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Fish P h y s io lo g y and B ioch em istry v o l. 7 n os 1-4 pp 343-349 (1989)
K u gler P u b lica tio n s, A m sterd a m /B erk eley
Studies on teleost corpuscles of Stannius: Physiological and biochemical
aspects of synthesis and release of hypocalcin in trout, goldfish and eel
Gert F lik, T eresa L ab ed z* , F loris P .J .G . L afeb er, S joerd E. W endelaar B onga an d P eter K .T . Pang*
D epartm ent o f A n im a l P h ysio lo g y, Faculty o f Science, U niversity o f N ijm egen, T oern ooiveld 25, 6525 E D
Nijmegen, The N etherlands; *D epartm en t o f P h ysiology , School o f M edicine U niversity o f A lberta,
Edm onton, A lb erta , C anada T6G 2H 7
Keywords: tro u t, g o ld fish , eel, fresh w ater, seaw ater, hypercalcem ia, hypocalcin, glycoprotein, secretory
p ro tein , c o n c an av a lin -A .
Abstract
H ypercalcem ia (in d u ced by C a C l2-injection o r seaw ater exposure o f th e fish) reduced the hypocalcin c o n ­
tent of corpuscles o f S tan n iu s (CS) in tro u t, goldfish and eel; con co m itan tly the synthetic activity o f CS o f
hypercalcem ic fish, as d eterm in ed in vitro , was enhanced. T he m onom eric form s o f prohypocalcin and o f
hypocalcin o f tro u t a n d go ld fish a re 32 and 28 kD A M r glycoprotein species respectively; those o f the eel
are 2 kD a bigger, viz. 34 an d 30 k D a respectively. M oreover, eel CS p ro d u ce in vitro an enigm atic 70 kD a
glycoprotein w ith affin ity for con can av alin -A . It is concluded th a t plasm a calcium levels control sto rag e a n d
synthesis rates o f hy p ocalcin in th e CS.
Introduction
The corpuscles o f S tan n iu s (CS) p ro d u c e w hat is
probably th e m ajo r hypocalcem ic calcitropic h o r­
mone in fish (Fenw ick 1982; W en d elaar B onga and
Pang 1986). T h e h o rm o n e , variously referred to as
hypocalcin o r teleocalcin, is a g ly co p ro tein (W agner
eta l. 1986; B u tk u s et al. 1987; L afeber et al. 1988).
Only recently, re p o rts have ap p e are d in the lite ra­
ture on the iso latio n an d N -term inal am ino acid
sequences o f h y p o calcins from sockeye salm on,
Oncorhynchus nerka (W agner et aL 1986), and
rainbow tro u t, Salm o gairdneri (L afeber et al.
1988). F o r th e A u s tra lia n eel, A ngu illa australisy
the presum ptive to ta l am ino acid sequence o f the
protein core o f th e hy pocalcin m olecule was eluci­
dated by cD N A tech n iques (B utkus et al. 1987). A
considerable ho m o lo gy was observed am o n g Nterm inal am ino acid sequences o f th e isolated
salm on, eel and tro u t hypocalcins an d this explains
the cross-reactivity o f hypocalcin sam ples in h eter­
ologous bioassays (P a n g et aL 1974; W endelaar
B onga et al. 1986; L afeb er e t al. 1988). C onverse­
ly, significant variation exists in th e m olecular size
o f the hypocalcin m olecules isolated fro m d iffe r­
ent species with reported m olecular radii ranging
from 39 kD a in salm on (W agner et aL 1986) to
approxim ately 54 kD a in rainbow tro u t (L afeber
et al. 1988). B oth W agner and colleagues and
L afeb er an d colleagues concluded from th eir s tu ­
dies th a t th e native hypocalcin m olecule is likely to
be a hom odim er. M ore recently, evidence h as been
given from studies o n in vitro synthesis o f hypocaicin by tro u t CS th a t hypocalcin is a 56 kD a dim eric
glycoprotein, w hich is processed from a 64 kD a
dim eric p ro h o rm o n e . These dim ers are highly sus­
ceptible to reducing agents such as dith io th reito l
an d 2-m ercap to -eth an o l (the use o f these agents is
F a cu lty o f S cien ce, U n iv ersity o f N ijm eg e n , T o c m o o iv c ld 25, 6525
C o rresp o n d en ce to : G ert F lik , D ep a rtm en t o f A n im a l P h y s io lo g y ,
ED N ijm eg en , T h e N eth erla n d s.
