence and absence of cAMP, respectively; , activityof protein kinase A. B: Effect of protein kinase C inhibitor peptide. S6 peptide analogue (0.1 mg/mL) was
phosphorylated by H1 histone kinase (0.35 mU) or protein kinase C (0.96 mU) under the respective standard
assay conditions described under Materials and Methods except that the inhibitor peptide was added at theindicated concentrations and Ca2+ (0.4 mM) plus phospholipid (8 µg/mL phosphatidylserine plus 0.8 µg/mL
diolein) or EGTA (0.5 mM) plus phospholipid suspension buffer were included as indicated below. Other
conditions were same as indicated in (A). and , activities of H1 histone kinase in the presence of Ca2+ plus
phospholipid and EGTA plus phospholipid suspension buffer, respectively; , activity of protein kinase C.
C: Effect of CKI-7 (inhibitor of CKI). H1 histone was phosphorylated by H1 histone kinase (0.25 mU) or
protein kinase C (0.48 mU) and α-casein was phosphorylated by CKI (0.7 mU) under the respective standard
assay condition described under Materials and Methods except that the inhibitor was added at the indicated concentrations. Other conditions were same as indicated in (A). , and : activities with H1 histone kinase,protein kinase C and CKI, respectively.

39

N. Kubota et al.

Table 4. Substrate specificity of H1 histone kinase and its comparison with other protein kinases
Substrate
H1 histonekinaseProteinkinase C

Relative kinase activity (%)
Proteincdc2
kinase A
KinaseCKI

CKII

H1 histone
100
100
100
100
1
6
H2B histone
35
44
391
14
1
4
Myelin basin protein
153
110
19
ND
0
3
α-Casein
0
ND
ND
ND
100
100
S6 peptide analogue
310
271
530
6
1
1
Kemptide
30
13
680
0
8
3
Na+ channel peptide
22
67
9
1
0
2
cdc2, cell division cycle 2; CKI, casein kinase I; CKII, casein kinase II; ND, not determined; protein kinase
A, cyclic AMP-dependent protein kinase; protein kinase C, Ca2+-phospholipid-dependent protein kinase.
Each protein kinase activity was measured as described under Materials and Methods except that theamount of enzyme and the incubation time were changed with respective enzymes as follows: H1 histonekinase, (0.3 mU, 30 min); protein kinase C, (0.96 mU, 10 min); protein kinase A, (2 mU, 5 min); cdc2 kinase,(0.3 mU, 30 min); CKI, (1.7 mU, 20 min); CKII, (0.8 mU, 20 min). Results are presented as % activities
taking that obtained using H1 histone as 100 for H1 histone kinase, protein kinase C, protein kinase A andcdc2 kinase or using α-casein as 100 for CKI and CKII.

The effects of various modulators on protein
kinase activities are shown in Tables 2 and 3,
and Fig. 8. The H1 histone kinase activity was
not significantly influenced by the addition ofcAMP, CaCl 2 plus calmodulin or heparin(Table 2). The data in Table 3 indicate that thephosphorylation by the purified H1 histonekinase proceeded in Ca 2+ and phospholipidindependent manner, in contrast to phosphorylation by protein kinase C. The kinase activity

was not significantly influenced by the additionof protein kinase A inhibitor peptide irrespective of the presence or absence of cAMP (Fig.
8A). The reaction was strongly inhibited by theaddition of protein kinase C inhibitor peptide
although at concentrations one order of magnitude higher than those for protein kinase C itself(Fig. 8B). This inhibitory effect was equallyobserved irrespective of either the presence orabsence of Ca2+ and phospholipid. The H1 histone kinase activity was moderately inhibited
by CKI-7 (Fig. 8C), a relatively specific inhibitor of CKI. In comparison, CKI-7 only weakly
inhibited protein kinase C (Chijiwa et al.,1989).
The substrate specificity of the purified H1
histone kinase was studied with various protein
or peptide substrates and compared with that ofother protein kinases (e.g., protein kinase A,
protein kinase C, cdc2 kinase, CKI and CKII)(Table 4). MBP and S6 peptide analogue were
phosphorylated more efficiently than H1 histone by our purified H1 histone kinase under thereaction conditions employed. In contrast, H2B
histone, Kemptide (a peptide substrate for protein kinase A) and a Na+ channel peptide were
not such effective substrates for the H1 histonekinase, because the efficiencies in phosphoryla-

