J. Cell Sd. 82, 73-84 (1986) 73Printed in Great Britain © The Company of Biologists Limited 1986

CALCIUM-TRANSPORT FUNCTION OF THE CHICKEMBRYONIC CHORIOALLANTOIC MEMBRANEI. IN VIVO AND IN VITRO CHARACTERIZATION

ROCKY S. TUAN*, MONICA J. CARSON, JUDITH A. JOZEFIAK,KATHY A. KNOWLES AND BARBARA A. SHOTWELLDepartment of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA

SUMMARYDuring chick embryonic development, the chorioallantoic membrane (CAM) is responsible for

the mobilization of shell calcium into the embryonic circulation. The calcium-transport function ofthe CAM was studied here by measuring CAM calcium uptake in vivo and in vitro. The in vivotechnique involved the use of an uptake chamber constructed on top of the CAM in situ. Thein vitro methods included two systems: CAM tissue disks and cell-free microsomal membranesisolated from the CAM. Analyses using these three assays show that calcium uptake by the CAMexhibited characteristics indicative of active transport, such as temperature dependence, satura-bility, energetic requirement and ion specificity. The data also show that calcium-uptake activitiesof the CAM increase as a function of embryonic age in a manner coincident with the increasedaccumulation of calcium by the developing embryo in ovo.

INTRODUCTION

The chorioallantoic membrane (CAM) of the chick embryo is the tissue respon-sible for translocating over 140 mg of eggshell calcium into the embryonic circulationduring development (Terepka et al. 1976). The CAM is formed as a result of theprogressive fusion of the chorionic and allantoic membranes so that, by incubationday 10, it completely surrounds the embryo and other contents of the egg andbecomes attached to the shell/shell membrane (Romanoff, 1961). The calcium-transport function of the CAM is highly developmentally regulated; activity beginsaround incubation day 12-13, rapidly increases in level thereafter, and reaches amaximal level around day 18-19 (Terepka et al. 1976; Tuan & Zrike, 1978). Thefunctionally active CAM exhibits a three-layered architecture, consisting of theectoderm, the mesoderm and the endoderm (Coleman & Terepka, 1972a), with theectoderm being directly adjacent to the calcium-rich shell membrane. Previous invitro studies carried out by Coleman & Terepka (19726) have shown that theectoderm, a columnar-like epithelium intercalated with a capillary bed (Narbaitz,1977), is the calcium-transporting region of the CAM. The transport activity of theCAM is highly specific for calcium, which is mobilized unidirectionally and in anenergy-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 transportchamber set-up (Garrison & Terepka, 1972a; Dunn, Graves & Fitzharris, 1981) forin vitro measurements, and the in vivo protocol involving the construction of anuptake chamber over the CAM in situ (Crooks & Simkiss, 1975; Tuan & Zrike,1978). Although much useful information on the kinetics and energetics of theCAM calcium-uptake/transport process has been and may be obtained using theseprocedures, they have inherently limited applicability for further analysis at asubcellular level, since whole tissues are used. In this work, we have further studiedthe 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 theexperimental validity of using these in vitro systems for studying CAM calciumtransport, on the basis of a detailed characterization and comparison with thepreviously established in vivo uptake assay system.

MATERIALS AND METHODS

Chick embryos and CAMFertilized white Leghorn chicken eggs were incubated at 37-5 °C in a humidified commercial egg-

incubator for the desired period of time. Whole CAM was harvested by dissecting it away from theshell membrane and was rinsed clear of adhering materials with cold physiological saline.

Preparation of microsomesAll 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-glasshomogenizer 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 thehomogenate at 11 000 g for 20 min. Microsomes were pelleted after centrifugation at 80 000 g for80 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 uptakeCalcium 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 of45Ca (1—3 /iCi; Amersham Corp.) was introduced into a transport chamber constructed on top ofthe CAM. After 15 min of incubation, the buffer was removed and the radioactivity retained by thesegment of the CAM underlining the chamber was determined. Calcium-uptake activities at 25CCwere expressed as mol calcium min"' cm"2.