344
a requirem ent in many biochemical analyses) and
appear as 28 and 32 kD a m onom ers in the presence
of these agents (Flik, unpublished data).
The studies presented here focus on the effects
of experim entally induced variations in plasm a cal­
cium concentrations on the storage and rate o f syn­
thesis o f hypocalcin by CS of rainbow trout, Salm o
gairdneri, goldfish, Carassius auratus, and N orth
Am erican eel, Anguilla rostrata LeSueur. F urther
studies on m olecular characteristics of the respec­
tive hypocalcins are also reported.
for three consecutive days. Injections o f NaCl solu­
tion o f identical osm olarity served as controls.
Four hours after the last injection, a blood sample
was taken by puncture o f the vessels o f the caudal
peduncle using a heparinized syringe with a 21-G
needle. Cells were separated from plasm a by cen­
trifugation (15 s, 9000 x g) and the plasm a stored
at -2 0 ° C until further analysis. The CS were re­
moved and prepared for incubation (see below).
Analytical procedures
Materials and methods
A nim als
Rainbow trout, Salmo gairdneri, of both sexes
ranging in body weight from 200 to 700 g, were
kept indoors under a photoperiod o f 16h light al­
ternating with 8h darkness in 1000 1 tanks supplied
with well-aerated, dechlorinated and filtered City
o f E dm onton tapw ater or in artificially prepared
seawater (Wimex sea salt) for at least 6 weeks. The
water tem perature was controlled at 10 ± 1°C, the
water pH was 7.4 and the Ca content o f the fresh­
water was 0.85 ± 0.12 m m ol.I-1 and o f the sea­
water 10 ± 0.5 m m ol.I-1 . The trout were fed daily
with P urina tro u t pellets. G oldfish, Carassius aura­
tus, weighing around 50 g, were kept in 1001 aquaria
supplied with a continuous flow o f City of E dm on­
ton tapw ater at 22°C and fed daily with Tetram in
tropical fish food. Eels, A nguilla rostrata Le
Sueur, weighing around 100 g were housed in 500 1
basins supplied with tapw ater of 18°C. The eels
were not fed.
Trishydroxym ethyl am inom ethane (Tris)-buffered (pH 7.4) 3-aminobenzoic acid ethyl ester (MS
222, Sigma; 0.5 g.I~') was used as an anesthetic
medium. The fish were killed by transection of the
spinal cord.
Calcium injection
Fish were injected intraperitoneally with 0.34
m ol.l-1 CaCl2 solution (100 /xl/100g fish per day)
Plasm a total calcium was determ ined with a com­
mercial calcium kit (Sigma); com bined calci­
um /phosphate standards were used as a reference.
Protein was estim ated with a commercial reagent
kit (Biorad) using bovine serum album in (BSA,
Biorad) as reference.
Labeling incubations
Imm ediately after killing the fish the CS were re­
moved and placed in H an k s’ balanced salt solution
(HBSS; Sigma, H13387). A dhering renal tissue was
carefully removed using a binocular microscope,
and the connective tissue capsule o f the CS incised.
CS tissue was weighed and pre-incubated for 60 min
in 500 /¿I HBSS at 22°C. The tissue was then trans­
ferred to 100 ¡A HBSS containing 1.85 to 3.70 MBq
o f the radiolabeled [35S]-cysteine (specific activity
4.58 GBq.m m ol; New E ngland Nuclear). At the
end o f the incubation the tissue was carefully rinsed
in HBSS (three times 500 ¿d). The tissue was homo­
genized in 1 ml of 50 m M acetic acid (HAc) in a
tight all-glass hom ogenizer and the homogenate
was centrifuged in an E ppendorf minifuge at 9000
X g for 10 min. A n aliquot o f the supernatant
(hereafter called ‘extract’) was used to determine
protein content and the rem ainder lyophilized and
stored at -8 0 ° C until further analysis.
Im m unoprécipitation and concanavaline-A
absorption
Indirect im m unoprécipitation o f hypocalcin anti-
345
M nCl2, M gC l2 and C aC l2, an d 1 m o l.I-1 NaCI
(R oelfzem a a n d V an E rp 1983). P ro d u c ts not
bound to C o n -A w ere rem oved w ith 5 w ashes of
Con-A b u ffer a n d p re cip ita tin g the C o n -A sepha­
rose by sh o rt c e n trifu g a tio n (15 s, 9000 x g). T he
m aterial b o u n d to C o n -A was dissociated from the
lectin by C o n -A b u ffe r m ade 0.3 m o l.I-1 in am ethyl-D -glucoside an d p re cip ita te d w ith trich lo ­
roacetic acid (10% w t/v o l, final co n c en tratio n ).
m in) and rinsed fo r at least tw o hours in flowing
dem ineralized w ater. S taining w’as p erfo rm ed in
the first fixative, w hich contained 2 g .l“ 1 C oom assie Blue R-250 (B iorad). T he gels w'ere destained in
the sam e aqueous solution o f m ethanol and HA c.
