J. Cell Sd. 82, 73-84 (1986)
73
Printed in Great Britain © The Company of Biologists Limited 1986
CALCIUM-TRANSPORT FUNCTION OF THE CHICK
EMBRYONIC CHORIOALLANTOIC MEMBRANE
I. IN VIVO AND IN VITRO CHARACTERIZATION
ROCKY S. TUAN*, MONICA J. CARSON, JUDITH A. JOZEFIAK,
KATHY A. KNOWLES AND BARBARA A. SHOTWELL
Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA
SUMMARY
During chick embryonic development, the chorioallantoic membrane (CAM) is responsible for
the mobilization of shell calcium into the embryonic circulation. The calcium-transport function of
the CAM was studied here by measuring CAM calcium uptake in vivo and in vitro. The in vivo
technique involved the use of an uptake chamber constructed on top of the CAM in situ. The
in vitro methods included two systems: CAM tissue disks and cell-free microsomal membranes
isolated from the CAM. Analyses using these three assays show that calcium uptake by the CAM
exhibited characteristics indicative of active transport, such as temperature dependence, saturability, energetic requirement and ion specificity. The data also show that calcium-uptake activities
of the CAM increase as a function of embryonic age in a manner coincident with the increased
accumulation of calcium by the developing embryo in ovo.
INTRODUCTION
The chorioallantoic membrane (CAM) of the chick embryo is the tissue responsible for translocating over 140 mg of eggshell calcium into the embryonic circulation
during development (Terepka et al. 1976). The CAM is formed as a result of the
progressive fusion of the chorionic and allantoic membranes so that, by incubation
day 10, it completely surrounds the embryo and other contents of the egg and
becomes attached to the shell/shell membrane (Romanoff, 1961). The calciumtransport function of the CAM is highly developmentally regulated; activity begins
around incubation day 12-13, rapidly increases in level thereafter, and reaches a
maximal level around day 18-19 (Terepka et al. 1976; Tuan & Zrike, 1978). The
functionally active CAM exhibits a three-layered architecture, consisting of the
ectoderm, the mesoderm and the endoderm (Coleman & Terepka, 1972a), with the
ectoderm being directly adjacent to the calcium-rich shell membrane. Previous in
vitro studies carried out by Coleman & Terepka (19726) have shown that the
ectoderm, a columnar-like epithelium intercalated with a capillary bed (Narbaitz,
1977), is the calcium-transporting region of the CAM. The transport activity of the
CAM is highly specific for calcium, which is mobilized unidirectionally and in an
energy-dependent manner (Garrison & Terepka, 1972a).
•Author for correspondence.
Key words: calcium transport, embryonic development, placental membrane.
74
R. S. Tuan and others
The assay systems previously used to study CAM calcium uptake (or transport)
have all been done with whole tissue preparations: e.g. the Ussing-type transport
chamber set-up (Garrison & Terepka, 1972a; Dunn, Graves & Fitzharris, 1981) for
in vitro measurements, and the in vivo protocol involving the construction of an
uptake chamber over the CAM in situ (Crooks & Simkiss, 1975; Tuan & Zrike,
1978). Although much useful information on the kinetics and energetics of the
CAM calcium-uptake/transport process has been and may be obtained using these
procedures, they have inherently limited applicability for further analysis at a
subcellular level, since whole tissues are used. In this work, we have further studied
the calcium-transport function of the CAM by means of two in vitro uptake assays
(CAM tissue disks and cell-free microsomal membranes). We have demonstrated the
experimental validity of using these in vitro systems for studying CAM calcium
transport, on the basis of a detailed characterization and comparison with the
previously established in vivo uptake assay system.
MATERIALS AND METHODS
Chick embryos and CAM
Fertilized white Leghorn chicken eggs were incubated at 37-5 °C in a humidified commercial eggincubator for the desired period of time. Whole CAM was harvested by dissecting it away from the
shell membrane and was rinsed clear of adhering materials with cold physiological saline.
Preparation of microsomes
All operations described below were performed at 4°C unless specified otherwise. A homogenate
of CAM was prepared as described previously (Tuan, 1979, 1985) using a Ten Broeck, all-glass
homogenizer in lOmM-imidazole, pH7-4, containing 50mM-KCl and 0-3M-sucrose (buffer A).
