RESUMEN
A cDNA encoding mosquito Armigeres subalbatus prophenol oxidase (As-pro-PO) was obtained by rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR) after Dirofilaria immitis inoculation. The 2205 bp As-pro-PO cDNA contains a 32 bp 5'-noncoding region, a 2055 bp open reading frame (685 amino acids), and a 118 bp 3'-noncoding region. Hydrophobic signal peptide for the endoplasmic reticulum targeting is not found in the NH2-terminal region. Two potential copper-binding domains, amino acids 197-245 and 345-412, are highly homologous to those of the other insect pro-POs. A 2.2 kb As-pro-PO transcript was identified by Northern blot analysis using D. immitis microfilariae-inoculated A. subalbatus. Both in situ hybridization and Northern blot analysis demonstrated that As-pro-PO mRNA was synthesized in mosquito haemocytes but not in other tissues, i.e. fat bodies, midguts and ovaries, etc.
Asunto(s)
Catecol Oxidasa/genética , Culicidae/enzimología , Dirofilaria immitis/fisiología , Precursores Enzimáticos/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Clonación Molecular , Culicidae/genética , Culicidae/parasitología , ADN Complementario , Perros , Femenino , Hibridación in Situ , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
We report the cloning of a gene, S2P, that encodes a putative metalloprotease required for intramembrane proteolysis of sterol-regulatory element-binding proteins (SREBPs) at Site-2. SREBPs are membrane-bound transcription factors that activate genes regulating cholesterol metabolism. The active NH2-terminal domains of SREBPs are released from membranes by sequential cleavage at two sites: Site-1, within the lumen of the endoplasmic reticulum; and Site-2, within a transmembrane segment. The human S2P gene was cloned by complementation of mutant CHO cells that cannot cleave SREBPs at Site-2 and are cholesterol auxotrophs. S2P defines a new family of polytopic membrane proteins that contain an HEXXH sequence characteristic of zinc metalloproteases. Mutation of the putative zinc-binding residues abolishes S2P activity. S2P encodes an unusual metalloprotease that cleaves proteins within transmembrane segments.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT , Proteínas de Unión al ADN/metabolismo , Endopeptidasas/genética , Endopeptidasas/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción , Animales , Células CHO/enzimología , Clonación Molecular , Cricetinae , Análisis Mutacional de ADN , ADN Complementario , Regulación Enzimológica de la Expresión Génica , Genes Reporteros , Prueba de Complementación Genética , Humanos , Luciferasas , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Metaloendopeptidasas/genética , Metaloendopeptidasas/metabolismo , Datos de Secuencia Molecular , Mutación/fisiología , Homología de Secuencia de Aminoácido , Proteína 1 de Unión a los Elementos Reguladores de Esteroles , TransfecciónRESUMEN
We have previously reported the preparation and characterization of unilamellar phosphatidylcholine vesicles from cholate-phospholipid micelles treated with the bile-salt sequestrant cholestyramine (Ventimiglia, J.B., Levesque, M.C., and Chang, T.Y. (1986) Anal. Biochem. 157, 323-330). We now describe a slightly modified procedure for forming unilamellar vesicles consisting of phosphatidylethanolamine, and the characterization of the resultant vesicles by gel exclusion chromatography. In contrast to phosphatidylcholine vesicles, the formation of phosphatidylethanolamine vesicles is highly pH dependent; pH 9.2 is superior to pH 8.1 or pH 7.1. Via the dialysis step, the final pH of the vesicles could be altered to be at 8.1 or at 7.1, although decreasing the pH from 9.2 resulted in the loss of approx. 20% of the total lipid as large aggregates. Residual cholate was still present in the resultant vesicles after cholestyramine treatment; the low levels of cholate, removable by dialysis, was found to stabilize the phosphatidylethanolamine vesicles formed at pH 8.1. These results suggest that the majority of the amino groups of the phosphatidylethanolamine molecules should either be in the deprotonated form, or be neutralized and/or restricted by the anionic cholate monomers in order to facilitate the vesicle formation. Phosphatidylethanolamine vesicles were found to be much more permeable to small ions than phosphatidylcholine vesicles. The incorporation of phosphatidylserine, but not phosphatidylinositol, into the phosphatidylethanolamine vesicles at 10% resulted in decreased permeability of the bilayer against the cobalt ion influx, suggesting cooperative and complementary packing of phosphatidylethanolamine and phosphatidylserine molecules within the bilayer.
Asunto(s)
Resina de Colestiramina , Coloides , Membrana Dobles de Lípidos , Micelas , Fosfatidilcolinas , Fosfatidiletanolaminas , Ácido Cólico , Ácidos Cólicos , Concentración de Iones de Hidrógeno , Permeabilidad , FosfolípidosRESUMEN
An unsaturated fatty acid-requiring mutant derived from Chinese hamster ovary (CHO) cells has been isolated and characterized. This mutant grows normally when oleate or other unsaturated fatty acids are supplemented in the growth medium. Unlike the wild-type CHO cells, growth stops when medium is deprived of unsaturated fatty acid. Whole cell pulse experiments with [14C]acetate or [14C]stearate indicate that the mutant is defective in unsaturated fatty acid synthesis. Enzyme assays in vitro show that the enzymatic defect of the mutant is localized to the microsomal stearoyl-CoA desaturase.