Purification and immunohistochemical analysis of calcium-binding
proteins expressed in the chick pineal gland
The vertebrate pineal gland is a main source of circulating
melatonin, which is secreted in a daily rhythmic manner
with a nocturnal peak. Especially in the case of the chick,
the pineal gland retains a circadian clock that drives the
daily rhythm in melatonin production even in dissociated
cells [1, 2]. Anatomical studies on the chick pineal gland
have described two types of morphologically distinct
pinealocytes, the follicular and the parafollicular pinealo-
cytes . The follicular pinealocytes are radially oriented
within each follicle, and protrude their rudimentary outer
segments into the follicular lumen, whereas the parafolli-
cular pinealocytes are located at the periphery of the
follicles. These two types of cells express several enzymes
constituting the melatonin synthesis pathway, including
N-acetyltransferase  and tryptophan hydroxylase .
Pinopsin, a pineal photoreceptive molecule , is expressed
in both types of cells: in the outer segments of the follicular
pinealocytes and in the string- and bulb-shaped cellular
structures of the parafollicular pinealocytes . These
observations, together with the light-dependent phase shift
of the daily rhythmic melatonin production in dissociated
cell culture [9–11], suggest that at least a subset of the
melatonin-secreting pinealocytes retains a circadian clock
system equipped with a photic-entrainment pathway.
Crucial roles of intracellular calcium ions (Ca2+) in the
function of the chick pineal cells have been demonstrated in
dissociated cell cultures. In chick pineal cells, Ca2+influx
increases at night through a cationic channel which is
regulated by the endogenous clock , and the nocturnal
increase in melatonin secretion is facilitated by intracellular
Ca2+[13, 14]. Pharmacological studies support the contri-
bution of calmodulin to the regulation of melatonin
production in dissociated pineal cell cultures [14–16]. In
contrast, immunohistochemical studies point to the absence
of calmodulin immunoreactivity in chick pinealocytes [17,
18]. This is a typical example of issues possibly based on the
lack of information at the protein level, and therefore we set
out to investigate the molecular identity of the chick pineal
Ca2+-binding proteins (CaBPs). Ca2+-binding proteins
having regulatory roles are termed ?Ca2+-sensor? proteins,
whereas those involved in stabilization of intracellular
concentration and/or its transport are termed
?Ca2+-buffer? proteins .
Generally, the Ca2+-sensor proteins are subjected to
large conformational changes upon Ca2+-binding to
expose the hydrophobic domain responsible for interaction
with their target proteins . This allows efficient and
comprehensive purification of a series of Ca2+-sensor
proteins by hydrophobic chromatography on a phenyl-
Sepharose column . Taking advantage of this proce-
dure, we were able to isolate five kinds of chick pineal
proteins which exhibited Ca2+-dependent elution from the
column, and they were identified by micro-sequencing
analysis as calmodulin, neurocalcin, sorcin, annexin II
and annexin V. Together with the protein level study,
Abstract: The pineal gland is a site of melatonin production, of which
intracellular calcium ions (Ca2+) are likely involved in various aspects. To
investigate the identity of molecules responsible for the Ca2+-dependent
processes in the pineal cells, we prepared a cellular extract from 2000 chick
pineal glands and isolated a series of Ca2+-binding proteins by taking
advantage of their Ca2+-dependent hydrophobic interaction with phenyl-
Sepharose beads. The proteins identified by micro-sequencing analysis
included calmodulin, neurocalcin, sorcin, annexin II and annexin V.
Immunohistochemical analysis of the chick pineal sections revealed that both
calmodulin and sorcin are expressed in the follicular and parafollicular
pinealocytes. On the other hand, neurocalcin was expressed in a few neuron-
like cells located predominantly in the parafollicular layer of the pineal
follicle. These results suggest that calmodulin and sorcin may contribute to
cellular functions in the chick pinealocytes.
Fumiko Shimizu, Kamon Sanada*
and Yoshitaka Fukada
Department of Biophysics and Biochemistry,
Graduate School of Science, The University of
Tokyo, Tokyo 113-0033, Japan
*Current address: Department of Pathology,
Harvard Medical School, 200 Longwood
Avenue, Boston, MA 02115, USA
Key words: calmodulin, chick pinealocytes,
circadian rhythm, melatonin, neurocalcin,
protein sequence analysis, sorcin
Address reprint request to Yoshitaka Fukada,
Department of Biophysics and Biochemistry,
Graduate School of Science, The University of
Tokyo, Hongo 7-3-1, Bunkyo-Ku, Tokyo 113-
Received October 11, 2002;
accepted December 10, 2002.