After gauze filtration, the post-mitochondrial supernatant was prepared by centrifugation of the
homogenate at 11 000 g for 20 min. Microsomes were pelleted after centrifugation at 80 000 g for
80 min and were suspended in 10 mM-imidazole, pH 7-0, containing 0-1 M-KC1 (buffer C) until use.
Occasionally, microsomes were washed by re-pelleting in buffer C at 80 000 £ for 80 min.
Assay of CAM calcium uptake
Calcium uptake in situ. The procedure used has been described (Crooks & Simkiss, 1975; Tuan
& Zrike, 1978; Tuan, 1980, 1983). Briefly, buffer containing 1 mM-CaCl2 and a tracer amount of
45
Ca (1—3 /iCi; Amersham Corp.) was introduced into a transport chamber constructed on top of
the CAM. After 15 min of incubation, the buffer was removed and the radioactivity retained by the
segment of the CAM underlining the chamber was determined. Calcium-uptake activities at 25CC
were expressed as mol calcium min"' cm" 2 .
Calcium uptake by tissue disks in vitro. After rinsing in physiological saline, freshly dissected
CAM was laid flat on a Petri dish and circular disks (1-27 cm2) were cut using a cork borer (no. 6).
Under standard assay conditions, uptake measurements were carried out in Hank's Balanced Salt
Solution containing 12-7/iM-CaClz and tracer quantities of '"CaC^. After bathing in the above
solution at 25CC with shaking for various periods of time (0-9 min), the tissue disks (in triplicates)
were removed, rinsed thoroughly with ice-cold physiological saline to remove unincorporated
radioactivity, solubilized with NCS (Amersham Corp.) and counted for radioactivity. Calciumuptake activities were expressed as mol calcium min" 1 cm" 2 .
Calcium uptake by cell-free microsomal membranes in vitro. Under standard conditions (Tuan,
1985), membrane preparations were assayed for calcium uptake in buffer C containing 5 mM-ATP,
0-lmM-CaCl 2 , and tracer quantities of 45 CaCl 2 ( - S x l ^ c t s m i n " 1 ml" 1 ) at 37°C. At the end of
specific intervals of incubation, a 100-/J sample of the mixture was immediately layered onto 200 /il
Ca transport by chorioallantoic
membrane.
I
75
of silicone oil (Nye Inc., New Bedford, MA; specific gravity = 1-041) contained in a 400 ^1 capacity
polypropylene microcentrifuge tube and centrifugation was carried out at 13 000 £ for 1 min. The
aqueous phase containing free, unincorporated calcium and the bulk of the silicone oil were
then removed by aspiration. The membrane pellet was solubilized in ACS II (Amersham) for
determination of incorporated radioactivity by liquid scintillation counting. As shown later in
Fig. 3B, microsomal calcium uptake measured by this method was kinetically linear for up to 3 min
of incubation, whereas controls in the absence of ATP did not exhibit any time-dependent calcium
uptake.
Isotopic determination of Ca2+/ATP ratio in microsomal calcium uptake
This was carried out by assaying the release of /-PO* from[y- 32 P]ATP (Amersham Corp.)
during microsomal calcium uptake. The procedure used was that of Seals et at. (1978), in which
sodium dodecyl sulphate was used to terminate microsomal calcium uptake in the presence of
[y- 32 P]ATP followed by extraction of the phosphomolybdate complex into xylene/isobutanol to
separate 32 P, from [y- P]ATP, and quantification by liquid scintillation counting. Calciumspecific ATP hydrolysis was based on the difference in hydrolysis in the presence and absence of
O'l mM-CaCl2 in the assay mixture.
Electron microscopy
Samples of CAM microsomes were processed for electron microscopy as described previously
(Tuan, 1979): fixation was with 2% glutaraldehyde in cacodylate buffer (pH7-4), postfixation
with osmium tetroxide, en bloc staining with uranyl acetate, and embedding in Epon. Ultrathin
sections were lead-stained and examined on a JEOL 100S electron microscope.
Protein assay
Protein was determined by the method of Lowry et a!. (1951) with bovine serum albumin
(Sigma Chemicals) as a standard.
