RESUMO
High-mobility group box-1 (HMGB1) protein acts as a transcription factor in the nucleus and also as a pro-inflammatory cytokine when released into extracellular fluids. The presence of higher levels of HMGB1 is reported in the gingival crevicular fluid from periodontal patients. Since the proliferation of bacteria within the periodontal pocket is closely involved in the exacerbation of periodontal disease, it is hypothesized that the periodontal pocket causes the release of HMGB1. Immunohistochemical staining of inflamed gingiva revealed that HMGB1 is exclusively dislocated from the nucleus to the cytoplasm in the pocket epithelium, whereas it is mainly present in the nucleus in the gingival epithelium. Butyric acid, an extracellular metabolite from periodontopathic bacteria populating the periodontal pocket, induced the passive release of HMGB1 as a result of eliciting necrosis in the human gingival epithelial cell line. Thus, the periodontal epithelium may provide a unique pathological setting for HMGB1 release by bacterial insult.
Assuntos
Ácido Butírico/farmacologia , Líquido do Sulco Gengival/química , Proteína HMGB1/metabolismo , Bolsa Periodontal/metabolismo , Fatores de Virulência/metabolismo , Adulto , Idoso , Estudos de Casos e Controles , Linhagem Celular , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Gengiva/citologia , Proteína HMGB1/análise , Humanos , Masculino , Pessoa de Meia-Idade , Necrose/metabolismo , Bolsa Periodontal/microbiologia , Bolsa Periodontal/patologia , Transporte Proteico , Espécies Reativas de Oxigênio/metabolismo , Fatores de Virulência/análiseRESUMO
IgG immune complexes trigger humoral immune responses by cross-linking of FcRs for IgG (FcgammaRs). In the present study, we investigated role of lipid rafts, glycolipid- and cholesterol-rich membrane microdomains, in the FcgammaR-mediated responses. In retinoic acid-differentiated HL-60 cells, cross-linking of FcgammaRs resulted in a marked increase in the tyrosine phosphorylation of FcgammaRIIa, p58(lyn), and p120(c-cbl), which was inhibited by a specific inhibitor of Src family protein tyrosine kinases. After cross-linking, FcgammaRs and tyrosine-phosphorylated proteins including p120(c-cbl) were found in the low-density detergent-resistant membrane (DRM) fractions isolated by sucrose-density gradient ultracentrifugation. The association of FcgammaRs as well as p120(c-cbl) with DRMs did not depend on the tyrosine phosphorylation. When endogenous cholesterol was reduced with methyl-beta-cyclodextrin, the cross-linking did not induce the association of FcgammaRs as well as p120(c-cbl) with DRMs. In addition, although the physical association between FcgammaRIIa and p58(lyn) was not impaired, the cross-linking did not induce the tyrosine phosphorylation. In human neutrophils, superoxide generation induced by opsonized zymosan or chemoattractant fMLP was not affected or increased, respectively, after the methyl-beta-cyclodextrin treatment, but the superoxide generation induced by the insoluble immune complex via FcgammaRII was markedly reduced. Accordingly, we conclude that the cross-linking-dependent association of FcgammaRII to lipid rafts is important for the activation of FcgammaRII-associated Src family protein tyrosine kinases to initiate the tyrosine phosphorylation cascade leading to superoxide generation.
