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Umeki et al., 1981; Tuan, 1982; MacManus et al., 1985a). The present paper describes the first isolation ofa 9000-Mr rat placental protein, by the use of the ...
Biochem. J. (1986) 235, 585-595 (Printed in Great Britain)

S85

The purification and complete amino acid sequence of the 9000-]Mr Ca2+-binding protein from rat placenta Identity with the vitaniin D-dependent intestinal Ca2+-binding protein John P. MAcMANUS,* David C. WATSON and Makato YAGUCHI Division of Biological Sciences, National Research Council of Canada, Ottawa, Ont. KIA OR6, Canada

A 9000-Mr Ca2+-binding protein was isolated from rat placenta and purified to homogeneity by h.p.l.c. procedures. The complete amino acid sequence was established for the 78-residue placental protein. A sequence analysis of a minor component of the rat intestinal Ca2+-binding protein (residues 4-78) and a tryptic peptide (residues 55-74), both purified by h.p.l.c., showed both proteins to be identical. Thus this placental 9000Mr Ca2+-binding protein is the same gene product as the intestinal Ca2+-binding protein whose synthesis is dependent on vitamin D.

INTRODUCTION In mammals vitamin D controls the synthesis of a Ca2+-binding protein of Mr approx. 10000 in the intestinal mucosa, this being regarded as the major Ca2+-transport protein (Wasserman & Fullmer, 1983). The protein has been isolated from bovine (Fullmer & Wasserman, 1973), human (Davie, 1981; Staun et al., 1983), pig (Hitchman et al., 1973) and rodent tissue (Bruns et al., 1977; Gleason & Lankford, 1981; Delorme et al., 1982; Christakos et al., 1984). The amino acid sequences of the bovine and pig proteins have been determined directly (Hofmann et al., 1979; Fullmer & Wasserman, 1980, 1981). However, only a partial amino acid sequence of the rat intestinal protein has been presented, from studies of mRNA sequences (Desplan et al., 1983) or translation in vitro (Leonard et al., 1984). These reports established that the intestinal CaBP structure, is highly conserved, and belongs to the multi-gene family of proteins that includes calmodulin, troponin C and parvalbumin (Goodman et al., 1979; Klee et al., 1980; Kretsinger, 1980; Baba et al., 1984). Antisera h-ave been generated against the rodent intestinal CaBP, and used to show the presence of a cross-reactive material in extracts of the placenta and visceral yolk-sac of mice and rats (Bruns et al., 1978, 1981; Marche et al., 1978; Delorme et al., 1979, 1982), and also in bone, kidney, lung and teeth (Thomasset et al., 1982; Delorme et al., 1983; Taylor et al., 1984). The equivalence of vitamin D-dependent Ca2+-binding proteins in various tissues on the basis of immunological cross-reactivity alone is further complicated by the presence in mammalian tissues, such as bone, brain, and kidney, of another protein, of Mr 28000, which may or may not be dependent on vitamin D, and which is akin to the intestinal Ca2+-binding protein of chickens (Delorme et al., 1983; Pansini & Christakos, 1984; Sonnenberg et al., 1984). Before accepting the identity of the rat intestinal and placental Ca2+-binding proteins proposed on immunoAbbreviation used: CaBP, 9000-Mr Ca2+-binding protein. * To whom correspondence should be addressed.

Vol. 235

logical grounds (Bruns et al., 1978, 1981; Marche et al., 1978; Delorme et al., 1979), it was considered necessary to isolate each of these proteins from their respective tissues, especially because of the presence in placenta of several proteins that bind Ca2+ (Bruns et al., 1978, 1981; Umeki et al., 1981; Tuan, 1982; MacManus et al., 1985a). The present paper describes the first isolation of a 9000-Mr rat placental protein, by the use of the h.p.l.c. procedures, and its amino acid sequence analysis in conjunction with that of the rat intestinal CaBP. MATERIALS AND METHODS Protein purification from placenta The Ca2+-binding proteins of placenta were purified from 200 g wet wt. of tissue dissected out of conceptus after 18-21 days of gestation in Sprague-Dawley rats Table 1. Purification of the 9000-M, Ca2 +-binding protein from rat placenta For experimental details see the text.