Reagents
All chemicals used were of reagent grade. Radiolabelled compounds and liquid scintillation
chemicals were purchased from Amersham Corp. (Chicago, IL). Oligomycin and dinitrophenol
were obtained from Sigma Chemicals.
RESULTS
CAM calcium uptake in vivo
Previous studies by Crooks & Simkiss (1975) and in this laboratory (Tuan & Zrike,
1978; Tuan, 1980, 1983) have demonstrated the validity and the usefulness of the
in situ uptake chamber technique for the study of CAM calcium uptake in vivo. In
this study, we have further applied this in vivo procedure to characterize the uptake
function with respect to ion specificity. As shown in Fig. 1, the CAM exhibited a
highly specific affinity for the uptake of calcium as demonstrated by the ability of
non-radioactive "^Ca to inhibit 45Ca uptake completely, whereas other divalent
cations were only partly effective or totally ineffective. The inhibitory effects of these
ions appeared to be dependent on how similar their ionic radii were to that of Ca 2+ ,
suggesting that active uptake required a specific ionic structure. It is noteworthy, as
shown in Fig. 1, that the ion specificity of CAM calcium uptake also bore remarkable
resemblance to the ion-binding affinity of the calcium-binding protein (CaBP) of the
76
R. S. Tuan and others
1-0
Ionic radii (A)
Fig. 1. Divalent cation specificity of calcium uptake by the CAM measured in vivo.
Chick embryos (17-day) were assayed in situ for CAM calcium uptake as described in
Materials and Methods. The uptake buffer in each test included an additional 1 mM of
each of the cations indicated and trace quantities of 4SCa. The uptake activities obtained
are expressed as percentages of that in the absence of additional non-radioactive cations
and are plotted against the ionic radii of the cations (Handbook of Chemistry, 1975). For
comparison, the ion specificity of the CAM CaBP (Tuan et al. 1978) is also shown here
(broken line).
CAM (Tuan et al. 1978), suggesting a possible functional link between calcium
uptake and the CaBP (see accompanying paper for more details).
Measurement of active CAM calcium uptake in vitro
Our main objective was to establish valid experimental conditions in vitro to assay
for CAM calcium uptake. From the analyses described below, the two systems
studied here, CAM tissue disks and CAM subcellular microsomal membrane
vesicles, were both found to exhibit genuine, active calcium uptake.
Tissue disks of CAM. As shown in Fig. 2, calcium uptake by CAM disks obtained
from day-17 chick embryos was linear for up to 6min of incubation at 25 °C and
under the experimental conditions used here. For all subsequent comparative
studies, uptake rates were routinely calculated on the basis of kinetics between 3 and
6min after the start of incubation. The energy-dependent nature of the CAM
calcium-uptake function was demonstrated by the substantially lowered rate of
uptake in the cold (0°C) and in the presence of energy poisons such as oligomycin
and dinitrophenol in the incubation buffer (Fig. 2, inset).
CAMmicrosomes. The microsomal membranes prepared from the CAM of 17-day
chick embryos were examined by electron microscopy, which revealed the predominant presence of intact, vesicular structures (Fig. 3A), probably originating from
both endoplasmic and plasma membranes as indicated by the presence of various
Ca transport by chorioallantoic membrane. I
11
marker enzymes (Tuan, 1979; also see accompanying paper). These microsomal
membranes were found to exhibit ATP-dependent uptake of calcium (Fig. 3B),
which was kinetically linear for up to 3min of incubation at 37°C. To verify that
the time-dependent accumulation of calcium by the membranes pelleted through
silicone oil was indeed a result of uptake, initial experiments included first layering
25 ^il of 1 mM-EDTA on top of the silicone oil before centnfugation, so that when the
microsomal mixture was added to the tube and spun any non-sequestered calcium
should be removed from the pelleted membranes by the EDTA. As shown in Fig. 3B
(inset), the rate of time-dependent calcium uptake was unaffected, although the
absolute amount of calcium associated with the membrane pellet was decreased
as a result of removal of non-specifically and externally adsorbed calcium. In all
subsequent comparative studies, microsomal calcium uptake was calculated from the
kinetics of net uptake during the first 2min of incubation. Furthermore, it was observed that CAM microsomal calcium uptake was temperature-dependent (Fig. 4).
This characteristic, taken together with those presented below, strongly suggested
the active nature of the process of microsomal calcium uptake.