Assuntos
Microdomínios da Membrana/metabolismo , Microdomínios da Membrana/fisiologia , Fosfotirosina/metabolismo , Receptores de IgG/metabolismo , Receptores de IgG/fisiologia , Superóxidos/metabolismo , Ubiquitina-Proteína Ligases , beta-Ciclodextrinas , Células Cultivadas , Ciclodextrinas/farmacologia , Detergentes/química , Inibidores Enzimáticos/farmacologia , Células HL-60 , Humanos , Fosforilação/efeitos dos fármacos , Transporte Proteico , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-cbl , Proteínas Proto-Oncogênicas pp60(c-src)/antagonistas & inibidores , Pirazóis/farmacologia , Pirimidinas/farmacologia , Transdução de Sinais , Tretinoína/farmacologia , Quinases da Família src/metabolismoRESUMO
In rabbit parotid acinar cells, the muscarinic cholinergic agonist methacholine induced an increase in the intracellular Ca(2+) concentration and provoked nitric oxide (NO) generation. Ca(2+)-mobilizing reagents such as thapsigargin and the Ca(2+) ionophore A23187 mimicked the effect of methacholine on NO generation. Methacholine-induced NO generation was inhibited by the removal of extracellular Ca(2+). Immunoblot analysis indicated that the antibody against the neuronal type of nitric oxide synthase (NOS) cross-reacted with NOS in the cytosol of rabbit parotid gland cells. Immunofluorescence testing showed that neuronal NOS is present in the cytosol of acinar cells but less in the ductal cells. NOS was purified approximately 8100-fold from the cytosolic fraction of rabbit parotid glands by chromatography on Sephacryl S-200, DEAE-Sephacel, and 29,59-ADP-Sepharose. The purified NOS was a NADPH- and tetrahydroxybiopterin-dependent enzyme and was activated by Ca(2+) within the physiological range in the presence of calmodulin. These results suggest that NO is generated by the activation of the neuronal type of NOS, which is regulated in rabbit parotid acinar cells by the increase in intracellular Ca(2+) levels induced by the activation of muscarinic receptors.
Assuntos
Cálcio/metabolismo , Óxido Nítrico/metabolismo , Glândula Parótida/citologia , Glândula Parótida/metabolismo , Animais , Calcimicina/farmacologia , Calmodulina/farmacologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Immunoblotting , Ionóforos/farmacologia , Cinética , Cloreto de Metacolina/farmacologia , Microscopia de Fluorescência , Agonistas Muscarínicos/farmacologia , NADP/farmacologia , Óxido Nítrico Sintase/metabolismo , Ligação Proteica , Coelhos , Ratos , Tapsigargina/farmacologia , Fatores de TempoRESUMO
Leukocyte cell surface antigen CD38 is a single-transmembrane protein whose extracellular domain has catalytic activity for NAD(+) glycohydrolase (NADase). We previously reported that b-series gangliosides inhibit the NADase activity of the extracellular domain of CD38 expressed as a fusion protein [Hara-Yokoyama, M., Kukimoto, I., Nishina, H., Kontani, K., Hirabayashi, Y., Irie, F., Sugiya, H., Furuyama, S., and Katada, T. (1996) J. Biol. Chem. 271, 12951-12955]. In the present study, we examined the effect of exogenous gangliosides on the NADase activity of CD38 on the surface of retinoic acid-treated human leukemic HL60 cells and CD38-transfected THP-1 cells. After incubation of the cells with G(T1b), inhibition of NADase activity was observed. The time course of inhibition was slower than that of the incorporation of G(T1b) into the cells, suggesting that incorporation into the cell membranes is a prerequisite for inhibition. Inhibition occurred efficiently when G(T1b) and CD38 were present on the same cells (cis interaction) rather than on different cells (trans interaction). Although gangliosides may affect localization of cell surface proteins, indirect immunofluorescence intensity due to CD38 was not affected after G(T1b) treatment. Comparison of the effect of G(T1b) and G(D1a) indicates that the tandem sialic acid residues linked to the internal galactose residue of the gangliotetraose core are crucial to the inhibition. These results suggest a novel role of complex gangliosides for the first time as cell surface inhibitors of CD38 through specific and cis interaction between the oligosaccharide moiety and the extracellular domain.
Assuntos
Antígenos de Diferenciação/metabolismo , Inibidores Enzimáticos/farmacologia , Gangliosídeos/farmacologia , N-Glicosil Hidrolases/antagonistas & inibidores , NAD+ Nucleosidase/metabolismo , ADP-Ribosil Ciclase , ADP-Ribosil Ciclase 1 , Antígenos CD/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Inibidores Enzimáticos/metabolismo , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/enzimologia , Espaço Extracelular/metabolismo , Citometria de Fluxo , Gangliosídeos/metabolismo , Gangliosídeos/fisiologia , Células HL-60 , Humanos , Hidrólise , Glicoproteínas de Membrana , N-Glicosil Hidrolases/metabolismo , Oligossacarídeos/metabolismo , Fatores de Tempo , Células Tumorais CultivadasRESUMO
The cell surface antigen CD38 is a multifunctional ectoenzyme that acts as an NAD(+) glycohydrolase, an ADP-ribosyl cyclase, and also a cyclic ADP-ribose hydrolase. The extracellular catalytic domain of CD38 was expressed as a fusion protein with maltose-binding protein, and was crystallized in the complex with a ganglioside, G(T1b), one of the possible physiological inhibitors of this ectoenzyme. Two different crystal forms were obtained using the hanging-drop vapor diffusion method with PEG 10,000 as the precipitant. One form diffracted up to 2.4 A resolution with synchrotron radiation at 100 K, but suffered serious X-ray damage. It belongs to the space group P2(1)2(1)2(1) with unit-cell parameters of a = 47.9, b = 94.9, c = 125.2 A. The other form is a thin plate, but the data sets were successfully collected up to 2.4 A resolution by use of synchrotron radiation at 100 K. The crystals belong to the space group P2(1) with unit-cell parameters of a = 57.4, b = 51.2, c = 101.1 A, and beta = 97.9 degrees, and contain one molecule per asymmetric unit with a VM value of 2.05 A(3)/Da.
