Intestinal Calcium-binding Protein Research Articles

Impact of the Phytoestrogen Content of Laboratory Animal Feed on the Gene Expression Profile of the Reproductive System in the Immature Female Rat

The effect of the dietary background of phytoestrogens on the outcome of rodent bioassays used to identify and assess the reproductive hazard of endocrine-disrupting chemicals is controversial. Phytoestrogens, including genistein, daidzein, and coumestrol, are fairly abundant in soybeans and alfalfa, common ingredients of laboratory animal diets. These compounds are weak agonists for the estrogen receptor (ER) and, when administered at sufficient doses, elicit an estrogenic response in vivo. In this study, we assessed the potential estrogenic effects of dietary phytoestrogens at the gene expression level, together with traditional biologic end points, using estrogen-responsive tissues of the immature female rat. We compared the gene expression profile of the uterus and ovaries, as a pool, obtained using a uterotrophic assay protocol, from intact prepubertal rats fed a casein-based diet (free from soy and alfalfa) or a regular rodent diet (Purina 5001) containing soy and alfalfa. Estrogenic potency of the phytoestrogen-containing diet was determined by analyzing uterine wet weight gain, luminal epithelial cell height, and gene expression profile in the uterus and ovaries. These were compared with the same parameters evaluated in animals exposed to a low dose of a potent ER agonist [0.1 μg/kg/day 17α-ethynyl estradiol (EE) for 4 days]. Exposure to dietary phytoestrogens or to a low dose of EE did not advance vaginal opening, increase uterine wet weight, or increase luminal epithelial cell height in animals fed either diet. Although there are genes whose expression differs in animals fed the soy/alfalfa-based diet versus the casein diet, those genes are not associated with estrogenic stimulation. The expression of genes well known to be estrogen regulated, such as progesterone receptor, intestinal calcium-binding protein, and complement component 3, is not affected by consumption of the soy/alfalfa-based diet when assessed by microarray or quantitative reverse transcriptase–polymerase chain reaction analysis. Our results indicate that although diet composition has an impact on gene expression in uterus and ovaries, it does not contribute to the effects of an ER agonist.

Pregnancy and lactation increase vitamin D-dependent intestinal membrane calcium adenosine triphosphatase and calcium binding protein messenger ribonucleic acid expression.

The calcium demands of pregnancy and lactation are known to up-regulate intestinal calcium absorption. Intestinal epithelial cells contain calcium ATPases and calcium binding proteins, which are believed to play important roles in intestinal calcium transport. However, the possible role of these two proteins in the up-regulation of intestinal calcium absorption observed in pregnancy and lactation is unknown. In this study, intestinal calcium ATPase (PMCA1), calcium binding protein (9K) (CaBP-9K), and vitamin D receptor (VDR) messenger RNA (mRNA) levels were determined by Northern analysis at different stages of pregnancy and early lactation in rats. Intestinal calcium ATPase and calcium binding protein mRNA levels did not differ significantly among nonpregnant rats and rats pregnant for 7 or 14 days. However, at 21 days gestation both calcium ATPase and calcium binding protein mRNA levels increased 2- to 3-fold. Calcium ATPase and calcium binding protein mRNA remained elevated at 7 days of lactation. Plasma 1,25-dihydroxyvitamin D3 (1,25-D3) concentration exhibited a similar pattern, rising markedly at 21 days gestation and remaining elevated in lactation. VDR mRNA levels did not change during the entire experiment. However, intestinal VDR content increased 2-fold in late pregnancy and lactation. These data suggest that transcription of calcium absorption factors is increased in late gestation and early lactation, perhaps mediated by increased plasma 1,25-dihydroxyvitamin D3 concentrations, and that the effects of gestation and lactation on VDR concentrations are probably posttranscriptional.

Classification and evolution of EF-hand proteins.

