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1.
Artigo em Inglês | MEDLINE | ID: mdl-18259068

RESUMO

Calbindin-D28k is a calcium-binding protein that belongs to the troponin C superfamily. It is expressed in many tissues, including brain, intestine, kidney and pancreas, and performs roles as both a calcium buffer and a calcium sensor and carries out diverse physiological functions of importance. In order to resolve the crystal structure of human calbindin-D28k and to gain a better understanding of its biological functions, recombinant human calbindin-D28k was crystallized at 291 K using PEG 3350 as precipitant and a 2.4 A resolution X-ray data set was collected from a single flash-cooled crystal (100 K). The crystal belonged to space group C2, with unit-cell parameters a = 108.1, b = 28.2, c = 70.6 A, beta = 107.8 degrees . The presence of one molecule per asymmetric unit is presumed, corresponding to a Matthews coefficient of 1.75 A(3) Da(-1).


Assuntos
Cálcio/química , Proteína G de Ligação ao Cálcio S100/química , Sequência de Bases , Calbindina 1 , Calbindinas , Clonagem Molecular , Cristalização , Cristalografia por Raios X , Primers do DNA , Humanos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteína G de Ligação ao Cálcio S100/genética , Proteína G de Ligação ao Cálcio S100/isolamento & purificação
2.
J Mol Biol ; 366(3): 965-75, 2007 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-17189639

RESUMO

The viral proteases have proven to be the most selective and useful for removing the fusion tags in fusion protein expression systems. As a key enzyme in the viral life-cycle, the main protease (M(pro)) is most attractive for drug design targeting the SARS coronavirus (SARS-CoV), the etiological agent responsible for the outbreak of severe acute respiratory syndrome (SARS) in 2003. In this study, SARS-CoV M(pro) was used to specifically remove the GST tag in a new fusion protein expression system. We report a new method to produce wild-type (WT) SARS-CoV M(pro) with authentic N and C termini, and compare the activity of WT protease with those of three different types of SARS-CoV M(pro) with additional residues at the N or C terminus. Our results show that additional residues at the N terminus, but not at the C terminus, of M(pro) are detrimental to enzyme activity. To explain this, the crystal structures of WT SARS-CoV M(pro) and its complex with a Michael acceptor inhibitor were determined to 1.6 Angstroms and 1.95 Angstroms resolution respectively. These crystal structures reveal that the first residue of this protease is important for sustaining the substrate-binding pocket and inhibitor binding. This study suggests that SARS-CoV M(pro) could serve as a new tag-cleavage endopeptidase for protein overproduction, and the WT SARS-CoV M(pro) is more appropriate for mechanistic characterization and inhibitor design.


Assuntos
Endopeptidases/metabolismo , Proteínas da Matriz Viral/biossíntese , Proteínas da Matriz Viral/metabolismo , Proteínas Virais/biossíntese , Calbindinas , Proteínas M de Coronavírus , Expressão Gênica , Vetores Genéticos , Glutationa Transferase/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteína G de Ligação ao Cálcio S100/isolamento & purificação , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave , Proteínas da Matriz Viral/antagonistas & inibidores , Proteínas da Matriz Viral/química
4.
Protein Sci ; 10(5): 927-33, 2001 May.
Artigo em Inglês | MEDLINE | ID: mdl-11316872

RESUMO

The structure of calbindin D(9k) with two substitutions was determined by X-ray crystallography at 1.8-A resolution. Unlike wild-type calbindin D(9k), which is a monomeric protein with two EF-hands, the structure of the mutated calbindin D(9k) reveals an intertwined dimer. In the dimer, two EF-hands of the monomers have exchanged places, and thus a 3D domain-swapped dimer has been formed. EF-hand I of molecule A is packed toward EF-hand II of molecule B and vice versa. The formation of a hydrophobic cluster, in a region linking the EF-hands, promotes the conversion of monomers to 3D domain-swapped dimers. We propose a mechanism by which domain swapping takes place via the apo form of calbindin D(9k). Once formed, the calbindin D(9k) dimers are remarkably stable, as with even larger misfolded aggregates like amyloids. Thus calbindin D(9k) dimers cannot be converted to monomers by dilution. However, heating can be used for conversion, indicating high energy barriers separating monomers from dimers.


