THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 258, No. 2. Issue of January 25, pp. 1305-1310, 1983 Printed in U.S.A.

Induction of Calcium-binding Protein before 1,25-Dihydroxyvitamin D1 Stimulation of Duodenal Calcium Uptake*

(Received for publication, July 21, 1982)

Charles W. Bishop$, Nancy C. Kendrick, and Hector F. DeLucag From the Deoartment of Biochemistrv. Colleee of Amicultural and Life Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53706

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Protein synthesis occurring before the onset of 1,25- dihydroxyvitamin D3 (1,%-(OH)zD3) stimulation of cal- cium uptake was examined in embryonic chick duo- dena by double label autoradiography. Duodena from 19-day-old embryos were cultured for 24 h. Duodena exposed to a saturating concentration of 1,25-(OH)~D3 during the last 2, 4, or 6 h were either cultured with [3H]leucine for the final 2 h or used for calcium uptake assays. Duodena not exposed to the hormone were either cultured with [‘4C]leucine for the final 2 h or used for calcium uptake assays. Calcium uptake by nonradiolabeled duodena was increased ( p < 0.05) only in tissues exposed to the hormone for 6 h. Tissue up- takes of [3mleucine and [l4C]1eucine were identical and linear with time. Soluble proteins were extracted from 3H- and “C-labeled tissues, mixed, and simultaneously resolved by two-dimensional polyacrylamide gel elec- trophoresis. Analysis of fluorographs and autoradi- ographs prepared from the double-labeled gels re- vealed a protein induced by 1,25-(OH)zD3 within 2 h of exposure to 1,25-(OH)zD3 and at least 2 h before the calcium uptake response. The induced protein co-mi- grated with purified chick calcium-binding protein dur- ing two-dimensional electrophoresis. These results demonstrate that 1) this protein is the calcium-binding protein and 2) calcium-binding protein is clearly syn- thesized in the embryonic duodenum before the calcium uptake response to 1,26-(OH)~D3 occurs.

Although chick intestinal calcium-binding protein has been intensively studied since it was discovered by Wasserman and Taylor (1) and Wasserman et al. (2), its exact physiological function remains unknown. The protein binds calcium with high affinity (2, 3) and is induced by vitamin Da and its more active metabolites (4,5), suggesting that its function is related to vitamin D-dependent intestinal calcium transport. This suggested function, however, is not consistently indicated by studies of the time course of calcium-binding protein appear- ance relative to the onset of the calcium uptake response (1, 6-17). Some of these studies (1, 6, 8-13) have demonstrated that the induction of calcium-binding protein biosynthesis

* This work was supported by Program Project Grant AM-14881 of the National Institutes of Health and by the Harry Steenbock Research Fund of the Wisconsin Alumni Research Foundation. 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.

$ Recipient of Postdoctoral Fellowship AM-06692-01 from the Na- tional Institutes of Health.

fj To whom all correspondence should be addressed. No reprints will be available from the authors.

precedes or coincides with increases in calcium uptake and the protein is therefore available to participate in the uptake process. In contrast, other studies (7, 14-17) have shown that calcium-binding protein does not appear until after calcium uptake is increased, indicating that it is not involved in the uptake process. In order to establish whether or not calcium- binding protein functions in intestinal calcium uptake, these conflicting results must be resolved.

We describe here the use of an extremely sensitive tech- nique for the detection of proteins induced by hormonal factors. Using these methods, the induction of calcium-binding protein prior to increased intestinal calcium uptake by 1,25- (OH)2Da’ in chick intestinal organ cultures can be clearly demonstrated.

EXPERIMENTAL PROCEDURES

Animals-Live chicken embryos (White Leghorns) were obtained from Sunnyside Hatcheries (Oregon, WI) a t 18 days of incubation. Before use in duodenal organ culture experiments, the embryos were maintained overnight at 37 “C in a humidified incubator.

