THE JOURNAL OF BIOLOGICAL CHEM~~Y Vol. 253, No. 4, Issue of February 25, pp. 1011-1016, 1978

Prmfed in U.S.A

Purification and Characterization of Calcium-binding Protein from Chick Chorioallantoic Membrane*

(Received for publication, September 13, 1977)

ROCKY S. TUAN,$ WILLIAM A. SCOTT, AND ZANVIL A. COHN

From the Laboratory of Cellular Physiology and Immunology, The Rockefeller University, New York, New York 10021

A procedure is described for the purification of the cal- cium-binding protein (CaBP) from the chorioallantoic membrane of the chick embryo. With this scheme, a HO- to ZOO-fold purification was achieved with a 40% yield. Char- acterization of the CaBP revealed that its properties differ from those of previously studied calcium-binding proteins. The CaBP has a molecular weight of 95,000 to 100,000 and appears to be composed of four subunits of identical molec- ular weight (22,000 to 25,000). The CaBP is a basic protein as indicated by its high electrophoretic mobility under acidic conditions and its relatively high isoelectric point of 8.06. The calcium-binding activity of the CaBP is sulfhydryl dependent and highly specific for calcium ions (10 high affinity sites, k,, = 2.35 x 10’ M-l; 100 to 120 low affinity sites, k, = 2.00 x lo5 M-l). Amino acid analysis indicated that the CaBP contains 2 to 10 residues of a modified amino acid, y-carboxyglutamate (y-CGlu). The presence of r-CGlu residues suggested that vitamin K may be involved in the expression of the CaBP in the chorioallantoic mem- brane.

Skeletal calcification in a vertebrate embryo requires the continuous acquisition of large amounts of calcium from extraembryonic sources. In the chick embryo, this calcium is supplied from the eggshell (1, 2) and is mobilized into the embryonic circulation by the placenta-like chorioallantoic membrane (3). The chorioallantoic membrane is an extraem- bryonic cellular membrane which, after the 10th to 11th day of incubation, completely surrounds the embryo and lines the internal surface of the porous acellular shell membrane adja- cent to the eggshell. The ectodermal epithelium of the cho- rioallantoic membrane faces the eggshell and contains numer- ous capillaries originating from the chorioallantoic circulation (4). Thus, the chorioallantoic membrane ectoderm represents the only cellular barrier between the calcium source, the eggshell, and the embryonic circulation. The chorioallantoic

* This work was supported in part by Grants HD 10299-01 and AI 07012 from the United States Public Health Service, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Present address, Department of Orthopaedic Surgery, Children’s Hospital Medical Center, Harvard Medical School, Boston, Massa- chusetts 02115

membrane is, therefore, the natural and logical tissue respon- sible for the mobilization of calcium from the eggshell into the embryo.

The pathway and mechanism of calcium transport by the chorioallantoic membrane has been the subject of several recent studies (5-12). These have shown that the chorioallan- toic membrane ectoderm carries out an active unidirectional transcellular transport of calcium from the eggshell to the embryonic circulation. The transport function is energy de- pendent, highly specific for calcium ions, and is not associated with a calcium-dependent ATPase activity. Furthermore, a distinctive feature of the chorioallantoic membrane calcium transport activity is its dependence on embryonic development (5, 10). The experimentally measured chorioallantoic mem- brane calcium transport rate (5) and the actual accumulation of calcium by the embryo (13) commence around the 12th to 14th day of incubation, increase rapidly thereafter, and reach maxima1 levels shortly before hatching on day 21. These observations thus indicate that the expression of calcium transport activity in the chorioallantoic membrane is a highly regulated function of embryonic development.

In a previous communication (14), we reported that extracts of calcium-transporting chorioallantoic membrane exhibit en- hanced calcium-binding activities which are attributable to a high molecular weight calcium-binding protein. Expression of the CaBPI occurs simultaneously with the onset of calcium absorption from the eggshell by the chorioallantoic membrane (5), and the temporal increase in CaBP activity is coincident with calcium deposition in the embryo (13). This strongly suggests that the CaBP may be important in the process of calcium transport in the chorioallantoic membrane.

To fully understand the role of this CaBP in the transport function of the chorioallantoic membrane, it is necessary to isolate the CaBP and study its properties with respect to those of the calcium transport activity of the chorioallantoic membrane. We report here the purification and characteriza- tion of the CaBP of the chorioallantoic membrane.