Assuntos
Proteína G de Ligação ao Cálcio S100/química , Proteína G de Ligação ao Cálcio S100/metabolismo , Substituição de Aminoácidos/genética , Amiloidose/metabolismo , Apoproteínas/química , Apoproteínas/genética , Apoproteínas/isolamento & purificação , Apoproteínas/metabolismo , Sítios de Ligação , Calbindinas , Cálcio/metabolismo , Cromatografia em Gel , Cristalografia por Raios X , Dimerização , Motivos EF Hand , Cinética , Metionina/genética , Metionina/metabolismo , Modelos Moleculares , Mutação/genética , Prolina/genética , Prolina/metabolismo , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteína G de Ligação ao Cálcio S100/genética , Proteína G de Ligação ao Cálcio S100/isolamento & purificação , Relação Estrutura-Atividade , Termodinâmica
5.
Biochemistry ; 40(5): 1257-64, 2001 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-11170451

RESUMO

Interactions that stabilize the native state of a protein have been studied by measuring the affinity between subdomain fragments with and without site-specific residue substitutions. A calbindin D(9k) variant with a single CNBr cleavage site at position 43 between its two EF-hand subdomains was used as a starting point for the study. Into this variant were introduced 11 site-specific substitutions involving hydrophobic core residues at the interface between the two EF-hands. The mutants were cleaved with CNBr to produce wild-type and mutated single-EF-hand fragments: EF1 (residues 1--43) and EF2 (residues 44--75). The interaction between the two EF-hands was studied using surface plasmon resonance (SPR) technology, which follows the rates of association and dissociation of the complex. Wild-type EF1 was immobilized on a dextran matrix, and the wild-type and mutated versions of EF2 were injected at several different concentrations. In another set of experiments, wild-type EF2 was immobilized and wild-type or mutant EF1 was injected. Dissociation rate constants ranged between 1.1 x 10(-5) and 1.0 x 10(-2) s(-1) and the association rate constants between 2 x 10(5) and 4.0 x 10(6) M(-1) s(-1). The affinity between EF1 and EF2 was as high as 3.6 x 10(11) M(-1) when none of them was mutated. For the 11 hydrophobic core mutants, a strong correlation (r = 0.999) was found between the affinity of EF1 for EF2 and the stability toward denaturation of the corresponding intact protein. The observed correlation implies that the factors governing the stability of the intact protein also contribute to the affinity of the bimolecular EF1-EF2 complex. In addition, the data presented here show that interactions among hydrophobic core residues are major contributors both to the affinity between the two EF-hand subdomains and to the stability of the intact domain.


Assuntos
Fragmentos de Peptídeos/química , Proteína G de Ligação ao Cálcio S100/química , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Animais , Calbindinas , Bovinos , Brometo de Cianogênio , Motivos EF Hand/genética , Humanos , Hidrólise , Cinética , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/metabolismo , Ligação Proteica/genética , Estrutura Terciária de Proteína , Ratos , Proteína G de Ligação ao Cálcio S100/isolamento & purificação , Proteína G de Ligação ao Cálcio S100/metabolismo , Homologia de Sequência de Aminoácidos , Espectrometria de Fluorescência , Ressonância de Plasmônio de Superfície , Termodinâmica
6.
Pharmacol Toxicol ; 87 Suppl 3: 5-30, 2000.
Artigo em Inglês | MEDLINE | ID: mdl-11097107

RESUMO

Calbindin-D28k is an intracellular protein with high affinity for calcium. In the kidney, this protein is exclusively localized in the distal tubule and in the proximal part of the collecting ducts. Functionally, calbindin-D28k is supposed to be involved in the regulation of the reabsorption of calcium and possibly magnesium in the distal nephron though the exact regulatory mechanisms are not yet known. Thus, several theories regarding the functional role of calbindin-D28k have been proposed: The carrier theory describes calbindin-D28k as a transport protein which binds calcium and then transports it from the luminal to the basolateralcell membrane. The buffer theory assumes that calbindin-D28k functions by binding calcium ions to prevent intracellular calcium concentrations from reaching toxic levels. The activator theory describes that calbindin-D28k increases the activity of calcium channels or the enzymatic activity of the Ca++-Mg++-ATPase in the luminal membrane and thereby increases the tubular reabsorption of calcium. The renal calbindin-D28k is dependent upon vitamin D. Pharmacological doses of the active vitamin D metabolite 1,25-(OH)2D increases the concentrations of renal calbindin-D28k, whereas the concentration of calbindin-D28k is low in conditions with reduced levels of circulating 1,25-(OH)2D. Likewise, plasma calcium concentrations, uremia and hypertension affect calbindin-D28k expression. However, several studies have rendered probable the effect of additional factors in the regulation of renal calbindin-D28k. The aim of the present dissertation therefore was to examine the regulation of renal calbindin-D28k in a series of physiological and pathophysiological conditions established in vivo in the rat. A possible correlation between hypertension and calbindin-D28k was examined in three models of experimental hypertension: the genetically defined spontaneous hypertensive rat, the salt-sensitive Dahl rat and the renovascular hypertensive rat. These three models clearly demonstrated three separate patterns in the calcium metabolism, but the studies were unable to support a role for calbindin-D28k in the development of hypertension. In all three models the development of hypertension caused an increased plasma 1,25-(OH)2D. This increase was accompanied by either unaltered or reduced levels of renal calbindin-D28k possibly secondary to a cellular resistance against 1,25-(OH)2D. Magnesium binds to calbindin-D28k with a relatively high affinity. The regulation of urinary magnesium excretion takes place in the distal tubule where calbindin-D28k is found in high concentrations. Therefore, a possible relation between magnesium and calbindin-D28k was examined. The studies demonstrated not previously known connections between magnesium intake, urinary magnesium excretion and renal calbindin-D28k which suggests that this protein is involved in the regulation of magnesium homeostasis by the kidney. Calcitonin increases the reabsorption of calcium in the distal tubule. Therefore, the effect ofcalcitonin on renal calbindin-D28k was examined both by eliminating the endogeneous calcitonin production by a selective thyroidectomy followed by an autotransplantation of the parathyroid glands and further by infusion of calcitonin. These studies demonstrated unchanged concentrations of renal calbindin-D28k. It was concluded that the increased calcium reabsorption induced by calcitonin in the distal tubule is not mediated by calbindin-D28k. Urinary calcium excretion is in part regulated by the action of PTH on calcium reabsorption in the distal nephron. Previous reports of increased expression of renal calbindin-D28k in uremic rats led us to suggest that secondary hyperparathyroidism associated with uremia induced the synthesis of renal calbindin-D28k. Therefore, the effect of PTH was examined in a study comprising selective parathyroidectomy and infusions of PTH, PTHrP, 1,25-(OH)2D and calcium. (ABSTRACT TRUNCATED)


