The purification and complete amino acid sequence of the 9000-Mr Ca2+-binding protein from rat placenta. Identity with the vitamin D-dependent intestinal Ca2+-binding protein.
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ABSTRACT:
SUBMITTER: MacManus JP
PROVIDER: S-EPMC1146724 | biostudies-other | 1986 Apr
REPOSITORIES: biostudies-other
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Project description:The Ca2+-binding protein (HCaBP) of the human placenta was studied with respect to its biochemical properties, tissue and cellular distribution, and possible involvement in placental Ca2+ transport. Optimal Ca2+ binding by the HCaBP occurs at pH 7-8 and in 100 mM-Na+ and 3 mM-Ca2+. The HCaBP possesses at least 10 Ca2+-binding sites with a Kd of 5 X 10(-6) M ([Ca2+]). Highly specific rabbit-derived anti-HCaBP antibodies were used for HCaBP immunoquantification and immunohistochemistry, which revealed that the HCaBP is localized in the chorionic villi and is primarily associated with the trophoblastic cells of the placenta. In addition, an 'in vitro' cell-free assay system for Ca2+ uptake was constructed with microsomal membranes isolated from term placental tissues. Ca2+ uptake by the placental microsomal fraction exhibited characteristics indicative of active Ca2+ transport such as temperature-dependence, saturability and energetic requirement. In this system, preincubation of microsomal membranes with anti-HCaBP antibodies inhibited Ca2+ uptake, suggesting that the HCaBP is functionally involved in placental membrane Ca2+ uptake.
| S-EPMC1144842 | biostudies-other
Project description:The single tyrosine residue in both pig and cow intestinal Ca2+-binding proteins fluoresces at 303 nm although the crystal structure of the cow protein shows a hydrogen bond between the hydroxy group of the tyrosine and glutamate-38 [Szebenyi & Moffat (1986) J. Biol. Chem. 261, 8761-8777]. The latter interaction suggests that tyrosinate fluorescence should dominate the emission spectra of these proteins. A fluorescence difference spectrum, produced by subtracting the spectrum of free tyrosine from the spectrum of the protein, gives a peak at 334 nm due to ionized tyrosine. That this component of the emission spectrum is not due to a tryptophan-containing contaminant is shown by its elimination when the protein is denatured by guanidine and when glutamate-38 is protonated. We conclude that, in solution, the tyrosine residue in this protein interacts occasionally with glutamate-38 but that a permanent hydrogen bond is not formed.
| S-EPMC1147898 | biostudies-other
Project description:A Ca2+-binding protein (CaBP) from human kidney was purified by two different procedures. The first involved heat-precipitation of a kidney cytosol fraction followed by gel filtration and chromatofocusing. This resulted in a 200-fold increase in the specific Ca2+-binding activity with a yield of 10%. A specific antibody was raised against the purified CaBP, as demonstrated by one precipitate in crossed immunoelectrophoresis of a kidney cytosol fraction. The antibody was coupled to Sepharose 4B and CaBP was then purified by immunoadsorbent chromatography. Applying this technique, a 500-fold purification of CaBP with a yield of 50% was obtained. Both preparations appeared homogeneous in crossed immunoelectrophoresis against a polyvalent antiserum and migrated as a single band corresponding to a mol.wt. of 26000 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. In gel filtration under non-denaturing conditions CaBP was eluted corresponding to a mol.wt. of 28000. The association constant for the high-affinity Ca2+-binding sites of CaBP was estimated by gel filtration to be 0.1 X 10(6)M-1, and the protein displayed Ca2+-dependent electrophoretic mobility, with more rapid anodic migration in the presence of EDTA. The protein eluted at a position corresponding to a pI of 4.5 in chromatofocusing. Immunochemical experiments with the specific antibody showed no cross-reaction between renal and intestinal CaBP.
| S-EPMC1153200 | biostudies-other
Project description:Rat intestinal Golgi-enriched membrane fractions take up Ca2+ by a vitamin D-dependent process that has been shown to recover within 15 min of repletion of vitamin D-deficient animals with intravenous 1,25-dihydroxycholecalciferol. The present paper reports studies characterizing the Ca2+-binding sites of these membrane fractions. Equilibrium binding of Ca2+ at concentrations between 5 and 400 microM showed significant decreases at all concentrations in membranes derived from vitamin D-deficient animals when compared with normal control-diet-fed animals. The predominant class of binding sites had a relatively high affinity for Ca2+ (KD approx. 3 microM). Vitamin D-deficiency did not change the affinity of this class of site, but decreased the number from 347 +/- 26 to 168 +/- 50 nmol of Ca2+ bound/mg of protein (means +/- S.D.). Mg2+ inhibited binding only at low Ca2+ concentrations, and the characteristics of this binding suggested positive co-operativity between two binding sites. Equimolar concentrations of Zn2+, La3+, Pb2+ and Mn2+ inhibited Ca2+ binding by over 50%. Increased ionic strength decreased Ca2+ binding by no more than half. Binding was maximal at pH 7.5 and half-maximal at pH 6.3. The large number of binding sites with relatively high affinity for Ca2+ suggests that it is unlikely that this binding is to any specific protein or to non-specific sites present on many proteins, and that the most likely sites are lipid molecules.
