Purification, calcium-binding properties, and conformational studies on a 28-kDa cholecalcin-like protein from bovine brain.

Journal of Biological Chemistry (Impact Factor: 4.57). 10/1985; 260(19):10662-70.
Source: PubMed


A large-scale preparation method for bovine brain 28-kDa cholecalcin-like protein is described. Flow dialysis binding studies revealed that the protein binds at least 3 mol of Ca2+/mol of protein. The protein undergoes conformational changes on binding calcium as shown by UV differential absorption spectroscopy, near and far UV circular dichroism, and intrinsic fluorescence. Circular dichroism (CD) studies in the far UV indicate an apparent increase in helical content in the presence of Ca2+. The effect of calcium on the protein structure is nearly maximum for 1 Ca2+ bound/protein molecule. UV differential absorption studies on the binding of the Ca2+ agonist Tb3+ and Tb3+ luminescence induced by energy Trp----Tb3+ transfer indicate that Tb3+ binds to two higher affinity Ca2+-binding sites. These sites are probably very close to the single Trp residue. Analysis of the fluorescence parameters of the single tryptophan residue in the apoprotein and its accessibility to ionic and neutral quenchers suggests that this residue is located in a highly hydrophobic domain on the protein surface.

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    • "Several lines of evidence point to a Ca 2+ sensor role for CB28. In vitro studies showed that CB28 undergoes a conformational change on binding Ca 2+ (Baudier et al. 1985; Gross et al. 1987; Winsky and Kuznicki 1996), and revealed Ca 2+ -dependent interactions with Ca 2+ - ATPase, alkaline phosphatase, mellitin and brain membrane fractions (Norman and Leathers 1982; Morgan et al. 1986; Winsky and Kuznicki 1995; La Bella et al. 1996). Ca 2+ -independent interactions of CB28 with phosphodiesterase, Ca 2+ -ATPase and a Na + transporter have also been reported (Reisner et al. 1992; Brunette et al. 1999). "
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    ABSTRACT: Recent attempts to understand the function of calbindin28kDa, a widely expressed calcium-binding protein, are confounded by uncertainties over its subcellular location. Using immunoblot analysis of rat brain subregions, we found that the proportion of particulate calbindin28kDa (24-43% of total) was independent of expression level and location. The association of calbindin28kDa with particulate structures appeared to be specific, since it persisted when soluble calbindin28kDa was sequestered by antibodies added before tissue disruption. Moreover, when exogenous calbindin28kDa was added during homogenisation of brain from calbindin28kDa-nullmutant mice, only 10% partitioned to the particulate fraction compared with 33% of endogenous calbindin28kDa in wildtype controls. Confocal microscopy showed that calbindin28kDa was predominantly extranuclear in all tissues analysed (i.e. various brain regions, isolated neurons, and dental enamel epithelium). Dual-label microscopy of neural dense particulate fractions confirmed the extranuclear location of calbindin28kDa and also showed that it partly colocalised with synaptosome and microtubule markers. Using sucrose step gradients, calbindin28kDa was separated from nuclei in parallel with synaptosome and endoplasmic reticulum markers. However, no association with the marker proteins (synaptophysin, ERp29, alpha/beta-tubulin) was detected by calbindin28kDa-immunoprecipitation analysis. Together these findings provide the first consistent picture that calbindin28kDa is located predominantly outside of the nucleus, irrespective of tissue type (neuronal vs. non-neuronal) and experimental approach (biochemical vs morphological). The evidence of a substantial, strong and specific association with insoluble cellular structures challenges the widely held view of calbindin28kDa as a mobile calcium buffer, and supports the existence of important alternative roles that involve target proteins.
    Cell and Tissue Research 12/2000; 302(2):171-80. DOI:10.1007/s004410000285 · 3.57 Impact Factor
  • Cell Calcium 03/1987; 8(1):1-41. DOI:10.1016/0143-4160(87)90034-0 · 3.51 Impact Factor
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    ABSTRACT: A Ca2+-binding protein named CAB-27 was purified from bovine brain 100,000 g supernatant. The protein has a molecular mass of 27,000 Da as determined by SDS/polyacrylamide-gel electrophoresis and 35,500 Da by sedimentation-coefficient and Stokes-radius analysis. The protein contains about 26% Glx and Asx and 13% basic residues. The acidic nature of the molecule is confirmed by its pI of 4.80. In the presence of 3 mM-MgCl2 and 150 mM-KCl, CAB-27 binds 2.0 mol of Ca2+/mol of protein, with an apparent Kd of 0.2 microM. Ca2+-binding is unaffected by prior incubation of the protein at 80 degrees C for 2 min. Brain contains about 130 mg of CAB-27/kg. Immunoblotting identified CAB-27 in several bovine tissues; it appears to be particularly rich in brain and kidney. In addition, CAB-27 is identified as an inhibitor of bovine pancreas phospholipase A2 in vitro. The inhibitory activity of CAB-27 was 20-fold less potent than lipocortin. On the basis of the Ca2+-binding properties, intracellular concentration and tissue distribution of this protein, we suggest that CAB-27 may be an important intracellular Ca2+ receptor.
    Biochemical Journal 07/1987; 244(2):401-8. DOI:10.1042/bj2440401 · 4.40 Impact Factor
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