Assuntos
Rim , Proteína G de Ligação ao Cálcio S100 , Animais , Calbindina 1 , Calbindinas , Cálcio/metabolismo , Hipertensão Renal/metabolismo , Rim/efeitos dos fármacos , Rim/metabolismo , Proteína G de Ligação ao Cálcio S100/química , Proteína G de Ligação ao Cálcio S100/isolamento & purificação , Proteína G de Ligação ao Cálcio S100/metabolismo , Proteína G de Ligação ao Cálcio S100/fisiologia , Vitamina D/metabolismo
7.
Protein Expr Purif ; 17(3): 387-91, 1999 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-10600456

RESUMO

Two efficient procedures for large-scale purification of calbindin D9k from porcine intestine by HPLC were developed. Both protocols start with heat treatment of the intestinal tissue followed by acetic acid extraction, a capture with alginic acid, NaCl precipitation of other proteins, and a concentration step on Amberlite XAD-2. In the first method, a single reverse-phase HPLC step completes the purification and results in milligram quantities of pure calbindin. In the second method, an additional ion exchange HPLC step was introduced, followed by a reverse-phase HPLC resulting in 100 milligram-scale preparations of homogeneous calbindin in a 56% yield from the Amberlite step. Both methods yielded a homogeneous metal-free apoprotein with a molecular weight of 8838.0 +/- 8.8 as analyzed by MALDI TOF mass spectrometry corresponding to N-acetylated porcine calbindin. The isolated apocalbindin was fully reconstituted with 2 molar equivalents of Ca(2+) and the protein displayed UV and fluorescence spectra characteristic of those of native calbindin D9k.


Assuntos
Intestinos/química , Proteína G de Ligação ao Cálcio S100/isolamento & purificação , Animais , Calbindinas , Cálcio/química , Cromatografia Líquida de Alta Pressão , Espectrometria de Massas , Desnaturação Proteica , Resinas Sintéticas/química , Proteína G de Ligação ao Cálcio S100/química , Cloreto de Sódio/química , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Suínos
8.
Protein Expr Purif ; 17(3): 465-76, 1999 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-10600467

RESUMO

Calretinin (CR) is a calcium-binding, neuronal protein of undefined function. Related proteins either buffer intracellular calcium concentrations or are involved in calcium-signaling pathways. We transformed three CR gene fragment sequences, corresponding to its three complementary domains (I-II, III-IV, and V-VI), into Pichia pastoris. High yields of extracellular expression, of more than 200 mg/liter, were achieved. Simple purification protocols provide high yields of homogenous proteins: dialysis and DEAE-cellulose chromatography for domains I-II and III-IV or ammonium sulfate precipitation and octyl-Sepharose chromatography for domain V-VI. To our knowledge, this is the first report of the expression of an EF-hand protein using P. pastoris. Direct comparison of the purified yields of domain I-II indicates a approximately 20-fold improvement over Escherichia coli. N-terminal amino acid sequencing confirmed our gene products and two anti-calretinin antibodies recognized the appropriate domains. All three CR domains bind (45)Ca and the domain containing EF-hands V and VI seems to have a lower calcium capacity than the other domains. Circular dichroism indicates a high helix content for each of the domains. Calcium-induced structural changes in the first two domains, followed by tryptophan fluorescence, correspond with previous studies, while tyrosine emission fluorescence indicates calcium-induced structural changes also occur in domain V-VI. The methods and expression levels achieved are suitable for future NMR labeling of the proteins, with (15)N and (13)C, and structure-function studies that will help to further understand CR function.