Calcium uptake by tissue disks in vitro. After rinsing in physiological saline, freshly dissectedCAM 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 SaltSolution containing 12-7/iM-CaClz and tracer quantities of '"CaC^. After bathing in the abovesolution 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 unincorporatedradioactivity, solubilized with NCS (Amersham Corp.) and counted for radioactivity. Calcium-uptake 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-CaCl2, and tracer quantities of 45CaCl2 ( - S x l ^ c t s m i n " 1 ml"1) at 37°C. At the end ofspecific 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 capacitypolypropylene microcentrifuge tube and centrifugation was carried out at 13 000 £ for 1 min. Theaqueous phase containing free, unincorporated calcium and the bulk of the silicone oil werethen removed by aspiration. The membrane pellet was solubilized in ACS II (Amersham) fordetermination of incorporated radioactivity by liquid scintillation counting. As shown later inFig. 3B, microsomal calcium uptake measured by this method was kinetically linear for up to 3 minof incubation, whereas controls in the absence of ATP did not exhibit any time-dependent calciumuptake.

Isotopic determination of Ca2+/ATP ratio in microsomal calcium uptakeThis was carried out by assaying the release of /-PO* from[y-32P]ATP (Amersham Corp.)

during microsomal calcium uptake. The procedure used was that of Seals et at. (1978), in whichsodium dodecyl sulphate was used to terminate microsomal calcium uptake in the presence of[y-32P]ATP followed by extraction of the phosphomolybdate complex into xylene/isobutanol toseparate 32P, from [y- P]ATP, and quantification by liquid scintillation counting. Calcium-specific ATP hydrolysis was based on the difference in hydrolysis in the presence and absence ofO'l mM-CaCl2 in the assay mixture.

Electron microscopySamples of CAM microsomes were processed for electron microscopy as described previously

(Tuan, 1979): fixation was with 2% glutaraldehyde in cacodylate buffer (pH7-4), postfixationwith osmium tetroxide, en bloc staining with uranyl acetate, and embedding in Epon. Ultrathinsections were lead-stained and examined on a JEOL 100S electron microscope.

Protein assayProtein was determined by the method of Lowry et a!. (1951) with bovine serum albumin

(Sigma Chemicals) as a standard.

ReagentsAll chemicals used were of reagent grade. Radiolabelled compounds and liquid scintillation

chemicals were purchased from Amersham Corp. (Chicago, IL). Oligomycin and dinitrophenolwere 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 thein situ uptake chamber technique for the study of CAM calcium uptake in vivo. Inthis study, we have further applied this in vivo procedure to characterize the uptakefunction with respect to ion specificity. As shown in Fig. 1, the CAM exhibited ahighly specific affinity for the uptake of calcium as demonstrated by the ability ofnon-radioactive "^Ca to inhibit 45Ca uptake completely, whereas other divalentcations were only partly effective or totally ineffective. The inhibitory effects of theseions appeared to be dependent on how similar their ionic radii were to that of Ca2+,suggesting that active uptake required a specific ionic structure. It is noteworthy, asshown in Fig. 1, that the ion specificity of CAM calcium uptake also bore remarkableresemblance 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 inMaterials and Methods. The uptake buffer in each test included an additional 1 mM ofeach of the cations indicated and trace quantities of 4SCa. The uptake activities obtainedare expressed as percentages of that in the absence of additional non-radioactive cationsand are plotted against the ionic radii of the cations (Handbook of Chemistry, 1975). Forcomparison, 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 calciumuptake 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 assayfor CAM calcium uptake. From the analyses described below, the two systemsstudied here, CAM tissue disks and CAM subcellular microsomal membranevesicles, were both found to exhibit genuine, active calcium uptake.

Tissue disks of CAM. As shown in Fig. 2, calcium uptake by CAM disks obtainedfrom day-17 chick embryos was linear for up to 6min of incubation at 25 °C andunder the experimental conditions used here. For all subsequent comparativestudies, uptake rates were routinely calculated on the basis of kinetics between 3 and6min after the start of incubation. The energy-dependent nature of the CAMcalcium-uptake function was demonstrated by the substantially lowered rate ofuptake in the cold (0°C) and in the presence of energy poisons such as oligomycinand dinitrophenol in the incubation buffer (Fig. 2, inset).