Forty-five distinct subfamilies of EF-hand proteins have been identified. They contain from two to eight EF-hands that are recognizable by amino acid sequence as being statistically similar to other EF-hand domains. All proteins within one subfamily are congruent to one another, i.e. the dendrogram computed from one of the EF-hand domains is similar, within statistical error, to the dendrogram computed from another(s) domain. Thirteen subfamilies--including Calmodulin, Troponin C, Essential light chain, Regulatory light chain--referred to collectively as CTER, are congruent with one another. They appear to have evolved from a single ur-domain by two cycles of gene duplication and fusion. The subfamilies of CTER subsequently evolved by gene duplications and speciations. The remaining 32 subfamilies do not show such general patterns of congruence; however, some--such as S100, intestinal calcium binding protein (calbindin 9 kd), and trichohylin--do not form congruent clusters of subfamilies. Nearly all of the domains 1, 3, 5, and 7 are most similar to other ODD domains. Correspondingly the EVEN numbered domains of all 45 subfamilies most closely resemble EVEN domains of other subfamilies. Many sequence and chemical characteristics do not show systemic trends by subfamily or species of host organisms; such homoplasy is widespread. Eighteen of the subfamilies are heterochimeric; in addition to multiple EF-hands they contain domains of other evolutionary origins.

The Highly Efficient Production of Full-Length and Mutant Rat Brain Calcium-Binding Proteins (Calbindins-D 28K) in a Bacterial Expression System

zWe expressed gram amounts of full-length and mutant rat brain calcium-binding proteins (calbindins-D 28K) lacking one or two "EF-hand" motifs in a bacterial expression system. The cDNA for the full-length rat calcium-binding protein was cloned into the NdeI and BamHI sites of the pET3a vector. Additionally, constructs of the rat brain calcium-binding protein lacking EF-hand 2 (Δ2 mutant), EF-hand 6 (Δ6 mutant), and EF-hands 2 and 6 (Δ2,6 mutant) were constructed using the same vector. These chimeric plasmids were used to transfect BL21(DE3) pLysS Escherichia coli cells. Following transformation, the cells were grown in the presence of isopropylthiogalactoside in order to induce bacterial T7 polymerase, which resulted in the production of large amounts of the proteins of interest in the bacterial cytosol. Expressed full-length and Δ2 and Δ2,6 mutant proteins represented 50% or more of total bacterial protein. The Δ6 protein was not expressed. Cell lysis followed by purification of the proteins on DEAE-cellulose routinely resulted in gram yields of the proteins. The purified proteins displayed the appropriate amino acid composition and amino-terminal amino acid sequence. When analyzed by matrix-assisted laser desorption mass spectrometry the proteins were found to have the appropriate molecular weights (within the accuracy limits of the instrument). The expressed proteins bound to a polyclonal antiserum raised against chick intestinal calcium-binding protein. In addition, the full-length, Δ2, and Δ2,6 mutants bound calcium as assessed by a 45Ca blotting procedure. The production of large amounts of readily purified vitamin D-dependent calcium-binding proteins should be useful in biophysical studies of the proteins.

Effects of methylprednisolone and uremia on renal and intestinal calbindin-D in the rat

The effects of glucocorticoids on renal and intestinal calcium binding protein (calbindin-D 28K and calbindin-D 9K) were examined in normal and uremic rats. Chronic uremic rats and normal controls were treated with either methylprednisolone (MP) 1.3 mg/kg/d or isotonic saline given as a continuous intraperitoneal infusion for 1 week before sacrifice. Renal calbindin-D 28K was measured by rocket immunoelectrophoresis and intestinal calbindin-D 9K was measured by an enzymelinked immunoadsorbent assay. Methylprednisolone treatment of chronic uremic rats increased plasma phosphate levels ( P < 0.05), but plasma calcium and 1, 25-dihydroxyvitamin D 3 were unchanged in all groups. MP treatment did not affect the renal calbindin-D 28K in either normal or uremic rats. In normal rats, MP treatment reduced intestinal calbindin-D 9K by 28% when compared to placebo ( P < 0.05). In contrast, chronic uremia increased renal calbindin-D 28K by 51% and 38% ( P < 0.001) in placebo and MP treated uremic rats, respectively, while intestinal calbindin-D 9K was unchanged. Thus, MP treatment and chronic uremia induced different changes in renal and intestinal calbindin-D of the rat suggesting that different mechanisms are involved in the regulation of these vitamin D dependent proteins.

