Intestinal Calcium-binding Protein Research Articles

Preparation of a photoaffinity probe for the vitamin D-dependent intestinal calcium binding protein: Evidence for a calcium dependent, specific interaction with intestinal alkaline phosphatase

A photoaffinity probe for the vitamin D-dependent chick intestinal calcium binding protein (CaBP) has been prepared by conjugation of methyl-4-azidobenzoimidate (MABI) to lactoperoxidase- 125I-iodinated CaBP to yield 125I-CaBP-MABI: [3 moles MABI per mole CaBP]. After incubation in vitro of 125I-CaBP-MABI (28,000 daltons) in model systems with bovine intestinal alkaline phosphatase (AP) (67,000 daltons), a UV light-dependent crosslinking occurred to yield a conjugate with a molecular weight of 95,000 (by SDS-gel electrophoresis); no crosslinking occurred with E. coli alkaline phosphatase. The formation of the 125I-CaBP-MABI-AP was found to occur only in the presence of calcium.

Cholecalciferol Requirements of Young Turkeys under Normal Conditions and during Recovery from Rickets

Day-old turkeys fed vitamin D-deficient diets became rachitic within 17–24 days. The symptoms included reductions in body weight, plasma calcium and inorganic phosphorus, plasma and intestinal calcium-binding protein (CaBP), plasma 25-hydroxycholecalciferol [25(OH)D3], bone ash, and kidney 25(OH)D3-24-hydroxylase and a rise in kidney 25(OH)D3-1-hydroxylase activity. Supplementation of the diet with 12.5 µg cholecalciferol per kilogram was sufficient to promote maximal body weight and normal plasma calcium, plasma phosphorus and bone ash. Feeding diets containing 250 or 1250 µg cholecalciferol per kilogram resulted in a reduced body weight. An increase in the concentration of plasma 25(OH)D3 with increasing dietary cholecalciferol concentration was observed. Feeding vitamin D-deficient rachitic birds for 4 days a diet containing 50 µg cholecalciferol per kilogram restored plasma calcium and phosphorus and bone ash. Body weight remained lower than that of the control for an additional 6-day period. Additional cholecalciferol, 25(OH)D3 or 1α-hydroxycholecalciferol in the diet, intramuscular injection of the vitamin D derivatives, or a highcalcium, high-phosphorus diet did not accelerate the recovery from the rachitic state.vitamin D metabolism deficiency requirements recovery calcium-binding protein turkey

Plasma parathyroid hormone levels and intestinal calcium binding protein in magnesium deficient rats.

Rats were pair fed a magnesium deficient or control diet. Plasma parathyroid hormone (PTH) was estimated by radioimmunoassay using synthetic 1-34 PTH and intestinal calcium-binding protein (CaBP) was quantified directly by RIA in proximal duodenum, distal ileum and medium jejunum. In magnesium depleted rats, plasma magnesium levels were significantly decreased, a fall in plasma phosphate paralleled the decrease in plasma magnesium and plasma calcium levels were significantly increased after 14 days of magnesium deficiency. A significant rise in plasma PTH was observed on day 7 and 14 after magnesium deficiency. This increase disappeared on day 20. During the whole experimental period, no significant differences in CaBP levels were observed between the two groups of rats. Thus it is difficult to postulate an increase in vitamin D-dependent calcium absorption to explain the hypercalcemia found in magnesium deficient rats. Neither can the hypercalcemia be readily explained by an increased bone calcium mobilisation due to transient PTH increase since previous results have reported decreased bone resorption in magnesium deficient rats under similar experimental conditions.

Immunocytochemical localization of the vitamin D-dependent calcium binding protein in chick duodenum.

The vitamin D-dependent calcium binding protein (CaBP) of chick duodenum has been localized by immunocytochemistry and by radioimmunoassay. Light microscopically, CaBP was seen to be present in the absorptive cells of the villi while in other cell types of the villi and the crypts, including goblet cells and endocrine cells, no CaBP was seen. At the electron microscopic level, CaBP was shown to be localized in the cytosol and the euchromatin of the nucleus but not in membrane-bounded cytoplasmic compartments. Quantitative evaluation of the immunocytochemical protein A-gold label showed that the terminal web and the cytosol of basal cellular regions were most highly labeled while the brush border was weakly labeled. The radioimmunoassay evaluation of intestinal subcellular fractions indicated that 96% of the homogenate CaBP is in the cytosol high-speed supernatant fraction. Collectively, these results support the hypothesis that the vitamin D-dependent intestinal CaBP may play a role in either regulation of intracellular calcium concentration or movement of calcium across the brush border membrane from the gut lumen.

