scholarly journals The 48 kDa Ca2+-binding protein of bovine brain

1987 ◽  
Vol 246 (1) ◽  
pp. 67-74 ◽  
Author(s):  
M Tokuda ◽  
N C Khanna ◽  
D M Waisman
Keyword(s):  
Molecular Mass ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Bovine Brain ◽  
Binding Properties ◽  
Tyrosine Residues ◽  
Tyrosine Protein Kinase ◽  
Stokes Radius ◽  
Phosphoamino Acid Analysis ◽  
Acidic Nature

A Ca2+-binding protein of molecular mass 48 kDa and named ‘CAB-48’ has been purified from bovine brain 100,000 g supernatant. About 30 mg of CAB-48 was purified from 1 kg of bovine brain. The protein has been characterized with respect to its physical, chemical and Ca2+-binding properties. It has an apparent molecular mass of 48 kDa by SDS/polyacrylamide-gel-electrophoresis and 75.2 kDa from sedimentation-velocity and Stokes-radius data. The acidic nature of the molecule is suggested by its pI of 4.7. In the presence of 3.0 mM-MgCl2 and 150 mM-KCl, CAB-48 binds 1.0 mol of Ca2+/mol of protein with an apparent Kd of 15 microM. A tyrosine protein kinase partially purified from rat spleen catalysed the incorporation of 0.73 mol of phosphate/mol of CAB-48, and phosphoamino acid analysis revealed that phosphorylation of CAB-48 was specific for tyrosine residues.

scholarly journals Purification and characterization of the 27,000 Da calcium-binding protein of bovine brain

1987 ◽  
Vol 244 (2) ◽  
pp. 401-408 ◽  
Author(s):  
M Tokuda ◽  
N C Khanna ◽  
D M Waisman
Keyword(s):  
Calcium Binding ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sedimentation Coefficient ◽  
Bovine Brain ◽  
Stokes Radius ◽  
Acidic Nature ◽  
Bovine Pancreas

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.

Rabphilin-3A, a putative target protein for smg p25A/rab3A p25 small GTP-binding protein related to synaptotagmin

1993 ◽  
Vol 13 (4) ◽  
pp. 2061-2068
Author(s):  
H Shirataki ◽  
K Kaibuchi ◽  
T Sakoda ◽  
S Kishida ◽  
T Yamaguchi ◽  
...  
Keyword(s):  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Bovine Brain ◽  
Target Protein ◽  
Dependent Manner ◽  
Gtp Binding Protein ◽  
Reading Frame ◽  
Gtp Binding ◽  
Putative Target

In a previous study (H. Shirataki, K. Kaibuchi, T. Yamaguchi, K. Wada, H. Horiuchi, and Y. Takai, J. Biol. Chem. 267:10946-10949, 1992), we highly purified from bovine brain crude membranes the putative target protein for smg p25A/rab3A p25, a ras p21-related small GTP-binding protein implicated in neurotransmitter release. In this study, we have isolated and sequenced the cDNA of this protein from a bovine brain cDNA library. The cDNA had an open reading frame encoding a protein of 704 amino acids with a calculated M(r) of 77,976. We tentatively refer to this protein as rabphilin-3A. Structural analysis of rabphilin-3A revealed the existence of two copies of an internal repeat that were homologous to the C2 domain of protein kinase C as described for synaptotagmin, which is known to be localized in the membrane of the synaptic vesicle and to bind to membrane phospholipid in a Ca(2+)-dependent manner. The isolated cDNA was expressed in COS7 cells, and the encoded protein was recognized with an anti-rabphilin-3A polyclonal antibody and was identical in size with rabphilin-3A purified from bovine brain by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Moreover, both rabphilin-3A purified from bovine brain and recombinant rabphilin-3A made a complex with the GTP gamma S-bound form of rab3A p25 but not with the GDP-bound form of rab3A p25. Immunoblot and Northern (RNA) blot analyses showed that rabphilin-3A was highly expressed in bovine and rat brains. These results indicate that rabphilin-3A is a novel protein that has C2 domains and selectively interacts with the GTP-bound form of rab3A p25.