MATERIALS AND METHODS

Embryos

Fertilized white Leghorn eggs were purchased from Shamrock Farms (North Brunswick, N.J.) and incubated at 37.5” in a humidi-

’ The abbreviations used are: CaBP, calcium-binding protein; pCMBS, p-chloromercuribenzene sulfonate; y-CGlu, y-carboxyglu- tamic acid; SDS, sodium dodecyl sulfate.

1011

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1012 Calcium-binding Protein of Chorioallantoic Membrane

lied egg incubator. Eggs were routinely examined by transillumi- nation for fertility and normal development of the embryo.

Reagents

All chemicals used were of analytical reagent grade.

Assay for Calcium-binding Activity

Calcium-binding activity was measured by the Chelex 100 (Bio- Rad) ion exchange method (14) in a Tris buffer which contained 13.7 mM TrisiHCl at pH 7.4, 0.12 M NaCI, 4.74 rnM KCI, 98.5 p~ glucose, 0.71 rnM dithiothreitol, and 0.02% sodium azide. Units of calcium-binding activity were calculated as (radioactivity in super- natant)/(radioactivity retained by resin) and expressed as a percent- age. The Chelex 100 calcium-binding assay was validated with a known calcium chelator, ethylenediaminetetraacetic acid. The ex- perimental results showed that a linear relationship exists between the measurable calcium-binding activity and the amount of EDTA in the assay mixture. The proportionality was maintained up to 150 units of calcium-binding activity. A similar linear relationship was also obtained with extracts of chorioallantoic membrane (see below).

Isolation and Purification of CaBP from Chorioallantoic Membrane

The chorioallantoic membrane of 19- to ZO-day-old chick embryos was used. All operations were carried out at 4” unless specified otherwise.

Step I: Preparation of Chorioallantoic Membrane Extracts -This was performed as described previously (14). The chorioallantoic membrane was homogenized in a Waring Blendor for four 15-s intervals with four volumes (v/w) of Tris buffer. The soluble cho- rioallantoic membrane extract is defined as the supernatant ob- tained after centrifugation at 31,000 x g for 30 min.

Step 2: Ammonium Sulfate Fractionation -Crystalline enzyme grade ammonium sulfate (Schwarz/Mann) was added slowly to the chorioallantoic membrane extract with stirring to achieve 40% salt saturation (243 g of ammonium sulfate per liter). After complete dissolution of the salt, the sample was allowed to stir for 30 min. The precipitate was removed by centrifugation at 13,000 x g for 20 min. Additional ammonium sulfate (63 g/liter) was added with stirring to reach 50% salt saturation. After stirring for at least 4 to 5 h, the precipitated CaBP-containing fraction was obtained by centrifugation at 13,000 x g for 20 min, redissolved in a minima1 volume of Tris buffer and dialyzed extensively against the same buffer to remove the residual ammonium sulfate. During the precip- itation procedure, the sample was maintained at pH 7.4 by dropwise addition of 1 N NaOH.

Step 3: Gel Filtration -This was performed on a Sephacryl S-ZOO (Pharmacia) column, 2.6 x 92 cm. Approximately 6 to 8 ml of the sample (4 to 6 mg of protein/ml) obtained from the ammonium sulfate step was applied to the column which was then eluted with Tris buffer at a flow rate of 40 ml/h. The column fractions were assayed for calcium-binding activity, and the fractions containing the activity, usually 15 to 20 fractions, were pooled. The CaBP activity contained in the pooled fractions was precipitated with 70% ammonium sulfate at pH 7.4 for 2 to 3 h, collected by centrifugation at 13,000 x g for 20 min, and redissolved in a minimal volume of Tris buffer. Ammonium sulfate was subsequently removed by exten- sive dialysis against Tris buffer.