Intestinal calcium-binding protein in the developing rat duodenum.

Summary: An immunoassay for rat intestinal calcium-binding protein (CaBP) employing antibody raised in chickens using purified rat intestinal CaBP has been used to measure immunoreactive CaBP in the duodenum of suckling and weanling rats. A 5-fold increase in immunoreactivity, associated with a 2 ½-fold increase in calcium-binding activity occurred at weaning, a period during which duodenal mucosal mass increased 2-fold and supernatant protein concentration increased 1 ½-fold. Speculation: The ontogeny of calcium-binding protein in the duodenum of young rats parallels the maturation of cholecalciferol hydroxylases and represents maturation of the intestine as an end organ responsive to the vitamin D endocrine system.

Immunocytochemical localization of vitamin D-dependent calcium-binding protein in renal tubules of rabbit, rat, and chick

Vitamin D-dependent calcium-binding protein (CaBP) was localized in tissue sections of kidneys from rabbits, rats, and chicks using antiserum specific for chick intestinal CaBP. In rabbit kidney, CaBP was present in all cells of the distal convoluted tubule and most cells of the connecting tubule. Fewer, but still a majority, of the cells of cortical collecting ducts contained CaBP. The intensity of immunochemical staining and the number of stained cells decreased markedly in medullary collecting ducts, and only a few collecting duct cells contained CaBP at the junction of the inner and outer medulla. In the rat kidney, CaBP was present in all distal convoluted tubule cells, but the immunochemical staining was less intense than in the rabbit. The protein also was found in most connecting tubule cells of the rat; however, only a few collecting duct cells in the superficial corte of the rat contained CaBP. CaBP was essentially absent from mid- to deep-cortical collecting duct cells, while a very few collecting duct cells always contained CaBP at the junction of the inner and outer stripes of the outer medulla. In the chick, CaBP was present in distal convoluted tubule cells as the distal convoluted tubule coursed adjacent to the central vein. CaBP was absent from chick collecting duct cells. In all three species CaBP was not detected in the other portions of the nephron.

Effect of vitamin D status on the equilibrium between occupied and unoccupied 1,25-dihydroxyvitamin D intestinal receptors in the chick.

The dynamic equilibrium between in vivo occupied and unoccupied 1,25-dihydroxyvitamin D(3)[1,25(OH)(2)D(3)] receptors of the chick intestinal mucosa was investigated by the exchange assay previously reported [(1980). J. Biol. Chem.255: 9534-9537]. These parameters and their correlation to biological response, i.e., the levels of intestinal vitamin D-dependent calcium binding protein (CaBP), were assessed under different physiological conditions. After a single 1,25(OH)(2)D(3) injection (3.25 nmol), occupied receptor levels increased sharply to a maximum between 1 and 2 h, followed by a rapid decline. A single dose of 1alpha-hydroxy-vitamin D(3) [1alpha(OH)D(3)], an analog that requires 25-hydroxylation for biological activity, resulted in a protracted, albeit lower, response with maximal receptor occupancy at 6 h and half maximal levels 24 h after injection. The intestinal receptor occupancy patterns mirrored the serum 1,25(OH)(2)D(3) levels after either 1,25(OH)(2)D(3) or 1alpha(OH)D(3) treatment. Additionally, time-course (half-life) of blood disappearance of 1,25(OH)(2)D(3) and occupied receptor levels were similar (1.9 and 2.3 h, respectively), suggesting that the amount of occupied 1,25(OH)(2)D(3) receptor is determined by a simple equilibrium between serum 1,25(OH)(2)D(3) and unoccupied receptors. A dose-response study after intramuscular 1,25(OH)(2)D(3) injection yielded a hyperbolic curve with an apparent plateau at 70% receptor occupancy, corresponding to 5 nmol 1,25(OH)(2)D(3) injected. Half-maximal occupancy was reached after a dose of 1 nmol 1,25(OH)(2)D(3), corresponding to 1.5 ng 1,25(OH)(2)D(3)/ml serum. From this value the apparent K(d) in vivo is 3.7 nM, which is similar to that determined in vitro. A 10-fold increase in the 1alpha(OH)D(3) dose resulted in less than a doubling of the levels of serum 1,25(OH)(2)D(3), occupied 1,25(OH)(2)D(3) receptors, or CaBP. Under all experimental conditions, there was a positive correlation between occupied receptor and CaBP levels; however, the slope of the lines depended on the times chosen for the assays due in part to the lag period for CaBP induction and its accumulation within the cell. Conversely, the correlation between serum 1,25-(OH)(2)D(3) levels and occupied receptor levels yielded a single regression line independent of the observation time. Short and long-term treatment with different vitamin D metabolites, estrogen, progesterone, or cortisol did not affect the levels of total intestinal 1,25(OH)(2)D(3) receptor. Under normal physiological conditions, only 10-15% of the total 1,25(OH)(2)D(3) receptor population was occupied by ligand. These studies provide a basis for further investigations of physiological and biochemical parameters of the vitamin D endocrine system and their clinical applications.