Chemicals-~-[4,5-”H]Leu (85 Ci/mmol), ~-[U-l~ClLeu (330 mCi/ mmol), and 45CaC12 were obtained from Amersham Corp. in aqueous solutions containing 2% ethanol. Dr. M. Uskokovic of Hoffmann- LaRoche generously supplied 1,25-(OH)zD:1, the purity and concen- tration of which were determined by its ultraviolet absorption spec- trum (ezM nm = 18,200 M” cm”). Sephadex G-25 and DEAE-Sephadex (A-25) were obtained from Pharmacia Fine Chemicals (Uppsala, Sweden), and Chelex 100 was obtained from Bio-Rad. All other reagents were of analytical grade.

Media-Waymouth’s 752/1 medium was obtained from GIBCO (Grand Island, NY). Low leucine medium, containing 75.8 p~ leucine, was prepared according to the published formulation for Waymouth’s 752/1 medium (18) by reducing the leucine concentration. The specific activity of leucine in low leucine medium prepared with [”]Leu was 6.6 mCi/pmol, while that for low leucine medium prepared with [“C] Leu was 0.33 mCi/pmol. Medium with 150 nM 1,25-(OH)2D:l was prepared by adding the hormone in ethanol. Equivalent volumes of ethanol alone were added to control medium. In all cases, the ethanol concentration in the medium never exceeded 0.1%. Penicillin (50 units/ml) and streptomycin (50 pg/ml), also obtained from GIBCO, were added to all media before use.

Duodenal Organ Cultures-Duodena excised from 19-day-old em- bryos were cultured for 24 h by the method of Corradino (10) as modified by Franceschi (19). All duodena were cultured in Way- mouth‘s 752/1 medium for the first 18 h and in low leucine medium for the remaining 6 h, with changes every 2 h. One group of duodena was exposed to 150 nM 1,25-(OH)2D3 for the entire 24 h period, while three groups were exposed to this saturating concentration of hor- mone only for the last 2, 4, or 6 h. During the final 2 h of culturing, nine duodena (three from each of the last three groups) were incu- bated in medium containing [’HILeu. A final group of duodena was never exposed to the hormone and served as a control group. Nine duodena from the control group were incubated in medium containing [I4C]Leu during the final 2 h of culturing. At the end of the culture period, duodena labeled with [”]Leu or [I4C]Leu were individually

I The abbreviation used is: 1,25-(OH)2D.%, 1,25-dihydroxyvitamin D3.

1305

1306 Calcium-binding Protein and Duodenal Calcium Uptake

rinsed with 4 "C buffer (50 mM PO,, 150 mM NaCl, pH 7.4), placed in separate vials, and frozen on dry ice. Nonradiolabeled duodena were used for calcium uptake assays.

Calcium Uptake Assay-Total calcium uptake by each nonradio- labeled duodenum was measured as previously described (19). Cal- cium uptake for each group of identically handled duodena was calculated as "Ca counts/min/mg of tissue/lO min & S.E. and ex- pressed as percentage of the calcium uptake determined for the control group.

Extraction of Soluble Proteins from Radiolabeled Duodena-The 18 duodena labeled with either ["]Leu or ["CILeu were grouped into 9 pairs. Each of three "H-labeled duodena cultured with 1,25- (0H)zD.g for 2 h was paired with a I4C-labeled duodenum which had been identically handled but not exposed to the hormone. Similarly, each of the six "H-labeled duodena exposed to 1,25-(OH)2D3 for 4 or 6 h was paired with an identically handled I4C-labeled duodenum not exposed to the hormone. During the extraction of the soluble proteins, the duodena of each pair were processed simultaneously, but sepa- rately.

Each radiolabeled duodenum was transferred to a Potter-Elvehjem homogenizer and individually homogenized in 1.5 ml of buffer (5 mM Tris, pH 7.4) with six strokes of a motor-driven pestle. The homoge- nates were centrifuged 100,000 X g for 45 min a t 4 "C. The 18 supernatants containing the extracted soluble proteins were re- covered.