Assuntos
Pichia/metabolismo , Proteína G de Ligação ao Cálcio S100/biossíntese , Sequência de Aminoácidos , Animais , Sequência de Bases , Calbindina 2 , Cálcio/metabolismo , Cromatografia , Dicroísmo Circular , Motivos EF Hand , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Vetores Genéticos , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteína G de Ligação ao Cálcio S100/química , Proteína G de Ligação ao Cálcio S100/isolamento & purificação , Análise de Sequência de Proteína
9.
Cell Transplant ; 8(1): 25-36, 1999.
Artigo em Inglês | MEDLINE | ID: mdl-10338273

RESUMO

Among the dopaminergic neurons in substantia nigra pars compacta and in the ventral tegmental area, subpopulations express the calcium-binding proteins calbindin (CB) and calretinin (CR), and the CB-containing neurons are supposed to be less prone to degeneration in Parkinson's disease. Glial cell line-derived neurotrophic factor (GDNF) is a potent survival factor for nigrostriatal dopaminergic neurons. Using free-floating roller-tube (FFRT) cultures derived from fetal rat (E14) ventral mesencephalon we found that GDNF (10 ng/ml) significantly increased the number of surviving tyrosine hydroxylase (TH)-immunoreactive neurons. The possible effects of GDNF treatment on CB-immunoreactive (CB-ir) and CR-ir neurons in such cultures were examined in the present study. The neuronal cell densities were measured by quantifying the numbers of CB-ir and CR-ir neurons in areas of sections through the most extensive parts of the spherical cultures. In 4-day-old and 8-day-old cultures GDNF treatment increased the density of CB-ir neurons by 50% and 59%, respectively. Partial co-existence of TH and CB was shown using the method of double immunolabeling. The density of CR-containing neurons was unaffected by GDNF treatment as confirmed by Western blotting for CR. Parallel effects of GDNF treatment were obtained for cultures of human fetal ventral mesencephalon (8 weeks postconception). In conclusion, our findings identify GDNF as a potent factor for fetal rat and human nigral CB-ir neurons able to promote their survival in culture. Referring to a suggested neuroprotective role of CB, the results may be of relevance in the context of neuronal transplantation of patients suffering from severe Parkinson's disease.


Assuntos
Fatores de Crescimento Neural/farmacologia , Proteínas do Tecido Nervoso/farmacologia , Proteína G de Ligação ao Cálcio S100/isolamento & purificação , Substância Negra/efeitos dos fármacos , Animais , Calbindina 2 , Calbindinas , Células Cultivadas , Feto , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Proteína Glial Fibrilar Ácida/isolamento & purificação , Humanos , Neurônios/química , Ratos , Ratos Sprague-Dawley , Substância Negra/química , Substância Negra/citologia
10.
Protein Expr Purif ; 15(3): 265-70, 1999 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-10092486

RESUMO

Calbindin D28k is a protein abundant in the mammalian central nervous system and in epithelial tissue involved in Ca2+ transport. Human calbindin D28k was cloned into a Pet3a vector and expressed in Escherichia coli. The protein was purified in three steps: (i) heat precipitation of bacterial proteins, (ii) ion-exchange chromatography on a DEAE-cellulose column in the presence of calcium, and (iii) ion-exchange chromatography on a DEAE-Sephacel column in the presence of EDTA. The protein was then supplemented with calcium and dialyzed against neutral water. The final yield was 20-50 mg of pure, homogeneous calcium-loaded calbindin D28k per liter of bacterial culture. The identity and purity of the protein were confirmed by immunoblotting, SDS-polyacrylamide gel electrophoresis, and agarose gel electrophoresis in the absence and presence of calcium and 1H NMR spectroscopy. The entire expression and purification protocol takes only 3 days and is easy to scale up and down. It was designed to minimize degradation and deamidation.