Sp. radioactivity , (c.p.m./ Purificamg of tion of protein) (fold) (mg)

Total protein

Homogenate 6675 Supernatant after heat 2200 treatment 65% satn. with (NH4)2SO4 320 DEAE-Sephacel 14 Sephadex G-50 2.8 H.p.l.c., reverse-phase, 1.6 pH 7.6 H.p.l.c. reverse-phase, 2.1* pH 2.5 *After freeze-drying.

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Fig. 1. Purification of placental Ca2+-binding protein (a) DEAE-Sephacel anion-exchange of proteins remaining in solution after adjustment to 65% saturation with (NH4)2SO4. X marks the place where the Chelex cation-exchange pre-column was disconnected and the start of the wash of the DEAE-Sephacel column with 100 mM-NaCl/5 mM-Tris/HCl buffer, pH 7.5. Y marks the place where gradient elution with NaCl (----) was started. A280; *-*, 45Ca radioactivity. (b) Gel filtration on a calibrated Sephadex G-50 column of the major Ca2+-binding protein in (a). A280; *-*, 45Ca radioactivity. (b) Determination of the apparent Mr of this Ca2+-binding protein. Mr standards were: a, ovalbumin (Mr 45000); b, chymotrypsinogen A (Mr 25000); c, ribonuclease (Mr 13 500); d, oncomodulin (Mr 11 700); e, aprotinin (Mr 6500); f, insulin B-chain (Mr 3 500). ,

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bred in this laboratory. The initial purification steps based on the isolation of a 108-residue Ca2+binding protein, oncomodulin, found in rat hepatoma (MacManus, 1980), and also placenta (MacManus et al., 1985a). A summary of the purification scheme is given in Table 1. Briefly, the placental tissue was homogenised in were

100 mM-NaCl/50 mM-KCl/0.5 mM-MgCl2/100 mMsodium acetate!

mm-phenylmethanesulphonyl

fluoride!

0.1 % 2-mercaptoethanol, pH 7.0 (buffer A). The temperature of the extract was raised in a water bath, and maintained between 65 and 80 °C for 5 min. The extract

rapidly cooled and centrifuged at 10000 g for 30 min. The supernatant was adjusted to 65 % saturation with (NH4)2SO4 (43 g/ 100 ml) and stirred for 1 h. The precipitated proteins were removed by centrifugation, and the supernatant containing the low-Mr components was dialysed extensively against distilled water. This extract was freeze-dried, the resulting powder redissolved in 100 ml of 5 mM-Tris/HCl buffer, pH 7.5, and the solution dialysed overnight against this same buffer. The dialysed material was passed first through a colum-n(l.5 cm x 30 cm) ofChelex-100 (Bio-Rad Laboratories, Mississauga, Ont., Canada) cation-exchange resin was

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Fig. 2. Further purification by h.p.l.c. of the placental Ca2+-binding protein from the purification step shown in Fig. l(b) (a) Reverse-phase h.p.l.c. on C-18 atpH 7.5. , A280; *-*, 45Ca radioactivity. (b) Demonstration of purity ofthe Ca2+-binding protein eluted at 34.5 min in (a) by reverse-phase h.p.l.c. on C-18 at pH 2.5. , A280. Amino acid analysis of this material is given in Table 2.

equilibrated with 5 mM-Tris/HCl buffer, pH 7.5, and then straight on to a tandem column (1.5 cm x 30 cm) of DEAE-Sephacel (Pharmacia, Dorval, P.Q. Canada). After the loading and washing of both columns with 5 mM-Tris/HCl buffer, pH 7.5, the cation-exchanger was disconnected, and elution of the adsorbed proteins on the DEAE column was commenced (Fig. la). Fractions containing Ca2+-binding material were located by the Chelex assay (Wasserman et al., 1968) and pooled for further purification by gel filtration on a column (1.5 cm x 90 cm) of Sephadex G-50 (Pharmacia) equilibrated in buffer A. The single Ca2+-binding peak (Fig. lb) was collected and freeze-dried. The freeze-dried material from the Sephadex G-50 was dissolved in 2 ml of 10 mM-Tris/HCI buffer, pH 7.6, and injected on to a C-18 reverse-phase h.p.l.c. column Vol. 235

