Biosynthesis Of Calcium-binding Protein Research Articles

1 alpha,25-Dihydroxyvitamin D-induced modification of a cytosolic protein in embryonic chick intestine.

Two-dimensional electrophoresis together with radiolabeling experiments was used to examine cytosolic proteins of embryonic chick duodenum for responses to 1,25-dihydroxyvitamin D3. 1,25-Dihydroxyvitamin D3 caused a striking decrease in [3H]leucine content of an 18,000-dalton protein (approximate pI, 5.1) after a 10-min pulse with radioisotope followed by a 4-h chase. Decreased [14C]leucine content of the same protein was also observed at various times following 1,25-dihydroxyvitamin D3 addition to culture media; a significant decrease in radiolabel incorporation occurred within 30 min after addition of the hormone. The results argue that 1,25-dihydroxyvitamin D3 causes either a decreased synthesis rate or a post-translational modification of this protein. This change joins the biosynthesis of calcium-binding protein as an early event in the response of chick embryonic intestine to 1,25-dihydroxyvitamin D3.

The early time course of calcium-binding protein induction by 1,25-dihydroxyvitamin D3 as determined by computer analysis of two-dimensional electrophoresis gels.

The time course of calcium-binding protein induction by 1,25-dihydroxyvitamin D3 was examined in embryonic chick duodena by single-label autoradiography. Duodena were excised from 19-day-old embryos, cultured for 24 h in defined medium, and exposed to 1,25-dihydroxyvitamin D3 at 1/2, 1, 1 1/2, 2, 2 1/2, 4, 6, and 20 h before the end of the culture period. Control duodena were identically handled but were not exposed to the hormone. All duodena were pulsed with [14C]leucine during the final 30 min of incubation. Cytosolic proteins, extracted from each radiolabeled tissue, were resolved by two-dimensional electrophoresis and autoradiographs were prepared from the resulting gels. Calcium-binding protein was detectable by autoradiography in duodena cultured with hormone for 1 h or more but not in the control duodena. The autoradiographs were converted to digital images by a scanning densitometer and entered into the Man-computer Interactive Data Access System. The total radioactivity incorporated into calcium-binding protein during the 30-min pulse was determined by computer analysis of the digitized autoradiographs, and the rate of calcium-binding protein biosynthesis was calculated. Duodena cultured with hormone for 1-20 h synthesized calcium-binding protein at rates of 129 +/- 17 to 8800 +/- 110 pg/mg of cytosolic protein/30 min. These rates demonstrate that calcium-binding protein is induced within the first hour of exposure to 1,25-dihydroxyvitamin D3, but in amounts too small to be detected by commonly used immunoassays.

Cell-free translation analysis of the vitamin D-dependent calcium binding protein mRNA activity present in total RNA and polysomal extracts from chick intestine

To further elucidate the mechanism(s) involved in the induction of a vitamin D-dependent intestinal calcium binding protein (CaBP), by the hormonally active form of vitamin D, 1,25-dihydroxyvitamin D 3 [1,25(OH) 2D 3], we have examined by cell-free translation CaBP-mRNA activity present in total cellular RNA extracts or polysomes obtained from chick small intestine. CaBP-mRNA activity was examined by immunochemical analysis of polypeptides completed upon addition of purified intestinal RNA or polysomes to a lysate system. Intestinal polysomes isolated from vitamin D-deficient chicks treated with vitamin D 3, 1,25(OH) 2D 3, or 1α-hydroxyvitamin D 3 were found to complete the synthesis of an immunoprecipitable protein which comigrated with authentic chick intestinal CaBP on SDS polyacrylamide gels. This product was found to represent up to 0.5–1.0% of the total polypeptides directed by polysomes isolated from treated birds but could not be detected among the products directed by intestinal polysomes obtained from vitamin D-deficient birds. A detectable level of polysomal CaBP-mRNA activity was present within 3 h and a maximal activity achieved at 9 h after administration of 1,25(OH) 2D 3. Increases in the levels of cytoplasmic CaBP (measured by radioimmunoassay) were first detected at 6 h, which was significantly later than the polysomal CaBP-mRNA activity. CaBP-mRNA activity in total cellular RNA isolated with guanidinium thiocyanate was also found to be dependent on treatment with vitamin D 3 or 1,25(OH) 2D 3 but represented only 0.1–0.2% of the total mRNA activity. Surprisingly, total RNA and poly(A) RNA were found to direct the synthesis and release of two additional polypeptides that were immunoprecipitated even with an affinity-purified antibody to CaBP. One, an approximately 11,000-dalton polypeptide, was clearly vitamin D dependent; the other, an approximately 38,000-dalton polypeptide, was not. The time course for appearance of CaBP-mRNA activity in total cell RNA extracts following 1,25(OH) 2D 3 treatment was found to be nearly identical to that observed in polysome preparations. These results indicate that induction of CaBP-biosynthesis results, at least in part, from a 1,25(OH) 2D 3-induced increase in the levels of total cellular CaBP-mRNA activity and are, therefore, consistent with a transcriptional regulation of CaBP biosynthesis by 1,25(OH) 2D 3.