Assuntos
Proteína G de Ligação ao Cálcio S100/biossíntese , Proteína G de Ligação ao Cálcio S100/isolamento & purificação , Sequência de Aminoácidos , Sequência de Bases , Calbindina 1 , Calbindinas , Cromatografia DEAE-Celulose , Clonagem Molecular/métodos , Sequência Consenso , Primers do DNA , Eletroforese em Gel de Ágar , Eletroforese em Gel de Poliacrilamida , Escherichia coli , Humanos , Immunoblotting , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Reação em Cadeia da Polimerase , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteína G de Ligação ao Cálcio S100/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
11.
Protein Expr Purif ; 9(2): 171-81, 1997 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-9056482

RESUMO

Chick calretinin has been previously expressed in Escherichia coli and purified to homogeneity [Cheung, W-T., Richards, D.E., and Rogers, J.H. (1993) Eur. J. Biochem. 215, 401-410]. In the present study we have developed an improved purification procedure, involving a heat precipitation step followed by DEAE-cellulose chromatography with calcium-dependent elution. Native calretinin was purified from chick brainstem using the same method as for the recombinant protein but with an added affinity chromatography step. Typically 30 g of brainstem yielded 350 micrograms of protein. Several differences between the two forms imply that the native protein is acetylated at the N-terminus but otherwise unmodified. The calcium binding activities of both forms of calretinin were measured by equilibrium dialysis with 45Ca in Ca2+/EGTA buffers. The recombinant form bound 4.9 +/- 0.12 calcium ions with Kd = 0.38 +/- 0.02 microM and the native form was not significantly different. Recombinant calretinin was used to study its interaction with other cations present in cells and it was found that calcium binding was affected by Mg2+. Calretinin appears to bind 4.69 +/- 0.13 magnesium ions with Kd = 4.5 mM. Mg2+ increased the apparent dissociation constant for Ca2+. The shift is consistent with competitive binding of Ca2+ and Mg2+ to the same five sites, but Mg2+ binding is too weak to interfere significantly with Ca2+ binding under physiological conditions.


Assuntos
Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteína G de Ligação ao Cálcio S100/química , Proteína G de Ligação ao Cálcio S100/isolamento & purificação , Aminoácidos/análise , Animais , Calbindina 2 , Cálcio/metabolismo , Galinhas , Clonagem Molecular , Ácido Ditionitrobenzoico , Vetores Genéticos/genética , Vetores Genéticos/isolamento & purificação , Focalização Isoelétrica , Magnésio/metabolismo , Espectrometria de Massas , Proteínas Recombinantes/metabolismo , Proteína G de Ligação ao Cálcio S100/genética , Proteína G de Ligação ao Cálcio S100/metabolismo , Análise de Sequência
12.
Eur J Biochem ; 239(3): 611-23, 1996 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-8774704

RESUMO

UNLABELLED: Enamel cells handle large amounts of calcium, particularly during the developmental phase (termed maturation) when dental enamel is hypermineralized. The extent of intracellular calcium burden, and the nature of calcium homeostasis machinery used to accommodate it, are largely unknown. Here, the calcium-binding capacity of enamel cell cytosol was found to increase during development, in parallel with the putative transcellular flux of calcium. At maturation, the abundance of calcium-binding proteins in enamel cells exceeded that in brain and other established calcium-oriented tissues, which implies a large calcium burden. A search for likely cytosolic calcium transporters revealed only one high-affinity calcium-binding protein (12 kDa, distinguished from alpha-parvalbumin) that was up-regulated during maturation, but its low abundance (0.02% of soluble protein) precluded a major calcium transport or cytoprotective role. Two low-affinity calcium-binding proteins up-regulated during maturation (by 1.8-fold and 2.1-fold respectively) were identified as calreticulin and endoplasmin, both residents of the endoplasmic reticulum. Together, calreticulin and endoplasmin constituted an exceptionally high proportion (5%) of soluble protein during maturation, which gives an inferred calcium capacity 67-fold higher than that of the principal cytosolic calcium-binding protein. 28-kDa calbindin. Evidence that endoplasmin expression varied inversely with serum calcium concentration, and that the inositol trisphosphate receptor also was highly expressed during maturation, supported the novel hypothesis that non-mitochondrial calcium stores play a major role in transcellular calcium transport. IN CONCLUSION: (a) enamel cells contain a general high abundance of calcium homeostasis proteins, consistent with a heavy intracellular calcium burden; (b) the expression pattern (phenotype) of calcium-binding proteins varies with enamel cell function; (c) enamel cells appear to contain unusually large non-mitochondrial calcium stores; (d) contrary to the prevailing view that calcium passes mainly through the cytosol of calcium-transporting cells, the findings imply a route through the endoplasmic reticulum. This study gives novel information about how a highly calcium-oriented tissue avoids calcium toxicity, and provides a new focus for investigations into the mechanisms of transcellular calcium transport.


