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1.
Eur J Biochem ; 268(7): 1972-81, 2001 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11277919

RESUMO

The human DRnm23 gene was identified by differential screening of a cDNA library obtained from chronic myeloid leukaemia-blast crisis primary cells. The over-expression of this gene inhibits differentiation and induces the apoptosis of myeloid precursor cell lines. We overproduced in bacteria a truncated form of the encoded protein lacking the first 17 N-terminal amino acids. This truncated protein was called nucleoside diphosphate (NDP) kinase CDelta. NDP kinase CDelta had similar kinetic properties to the major human NDP kinases A and B, but was significantly more stable to denaturation by urea and heat. Analysis of denaturation by urea, using size exclusion chromatography, indicated unfolding without the dissociation of subunits, whereas renaturation occurred via a folded monomer. The stability of the protein depended primarily on subunit interactions. Homology modelling of the structure of NDP kinase CDelta, based on the crystal structure of NDP kinase B, indicated that NDP kinase CDelta had several additional stabilizing interactions. The overall structure of the two enzymes appears to be identical because NDP kinase CDelta readily formed mixed hexamers with NDP kinase A. It is possible that mixed hexamers can be observed in vivo.


Assuntos
Isoenzimas/genética , Isoenzimas/metabolismo , Núcleosídeo-Difosfato Quinase/genética , Núcleosídeo-Difosfato Quinase/metabolismo , Sequência de Aminoácidos , Western Blotting , Catálise , Estabilidade Enzimática , Temperatura Alta , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Nucleosídeo NM23 Difosfato Quinases , Neuroblastoma/enzimologia , Desnaturação Proteica , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Células Tumorais Cultivadas , Ureia/farmacologia
2.
J Biol Chem ; 272(25): 15599-602, 1997 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-9188446

RESUMO

The point mutation serine 120 to glycine in the human nucleoside diphosphate kinase A has been identified in several aggressive neuroblastomas (Chang, C. L., Zhu, X. X., Thoraval, D. H., Ungar, D., Rawwas, J., Hora, N., Strahler, J. R., Hanash, S. M. & Radany, E. (1994) Nature 370, 335-336). We expressed in bacteria and purified wild-type and S120G mutant nucleoside diphosphate kinase A. The mutant enzyme had enzymatic and structural properties similar to the wild-type enzyme, whereas its stability to denaturation by heat and urea was markedly reduced. More importantly, upon renaturation of the urea-denatured mutant protein, a folding intermediate accumulated, having the characteristics of a molten globule. It had no tertiary structure, as shown by near UV circular dichroism, whereas the secondary structure was substantially recovered. The hydrophobic probe 8-anilino-1-naphthalene sulfonate bound to the intermediate species with an increase in fluorescence intensity and a blue shift. The hydrodynamic size was between that expected for a folded and an unfolded monomer. Finally, electrophoresis in a transverse urea gradient displayed no renaturation curve, and the protein showed the tendency to aggregate at the lowest urea concentrations. The existence of a molten globule folding intermediates resulting from an altered folding in the mutated protein might be related to the aggressiveness of neuroblastomas.


Assuntos
Antígenos de Neoplasias/genética , Proteínas Monoméricas de Ligação ao GTP , Neuroblastoma/enzimologia , Núcleosídeo-Difosfato Quinase/genética , Mutação Puntual , Dobramento de Proteína , Fatores de Transcrição/genética , Antígenos de Neoplasias/química , Cromatografia em Gel , Dicroísmo Circular , Glicina , Humanos , Cinética , Peso Molecular , Nucleosídeo NM23 Difosfato Quinases , Neuroblastoma/genética , Núcleosídeo-Difosfato Quinase/química , Serina , Fatores de Transcrição/química
3.
J Mol Biol ; 242(4): 599-603, 1994 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-7932716

RESUMO

The cloning of murine tryptophanyl-tRNA synthetase revealed the existence of at least three messenger RNAs able to code for this enzyme. In most of the tissues tested, two major mRNA species were detected. They are produced by alternative polyadenylation and they share the same open reading frame. The deduced peptide sequence is highly homologous to bovine and human tryptophanyl-tRNA synthetases. In embryonic stem cells, a third type of mRNA was characterized. Surprisingly, this mRNA contains, at the C terminus of the open reading frame, a sequence coding for six additional amino acids. Southern blot and polymerase chain reaction analysis showed that the two open reading frames are encoded by the same gene. Thus, alternative splicing may generate two tryptophanyl-tRNA synthetase isoforms. This phenomenon is the first reported case for an aminoacyl-tRNA synthetase mRNA with two open reading frame isoforms. Moreover, to our knowledge, this is the first time that a peptide addition to the COOH terminus of a protein, by mRNA alternative splicing, is described. The extra hexapeptide, Cys-Phe-Cys-Phe-Asp-Thr COOH, resembles the consensus sequence found in C termini of Ras proteins.