Assuntos
Antígenos CD , Antígenos de Diferenciação/química , Antígenos de Diferenciação/metabolismo , Gangliosídeos/química , Gangliosídeos/metabolismo , NAD+ Nucleosidase/química , NAD+ Nucleosidase/metabolismo , ADP-Ribosil Ciclase , ADP-Ribosil Ciclase 1 , Antígenos de Diferenciação/genética , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Cristalização , Proteínas Ligantes de Maltose , NAD+ Nucleosidase/genética , Conformação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Difração de Raios XRESUMO
Amylase release from parotid acinar cells is mainly induced by the accumulation of intracellular cAMP, presumably through the phosphorylation of substrates by cAMP-dependent protein kinase (PKA). However, the molecular mechanisms of this process are not clear. In a previous study (Fujita-Yoshigaki, J., Dohke, Y., Hara-Yokoyama, M., Kamata, Y., Kozaki, S., Furuyama, S., and Sugiya, H. (1996) J. Biol. Chem. 271, 13130-13134), we reported that vesicle-associated membrane protein 2 (VAMP2) is localized at the secretory granule membrane and is involved in cAMP-induced amylase secretion. To study the formation of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex containing VAMP2 in parotid acinar cells, we prepared rabbit polyclonal antibody against the peptide corresponding to Arg(47)-Asp(64) of VAMP2 (anti-SER4256). The recognition site of anti-SER4256 overlaps the domain involved in binding target membrane SNAREs (t-SNARES). Then we examined the condition of VAMP2 by immunoprecipitation with anti-SER4256. VAMP2 was not included in the immunoprecipitate from solubilized granule membrane fraction under the control conditions, but incubation with cytosolic fraction and cAMP caused immunoprecipitation of VAMP2. The effect of cytosolic fraction and cAMP was reduced by addition of PKA inhibitor H89. Addition of both the catalytic subunit of PKA and the cytosolic fraction allowed immunoprecipitation of VAMP2, whereas the PKA catalytic subunit alone did not. These results suggest that () the t-SNARE binding region of VAMP2 is masked by some protein X and activation of PKA caused the dissociation of X from VAMP2; and () the effect of PKA is not direct phosphorylation of X, but works through phosphorylation of some other cytosolic protein.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas de Membrana/metabolismo , Glândula Parótida/metabolismo , Proteínas de Transporte Vesicular , Animais , Cálcio/metabolismo , Membrana Celular/metabolismo , AMP Cíclico/metabolismo , Ativação Enzimática , Glândula Parótida/citologia , Testes de Precipitina , Ligação Proteica , Proteínas Qa-SNARE , Proteínas R-SNARE , Ratos , Proteínas SNARERESUMO
Rat parotid acinar cells secrete amylase through the stimulation of beta-adrenoceptors followed by accumulation of intracellular cAMP. However, it remains unclear at the molecular level how secretory granules fuse with the apical membranes. We have examined whether SNARE proteins are involved in exocytosis in the salivary glands, and have found that one of the SNARE proteins, VAMP-2, is localized at the secretory granule membrane of rat parotid acinar cells. Moreover, botulinum neurotoxin B, which has endoprotease activity that cleaves VAMP-2, inhibited cAMP-dependent amylase release but did not inhibit basal secretion in the absence of cAMP. These results suggest that VAMP-2 is essential for cAMP-regulated exocytosis in rat parotid acinar cells. In contrast, both neurotoxins A and C1 (endoproteases that cleave SNAP-25 and syntaxin 1 respectively) failed to inhibit cAMP-dependent amylase release. Therefore, neither SNAP-25 nor syntaxin 1 are involved in amylase secretion in the parotid glands. Clarification of the mechanism of secretion will require the identification of proteins that interact and function cooperatively with VAMP-2. This approach may also reveal details of the molecular mechanism by which the cAMP facilitates secretion in other systems, including neurotransmission.