Uptake and Incorporation of Radiolabeled Leucine-Tissue up- take of radiolabeled leucine was monitored during the final 2 h of culturing. At 30-min intervals, triplicate samples of culture medium were analyzed by liquid scintillation spectrometry, and the percentage of radiolabel removed from the medium at each sampling time was calculated. Incorporation of radiolabeled leucine into newly synthe- sized proteins was estimated by analysis of protein precipitated by addition of trichloracetic acid. An aliquot of each extract was com- bined with an equal volume of 10% trichloroacetic acid prechilled to 4 "C. The precipitated proteins were washed twice with 5% trichlo- roacetic acid and dissolved in 0.2 N NaOH. Triplicate aliquots of this solution were analyzed for protein content by the Lowry method (20) and for radioactivity by liquid scintillation spectrometry. Radiolabel incorporation was expressed as disintegrations/min/mg of precipi- tated protein.

analyzed for total protein content by the Bradford method (21) and Two-dimensional Electrophoresis-Soluble protein extracts were

for total radioactivity by liquid scintillation spectrometry. Each ex- tract was lyophilized and resuspended in sodium dodecyl sulfate-free lysis buffer (22) to a final concentration of 6 mg/ml. The resulting concentrates were paired (see above) and combined to form protein mixtures in which the ratio of "H to I4C disintegrations/min was exactly 101. A 15.6-pl sample containing 94 pg of protein (2.0 X IO6 "H dpm and 2.0 X lo5 I4C dpm) was removed from each of the nine mixtures and resolved by the O'Farrell method of two-dimensional polyacrylamide gel electrophoresis (22). All gels (120 X 150 X 0.75 mm) were standardized internally during electrophoresis in the second dimension (molecular weight) with myosin (220,000), phosphorylase a (94,000), catalase (60,000), and actin (43,000). Fifteen-pl aliquots of the remaining portion of concentrates containing 3H-labeled proteins were each mixed with 1 pg of purified chick vitamin D3-dependent intestinal calcium-binding protein and resolved by two-dimensional electrophoresis.

Fluorography and Autoradiography-Two-dimensional gels were stained with Coomassie blue (22), impregnated with 2,5-diphenylox- azole (23), and dried under vacuum. Each gel containing a mixture of 'H- and I4C-labeled proteins was exposed sequentially to preflashed Kodak X-Omat AR film for 72 h a t -70 "C and to Kodak No Screen film for 35 h a t 22 "C (24). Gels containing only "-labeled proteins were exposed to X-Omat AR film for 48 h at -70 "C but not to NO Screen fdm.

Purification of Calcium- binding Protein-Duodena excised from 4-week-old White Leghorn chicks (Sunnyside Hatcheries, Oregon, WI) were slit open lengthwise and rinsed with 4 "C buffer (14 mM Tris, 150 mM NaCI, 5 mM KCl, 1 mM 2-mercaptoethanol, pH 7.4). The mucosa was scraped free of the underlying muscle at 4 "C with glass slides, washed with buffer, and homogenized in an equal volume of buffer with a Polytron (Brinkmann Instruments). The supernatant remaining after high speed centrifugation of the homogenate (100,000 X g for 2 h at 4 "C) was desalted by passage through a Sephadex G- 25 column (2.5 x 85 cm) equilibrated with Tris buffer containing 50 mM NaCI. Fractions containing protein as determined by the Bradford

method (21) were pooled, heated to 60 "C for 20 min, and chilled to 4 "C.

Proteins not precipitated from solution by heat treatment were recovered as the supernatant solution following ultracentrifugation (100,000 X g for 45 min a t 4 "C). The recovered supernatant solution was applied without concentration to a column (1.5 X 90 cm) of DEAE-Sephadex (A-25) equilibrated a t 4 "C with Tris buffer contain- ing 50 mM NaCI. Proteins were eluted from the column with 200 ml of Tris buffer containing 50 mM NaCl followed by a 500-ml NaCl gradient (50 to 300 mM) and 200 ml of Tris buffer containing 300 mM NaC1. The flow rate was 8 ml/cmz and 10.5-1111 fractions were col- lected. All fractions were analyzed for calcium-binding activity by Chelex ion exchange assay (2), NaCl concentration using a conductiv- ity meter (London Company, Cleveland, OH), and protein content by the Bradford method (21).

