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S Christakos

Publications and source records attributed to S Christakos.

At least 91 records · Page 5Linked to original sources
PubMed1989

Transcriptional regulation and chromosomal assignment of the mammalian calbindin-D28k gene.

To determine whether 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] regulates transcription of the rat renal calbindin-D28k gene, the rate of calbindin-D28k mRNA synthesis was measured directly in nuclei using the in vitro nuclear transcription assay. Nuclei were prepared from kidneys of vitamin D-deficient rats at various times after a single ip injection of 1,25-(OH)2D3, and transcription was allowed to proceed in vitro in the presence of [32P]UTP for 30 min at 29 C, at which time the incorporation of UTP into trichloroacetic acid-precipitable material was optimal. Incorporation of UTP was decreased by 64.6% by alpha-amanitin, which selectively inhibits polymerase II. Purified [32P]RNA was analyzed for newly synthesized calbindin-D-28k gene transcripts by hybridization to calbindin-D28k cDNA immobilized on nitrocellulose filters. Using this assay we found that the first significant increase in calbindin-D28k gene transcription occurred at 1 h, and the peak of transcriptional activity occurred at 2 h. Within 12 h of 1,25-(OH)2D3 treatment, calbindin-D28k gene transcription returned to control levels. Using Northern blot analysis, a significant increase in calbindin-D RNA was first observed 2 h after hormone administration, reaching a maximum at 12 h. Renal calbindin-D28k protein levels are significantly increased by 3 h and reach a maximum value 48 h after hormone administration. Our results suggest that the early increase in renal calbindin-D28k may be due to transcriptional regulation. The long time lag between transcription and the peak of calbindin mRNA and calbindin protein accumulation may reflect the involvement of post-transcriptional mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Analysis of rat vitamin D-dependent calbindin-D28k gene expression.

We report the use of a cloned cDNA for mammalian calbindin-D28k (28-kDa vitamin D-dependent calcium-binding protein) to study the expression of the rat calbindin gene. Tissue distribution studies, using Northern analysis, indicated that calbindin-D28k-mRNA is detected in rat kidney and brain but is not detected in rat intestine, testes, bone, pancreas, liver, lung, or skeletal muscle. Both rat kidney and brain contain three RNA species (1.9, 2.8, and 3.2 kilobase pairs). The regulation of the gene was characterized by both Northern and slot blot analysis. Hormonal regulation, developmental expression of calbindin-D28k-mRNA, and the effect of dietary alteration were examined. In the kidney all three species of mRNA were dependent on the presence of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) for their induction. The time course of induction of renal calbindin-D28k-mRNA indicated that a significant increase in calbindin-D-mRNA was detectable as early as 2 h following a single injection of 1,25-(OH)2D3 (200 ng/100 g of body weight), reaching a maximum at 12 h. Unlike the kidney high levels of calbindin-D28k-mRNA were observed in the brain of vitamin D-deficient rats. The concentration of calbindin-D28k-mRNA in brain was unchanged after 1,25-(OH)2D3 administration. Developmental studies indicated that calbindin-D-mRNA in rat kidney and brain is present prior to birth but is developmentally regulated in a tissue-specific manner. The most pronounced changes in the abundance of renal calbindin-D28k-mRNA occur between birth and 1 week of age. Unlike the kidney a large increase in brain calbindin-D28k-mRNA occurs at a later time, between 1 and 2 weeks of age (the period of major synapse formation). In dietary alteration studies results of Northern blot analysis indicate that low dietary phosphorus results in increased calbindin-D-mRNA in kidney but not in brain. These studies represent the first analysis of the rat calbindin-D28k gene and its regulation in vivo. Our findings suggest that in rat kidney and brain there are significant differences both in the expression of the gene for calbindin-D28k and its regulation by 1,25-(OH)2D3.

Aging↗
PubMed1988

Molecular cloning of mammalian 28,000 Mr vitamin D-dependent calcium binding protein (calbindin-D28K): expression of calbindin-D28K RNAs in rodent brain and kidney.