Intestinal and renal calcium-binding protein in rats with experimental short bowel syndrome

Intestinal calcium-binding protein (calbindin-D9K) and renal calcium-binding protein (calbindin-D28K) levels were measured by enzyme-linked immunoadsorbent assays in rats with short bowel syndrome induced by resection of about 85% of the small intestine. Rats with short bowel syndrome had significantly lower mucosal concentrations of calbindinD9K (P < 0.001) and a parallel reduction of both intestinal calcium absorption (P < 0.001) andp-1,25-dihydroxyvitamin D (P < 0.01) in spite of a general hypertrophy of the duodenal mucosa. Interestingly, the renal concentration of calbindin-D28K was significantly elevated (P < 0.05) in rats with short bowel syndrome, a change apparently related to factors independent of vitamin D.

Measurement of Calbindin-D9K in Small Intestinal Biopsy Specimens of Children

The vitamin D dependent intestinal calcium-binding protein (calbindin-D9K) was measured by an enzyme-linked immunoadsorbent assay in small intestinal biopsy specimens from 10 children (aged 15-126 months). The aim was to study the relationship between calcium-binding protein and age, bone age, and height. The patients were examined due to complaints of chronic diarrhea, but no evidence of malabsorption was found. The amount of calbindin-D9K per mg of soluble protein in the small intestinal biopsy specimens was higher than previously studied in normal adults. Calbindin-D9K correlated inversely with chronological age, bone age, and height of the children (p = -0.87, rho = -0.66, and rho = -0.66; p less than 0.05). A direct correlation was found between calbindin-D9K and intestinal alkaline phosphatase activity (p = 0.60; p less than 0.05). The decline in calbindin-D9K may indicate that the active vitamin D dependent intestinal calcium absorption decreases during childhood.

Renal and intestinal calcium-binding proteins in normal and uraemic rats

The effect of chronic uraemia on the concentrations of the 28 kDa renal and 9 kDa intestinal calcium-binding proteins (calbindin-D28K and calbindin-D9K) was investigated in rats. Calbindin-D9K was measured by a competitive enzyme-linked immunoadsorbent assay and calbindin-D28K by rocket immunoelectrophoresis. Chronic uraemia was induced by 5/6 nephrectomy and the results were compared to sham-operated animals. Rats were fed on a diet containing 0.9% calcium and 1.2% phosphorous. Plasma creatinine and plasma urea were elevated in the nephrectomized rats (p less than 0.001), while plasma-1,25-dihydroxycalciferol vitamin D and fractional calcium absorption were unchanged. Plasma parathyroid hormone was significantly elevated in the uraemic rats. The concentration of calbindin-D28K in renal tissue was increased (p less than 0.001) in rats with chronic uraemia and a direct correlation was found between renal calbindin-D28K and plasma urea (p less than 0.05). Intestinal calbindin-D9K correlated inversely with plasma creatinine (p less than 0.05), but the mean level of calbindin-D9K was unchanged in this model of moderate chronic uraemia. Thus, different regulatory mechanisms control levels of calbindin-D9K and calbindin-D28K.

The epidermolytic toxins are serine proteases

Certain strains of Staphylococcus aureus usually belonging to phage group II produce epidermolytic toxins (ETA and ETB) which cause intraepidermal splitting in mice, neonates and occasionally adults. Amino acid sequences of ETA and ETB have been reported but the mechanism of epidermolysis remains unknown. A search of the NBRF-PIR computer database showed the toxins to have significant sequence similarity with staphylococcal V8 protease and that the catalytic triad of V8 protease is present in ETA and ETB. Comparison of ETA, ETB and V8 protease with other members of the trypsin-like serine protease family revealed little homology save for the immediate vicinity of the residues constituting the catalytic triad. The toxins, therefore, exhibit a distant relationship to mammalian serine proteases. A potential Ca 2+-binding loop was identified in ETA (but not ETB) on the basis of sequence similarity with the second calcium-binding loop of rat intestinal calcium-binding protein. Epidermolysis produced by ETA in the mouse bioassay was shown to be inhibited by the presence of EDTA consistent with a Ca 2+-dependent mechanism.