Regulation of calcium-binding protein in mouse placenta and intestine.

To study the developmental controls for both intestinal and placental calcium-binding protein (CaBP), we have altered mineral metabolism by feeding pregnant mice diets high in calcium and strontium. The effects of these dietary changes on CaBP content of maternal intestine and placenta and also on fetal mineral accumulation have been measured. Feeding high-calcium diets to pregnant mice decreased CaBP content in both maternal intestine and placenta (45 and 58%, respectively). Under these conditions of maternal hypercalcemia, fetal mineral accumulation was unchanged. In experiments with strontium-containing diets, CaBP content was reduced in both maternal intestine and placenta (66 and 67%, respectively), and fetal mineral accumulation was markedly reduced (70%). Administration of 1,25-dihydroxycholecalciferol [1,25(OH)2D] to the strontium-fed mice allowed the gestational rise in maternal intestinal CaBP to proceed to normal levels or above; however, the level of placental CaBP was not affected. Similarly, administration of 1,25(OH)2D to pregnant mice on a normal diet increased maternal intestinal CaBP, but had no effect upon placental CaBP. Thus, administration of 1,25(OH)2D to the mother, in amounts sufficient for a maternal intestinal response, was not a sufficient condition to increase placental production of CaBP.

Intestinal calcium-binding protein in the baboon ( Papio cynocephalus)

1. 1. Calcium-binding protein (CaBP) has been isolated from baboon ( Papio cynocephalus) intestinal mucosa by gel filtration and ion-exchange chromatography. 2. 2. Similarity in electrophoretic behavior, size and charge and immunologic structure are demonstrated between the baboon CaBP and CaBPs isolated from other species. 3. 3. Baboon intestinal CaBP is resistant to neuraminidase digestion.

Calcium Metabolism in Glucocorticoid-Treated Pony Foals

Pony foals were injected intramuscularly with 0, 0.5, or 5.0 mg dexamethasone per 100 kg body weight daily for up to 11 months. True absorption of dietary calcium was inhibited by treatment at 18 days (57% versus 14% of intake; P < .01) and 10 months (72% versus 55% versus 43%; P < .01), but not at 2 or 7 months, and was independent of age and treatment related decreases in intestinal calcium binding protein activities. Uninary excretion of calcium was increased by treatment through 7 months but decreased at 10 months. Treatment for 18 days resulted in total calcium excretions equivalent to 127% of dietary intake. All animals were in positive calcium balance thereafter. Endogenous fecal calcium excretion was not affected by treatment, age, or sex, averaging 18.0 mg per kg body weight per day. At 2 months, rates of calcium deposition into bone were 13.02, 7.01 and 6.83 g per 100 kg per day in ponies treated with 0, 0.5, and 5.0 mg per 100 kg, respectively. Corresponding rates of calcium removal from bone were 5.41, 1.88, and 2.69 g per 100 kg per day. These rates remained significantly inhibited in the treated ponies at 7 and 10 months. The net effect of dexamethasone treatment was an inhibition of bone turnover: calcium removal was suppressed more than calcium deposition.calcium metabolism calcium kinetics bone turnover equine glucocorticoid treatment

Regulation of calcium-binding protein messenger RNA by 1,25-dihydroxycholecalciferol.

