The 48 kDa Ca2+-binding protein of bovine brain.
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ABSTRACT:
SUBMITTER: Tokuda M
PROVIDER: S-EPMC1148240 | biostudies-other | 1987 Aug
REPOSITORIES: biostudies-other
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Project description:The single tyrosine residue in S-100b protein was nitrated by treatment with tetranitromethane in 0.1 M-Tris/HCl buffer, pH 8.0, containing 2 mM-EDTA. The nitrated protein did not differ significantly in secondary structure from its native unmodified counterpart, as revealed by far-u.v. c.d. measurements. The effect of Ca2+ on the modified protein was different from that on the native protein, e.g. addition of Ca2+ resulted in a loss of helical content from 55 to 47% with the native protein whereas Ca2+ had no significant effect on the gross conformation of the nitrated derivative. Near-u.v. c.d. studies also indicated a very minimal effect on the tyrosine residue and this was also reflected in the u.v.-absorption difference spectrum. Polyacrylamide-gel electrophoresis in the absence of SDS showed the nitrated S-100b to move faster in the presence of EDTA compared with the calcium-bound state, suggesting that the modified protein does bind Ca2+ although it does not undergo a major conformational change in response to Ca2+ addition. In contradistinction, Zn2+ binding was not influenced by nitration, as demonstrated by aromatic c.d. and u.v.-difference spectroscopy. It is clear from this study that the single tyrosine residue in S-100b is critical to sense the Ca2+-induced conformational changes in the protein.
| S-EPMC1147196 | biostudies-other
Project description:A set of four proteins, termed calcimedins, are isolatable from smooth, cardiac and skeletal muscle by using a fluphenazine-Sepharose affinity column. The calcimedins show apparent Mr values of 67,000, 35,000, 33,000 and 30,000 by SDS/polyacrylamide-gel electrophoresis. The 67,000-Mr calcimedin (67 kDa calcimedin) has now been purified to homogeneity by using DEAE-cellulose chromatography followed by Ca2+-dependent binding to phenyl-Sepharose. The amino acid analysis of the 67 kDa calcimedin shows this protein does not contain trimethyl-lysine but does contain 2 mol of tryptophan/mol of protein. The 67 kDa calcimedin shows positive ellipticity in the near-u.v. range with c.d. Ca2+-binding studies indicate one high-affinity Ca2+-binding site with Kd 0.4 microM. The data show that the 67 kDa calcimedin is distinct from other Ca2+-binding proteins described to date.
| S-EPMC1147096 | biostudies-other
Project description:Several bovine brain proteins have been found to interact with a hydrophobic chromatography resin (phenyl-Sepharose CL-4B) in a Ca2+-dependent manner. These include calmodulin, the Ca2+/phospholipid-dependent protein kinase (protein kinase C) and a novel Ca2+-binding protein that has now been purified to electrophoretic homogeneity. This latter protein is acidic (pI 5.1) and, like calmodulin and some other high-affinity Ca2+-binding proteins, exhibits a Ca2+-dependent mobility shift on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, with an apparent Mr of 22 000 in the absence of Ca2+ and Mr 21 000 in the presence of Ca2+. This novel calciprotein is distinct from known Ca2+-binding proteins on the basis of Mr under denaturing conditions, Cleveland peptide mapping and amino acid composition analysis. It may be a member of the calmodulin superfamily of Ca2+-binding proteins. This calciprotein does not activate two calmodulin-dependent enzymes, namely cyclic nucleotide phosphodiesterase and myosin light-chain kinase, nor does it have any effect on protein kinase C. It may be a Ca2+-dependent regulatory protein of an as-yet-undefined enzymic activity. The Ca2+/phospholipid-dependent protein kinase is also readily purified by Ca2+-dependent hydrophobic-interaction chromatography followed by ion-exchange chromatography, during which it is easily separated from calmodulin. A preparation of protein kinase C that lacks contaminating kinase or phosphatase activities is thereby obtained rapidly and simply. Such a preparation is ideal for the study of phosphorylation reactions catalysed in vitro by protein kinase C.