Fig. 9. One-dimensional peptide mapping of tryptic
phosphopeptides derived from radioactive H1 histone. The sample phosphorylated each with cdc2
kinase (A); purified H1 histone kinase (B); protein
kinase C (C); protein kinase A (D) was analyzed as
described under Materials and Methods.

40

H1 histone kinase purified from skeletal muscle

tion decreased to 22–35% comparedwith that of H1 histone. α-Casein (astandard substrate for casein kinase)
was not phosphorylated by our kinase. From the results in Table 4, thesubstrate specificity of the purified
H1 histone kinase was clearly different from that of protein kinase A,
cdc2 kinase and CKI and CKII, butsimilar to that of protein kinase C.
To clarify the catalytic specificity of the H1 histone kinase, peptide
mapping of the 32P-labeled H1 histone
was performed. At first, one-dimensional peptide mapping was examin- Fig. 10. Two-dimensional peptide mapping of tryptic phosphopeptides of radioactive H1 histone. Each H1 histone phosphoed using H1 histone preparations rylated separately with either H1 histone kinase or protein
partially phosphorylated by the H1 kinase C was analyzed as described under Materials andhistone kinase, protein kinase A, Methods. Direction 1, high-voltage electrophoresis; Direction 2,
protein kinase C and cdc2 kinase, re- ascending thin layer chromatography. A: H1 histone kinase; B:
spectively, for comparative studies. protein kinase C; C: co-chromatography of H1 histone kinaseand protein kinase C.
The autoradiogram of the tryptic
phosphopeptide maps is shown in
Fig. 9. The pattern of the radioactive peptides obtained using H1 histone kinase and protein
obtained using the H1 histone kinase was kinase C were examined using the two-dimendifferent from those obtained by using protein sional peptide mapping. Both autoradiograms
kinase A and cdc2 kinase, but was similar to showed one major and several minor spots (Figs.
that obtained by using protein kinase C. In 10A and B) and co-chromatography (Fig. 10C)
order to compare each functional specificity indicates that the major spots are identical.
more precisely, the tryptic phosphopeptides
Phosphoamino acid analysis of H1 histone
indicated that radioactive phosphate was primarily incorporated into serine residue(s) (Fig.
11). When the radioactive H1 histone waschemically cleaved at Tyr-74 with N-bromosuccinimide, most of the radioactivity was recovered in a 15 kDa polypeptide derived fromthe carboxyl terminus (Fig. 12). Negligible raFig. 11. Analysis of phosdioactivity was detected on the 6 kDa polypepphorylated amino acid oftide derived from the amino terminus. These
H1 histone. H1 histone wasphosphorylated by H1 hisresults clearly indicate that the serine residue(s)tone kinase and phosin the carboxyl-terminal domain of H1 histonephorylated amino acids ofare phosphorylated by the purified H1 histone
the modified histone werekinase. The high absorbance at 230 nm detectanalyzed as described under
ed around the low molecular mass region isMaterials and Methods. The
positions of phosphoamino
most likely due to unreacted reagent and acetic
acids are indicated on theacid used as a solvent.
right side of the figure. Pi,
inorganic phosphate; P-Ser,
phosphoserine; P-Thr,
phosphothreonine; P-Tyr,phosphotyrosine.

41

N. Kubota et al.

(cpm)
0.3

1000

C-term0.2

OD at 230 nm

Radioactivity

800

600

N-term

400
0.1

200

0

Fig. 12. Analysis ofphosphorylated domainof H1 histone by Nbromosuccinimide treatment. Phosphorylated
H1 histone was analyzed
as described under Materials and Methods.
The elution positions ofcarboxyl-terminal (Cterm) and amino-terminal (N-term) domains
are indicated by arrows.
Γ, total radioactivity ineach fraction; —, absorbance at 230 nm.