Assuntos
Calcificação Fisiológica/fisiologia , Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Esmalte Dentário/metabolismo , Homeostase/fisiologia , Animais , Transporte Biológico , Calbindinas , Canais de Cálcio/metabolismo , Cálcio da Dieta/farmacologia , Proteínas de Ligação ao Cálcio/isolamento & purificação , Calreticulina , Esmalte Dentário/citologia , Retículo Endoplasmático/metabolismo , Epitélio/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Receptores de Inositol 1,4,5-Trifosfato , Glicoproteínas de Membrana/isolamento & purificação , Dente Molar , Ratos , Ratos Wistar , Receptores Citoplasmáticos e Nucleares/metabolismo , Ribonucleoproteínas/isolamento & purificação , Proteína G de Ligação ao Cálcio S100/isolamento & purificação
13.
Biochem Mol Biol Int ; 38(6): 1199-210, 1996 May.
Artigo em Inglês | MEDLINE | ID: mdl-8739042

RESUMO

Calbindin D28K (CB), a cytosolic calcium binding protein (CBP), forms a macromolecular complex with the polypeptide mellitin (ME) the absence of calcium, which can be reversibly dissociated by the addition of Ca2+. The molar ratio of CB:ME constituted in this complex is 1:4, suggesting that CB interacts with the tetrameric form of ME. Like free tetrameric ME, the CB:ME complex does not migrate into 15% non-denaturing polyacrylamide electrophoretic gels, although both constituents migrate normally after irreversible complex denaturation by heating in sodium dodecyl sulphate (SDS). The interaction of these two proteins can be distinguished from the association of calmodulin (CM) with ME, which forms a reversibly dissociable, equimolar complex in the presence of Ca2+ and a stable non-migrating complex (molar ratio = 1:12) in its absence. Thus, CB and CM appear to bind ME under different Ca2+ regulatory control, suggesting possible roles for CB as a Ca(2+)-dependent regulatory binding protein.


Assuntos
Cálcio/farmacologia , Meliteno/metabolismo , Proteína G de Ligação ao Cálcio S100/metabolismo , Animais , Sequência de Bases , Química Encefálica , Calbindina 1 , Calbindinas , Calmodulina/metabolismo , Bovinos , Eletroforese em Gel de Poliacrilamida , Cinética , Dados de Sequência Molecular , Ratos , Proteína G de Ligação ao Cálcio S100/isolamento & purificação
14.
Eur J Biochem ; 230(1): 68-79, 1995 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-7601126

RESUMO

Enamel cells are likely to experience heavy demands for intracellular calcium homeostasis during the secretion and hypermineralization of dental enamel. Here, the two major high-affinity calcium-binding proteins in rat enamel epithelium were identified as calbindin28kDa and calmodulin, using a microscale approach. Both proteins were hyperabundant, totalling up to 2% of the soluble protein and surpassing the amounts in cerebellum, the benchmark tissue. Calbindin28kDa and calmodulin accounted for 26% of the total calcium-binding capacity in enamel cell cytosol, under near physiological conditions. Numerous calmodulin-binding proteins were detected with an overlay assay, indicating that calmodulin has multiple major targets in enamel cells. The calcium/calmodulin-regulated protein phosphatase, calcineurin, was identified as a principal calmodulin target constituting 0.1% of the soluble protein. Calmodulin and calcineurin were expressed constitutively, implying continued heavy usage of calcium/calmodulin-based and phosphorylation-based signalling events throughout enamel cell development. Calbindin28kDa, in contrast, was expressed at fourfold higher levels in secretion-phase cells than during the calcium-intensive hypermineralization phase, unexpectedly pointing to an important role associated with secretion. Supporting this notion, immunoblots revealed that 33% of total (SDS-soluble) calbindin28kDa was in the particulate fraction and predominantly associated with the Triton-insoluble cytoskeleton. Solubilisation of cytoskeletal calbindin28kDa required high concentrations of NaCl or urea, indicating the existence of a high-affinity target ligand. The unusual abundance of calmodulin, calbindin28kDa and calcineurin demonstrated here provides the first molecular evidence that enamal cells possess a strong capability for intracellular calcium homeostasis. Since none of these proteins was up-regulated during enamel hypermineralization, it appears that other calcium-binding proteins are primarily involved in the putative transcellular passage of calcium.


Assuntos
Proteínas de Ligação a Calmodulina/análise , Calmodulina/análise , Esmalte Dentário/química , Fosfoproteínas Fosfatases/análise , Proteína G de Ligação ao Cálcio S100/análise , Animais , Calbindinas , Calcineurina , Cálcio/metabolismo , Calmodulina/metabolismo , Citosol/química , Peso Molecular , Coelhos , Ratos , Proteína G de Ligação ao Cálcio S100/isolamento & purificação , Proteína G de Ligação ao Cálcio S100/fisiologia
15.
Vis Neurosci ; 12(2): 301-7, 1995.
Artigo em Inglês | MEDLINE | ID: mdl-7786851