1,25-Dihydroxycholecalciferol modulates the synthesis of the intestinal calcium-binding protein. To determine if this effect is due to an increase in calcium-binding protein mRNA activity, we measured total cytoplasmic protein-specific mRNA activity from chick intestine in an in vitro wheat germ translation system. 1,25-Dihydroxycholecalciferol enhances calcium-binding protein specific mRNA activity. The sterol does not induce a general increase in mRNA synthesis since the concentration of only a few proteins increased. Thus 1,25-dihydroxycholecalciferol regulates only a subset of genes and specifically affects a transcriptional process involving calcium-binding protein synthesis.

Primary translation product of mRNA coding for rat duodenal vitamin D-dependent calcium-binding protein

The intestinal mucosa of several species contains vitamin D-dependent calcium-binding proteins (CaBP) which may be involved in the complex process of calcium transport [ 11. The cytosolic CaBP (11 500 Mr) [2,3] of the rat duodenum accounts for 2% of the soluble mucosal protein [4]. It also exhibits considerable specificity since it has no immuno-crossreactivity with either other mammalian intestinal CaBPs [5], including porcine CaBP [5-71, or the larger (28 000 Mr) [8] vitamin D-dependent CaBP of the chick duodenal mucosa [ 51. The induction of intestinal 28 000 Mr CaBP synthesis by active messenger RNA (mRNA) has been studied in the chick [9-121. It appears to be dependent on the hormonal metabolite of vitamin D, 1,25-dihydroxycholecalciferol (l,25-(OH)2D3) [ 111. In the rat there is evidence indicating that the 11 500 Mr duodenal CaBP concentration measured by using radioimmunoassay is dependent upon 1,25-(OH)2Da [13,14]. To date, however, there is no report of its de novo synthesis. To further study the regulation of rat duodenal CaBP production we have undertaken an analysis of the expression of the relevant gene(s). This report deals with the identification of the primary translation product of the mRNA coding for rat duodenal CaBP.

Calcium-dependent interaction of S100b, troponin C, and calmodulin with an immobilized phenothiazine.

We have purified the brain-specific protein S100b by affinity-based adsorption chromatography on phenothiazine-Sepharose conjugates and studied the interaction of this and other calcium-modulated proteins with the immobilized antipsychotic drug. Bovine brain calmodulin, rabbit skeletal muscle troponin C, and bovine brain S100b bind to phenothiazine-Sepharose in a calcium-dependent manner. These three proteins competitively inhibit the calcium-dependent binding of 125I-labeled chicken gizzard calmodulin to the immobilized drug. However, carp parvalbumin and chicken intestinal vitamin D-dependent calcium binding protein do not inhibit the phenothiazine--calmodulin interaction. These results suggest that the known amino acid sequence homology among calmodulin, troponin C, and S100b may be reflected in a similar functional domain present in these proteins but absent in parvalbumin and vitamin D-dependent protein.

Rat intestinal calcium-binding protein: Rapid purification with AG MP-1 ion-exchange chromatography

Rat intestinal calcium-binding protein has been purified using standard preparative methods that include heat treatment and gel-filtration chromatography. Final purification was achieved using ion-exchange chromatography with the macroporous ion exchanger AG MP-1. Substitution of this step for preparative polyacrylamide slab gel electrophoresis allowed purification of milligram quantities of homogeneous calcium-binding protein from larger amounts of the partially purified preparation without dilution. The calcium-binding protein thus purified is heat stable, has a molecular weight of 11,500, and has electrophoretic behavior characteristic of rat intestinal calcium-binding protein purified by other techniques.

Vitamin D3 metabolite injections to thyroparathyroidectomized pregnant rats: effects on calcium-binding proteins of maternal duodenum and of fetoplacental unit.