Assuntos
AMP Cíclico/fisiologia , Exocitose/fisiologia , Proteínas de Membrana/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Glândulas Salivares/química , Proteínas de Transporte Vesicular , Animais , Proteínas R-SNARE , Proteínas SNARE , Glândulas Salivares/citologia , Glândulas Salivares/fisiologia , Sintaxina 1RESUMO
ADP-ribosylation factor (Arf) is a 20 kDa polypeptide that is a member of the Ras superfamily of small molecular mass GTP-binding proteins. In addition to an essential role of Arf1 in vesicle budding, recent observations suggest a role for Arf6 in calcium-dependent exocytosis in bovine adrenal chromaffin cells. In rat parotid acinar cells, exocytosis is cAMP-dependent and our findings suggest an interaction of Arf1 with the secretory granules. We describe here the structural and functional background to the Arf proteins focusing on their role in rat parotid acinar cells.
Assuntos
Difosfato de Adenosina/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Glândula Parótida/citologia , Glândula Parótida/metabolismo , Fator 1 de Ribosilação do ADP , Fatores de Ribosilação do ADP , Animais , Exocitose/fisiologia , RatosRESUMO
Guanosine 3',5'-cyclic monophosphate (cGMP) is a second messenger generated in response to hormones or neurotransmitters in various tissues and cells. In parotid acinar cells, the activation of muscarinic cholinergic and beta-adrenergic receptors induces an increase in intracellular cGMP. However, the mechanism of cGMP production in parotid acinar cells has not been well elucidated. cGMP production is induced by the activation of guanylyl cyclases, which are directly activated by nitric oxide (NO). NO plays an important role as an inter- and intracellular signal molecule in various organs and cells. Biosynthesis of NO is catalyzed by NO synthase (NOS), and NO generation is controlled by the regulation of NOS activity, for example by Ca2+. We have studied the regulation of NOS activity, NO generation and cGMP production in rabbit parotid acinar cells, and have demonstrated a functional Ca2+-NO-cGMP signaling pathway.
Assuntos
Sinalização do Cálcio/fisiologia , GMP Cíclico/metabolismo , Óxido Nítrico/metabolismo , Glândula Parótida/citologia , Glândula Parótida/fisiologia , Animais , CoelhosRESUMO
We investigated the interaction of ADP-ribosylation factor (Arf) with the secretory granules in rat parotid acinar cells. The 20. 5-kDa small-molecular-mass GTP-binding protein in the cytosolic fraction of rat parotid acinar cells was identified as ADP-ribosylation factor1 by using a pan-Arf monoclonal antibody and isotype-specific polyclonal antibodies for Arf proteins 1, 3, 5, and 6. Incubation of the cytosolic fraction with isolated secretory granule membranes in the presence of GTPgammaS resulted in the translocation of Arf1 from the cytosolic fraction to the secretory granule membranes. The translocation was not observed in the presence of GDPbetaS in place of GTPgammaS, indicating that the process is GTP-dependent. The immunoelectron microscopy experiment confirmed Arf1 is translocated to the secretory granules. A prior treatment of the granule membranes with trypsin inhibited the translocation of Arf1 at 2 mM Mg2+, but had no effect in the absence of Mg2+ (condition of spontaneous conversion of Arf-GDP to Arf-GTP). Thus, the trypsin-sensitive nucleotide exchange activity for Arf1 is probably associated with the secretory granule membranes. These results demonstrate Arf1 translocates to the secretory granules in rat parotid acinar cells.