Halothane and Isoflurane Alter the Calcium sup 2+ Binding Properties of Calmodulin

1995 ◽  
Vol 83 (1) ◽  
pp. 120-126. ◽  
Author(s):  
Aaron Levin ◽  
Thomas J. J. Blanck
Keyword(s):  
Binding Protein ◽  
Fluorescence Emission ◽  
Volatile Anesthetic ◽  
Volatile Anesthetics ◽  
Bovine Brain ◽  
Hill Coefficient ◽  
Excitation Wavelength ◽  
Binding Properties ◽  
Fluorescence Measurements ◽  
The Hill

Background Ca2+ plays an important role in signal transduction and anesthetic mechanisms. To date, no one has observed a direct effect of volatile anesthetics on a Ca(2+)-binding protein. We therefore examined the effects of halothane and isoflurane on the Ca(2+)-binding properties of bovine brain calmodulin. Methods The fluorescence emission of calmodulin was obtained over a range of Ca2+ concentrations (10(-7)-10(-4)M) in the presence and absence of halothane and isoflurane. The intrinsic tyrosine fluorescence of calmodulin was measured at an excitation wavelength of 280 nm and an emission wavelength of 320 nm. Fluorescence measurements were carried out in 50 mM hydroxyethylpiperazineethane sulfonic acid, 100 mM KC1, and 2 mM ethyleneglycol-bis-(beta-aminoethyl ether) tetraacetic acid at pH 7.0 and 37 degrees C. Experiments were performed in polytetrafluorethylene-sealed cuvettes so that the volatile anesthetic concentrations remained constant. The titration data were analyzed in two ways. The data were fit to the Hill equation by using nonlinear regression analysis to derive the Hill coefficient and the dissociation constant. The data were also analyzed by two-way analysis of variance with multiple comparisons to determine statistically significant effects. Volatile anesthetic concentrations were measured by gas chromatography. Results The presence of volatile anesthetics altered the Ca(2+)-binding affinity of calmodulin in a dose-dependent fashion. At 0.57% (0.25 mM) halothane and 1.7% (0.66 mM) isoflurane, the affinity of calmodulin for Ca2+ relative to control was decreased. However, at higher concentrations of both anesthetics, the affinity for Ca2+ was increased. When the volatile anesthetics were allowed to evaporate from the experimental solutions, the observed rightward shift of the calmodulin-Ca2+ binding curve for Ca2+ at low concentrations of the anesthetics returned to the control position. The leftward shift seen at high concentrations of the anesthetics was irreversible after evaporation of 8.7% (3.3 mM) isoflurane and 5.7% (2.5 mM) halothane. Conclusions These data demonstrate a complex interaction of two hydrophobic volatile anesthetics with calmodulin. A biphasic effect was observed both for halothane and for isoflurane. Calmodulin, an EF-hand Ca(2+)-binding protein, undergoes a conformational shift when binding Ca2+, exposing several hydrophobic residues. These residues may be sites at which the anesthetics act.

scholarly journals Identification and purification from bovine brain of a guanine-nucleotide-binding protein distinct from Gs, Gi and Go

1987 ◽  
Vol 246 (2) ◽  
pp. 431-439 ◽  
Author(s):  
G L Waldo ◽  
T Evans ◽  
E D Fraser ◽  
J K Northup ◽  
M W Martin ◽  
...  
Keyword(s):  
Gel Electrophoresis ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Binding Activity ◽  
Bovine Brain ◽  
Guanine Nucleotide ◽  
Guanine Nucleotide Binding Protein ◽  
Gtp Binding ◽  
Guanine Nucleotide Binding ◽  
The Brain

A guanine-nucleotide-binding protein (G-protein) was purified from cholate extracts of bovine brain membranes by sequential DEAE-Sephacel, Ultrogel AcA-34, heptylamine-Sepharose and Sephadex G-150 chromatography. Guanosine 5′-[gamma-[35S]thio]triphosphate (GTP[35S])-binding activity copurified with a 25,000 Da peptide and a 35,000-36,000 Da protein doublet. Neither pertussis toxin nor cholera toxin catalysed the ADP-ribosylation of a protein associated with the GTP[35S]-binding activity. Photoaffinity labelling of the purified protein with 8-azido[gamma-32P]GTP indicated that the GTP-binding site resides on the 25,000 Da protein. The 35,000-36,000 Da protein doublet was electrophoretically indistinguishable from the beta-subunits of other GTP-binding proteins, and the 36,000 Da protein was recognized by antiserum to oligomeric Gt. The purified protein specifically bound 17.2 nmol of GTP[35S]/mg of protein. The Kd of the binding site for radioligand was approx. 15 nM. The brain GTP-binding protein co-migrated during SDS/polyacrylamide-gel electrophoresis with a GTP-binding protein, named Gp, purified from human placenta [Evans, Brown, Fraser & Northup (1986) J. Biol. Chem. 261, 7052-7059], and cross-reacted with antiserum raised against the placental protein, but not with antiserum raised to brain Go. SDS/polyacrylamide-gel electrophoresis of the brain and placental GTP-binding proteins in the presence of Staphylococcus aureus V8 protease yielded identical peptide maps.

scholarly journals Ca2+ and Zn2+-binding properties of nitrated S-100b protein from bovine brain