(4.1 mm x 250 mm; Synchrom, Linden, IN, U.S.A.) also equilibrated in 10 mM-Tris/HCl buffer, pH 7.6. The proteins were eluted with an increasing concentration (10% per min for 50 min) of propan- 1 -ol, and monitored for Ca2+-binding activity. The peaks of interest were freeze-dried and dissolved in 0.1% trifluoroacetic acid. The dissolved material was injected on to another C-18 h.p.l.c. column equilibrated with 0.1% trifluoroacetic acid, and eluted with an increasing concentration of propan- 1-ol in 0.100 trifluoroacetic acid. The single major absorbance peak, eluted at 40.5 min (Fig. 2b), was collected and freeze-dried. Protein purification from intestinal mucosa In a similar fashion, 50 g wet wt. of intestinal mucosa that had been scraped from the first 10 cm of rat

588

duodenum was extracted, and the proteins were separated by DEAE-Sephacel anion-exchange, gelpermeation and h.p.l.c. steps as described above.

Peptides produced by trypsin and acid cleavage The materials that had been freeze-dried after h.p.l.c. in 0.1% trifluoroacetic acid from placenta or intestine were dissolved (1 mg/200 1I) in 50 mM-NH4HCO3, pH 8.0, and decalcified by addition of saturated trichloroacetic acid to a final concentration of 20% (w/v). The precipitated protein was kept on ice for 15 min, and collected by centrifugation. The pellet was washed in of 50 mm20% trichloroacetic acid, resuspended in 200 10 NH4HCO3/1 mM-EGTA, pH 8.0, and the pH adjusted to 8.0 by dropwise addition of aq. NH3. Without this decalcification these, and other, Ca2+-binding proteins are very resistant to the action of trypsin (Klee et al., 1980; MacManus et al., 1983). The cleavage by trypsin and the method of separation of the resulting peptides by h.p.l.c. has been described previously (MacManus et al., 1983). Cleavage at aspartate residues was performed by mild acid hydrolysis by incubation of protein in 0.25 M-acetic acid at 100 °C in vacuo for 10 h as described previously (MacManus et al., 1983). Amino acid composition and sequence analysis Amino acid composition analysis was performed after hydrolysis in 6 M-HCl at 100 °C in vacuo for 24, 48 and 72 h (MacManus et al., 1983). Sequence analysis of tryptic peptides by liquid-phase methods was as described previously (MacManus et al., 1983). Other peptides were sequenced by automated gas-phase methods with a Applied Biosystems 470 A sequencer by dissolving 1-5 nmol in 30 u1l of distilled water and applying the sample to a cartridge filter containing 1 mg of precycled Polybrene. Identification of amino acid derivatives was by h.p.l.c. (MacManus et al., 1983). Analysis of the N-terminal tripeptide The putative N-terminal tripeptide was obtained from tryptically cleaved protein as described above, but with isocratic elution of the h.p.l.c. column (detailed results not shown) to resolve the peptides eluted in the first 4 min of Fig. 4. Amino acid analysis identified the putative N-terminal tripeptide (Ala1,Lys1,Ser1). A 10 nmol portion of this peptide was dissolved in 50 mM-NH4HCO3, pH 8.0, and subjected to digestion with carboxypeptidases A and B (Sigma Chemical Co., St. Louis, MO, U.S.A.) at a final concentration ratio of 1:25 (w/w) at 37 °C (Ambler, 1972). Samples (50 ll) were removed at 1, 2, 4, 6 and 24 h, and, after the addition of 10 1 of acetic acid, were then freeze-dried. After several cycles of dissolution in 300 ,l of water and re-freeze-drying, the samples were subjected to amino acid analysis. RESULTS Protein purification The purificaiton of the major low-Mr rat placental Ca2+-binding protein is outlined in Table 1. Two Ca2+-binding entities were observed after DEAE-cellulose chromatography, in tubes 14-18 and 27-29 (Fig. 1 a). The latter, minor, Ca2+-binding material was oncomodulin, a

J. P. MacManus, D. C. Watson and M. Yaguchi Table 2. Amino acid composition of the 9000Mr placental

Ca2+-binding protein

Residues were analysed from protein hydrolysed for 24, 48 and 72 h. Corrections were made for hydrolytic loss by extrapolation of values to zero time.