Step 4: Preparative Isoelectric Focusing -This was performed as described by Haglund (15) with a llO-ml isoelectric focusing column (LKB) in 0 to 40% sucrose gradient containing 2% ampholytes (LKB Ampholine, pH 7 to 9) and 0.71 rnM dithiothreitol. The sample (10 to 15 mg of protein) obtained from the gel filtration step was introduced into the gradient solutions, and the gradient was formed at approx- imately 100 ml/h. Electrofocusing was carried out with the cathode at the bottom of the column for 40 to 44 h at 700 V. The column was then eluted at a rate of 200 ml/h, and fractions of 1.8 ml were collected. The pH and calcium-binding activity of each fraction were measured immediately. The fractions containing CaBP activity were pooled and dialyzed extensively (at least 48 h) against 1 N NaCl, 0.71 mM dithiothreitol to remove most of the protein-bound ampholytes. Complete removal of the ampholytes was achieved by precipitating the CaBP with 70% ammonium sulfate followed by centrifugation at 13,000 x g for 20 min. The purified CaBP was redissolved in Tris buffer and dialyzed against the same buffer.

Protein Determination Protein was estimated by the method of Lowry et al. (16) with

bovine serum albumin (Armour) as a standard.

Polyacrylamide Gel Electrophoresis

Nondenaturing Acid Ge( System -The procedure used was a modification of the method of Panyim and Chalkley (17). The electrophoresis buffer contained 5% acetic acid and 2 rnM EDTA, pH 3.2. Samples containing approximately 5 mg of protein/ml were adjusted to pH 3.2 with acetic acid and applied in 20- to 30-~1 volumes to the gel slab (7.5% acrylamide, 17 to 18 cm). Electropho- resis was carried out at room temperature toward the cathode at a constant current of 2 mA/sample.

Sodium Dodecyl Sulfate-denaturing Gel System -The procedure was modified from that used by Laemmli and Favre (18) for discon- tinuous gel electrophoresis in the presence of SDS. The gel slab (17 to 18 cm) consisted of a stacking gel (6% acrylamide, 1.5 to 2.0 cm) and a separating gel (10% acrylamide, 15 to 16 cm). Fifty micrograms of each denatured protein sample were applied to the gel. Electro- phoresis was carried out at room temperature in a buffer containing 0.005 M Tris/HCl, pH 8.6, 0.38 M glycine, 0.1% SDS at 60 V until the samples reached the separating gel and was continued at a constant voltage of 120 V for 2 to 2i12 h. For molecular weight determinations, the gel was calibrated with molecular weight markers (nos. 44223 and 44230) obtained from BDH Chemicals

The gel slabs were routinely stained with Coomassie blue accord- ing to standard procedures (17, 18).

Analytical Gel Filtration

The molecular weight of the purified CaBP was determined by gel filtration on a column (1.5 x 28 cm) of Ultrogel AcA 44 (LKB). The column was eluted with Tris buffer at 3.2 ml/h and was calibrated with the following molecular weight markers (Pharma- cia): ribonuclease (13,700 daltons), chymotrypsinogen A (25,000), ovalbumin (45,000), and aldolase (158,000). The elution profile was monitored at 280 nm. Fractions of 1.0 ml each were collected and assayed for calcium-binding activity by the Chelex 100 method.

Determination of Binding Constants of CaBP

The calcium-binding constants of the CaBP in Tris buffer were determined with a calcium-specific electrode (Calcium Selectrode, Radiometer). The response (mV) of the calcium-specific electrode was calibrated with standard CaCl, solutions in Tris buffer and was linear with respect to calcium concentrations from lo-” to IO-’ M.

For measurements of calcium binding, 3.0 ml of an approximately 3 to 5 rnM standard CaCI, solution in Tris buffer was titrated with a solution of purified CaBP of known concentration (-0.5 mg/ml in Tris buffer) at 25”. After each addition of CaBP, the solution was allowed to equilibrate for at least 3 to 5 min with slow stirring before the reading (mV1 was recorded from the pH meter. Based on the calibrated curve, the mV readings were converted to the corre- sponding calcium concentrations in the solution. The data were plotted according to Scatchard (19).

Amino Acid Analysis

The CaBP was dialyzed exhaustively against distilled water, lyophilized, and hydrolyzed in (a) 6 N HCl in cacao (20) or (b) 2 N KOH under N, at 110” for 22 to 24 h (21). The hydrolysates were dried under reduced pressure and then dissolved in 0.2 M sodium citrate buffer, pH 2.2, and analyzed on a Beckman-Spinco model 121 amino acid analyzer.