RESUMO

Calbindin D-28K is a calcium-binding protein found in the cone but not rod photoreceptor cells in the retinas of a variety of species. Recent studies of the monkey retina indicated that calbindin D-28K may be expressed preferentially in non-foveal regions of the retina. In the current studies of human retinas, immunohistochemical experiments demonstrated that calbindin D-28K is reduced or absent in the fovea and parafovea, but prevalent in the perifovea and periphery. These findings were supported by the quantification of calbindin D-28K in 1-mm trephine punches obtained from different regions of the human retina. The specificity of the anti-calbindin D-28K antibodies used in these studies was confirmed by Western blot analysis using purified calbindin D-28K. The protein was purified from retinal tissue and its identity confirmed by partial amino-acid sequence analysis. The expression of calbindin D-28K did not correlate with the spectral properties of the cones, rather to their position in the retina. The study of spatially expressed genes, like the one encoding calbindin D-28K, may help explain the patterns of retinal degeneration seen in some human cone-rod dystrophies.


Assuntos
Retina/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Proteína G de Ligação ao Cálcio S100/metabolismo , Idoso , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Especificidade de Anticorpos , Western Blotting , Calbindinas , Eletroforese em Gel de Poliacrilamida , Feminino , Imunofluorescência , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/isolamento & purificação , Proteínas do Tecido Nervoso/metabolismo , Fragmentos de Peptídeos/química , Retina/química , Pigmentos da Retina/metabolismo , Proteína G de Ligação ao Cálcio S100/química , Proteína G de Ligação ao Cálcio S100/isolamento & purificação
16.
Biochem Mol Biol Int ; 33(4): 713-21, 1994 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-7981659

RESUMO

The ability of rat calretinin to bind to hydrophobic resins in a Ca(2+)-dependent manner was examined. Both native calretinin present in cerebellum extract and purified recombinant calretinin bound similarly to hydrophobic resins such as phenyl-, hexyl-, octyl-, and W7-agarose. Hydrophobic interactions of calretinin were partially Ca(2+)-dependent since 1/3 of bound protein was released from the resins by EGTA under varied conditions. Some calretinin tryptic fragments bound to octyl-agarose in a manner similar to uncleaved calretinin, while others bound to the resin in a Ca(2+)-independent manner. These and other results suggest that calretinin has several hydrophobic regions of varied strength and sensitivity to Ca2+. It is proposed that the local changes in hydrophobicity induced by Ca2+ binding might be relevant for calretinin functions.


Assuntos
Cálcio/fisiologia , Resinas Vegetais/metabolismo , Proteína G de Ligação ao Cálcio S100/metabolismo , Animais , Calbindina 2 , Cerebelo/química , Fenômenos Químicos , Físico-Química , Ácido Egtázico/farmacologia , Ligação Proteica , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Proteína G de Ligação ao Cálcio S100/isolamento & purificação
17.
Biochim Biophys Acta ; 1200(2): 191-6, 1994 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-8031840

RESUMO

Analyses of the calcium-binding protein, calbindin9kDa, purified to apparent homogeneity (SDS-PAGE) from rat duodenum, revealed variable contamination by two other 9 kDa proteins (up to 0.2 mol equivalent each) which were identified as ubiquitin and its C-terminal variant, des-Gly-Gly-ubiquitin. We found that the co-purification of these proteins did not reflect a tight molecular interaction but instead their unexpectedly similar physical characteristics in nondenaturing conditions. Like calbindin9kDa, free ubiquitin was abundant (1% and 0.4% of soluble protein, respectively) in duodenum mucosa of 7-8-week-old rats and its concentration varied daily and with feeding status. In rats fed from midnight to 8.30 a.m., the ubiquitin concentration was specifically higher at 10 pm than at 10 a.m. (11.2 +/- 0.7 and 7.7 +/- 0.8 nmol per g wet weight, respectively, P < 0.02), whereas calbindin9kDa tended towards an opposite variation (18.0 +/- 1.9 and 21.8 +/- 1.7 nmol per g, respectively). Based on its unusually high abundance and novel feeding-related variations, ubiquitin must have an important functional role in the rat duodenum which is distinctly regulated from the calcium transport-associated role of calbindin9kDa.


