RESUMEN
Expression of aquaporins (AQP) and water permeability were studied in Xenopus laevis oocytes and immobilized glial cells by a pulsed-field gradient spin echo NMR technique and a photometric swelling assay. Oocytes injected with poly(A) RNA from C6-BU-1 cells showed increased swelling behavior under hypoosmotic stress due to expressed water channels as compared to control oocytes. The swelling could be reversibly inhibited by HgCl2. Furthermore, the intracellular relaxation time and the apparent intracellular diffusion coefficient of water in oocytes were determined by diffusion-weighted 1H NMR experiments to be T2=36 ms and Dapp, intra=0.18x10-3 mm2/s. In immobilized C6 and F98 cells the mean exchange time of intracellular water was found to be 51 ms which increased to 75 ms upon chronic treatment (4 days) in hypertonic medium. Additional hybrid depletion experiments with antisense oligonucleotides directed against AQP1 were performed on oocytes and C6 cells. Moreover, different water channel subtypes of glial cells were assessed by a reverse transcriptase polymerase chain reaction assay. With this, the mRNA encoding AQP1 could be detected in primary cultures and glial cell lines, whereas AQP4 mRNA was found in astroglia-rich primary cultures, but not in F98 and C6 cells. Our results show that water permeability in glial cells is mainly mediated by water channels which play an important role in the regulation of water flow in brain under normal and pathological conditions.
Asunto(s)
Acuaporinas/metabolismo , Permeabilidad de la Membrana Celular , Agua/metabolismo , Animales , Acuaporina 1 , Acuaporina 4 , Acuaporinas/genética , Acuaporinas/aislamiento & purificación , Transporte Biológico , Tamaño de la Célula , Células Cultivadas , Difusión , Neuroglía/citología , Neuroglía/metabolismo , Neuronas/citología , Neuronas/metabolismo , Resonancia Magnética Nuclear Biomolecular , Oligonucleótidos Antisentido , Oocitos/citología , Oocitos/metabolismo , Fotometría/métodos , Reacción en Cadena de la Polimerasa , Ratas , Xenopus laevisRESUMEN
The assembly of a heterodimeric luciferase was studied after de novo synthesis of corresponding precursor proteins in reticulocyte lysate and concomitant transport into dog pancreas microsomes. This cytosolic luciferase from a prokaryotic organism (Vibrio harveyi) was specifically used as a model protein to investigate (i) whether the eukaryotic cytosol and the microsomal lumen have similar folding capabilities and (ii) whether the requirements of a polypeptide for certain molecular chaperones and folding catalysts are determined by the polypeptide or the intracellular compartment. The two luciferase subunits were fused to the preprolactin signal peptide. Data indicate that efficient assembly of luciferase occurs in the mammalian microsomes. Furthermore, it was observed that luciferase assembly can be separated in time from synthesis and membrane transport, depends on ATP hydrolysis, is partially sensitive to cyclosporin A and FK506, and in the absence of lumenal proteins is less efficient as compared with the presence of lumenal proteins. Thus, heterodimeric luciferase depends on functionally related molecular chaperones and folding catalysts during its assembly in either the eukaryotic cytosol or the microsomal lumen.
Asunto(s)
Isomerasas de Aminoácido/metabolismo , Proteínas Portadoras/metabolismo , Luciferasas/metabolismo , Microsomas/metabolismo , Chaperonas Moleculares/metabolismo , Pliegue de Proteína , Adenosina Trifosfato/metabolismo , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Transporte Biológico , Sistema Libre de Células , Ciclosporina/farmacología , Perros , Hidrólisis , Luciferasas/genética , Páncreas , Isomerasa de Peptidilprolil , Biosíntesis de Proteínas , Conformación Proteica , Proteolípidos/metabolismo , Conejos , Proteínas Recombinantes/metabolismo , Reticulocitos , Estereoisomerismo , Tacrolimus/farmacologíaRESUMEN
Folding of polypeptides emerging from the protein translocase in the membrane of mammalian microsomes was analyzed after synthesis of corresponding precursor proteins in a mammalian translation system. Firefly luciferase was used as a model protein; the corresponding hybrid precursor contained the preprolactin signal peptide. The rates and efficiencies of folding of luciferase in microsomes were compared with those of folding of luciferase in the cytosol. Furthermore, folding of luciferase in microsomes was compared with that in proteoliposomes, i.e. in the absence of luminal molecular chaperones and folding catalysts. Folding in microsomes was less efficient compared with folding in the cytosol. Folding in the absence of luminal proteins was more efficient compared with folding in their presence and identical to folding in the cytosol. Thus, firefly luciferase emerging from translocase can efficiently fold to its native conformation without chaperoning by any luminal proteins. There may be molecular chaperones present in the microsomal membrane that can efficiently substitute for the cytosolic chaperone machinery comprising Hsp40, Hsp60, and Hsp70 with respect to folding of firefly luciferase.