| S-EPMC1144404 | biostudies-other
Project description:We have previously described the use of Ca2+-dependent hydrophobic-interaction chromatography to isolate the Ca2+ + phospholipid-dependent protein kinase (protein kinase C) and a novel heat-stable 21 000-Mr Ca2+-binding protein from bovine brain [Walsh, Valentine, Ngai, Carruthers & Hollenberg (1984) Biochem. J. 224, 117-127]. The procedure described for purification of the 21 000-Mr calciprotein to electrophoretic homogeneity has been modified to permit the large-scale isolation of this Ca2+-binding protein, enabling further structural and functional characterization. The 21 000-Mr calciprotein was shown by equilibrium dialysis to bind approx. 1 mol of Ca2+/mol, with apparent Kd approx. 1 microM. The modified large-scale purification procedure revealed three additional, previously unidentified, Ca2+-binding proteins of Mr 17 000, 18 400 and 26 000. The 17 000-Mr and 18 400-Mr Ca2+-binding proteins are heat-stable, whereas the 26 000-Mr Ca2+-binding protein is heat-labile. Use of the transblot/45CaCl2 overlay technique [Maruyama, Mikawa & Ebashi (1984) J. Biochem. (Tokyo) 95, 511-519] suggests that the 18 400-Mr and 21 000-Mr Ca2+-binding proteins are high-affinity Ca2+-binding proteins, whereas the 17 000-Mr Ca2+-binding protein has a relatively low affinity for Ca2+. Consistent with this observation, the 18 400-Mr and 21 000-Mr Ca2+-binding proteins exhibit a Ca2+-dependent mobility shift on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, whereas the 17 000-Mr Ca2+-binding protein does not. The amino acid compositions of the 17 000-Mr, 18 400-Mr and 21 000-Mr Ca2+-binding proteins show some similarities to each other and to calmodulin and other members of the calmodulin superfamily; however, they are clearly distinct and novel calciproteins. In functional terms, none of the 17 000-Mr, 18 400-Mr or 21 000-Mr Ca2+-binding proteins activates either cyclic nucleotide phosphodiesterase or myosin light-chain kinase, both calmodulin-activated enzymes. However, the 17 000-Mr Ca2+-binding protein is a potent inhibitor of protein kinase C. It may therefore serve to regulate the activity of this important enzyme at elevated cytosolic Ca2+ concentrations.
| S-EPMC1152916 | 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:Conformational changes in the beta-subunit of the bovine brain Ca2+-binding protein S100b (S100-beta) accompanying Ca2+ binding were investigated by analysis of the spectroscopic properties of the single tyrosine residue (Tyr17 beta) and flow-dialysis binding experiments. S100-beta binds Ca2+ sequentially at two sites to change the conformation of the protein. The first Ca2+ ion binds to site II beta, a typical Ca2+-binding site in the C-terminal region, and it does not significantly perturb the proximal environment of Tyr17 beta. After the first site is occupied, another Ca2+ ion binds to the N-terminal Ca2+-binding site, I beta, and strengthens a hydrogen bond between Tyr17 beta and a neighbouring carboxylate acceptor group, which results in a large increase in the Tyr17 beta fluorescence spectrum half-width and a positive absorption and c.d. signal between 290 and 275 nm. Ca2+ binding to the S100b.Zn2+6 complex, studied by flow-dialysis and fluorescence measurements showed that, although Zn2+ ions increase the affinity of S100b protein for Ca2+, the Ca2+-binding sequence was not changed. Tb3+ (terbium ion) binding studies on the S100b.Zn2+6 complex proved that Tb3+ antagonizes only Ca2+ binding site II beta and confirmed the sequential occupation of Ca2+-binding sites on the S100b.Zn2+6 complex.