| S-EPMC1153347 | biostudies-other
Project description:The physicochemical properties of a novel Mr-21 000 Ca2+-binding protein isolated from bovine brain were investigated. The protein exhibited a partial specific volume of 0.724 ml/g, a degree of hydration of 0.47 g of water/g of protein and a mean residue weight of 119. Sedimentation equilibrium analysis revealed Mr = 22 600 in the absence of Ca2+; Ca2+ binding appeared to induce dimerization of the molecule. Size-exclusion chromatography indicated a compacting of the molecule on binding of Ca2+: the Stokes radius decreased from 2.75 nm in the absence of Ca2+ to 2.56 nm in its presence. Far-u.v.c.d. spectroscopy showed the apoprotein to be composed of 44% alpha-helix, 18% beta-pleated sheet and 38% random coil. Addition of either KCl (0.1 M) plus Mg2+ (1 mM), or Ca2+ (2 mM), changed the conformation to 49% alpha-helix, 18% beta-pleated sheet and 33% random coil. Near-u.v.c.d. and u.v. difference spectroscopy both indicated perturbations in the environments of all three types of aromatic amino acids on binding of Ca2+. Ca2+ binding also resulted in a 30% enhancement in the tryptophan fluorescence emission intensity. Ca2+ titration of the far-u.v.c.d. and fluorescence enhancement provided KD values of 9.91 microM and 4.68 microM respectively. Finally, the protein was shown to bind Zn2+ with KD = 1.44 microM (no Mg2+) and 1.82 microM (+ Mg2+). These observations strongly support the possibility that this novel Ca2+-binding protein resembles calmodulin and related Ca2+-binding proteins and undergoes a conformational change on binding of Ca2+ which reflects a physiological role in Ca2+-mediated regulation of brain function.
| S-EPMC1152917 | biostudies-other
Project description:The cation-binding properties of the vitamin D-dependent Ca2+-binding protein from pig duodenum were investigated, mainly by flow dialysis. The protein bound two Ca2+ ions with high affinity, and Mg2+, Mn2+ and K+ were all bound competitively with Ca2+ at both sites. The sites were distinguished by their different affinities for Mn2+, the one with the higher affinity being designated A (Kd 0.61 +/- 0.02 microM) and the other B (Kd 50 +/- 6 microM). Competitive binding studies allied to fluorimetric titration with Mg2+ showed that site A bound Ca2+, Mg2+ and K+ with Kd values of 4.7 +/- 0.8 nM, 94 +/- 18 microM and 1.6 +/- 0.3 mM respectively, and site B bound the same three cations with Kd values of 6.3 +/- 1.8 nM, 127 +/- 38 microM and 2.1 +/- 0.6 mM. For the binding of these cations, therefore, there was no significant difference between the two sites. In the presence of 1 mM-Mg2+ and 150 mM-K+, both sites bound Ca2+ with an apparent Kd of 0.5 microM. The cation-binding properties were discussed relative to those of parvalbumin, troponin C and the vitamin D-dependent Ca2+-binding protein from chick duodenum.
| S-EPMC1153475 | biostudies-other
Project description:A 68 000-Mr protein is a major component of a Nonidet P-40-insoluble fraction of lymphocyte plasma membrane prepared from human B lymphoblastoid cells ( BRI 8) and pig mesenteric lymph nodes. The association of the protein with the detergent-insoluble complex depends on free Ca2+ concentrations of greater than 10 microM. The human and pig 68 000-Mr proteins were purified and appear to be homologous on the basis of amino acid composition and peptide mapping. The protein is monomeric, has pI 5.8 and a single high-affinity Ca2+-binding site (KD 1.2 microM). The results are discussed in terms of the possible role of the 68 000-Mr protein as an intracellular Ca2+ receptor in lymphocytes.
| S-EPMC1153478 | biostudies-other
Project description:Ca2+ binding and concentrations of non-esterified fatty acids and phospholipids were compared in membrane fractions of rat small intestine. These fractions differed in density and were enriched for galactosyltransferase activity, a Golgi-membrane marker. Ca2+ binding was highest in the Golgi subfraction with the least density, as were the concentrations of both non-esterified fatty acids and phospholipids; galactosyltransferase activity was distributed differently. The large amount of non-esterified fatty acids was sufficient to account for a 2:1 complex of fatty acid-Ca2+. In vitamin D-deficient animals, the yield of protein in the lightest subfractions was decreased, but Ca2+ binding per mg of protein was further decreased to about 60%. In Golgi fractions from vitamin D-deficient animals, Ca2+ binding and the concentration of non-esterified fatty acids were decreased in parallel, but phospholipids were not significantly changed. There was a close correlation between Golgi Ca2+ binding and non-esterified fatty acid concentrations (r = 0.89; P less than 0.001). Non-esterified fatty acids, which are unusually prevalent in these membrane fractions, are likely to be the binding sites that account for this vitamin D-dependent Ca2+ uptake.
| S-EPMC1153348 | biostudies-other
Project description:The calcineurin homologous protein (CHP) belongs to an evolutionarily conserved Ca(2+)-binding protein subfamily. The CHP subfamily is composed of CHP1, CHP2, and CHP3, which in vertebrates share significant homology at the protein level with each other and between other Ca(2+)-binding proteins. The CHP structure consists of two globular domains containing from one to four EF-hand structural motifs (calcium-binding regions composed of two helixes, E and F, joined by a loop), the myristoylation, and nuclear export signals. These structural features are essential for the function of the three members of the CHP subfamily. Indeed, CHP1-CHP3 have multiple and diverse essential functions, ranging from the regulation of the plasma membrane Na(+)/H(+) exchanger protein function, to carrier vesicle trafficking and gene transcription. The diverse functions attributed to the CHP subfamily rendered an understanding of its action highly complex and often controversial. This review provides a comprehensive and organized examination of the properties and physiological roles of the CHP subfamily with a view to revealing a link between CHP diverse functions.
| S-EPMC3404583 | biostudies-literature
Project description: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.
| S-EPMC282647 | biostudies-other