Developmental changes of Ca2+, PO4, and calcitriol metabolism in spontaneously hypertensive rats

We have measured Ca and P balance, serum calcitriol, and vitamin D-dependent intestinal calcium-binding protein (CaBP9k) in the spontaneously hypertensive rat (SHR) and its normotensive control, the Wistar-Kyoto rat (WKY) at 4-5 and again at 13-14 wk of age. In rats on a 1% Ca diet, P balance was significantly more positive in the 5-wk-old SHR than WKY (P less than 0.01); Ca balance tended to be greater in the 5-wk-old SHR. In contrast, in the 14-wk-old SHR, P and Ca balance were less positive than in the WKY (P was 5.8 +/- 1.3 vs. 13 +/- 1.7 mg/day, P less than 0.01, and Ca was 53 +/- 4.6 vs. 67 +/- 2.7 mg/day, P less than 0.05). On the 1% Ca diet, plasma calcitriol levels of the 5-wk-old SHR were higher than those of the WKY (58 +/- 3.2 vs. 40 +/- 2.1 pg/ml, P less than 0.002), whereas at 12 wk there was no difference. On Ca-deficient (0.1%) diets, plasma calcitriol was increased in 5-wk-old and 12-wk-old SHR and WKY, compared with the 1% Ca diet (P less than 0.001). Calcitriol metabolic clearance rate was the same in the 13-wk-old SHR and WKY on either Ca diet. Intestinal CaBP9k content of the 5-wk-old (38 +/- 2.6 vs. 40 +/- 3.9 micrograms/mg) and the 12-wk-old (11 +/- 1.6 vs. 14 +/- 1.4 micrograms/mg) SHR and WKY were similar on the 1% Ca diet.(ABSTRACT TRUNCATED AT 250 WORDS)

Elevated 1,25-dihydroxyvitamin D3 and intestinal calbindin-D9k in the toothless rat

The toothless (tl) rat is a nonlethal osteopetrotic mutation characterized by systemic skeletal sclerosis, growth plate morphology suggestive of rickets, and morphological evidence of reduced osteoclastic bone resorption. Vitamin D metabolites, serum calcium and phosphorus levels, and the developmental appearance of vitamin D-dependent intestinal calcium binding protein (calbindin-D9k) was studied in normal and mutant rats of tl stock from 7 to 35 days of age. 1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] was found to be significantly elevated in mutant animals by 7 days of age (71 +/- 9 pM, tl/tl vs. 24 +/- 8 pM, +/?) and continued to increase to a peak of 428 pM at the time of weaning. This was 240% higher than normals at this period. The elevated levels of 1,25-(OH)2D3 stimulated a significant and precocious appearance of intestinal calbindin-D9k in mutants, beginning by 14 days of age and reaching their peak levels at 21 days postpartum (25.6 +/- 1.7 micrograms/mg protein, tl/tl vs. 16.4 +/- 1.5 micrograms/mg protein, +/?). The cause of the elevated circulating levels of 1,25-(OH)2D3 in tl rats is unknown but may be due to the low serum phosphorus levels present in these animals.