We report the isolation of a cloned cDNA for the mammalian 28,000 Mr vitamin D-dependent calcium binding protein (calbindin-D28K; CaBP28K) by immunological screening of a lambda gt11 bacterial expression library. The library contained cDNAs copied from poly(A)RNA of adult mouse cerebellum. We confirmed the identity of the CaBP28K cDNA by comparing its DNA sequence with that of chick CaBP28K cDNA. In the coding region, 79% of the mouse cDNA sequence was identical to the reported sequence of CaBP28K cDNA derived from chicken intestine. Rat brain and kidney each contain three species of poly(A)RNA that hybridize to CaBP28K cDNA--a major species of 1.9 kb, and rarer components of 2.8 kb and 3.2 kb. All three RNAs appear to be transcribed from a single gene. The ratios of these CaBP28K RNAs were the same in brain and kidney. In the cerebellum, in situ hybridization reveals that CaBP28K RNAs are confined to Purkinje neurons.

Animals↗
PubMed1988

Modulation of rat calbindin-D28 gene expression by 1,25-dihydroxyvitamin D3 and dietary alteration.

We have used a specific cDNA to the mammalian 28,000 Mr vitamin D-dependent calcium binding protein (calbindin-D28k) to study the regulation of the expression of this mRNA in rat kidney and brain. The effects of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) and dietary alteration on genomic expression were characterized by both Northern and slot blot analysis. Administration of 1,25-(OH)2D3 for 7 days (25 ng/day) to vitamin D-deficient rats resulted in a marked increase in renal calbindin-DmRNA, renal calbindin, and serum calcium. When vitamin D-deficient rats were supplemented for 10 days with calcium (3% calcium gluconate in the water, 2% calcium in the diet) serum calcium levels were similar to the levels observed in the 1,25-(OH)2D3-treated rats. However, in the calcium-supplemented rats the levels of renal calbindin and renal calbindin mRNA were similar to the levels observed in the vitamin D-deficient rats, suggesting that calcium alone without vitamin D does not regulate renal calbindin gene expression in vivo. In dietary alteration studies in vitamin D-replete rats, renal calbindin protein and mRNA increased 2.5-fold in rats fed diets low in phosphate providing evidence that in the rat the nutritional induction of calbindin is accompanied by a corresponding alteration in the concentration of its specific mRNA. Under low dietary calcium conditions, the levels of renal calbindin protein and mRNA were similar to the levels observed in control rats, although 1,25-(OH)2D3 serum levels were markedly elevated, suggesting that factors in addition to 1,25-(OH)2D3 can modulate renal calbindin gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

A quantitative immunobinding assay for vitamin D-dependent calcium-binding protein (calbindin-D28k) using nitrocellulose filters.

A sensitive dot immunobinding assay has been developed for the quantitative determination of vitamin D-dependent calcium-binding protein (calbindin-D28k; CaBP) in rat and human kidney and brain. Protein samples are spotted onto nitrocellulose sheets, fixed, and then rinsed with Tris-buffered saline. The remaining protein binding sites are blocked with bovine serum albumin, gelatin, or nonfat dry milk protein and the filters are then incubated sequentially with antiserum to calbindin-D28k (1:500 dilution) and 125I-protein A (200,000 cpm/ml). After washing, the radioactivity bound to each sample is quantitated by counting in a gamma counter. The sensitivity of the assay is such that 10 ng calbindin-D28k can be accurately quantitated. The highest levels of CaBP were detected in kidney (7.8 +/- 0.5 micrograms/mg protein) and cerebellum (22.1 +/- 1.4 micrograms/mg protein). Ten micrograms calmodulin, lactalbumin, or parvalbumin and 100 micrograms liver extract showed no reactivity in the assay. The assay is precise (intraassay variability, 4.0%) and reproducible (interassay variability, 8.8%). There was good agreement between the data in this assay and the data we obtained using radioimmunoassay (RIA). The assay has several advantages over the RIA. Iodination of pure antigen is not required and it is possible to detect membrane-bound and insoluble antigens using this assay. Also, the antiserum and 125I-protein A solutions can be saved and reused. This assay represents a major modification of the original immunobinding assays which used the less sensitive peroxidase stain. It is also an improvement over previous 125I immunobinding assays which were not quantitative but were used as antigen "spot tests" or which required iodination of the antibody.