Amino acid composition (mol of residue/mol) Determined

Asx Thr Ser Glx Pro

Gly Ala

Cys Val Met Ile Leu Tyr Phe His

24h

48 h

72 h

Assumed

7.21 1.06 9.64 14.81 3.09 3.10 4.05 0 1.98 0.95 2.02 10.17 2.10 6.12 0 11.84 0

7.30 0.97 8.22 14.76 3.15 3.18 4.10 0 2.03 0.93 2.02 10.14 2.09 6.19 0 11.87 0

7.18 0.89 7.42 14.81 3.14 3.13 4.02 0 2.24 0.90 2.07 10.30 1.90 6.08 0 11.76 0

7 1 10 15 3 3 4 0 2 1

Lys Arg Trp Total * From sequence.

2 10 2 6 0 12 0

-0* 78

108-residue protein found originally in tumours (MacManus et al., 1983, 1985a). The fractions containing the former, major, Ca2+-binding material were pooled, and further purified by gel filtration (Fig. lb) on a calibrated column of Sephadex G-50 (Fig. lc). A single Ca2+-binding peak eluted at an apparent Mr of 9000. When this 9000 Mr Ca2+-bindingmaterial was subjected to h.p.l.c. at neutral pH, a single peak of Ca2+-binding activity was found, co-eluted with a major 280 nmabsorbance peak at 34.5 min (Fig. 2a). This Ca2+-binding protein was collected and further purified by h.p.l.c. in 0.1 % trifluoroacetic acid, resulting in a single major 280 nm-absorbance peak at 40 5 min (Fig. 2b). The amino acid composition of this placental CaBP (Table 2) bore some resemblance to the estimates reported for rat intestinal CaBP in the high proportion of acidic amino acids, and the absence of cysteine, histidine and arginine (Christakos et al., 1984; Delorme et al., 1983). The isolation of Caa2+-binding material from rat intestinal mucosa yielded a more complicated picture than that from placenta. The 9000Mr Ca2+-binding material from Sephadex G-50 yielded more than one Ca2+-binding entity when chromatographed by reversephase h.p.l.c. at neutral pH (Fig. 3a). Three 280 nmabsorbance peaks were observed that had the ability to bind Ca2+, the first being eluted at 33.0 min (peak I), the second at 34.0 min (peak II) and the third at 35.5 min (peak III). Each peak was collected and chromatographed separately by reverse-phase h.p.l.c. in 0.1 % trifluoroacetic acid. Under these conditions peak I material was eluted at 38.0 min (result not shown), peak II material, the major 1986

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Fig. 3. Purification by h.p.l.c. of intestinal CaBP obtained from the Sephadex G-50 step (see the Materials and methods section) (a) Reverse-phase h.p.l.c. on C- 18 at pH 7.5, showing three peaks of Ca2+-binding activity. , A280; *-*, 45Ca radioactivity. (b) Furtiher purification on C-18 at pH 2.5 of peak II from (a). , A280. , A280. (c) Peak III from (a) at pH 2.5.

Vol. 235

J. P. MacManus, D. C. Watson and M. Yaguchi

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Fig. 4. Tryptic peptide map by h.p.l.c. of placental (a) and intestinal (b) CaBPs The amino acid compositions of these peptides are given in Table 3.

entity, was eluted at 40.5 min (Fig. 3b), and peak III material, the minor entity, was eluted at 41.5 min (Fig. 3c). This minor intestinal Ca2+-binding entity proved to be very useful in the elucidation of the primary structure of these proteins-(see below).

Amino acid sequence analysis

The h.p.l.c.-pure placental Ca2+-binding protein (Fig. 2b) was subjected to several cycles of Edman degradation with no resulting amino acid phenylthiohydantoin 1986