RESULTS

Purification of &BP -A simple and reproducible purifica- tion scheme for the CaBP of the chorioallantoic membrane was developed (see under “Materials and Methods”). The entire chorioallantoic membrane from 19- to ZO-day-old chick embryos was used as starting material, including that part of the chorioallantoic membrane (polar region) which lies under- neath the air space and is separated from the eggshell. No difference in the levels of calcium-binding activity per mg of protein in extracts from this region of the chorioallantoic membrane and in extracts from the equatorial region of the chorioallantoic membrane was observed. The results of a typical purification are given in Table I. The procedure has been repeated at least 50 times. In every case, a 170- to 200- fold purification of the CaBP was obtained with a yield of approximately 40%.

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Recoveries of calcium-binding activity during purification were low (<lo%) in the absence of reducing agents. Therefore, dithiothreitol (0.71 mM) was included in all buffers to main- tain the stability of the CaBP. Likewise, the purified CaBP also appeared to be stable at 4” in Tris buffer with 0.71 mM dithiothreitol. More than 95% of the calcium-binding activity was retained after storage for 2 weeks under these conditions. However, in the absence of dithiothreitol, all activity of the purified CaBP was lost in less than 2 days at 4”. Samples of CaBP stored frozen at -20” usually showed signs of floccula- tion and loss of activity after thawing, whereas the activity was completely destroyed at temperatures higher than 50 to 60”.

The dependence of the CaBP activity on dithiothreitol suggested that one or more sulfhydryl groups are essential

TABLE I

Purification of chorioallantoic membrane CaBP

Purlficatlon step Volume Tota1 Total Specific Y,eld protein’ actlvityb activity

“;;&:r- tmn

ml w units unitslmg %

Chorioallantoic 300 660 4950 7.5 100 1 membrane ex- tract’

Ammonium sulfate 15 63.4 2822 44.5 57.0 6.0 precipitation

Gel filtration 76 23.1 3138 135.8 63.4 19.0 Isoelectric focusing 16.2 1.3 1772 1363.1 35.8 181.7

a Protein was determined by the method of Lowry et al. (16). ’ Calcium-binding activity was determined by the Chelex 100

assay (14). c The chorioallantoic membrane extract was prepared from the

chorioallantoic membrane of 19- to 20-day-old chick embryos.

I 0

I 2 4 6 4

PCMBS (mtvl)

for calcium binding. This is supported by the inhibitory effect of the sulfhydryl-binding agent, p-chloromercuribenzene sul- fonate, on the calcium-binding activity in chorioallantoic membrane extracts. As shown in Fig. 1, more than 50% of the activity in chorioallantoic membrane extracts was inhibited by 6 mMpCMBS. A similar inhibitory effect was also observed with comparable levels of mersalyl acid, another sulfhydryl- binding agent.

Molecular Weight of CaBP - Elution profiles from Ultrogel AcA 44 revealed a single coincident peak of activity and protein. By calibration of similar columns with marker pro- teins of known molecular weights, the molecular weight of the CaBP was estimated to be in the range of 95,000 to 100,000.

Isoelectric Point of CaBP -The isoelectric point (~1) of the CaBP was determined by column isoelectric focusing of the purified CaBP in a pH gradient of 7 to 9. The elution profile showed a single, sharp calcium-binding activity peak. Based on fifteen separate determinations with different batches of purified CaBP, the average p1 of the CaBP was 8.06 + 0.10.

Electrophoretic Mobility of CaBP - The electrophoretic be- havior of the CaBP was studied with a nondenaturing acidic gel electrophoresis system. Due to its basicity, the CaBP was expected to exhibit a high electrophoretic mobility toward the cathode under acidic conditions. Fig. 2A shows the electropho- retie patterns of a crude 19- to 20-day-old chorioallantoic membrane extract prior to purification and of the purified CaBP preparation. As expected, the purified CaBP migrated as a single band of protein with an R,,, of 0.86.

The acidic gel electrophoresis system was also employed to study the course of expression of the CaBP in the chorioallan- toic membrane during embryonic development. Fig. 3 shows the electrophoretic patterns of chorioallantoic membrane ex-

FIG. 1 (left). Effect ofpCMBS on the calcium-bindirlg activity in the chorioallantoic membrane. The chorioallantoic membrane ex- tract (4.7 mg of protein/ml) was prepared from 19-day-old embryos and was incubated with the indicated concentrations of pCMBS for 15 min at room temperature. The calcium-binding activities of the pCMBS-treated chorioallantoic membrane extracts were determined by the Chelex 100 assay (14) and expressed as a percentage of the untreated chorioallantoic membrane extract. Controls usingpCMBS alone did not exhibit any calcium-binding activity.