Approximately 70% of the protein recovered from the column was eluted in the void volume but contained negligible calcium-binding activity. High calcium-binding activity was detected in 17 fractions containing 185 to 250 mM NaCl. These fractions were pooled and diluted with NaC1-free Tris buffer to obtain a final NaCl concentration of 50 mM. The pooled and diluted fractions containing high calcium- binding activity were applied (without concentration) to another column (1.5 X 90 cm) of DEAE-Sephadex (A-25) equilibrated a t 4 "C with Tris buffer containing 50 mM NaCl and 1 mM CaCL Develop- ment of this column proceeded exactly as described for the preceding column except that 1 mM CaCL was included in all buffers. The collected fractions were analyzed for calcium-binding activity, NaCl concentration, and protein content. All of the calcium-binding activity was eluted in 8 fractions containing 70 to 140 mM NaC1. Two-dimen- sional gel electrophoresis of this material revealed only one protein having a molecular weight of approximately 27,000 and an apparent PI of 4.2, that is, vitamin D-dependent calcium-binding protein (2).

RESULTS

Time of Onset of the Calcium Uptake Response to 1,25- (OH)zD3-Three groups of eight duodena were cultured for 24 h and exposed to medium containing a saturating concentra- tion of 1,25-(OH)2D3 a t various times before the end of the culture period. As shown in Fig. lA, calcium uptake for duodena exposed to 1,25-(OH)2D:3 for 2 h was not increased over that for an identically handled control group. In contrast, uptake by duodena exposed to the hormone for 6 or 24 h was increased ( p < 0.05) over that of the control group, the response observed a t 24 h being more than double that ob- served at 6 h. The results of this experiment indicated that the calcium uptake response was first detectable between 2 and 6 h of continuous exposure to the hormone.

Y laoC A * T 1 6

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0 2 4 6

Calcium- binding Protein and Duodenal Calcium Uptake 1307

"H- and I4C-labe1ed proteins. This fluorograph, like all others in this experiment, was prepared with Kodak X-Omat AR fdm which detects "H and "'C equally well when the isotopes are present in a 1O:l dpm ratio (25). The area immediately surrounding the circled protein at the lower right is redis- played in Fig. 4A. Shown in Fig. 4B is the matching area of an autoradiograph produced from the same gel. This autoradi- ograph, like all others in this experiment, was prepared with Kodak No Screen film which detects I4C but not "H. The protein identified by the arrow was detected by fluorography but not autoradiography, indicating that it was present only in the "H-labeled tissue. As the "H-labeled proteins contained in this gel were extracted from a duodenum exposed to 1,25-

20 - d z 18-

4 16-

14-

m 2

8 1 2 - Ir. LL 10- 0

1 1 1 1 1 1 1 1 1 1 1 1

0 30 60 90 120 INCUBATION TIME (min)

FIG. 2. Tissue uptake of radiolabeled leucine. Duodena were cultured for a total of 24 h and incubated in the presence of radiola- beled leucine during the final 2 h. Three groups of three duodena exposed to 150 nM 1,25-(OH)dh for 2,4 or 6 h were incubated together in a single Petri dish containing medium with [:'H]Leu. Three control groups not exposed to the hormone were similarly incubated in medium with ['.'ClLeu. At 30-min intervals, samples of culture me- dium were removed from the two Petri dishes containing [:'H]Leu and [I4C1Leu, respectively, and analyzed by liquid scintillation spectrom- etry. Uptake of [:'H]Leu by 1,25-(OH)nD:,-treated duodena (A) and ["ClLeu by controls (0) was determined by calculating the percent- age of radiolabel (X f S.E.; n = 3) removed from the medium.