J. Pineal Res. 2003; 34:208–216
Copyright ? Blackwell Munksgaard, 2003
Journal of Pineal Research
immunohistochemical analysis revealed that calmodulin
and sorcin are expressed in most of the follicular and
parafollicular pinealocytes, supporting their active roles in
Materials and methods
Isolation of calcium-binding proteins
Animals used in this study were treated in accordance with
the guidelines of The University of Tokyo. Newly hatched
male chicks (Gallus gallus) were purchased from local
suppliers and killed by decapitation on day 1–3 during the
daytime. Their pineal glands were isolated, frozen with
liquid nitrogen, and stored at )80?C until use. After
thawing quickly at 37?C, the pineal glands (2,120 glands,
4.75 g wet weight in total) were homogenized with 40 mL
of an extraction buffer (10 mm Tris–HCl, 2 mm ethylene-
5 mm MgCl2, 1 mm DTT, 4 lg/mL leupeptin, 4 lg/mL
aprotinin, 0.5 mm benzamidine; pH 8.0) using a glass–glass
homogenizer. The homogenate
125,000 g for 30 min at 4?C and the supernatant was
stored. The precipitate was homogenized again with 50 mL
of the extraction buffer and the homogenate was centri-
fuged. The supernatants obtained from the two centrifuga-
supplemented with CaCl2 so as to yield a free Ca2+
concentration of 2 mm.
Calcium-dependent hydrophobic-interaction chromatog-
raphy on phenyl-Sepharose column was performed accord-
ing to the method of Gopalakrishna and Anderson (1982)
 with some modifications as described previously .
The phenyl-Sepharose CL-4B (Amersham Biosciences
Corp., NJ, USA) column (7 mm ID · 52 mm) was pre-
equilibrated with a high-Ca2+buffer (10 mm Tris–HCl,
2 mm EGTA, 4 mm CaCl2, 1 mm DTT, 4 lg/mL leupeptin,
4 lg/mL aprotinin, 0.5 mm benzamidine; pH 8.0), and then
the pineal extract was applied to the column at a flow rate of
28 mL/hr. After washing with the high-Ca2+
proteins were eluted from the column with a low-Ca2+
buffer(10 mmTris–HCl,2 mmEGTA,1 mmDTT,4 lg/mL
leupeptin,4 lg/mLaprotinin,0.5 mmbenzamidine;pH 8.0).
Amino acid sequence analysis of calcium-binding
A phenyl-Sepharose fraction (Fig. 1, fraction 3) containing
the protein with an apparent molecular weight of 20,000
(20K protein) was subjected to an anion-exchanger column
Mono Q FPLC (fast protein liquid chromatography;
Amersham Biosciences Corp.), and proteins were eluted
with a linear gradient of 0–1 m NaCl (25 mm/min) in
10 mm Tris–HCl buffer (pH 8.0) at a flow rate of 2 mL/
min. The 20K protein eluted from the column forming a
peak at a position of 420 mm NaCl. This peak fraction was
then subjected to reversed-phase high performance liquid
chromatography (HPLC) (model 600E, Waters) on a
Cosmocil 5C18-P300 column (Nacalai Tesque, Inc., Kyoto,
Japan), and proteins were eluted with a linear gradient of
10–70% acetonitrile (1%/min) in 0.1% trifluoroacetic acid
at a flow rate of 1 mL/min. The 20K protein that eluted at a
position of 47% acetonitrile was collected and lyophilized.
Because the purified 20K protein was resistant to Edman
degradation, theprotein (approximately
denatured with 8 m urea in 110 lL of 50 mm Tris–HCl
buffer (pH 9.0) for 30 min at 37?C, and then digested with
40 lg) was
Fig. 1. Isolation of the chick pineal calcium-binding proteins.
Upper panel, Elution profile of the pineal proteins from the phenyl-
Sepharose column. Soluble proteins were extracted from 2,120
chick pineal glands and applied to the column in the presence of
2 mm free Ca2+. After washing the column with a high Ca2+buffer
(open bar in the horizontal axis), the bound proteins were eluted
with a low-Ca2+buffer containing 2 mm EGTA (solid bar). Bot-
tom panel, SDS–PAGE of the phenyl-Sepharose fractions. Aliquots
(1/50 volume) of the flow-through fraction (FT), and eluate frac-
tions (1–7) were electrophoresed in a SDS-polyacrylamide gel
(13%) and stained with Coomassie Brilliant Blue. The partial
amino acid sequences of the proteins (indicated with arrowheads)
were determined by peptide sequencing, and the protein band
indicated with an arrow was immunostained with an anti-visinin
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