F or au to ra d io g ra p h y the fixed gels were im p reg n a t­
ed with P V O /P O P O P according to the m eth o d of
B onner and Laskey (1974). B efore drying (B iorad
slab dryer), the gels were d eh y d rated for 8 to 12h in
50% (vol/vol) m ethanol in w ater co n tain in g 3%
(vol/vol) glycerol. P reflashed K odak X A R -5 Xray film was used for au to ra d io g rap h y ; exposure
tim e was 2 to 72h at - 8 0 ° C . In som e cases gels
were sliced (2 m m ) and the radioactivity in the gel
d eterm ined by liquid scintillation co u n tin g . To this
end, 0.5 mi H 20 2 was added to the gel slice, fol­
lowed by in cu b atio n fo r 12h at 60°C . Next 4 ml o f
Scintiverse (Fisher) was added and the rad io a ctiv i­
ty determ ined in a LKB R ackbeta LSC equipped
with a dpm p ro g ram . S tained gels an d a u to ra d io ­
graphs were scanned densitom etrically w ith a LKB
2202 U ltroscan Laser D ensitom eter equipped with
a LKB 2220 R ecording In teg rato r.
Separation tech n iq u es
Enzyme-linked immunosorbent assay (ELISA)
To estim ate the relative m olecular w eight o f the CS
products, sam ples w ere sep a rated by SDS poly ­
acrylam ide slab gel electro p h o resis (S D S -P A G E )
using a B iorad P ro te a n II o r M ini P ro te a n II Slab
Cell, follow ing th e p ro to c o l o f L aem m li (1970).
The m ark ers used fo r m o lecu lar w eight were p re­
stained S D S -P A G E sta n d a rd s (B iorad; lysozym e,
17 kD A , so y b ean try p sin in h ib ito r, 27 kD a, c a r­
bonic an h y d rase, 39 k D a , ov alb u m in , 50 kD a, b o ­
vine serum alb u m in , 75 kD a, an d p h o sp h o ry lase B,
130 kD a) th a t w ere m ixed w ith [ 14C ]-m ethylated
protein m ark ers (A m ersham pic., C F A .626; lysozyme, 14.3 k D a, ca rb o n ic an h y d rase, 30 kD a,
ovalbum in, 46 kD a, bovine serum alb u m in , 69
kD a, p h o sp h o ry lase B, 92.5 k D a , an d m yosin, 200
kDa).
T o determ ine to tal hypocalcin antigenic activity in
CS extracts an E L IS A was used th at has been de­
scribed in detail (K aneko e t al. 1988). M ic ro titra ­
tion plates were coated w ith 200 ¡A o f serial dilu ­
tions o f purified tro u t hypocalcin or tissue extract.
A lkaline p h o sp h atase co n ju g ated IgG was the sec­
o nd antibody; alkaline p h o sp h atase activity was
determ ined on the basis o f p ara-n itro p h en y l p h o s­
p h a te hydrolysis, determ ined sp ec tro p h o to m e tri-
gen was carried out by th e p ro ced u re o f A nderson
and Blobel (1983) using a w ell-characterized a n ti­
serum fo r tro u t h y p o calcin (R A D H 1; K aneko et al.
1988) and P ro te in A S ep h aro se CL-4B (P h arm acia)
as a so lid -p h ase im m u n o a d so rb e n t. Lyophilized
CS extract was re co n stitu ted w ith distilled w ater
and sodium dodecyl su lp h ate (SDS) was used as
detergent in the im m u n o p récip itatio n procedure.
G lycoprotein iso lation was carried o u t using
C oncanavaline-A sep h aro se (P h arm a cia) as a
solid-phase a d so rb e n t. R eco n stitu ted CS extract
was mixed o v ern ig h t w ith 10 volum es C on-A se­
pharose in a ‘C o n -A b u ffe r’ (50% v o l/v o l) consist­
ing of 15 m M T ris-H C l (pH 7.4), 1 m m o l.I-1 each
Gels were fixed in 40% (v o l/v o l) m ethanol: 10%
(vol/vol) acetic acid (H A c) in w a te r ( lh ) , 5% (v o l/
vol) m eth an o l:7 % (v o l/v o l) H A c in w ater (lh ) and
10% (v o l/v o l) g lu tara ld e h y d e (B D H ) in w ater (30
cally as the change in A 405.