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derivative, showing that the N-terminus was blocked. Tryptic peptides were generated from this placental protein, and also from the intestinal peak II material (Fig. 3b). These tryptic peptides were separated by h.p.l.c. (Fig. 4), and the amino acid compositions of these peptides were obtained (Table 3). The tryptic peptide profiles in Figs. 4(a) and 4(b) were similar but not identical. However, when the amino acid compositions were examined these differences were seen to be more apparent than real. Peptide P5 from the placental material appeared to have an uncleaved lysine residue, which was totally cleaved in the possibly equivalent intestinal peptide, leading to the relatively larger amounts of peptides Il and 13. The amino acid sequences of these placental tryptic peptides were obtained, but other cleavages of the intact protein were required before alignment was possible. Tryptic peptide P1-2 gave two sequences corresponding to residues 29-32 and 76-78, as determined by overlap with peptides A3 and A4 (see Fig. 6). The next cleavage of the placental Ca2+-binding protein was with mild acid, and the resulting peptides were separated by h.p.l.c. (Fig. 5). Both their amino acid compositions (Table 4) and sequences were obtained. The sequence analysis of these large peptides AL, A3 and A4, in conjunction with the sequences of the tryptic peptides, and referral to the published bovine and pig intestinal CaBP sequences (Hofmann et al., 1979; Fullmer & Wasserman, 1980, 1981), permitted the alignment shown in Fig. 6 for residues 4-78 of the placental protein. A comparison of the amino acid compositions of peptide A2 (Table 4), the tryptic peptides (Table 3) and the intact protein (Table 2) indicated that a remaining putative N-terminal tripeptide of composition (Ala1,Lys1,Ser,) was missing. Such a peptide was recovered by repeating a tryptic peptide separation such as that illustrated in Fig. 4, but with isocratic elution for the first 6 min (detailed results not shown). When it was subjected to carbo yeptidase digestion, lysine was liberated first, followed by serine (Fig. 7). No alanine was detected, even after 24 h digestion. The conclusion of these experiments was that the N-terminal sequence was blocked (presumably acetylated) Ala-Ser-Lys (Fig. 6). Because of the similarity of composition of the tryptic peptides of the intestinal CaBP to those of the placental CaBP (Table 3), it was decided that sequence analysis was redundant except for peptide 18. However, use was made of the minor (peak III) intestinal Ca2+-binding entity (Fig. 3c). This polypeptide was analysed in the gas-phase sequencer for 57 cycles, at which point the yield of the amino acid derivative fell below detectable levels. However, this analysis yielded information for residues 4-61, which, in conjunction with the sequence of tryptic peptide 18, gave overlap with all the mild-acid cleavage points, and most of the tryptic cleavage points (Fig. 6). This analysis unequivocally showed that the intestinal and placental Ca2+-binding proteins were identical for residues 4-74. The inclusion of evidence from the identical h.p.l.c. retention time of tryptic peptides from both proteins (P1-2 and 11-2), and their identical amino acid compositions (Table 3), in conjunction with the composition of the presumptive intestinal N-terminal tripeptides (residues 1-3) (Fig. 6), strongly indicate the identity of both proteins in all 78 residues.

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Fig. 5. Peptides purified by h.p.l.c. from placental CaBP cleaved by mild acid The amino acid compositions of these peptides are given in Table 4. Table 4.. Amino acid compositions of peptides generated from placental CaBP by mild acid hydrolysis (see Fig. 5)

Amino acid composition (mol of residue/mol)

Residues ... Peptide ...

51-56 Al

Asx Thr Ser

1.0

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1.1

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1-21 A2

2.9 4.3 1.1 1.4 2.9

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23-49 A4

1.9 4.8

1.2 0.9 4.7 5.4 1.9

1.3 1.8

1.0

0.9 0.8 2.1

0.9 0.9 1.0

1.1 5.1

1.3 0.8 2.9 2.1

0.8 6.8 1.2 3.3

DISCUSSION The primordial gene of the Ca2+-binding protein multigene family is regarded as having consisted of coding sequences for one domain: a helix, a Ca2+-binding loop of oxygen-;.donating side chains, and another flanking helix (Goodman et al., 1979; Kretsinger, 1980;