Fetomaternal relationships with respect to vitamin D metabolism were investigated in thyroparathyroidectomized (TPTX) pregnant rats, with or without treatment with different vitamin D3 metabolites. Calcium-binding protein (CaBP) in maternal duodenum was used as an index of 1,25-(OH)2D3 status of the mother. Pregnant rats were TPTX on day 12.5 and CaBP was measured on 21.5 days of gestation by RIA in maternal duodenal mucosa and in the fetoplacental unit (placenta, fetal membranes, and fetal intestine). In the duodenum of TPTX mothers, the CaBP concentration was reduced by 50%. This fall was associated with a decrease of 1,25-(OH)2D in maternal plasma. CaBP in maternal duodenum increased by the administration of 1,25-(OH)2D3 or 1,24,25-(OH)3D3. In contrast, 24,25-(OH)2D3 injections to TPTX mothers were ineffective. In both placenta and fetal membranes, CaBPs decreased by 20% in TPTX mothers and were normalized only in 1.25-(OH)2D3-treated TPTX mothers. In the fetal intestine, CaBP variations paralleled those of maternal duodenal CaBP. The data indicate that plasma levels of 1,25-(OH)2D in TPTX pregnant rats are partly under the control of maternal parathyroid glands, and they support that even in pregnancy, the CaBP concentration in maternal duodenum may well reflect the 1,25-(OH)2D status of the mother. The CaBP synthesis in placenta and fetal membranes are vitamin D-dependent, and their regulation differs from that of intestinal CaBP. It app]ears that 1 alpha-hydroxylase activities of the fetoplacental unit (placenta and fetal kidney) are blunted in TPTX animals and that CaBP synthesis in the fetus depends on the presence of 1 alpha-hydroxylated vitamin D3 metabolites in the mother.

The amino acid sequence of bovine intestinal calcium-binding protein.

The complete amino acid sequence of the vitamin D-dependent bovine intestinal calcium-binding protein (minor A component) has been determined: Lys-Ser-Pro-Glu-Glu-Leu-Lys-Gly-Ile-Phe-Glu-Lys-Tyr-Ala-Ala-Lys-Glu-Gly-Asp-Pro-Asn-Gln-Leu-Ser-Lys-Glu-Glu-Leu-Lys-Leu-Leu-Leu-Gln-Thr-Glu-Phe-Pro-Ser-Leu-Le u-Lys-Gly-Pro-Ser-Thr-Leu-Asp-Glu-Leu-Phe-Glu-Glu-Leu-Asp-Lys-Asn-Gly-Asp-Gly-Glu-Val-Ser-Phe-Glu-Glu-Phe-Gln-Val-Leu-Val-Lys-Lys-Ile-Ser-Gln-OH. It is a 75-residue protein (computed Mr = 8501), contains a single Tyr, and is devoid of Cys, Met, Trp, His, and Arg. The bulk of the sequence was determined by automated sequencing of: (i) the intact protein for 20 cycles; (ii) a large N-bromosuccinimide peptide for 37 cycles; (iii) a tryptic peptide (29 cycles), isolated by high performance liquid chromatography. Also described is a highly sensitive and rapid procedure for peptide mapping by high performance liquid chromatography.

Vitamin D-dependent calcium-binding protein. Immunochemical studies and synthesis by placental tissue in vitro.

A protein similar to rat intestinal calcium-binding protein (CaBP) has been identified in both mouse placenta and mouse small intestine. The mouse protein had a molecular weight of approximately 10,000, exhibited cation-binding properties, and demonstrated immunologic identity with vitamin D-dependent rat CaBP. Under normal dietary conditions, the concentrations of CaBP in mouse placenta and intestine increased 6- and 3-fold, respectively, during the third trimester of pregnancy in parallel with the fetal demands for skeletal mineral. Studies of in vitro protein synthesis indicated that CaBP was synthesized by placental tissue. Slices of mouse or rat placental tissue (12-18-day gestation) were incubated with [3H]leucine and the biosynthesis of placental CaBP was quantified by an immunoprecipitation method using rabbit antiserum to rat intestinal CaBP. Sodium dodecyl sulfate gel electrophoresis of the radioactive immune complex revealed a single 3H-labeled peak corresponding to the molecular weight of rat and mouse CaBP (10,050). The amount of CaBP synthesized by mouse placental tissue was dependent upon gestational age of the placenta and reflected the in vivo changes in placental CaBP content observed during gestation. These data indicate that CaBP is synthesized by placenta and provide an in vitro model for studying the developmental control of placental CaBP synthesis.