Animals↗
PubMed1987

Calcium binding protein in squid brain: biochemical similarity to the 28,000-Mr vitamin D-dependent calcium binding protein (calbindin-D28k).

A calcium binding protein that is biochemically similar to vertebrate 28,000-Mr vitamin D-dependent calcium binding protein (calbindin-D28k) has been purified from squid brain. Squid brain calbindin was found to have an isoelectric point of 5.0, was heat stable up to 60 degrees C, and showed increased electrophoretic mobility in the presence of chelator. Amino acid analysis revealed a high content of glutamic and aspartic acids and a low level of methionine, histidine, and tyrosine, a finding similar but not identical to the composition of vertebrate calbindin-D28k. The molecular weight of the squid protein, determined by Ferguson plot analysis of data obtained from sodium dodecyl sulfate-gel electrophoresis, was calculated to be 25,700, as compared with 27,800 for rat renal calbindin. Immunocytochemical analysis demonstrated immunoreactive protein in a selected population of neurons and fibers in several areas of the molluscan nervous system. This study represents the first purification from an invertebrate of a calcium binding protein that is biochemically similar to vitamin D-dependent calcium binding protein. These results demonstrate that calbindin, although not identical in vertebrates and cephalopods, may be phylogenetically conserved in structure. The restricted distribution of immunoreactive calbindin in both the cephalopod and mammalian brain suggests that the function of neuronal calbindin may also be conserved in evolution.

Amino Acids↗
PubMed1987

Vitamin D-dependent calcium binding proteins in the kidney and intestine of the X-linked hypophosphatemic mouse: changes with age and responses to 1,25-dihydroxycholecalciferol.

We have previously observed decreased intestinal 9 kilodalton (kd) vitamin D-dependent calcium binding protein (CaBP) and decreased calcium absorption in juvenile X-linked hypophosphatemic (Hyp) mice. The present studies were undertaken to examine whether the kidney CaBPs (9 kd and 28 kd) are also affected in young Hyp mice and to investigate the ability of 1,25-dihydroxycholecalciferol [1,25-(OH)2D3] to increase CaBP in the intestine and kidney. The 28 kd CaBP and the 9 kd CaBP were measured in the kidneys and the 9 kd CaBP in the intestines of normal and Hyp mice from 1 week to 40 weeks of age. At all times between 3 and 6 weeks, intestinal CaBP in Hyp mice was decreased by more than 50% (P less than 0.005-0.001) and no significant decrease was present in the adult Hyp mice (12 and 40 weeks of age). By contrast, both kidney CaBPs were decreased only slightly in young Hyp mice. Between 1 and 6 weeks of age, the 9 kd CaBP in Hyp mice was 82% +/- 4% of control (P less than 0.001) and the 28 kd protein was 89% +/- 3% of control (P less than 0.001). Minipumps containing 1,25-(OH)2D3 or vehicle were implanted in 4-week and 13-week-old Hyp mice for 3 days to provide a dose of 0.12 micrograms/kg mouse X day. The 9 kd CaBP was increased approximately 3-fold (P less than 0.001) by 1,25-(OH)2D3 in the intestines of Hyp mice at both ages. The 9 kd kidney CaBP in Hyp mice also was increased by 1,25-(OH)2D3 treatment at both ages, but only by 33-52%. The 28 kd CaBP in the kidney was not affected by 1,25-(OH)2D3 treatment of Hyp mice at either age. We conclude that (9 kd and 28 kd) CaBPs levels in both intestine and kidney are decreased in juvenile Hyp mice although to much different degrees. The administration of 1,25-(OH)2D3 to Hyp mice increases the 9 kd CaBP in both intestine and kidneys, whereas the renal 28 kd CaBP is unaffected.

Aging↗
PubMed1986

Specific in vitro activation of Ca,Mg-ATPase by vitamin D-dependent rat renal calcium binding protein (calbindin D28K).