0.0
50

100

150

200

(mL)

Elution volume
Discussion

In order to obtain further information on the
enzymatic properties of this H1 histone kinase,the effects of various types of protein kinase
modulators (Tables 2 and 3, and Fig. 8), thesubstrate specificity (Table 4) and the catalytic
specificity (Figs. 9–11) were examined andcompared with those of the representative protein serine/threonine kinases. The results obtained in these experiments revealed that the featuresof the purified H1 histone kinase were clearly
different from those of protein kinase A, cdc2
kinase, CKI, CKII and calmodulin-dependent
protein kinase, but similar to those of protein
kinase C except for the requirement for Ca2+
and phospholipid on this kinase activity. The
Km value for H1 histone by the histone kinase
was slightly lower than that by protein kinase C(Hashimoto et al., 1996). The analyses on thephosphopeptides and the phosphoamino aciddemonstrated that the H1 histone kinase phosphorylated serine residue(s) of the carboxylterminal region generated by N-bromosuccinimide treatment as well as protein kinase C

In this paper, we reported the partial purification procedure and properties of one of the
major H1 histone kinases recovered when theproteins obtained during the preparation of the
microsomal fraction of rabbit skeletal muscle
were separated on the DEAE-Sephacel column(Fig. 1). After the anion-exchange column, theenzyme was further purified by the column
chromatographies on H1 histone-Sepharose andhydroxyapatite (Figs. 2 and 3). Finally, thiskinase was purified by about 540-fold with anapproximate yield of 80% (Table 1), although
the final preparation did not seem to be homogeneous. The molecular mass of the purified
H1 histone kinase was estimated to be 45,000–
50,000 Da by gel-filtration and in-gel assay
methods. Consistent with this results, themolecular mass of 47,700 Da was also calculated with its Stokes radius and sedimentation
coefficient (Siegel and Monty, 1966) (data notshown).
42

H1 histone kinase purified from skeletal muscle(Jakes et al., 1988). These results suggest apossibility that the purified enzyme may be aproteolyzed product of protein kinase C (Takai
et al., 1977; Hashimoto and Yamamura, 1989).
However, some properties of this kinase weredistinguishable from those of the proteolyzed
protein kinase C. For example, the H1 histonekinase was recovered from the DEAE-Sephacelcolumn by the buffer at pH 7.5 containing 100
mM NaCl, whereas the catalytic fragment ofprotein kinase C is usually eluted from the sameanion-exchange column by the buffer containing higher salt concentrations (Takai et al.,1977).
The catalytic fragment of protein kinase C
was usually produced by the limited proteolysisof this enzyme by some protease such as calpain(Kishimoto et al., 1983) or trypsin (Huang et al.,1989) in in vitro reaction systems as well as cellular systems stimulated with tumor-promoting
phorbol ester such as 12-O-tetradecanoylphorbol 13-acetate (TPA) (Nishizuka, 1989).
Although the physiological significance on thegeneration of the catalytic fragment of protein
kinase C has not been well confirmed, it has
been assumed to be temporarily-produced intermediate during the process of down-regulation(Nishizuka, 1989) or an active kinase with a
special role for recognizing specific protein(s)for cellular regulation (Pontremoli et al., 1987).
In the case of protein kinase C, the translocationfrom cytosol to membrane or other organelle
has been demonstrated after stimulation withvarious effectors (Disatnik et al., 1994). It
seems to be plausible to assume that the translocated kinase may be subjected to limited
proteolysis by endogenous protease.
It has been well known that protein kinase C
plays important roles in signal transduction
pathways through phosphorylation of various
intracellular and membrane-bound proteins aswell as protein kinase A (Nishizuka, 1989). Although the relation between the H1 histonekinase and protein kinase C is not clear at thistime, we expect that the histone kinase may participate in the regulation of some microsomal
functions. Further purification and structural
analysis as well as the studies on the mechanismof activation and the survey for its physiologi-