TAKE I Incorporation of radiolabeled leucine into newly synthesized

proteins Soluble proteins were extracted from "H-labeled duodena exposed

to 1,25-(OH)2D:l for 2, 4, or 6 h and from 'IC-labeled duodena not exposed to the hormone (controls). Incorporation of radiolabeled leucine into the extracted proteins was determined as described under "ExDerimental Procedures."

Source of soluble pro- :$$$E-- Radiolabel incorpo- n cine in ration into soluble teins ture medium proteins

mCi/gmol 10 X dpm/mE ? S.E. "H-labeled duodena 9 6.60 40.0 f 2.2 "C-labeled duodena

(controls) 0.33 1.94 f 0.06

In a second experiment, the time of onset of the calcium uptake response to 1,25-(OH)?D:, was more precisely deter- mined. Three groups of eight duodena were cultured for 24 h under the same experimental conditions and were continu- ously exposed to medium containing 1,25-(OH)2D:1 for the final 2, 4, or 6 h, respectively. As shown in Fig. lB, calcium uptake by duodena exposed to the hormone for either 2 or 4 h was unchanged relative to that of a control group. Calcium uptake by duodena exposed to 1,25-(OH)2D:l for 6 h was significantly increased ( p < 0.05) and comparable to that observed in the previous experiment, demonstrating that this hormonal response was first detectable between 4 and 6 h.

Uptake and Incorporation of Radiolabeled Leucine-Up- take of radiolabeled leucine by cultured duodena is displayed in Fig. 2. Uptake of [:'H]Leu by duodena exposed to 1,25- (OH)2D:I was linear with time and essentially identical with the uptake of ['4C]Leu by duodena not exposed to the hor- mone. Incorporation of radiolabeled leucine into the soluble proteins is summarized in Table I.

Fluorographs and Autoradiographs-Shown in Fig. 3 is a fluorograph produced from one of the gels containing both

-' TC' "0

4

" " - - " FIG. 3. Fluorograph of a two-dimensional double-labeled

gel. "H-labeled proteins extracted from a duodenum treated with 1,25-(OH)zD,, for 6 h were combined with 'IC-labeled proteins ex- tracted from a control duodenum not treated with the hormone. This protein mixture, containing 2.0 X IO'; "H dpm and 2.0 X IO5 "C dpm, was resolved by two-dimensional polyacrylamide gel electrophoresis, and the fluorograph displayed was produced (see "Experimental Procedures"). Proteins having isoelectric points of approximately 8 lie along the left border, and those with approximately 4 lie along the right border. Proteins having molecular weights in excess of 220,000 remain unresolved along the top edge, and those having molecular weights less than 17,000 lie in running front at the bottom. The area immediately surrounding the circledprotein is redisplayed in Fig. 4A.

FIG. 4. Detection of a hormonally responsive protein in a duodenum exposed to 1,25-(OH)*D, for 6 h. The area of the fluorograph shown in Fig. 3 containing the circled protein is redis- played (A) with the matching area of an autoradiograph ( B ) produced from the same gel (see "Experimental Procedures"). This gel con- tained "H-labeled proteins from a duodenum treated with 1,25- (OH)pD:, for 6 h and '"C-labeled proteins from a control duodenum. The protein marked by the arrou (identical with the circled protein in Fig. 3) was detected only by fluorography, indicating that it was present in the 1,25-(OH)2D.I-treated duodenum but not in the control.