Assuntos
Proteínas de Transporte/metabolismo , Grânulos Citoplasmáticos/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Glândula Parótida/metabolismo , Fator 1 de Ribosilação do ADP , Fatores de Ribosilação do ADP , Animais , Transporte Biológico , Proteínas de Transporte/química , Bovinos , Fracionamento Celular , Proteína Coatomer , Grânulos Citoplasmáticos/química , Proteínas de Ligação ao GTP/química , Nucleotídeos de Guanina/metabolismo , Humanos , Masculino , Proteínas de Membrana/metabolismo , Microscopia Imunoeletrônica , Glândula Parótida/química , Glândula Parótida/citologia , Ratos , Ratos Sprague-DawleyRESUMO
We investigated the mechanism of guanosine 3',5'-monophosphate (cGMP) production in rabbit parotid acinar cells. Methacholine, a muscarinic cholinergic agonist, stimulated cGMP production in a dose-dependent manner but not isoproterenol, a beta-adrenergic receptor stimulant. Methacholine-stimulated cGMP production has been suggested to be coupled to Ca2+ mobilization, because intracellular Ca2+ elevating reagents, such as thapsigargin and the Ca2+ ionophore A23187, mimicked the effect of methacholine. The cGMP production induced by Ca2+ mobilization has also been suggested to be coupled to nitric oxide (NO) generation because the effects of methacholine, thapsigargin and A23187 on cGMP production were blocked by NG-nitro-L-arginine methyl ester (L-NAME), a specific inhibitor of nitric oxide synthase (NOS), and hemoglobin, a scavenger of nitric oxide (NO). Sodium nitroprusside (SNP), a NO donor, stimulated cGMP production. Furthermore, methacholine stimulated NO generation, and NOS activity in the cytosolic fraction in rabbit parotid acinar cells was exclusively dependent on Ca2+. These findings suggest that cGMP production induced by the activation of muscarinic cholinergic receptors is coupled to NO generation via Ca2+ mobilization.
Assuntos
Cálcio/metabolismo , GMP Cíclico/biossíntese , Óxido Nítrico/biossíntese , Glândula Parótida/metabolismo , Aminoquinolinas/farmacologia , Animais , Arginina/metabolismo , Calcimicina/farmacologia , Cálcio/farmacologia , Citrulina/metabolismo , Inibidores Enzimáticos/metabolismo , Guanilato Ciclase/farmacologia , Cloreto de Metacolina/farmacologia , Modelos Biológicos , NG-Nitroarginina Metil Éster/farmacologia , Coelhos , Receptores Muscarínicos/metabolismo , Tapsigargina/farmacologia , Fatores de TempoRESUMO
We have recently reported that gangliosides act as inhibitors of ADP-ribosyltransferases and NAD+ glycohydrolases (NADase) of pertussis toxin and the C3 exoenzyme from Clostridium botulinum (Hara-Yokoyama, M., Hirabayashi, Y., Irie, F., Syuto, B., Moriishi, K., Sugiya, H., and Furuyama, S. (1995) J. Biol. Chem. 270, 8115-8121). Here, we investigated the effect of gangliosides on the enzymatic activity of leukocyte cell surface antigen CD38, which is identified as an ecto-NADase (Kontani, K., Nishina, H., Ohoka, Y., Takahashi, K., and Katada, T. (1993) J. Biol. Chem. 268, 16895-16898). Gangliosides GM1a and GQ1balpha inhibited the NADase activity in the immunoprecipitate of anti-CD38 antibody from the membrane extract of retinoic acid-treated human leukemic HL-60 cells. Gangliosides also inhibited the NADase activity of the extracellular domain of CD38 antigen that was deprived of the transmembrane domain and was expressed in Escherichia coli as a fusion protein with maltose-binding protein (MBP-CD38). The order of the inhibitory effect of purified ganglioside species on the NADase activity on MBP-CD38 was as follows: GQ1balpha > GT1b, GQ1b > GD1a, GD1b, GM1a, GM1b, GD3, GM3. GQ1balpha inhibited the NADase of MBP-CD38 in a noncompetitive manner versus NAD+ with a Ki value of about 0.3 microM. Neither ceramide nor the oligosaccharide moiety of GQ1balpha had an effect on the NADase activity. GQ1balpha, GT1b, and GQ1b also efficiently inhibited the ADP-ribosyl cyclase activity of MBP-CD38. At present, gangliosides are the only endogenous species that can block the enzymatic activity of CD38 antigen. The present results suggest a potential role of gangliosides as inhibitors of the ecto-NADases.