Lonomycin-regulated phosphorylation of the myeloid calcium-binding protein p14

Two associated calcium-binding proteins (CaBPs) have recently been identified specifically in cells of myeloid origin. These proteins have relative molecular masses (Mr) of 8,000 and 14,000 and are variously referred to as the cystic fibrosis antigen, the L1 light chain, MRP-8 or p8, and the L1 heavy chain, MRP14 or p14, respectively. The expression of p8 and p14 seems to be confined to a specific stage of myeloid cell differentiation, because both proteins are expressed in circulating neutrophils and monocytes but not in normal tissue macrophages. In chronic inflammatory conditions, however, such as rheumatoid arthritis, macrophages in affected tissues express both p8 and p14. These proteins are members of a family of CaBPs of low Mr, which include S-100 alpha and beta proteins, calcyclin (2A9), intestinal CaBP and p11. All the proteins have an Mr of approximately 10,000 with the exception of p14 which has a longer C-terminal sequence after the second calcium-binding domain. Little is known about their function, although by analogy with calmodulin they could be molecules involved in intracellular signalling that are activated by an increase in the intracellular Ca2+ concentration ([Ca2+]). Here we report that p14 is phosphorylated in both monocytes and neutrophils. The level of p14 phosphorylation can be increased by elevating the [Ca2+]i using the ionophore ionomycin, but is not affected by activation of protein kinase C using phorbol 12,13-dibutyrate. The phosphorylated residue is threonine at position 113, which is the penultimate amino acid in p14 and contained in the longer 'tail' sequence. Part of this sequence is identical to the neutrophil immobilizing factors NIF-1 and NIF-2, indicating that the phosphorylation event could have a role in the generation of NIF activity in the p14 protein.

Plasma membrane calcium pump and 28-kDa calcium binding protein in cells of rat kidney distal tubules

In an effort to extend our studies on Ca2+ pumps to animal models, we developed a new monoclonal antibody (5F10) prepared against the human erythrocyte Ca2+-Mg2+-adenosinetriphosphatase (ATPase) that recognizes a protein of approximately 140 kDa in rat kidney homogenates. Enzyme-linked immunosorbent assays show that monoclonal antibody 5F10 binds purified Ca2+-Mg2+-ATPase and rat kidney membrane extracts in a concentration-dependent manner. In paraffin-embedded tissue sections, antibody 5F10 binds to an epitope in the basolateral membranes of rat kidney distal convoluted tubule principal cells. The antibody does not bind to intercalated cells. The latter cells were characterized by the presence of large amounts of carbonic anhydrase C. Polyclonal antibodies directed against chick intestinal 28-kDa vitamin D-dependent calcium binding protein (28-kDa CaBP) also bind epitopes in distal convoluted tubule cells, connecting tubules, and portions of collecting duct but not intercalated cells. Western blot and 45Ca blot analysis of renal cytosolic proteins showed that the polyclonal 28-kDa CaBP-directed antibody detects a protein which also binds calcium. Western blot analysis with monoclonal antibody 5F10 shows binding to both the authentic purified erythrocyte Ca2+ pump (approximately 138 kDa) and to tryptic fragments of this pump. Antibody JA3, previously used for staining of human kidney tubules, reacts with a different set of tryptic fragments, showing that the two antibodies are directed against different regions or conformational determinants on the pump molecule. We show that Ca2+-Mg2+-ATPase and 28-kDa CaBP are present in the principal cells of the distal convoluted tubule of the rat and are absent in intercalated cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Epidermal Growth Factor Increases Intestinal Calbindin-D9kand 1,25-Dihydroxyvitamin D Receptors in Neonatal Rats*

Epidermal growth factor (EGF) has been reported to increase intestinal calcium absorption in suckling rats. The mechanism of this effect is unknown, as are the roles of vitamin D-dependent and independent pathways. The present studies were undertaken to investigate the ability of EGF to accelerate the postnatal induction of the vitamin D-dependent intestinal calcium-binding protein, calbindin-D9k. Subcutaneous administration of EGF increased duodenal calbindin-D9k in suckling rats by more than 100% (P less than 0.001). The effect of EGF was not seen in older weaned animals or when EGF was given to suckling rats by gavage. Administration of EGF simulated the changes of normal development. 1) It increased calbindin-D9k, and the effect was greater in proximal than distal duodenum. 2) EGF increased alkaline phosphatase activity to the same extent in proximal and distal duodenum. 3) EGF increased sucrase more markedly in distal than in proximal epithelium. Maximal and half-maximal effects of EGF on each of these proteins were observed at twice daily doses of 0.1 and 0.04 microgram/g BW, respectively. 4) EGF at the maximally effective dose produced a small (30%) but statistically significant (P less than 0.005) increase in serum 1,25-dihydroxyvitamin D. 5) Most importantly, EGF treatment resulted in a 2-fold increase in intestinal 1,25-dihydroxyvitamin D receptors (VDR) in the proximal segments of the small intestine (P less than 0.001). EGF effects on calbindin-D9k and VDR were specific for the intestine, as EGF did not change kidney calbindin-D9k or kidney VDR. Thus, EGF was able to prematurely initiate a complex series of molecular changes that occur during normal development. The mechanism of EGF's action to stimulate calcium absorption appears to involve a maturation effect on the vitamin D-dependent pathway.