I
•c
3
0
2
4
Time (min)
6
Fig. 2. Kinetics of calcium uptake by CAM tissue disks. The assay was carried out at
25°C using tissue disks (l'27cm z ) of the CAM of 17-day embryos as described in
Materials and Methods. All data were the mean ± S.E.M. of three to four separate
experiments (with triplicates for all time points in each experiment). Inset: effect of low
temperature and metabolic poisons on calcium uptake. In this experiment, the CAM
disks from 17-day embryos were first pre-incubated either at 0°C or in the presence of
oligomycin ( 1 X 1 0 ~ 5 M ) or dinitrophenol (DNP; 1 X 1 0 ~ 4 M ) for 30min and then assayed
for calcium uptake at low temperature or with poisons. The activities (uptake rates) are
the mean ± S.E.M. of triplicates expressed relative to that of control.
78
R. S. Tuan and others
200
0
1
2
Time (min)
3
4
Fig. 3. A. Ultrastructure of CAM microsomes. Electron microscopy of CAM microsomes isolated from 17-day embryos revealed abundant vesicular structures of varying
sizes (0-1—0-3 fim diameter). Both ribosome-bearing endoplasmic membranes and smooth
vesicles are present. Bar, 1 ^m. B. Kinetics of calcium uptake by CAM microsomal
membranes. The assay was carried out at 37°C using microsomes prepared from 17-day
to 18-day chick embryos as described in Materials and Methods. ( •
• ) Calcium
uptake in the presence of 5 mM ATP; (O
O) calcium uptake in the absence of ATP.
For comparison, all values of calcium uptake (nmol Ca 2 + /mg microsomal protein) were
expressed as percentages of the value at 2 min (== 1-4nmol Ca 2 + mgprotein" 1 ). The data
represent the mean ± S.E.M. of four experiments with triplicate or quadruplicate for all
time points in each experiment. Inset: effect of centrifugation through EDTA on the
measurement of calcium uptake by CAM microsomes. The microsomal suspension was
centrifuged through a layer of 2 mM EDTA lying on top of the silicone oil as described in
the text. Although the overall radioactivity levels were lower, the rate of microsomal
calcium accumulation remained unaltered compared to controls. (Note: in the above
experiments, the mean microsomal calcium uptake rate was l S ^ p m o l s " 1 mgprotein" 1 ).
Ca transport by chorioallantoic membrane. I
79
Characteristics of CAM calcium uptake in vitro
Calcium dependence. In the cell-free microsomal system, calcium-uptake activity
appeared to be linearly dependent on [Ca2+] up to 2mM, above which saturation
occurred (Fig. 5). Lineweaver-Burke analysis of the data obtained from microsomes
isolated from 17-day embryos yielded a/Cm value of approximately 0-5 mM and a Vmax
of 15 pmols" 1 mg protein"1. Similarly, calcium uptake by CAM tissue disks was also
6
T
'53
o
o.
00
-E
4
|
'S
10
20
30
Temperature (°C)
40
Fig. 4. Effect of incubation temperature on calcium uptake by CAM microsomal
membranes. Microsomes were obtained from the CAM of 15-day to 16-day embryos and
assayed for calcium uptake as described in Materials and Methods at the indicated
temperatures.
200
a
3
100
S
'S
0
1
2
3
[Ca2+] (IDM)
4
5
Fig. 5. Calcium-uptake activity of CAM microsomal membranes as a function of [Ca z + ].
Microsomes were obtained from the CAM of 17-day embryos and assayed for calciumuptake activity as described in Materials and Methods at the indicated calcium
concentrations. All activities are expressed as percentage values of that at 1 mM-Ca2+.
80
R. S. Tuan and others
linearly dependent on [Ca2+] and saturable at l-2mM-Ca2+ with a Km value of
approximately 0-3 mM. This latter rinding corresponded well with those reported by
Terepka et al. (1969) and by Garrison & Terepka (19726) who also observed
saturation at [Ca 2+ ] ^ 1 mM and Km of 0-28 mM in CAM calcium transport studies
with the Ussing chamber method.