Baba et al., 1984). By gene duplication and deletion the separate genes for the four-domain calmodulin and

troponin C, the three-domain parvalbumin and oncomodulin and the two-domain intestinal CaBP and S-100 protein are said to have arisen (Goodman et al., 1979; Kretsinger, 1980; Baba et al., 1984). The primary and secondary structures of these proteins are very similar, consisting of units of these 30-residue helix-loop-helix domains jointed by hinge sections (Goodman et al., 1979; Kretsinger, 1980; Gariepy & Hodges, 1983). Moreover, the tertiary structures estimated by X-ray diffraction of parvalbumin and the intestinal CaBP also show similarities (Kretsinger, 1980; Szebenyi & Moffat, 1983). These similarities of different small Ca2+-binding proteins suggest that antibodies directed against sequence or conformational antigenic determinants on any one of the family could also cross-react with similar epitopes on other family members. Indeed, several independent anti-calmodulin sera also cross-reacted with troponin C (Chafouleas et al., 1979; MacManus et al., 1981; Van Eldik & Watterson, 1981) and some bacterial proteins (Harmon et al., 1985), anti-parvalbumin sera with another parvalbumin-like protein in human tumour cells (Pfyffer et al., 1984), and anti-(S-100 protein) sera with both protein S-100a and protein S-100/J, which are separate gene products (Hidaka et al., 1983; Van Eldik et al., 1984). Confusion can also arise in reverse fashion. Antisera generated separately were supposed to have reacted with a skin Ca2+-binding protein (Saurat et al., 1981; Pavlovitch et al., 1983). However, when isolated, 1986

Rat placental Ca2+-binding protein

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T T T 15 5 I0 f 20 Ac- N-Alo-Ser-Lys -Lys-Ser-Pro-Glu-Glu-Met -Lys-Ser-Ile-Phe-Gln-Lys-Tyr-Alo-Alo- Lys -Glu____-P2 ^

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Fig. 6. Identitity of the amino acid sequences of rat placental and intestinal CaBPs with overlapping peptides used for analysis The continuous lines indicate sequences obtained by automated degradation of placental peptides, and the broken lines indicate sequences assumed from composition analysis and overlap. The broken arrowed line is the sequence from carboxypeptidase digestion. The continuous arrowed line is the sequence obtained from the minor peptide of the intestinal CaBP (peak III in Fig. 3c). Abbreviations for cleavage points are: A, mild acid; T, trypsin.

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For details see the text.

skin Ca2+-binding protein turned out to be parvalbumin (MacManus et al., 1985b). Anti-(intestinal CaBP) sera had been used to show the presence of a Ca2+-binding entity in rodent placenta, and the identity of both proteins was suggested (Bruns et al., 1981; Delorme et al., 1982). But other members of the Ca2+-binding protein family occur in placenta, e.g. calmodulin (Umeki et al., 1981) and oncomodulin (MacManus et al., 1985a). In addition, a large Vol. 235

Ca2+-binding protein has also been described (Tuan, 1982). Any one of these proteins, their breakdown products or an unknown protein have the potential of cross-reaction with antisera directed against the small intestinal CaBP. In the present work this immunoreactive Ca2+-binding entity has been isolated and purified to homogeneity for the first time from rat placenta. It has an Mr of 9000 and cross-reacts with both a rabbit and a chicken anti(intestinal CaBP) serum (J. P. MacManus, unpublished work). It has a primary structure of 78 residues with N-acetylalanine as the probable N-terminal amino acid. The rat intestinal CaBP has also been isolated and its primary structure also directly established. Both proteins are identical for residues 4-74, and probably in entirety for residues 1-78. The helix-loop-helix Ca2+-binding domains can easily be discerned (Fig. 8), and are homologues to the domains in the bovine and pig molecules. The rat protein has 62 of 78 residues identical with the bovine molecule, and 67 identical with the pig molecule. A partial sequence of the rat protein has been presented from studies using intestinal nucleic acids (Desplan et al., 1983; Leonard et al., 1984). Several differences in this translated sequence can now be seen, most obviously residue 60 being a glycine in the Ca2+-binding loop, not an aspartate. Also, residue 54 is a lysine residue, not a glutamate (Fig. 8). This extra lysine residue in the rat protein compared with both bovine and pig proteins means tryptic peptide mapping would not be a conclusive fingerprint of homology. The N-terminal amino acid is shown to be an alanine residue, not a serine. Finally, the

J P. MacManus, D. C. Watson and M. Yaguchi

594

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Fig. 8. Comparison of the sequence of rat placental and intestinal CaBPs with those of bovine and pig intestinal CaBPs The heavy type shows the rat sequence established in Fig. 6. The residues above this sequence are those different from the previous partial studies (Desplan et al., 1983; Leonard et al., 1984). The residues below this heavy type are those different in the bovine (Fullmer & Wasserman, 1980, 1981) and pig (Hofmann et al., 1979) proteins. The Ca2+-binding ligands are underlined, with the flanking helices underlined by squares.