Assuntos
Antígenos CD , Antígenos de Diferenciação/efeitos dos fármacos , Gangliosídeos/farmacologia , N-Glicosil Hidrolases/efeitos dos fármacos , NAD+ Nucleosidase/antagonistas & inibidores , ADP-Ribosil Ciclase , ADP-Ribosil Ciclase 1 , Animais , Aplysia , Bovinos , Ceramidas/química , Gangliosídeos/química , Células HL-60 , Humanos , Hidrólise , Glicoproteínas de Membrana , NAD/metabolismo , Oligossacarídeos/químicaRESUMO
Amylase exocytosis of the parotid gland is mediated by intracellular cAMP. To investigate whether cAMP-dependent secretion has a mechanism similar to that of regulated neuroexocytosis, we examined the expression of synaptosome-associated proteins. In rat parotid acinar cells, we found 25 (p25) and 18 kDa (p18) proteins reacted with antibodies against Rab3A and vesicle-associated membrane protein 2 (VAMP-2), respectively. On the other hand, syntaxin 1 and SNAP-25, which interact with VAMP-2 at synapses, were undetectable. Rab3A-like p25 and VAMP-2-like p18 were also expressed in other exocrine acinar cells. The latter was localized at secretory granule membranes, and the former was detected in secretory granule and cytosolic fractions. The antibody against VAMP-2 used in this study did not react with cellubrevin, and p18 was cleaved with botulinum neurotoxin B. Thus, we identified p18 as VAMP-2. Botulinum neurotoxin B inhibited the cAMP-induced amylase release from streptolysin O-permeabilized acinar cells. Therefore, VAMP-2 is required for cAMP-regulated amylase release in rat parotid acinar cells. This is the first report that VAMP-2 is involved in regulated exocytosis that is independent of Ca2+.
Assuntos
Amilases/antagonistas & inibidores , Toxinas Botulínicas/farmacologia , AMP Cíclico/metabolismo , Exocitose , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Glândula Parótida/efeitos dos fármacos , Amilases/metabolismo , Animais , Membrana Celular/metabolismo , Citosol/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Hidrólise , Masculino , Glândula Parótida/citologia , Glândula Parótida/metabolismo , Proteínas R-SNARE , Ratos , Ratos Sprague-Dawley , Proteínas rab3 de Ligação ao GTPRESUMO
We have previously reported the presence of an endogenous inhibitory activity in bovine brain for the ADP-ribosylation of GTP-binding proteins catalyzed by pertussis toxin (PT) (Hara-Yokoyama, M., and Furuyama, S. (1989) Biochem. Biophys. Res. Commun. 160, 67-71). In the present study, we identified the inhibitor as a ganglioside. The screening of various gangliosides revealed that GQ1b alpha most effectively inhibited the ADP-ribosyltransferase activities of both the holoenzyme and the catalytic subunit of PT. GQ1b alpha is a ganglioside newly identified as one of the antigens recognized by the cholinergic neuron-specific antibody, anti-Chol-1 alpha (Hirabayashi, Y., Nakao, T., Irie, F., Whittaker, V.P., Kon, K., and Ando, S. (1992) J. Biol. Chem. 267, 12973-12978). GQ1b alpha also inhibited the PT-catalyzed NAD+ glycohydrolysis. Unlike PT activity, the ADP-ribosylation and the NAD+ glycohydrolysis catalyzed by the C3 exoenzyme from Clostridium botulinum type C were inhibited by GT1b and GQ1b. The ADP-ribosylation catalyzed by either PT or the C3 exoenzyme was not inhibited by ceramide, galactocerebroside, or sialic acid. In addition to the inhibitory action of gangliosides on ADP-ribosylation, the importance of gangliosides as regulators of NAD+ metabolism is discussed.