Biosynthesis of calcium-binding protein of chick embryonic chorioallantoic membrane: In vitro organ culture and cell-free translation

Biosynthesis of the calcium-binding protein (CaBP) of the chick embryonic chorioallantoic membrane (CAM) was studied using in vitro organ culture and cell-free translation. The organ culture studies showed: 1) The CaBP is a relatively stable protein ( t 1 2 = 60 h ); 2) Biosynthesis of the CaBP involves microsomes and includes two posttranslational modifications, glycosylation and γ-glutamyl carboxylation; and 3) During embryonic development, a single species of the CaBP is expressed in the CAM. Cell-free translation of total CAM mRNA, including CaBP mRNA, was achieved in a rabbit reticulocyte lysate system using [ 35S]Met as a tracer. Based on the properties of the nascent CaBP polypeptide translated in the presence or absence of microsomal membranes, the early stages of CaBP synthesis appear to be: translation of CaBP mRNA on membrane-bound polysomes, insertion and translocation of the nascent polypeptide across microsomal membranes, and co-translational cleavage of a signal sequence.

A fluorinated vitamin D 3 analog with biopotency greater than 1α,25-dihydroxy vitamin D 3

Vitamin D metabolites and analogs induce de novo synthesis of a specific calcium-binding protein in embryonic chick duodenum maintained in organ culture. Using calcium-binding protein biosynthesis as a specific and sensitive biochemical indicator of intrinsic biopotency, 24,24-difluoro-1α,25-dihydroxy vitamin D 3 was found to be approximately four times more potent on a molar basis than the most active, naturally occurring metabolite, 1α,25-dihydroxy vitamin D 3.

Calcium-binding protein biosynthesis in the rat: Regulation by calcium and 1,25-dihydroxyvitamin D 3

Vitamin D-replete rats on high or low calcium diets received by intraperitoneal injection varying doses (62.5–750 ng/animal) of 1,25-dihydroxyvitamin D 3 (1,25-(OH) 2-D 3). The animals on the high calcium diet showed a progressive, dose-dependent response to the vitamin D metabolite; their duodenal levels of the cytosolic calcium-binding protein increased from about 50 nmol Ca bound/g mucosa to nearly 100 nmol Ca bound/g mucosa. The animals on the low calcium diet, whose calcium-binding protein base levels were about 100 nmol Ca bound/g mucosa, showed no response to the metabolite and their calcium-binding protein levels remained unchanged even with high doses of 1,25-(OH) 2-D 3. Vitamin D-deficient animals on a high calcium diet from weaning gave the same quantitative response to progressive doses of 1,25-(OH) 2-D 3 as the replete animals, but the time and rate at which calcium-binding protein reached its maximum level in the intestine differed in the two groups. In the replete animals the calcium-binding protein response to 1,25-(OH) 2-D 3 was completed by 1 h after treatment, whereas in the deficient rats the maximum response was not attained until 16–20 h. Plasma calcium responses to exogenous 1,25-(OH) 2-D 3 occurred before the calcium-binding protein response in D-deficient animals and after in replete animals. The plasma calcium response therefore is not an index of the molecular response to vitamin D. It is concluded that the extent of the calcium-binding protein response to exogenous 1,25-(OH) 2-D 3 is a function of the calcium status of the D-replete animal and that there exists an upper limit of this response. The time and rate of the calcium-binding protein response, on the other hand, depend on prior vitamin D status and may reflect a post-transcriptional as well as a transcriptional role of the metabolite in the intestinal cell.