Methylation of the vitamin D-dependent CaBP gene (calbindin 9 Kd) does not mediate tissue or vitamin D regulation

The vitamin D-dependent intestinal calcium binding protein gene is predominantly expressed in the intestine. In this report we have examined the possibility that methylation of the gene might play a role in its tissue-specific expression employing genomic Southern analysis. None of the Hpa II and Hha I sites examined by the indicated probes in and around the gene were found to be methylated in the intestine, kidney and liver. No change in the methylation of these sites was detected in response to 1,25-dihydroxy-vitamin D3 administration to vitamin D-deficient rats under conditions which stimulate the expression of the gene. These results indicate that the rat intestinal calcium binding protein gene is not methylated in these tissues, at the indicated sites and, therefore, methylation seems not to be involved in the regulation of this gene's expression.

Conformation change of the intestinal calcium-binding protein induced by phospholipids in the presence and absence of calcium

Effects of phospholipids (PL's) and lyso-PL's on the conformation of the porcine intestinal calcium-binding protein (CaBP) were studied fluorometrically with 1-(dimethylamino)naphthalene-5-sulfonyl-(DNS-) labeled CaBP. The fluorescence intensity of DNS-labeled CaBP was much higher in the presence of excess EGTA than in its Ca2+-bound state. In the absence of free Ca2+ (with 1 mM EGTA) the fluorescence of the labeled CaBP was greatly enhanced by addition of lysophosphatidylcholine (lyso-PC), lysophosphatidylserine (lyso-PS), or lysophosphatidylinositol (lyso-PI). With addition of 25 microM Ca2+ the enhancement of the fluorescence by these lyso-PL's was depressed; especially that due to lyso-PC became small. Lysophosphatidylethanolamine (lyso-PE), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylethanolamine (PE), and mono- and dipalmitoylglycerols had no or much less effect on the fluorescence in the presence and absence of Ca2+. Lyso-PC attenuated in a concentration-dependent manner the quenching of the fluorescence of the DNS-CaBP by high temperatures and increase of ionic strength in the presence of EGTA. Lyso-PL's generally protected the CaBP from digestion with proteases in the presence of EGTA. These experimental results suggest that particular lyso-PL's have Ca2+-sensitive interaction with the porcine CaBP and induce conformation change of the CaBP molecules.

The extrinsic 33 kDa polypeptide of the oxygen-evolving complex of photosystem II is a putative calcium-binding protein and is encoded by a multi-gene family in pea

The extrinsic 33 kDa polypeptide of the water-oxidizing complex has been extracted from pea photosystem II particles by washing with alkaline-Tris and purified by ion-exchange chromatography. The N-terminal amino acid sequence has been determined, and specific antisera have been raised in rabbits and used to screen a pea leaf cDNA library in λgt11. Determination of the nucleotide sequence of positive clones revealed an essentially full-length cDNA for the 33 kDa polypeptide, the deduced amino acid sequence showing it to code for a mature protein of 248 amino acids with an N-terminal transit peptide of 81 amino acids. The protein showed a high degree of conservation with previously reported sequences for the 33 kDa protein from other species and the sequence contained a putative Ca(2+)-binding site with homology to mammalian intestinal calcium-binding proteins. Northern analysis of total pea RNA indicated a message of approximately 1.4 kb, in good agreement with the size of the cDNA obtained at 1.3 kbp. Southern blots of genomic DNA probed with the labelled cDNA give rise to several bands suggesting that the 33 kDa polypeptide is coded by a multi-gene family.