CAMmicrosomes. The microsomal membranes prepared from the CAM of 17-daychick embryos were examined by electron microscopy, which revealed the predomi-nant presence of intact, vesicular structures (Fig. 3A), probably originating fromboth 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 microsomalmembranes 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 thatthe time-dependent accumulation of calcium by the membranes pelleted throughsilicone oil was indeed a result of uptake, initial experiments included first layering25 il of 1 mM-EDTA on top of the silicone oil before centnfugation, so that when themicrosomal mixture was added to the tube and spun any non-sequestered calciumshould 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 theabsolute amount of calcium associated with the membrane pellet was decreasedas a result of removal of non-specifically and externally adsorbed calcium. In allsubsequent comparative studies, microsomal calcium uptake was calculated from thekinetics of net uptake during the first 2min of incubation. Furthermore, it was ob-served that CAM microsomal calcium uptake was temperature-dependent (Fig. 4).This characteristic, taken together with those presented below, strongly suggestedthe active nature of the process of microsomal calcium uptake.

I•c

3

0 2 4 6Time (min)

Fig. 2. Kinetics of calcium uptake by CAM tissue disks. The assay was carried out at25°C using tissue disks (l'27cmz) of the CAM of 17-day embryos as described inMaterials and Methods. All data were the mean ± S.E.M. of three to four separateexperiments (with triplicates for all time points in each experiment). Inset: effect of lowtemperature and metabolic poisons on calcium uptake. In this experiment, the CAMdisks from 17-day embryos were first pre-incubated either at 0°C or in the presence ofoligomycin ( 1 X 1 0 ~ 5 M ) or dinitrophenol (DNP; 1 X 1 0 ~ 4 M ) for 30min and then assayedfor calcium uptake at low temperature or with poisons. The activities (uptake rates) arethe mean ± S.E.M. of triplicates expressed relative to that of control.

78 R. S. Tuan and others

200

0 1 2 3 4Time (min)

Fig. 3. A. Ultrastructure of CAM microsomes. Electron microscopy of CAM micro-somes isolated from 17-day embryos revealed abundant vesicular structures of varyingsizes (0-1—0-3 fim diameter). Both ribosome-bearing endoplasmic membranes and smoothvesicles are present. Bar, 1 ^m. B. Kinetics of calcium uptake by CAM microsomalmembranes. The assay was carried out at 37°C using microsomes prepared from 17-dayto 18-day chick embryos as described in Materials and Methods. ( • • ) Calciumuptake in the presence of 5 mM ATP; (O O) calcium uptake in the absence of ATP.For comparison, all values of calcium uptake (nmol Ca2+/mg microsomal protein) wereexpressed as percentages of the value at 2 min (== 1-4nmol Ca2+mgprotein"1). The datarepresent the mean ± S.E.M. of four experiments with triplicate or quadruplicate for alltime points in each experiment. Inset: effect of centrifugation through EDTA on themeasurement of calcium uptake by CAM microsomes. The microsomal suspension wascentrifuged through a layer of 2 mM EDTA lying on top of the silicone oil as described inthe text. Although the overall radioactivity levels were lower, the rate of microsomalcalcium accumulation remained unaltered compared to controls. (Note: in the aboveexperiments, the mean microsomal calcium uptake rate was lS^pmols" 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 activityappeared to be linearly dependent on [Ca2+] up to 2mM, above which saturationoccurred (Fig. 5). Lineweaver-Burke analysis of the data obtained from microsomesisolated 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

T 6

'53oo.00

- E 4

|'S

10 20 30Temperature (°C)

40

Fig. 4. Effect of incubation temperature on calcium uptake by CAM microsomalmembranes. Microsomes were obtained from the CAM of 15-day to 16-day embryos andassayed for calcium uptake as described in Materials and Methods at the indicatedtemperatures.

200

a3

S

'S

100

0 1 2 3 4 5[Ca2+] (IDM)

Fig. 5. Calcium-uptake activity of CAM microsomal membranes as a function of [Caz +].Microsomes were obtained from the CAM of 17-day embryos and assayed for calcium-uptake activity as described in Materials and Methods at the indicated calciumconcentrations. 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 ofapproximately 0-3 mM. This latter rinding corresponded well with those reported byTerepka et al. (1969) and by Garrison & Terepka (19726) who also observedsaturation at [Ca2+] ^ 1 mM and Km of 0-28 mM in CAM calcium transport studieswith the Ussing chamber method.