Ionic requirement. Calcium uptake by the CAM appeared to be dependent on
external sodium. As shown in Table 1, lowering [Na + ] in the bathing buffer by
partial or total substitution of NaCl with choline chloride resulted in decreased
calcium uptake by CAM tissue disks. This rinding was consistent with that previously reported by Terepka et al. (1976), who also observed [Na + ] dependence of
calcium transport in Ussing chamber studies of whole CAM in vitro. We also
observed that both CAM tissue disks and cell-free microsomes exhibited decreased
uptake activity when treated with ouabain, the Na + ,K + -ATPase inhibitor (Table 1),
further suggesting that proper Na + (and/or K + ) balance was probably essential for
functional calcium uptake by the CAM.
To gain further insight into the nature of the Na + (and/or K + ) requirement in
calcium uptake, we made use of the cell-free microsomes and carried out analysis of
calcium uptake under the following experimental conditions: (1) pre-loading microsomes with buffer in which K + was substituted with either Na + or choline; and
Table 1. Ionic requirement of CAM calcium uptake
Relative calcium-uptake activity (%)*
Treatment
CAM tissue disks
100
Control
Ouabain (/ZM)f
+
Substitution of Na with choline
in uptake buffer
Pre-loading microsomes withf
Substitution of K + in uptake
buffer with
0-1
1
10
100% choline
20 % choline
10 % choline
Na +
Choline
Na +
Choline
42 ± 2 (2)
61 ± 6 (2)
59 ± 5 (2)
110±10(2)
—
—
CAM microsomes
100
48±1 (1)
45 ±2(1)
58 ± 3 (2)
—
86 ± 2 (2)
98 ± 8 (2)
33 ± 18 (2)
44± 13 (2)
• Calcium-uptake activities were assayed based on kinetic measurements as described in
Materials and Methods and are expressed as percentages (±S.E.) of control values. In these
measurements, triplicate samples were used in CAM tissue disk experiments for all time points (see
Fig. 2) and quadruplicates were used for the microsomal experiments (see Fig. 3B). The number
of experiments, each using =20 embryos (day 16-17), for the data points is indicated in parenthesis. The ranges of control values of calcium uptake in these experiments were: 40-50pmol
min~ 1 cm~ 2 (tissue disks) and 8-14 pmol s~' mgprotein" 1 (microsomes).
f Samples were pre-incubated for 15-30min (25°C for tissue disks, 4°C for microsomes) in
uptake buffer containing ouabain at the indicated concentrations immediately before assay and
were compared with controls incubated similarly in the absence of ouabain.
| Microsomes were pre-loaded by homogenization and suspension of the membrane pellet in
buffer C in which KC1 was replaced with equimolar NaCl or choline chloride.
Ca transport by chorioallantoic membrane. I
(2) suspending control, K+-loaded microsomes in buffers containing either Na + or
choline in place of K + . As shown in Table 1, the results appeared to indicate that the
presence of external K + was the only specific requirement for functional calcium
uptake. Perturbation of this K + balance probably resulted in the substantial decrease
of microsomal calcium-uptake activity in the presence of ouabain.
ATP dependence. Calcium uptake by the cell-free microsomes was absolutely
dependent on ATP. Substitution of ATP with either GTP or ADP at identical
concentrations resulted in significantly diminished calcium-uptake activity (5-30 %
of control) by CAM microsomes. The stoichiometry of the bioenergetic requirement
of microsomal calcium uptake was also determined from simultaneous analyses of the
rates of calcium uptake and ATP hydrolysis (see Materials and Methods). These
experiments yielded an ATP/Ca z+ ratio of 4-6 (three experiments), indicating an
apparent inefficiency as compared to other ion-transporting membrane systems, such
as the ubiquitous Na + /K + -transporting system of the plasma membrane (Wilson,
1978) and the calcium-uptake system of the mitochondrion (Malstrom & Carafoli,
1979) and the sarcoplasmic reticulum (Wilson, 1978). It is noteworthy that Garrison
& Terepka (1972a,6) previously reported a Ca /O2 ratio of —0-45 with CAM
tissues studied in an Ussing transport chamber, also indicating that the CAM calciumtransport system had a much higher energy requirement compared with other active
transport systems (e.g. the Ca2+/C>2 ratio for isolated mitochondrion = 12; Chance,
1965).