cDNA synthesized can now be correctly assigned as coding for residues 10-78, and not 7-75 as originally stated (Desplan et al., 1983). The results of the present study means that use of anti-(intestinal CaBP) sera with placental tissue can now be more confidently interpreted, since the same gene product is expressed in both intestinal mucosa and placental labyrinth. The synthesis of CaBP in intestinal mucosa is dependent on vitamin D, and the protein is regarded as the main Ca2+- transport protein (Wasserman & Fullmer, 1983). However, the synthesis of the placental CaBP has not been shown conclusively to be dependent on vitamin D (Marche et al., 1978; Garel et al., 1981; Bruns et al., 1982), and the steroid seems to be unnecessary for active Ca2+ transport across the placenta (Halloran & DeLuca, 1983; Brommage & DeLuca, 1984). Maybe the expression of the CaBP gene is controlled differently in placenta and intestine. Immunohistochemistry has shown that the placental CaBP is present in the labyrinth, the area offoeto-maternal exchange (Marche et al., 1978; Delorme et al., 1979; Bruns et al., 1984), and also in the visceral yolk-sac, an organ that also has exchange properties (Delorme et al., 1983). Thus, in both intestine and conceptus, CaBP is located in areas of entry of Ca2+ into the organism. In contrast, another placental Ca2+-binding protein, oncomodulin, is not synthesized in visceral yolk-sac, and predominates in the placental spongiotrophoblast, not the labyrinth (MacManus et al., 1985a). A comparative study of developmental control of these two separate, but structurally related, Ca2+-binding proteins promises to be rewarding. Our thanks are due to B. M. Braceland for providing extracts of placental and intestinal tissues. The provision of antisera by Dr. M. E. H. Bruns, University of Virginia, Charlottesville, VA, U.S.A., and Dr. W. A. Gleason, University of Texas, Houston, TX, U.S.A., is gratefully acknowledged. The helpful comments of Dr. C. S. Fullmer, Cornell University, Ithaca, NY, U.S.A., were much appreciated. Our thanks are due to D. J. Gillan for the Figures and to K. O'Connor for the final manuscript. This paper is N.R.C.C. Publication no. 25415.

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Hofmann, T., Kawakami, M., Hitchman, A. J., Harrison, J. E. & Dorrington, K. J. (1979) Can. J. Biochem. 57, 737-748 Klee, C. B., Crouch, T. M. & Richman, P. G. (1980) Annu. Rev. Biochem. 49, 489-515 Kretsinger, R. H. (1980) CRC Crit. Rev. Biochem. 8, 119-174 Leonard, W. J., Strauss, A. W., Go, M. F., Alpers, D. M. & Gordon, J. I. (1984) Eur. J. Biochem. 139, 561-571 MacManus, J. P. (1980) Biochim. Biophys. Acta 621, 296-304 MacManus, J. P., Braceland, B. M., Rixon, R. H., Whitfield, J. F. & Morris, H. P. (1981) FEBS Lett. 133, 99-102 MacManus, J. P., Watson, D. C. & Yaguchi, M. (1983) Eur. J. Biochem. 136, 9-17 MacManus, J. P., Brewer, L. M. & Whitfield, J. F. (1985a) Cancer Lett. 27, 145-151 MacManus, J. P., Watson, D. C. & Yaguchi, M. (1985b) Biochem. J. 229, 39-45 Marche, P., Delorme, A. & Gleizes, P. C. (1978) Life Sci. 23, 2555-2562 Pansini, A. R. & Christakos, S. (1984) J. Biol. Chem. 259, 9735-9741 Pavlovitch, J. H., Didierjean, L., Rizk, M., Balsan, S. & Saurat, J. H. (1983) Am. J. Physiol.244, C50-C57 Pfyffer, G. E., Haemmerli, G. & Heizmann, C. W. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 6632-6636 Saurat, J. H., Didierjean, L., Pavlovitch, J. H., Laouari, D. & Balsan, S. (1981) J. Invest. Dermatol. 76, 221-223 Received 12 July 1985/18 November 1985; accepted 18 December 1985

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