RESUMEN
The branched respiratory chain in mitochondria from the halotolerant yeast Debaryomyces hansenii contains the classical complexes I, II, III and IV plus a cyanide-insensitive, AMP-activated, alternative-oxidase (AOX). Two additional alternative oxidoreductases were found in this organism: an alternative NADH dehydrogenase (NDH2e) and a mitochondrial isoform of glycerol-phosphate dehydrogenase (MitGPDH). These monomeric enzymes lack proton pump activity. They are located on the outer face of the inner mitochondrial membrane. NDH2e oxidizes exogenous NADH in a rotenone-insensitive, flavone-sensitive, process. AOX seems to be constitutive; nonetheless, most electrons are transferred to the cytochromic pathway. Respiratory supercomplexes containing complexes I, III and IV in different stoichiometries were detected. Dimeric complex V was also detected. In-gel activity of NADH dehydrogenase, mass spectrometry, and cytochrome c oxidase and ATPase activities led to determine the composition of the putative supercomplexes. Molecular weights were estimated by comparison with those from the yeast Y. lipolytica and they were IV2, I-IV, III2-IV4, V2, I-III2, I-III2-IV, I-III2-IV2, I-III2-IV3 and I-III2-IV4. Binding of the alternative enzymes to supercomplexes was not detected. This is the first report on the structure and organization of the mitochondrial respiratory chain from D. hansenii.
Asunto(s)
Complejo I de Transporte de Electrón/química , Transporte de Electrón , Glicerolfosfato Deshidrogenasa/química , NADH Deshidrogenasa/química , Oxidorreductasas/química , Secuencia de Aminoácidos , Respiración de la Célula/fisiología , Debaryomyces/enzimología , Complejo I de Transporte de Electrón/metabolismo , Glicerolfosfato Deshidrogenasa/fisiología , Mitocondrias/enzimología , Mitocondrias/metabolismo , Membranas Mitocondriales/química , Membranas Mitocondriales/enzimología , Proteínas Mitocondriales/química , Proteínas Mitocondriales/metabolismo , NADH Deshidrogenasa/fisiología , Oxidación-Reducción , Oxidorreductasas/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismoRESUMEN
Las proteínas y los lípidos juegan un rol importante en la estructura y función de las membranas celulares y subcelulares. Diversos investigadores han estudiado acerca de los lípidos que componen la membrana mitocondrial de muchos organismos y han encontrado principalmente fosfolípidos (cardiolipina, fosfatidiletanolamina, fosfatidilserina) y colesterol. La composición lipídica de la membrana mitocondrial interna (mmi) de ningún helminto se ha determinado hasta ahora. Ascaris suum, parásito helminto que habita el intestino delgado del cerdo y metaboliza los carbohidratos hasta ácidos grasos de cadena corta en condiciones anaeróbicas, ha sido utilizado como modelo en estudios de bioquímica comparativa. En este estudio, se aisló mediante centrifugación diferencial en sacarosa, la fracción mitocondrial del músculo de Ascaris y luego se separó la mmi por centrifugación en presencia de fosfato de potasio 10mM. La fracción de lípidos de la mmi fue extraída por el método de Folch y aplicada en cromatoplacas para Cromatografía en Capa Fina (CCF). Mediante ensayos enzimáticos para la determinación indirecta de colesterol y triacilglicéridos, y con CCF se encontró que colesterol, fosfolípidos y otro lípido no identificado, están presentes en la membrana interna de la mitocondria del parásito. La fracción de fosfolípidos separada por CCF fue extraída de la sílica gel con cloroformo: metanol: H2O (30:50:20) y analizada en Cromatografía de Gases (CG) para determinar su composición en ácidos grasos. Los ácidos oleico, palmítico, linoleico y esteárico se encontraron en: 8,38; 7,98; 6,39 y 6,28%, respectivamente. Otros ácidos como el linolénico y mirístico, estaban en menor proporción (1,65 y 0,50%, respectivamente), similar a lo reportado en otras investigaciones referidas a la composición lipídica mitocondrial de otros organismos, lo que sugiere un papel importante de estos ácidos grasos para la función mitocondrial.
The lipids and proteins play an important role in cellular and subcellular membranes structure and function. Several researchers have studied mitochondrial membrane lipids from many organisms, observing mainly phospholipids (cardiolipin, phosphatidilethanolamine, phosphatidilserine) and cholesterol. The lipidic composition of inner mitochondrial membrane (mmi) of none helminthes has been determinate. Ascaris suum, one helminthe parasite that inhabits the small intestine of pig and metabolizes carbohydrates to short chain volatile fatty acids of in aerobic conditions, has been used as a model in comparative biochemistry studies. In this study, the mitochondrial fraction from Ascaris muscle was isolated by means of differential centrifugation in sucrose and the mmi was separated by centrifugation in 10mM Potassium Phosphate. The mmi lipidic fraction was extracted by Folch method and applied on plate for Thin Layer Chromatography (TLC). By mean of indirect enzymatic assays for cholesterol and triglycerides determination, and with TLC, cholesterol, phospholipid and other unknown lipid were found in parasite inner membrane. The fraction of phospholipids separated by TLC was extracted from silica gel with chlorophorm: methanol: H2O (30:50:20) and analyzed by Gas Chromatography (GC) to determine fatty acids composition. Oleic, palmitic, linoleic and stearic acid were present in 8.38; 7.98; 6.39 and 6.28%, respectively. Others acids as linolenic and miristic were found in minor proportion (1.65 and 0.50%), in concordance with reports of researches about mitochondrial lipidic composition in other organisms that suggests an important role of these fatty acids in mitochondrial function.