The vitamin D-dependent, calcium-binding protein from rat kidney, calbindin D28k (renal CaBP) specifically stimulates Ca,Mg-ATPase activity of human erythrocyte plasma membranes in a dose-dependent, calcium-sensitive manner. This stimulation was about two-fold compared to a three-fold stimulation by calmodulin. The effect was specific since other calcium-binding proteins and low molecular weight proteins did not stimulate Ca,Mg-ATPase activity. Renal CaBP did not stimulate cyclic nucleotide phosphodiesterase at concentrations greater than those which stimulated Ca,Mg-ATPase activity. This is the first report of a specific in vitro effect of renal CaBP on an enzyme system.

Animals↗
PubMed1986

Calcium-binding protein (28,000 Mr calbindin-D28k) in kidneys of the bullfrog Rana catesbeiana during metamorphosis.

A protein of approximately 28,000 relative molecular mass (Mr) cross-reacting with antiserum against the 28,000-Mr rat renal calcium-binding protein (calbindin-D28k) has been localized in the kidney of a salientian amphibian, Rana catesbeiana. Cells reactive for calbindin-D28k were found in the distal tubule at all stages of metamorphosis by the unlabeled antibody peroxidase-antiperoxidase technique. Adult kidneys appeared to have more calbindin-D28k-positive cells. The renal corpuscle, neck, and proximal tubule were negative. An immunoreactive 28,000-Mr band that comigrated with the band of calbindin-D28k was visualized by the immunoblot technique. The finding of the 28,000-Mr calbindin-D in the anamniotic kidney demonstrates that this calcium-binding protein (CaBP) is phylogenetically older than our previous studies of higher vertebrates had revealed (Rhoten et al., 1985). Although the function of calbindin-D28k in the distal nephron is unknown, this CaBP can now be presumed to have functional significance in the mesonephric as well as the metanephric kidney.

Animals↗
PubMed1986

Immunocytochemical localization and immunological characterization of vitamin D-dependent calcium-binding protein in the bullfrog cerebellum.

Antiserum prepared against rat renal calcium-binding protein (CaBP) was used with the unlabeled antibody peroxidase-antiperoxidase (PAP) technique to localize the 28,000 molecular weight CaBP in the cerebellum of the bullfrog, Rana catesbeiana. Whole brains of premetamorphic tadpoles and adults were fixed in Bouin's solution for 2 or 24 h and embedded in paraffin. 8-microns parasagittal sections were prepared and treated by the PAP method. Purkinje cells of the cerebellum in tadpoles and adults were specifically stained for CaBP. In the premetamorphic corpus cerebelli, the stained Purkinje cells corresponded to the precociously developed Purkinje cells described previously. In the auricular lobe region of the cerebellum mature Purkinje cells were stained. In addition, smaller stained cells were seen. The latter were presumed to be immature Purkinje cells that would mature at the time of metamorphosis. Immunoblot procedure demonstrated cross-reactivity for the ranid brains in the 28,000 molecular weight region. This immunoreactive band comigrated with the immunoreactive band observed with purified rat renal CaBP. Although the exact functional significance of CaBP is unknown at this time, our immunocytochemical and immunological findings indicate that CaBP is an excellent marker for studies of Purkinje cell maturation.

Animals↗
PubMed1986

1,25-Dihydroxyvitamin D3 treatment results in increased choline acetyltransferase activity in specific brain nuclei.

To better understand the role of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] in brain function, the level of calcium-binding protein (CaBP) and the activities of choline acetyltransferase (CAT) and monoamine oxidase were measured in discrete brain nuclei of vitamin D-deficient and -replete male rats. The nuclei sampled were those in which receptors for 1,25-(OH)2D3 and/or vitamin D-dependent CaBP have been localized. Significant elevations in CAT activity were found in the arcuatemedian eminence and in the bed nucleus of the stria terminalis of rats made vitamin D replete by eight daily ip injections of 100 or 200 ng 1,25-(OH)2D3 as well as by constant intraventricular (ivi) infusion of 25 ng 1,25-(OH)2D3 for 7 days. The percent increase ranged from 12-45% and was related to the ip dose administered. Constant ivi of 2 mM CA2+ or 125 ng 25-hydroxyvitamin D3/day for 7 days did not alter CAT activity. No significant changes in monoamine oxidase or CaBP in discrete brain nuclei were observed with vitamin D repletion. Since the arcuate-median eminence of the hypothalamus is an important regulatory site in the neuroendocrine control of reproduction, serum testosterone was measured. Serum testosterone levels were abnormally low in the vitamin D-deficient animals, but increased 2- to 5-fold to normal values in those rats made vitamin D replete by constant ivi of 25 ng 1,25-(OH)2D3 or by ip injection of 100 or 200 ng 1,25-(OH)2D3. Patterns of serum LH paralleled those for testosterone. Our results suggest that 1,25-(OH)2D3 effects cholinergic activity in several discrete brain regions and may play a role in the neuroendocrine regulation of certain aspects of anterior pituitary gland function.