Statistics
Significance o f differences (p < 0.025) between
tre a tm e n t groups was assessed by the M annW hitney U -test.
346
PROTEIN EXTRACTED
cpm/mg wt wt
X 1 0 '3
Fig. 1. P rotein extracted from trout corp u scles o f Stan n iu s (C S)
as a fu n ction o f the wet w eight o f the C S. C aC ^ -treatm en t
(values represented by triangles) resulted in sign ifican tly low er
(M an n -W h itn ey-test, p < 0.0 0 1 ) p rotein con ten t o f the C S co m ­
pared with fresh w ater con trols (d o ts). T h e regression is defined
as: extracted p rotein = 3 6 .1 4 x (wet w eight C S , in m g) ¡ig (p < 0 .0 0 1 ).
1.94
Fig. 3. S D S -P A G E an alysis (g els w ere sliced in 2 m m sam ples
CS ir.HC
g/aP
( o f im m u n op recip itated p rod u cts syn th esized b y CS o f CaC l2in jected (so lid line) and N aC l-in jected
0.4 J
(d o tted line) trout,
form ed during a 4h in cu b ation w ith [35S ]-C ys in H B S S . The
interrupted line represents the rad ioactivity recovered from the
supernatant b y T C A -p recip itation after im m u n op recip itation .
N otice the increased lab elin g brought a b ou t by C a C l 2 injec­
tion s. For com p arison the data h ave been n orm alized to the wet
w eight o f th e CS sam p les. P o sitio n s o f m arker p ro tein s are in­
d icated on the X -axis.
OJ
2
3
4
PLASMA Ca
5
mM
Fig. 2. C orp u scle o f S tannius (CS) im m u n oreactive h yp ocalcin
(ir-H C , determ ined by E L IS A ) con ten t and plasm a calcium lev­
els in freshw ater rainbow trout, gold fish and eel. T he ir-H C co n ­
tent o f CS extracts is n egatively correlated w ith the plasm a cal­
ciu m level o f th e fish . H yp ercalcem ia w a s in d u ced b y C aC l2-injectio n s (see M aterials and M eth o d s section ).
Results
As shown in Fig. 1, significantly less protein was ex­
tracted from CS of trout injected with CaCl2. Mac-
roscopically, the glands o f CaCl2-injected fish ap­
peared blue and translucent compared to the
opaque white o f the glands in the controls.
T he CS im m unoreactive hypocalcin content de­
term ined by E L IS A is inversely related to the plas­
m a calcium concentration in goldfish, eel and
tro u t (Fig. 2).
After a 4h labeling o f C S from NaCl-injected
control fish or C aC l2-injected fish with [3SS]-cysteine, both 32 and 28 kDa, RADH-I-immunoprecipitable Mr species are formed. C a C l 2 -treatment
o f the fish more than doubled (207 ± 21% , n = 6,
p < 0.001) the amount o f immunoprecipitable
products formed during such an incubation (com­
parable amounts o f CS wet weight o f CaCl2-injected and N aC l-injected fish were incubated under
identical conditions). Analysis o f the supernatant
remaining after immuno-precipitation indicated
347
A
B
C
A%
69 _
46 _
*
1 4 .3 _
F/g. 4. S D S -P A G E a n a ly sis (au torad iograp h ) o f con can avaline-A adsorbed CS p ro d u cts form ed after lab elin g o f 4h with
[35S]-Cys in H B S S . I.an e A g o ld fish C S , lane B trout C S, lane
C eel CS. M r valu es o f m arker protein s are in d icated on the left.
Mr
that virtually all new ly-synthetized products were
precipitated (IP ctr; Fig. 3).
Fig. 4 shows the concanavalin-A adsorbable
[35S]-cysteine labeled p roducts form ed during a 4h
labeling o f CS o f tro u t, goldfish and eel. For rea­
sons of co m p ariso n , sim ilar am o u n ts of wet weight
tissue were in cu b ated under identical conditions.