cal target(s) seem to be important for more detailed understanding of this H1 histone kinase.
Acknowledgments: We are grateful to Dr. Y. Nishizuka,
the President of Kobe University, for his encouragement and support.
This work was supported in part by Grants-inAid No. 08670178 for General Scientific Researchand No. 07307003 for Co-operative Research from
the Ministry of Education, Science, Sports and Culture of Japan.
References
1 Beemon K, Hunter T. Characterization of Rous
sarcoma virus src gene products synthesized invitro. J Virol 1978;28:551–566.
2 Bradford MM. A rapid and sensitive method forthe quantitation of microgram quantities ofprotein utilizing the principle of protein-dye
binding. Anal Biochem 1976;72:248–254.
3 Bustin M, Cole RD. Bisection of a lysine-richhistone by N-bromosuccinimide. J Biol Chem
1969;244:5291-5294.
4 Cheng H-C, Kemp BE, Pearson RB, Smith AJ,
Misconi L, Van Patten SM, et al. A potent synthetic peptide inhibitor of the cAMP-dependent
protein kinase. J Biol Chem 1986;261:989–992.
5 Chijiwa T, Hagiwara M, Hidaka H. A newly synthesized selective casein kinase I inhibitor, N-(2aminoethyl)-5-chloroisoquinoline-8-sulfonamide, and affinity purification of casein kinase I
from bovine testis. J Biol Chem 1989;264:4924–
4927.
6 Disatnik M-H, Buraggi G, Mochly-Rosen D.
Localization of protein kinase C isozymes in
cardiac myocytes. Exp Cell Res 1994;210:287–
297.
7 Edelman AM, Blumenthal DK, Krebs EG. Protein serine/threonine kinases. Ann Rev Biochem
1987;56:567–613.
8 Hashimoto E, Takeda M, Nishizuka Y, Hamana
K, Iwai K. Studies on the sites in histones phosphorylated by adenosine 3':5'-monophosphatedependent and guanosine 3':5'-monophosphatedependent protein kinases. J Biol Chem 1976;
251:6287–6293.
9 Hashimoto E, Mizuta K, Yamamura H. Proteaseactivated protein kinase in rat liver plasma membrane. Biochem Biophys Res Commun 1985;
131:246–254.
10 Hashimoto E, Yamamura H. Further studies on
the ionic strength-dependent proteolytic activation of protein kinase C in rat liver plasma membrane by endogenous trypsin-like protease. J
Biochem 1989;106:1041–1048.
11 Hashimoto E, Takeuchi F, Yamamura H. Protease-43

N. Kubota et al.

phosphorylation of a Mr 25,000 protein, a putative protein phosphatase 2A modulator, by casein
kinase I, and analysis of multiple endogenous
phosphates. J Biochem 1996;119:1004–1013.
24 Mahoney CW, Nakanishi N, Ohashi M. Phosphoamino acid analysis by semidry electrophoresis on cellulose thin-layer plates using thePharmacia/LKB Multiphor or Atto flatbed apparatus. Anal Biochem 1996;238:96–98.
25 Naka M, Nishikawa M, Adelstein RS, Hidaka H.
Phorbol ester-induced activation of humanplatelets is associated with protein kinase C phosphorylation of myosin light chains. Nature 1983;
306:490–492.
26 Nishizuka Y. Studies and prospectives of theprotein kinase C family for cellular regulation.
Cancer 1989;63:1892–1903.
27 Nishizuka Y. Protein kinase C and lipid signaling for sustained cellular responses. FASEB J
1995;9:484–496.
28 Pontremoli S, Melloni E, Michetti M, Sparatore
B, Salamino F, Sacco O, et al. Phosphorylationand proteolytic modification of specific cytoskeletal proteins in human neutrophils stimulatedby phorbol 12-myristate 13-acetate. Proc Natl
Acad Sci USA 1987;84:3604–3608.
29 Rubin CS, Erlichman J, Rosen OM. Cyclic
AMP-dependent protein kinase from bovine heart
muscle. Methods Enzymol 1974;38:308–315.
30 Sekiguchi K, Tsukuda M, Ase K, Kikkawa U,
Nishizuka Y. Mode of activation and kinetic properties of three distinct forms of protein kinase C
from rat brain. J Biochem 1988;103:759–765.
31 Siegel LM, Monty KJ. Determination of molecular weights and frictional ratios of proteins in
impure systems by use of gel filtration and density gradient centrifugation. Application to crude
preparations of sulfite and hydroxylamine reductases. Biochim Biophys Acta 1966;112:346–362.
32 Takai Y, Kishimoto A, Inoue M, Nishizuka Y.
Studies on a cyclic nucleotide-independent protein kinase and its proenzyme in mammaliantissues. I. Purification and characterization of anactive enzyme from bovine cerebellum. J Biol
Chem 1977;252:7603–7609.
33 Trimmer JS, Cooperman SS, Tomiko SA, Zhou JY,
Crean SM, Boyle MB, et al. Primary structure andfunctional expression of a mammalian skeletal
muscle sodium channel. Neuron 1989;3:33–49.
34 Wettenhall REH, Morgan FJ. Phosphorylation ofhepatic ribosomal protein S6 on 80 and 40 S ribosomes. Primary structure of S6 in the region of
the major phosphorylation sites for cAMPdependent protein kinases. J Biol Chem 1984;
259:2084–2091.