Ionic requirement. Calcium uptake by the CAM appeared to be dependent onexternal sodium. As shown in Table 1, lowering [Na+] in the bathing buffer bypartial or total substitution of NaCl with choline chloride resulted in decreasedcalcium uptake by CAM tissue disks. This rinding was consistent with that pre-viously reported by Terepka et al. (1976), who also observed [Na+] dependence ofcalcium transport in Ussing chamber studies of whole CAM in vitro. We alsoobserved that both CAM tissue disks and cell-free microsomes exhibited decreaseduptake activity when treated with ouabain, the Na+,K+-ATPase inhibitor (Table 1),further suggesting that proper Na+ (and/or K+) balance was probably essential forfunctional calcium uptake by the CAM.

To gain further insight into the nature of the Na+ (and/or K+) requirement incalcium uptake, we made use of the cell-free microsomes and carried out analysis ofcalcium uptake under the following experimental conditions: (1) pre-loading micro-somes with buffer in which K+ was substituted with either Na+ or choline; and

Table 1. Ionic requirement of CAM calcium uptake

Treatment

Control

Ouabain (/ZM)f

Substitution of Na+ with cholinein uptake buffer

Pre-loading microsomes withf

Substitution of K+ in uptakebuffer with

0-11

10

100% choline20 % choline10 % choline

Na+

Choline

Na+

Choline

Relative calcium-uptake activity (%)*

CAM tissue disks

100

42 ± 2 (2)61 ±6 (2)59 ± 5 (2)

110±10(2)—

—

CAM microsomes

10048±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 inMaterials and Methods and are expressed as percentages (±S.E.) of control values. In thesemeasurements, triplicate samples were used in CAM tissue disk experiments for all time points (seeFig. 2) and quadruplicates were used for the microsomal experiments (see Fig. 3B). The numberof experiments, each using =20 embryos (day 16-17), for the data points is indicated in par-enthesis. The ranges of control values of calcium uptake in these experiments were: 40-50pmolmin~1cm~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) inuptake buffer containing ouabain at the indicated concentrations immediately before assay andwere compared with controls incubated similarly in the absence of ouabain.

| Microsomes were pre-loaded by homogenization and suspension of the membrane pellet inbuffer C in which KC1 was replaced with equimolar NaCl or choline chloride.

Ca transport by chorioallantoic membrane. I 81

(2) suspending control, K+-loaded microsomes in buffers containing either Na+ orcholine in place of K+. As shown in Table 1, the results appeared to indicate that thepresence of external K+ was the only specific requirement for functional calciumuptake. Perturbation of this K+ balance probably resulted in the substantial decreaseof microsomal calcium-uptake activity in the presence of ouabain.

ATP dependence. Calcium uptake by the cell-free microsomes was absolutelydependent on ATP. Substitution of ATP with either GTP or ADP at identicalconcentrations resulted in significantly diminished calcium-uptake activity (5-30 %of control) by CAM microsomes. The stoichiometry of the bioenergetic requirementof microsomal calcium uptake was also determined from simultaneous analyses of therates of calcium uptake and ATP hydrolysis (see Materials and Methods). Theseexperiments yielded an ATP/Caz+ ratio of 4-6 (three experiments), indicating anapparent inefficiency as compared to other ion-transporting membrane systems, suchas 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 CAMtissues studied in an Ussing transport chamber, also indicating that the CAM calcium-transport system had a much higher energy requirement compared with other activetransport 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 embryonicage (Terepka et al. 1976; Tuan & Zrike, 1978), we next measured in vitro calciumuptake by CAM tissue disks and microsomal membranes of embryos at various stagesof development in order to assess the relevance of the two in vitro systems to CAMcalcium transport in vivo. As shown in Figs 6A,B, the age profiles of CAM uptakein 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 wasdevelopment-specific and occurred around incubation days 12—14.

DISCUSSION

In this investigation, we have characterized the process of calcium uptake by thechick embryonic CAM using both in vivo and in vitro methods. Our resultsdemonstrate the active nature, and the ion specificity and dependence of the trans-port function, and further confirm its development-specific pattern of expressionduring chick embryonic development.