After extraction o f the products from the CS,
equivalent am o u n ts o f protein were subjected to
concanavalin-A a d so rp tio n . F or tro u t and gold­
fish 32 and 28 kD a M r species were recovered. The
eel yielded 70, 34 and 30 kD a M r species.
In Fig. 5, CS synthetic activities o f freshw ater
and of seaw ater tro u t are com pared. P lasm a calci­
um levels in the seaw ater tro u t were significantly
elevated com pared with freshw ater tro u t (2.67 ±
0.12 and 3.05 ± 0.08 for freshw ater and seaw ater
trout, respectively; n = 6, p < 0.01). In both
groups 32 an d 28 kD a M r species are the conspic­
uous products labeled by [35S]-cysteine during a
4h incubation. M acroscopically, the CS o f sea­
water tro u t appeared translucent and resem bled
those o f C aC l2-injected tro u t. T he synthetic activi­
ty o f seaw ater tro u t CS exceeded th at o f freshw ater
trout by a facto r 2.1 ± 0.2 (n = 5, p < 0.001; com ­
parison o f th e to tal radioactivity in the 32 and 28
Fig. 5. S D S -P A G E analysis (scan o f au toradiograph ) o f CS
p rod u cts labeled during 4h in cu b ation w ith [35S ]-C ys in H B SS.
C S o f freshw ater fish (solid line) were com p ared w ith C S o f se a ­
w ater fish (dotted lin e). Y -axis: relative ab sorb an ce (A ^o); the
height o f the 32 k D a peak w as d esign ated 100% A . M r values
o f the m ajor p rod u cts form ed are indicated on the X -axis.
kD a peaks). R em arkable differences in peak
height were consistently observed with the 32 kD a
peak o f seaw ater tro u t CS surpassing that o f fresh­
w ater tro u t CS by 34% and the 28 kD a peak o f sea­
w ater tro u t surpassing that o f freshw ater tro u t CS
by 60% . T ogether these observations indicate e n ­
hanced processing o f hypocalcin in hypercalcem ic
tro u t (com parison with freshw ater trout).
Discussion
From the d a ta presented in this paper it is concluded
th a t stored hypocalcin is reduced in response to ex­
perim entally induced, m ild hypercalcem ia in fresh­
w ater tro u t, goldfish and eel. CS taken from hyper­
calcem ic fish show ed increased synthetic activity in
vitro, as the am ounts o f new ly-synthetized, iram u-
348
noprecipitable hypocalcin produced during 4h la­
beling doubled com pared with control freshw ater
fish. T ro u t and goldfish prohypocalcin and hypo­
calcin have com parable m olecular radii (32 and 28
kD a, respectively, for the reduced, m onom eric
form o f the molecules). T he molecules o f eel differ
from those o f tro u t and goldfish, having a 34 and
30 kD a m olecular radius respectively. Also eel CS
produce significant am ounts o f a 70 kD a M r gly­
coprotein species with affinity for concanavalin-A .
Experim ents with seaw ater tro u t CS provide evi­
dence that an elevation o f plasm a calcium levels in­
duced by exposure to high calcium w ater also stim ­
ulates the rate o f hypocalcin synthesis o f the CS.
The inverse relationship between the hypocalcin
content o f the CS and plasm a calcium levels is in
perfect agreem ent with the presum ptive role o f the
CS in hypocalcemic control. The different m acroscopical appearance o f activated CS (blue and
‘translucent’ rather than white and opaque), is con­
co rdant with o u r observation o f significantly de­
creased am ounts of extractable protein in the CS
o f C aC l2-treated fish. This latter observation also
corroborates (electron) m icroscope studies on the
glands by Lopez et al. (1984) and by Lafeber and
P erry (1988), who showed significant degranula­
tion o f the glands o f eel and tro u t after experim en­
tal hypercalcem ia. Thus we m ay conclude that
degranulation o f the gland is reflected by the de­
creased content o f extractable protein. In a recent
report we have shown th at the CS o f freshwater
fish norm ally store an abundance o f RA D H -Iim m unoreactive hypocalcin (W endelaar Bonga
et al. 1988). Already in 1980, A ida and colleagues
reported th at coho salm on CS degranulate in re­
sponse to high calcium levels in vitro and develop
m ore extensive rough endoplasm ic reticulum and
Golgi systems; degranulation was a result o f in­
creased exocytosis. From these results it was ten ta­
tively concluded that plasm a calcium levels may
control CS synthetic and secretory activities. O ur
present observations corroborate the conclusion
that the CS are under direct control by plasm a cal­
cium levels, as mild hypercalcem ia such as induced
by a C aC l2-injection and by exposure o f the fish
to seawater dim inished storage and enhanced hypo­
calcin synthesis rates o f the CS. It is o u r experience
that care should be taken with the dose o f C aC l2
used to induce hypercalcem ia. D oubling th e dose
used in this study (i.e. injections of the sam e volume
but 0.68 M instead o f 0.34 M C aC l2) inhibits CS
synthetic activity (Flik, unpublished data).