activated form of protein kinase C with Mr 80,000
generated from rat liver plasma membrane bytrypsin-like protease. Biochem Int 1990a;21:949957.
12 Hashimoto E, Tanaka Y, Yamamura H. Mechanism of proteolytic activation of rat liver protein
kinase C generating Ca2+-phospholipid-independent form with apparent molecular mass of
80,000. Int J Biochem 1990b;22:865–871.
13 Hashimoto E, Takeuchi F, Tanaka Y, Yamamura
H. Purification and phosphorylation of a M r
25,000 protein, an effective phosphate acceptor
for casein kinase II and protein kinase C, detectedin the cytosolic fraction of Xenopus laevis oocytes.
J Biochem 1995;118:453–460.
14 Hashimoto E, Kobayashi N, Kubota N, Tanaka
Y, Yamamura H. Phosphorylated sites of M r
25,000 protein, a putative protein phosphatase 2A
modulator, and phosphorylation of the synthetic
peptide containing these sites by protein kinase
C. J Biochem 1996;119:626–632.
15 Huang FL, Yoshida Y, Cunha-Melo JR, Beaven
MA, Huang K-P. Differential down-regulation ofprotein kinase C isozymes. J Biol Chem 1989;
264:4238–4243.
16 House C, Kemp BE. Protein kinase C contains a
pseudosubstrate prototope in its regulatory domain.
Science 1987;238:1726–1728.
17 Inui M, Saito A, Fleischer S. Purification of theryanodine receptor and identity with feet structures of junctional terminal cisternae of sarcoplasmic reticulum from fast skeletal muscle. J
Biol Chem 1987;262: 1740–1747.
18 Jakes S, Hastings TG, Reimann EM, Schlender
KK. Identification of the phoshposerine residue
in histone H1 phosphorylated by protein kinase
C. FEBS Lett 1988;234:31–34.
19 Kameshita I, Fujisawa H. A sensitive method for
detection of calmodulin-dependent protein kinase
II activity in sodium dodecyl sulfate-polyacrylamide gel. Anal Biochem 1989;183: 139–143.
20 Kishimoto A, Kajikawa N, Shiota M, Nishizuka Y.
Proteolytic activation of calcium-activated,
phospholipid-dependent protein kinase by calciumdependent neutral protease. J Biol Chem 1983;258:
1156–1164.
21 Kishimoto A, Mikawa K, Hashimoto K, Yasuda
I, Tanaka S, Tominaga M, et al. Limited proteolysis of protein kinase C subspecies by calciumdependent neutral protease (calpain). J Biol
Chem 1989;264:4088–4092.
22 Laemmli U. K. Cleavage of structural proteinsduring the assembly of the head of bacteriophage
T4. Nature 1970; 227:680–685.
23 Liu Y, Kohara H, Hashimoto E. Studies on the(Received December 19, 1997, Accepted January 9, 1998)

44