Assuntos
ADP Ribose Transferases/metabolismo , Toxinas Botulínicas , Gangliosídeos/farmacologia , Toxina Pertussis , Inibidores de Poli(ADP-Ribose) Polimerases , Fatores de Virulência de Bordetella/metabolismo , Animais , Encéfalo/metabolismo , Sequência de Carboidratos , Catálise , Bovinos , Ceramidas/farmacologia , Ácido Cólico , Ácidos Cólicos/metabolismo , Galactosilceramidas/farmacologia , Gangliosídeos/química , Dados de Sequência Molecular , Ácido N-Acetilneuramínico , Ácidos Siálicos/farmacologia , Fatores de Virulência de Bordetella/químicaRESUMO
The particulate proteins of 34, 26, and 22 kDa are phosphorylated on serine residues in the rat parotid gland by activation of the cAMP-mediated signaling system. The 34 kDa protein was identified as ribosomal protein S6 by immunoprecipitation with anti S6 peptide antibody. The dephosphorylation of S6 was observed by incubation of the particulate fraction of the saponin-permeabilized cells labeled with [gamma-32P]ATP in the presence of cAMP/3-isobutyl-1-methylxanthine. The dephosphorylation of S6 was inhibited by either okadaic acid, a potent inhibitor of protein phosphatase, or Zn2+, however, neither Ca2+ nor Mg2+ showed significant effect. S6 phosphatase activities detected by using the 32P-labeled S6 peptide as a substrate were inhibited by both okadaic acid and Zn2+. These results suggest that the dephosphorylation of S6 is mediated by the okadaic acid and Zn(2+)-sensitive phosphatases in the rat parotid gland.
Assuntos
Éteres Cíclicos/farmacologia , Glândula Parótida/enzimologia , Fosfoproteínas Fosfatases/metabolismo , Proteínas Ribossômicas/metabolismo , Zinco/farmacologia , Sequência de Aminoácidos , Animais , Cálcio/farmacologia , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Magnésio/farmacologia , Dados de Sequência Molecular , Ácido Okadáico , Glândula Parótida/citologia , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Proteína S6 Ribossômica , Transdução de SinaisRESUMO
1. Adenylylation, a posttranslational modification of proteins, was investigated in saponin-permeabilized acinar cells of the rat parotid gland. 2. When cells were incubated with [2,8-3H]ATP, several proteins, including a 26 kDa protein in the particulate fraction, were labeled. 3. Upon incubation of cells with [alpha-32P]ATP in the presence of cAMP and 3-isobutyl-1-methylxanthine, 32P-labeling of the 26 kDa protein was observed. 4. After treatment with snake venom phosphodiesterase, [32P]AMP was released from the 26 kDa protein. Such release was not observed when cells were labeled with [gamma-32P]ATP. 5. The 32P-labeling pattern of proteins with [alpha-32P]ATP was clearly different from that with [adenylate-32P]NAD+. 6. The results suggest that the 26 kDa protein is one of the adenylylation substrates in rat parotid acinar cells.
Assuntos
Trifosfato de Adenosina/metabolismo , Glândula Parótida/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo , 1-Metil-3-Isobutilxantina/farmacologia , Adenosina Difosfato Ribose/metabolismo , Animais , Autorradiografia , Permeabilidade da Membrana Celular , AMP Cíclico/farmacologia , Eletroforese em Gel de Poliacrilamida , Marcação por Isótopo , Peso Molecular , Glândula Parótida/citologia , Glândula Parótida/efeitos dos fármacos , Diester Fosfórico Hidrolases/farmacologia , Radioisótopos de Fósforo , Proteínas/química , Ratos , Venenos de Serpentes/enzimologia , Especificidade por SubstratoRESUMO
Phosphorylation of three particulate proteins with molecular masses of 34, 26, and 22 kDa was stimulated in the presence of cyclic AMP/3-isobutyl-1-methylxanthine in saponin-permeabilized rat parotid acinar cells. When the particulate fraction isolated from the cells labeled with [gamma-32p]ATP was incubated at 30 degrees C, dephosphorylation of the 26 kDa phosphoprotein occurred in the presence of Mg2+ or Mn2+. Okadaic acid had no effect on the Mg(2+)-dependent dephosphorylation of the 26 kDa phosphoprotein. Addition of the recombinant type 2C phosphatase, Mg(2+)-dependent and okadaic acid-insensitive phosphatase, caused a remarkable dephosphorylation of the 26 kDa phosphoprotein. These observations strongly suggest type 2C phosphatase is involved in the dephosphorylation of the 26 kDa phosphoprotein.