Calcium-binding Protein and Duodenal Calcium Uptake 1309

FIG. 9. Co-migration of the 1.25-(OH)2D3 responsive protein with calcium-binding protein. The gel shown in Fig. 8 was im- pregnated with 2,5-diphenyloxazole (23), dried, and exposed to Kodak X-Omat AR film for 48 h at -70 "C. The area of the resulting fluorograph in which the hormonally responsive protein was located (Figs. 3 and 4) is shown here. This protein can be seen surrounding the punched hole which marked the exact position of calcium binding- protein.

phy, indicating that it was present only in the "H-labeled tissue. As "H-labeled proteins contained in this gel were ex- tracted from a duodenum exposed to 1,25-(OH)zD:, for only 4 h, this protein appeared to be hormonally induced before an increase in calcium uptake was observed. The protein was easily detected in all three gels containing "H-labeled proteins from duodena exposed to hormone for 4 h. I t was also detect- able in the fluorographs prepared from all three gels contain- ing "H-labeled proteins from duodena exposed to hormone for only 2 h, one of which is shown in Fig. 6.

To examine the electrophoretic mobility of this hormonally responsive protein relative to calcium-binding protein, non- radiolabeled calcium-binding protein was purified from 4- week-old chicks and subjected to two-dimensional electropho- resis. Fig. 7 shows the resulting gel stained with Coomassie blue. Calcium-binding protein can be seen at the lower right relative to molecular weight standards that appear as hori- zontal lines. An identically prepared gel also stained with Coomassie blue containing both nonradiolabeled calcium- binding protein and "H-labeled proteins extracted from a duodenum exposed to 1,25-(OH)2D3 for 6 h is shown in Fig. 8. Calcium-binding protein, easily identified by reference to the standardized gel (Fig. 7), was marked by punching a small hole in the gel. A fluorograph was then prepared from the same gel, which contained a hole at the position occupied by calcium-binding protein. The area of the fluorograph in which the unidentified hormonally responsive protein was located is shown in Fig. 9. This protein can be seen at the site of the punched hole, demonstrating that it co-migrated exactly with calcium-binding protein.

DISCUSSION

The experiments presented above demonstrate that cul- tured duodena from 19-day-old chick embryos rapidly synthe- size a protein in response to 1,25-(OH)2D3. This protein was consistently detected in duodena exposed to 1,25-(OH)zD:i for only 2 h and was more apparent in duodena exposed to hormone for longer periods of time. Its synthesis was examined in relation to the calcium uptake response. Under the cultur- ing conditions used, the embryonic duodena did not show increased calcium uptake until after 4 h of continuous expo- sure to a saturating concentration of 1,25-(OH)2D3. By 6 h of exposure to the hormone, calcium uptake was increased by

approximately 30% over control values. By 24 h, calcium uptake was further increased to more than 70% above control values. These results are consistent with a previous report from our laboratory concerning calcium uptake by cultured duodena from 19-day-old chick embryos (19) which showed that increases in calcium uptake were not detectable before 4.5 h of exposure to a saturating concentration of hormone, but increases of approximately 30 and 60% occurred by 6 and 24 h, respectively. Since the protein identified in the present study was detected in duodena exposed to 1,25-(OH)~Ds for only 2 h, its synthesis preceded the onset of the calcium uptake response by at least 2 h.

The 1,25-(OH)2Dn-re~p~n~ive protein was detected by dou- ble label autoradiography (25). In this technique, double- labeled gels were exposed sequentially to X-Omat AR and No Screen films. During exposure to the X-Omat AR films, both isotopes contributed equally to the production of photo- graphic images, "H by fluorography and I4C by a combination of fluorography and direct autoradiography. In contrast, the No Screen films were completely insensitive to fluorography under the conditions used and recorded images in response to

C by direct autoradiography alone. Images recorded on any pair of films exposed to the same gel were completely super- imposible, thereby allowing direct comparison of identical image areas. Given that the exposures of the films were appropriately selected, consistent detection of a protein image on X-Omat AR fiims without its detection at the same location on matching No Screen f ims demonstrated that 1) this pro- tein was present in the gels only in "H-labeled form, and 2) it was induced in response to 1,25-(OH)zD~. As the identified protein was invariably detected only on X-Omat AR fims, it followed that this protein was hormonally induced.