Calmodulin binding to the intestinal brush-border membrane: Comparison to other calcium-binding proteins

The intestinal brush-border membrane contains a high concentration of calmodulin bound to a 105,000 dalton (105 kDa) protein. Binding of radioiodinated calmodulin to this protein does not require calcium but is inhibited by trifluoperazine and excess unlabelled calmodulin. Recent evidence suggests that the 105 kDa protein in conjunction with calmodulin may be involved in the regulation of calcium transport across the brush-border membrane. In this report, we evaluated the binding of the 105 kDa protein to other radioiodinated calcium-binding proteins including the vitamin D-dependent intestinal calcium-binding protein. We observed that troponin C and S100 beta protein both bound strongly to the 105 kDa protein. The binding of S100 beta was inhibited by EGTA, but was little affected by trifluoperazine and excess unlabelled S100 beta, whereas that of troponin C was inhibited by trifluoperazine and excess unlabelled troponin C, but was little affected by EGTA. Both troponin C and S100 beta bound to a large number of proteins to which calmodulin did not bind. The vitamin D-dependent calcium-binding protein (calbindin) from chick intestine and rat kidney also bound to the 105 kDa protein, albeit more weakly than troponin C, S100 beta and calmodulin. The binding of the calbindins was increased by EGTA and was little affected by trifluoperazine and excess unlabelled calbindin. Parvalbumin, rat osteocalcin, and alpha-lactalbumin showed little binding to any brush-border membrane protein. Our results indicate that the 105 kDa calmodulin-binding protein of the intestinal brush border can bind to a variety of calcium-binding proteins all of which contain homologous regions thought to be the calcium-binding sites. Only the binding of troponin C resembles the binding of calmodulin, however, in being inhibited by trifluoperazine and excess unlabelled ligand. The functional significance of these observations in terms of regulating calcium transport across the brush-border membrane remains to be established.

Structure and nucleotide sequence of the rat intestinal vitamin D-dependent calcium binding protein gene.

The vitamin D-dependent intestinal calcium binding protein (ICaBP, 9 kDa) is under transcriptional regulation by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], the hormonal active form of the vitamin. To study the mechanism of gene regulation by 1,25-(OH)2D3, we isolated the rat ICaBP gene by using a cDNA probe. Its nucleotide sequence revealed 3 exons separated by 2 introns within approximately 3 kilobases. The first exon represents only noncoding sequences, while the second and third encode the two calcium binding domains of the protein. The gene contains a 15-base-pair imperfect palindrome in the first intron that shows high homology to the estrogen-responsive element. This sequence may represent the vitamin D-responsive element involved in the regulation of the ICaBP gene. The second intron shows an 84-base-pair-long simple nucleotide repeat that implicates Z-DNA formation. Genomic Southern analysis shows that the rat gene is represented as a single copy.

Instability of purified 9-kDa porcine intestinal calcium-binding protein in the absence of calcium

Purified 9-kDa porcine intestinal calcium-binding protein (ICaBP, Calbindin D 9K) is unstable when stored at 4 °C in the absence of Ca(II). Cleavage of the polypeptide occurs producing ~5.2- and 3.7-kDa fragments. The former dimerizes giving a species which migrates on sodium dodecylsulfate-urea gels with an M r 13,700, in contrast to the observed M r 11,000 for native ICaBP. The fragmentation also results in an irreversible loss of high affinity Ca(II) binding and an alteration of the ultraviolet absorption spectrum. The presence of Ca(II) bound to ICaBP prevents fragmentation. The dimer can be isolated from aged preparations of apo-ICaBP and is stable on further storage with or without Ca(II) present. The observed molecular weights of the fragments along with the amino acid analysis and ultraviolet spectra of the dimer suggest cleavage of the polypeptide chain of ICaBP in the vicinity of residue 49.