Assuntos
Isoenzimas/metabolismo , Glândula Parótida/metabolismo , Fosfoproteínas/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Permeabilidade da Membrana Celular , AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , Ácido Edético/farmacologia , Ácido Egtázico/farmacologia , Éteres Cíclicos/farmacologia , Técnicas In Vitro , Isoenzimas/antagonistas & inibidores , Isoenzimas/isolamento & purificação , Magnésio/farmacologia , Peso Molecular , Ácido Okadáico , Glândula Parótida/citologia , Glândula Parótida/efeitos dos fármacos , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/isolamento & purificação , Ratos , Proteínas Recombinantes/metabolismoRESUMO
The gene for the Glu-tRNA synthetase from an extreme thermophile, Thermus thermophilus HB8, was isolated using a synthetic oligonucleotide probe coding for the N-terminal amino acid sequence of Glu-tRNA synthetase. Nucleotide-sequence analysis revealed an open reading frame coding for a protein composed of 468 amino acid residues (Mr 53,901). Codon usage in the T. thermophilus Glu-tRNA synthetase gene was in fact similar to the characteristic usages in the genes for proteins from bacteria of genus Thermus: the G + C content in the third position of the codons was as high as 94%. In contrast, the amino acid sequence of T. thermophilus Glu-tRNA synthetase showed high similarity with bacterial Glu-tRNA synthetases (35-45% identity); the sequences of the binding sites for ATP and for the 3' terminus of tRNA(Glu) are highly conserved. The Glu-tRNA synthetase gene was efficiently expressed in Escherichia coli under the control of the tac promoter. The recombinant T. thermophilus Glu-tRNA synthetase was extremely thermostable and was purified to homogeneity by heat treatment and three-step column chromatography. Single crystals of T. thermophilus Glu-tRNA synthetase were obtained from poly(ethylene glycol) 6000 solution by a vapor-diffusion technique. The crystals diffract X-rays beyond 0.35 nm. The crystal belongs to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters of a = 8.64 nm, b = 8.86 nm and c = 8.49 nm.
Assuntos
Glutamato-tRNA Ligase/genética , Thermus thermophilus/enzimologia , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , Códon , Cristalização , DNA Bacteriano/genética , Eletroforese em Gel de Poliacrilamida , Escherichia coli/enzimologia , Expressão Gênica , Glutamato-tRNA Ligase/metabolismo , Dados de Sequência Molecular , Plasmídeos , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , Difração de Raios XRESUMO
When saponin-permeabilized rat parotid acinar cells were incubated with [adenylate-32P]NAD+, labelling of proteins (33, 27 and 23 kDa) in particulate fractions of the cells was stimulated by isoproterenol. The effect of isoproterenol was completely blocked by a beta-antagonist. Both forskolin or cAMP mimicked the effect of isoproterenol on the labelling. However, an inhibitor of cAMPdPK failed to induce complete inhibition of the effects of isoproterenol, forskolin and cAMP. When the labelled proteins were treated with snake venom phosphodiesterase, neither [32P]5'-AMP nor [32P]phosphoribosyladenosine was released. These results suggest that covalent modification of proteins with NAD+, which is distinct from ADP-ribosylation and cAMPdPK-dependent phosphorylation, is coupled to beta-receptor-cAMP signalling system in rat parotid acinar cells.
Assuntos
Isoproterenol/farmacologia , NAD/metabolismo , Glândula Parótida/metabolismo , Amilases/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Colforsina/farmacologia , AMP Cíclico/farmacologia , Glândula Parótida/efeitos dos fármacos , Fosfodiesterase I , Diester Fosfórico Hidrolases , Radioisótopos de Fósforo , Propranolol/farmacologia , Saponinas/farmacologiaRESUMO
The conformation change of Thermus thermophilus tRNA(1Ile) upon complex formation with T. thermophilus elongation factor Tu (EF-Tu) was studied by analysis of the circular dichroism (CD) bands at 315 nm (due to the 2-thioribothymidine residue in the T-loop) and at 295 nm (due to the core structure of tRNA). Formation of the ternary complex of isoleucyl-tRNA(1Ile) and EF-Tu.GTP increased the intensities of these CD bands, indicating stabilization of the association between the T-loop and the D-loop and also a significant conformation change of the core region. Upon complex formation of EF-Tu.GTP and uncharged tRNA, however, the conformation of the core region is not changed, while the association of the two loops is still stabilized. On the other hand, the binding with EF-Tu.GDP does not appreciably affect the conformation of isoleucyl-tRNA or uncharged tRNA. These indicate the importance of the gamma-phosphate group of GTP and the aminoacyl group in the formation of the active complex of aminoacyl-tRNA and EF-Tu.GTP.