556 INTESTINAL CALCIUM-BINDING PROTEIN IN THE DEVELOPING RAT DUODENUM

To monitor the maturation of the Vitamin D dependent active calcium transport mechanism in neonatal rat intestine, calciumbinding protein (CaBP) was measured in duodena of suckling and weanling rats. Duodena from pylorus to ligament of Treitz were removed from pups from 1 to 7 weeks old, weighed, rinsed and homogenized. The 30,000 xg supernate of each homogenate was assayed for calcium-binding activity using the Chelex-100 competitive radiocalcium binding assay, for CaBP by radial immunodiffusion using an antibody raised in chickens against purified rat intestinal CaBP, and for total protein. The marked enhancement of calcium binding activity and immunoreactive CaBP noted at weaning (Wk 3), corresponds with 1) enhancement and localization of active calcium transport characterizing functional maturation of the neonatal rat intestine and 2) maturation of the vitamin D endocrine system.

Relationships between Cholecalciferol Metabolism and Growth in Chicks as Modified by Age, Breed and Diet

Fast-growing heavy (White Rock) chicks, fed a vitamin D-deficient diet, exhibited a higher activity of kidney 25-hydroxycholecalciferol-1-hydroxylase (1-hydroxylase) than slow-growing light (White Leghorn × Rhode Island Red) chicks fed the same diet. 1-Hydroxylase and duodenal calcium-binding protein (CaBP) declined with age. Feeding of low energy diets with or without vitamin D resulted in a slower rate of growth and reduced 1-hydroxylase activity and CaBP concentration. Severe dietary restriction of either calcium or phosphorus resulted in a lower growth rate as well as a duodenal CaBP as compared to a moderate mineral restriction. The severe dietary calcium restriction also resulted in a lower 1-hydroxylase activity than that resulting from a moderate restriction. The results clearly indicate that high 1-hydroxylase activity and a high intestinal CaBP are associated with a high growth rate.Vitamin D deficiency calcium-binding protein

Determination of the rates of synthesis and degradation of vitamin D-dependent chick intestinal and renal calcium-binding proteins

Vitamin D 3 and its biologically active metabolite 1,25-dihydroxyvitamin D 3 [1,25(OH) 2D 3] are shown to induce in the chick intestine and kidney the biosynthesis of a calcium binding protein (CaBP). In vitamin D 3-replete chickens raised under adequate dietary calcium (Ca) and phosphorus (P) conditions, the steady-state level of intestinal CaBP (30–50 g/mg protein) is 5- to 20-fold greater than that of renal CaBP. Whereas dietary phosphorus restriction is known to elevate both intestinal and renal CaBP levels, dietary calcium restriction elevates only intestinal CaBP. The present study reports the rates of biosynthesis in vivo and in vitro, and of biodegradation in vivo, of both intestinal and renal CaBP after administration of vitamin D 3 or 1,25(OH) 2D 3 to rachitic chicks. The apparent rate constant of degradation for intestinal CaBP was 0.024 h −1 ( t 1 2 = 29 h ) and that for renal CaBP was 0.019 h −1 ( t 1 2 = 36 h ) while total cellular soluble protein in the intestine and kidney had half-lives of 43 and 70 h, respectively. The time course of induction of the synthesis of CaBP was determined in intestine and kidney after administration of a physiological dose of 1,25(OH) 2D 3 to rachitic chicks. Intestinal CaBP synthesis was detectable by 3 hours, reached a maximal rate by 10 hours, and sharply decayed by 16–20 hours. The time course of induction of renal CaBP synthesis was very similar, although the rate of renal CaBP synthesis was readily detectable at the initial time of administration of 1,25(OH) 2D 3. The relative rates of synthesis of CaBP in the intestine and kidney under a variety of dietary Ca and P conditions in the vitamin D 3-replete chick exactly paralleled the steady-state level of CaBP in these two tissues. These results are consistent with a model in which the steady-state levels of intestinal and renal CaBP are solely determined by their respective rates of biosynthesis; the CaBP biosynthetic capability, in turn, is regulated by the availability of 1,25(OH) 2D 3 to each target organ.