Developmental expression of CAM calcium-uptake activity
Since the CAM calcium-transport function is expressed as a function of embryonic
age (Terepka et al. 1976; Tuan & Zrike, 1978), we next measured in vitro calcium
uptake by CAM tissue disks and microsomal membranes of embryos at various stages
of development in order to assess the relevance of the two in vitro systems to CAM
calcium transport in vivo. As shown in Figs 6A,B, the age profiles of CAM uptake
in vitro compared favourably with that obtained from measurements in ovo (Tuan &
Zrike, 1978) and that calculated from total embryonic calcium contents (Romanoff,
1967). Specifically, all profiles indicated that onset of CAM calcium uptake was
development-specific and occurred around incubation days 12—14.
DISCUSSION
In this investigation, we have characterized the process of calcium uptake by the
chick embryonic CAM using both in vivo and in vitro methods. Our results
demonstrate the active nature, and the ion specificity and dependence of the transport function, and further confirm its development-specific pattern of expression
during chick embryonic development.
Our main objective here was to devise in vitro methods that would permit the
analysis of the CAM calcium-transport function at a subcellular and molecular level.
The experimental evidence presented here clearly shows that the two in vitro assays
81
82
R. S. Tuan and others
18
20
Age (days)
Fig. 6. A. In vitro calcium-uptake activity of the CAM as a function of embryonic
development. Microsomes and tissue disks were prepared from the CAM obtained from
embryos at various developmental stages and assayed for calcium uptake as described
in Materials and Methods. B. In vivo calcium uptake activity of the CAM and the
accumulation of calcium by the developing embryo as a function of embryonic development. The data for CAM calcium uptake in vivo were from Tuan & Zrike (1978) and
those for embryonic calcium content from Romanoff (1967).
provide valid measurements of active calcium uptake by the CAM. It is particularly
noteworthy that CAM calcium uptake measured by in vitro methods exhibits
activity-development profiles similar to those based on in vivo measurements and
total embryonic calcium contents, thereby strbngly indicating the physiological
relevance of these in vitro data. In fact, assuming the in vitro CAM tissue disk
calcium-uptake rate (at the saturating level of 1 mM-Ca2+) and a total egg surface area
of ~60cm , the amount of daily calcium accumulation by the chick embryo at
incubation day 15 is estimated to be ~10mg, which compares favourably with the
actual value of ~15 mgday" 1 based on daily total embryonic calcium measurements
Ca transport by chorioallantoic membrane. I
83
(Romanoff, 1967) or the value of ~7mgday~ based on the in vivo uptake assay
(Crooks & Simkiss, 1975).
The ready availability and relatively simple morphology of the CAM and the
developmentally regulated characteristics of its calcium transport function have
made the CAM an attractive and useful experimental model for the study of
transcellular calcium transport (Terepka et al. 1976). Although much information
has been accumulated from the work in several laboratories, the mechanism and
mode of regulation of the CAM calcium-transport function remain to be resolved.
Results from our laboratory strongly suggest that three biochemical moieties, a
calcium-binding protein (Tuan & Scott, 1977), a Caz+-activated ATPase (Tuan &
Knowles, 1984) and carbonic anhydrase (Tuan, 1984; Tuan & Zrike, 1978) are
probably functional components of the CAM calcium-transport machinery. As
demonstrated in the accompanying paper, the in vitro assay systems devised here can
be used to test directly the functional involvement of these components.
This work was supported in part by grants from the National Institutes of Health (HD 15306,
HD15822, and HD 17887) and the National Foundation-March of Dimes Birth Defects
Foundation (Basil O'Connor Starter Research grant 5-343 and Basic Research grant 1-939).
REFERENCES
CHANCE, B. (1965). The energy linked reaction of calcium with mitochondrion. J. biol. Chetn. 240,
2729-2748.
COLEMAN, J. & TEREPKA, A. (1972a). Fine structural changes associated with the onset of calcium
sodium, and water transport by the chicken chorioallantoic membrane. J. Membr. Biol. 7,
111-127.
COLEMAN, J. & TEREPKA, A. (19726). Electron probe analysis of the calcium distribution in cells of
the embryonic chick chorioallantoic membrane. II. Demonstration of intracellular location
during active transcellular transport. J . Histochem. Cytochem. 20, 414-424.
CROOKS, J. & SIMKISS, K. (1975). Calcium transport by the chick chorioallantois in vivo. Q.J. exp.