Stimulation of a Cd-binding protein, and inhibition of the vitamin D-dependent calcium-binding protein, by zinc or cadmium in organ-cultured embryonic chick duodenum

Embryonic chick duodenum maintained in organ culture responds to 1α,25-dihydroxy vitamin D 3 in the culture medium by de novo synthesis of a specific calcium-binding protein (CaBP). The addition of Cd 2+(3–5 × 10 −5 m) or Zn 2+(10 −5-10 −4 m) to the medium inhibited CaBP, but stimulated biosynthesis of a Cd-binding protein (CdBP). CdBP in duodenal homogenate supernatants was assessed in two ways: first, by its 109Cd-binding activity ( 109CdBA) using a competitive ion exchange procedure; and, second, by the extent of [ 35S]-cystine incorporation into a specific peak or band after gel filtration or analytical polyacrylamide disc gel electrophoresis, respectively. Regardless of whether cadmium- or zinc-stimulated, the 35S-labeled CdBP—the only protein significantly labeled under the conditions employed—migrated identically upon gel filtration and electrophoresis, and comigrated with purified chick liver Cd-metallothionein. Neither actinomycin D nor α-amanitin, in concentrations sufficient to severely inhibit CaBP, significantly reduced CdBP production. However, cycloheximide did inhibit either Cd 2+- or Zn 2+-stimulated CdBP by about 50% at an inhibitor concentration which abolished CaBP. The inhibitor studies, coupled with the observations of extensive incorporation of [ 35S]cystine into CdBP, suggest that the metals stimulated biosynthesis by a mechanism operating at the translational level. The organcultured duodenum seems well suited for studies of the regulation of CdBP biosynthesis especially since it responds predictably to the steroid hormone, 1α,25-dihydroxy vitamin D 3, in the induction of another specific protein, CaBP, at the transcriptional level. The biosynthesis of CaBP thus may serve as a convenient control in studies of CdBP production under various experimental conditions.

Metabolic studies of the cytosolic calcium-binding protein of the rat.

Biosynthesis of the calcium-binding protein (CaBP) found in mucosal scrapings or isolated cells of rat intestinal mucosa (1) requires 1,25-dihydroxyvitamin D3 (1,25-(OH) 2-D3), the dihydroxylated metabolite of vitamin D (2). Studies of this process have suggested (3,4) that it is akin to steroid hormone induced protein synthesis, involving a sequence of two cytosolic and one nuclear receptor for 1,25-(OH) 2-D3, with CaBP synthesis the ultimate result. Cell-free translation of the chick CaBP, a larger molecule, and of the pig CaBP (MW ≃ 10,000, like that of the rat [5]) has been effected (6,7). In addition to an absolute requirement for 1,25-(OH)2-D3, CaBP biosynthesis is also regulated by calcium, inasmuch as animals on high calcium intakes have low levels of CaBP, while low calcium intakes are associated with much higher CaBP levels (5,8,9). Moreover, in vitro studies with isolated intestinal cells have suggested (10) that raising the calcium concentration of the medium depresses the amount of CaBP that can be synthesized. In addition, the calcium status of the whole animal affects the amount of circulating 1,25-(OH)2-D3, with larger amounts circulating and reaching the intestinal target cells in animals on low calcium intakes (11).

Calcium-binding protein of intestine: Induction by biologically significant cholecalciferol-like steroids in vitro