A 2 kDa difference is found between the Mr of
eel prohypocalcin and hypocalcin m olecules and
the corresponding molecules o f the tro u t and gold­
fish. We tentatively conclude th at this difference
derives from differences in th e protein core o f the
glycoproteins. Butkus e ta l. (1987) and Flik (unpub­
lished data) have show n for eel and tro u t, respec­
tively, th at the glyco-m oiety o f the hypocalcins of
these species com prises 4 kD a o f the native m ono­
m eric molecules. Since the N -term inal am ino acid
sequences o f eel and tro u t hypocalcin are highly
conserved (see Lafeber et al. 1988), this extra 2
kDa protein m oiety in the eel hypocalcin m ust be
situated in the C -term inal region o f the molecule.
These observations seem to ju stify fu tu re research
on total sequences o f fish hypocalcins.
A t first sight, one m ight conclude th at the 70
kD a M r species produced by the eel CS is a secre­
tory protein. Secretory proteins (e.g. chrom ogranin A and SP-I), which are considered im portant
m arkers for neuroectoderm al cells, have been local­
ized im m unocytochem ically in eel CS hypocalcin
granules (Tisserand-Jochem et al. 1987). T hen, in
addition to the direct control o f the CS by plasma
calcium levels, a characteristic shared by the CS
and calcitropic glands o f the higher vertebrates,
the CS m ight also have the sam e em bryological ori­
gin. Secretory proteins o f higher vertebrates have
m olecular radii o f around 70 kD a (W ohlfarter et
al. 1988), and are all glycoproteins with O-linked
sugar residues (M ajzoub et al. 1982). But such
glycoproteins lack affinity for the lectin concana­
valin-A. W e conclude therefore, th a t th e 70 kDa
glycoprotein o f the eel CS, which was collected by
concanavalin-A ad sorption, is not a secretory pro­
tein but we do not exclude the possible presence of
a secretory protein in (eel) CS, as antisera, albeit
polyclonals, to chrom ogranin-A and SP-I do in­
deed crossreact with eel and tro u t hypocalcin con­
taining granules (unpublished observations).
W e conclude th at hypocalcin produced by teleost
CS is a unique hypocalcaem ic horm one. Newly-
349
synthesized hypocalcin is a dim eric glycoprotein,
that may show interspecies variatio n in the size o f
the protein core of th e m olecule. D uring processing
of the native dim eric p ro h o rm o n e , an 8 kD a protein-moiety is cleaved o ff to form a 56 kD a (trout
and goldfish) o r 60 kD a (eel) hypocalcin.
K an ek o, T ., F raser, R .A ., L ab ed z, T ., H arvey, S ., L afeb er,
F .P J .G . and P a n g , P .K .T . 1988. C h aracterization o f a n ­
tisera raised against h y p o ca lcin (teleoscalcin ) purified from
corp u scles o f Stan n iu s o f rainbow trou t, S a lm o g a ird n eri.
G en . C om p . E n d o crin o l. 69: 2 3 8 - 2 4 5 .
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B o n g a , S .E . 1988. Id en tific a tio n o f h yp ocalcin (teleocalcin )
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The experim ents were carried out in the D e p art­
ment of Physiology, U niversity o f A lberta, and
were generously su p p o rted by the N ational Sci­
ences and Research C ouncil o f C a n a d a (G rant
NSERC A3639 to D r P .K .T . P ang) an d the A lberta
Heritage F o u n d atio n fo r M edical R esearch (G rant
AHFMR EG 6972). G. Flik received a travel grant
from the N etherlands O rganization for the a d ­
vancement o f P u re R esearch (N W O ). T he authors
thanks M rs A . Neil fo r skilful assistance and excel­
lent organization o f fish h usbandry; Drs P . Verbost
(Nijmegen) an d D . F akcre (E dm onton) are ac­
knowledged for stim ulating discussions during
these studies. Special th a n k s go to D r J .C . Fenwick
(Ottawa) for the shipm ent o f eels from O ttaw a to
Edm onton.
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