Animals↗
PubMed1986

Expression of monocyte-specific oncogenes c-fos and c-fms in HL60 cells treated with vitamin D3 analogs correlates with inhibition of DNA synthesis and reduced calmodulin concentration.

Monocytic differentiation of cultured leukemic cells HL60 can be induced by a variety of compounds including analogs of vitamin D3. We studied the expression of oncogenes c-fos, c-fms, c-fes, c-myb, c-myc, and c-Ha-ras during this process, and attempted to relate these and a conventional marker of monocytic differentiation, the nonspecific esterase activity, to changes in cytosol levels of the Ca2+-binding proteins. The analogs of vitamin D3, 1 alpha,25-dihydroxycholecalciferol, 1 alpha,25-dihydroxy-26,27-hexafluorocholecalciferol, and 1 alpha,25-dihydroxy-24R-fluorocholecalciferol, reduced the total calcium binding activity and the cellular levels of calmodulin, but calbindin D28k could not be detected in HL 60 cells. A correlation was evident between the potency of these compounds as inducers of differentiation demonstrated by nonspecific esterase activity, the degree of reduction in calmodulin concentration, the rate of the inhibition of DNA synthesis and of expression of c-myc and c-myb genes, and the induction of the expression of oncogenes c-fos and c-fms. These experiments indicate that the events which underlie monocytic differentiation of HL 60 cells can be observed to occur in discrete steps which include an inhibition of DNA synthesis, a reduction of cellular levels of calmodulin, and altered expression of several oncogenes.

Annexin A6↗
PubMed1985

Effect of vitamin D deficiency on the composition and in vitro labeling of rat kidney proteins.

Densitometric analysis of single-dimension gels consistently demonstrated that, in addition to rat renal calcium binding protein (CaBP) (Mr 28,000), two other kidney proteins of Mr 16,500 and Mr 18,000 were significantly enriched in their contents in the vitamin D-replete rat. Partial characterization of the Mr 18,000 and 16,500 proteins revealed that these proteins were heat-stable and distinct from calmodulin, as determined by their inability to undergo the calcium-dependent mobility shift in sodium dodecyl sulfate gels which is characteristic of calmodulin. The Mr 16,500 and Mr 18,000 kidney proteins did not cross-react with rat renal or rat intestinal CaBP antisera, as assessed by radioimmunoassay and Western blot analysis. A comparison of peptide maps of tryptic digests of these proteins and purified rat renal CaBP, as analyzed by high-pressure liquid chromatography, revealed no apparent homology. Protein synthesis studies using [35S]methionine and short-term tissue culture of kidney cortex fragments indicated that the most pronounced effect of vitamin D or 1,25 dihydroxyvitamin D3 was increased synthesis of the Mr 28,000 protein (3.2- to 4.6-fold increase compared to -D rats, P less than 0.001). Synthesis of a Mr 54,500 protein increased by 1.3- to 1.5-fold (P less than 0.05) and [35S]methionine incorporation into a Mr 66,000 protein decreased by 1.2- to 1.3-fold (P less than 0.05) in +D rats. This study represents the first detailed characterization of the effects of vitamin D on the composition and synthesis of rat kidney proteins. The data indicate that the most significant effect of vitamin D on kidney proteins is increased synthesis of the Mr 28,000 CaBP, suggesting that a major role of vitamin D in renal function is regulation of calcium transport at the distal tubule. However, dietary vitamin D or 1,25(OH)2D3 can influence the expression as well as the suppression of other specific kidney proteins.