Vitamin D (cholecalciferol-like) steroids added to the culture medium induce a specific calcium-binding protein (CaBP) ‡ in embryonic chick duodenum maintained in organ culture. Responses of the isolated duodenum faithfully mimic several other cholecalciferol-dependent responses observable in vivo attesting to their physiological significance. Therefore, this system provides a relevant bioassay, i.e. a physiological response (CaBP-induction) in a principal target organ, for the study of structure-activity relationships of biologically-significant cholecalciferol-like steroids. The several cholecalciferol-like steroids normally circulating in the intact animal, including cholecalciferol itself, 25-hydroxycholecalciferol, 24R,25-dihydroxycholecalciferol and 1α,25-dihydroxycholecalciferol, induced CaBP biosynthesis in vitro at or below physiological concentrations. This clearly demonstrates the relative rather than absolute specificity of the intestine for cholecalciferol-like steroids in the induction of CaBP. Maximal activity was observed with either 1α-hydroxycholecalciferol (not a native steroid), 1α,25-dihydroxycholecalciferol or 1α,24R,25-trihydroxycholecalciferol indicating a requirement for the 1α-hydroxyl function but suggesting the non-essentiality of the 25-hydroxyl function. The cis triene structure and an intact side-chain were also found to be essential to optimal activity. Preliminary studies with impure analogs suggest an absolute requirement for the 3β-hydroxyl group. The minimal structure inducing CaBP among naturally occurring steroids is cholecalciferol itself.

Vitamin d-stimulated intestinal calcium absorption may not involve calcium-binding protein directly.

ONE of the earliest responses of chick intestine to cholecalciferol (vitamin D3) is the appearance of a soluble calcium-binding protein (CaBP) for which current evidence indicates a role in the intestinal absorption of calcium1,2. CaBP, which has never been detected in the intestine of cholecalciferol-deficient chickens, is synthesised de novo in response to cholecalciferol, apparently by the induction of the transcription of a specific mRNA for CaBP (ref. 3). In the chick CaBP biosynthesis is initiated after a lag of about 7 h after physiological doses of the vitamin. A correlation between the appearance of CaBP and the first detectable increase in calcium absorption in response to cholecalciferol was observed when CaBP was measured using immunological procedures4,5 but not using the less sensitive Chelex assay6. Consequently it has been generally accepted that CaBP plays a major role in chicks in cholecalciferol-stimulated calcium transport, but by a mechanism which has still to be described. These changes in intestinal CaBP levels and in calcium absorption are brought about by 1,25-dihydroxycholecalciferol (1,25-(OH)2D3), the hormonal metabolite of D3. We report here the sequence and time scale of changes in calcium absorption and CaBP levels in the intestine in response to 1,25-(OH)2D3. By comparison with events observed after cholecalciferol administration, the changes in response to the hormone turn out to be much more rapid and to have different rates of decay, with calcium transport being stimulated before CaBP can be detected immunologically.

The induction of calcium-binding protein biosynthesis by vitamin D 3 and 25-hydroxycholecalciferol.

The induction of calcium-binding protein biosynthesis by vitamin D 3 and 25-hydroxycholecalciferol.

The induction of calcium binding protein biosynthesis in intestine by vitamin D 3

The effect of vitamin D 3 on de novo biosynthesis of the calcium-binding protein of chick intestinal mucosa, and the temporal relationship between such synthesis and the stimulation of intestinal calcium transport by the vitamin was investigated. When doses of 20 IU of vitamin D 3 were administered to either frankly D-deficient (“rachitic”) or partially D-deficient (“marginal”) chicks, the rate of biosynthesis of Ca-binding protein as measured by incorporation of [ 3H]leucine was increased. In rachitic chicks, incorporation into Ca-binding protein was initiated from an undetectable to a substantial rate in 24 h. In marginal chicks, a three to four-fold increase in incorporation rate was seen in 12 h. Incorporation of isotope into total mucosal protein was unaffected at the 20 I.U. dose level in either type of chick, but was stimulated 50 % in 24 h by treatment of rachitic chicks with high doses of 500 I.U. This indicated that large doses of the vitamin may elicit rapid non-specific stimulatory effects on growth and metabolism, in contrast to the apparently specific effects on calcium transport of small doses. Simultaneous measurement of the effect of vitamin D on calcium transport and rate of Ca-binding protein biosynthesis in marginal chicks indicated that the rate of Ca-binding protein biosynthesis was stimulated by the vitamin several hours before any effect on transport could be detected. 9 h after the administration of 20 I.U., [ 3H]leucine incorporation into pure Ca-binding protein was stimulated two to three-fold, with no effect whatsoever on calcium transport. Ca-binding protein content in the mucosal tissue appeared to increase in parallel with the rate of calcium transport. These data are consistent with the hypothesis that vitamin D acts at the genetic level, and also suggests that Ca-binding protein may play a primary role in vitamin D-mediated calcium transport.