Our main objective here was to devise in vitro methods that would permit theanalysis 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

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 embryonicdevelopment. Microsomes and tissue disks were prepared from the CAM obtained fromembryos at various developmental stages and assayed for calcium uptake as describedin Materials and Methods. B. In vivo calcium uptake activity of the CAM and theaccumulation of calcium by the developing embryo as a function of embryonic develop-ment. The data for CAM calcium uptake in vivo were from Tuan & Zrike (1978) andthose for embryonic calcium content from Romanoff (1967).

provide valid measurements of active calcium uptake by the CAM. It is particularlynoteworthy that CAM calcium uptake measured by in vitro methods exhibitsactivity-development profiles similar to those based on in vivo measurements andtotal embryonic calcium contents, thereby strbngly indicating the physiologicalrelevance of these in vitro data. In fact, assuming the in vitro CAM tissue diskcalcium-uptake rate (at the saturating level of 1 mM-Ca2+) and a total egg surface areaof ~60cm , the amount of daily calcium accumulation by the chick embryo atincubation day 15 is estimated to be ~10mg, which compares favourably with theactual 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 thedevelopmentally regulated characteristics of its calcium transport function havemade the CAM an attractive and useful experimental model for the study oftranscellular calcium transport (Terepka et al. 1976). Although much informationhas been accumulated from the work in several laboratories, the mechanism andmode of regulation of the CAM calcium-transport function remain to be resolved.Results from our laboratory strongly suggest that three biochemical moieties, acalcium-binding protein (Tuan & Scott, 1977), a Caz+-activated ATPase (Tuan &Knowles, 1984) and carbonic anhydrase (Tuan, 1984; Tuan & Zrike, 1978) areprobably functional components of the CAM calcium-transport machinery. Asdemonstrated in the accompanying paper, the in vitro assay systems devised here canbe 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 DefectsFoundation (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 calciumsodium, 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 ofthe embryonic chick chorioallantoic membrane. II. Demonstration of intracellular locationduring 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 chickchorioallantoic 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 transportin the isolated chick chorioallantoic membrane. J. Membr. Biol. 7, 128-145.

GARRISON, J. & TEREPKA, A. (19726). The interrelationships between sodium ion, calciumtransport, 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: CRCPress.

LOWRY, O., ROSEBROUGH, N., FARR, A. & RANDALL, R. (1951). Protein measurement with theFolin phenol reagent. J. biol. Chem. 193, 265-275.

MALSTROM, K. & CARAFOU, E. (1979). Calcium transport in mitochondria. In MembraneBiochemistry (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 ofcalcium. Symp. Soc. exp. Biol. 30, 117-140.

TEREPKA, A., STEWART, M. & MERKEL, N. (1969). Transport function of the chick chorio-allantoic 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 embryonicchorioallantoic membrane. J . biol. Chem. 254, 1356-1364.

TUAN, R. (1980). Calcium transport and related functions in the chorioallantoic membrane ofcultured shell-less chick embryos. Devi Biol. 74, 196-204.

TUAN, R. (1983). Supplemented eggshell restores calcium transport in chorioallantoic membraneof cultured shell-less chick embryos. J. Embryol. exp. Morph. 74, 119-131.

TUAN, R. (1984). Carbonic anhydrase and calcium transport function of the chick embryonicchorioallantoic membrane. Ann. N.Y. Acad. Sci. 429, 459-472.

TUAN, R. (1985). Ca2+-binding protein of the human placenta: Characterization, immunohisto-chemical localization and functional involvement in Caz+ transport. Biochem.J. 227, 317-326.

TUAN, R. & KNOWLES, K. (1984). Calcium-activated ATPase of the chick embryonic chorio-allantoic membrane. Identification, developmental expression, and topographic relationshipwith calcium-binding protein. J . biol. Chem. 259, 2754-2763.

TUAN, R. & SCOTT, A. (1977). Calcium-binding protein of chorioallantoic membrane: Identifi-cation 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-bindingprotein in the chick chorioallantoic membrane. .7. biol. Chem. 253, 1011-1016.

TUAN, R. & ZRIKE, J. (1978). Functional involvement of carbonic anhydrase in calcium transportof 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)