Assuntos
Processamento Alternativo , Isoenzimas/genética , Triptofano-tRNA Ligase/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sondas de DNA , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/metabolismo , Células-Tronco
4.
Biol Cell ; 60(3): 245-53, 1987.
Artigo em Inglês | MEDLINE | ID: mdl-2962682

RESUMO

The role of Ca2+-ATPase as the driving force for active calcium uptake, involved in the relaxation of smooth muscle, was studied. It was shown by immunocytochemistry that Ca2+-ATPase activity was localized at the plasma membrane level of longitudinal smooth muscle of pregnant rat uteri (18-20 days). To study calcium regulation in uterine longitudinal smooth muscle, 2 microsomal fractions (F1 and F2) were obtained, enriched in plasma membrane material (Lalanne et al., 1984, in: Calcium Regulation in Smooth Muscles. INSERM series, 124, pp. 283-292). In the present paper this material is characterized at both morphologic and cytochemical levels. Both fractions are ultrastructurally heterogeneous: (a) thin sections clearly show 2 populations that differ in vesicular shape and size; (b) negative staining also shows differences in membrane structure, which could be related to biochemical differences and/or to the well known heterogeneity of the plasma membrane. Two reactions (PATAg and concanavalin A-biotin-avidin-ferritin), allowing visualization of cell coat glycans, were performed on F1 and F2 and on thin sections of longitudinal smooth muscle. Plasma membrane and almost all the vesicles of F1 and F2 are reactive. It is concluded that these 2 fractions are characteristic enough for studying, at the molecular level, the ability of plasma membrane to control calcium circulation in uterine smooth muscle.


Assuntos
ATPases Transportadoras de Cálcio/metabolismo , Membrana Celular/enzimologia , Músculo Liso/enzimologia , Útero/enzimologia , Animais , Fracionamento Celular , Membrana Celular/ultraestrutura , Feminino , Histocitoquímica , Microscopia Eletrônica , Músculo Liso/ultraestrutura , Ratos , Receptores de Concanavalina A/metabolismo , Útero/ultraestrutura
5.
Biochem Biophys Res Commun ; 128(1): 113-9, 1985 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-3157377

RESUMO

This work is the first biochemical study of skeletal muscle membranes isolated from mice displaying an inherited neuromuscular disease: MedJ strains. It is focused on the research of a possible alteration of membrane biological activities related to this disease. We describe a procedure which allows the simultaneous preparation of mitochondrial and microsomal fractions from a small amount of skeletal muscle. When EGTA and BSA are present in the buffers, functional mitochondria can be prepared. Under these conditions we found that no major modification occurs for this disease at the mitochondrial inner membrane level. A dramatic impairment of a calcium active transport activity found in the microsomal fraction obtained from MedJ is noticed, suggesting that some modification may occur at this level.


Assuntos
Membranas Intracelulares/ultraestrutura , Músculos/ultraestrutura , Doenças Neuromusculares/patologia , Animais , Cálcio/metabolismo , ATPases Transportadoras de Cálcio/metabolismo , Fracionamento Celular , Camundongos , Camundongos Mutantes , Microssomos/ultraestrutura , Mitocôndrias Musculares/ultraestrutura , Músculos/enzimologia , Consumo de Oxigênio
7.
Biochimie ; 58(10): 1159-65, 1976.
Artigo em Inglês | MEDLINE | ID: mdl-1009171

RESUMO

tRNATrp from beef lever has been purified by classical chromatographical methods. Total tRNA, prepared on a large scale (total aminoacid acceptance 1280 pmol/A260 unit) was submitted to chromatography on benzoylated-DEAE cellulose, then DEAE Sephadex. The major species accepting tryptophan issued from the second chromatography was aminoacylated with [14C] tryptophan and chromatographed on benzoylated-DEAE cellulose. The tRNA carrying the radioactive label was eluted in the ethanolic region. After stripping, the resulting tRNATrp has an acceptance of 1800 pmol/A260 unit. No isoacceptors could be demonstrated by chromatography of the pure species on RCP 5 in 6 M urea. The yield in pure tRNATrp was currently in the range of 25 to 30 percent of the total tryptophan acceptance of the starting curde tRNA.


Assuntos
Fígado/metabolismo , RNA de Transferência/isolamento & purificação , Aminoácidos/metabolismo , Animais , Bovinos , Isoleucina , Metionina , RNA de Transferência/metabolismo , Triptofano , Valina
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