Assuntos
Duodeno/metabolismo , Proteína G de Ligação ao Cálcio S100/metabolismo , Ubiquitinas/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Calbindinas , Cromatografia por Troca Iônica , Ingestão de Alimentos , Expressão Gênica , Dados de Sequência Molecular , Ratos , Proteína G de Ligação ao Cálcio S100/química , Proteína G de Ligação ao Cálcio S100/isolamento & purificação , Ubiquitinas/química , Ubiquitinas/isolamento & purificação
18.
Protein Expr Purif ; 5(2): 187-91, 1994 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-8054853

RESUMO

Rat calretinin coding region was subcloned into a prokaryotic expression vector (pGEX). The glutathione-S-transferase:calretinin fusion protein produced in Escherichia coli was purified on a glutathione-Sepharose affinity column. Recombinant rat calretinin was cleaved on the column by thrombin, eluted, and purified to homogeneity using DEAE-cellulose chromatography. Recombinant and native rat calretinin performed the same on DEAE columns, denaturing polyacrylamide gel electrophoresis (SDS-PAGE), Western blots, and 45Ca overlay on nitrocellulose blots. The recombinant calretinin migrated similarly to the more basic (pI 5.3) of two forms of native calretinin demonstrated by two-dimensional SDS-PAGE. Calcium binding equilibria revealed identical apparent binding affinity and capacity. Difference(s) between native and recombinant did not affect the binding of calcium to calretinin or antibody recognition. Thus recombinant calretinin may be useful in the elucidation of possible cellular targets of native calretinin.


Assuntos
Proteínas Recombinantes de Fusão/genética , Proteína G de Ligação ao Cálcio S100/genética , Animais , Sequência de Bases , Western Blotting , Calbindina 2 , Cálcio/metabolismo , Cromatografia DEAE-Celulose , Eletroforese em Gel Bidimensional , Escherichia coli , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Vetores Genéticos , Isopropiltiogalactosídeo/farmacologia , Dados de Sequência Molecular , Ratos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteína G de Ligação ao Cálcio S100/biossíntese , Proteína G de Ligação ao Cálcio S100/isolamento & purificação
19.
Arch Biochem Biophys ; 308(1): 311-7, 1994 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-8311469

RESUMO

We expressed gram amounts of full-length and mutant rat brain calcium-binding proteins (calbindins-D28K) lacking one or two "EF-hand" motifs in a bacterial expression system. The cDNA for the full-length rat calcium-binding protein was cloned into the NdeI and BamHI sites of the pET3a vector. Additionally, constructs of the rat brain calcium-binding protein lacking EF-hand 2 (delta 2 mutant), EF-hand 6 (delta 6 mutant), and EF-hands 2 and 6 (delta 2, 6 mutant) were constructed using the same vector. These chimeric plasmids were used to transfect BL21 (DE3) pLysS Escherichia coli cells. Following transformation, the cells were grown in the presence of isopropylthiogalactoside in order to induce bacterial T7 polymerase, which resulted in the production of large amounts of the proteins of interest in the bacterial cytosol. Expressed full-length and delta 2 and delta 2,6 mutant proteins represented 50% or more of total bacterial protein. The delta 6 protein was not expressed. Cell lysis followed by purification of the proteins on DEAE-cellulose routinely resulted in gram yields of the proteins. The purified proteins displayed the appropriate amino acid composition and amino-terminal amino acid sequence. When analyzed by matrix-assisted laser desorption mass spectrometry the proteins were found to have the appropriate molecular weights (within the accuracy limits of the instrument). The expressed proteins bound to a polyclonal antiserum raised against chick intestinal calcium-binding protein. In addition, the full-length, delta 2, and delta 2,6 mutants bound calcium as assessed by a 45Ca blotting procedure. The production of large amounts of readily purified vitamin D-dependent calcium-binding proteins should be useful in biophysical studies of the proteins.


Assuntos
Encéfalo/metabolismo , Proteínas Recombinantes/biossíntese , Proteína G de Ligação ao Cálcio S100/biossíntese , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Calbindinas , Quimera , Cromatografia em Gel , Cromatografia por Troca Iônica , Clonagem Molecular , Primers do DNA , Escherichia coli , Vetores Genéticos , Cinética , Dados de Sequência Molecular , Plasmídeos , Reação em Cadeia da Polimerase , Ratos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Proteína G de Ligação ao Cálcio S100/isolamento & purificação , Proteína G de Ligação ao Cálcio S100/metabolismo
20.
Cell Calcium ; 14(9): 639-48, 1993 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-8242719

RESUMO

We have purified recombinant human calretinin (CR) from Escherichia coli lysates and have produced a polyclonal antiserum against it. The antiserum recognizes determinants conserved in fish, chicken, rat, monkey and human CR. We show its use in the qualitative detection of CR by different methods of immunohistochemistry as well as in the detection of CR on immunoblots.


Assuntos
Anticorpos , Química Encefálica , Proteína G de Ligação ao Cálcio S100/imunologia , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos , Sequência de Bases , Calbindina 2 , Galinhas , Escherichia coli , Peixes , Haplorrinos , Humanos , Dados de Sequência Molecular , Ratos , Proteína G de Ligação ao Cálcio S100/química , Proteína G de Ligação ao Cálcio S100/genética , Proteína G de Ligação ao Cálcio S100/isolamento & purificação
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