FIG. 2 (center and right). A, gel electrophoresis of chorioallantoic membrane (CAM) extract and purified CaBP. This was performed

71.5K- 53.0 = 42.9 #

28.6K- 14.3K-

Mol.

wt. in the nondenaturing acid gel system with the cathode at the bottom of the gel as described under “Materials and Methods.” An extract of 19- to 20-day-old chorioallantoic membrane (100 pg of protein) and 50 pg of purified CaBP were used. The CaBP gel was intentionally overloaded to reveal the absence of other protein components, and the location of the CaBP is indicated (arrow). B, SDS gel electrophoresis of purified CaBP. Twenty micrograms of purified CaBP were used, and electrophoresis was carried out as described under “Materials and Methods.” The molecular weights of standards are as indicated (K, 10”).

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Calcium-binding Protein of Chorioallantoic Membrane

CaBP

lid lad 17d md FIG. 3. Age profile of electrophoretic patterns of chorioallantoic

membrane extract. Extracts of chorioallantoic membrane were pre- pared from embryos of the indicated ages (days of incubation) as described under “Materials and Methods.” The protein load was 100 pg each, and electrophoresis was carried out toward the cathode in the nondenaturing acid gel system described under “Materials and Methods.”

tracts prepared from embryos at different stages. The age- dependent appearance of the fast moving protein band sug- gests that an increase in the absolute amount of the CaBP occurs as a function of embryonic development.

Subunit Composition of CaBP-The subunit structure of the CaBP was investigated by means of SDS polyacrylamide gel electrophoresis. As shown in Fig. 2B, the purified CaBP, denatured in SDS and dithiothreitol, migrated as a single, sharp band of protein with a mobility corresponding to a molecular weight of 22,000 to 25,000, relative to marker proteins. Based on a molecular weight of 95,000 to 100,000 for the native protein determined by gel filtration, the CaBP appears to be composed of four subunits of identical size. Gradual oxidation of the denatured CaBP in air resulted in the appearance of dimer (47,000 to 50,000 M,)- and tetramer (70,000 to 95,000)-like bands upon electrophoresis.

Amino Acid Composition of CaBP-The data from amino acid analysis of an acid hydrolysate of the CaBP are shown in Table II. The CaBP contains large amounts of acidic (73 aspartic acid and 68 glutamic acid) as well as basic (34 lysine and 63 arginine) residues. The number of cysteine residues in the CaBP represents an estimate only since analyses of per-formic ,acid-oxidized samples (22) were not performed.

An interesting finding was that the CaBP contains 2 to 10 residues of a modified amino acid, y-carboxyglutamic acid. The y-CGlu content of the CaBP was determined from the analysis of an alkaline hydrolysate of the CaBP by the method of Hauschka et al. (21). Based on its stability in alkali and its complete conversion into glutamic acid under acidic conditions (21), the y-CGlu of the CaBP was quantified by comparing the y-CGlu and glutamic acid contents obtained from both the acid and alkaline analyses.

Calcium-binding Affinity of CaBP - Initial investigations showed, that the calcium-binding activity in chorioallantoic membrane extracts was highly specific for calcium ions. Fig. 4 shows the relative effectiveness of various divalent cations in inhibiting the binding of Wa by chorioallantoic membrane

TABLE II

Ammo cwd compositzon of &BP

CaPB was hydrolyzed in 6 N HCl. The values reported have not been corrected for any destruction which may have occurred during acid hydrolysis.

Amino acid Residues/Molecule

Aspartic acid 73 Threonine 33 Serine 33 Glutamic acid 68 Glycine 47 Alanine 59 Cysteine (2) Valine 40 Methionine 11 Isoleucine 36 Leucine 78 Tyrosine 38 Phenylalanine 60 Histidine 23 Lysine 34 Arginine 63 y-CGlu” 2-10

n For the determination of y-CGlu, the CaBP was hydrolyzed in 2 N KOH (see “Materials and Methods”). The y-CGlu content repre- sents the data from three separate determinations.