1986 ◽  
Vol 238 (3) ◽  
pp. 715-719 ◽  
Author(s):  
R S Mani ◽  
C M Kay
Keyword(s):  
Conformational Changes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Difference Spectrum ◽  
Bound State ◽  
Bovine Brain ◽  
Tyrosine Residue ◽  
Difference Spectroscopy ◽  
Native Protein ◽  
Binding Properties ◽  
Helical Content

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.

scholarly journals Identification of Na+,Pi-binding protein in kidney and intestinal brush-border membranes

1988 ◽  
Vol 255 (1) ◽  
pp. 185-191 ◽  
Author(s):  
H Debiec ◽  
R Lorenc
Keyword(s):  
Molecular Mass ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Membrane Vesicles ◽  
High Specificity ◽  
Brush Border Membranes ◽  
Intestinal Brush Border

An Na+, Pi-binding protein has been extracted from kidney and intestinal brush-border membranes with an organic solvent and has been purified by Kieselghur and Sephadex LH-60 chromatography. The molecular mass of this protein has been estimated to be about 155 kDa as determined by gel-filtration chromatography on Sepharose 2B. Under denaturing conditions, polyacrylamide-gel electrophoresis revealed a monomer of molecular mass about 70 kDa. The protein has high specificity and high affinity for Pi [K0.5 (concentration at which half-maximal binding is observed) near 10 microM]. Na2+ binding also exhibits saturation behaviour, with a K0.5 near 7.5 mM. Pi binding is inhibited by known inhibitors of Pi transport in brush-border membrane vesicles. It appears that this protein could be involved in Na+/Pi co-transport across the renal and intestinal brush-border membranes.

scholarly journals Purification and characterization of an endogenous inhibitor of the sialyltransferase CMP-N-acetylneuraminate: lactosylceramide α2,6-N-acetylneuraminyltransferase (EC 2.4.99.-)

1988 ◽  
Vol 254 (2) ◽  
pp. 559-565 ◽  
Author(s):  
I Albarracin ◽  
F E Lassaga ◽  
R Caputto
Keyword(s):  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Brain Homogenate ◽  
Bovine Brain ◽  
Endogenous Inhibitor ◽  
Purification And Characterization ◽  
Heat Stable ◽  
Heat Stable Protein ◽  
Acidic Nature

The presence in the 100,000 g supernatant of rat brain homogenate of an inhibitor of the sialyltransferase has been confirmed. It is also present in chicken and bovine brain and in other rat and bovine organs. The inhibitor has been purified, a preparation with a specific activity 130-fold higher than that of the original 100,000 g supernatant of brain being obtained. It runs as a single peak in polyacrylamide-gel electrophoresis; when run in the presence of SDS, two components appeared. The apparent Mr of the components were 14,800 and 22,400. The inhibitor has been characterized as a heat-stable protein of acidic nature. It has effect on the glycolipid and the glycoprotein sialyltransferase activities but has no effect on the galactosaminyltransferase activity.

scholarly journals Isolation, purification and characterization of an iron-binding protein from the horseshoe crab (Limulus polyphemus)

1988 ◽  
Vol 252 (1) ◽  
pp. 151-157 ◽  
Author(s):  
R Topham ◽  
B Cooper ◽  
S Tesh ◽  
G Godette ◽  
C Bonaventura ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Horseshoe Crab ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Limulus Polyphemus ◽  
Evolutionary Development ◽  
Iron Binding ◽  
Oxygen Carriers ◽  
Iron Binding Protein

The presence of an iron-binding protein in the haemolymph of the horseshoe crab, Limulus polyphemus, was detected by gel filtration of 59Fe-labelled haemolymph. Lysis of amoebocytes did not change the amount of iron-binding protein in haemolymph samples. The protein was purified to homogeneity by ion-exchange chromatography. The molecular mass of the purified protein was estimated to be 282,000 +/- 10,000 Da by gel filtration and analytical ultracentrifugation. SDS/polyacrylamide-gel electrophoresis demonstrated that the protein is composed of ten subunits having a molecular mass of 28,000 +/- 2,000 Da. The purified, unlabelled protein efficiently sequestered 59Fe in the absence of haemolymph indicating that no other haemolymph factors are required for the incorporation of iron into the protein. No 59Fe was removed from the purified protein with EDTA or 2,2′-bipyridyl. Partial removal of 59Fe was achieved by dialysis with nitrilotriacetic acid or desferal. Analysis of the iron-loaded protein indicated that each subunit has the capacity to bind two iron atoms with high affinity. The isolation of an iron-binding protein from L. polyphemus supports the proposal that such proteins are an ancient evolutionary development not necessarily linked to the appearance of iron proteins which serve as oxygen carriers.

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