Exposures of the films were carefully selected so that the faintest images produced on one film were also produced on the other, with a few exceptions.' However, the X-Omat AR films were always more highly exposed than the No Screen films (Figs. 4 and 5). This difference in exposures, which could be misconstrued as the cause for differential detection of a few proteins, was required since both isotopes contributed to image formation on the X-Omat AR films but only one formed the image recorded on the No Screen fiims.

Since the protein identified in this report was induced by 1,25-(OH)& a strong possibility existed that it was the vitamin Dn-dependent intestinal calcium-binding protein (2). Two observations further supported this possibility. 1) The identified protein was extracted from duodenal homogenates as a soluble protein, and 2) it migrated during two-dimensional electrophoresis as a protein having a molecular weight of 27,000 and an apparent PI of 4.2. The electrophoretic mobility of this protein was therefore examined relative to calcium- binding protein purified from 4-week-old chicks, with the result that both proteins co-migrated identically during two- dimensional electrophoresis. This result led to the further conclusion that the protein is indeed calcium-binding protein.

The experiments presented above, when considered as a whole, support the conclusion that calcium-binding protein is synthesized in duodena from 19-day-old chick embryos within 2 h of exposure to 1,25-(OH)2Ds and at least 2 h before the onset of the calcium uptake response. This conclusion is in agreement with two other studies using embryonic chick duodena which demonstrated that calcium-binding protein was induced in 1,25-(OH)~Ds-treated duodena without stim- ulation of calcium uptake (10, 11). This conclusion, however,

These exceptions, aside from the protein in question, are presently being studied in greater detail and will be discussed in a future publication.

14

1310 Calcium-binding Protein and Duodenal Calcium Uptake

is a t variance with other studies of 1,25-(OH)zDs action using 3- or 4-week-old chicks. In two of these studies (14, 17), in vitro synthesis of calcium-binding protein by isolated duo- denal polysomes was detected by immunoprecipitation at all times when calcium transport was significantZy increased, beginning at 2 h; but calcium-binding protein synthesis was not detected before increases in calcium transport. These and other studies (15, 16) also demonstrated that calcium trans- port was increased by 1,25-(OH)2D3 several hours before cal- cium-binding protein was detected in duodenal homogenates by immunoelectrophoresis, immunodiffusion, or the Chelex ion exchange assay.

To reconcile the conclusion of the present study with the results from studies of 4-week-old chicks, we postulate that smaller amounts of calcium-binding protein can be detected by double label autoradiography than by immunological or ion exchange assays. The absolute lower limit of detection for immunoelectrophoresis, immunoprecipitation, and immuno- diffusion is approximately 1 pg of calcium-binding protein/g of mucosa, wet weight (13, 26, 27), while that of the Chelex ion exchange assay is considerably higher (6). The absolute lower limit of detection for calcium-binding protein by double label autoradiography is currently under investigation in our laboratory. Preliminary data3 indicate that it is a t least 1 order of magnitude below that for the immunoassays mentioned under the conditions selected for the present experiments. It is conceivable, therefore, that calcium-binding protein is syn- thesized in the duodena of 4-week-old chicks before 1,25- (OH)2D3 stimulation of calcium transport in quantities too small to be detected by immunological or ion exchange tech- niques.

Although the synthesis of trace amounts of calcium-binding protein prior to stimulation of calcium uptake is, a t present, of unknown physiological significance, it is clear from these experiments that the induction of calcium-binding protein in the embryonic duodenum is not a secondary response to 1,25- (OH)2Ds stimulation of calcium uptake. The detection of calcium-binding protein biosynthesis after only 2 h of exposure to 1,25-(OH)2D3 suggests instead that its induction is a pri- mary hormonal response which may yet be directly related to the calcium transport response.

'IC. W. Bishop, N. C. Kendrick, and H. F. DeLuca, unpublished observations.

Acknowledgments-We wish to thank Dr. Robert Brommage for his helpful comments regarding the experimental design and for his thoughtful review of the manuscript. The assistance of Dr. Gail Thompson in this latter regard is also greatly appreciated.

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