‘Sn ~ Nii

V 80

Mn

Zne $fl Sr

co

Ionic radii (11) FIG. 4. Ion specificity of the chorioallantoic membrane calcium-

binding activity. The 19- to 20-day chorioallantoic membrane extract used was enriched by 40 to 50% ammonium sulfate precipitation (see under “Materials and Methods”) and contained 4.7 mg of protein/ml. The Chelex 100 calcium-binding assay mixtures con- tained 0.1 ml of the chorioallantoic membrane sample, 0.43 jzM WaCI,, and 1.67 rnM of the cations indicated. The calcium-binding activities obtained are expressed as a percentage of that in the absence of additional nonradioactive cations and are plotted against the ionic radii of the cations (23).

extract as measured with the Chelex 100 assay. Nonradioac- tive -“‘Ca completely displaced %a, whereas other divalent cations were either totally ineffective or only partially effec- tive in this respect. The following ion affinity series was obtained from these data: Ca > Cd > Sr, Zn > Mn > Ba, Ni > Sn, Co > Mg.

The binding affinity of the chorioallantoic membrane CaBP for calcium was further studied with the purified preparations of CaBP. The calcium-binding constants of the CaBP were determined with a calcium-specific electrode (see under “Ma- terials and Methods”). From the Scatchard plot of the binding data shown in Fig. 5, two classes of calcium-binding sites on the CaBP are apparent. Ten high affinity sites with a binding constant of 2.35 x 10’ M-’ and 100 to 120 low affinity sites with a binding constant of 2.00 x lo5 M-I were calculated.

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Calcium-binding Protein of Chorioallantoic Membrane 1015

ICal,/[CaBPl FIG. 5. Scatchard plot of calcium binding by the CaBP. The

binding data obtained as described under “Materials and Methods” are represented as follows: [Cal* = concentration of bound calcium, [Cal, = concentration of free ionic calcium, and [CaBPI = concentra- tion of CaBP. From the Scatchard plot, the binding constants were determined from the slopes of the lines, and the number of binding sites from the intercepts on the abscissa (19).

DISCUSSION

The relatively simple four-step purification procedure repro- ducibly yielded 180- to 200-fold purification of the CaBP from the chorioallantoic membrane of 19- to 20-day-old chick em- bryos. The isoelectric focusing step of the purification scheme takes advantage of the relatively high isoelectric point of the CaBP to obtain a highly purified preparation. The chromato- graphic and electrophoretic properties of the purified CaBP indicated the absence of any major protein contaminants.

Several molecular properties of the CaBP have been char- acterized. The high isoelectric point (8.06) of the CaBP is compatible with its amino acid composition (Table II). The p1 of the CaBP is estimated to be approximately 7.5 by the method of Tanford and Hauenstein (24), based on the amino acid composition and the respective pK, of the amino acids. This value is sufficiently close to the actual pI of 8.06, considering the approximations involved in such a calculation (24). The basicity of the chorioallantoic membrane CaBP is not due to a lack of acidic side chains but rather the presence of equally abundant basic amino acid residues, such as lysine and arginine, in the molecule. Furthermore, as pointed out by Kretsinger (251, there appears to be no correlation between the acidity of various calcium-binding proteins and their calcium affinity. The high pI of the chorioallantoic membrane CaBP is therefore not necessarily inconsistent with its cation- binding activity.

The CaBP has a molecular weight of 95,000 to 100,000 and appears to consist of four subunits of identical size (22,000 to 25,000). The interaction of these subunits to form the native CaBP appears to be partially disulfide-dependent, but the exact mechanism is not known. In addition, it remains to be established whether other factors, such as calcium ions, are also involved in the subunit assembly of the CaBP and whether the subunits are indeed identical.

The cation-binding activity of the CaBP is highly specific for calcium ions (Fig. 31, suggesting that the CaBP possesses binding sites with a high degree of stereospecificity and that calcium binding by the CaBP is not a result of simple

electrostatic or ionic interactions (26). The specificity of the CaBP for calcium ions is further affirmed by its high binding constants (high affinity, k, = 2.35 x 10’ M-‘; low affinity, k,, = 2.0 x lo5 M-‘1 and large number of binding sites (10 high affinity sites, 100 low affinity sites). It may be pointed out that, since the soiubilized calcium from the eggshell is contin- uously available to the chorioallantoic membrane in situ, the high calcium affinity of the CaBP should guarantee that it is readily bound with calcium ions.