Physiol. 60, 55-63.
DUNN, B., GRAVES, J. & FITZHARRIS, T. (1981). Active calcium transport in the chick
chorioallantoic membrane requires interaction with the shell membrane and/or shell calcium.
Devi Biol. 88, 259-268.
GARRISON, J. & TEREPKA, A. (1972a). Calcium-stimulated respiration and active calcium transport
in the isolated chick chorioallantoic membrane. J. Membr. Biol. 7, 128-145.
GARRISON, J. & TEREPKA, A. (19726). The interrelationships between sodium ion, calcium
transport, and oxygen utilization in the isolated chick chorioallantoic membrane. J. Membr. Biol.
7, 146-163.
Handbook of Chemistry (1975). 60th edn (ed. R. C. Weast), pp. F214-F215. Boca Raton: CRC
Press.
LOWRY, O., ROSEBROUGH, N., FARR, A. & RANDALL, R. (1951). Protein measurement with the
Folin phenol reagent. J. biol. Chem. 193, 265-275.
MALSTROM, K. & CARAFOU, E. (1979). Calcium transport in mitochondria. In Membrane
Biochemistry (ed. E. Carafoli & G. Semenza), pp. 103-112. Berlin: Springer-Verlag.
NARBAITZ, R. (1977). Structure of the intra-chorionic blood sinus in the chick embryo. J. Anat.
124, 347-354.
ROMANOFF, A. L. (1961). The Avian Embryo, pp. 1081-1140. New York: Macmillan.
ROMANOFF, A. L. (1967). Biochemistry of the Avian Embryo, p. 39. New York: John Wiley and
Sons.
SEALS, J., MCDONALD, J., BRUNS, D. & JARETT, L. (1978). A sensitive and precise isotopic assay
of ATPase activity. Analyt. Biochem. 90, 785-795.
84
R. S. Tuan and others
TEREPKA, A., COLEMAN, J., ARMBRECHT, H. & GUNTER, T. (1976). Transcellular transport of
calcium. Symp. Soc. exp. Biol. 30, 117-140.
TEREPKA, A., STEWART, M. & MERKEL, N. (1969). Transport function of the chick chorioallantoic membrane. II. Active calcium transport, in vitro. Expl Cell Res. 58, 107-117.
TUAN, R. (1979). Vitamin K-dependent y-glutamyl carboxylase activity in the chick embryonic
chorioallantoic membrane. J . biol. Chem. 254, 1356-1364.
TUAN, R. (1980). Calcium transport and related functions in the chorioallantoic membrane of
cultured shell-less chick embryos. Devi Biol. 74, 196-204.
TUAN, R. (1983). Supplemented eggshell restores calcium transport in chorioallantoic membrane
of cultured shell-less chick embryos. J. Embryol. exp. Morph. 74, 119-131.
TUAN, R. (1984). Carbonic anhydrase and calcium transport function of the chick embryonic
chorioallantoic membrane. Ann. N.Y. Acad. Sci. 429, 459-472.
TUAN, R. (1985). Ca 2+ -binding protein of the human placenta: Characterization, immunohistochemical localization and functional involvement in Ca z+ transport. Biochem.J. 227, 317-326.
TUAN, R. & KNOWLES, K. (1984). Calcium-activated ATPase of the chick embryonic chorioallantoic membrane. Identification, developmental expression, and topographic relationship
with calcium-binding protein. J . biol. Chem. 259, 2754-2763.
TUAN, R. & SCOTT, A. (1977). Calcium-binding protein of chorioallantoic membrane: Identification and developmental expression. Proc. natn. Acad. Sci. U.SA. 74, 1946—1949.
TUAN, R., SCOTT, W. & COHN, Z. (1978). Purification and characterization of a calcium-binding
protein in the chick chorioallantoic membrane. .7. biol. Chem. 253, 1011-1016.
TUAN, R. & ZRIKE, J. (1978). Functional involvement of carbonic anhydrase in calcium transport
of the chick chorioallantoic membrane. Biochem.J. 176, 67-74.
WILSON, D. (1978). Cellular transport mechanisms. A. Rev. Biochem. 47, 933-965.
{Received 17 July 1985 -Accepted
2 October 1985)