| S-EPMC1133549 | biostudies-other
Project description:UNLABELLED:The PET radioligand (11)C-PBR28 targets translocator protein (18 kDa) (TSPO) and is a potential marker of neuroimmune activation in vivo. Although several patient populations have been studied using (11)C-PBR28, no investigators have studied cognitively impaired patients who would require anesthesia for the PET procedure, nor have any reports investigated the effects that anesthesia may have on radioligand uptake. The purpose of this study was to determine whether the anesthetic propofol alters brain uptake of (11)C-PBR28 in healthy subjects. METHODS:Ten healthy subjects (5 men; 5 women) each underwent 2 dynamic brain PET scans on the same day, first at baseline and then with intravenous propofol anesthesia. The subjects were injected with 680 ± 14 MBq (mean ± SD) of (11)C-PBR28 for each PET scan. Brain uptake was measured as total distribution volume (V(T)) using the Logan plot and metabolite-corrected arterial input function. RESULTS:Propofol decreased V(T), which corrects for any alteration of metabolism of the radioligand, by about 26% (P = 0.011). In line with the decrease in V(T), brain time-activity curves showed decreases of about 20% despite a 13% increase in plasma area under the curve with propofol. Reduction of V(T) with propofol was observed across all brain regions, with no significant region X condition interaction (P = 0.40). CONCLUSION:Propofol anesthesia reduces the V(T) of (11)C-PBR28 by about 26% in the brains of healthy human subjects. Given this finding, future studies will measure neuroimmune activation in the brains of autistic volunteers and their age and sex-matched healthy controls using propofol anesthesia. We recommend that future PET studies using (11)C-PBR28 and concomitant propofol anesthesia, as would be required in impaired populations, include a control arm to account for the effects of propofol on brain measurements of TSPO.
| S-EPMC3777621 | biostudies-literature
Project description:The 67 k calcimedin is a Ca2+-binding protein present in both muscle cells and peritoneal macrophages. Many tissues, including lymphoid tissues, liver and lymphocytes, have been shown to contain Ca2+-binding proteins of similar molecular size, such as the p67(67 kDa) calelectrin or the 68 kDa lymphocyte protein. We have tested affinity-purified antibodies raised to the smooth-muscle 67 k calcimedin in these several tissues and here report that the 67 k calcimedin is not detectable in liver, thymus, spleen or thymic lymphocytes. These findings support recent biochemical evidence, discussed here, suggesting that the 67 k calcimedin is a protein different from calelectrin and the 68 kDa lymphocyte protein. The more limited tissue distribution of the 67 k calcimedin, which includes muscle and macrophages, suggests that the 67 k calcimedin may function in Ca2+-mediated events special to these cell types. The affinity-purified antibodies to the 67 k calcimedin will be useful in obtaining information concerning the special roles of this Ca2+-binding protein in these cells.
| S-EPMC1148979 | biostudies-other
Project description:Recent reports have shown that there exists in mammalian brain a number of heat-stable Ca2(+)-binding proteins that are distinct from calmodulin [McDonald & Walsh (1985) Biochem. J. 232, 559-567]. We have attempted to characterize equivalent Ca2(+)-binding proteins from Drosophila. Affigel-phenothiazine chromatography, which can be used to purify calmodulin and other Ca2(+)-binding proteins, allowed the identification of a possible heat-stable 23 kDa Ca2(+)-binding protein. A purification procedure for this protein has been devised. Purified 23 kDa protein shows characteristics typical of a Ca2(+)-binding protein; there is a mobility shift on SDS/polyacrylamide gels in the presence of EGTA, and Western blotting, followed by the use of the 45Ca2+ overlay technique, confirms that the 23 kDa protein does bind Ca2+. 45Ca2+ binding studies indicate that this protein binds 1 mol of Ca2+/mol of protein, with Kd 1.9 microM. A single band with pI 5.2 is obtained on isoelectric focusing. Analysis of Western blots of Drosophila tissues probed with antibodies to the Ca2(+)-binding protein indicates that it has a widespread distribution, but is absent from muscle tissue. The antibodies also cross-react with a protein of identical molecular mass in extracts of sheep brain. The possible similarity between this Drosophila Ca2(+)-binding protein and mammalian proteins is discussed, and comparison is made between this Drosophila protein and other Ca2(+)-binding proteins purified from vertebrates.
| S-EPMC1149613 | biostudies-other
Project description:The purification to homogeneity of a 60 kDa phosphoinositide-specific phospholipase C from bovine brain cytosol is reported here. This enzyme exhibits the same properties, in terms of response to Ca2+, as does the cytosolic activity in a variety of cell types. We show here that Ca2+ does not appear to modulate the binding of the enzyme to the substrate, but induces dramatic changes in its secondary structure. Therefore we suggest that a decrease in the alpha-helix content of this enzyme correlates with its ability to be activated by Ca2+.
| S-EPMC1135313 | biostudies-other