At present there are more than 70 proteins which have been reported to possess specific and/or physiologically signif- icant calcium-binding activity (25). Many of these proteins are associated with calcium-transporting systems, including the epithelia of the intestinal mucosa (271, the kidney tubules (281, the parathyroid gland (291, the pancreas (301, and the avian shell gland (31). Calcium-binding proteins are also present in several intracellular calcium-regulating systems, e.g. the mitochondrion and the sarcoplasmic reticulum (see references in Ref. 25).

Our findings reveal that the calcium-transporting chorioal- lantoic membrane of the chick embryo contains a unique CaBP. The expression of the CaBP in the chorioallantoic membrane is developmentally dependent (14) and correlates temporally with the accumulation of calcium by the embryo (13) and with the transport activity of the chorioallantoic membrane (5). Unlike most CaBP’s (251, the chorioallantoic membrane CaBP has a remarkably high p1. The chorioallan- toic membrane CaBP exhibits no calcium-dependent ATPase activity (14) and therefore differs from a host of calcium- dependent ATPases present in the mitochondrion and the sarcoplasmic reticulum (25). The high molecular weight and calcium affinities of the chorioallantoic membrane CaBP also distinguish it from a variety of well characterized CaBP’s, such as troponin (321, calsequestrin (331, and phosphovitin (34).

One of the most extensively studied CaBP associated with transepithelial calcium transport is the vitamin D-dependent CaBP of the chick intestinal mucosa (27). In rachitic chicks, vitamin D-mediated stimulation of intestinal calcium trans- port is also accompanied by the concomitant expression of a CaBP. However, the properties of the intestinal CaBP of the adult chick differ significantly from those of the chorioallan- toic membrane CaBP of the chick embryo. The intestinal CaBP consists of a single polypeptide and has a molecular weight of 28,000 and a p1 of 4.0. Two kinds of calcium-binding sites are present on the intestinal CaBP: 4 high affinity sites, k,, = 2 x 10” M-‘; 30 low affinity sites, k,, = 10 to 100 Mm’. The amino acid composition of the intestinal CaBP (43) also differs significantly from that of the chorioallantoic membrane CaBP (Table II). Moreover, antiserum against the intestinal CaBP does not cross-react with chorioallantoic membrane extracts (11). These differences are of particular interest in that they may reflect different modes of expression or regulation, or both, of these CaBP’s.

Amino acid analysis of the CaBP showed that it contains 2 to 10 y-CGlu residues/molecule. The calcium-binding amino acid, y-CGlu, has recently been shown to be formed by a post- translational, vitamin K-dependent enzymatic modification of glutamate residues on protein molecules (35, 42, 44). The presence of y-CGlu in the CaBP therefore suggests that it may also be an activation product of the vitamin K-dependent carboxylation system. Various y-CGlu-containing proteins, such as prothrombin (36) and the bone calcium-binding pro- tein (37), have been shown to be activated in this manner. In

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1016 Calcium-binding Protein of Chorioallantoic Membrane

these proteins, the y-CGlu residues are responsible for the calcium-binding activity. However, the relatively high cal- cium-binding constants and large number of calcium-binding sites of the CaBP, compared to those of other y-CGlu-contain- ing proteins such as prothrombin (10 binding sites, k, = lo4 M-‘1 (251, indicate that a direct role of y-CGlu residues in the actual calcium-binding activity for the CaBP is not likely.

The recent discoveries of several y-CGlu-containing proteins in various tissues (21, 38-41) have generated much interest in the complex metabolic function of vitamin K. The findings reported here suggest that vitamin K may be involved in regulating the activity of the CaBP of the chorioallantoic membrane. Our recent investigation’ has indeed confirmed that the expression of the CaBP in the chorioallantoic mem- brane is vitamin K inducible. The exact molecular mechanism of action of vitamin K in the expression of the calcium transport activity in the chorioallantoic membrane remains, however, to be resolved.

Acknowledgments -We wish to thank Drs. Peter Blackburn and Jane Lian for assistance in the amino acid analyses, Ms. Joanne Zrike for excellent technical assistance, and Ms. Lovice Weller for typing the manuscript.

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R S Tuan, W A Scott and Z A Cohnchorioallantoic membrane.

Purification and characterization of calcium-binding protein from chick

1978, 253:1011-1016.J. Biol. Chem. 

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