Animals↗
PubMed1985

Presence and localization of two vitamin D-dependent calcium binding proteins in kidneys of higher vertebrates.

We looked for the presence of vitamin D-dependent intestinal calcium binding protein (CaBP), relative molecular mass (mol wt) about 10,000, in kidneys of mammals, birds, and reptiles, and compared the localization of this 10,000 mol wt CaBP with the localization of the 28,000 mol wt vitamin D-dependent CaBP. Antiserum directed against rat intestinal CaBP (RICaBP) was used with the unlabeled antibody peroxidase-antiperoxidase technique to localize the 10,000 mol wt CaBP. Kidneys of adult Swiss and DBA/2J mice were positive for the 10,000 mol wt CaBP, whereas kidneys of adult rat, chicken, and two species of lizard were negative. Extracts of adult rat kidney showed no detectable immunoreactivity with the antiserum to RICaBP by radial immunodiffusion assay. However, kidneys of postnatal rats (12 days old) exhibited limited immunocytochemical reactivity and relatively low immunoreactivity (about 1 micrograms/mg protein) for 10,000 mol wt CaBP. In both strains of mice, 10,000 mol wt CaBP was localized predominantly to the distal convoluted tubules, connecting tubules, and collecting tubules of the cortical labyrinth. In 12-day-old rats 10,000 mol wt CaBP was localized to the distal convoluted tubules and cortical ascending thick limbs. Absorption of the antiserum with electrophoretically pure RICaBP eliminated specific reactivity. By dual color staining of mouse kidneys, the population of cells reactive for the 10,000 mol wt CaBP was found to be similar, but not identical, to that population of cells reactive for the 28,000 mol wt CaBP. Absorption of the antiserum to RICaBP with either rat renal CaBP or chicken intestinal CaBP (both 28,000 mol wt CaBPs) had no affect on the positive reactivity of the distal mouse nephron for RICaBP. Conversely, absorption of the antiserum to rat renal CaBP with the 10,000 mol wt RICaBP had no affect on reactivity for the 28,000 mol wt CaBP. Thus, two immunologically distinct CaBPs, mol wt 10,000 and 28,000, are present in the adult mouse nephron; whereas, kidneys of adult rats, chickens, and saurian reptiles apparently contain significant levels of only 28,000 mol wt CaBP. Since the 12-day-old rat nephron contains both the 28,000 mol wt and the 10,000 mol wt CaBP it appears that in the kidney, ontogenetically, and perhaps evolutionarily as well, the 28,000 mol wt CaBP is more highly conserved.

Animals↗
PubMed1985

Cell-free translational analysis of messenger ribonucleic acid coding for vitamin D-dependent rat renal calcium-binding protein.

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] has been found to exert its effects in a manner entirely analogous to that of other steroid hormones and is known to induce the synthesis of a calcium-binding protein (CaBP). The effects of 1,25-(OH)2D3 and dietary alteration on genomic expression in rat kidney were studied by in vitro translation of poly(A+)-containing RNA and by immunoprecipitation. Poly(A+)RNA from rat kidneys was translated in a rabbit reticulocyte lysate system in the presence of [35S]methionine, and the renal CaBP mRNA translation product was identified and quantitated by specific immunoprecipitation. Total translation products and specific immunoprecipitable products were visualized on sodium dodecyl sulfate-gels, followed by fluorography. After the addition of affinity purified rat renal CaBP antiserum to the 35S-labeled translation products, only one protein band, electrophoretically indistinguishable from that of purified renal CaBP (mol wt, 28,000), was observed. When 10 micrograms purified renal CaBP were added to the translation product before addition of the antiserum, immunoprecipitation of the 35S-labeled 28,000 mol wt protein was not observed. A comparison of the peptides produced after limited digestion with trypsin of 125I-labeled CaBP and [3H]tyrosine-labeled translation product indicated a good coincidence of peaks from purified 125I-labeled CaBP and the immunoprecipitated translation product, suggesting that the immunoprecipitated translation product is indeed vitamin D-dependent renal CaBP. When 100 ng 1,25-(OH)2D3 were injected for 7 days to 8-week-old vitamin D-deficient rats, there was a 4-fold increase in CaBP mRNA levels in the kidney (quantitated by densitometry of immunoprecipitates analyzed on sodium dodecyl sulfate-polyacrylamide gels). This increase in mRNA was accompanied by a corresponding increase in the concentration of renal CaBP, as measured by RIA, thus establishing a bridge between CaBP and the putative transcriptional effect of 1,25-(OH)2D3 in rat kidney. Similarly, both the concentration of renal CaBP and renal CaBP mRNA levels increased 4-fold in rats fed low phosphorus diets, increased 2-fold in rats fed low calcium diets, and decreased 67% in rats fed low sodium diets, providing evidence that the nutritional induction or decrease in renal CaBP is accompanied by a corresponding alteration in the concentration of its specific translatable mRNA. These results are consistent with a transcriptional control mechanism for the induction of renal CaBP.

Animals↗
PubMed1984

Vitamin D-dependent calcium-binding protein in rat kidney. Purification and physiocochemical and immunological characterization.

A vitamin D-dependent Mr = 28,000 calcium-binding protein (CaBP) has been isolated from rat kidney. Rat renal CaBP was purified from heat-treated post-mitochondrial supernatants by gel filtration on Sephadex G-100 followed by preparative gel electrophoresis. The specific properties and characteristics of the protein were examined. Rat renal CaBP was found to have a pI of 4.8 and showed increased electrophoretic mobility during polyacrylamide gel electrophoresis in the presence of 1 mM EDTA. Amino acid analysis of renal CaBP revealed a high content of glutamic and aspartic acids and a low level of methionine, histidine, cysteine, and tyrosine, similar but not identical to chick intestinal CaBP. Circular dichroism studies indicated that the alpha-helical content of renal CaBP was of the order of 31% and was changed to a minor degree by the addition of calcium. A study of the thermal stability indicated that renal CaBP is heat-stable up to 75 degrees C. Binding studies utilizing the technique of equilibrium dialysis established a dissociation constant of 2.1 X 10(-6) M and binding sites with a capacity of approximately 4 mol Ca2+/mol of CaBP. Immunologically, using Ouchterlony immunodiffusion, a precipitin line which joined with total coalescence with that due to purified renal CaBP was observed with extracts of rat brain and kidney but not with extracts of rat parathyroid, skeletal muscle, myocardium, bone, pancreas, intestine, and liver. In summary, these studies present the first purification and characterization of vitamin D-dependent rat renal calcium-binding protein. The antibody as well as the protein will be useful for the development of a radioimmunoassay and for the determination of the molecular mechanisms of induction of renal calcium-binding protein.

Amino Acids↗
PubMed1984

Calmodulin and rat vitamin D-dependent calcium-binding proteins: biochemical and immunochemical comparison.

Purified vitamin D-dependent rat intestinal (Mr 10,000) and rat renal (Mr 28,000) calcium-binding proteins (CaBPs) have been compared to vertebrate calmodulin, and the vitamin D-dependent CaBPs have been found to be distinct from calmodulin by biochemical and immunochemical criteria. Rat renal and rat intestinal CaBPs do not stimulate 3',5'-cyclic nucleotide phosphodiesterase, do not compete with iodinated calmodulin for binding to phenothiazine-Sepharose conjugates, do not cross-react immunochemically, and do not contain N epsilon-trimethyllysine. In addition, although calmodulin exhibits a characteristic calcium-dependent mobility shift on polyacrylamide gels in the presence of sodium dodecyl sulfate, a similar mobility shift is not observed for the vitamin D-dependent CaBPs. Immunocytochemically, calmodulin has a widespread localization in the kidney, whereas CaBP is present specifically in the distal tubules of the kidney. These localizations suggest a specialized role for CaBP in the kidney. Thus, although the vitamin D-dependent CaBPs and calmodulin are similar in that they are small, acidic, calcium-binding proteins, these two classes of proteins are biochemically and immunochemically distinct.

3',5'-Cyclic-AMP Phosphodiesterases↗