RESUMEN
Two types of binding sites for hexokinase, designated as Type A or Type B sites, have been shown to coexist on brain mitochondria. The ratio of these sites varies between species. HK1 attaches by reversibly binding to the voltage dependent anion channel (VDAC). Regarding the nature of hexokinase binding sites, we investigated if it was linked to distinct VDAC interactomes. We approached this question by 2D BN/SDS-PAGE of mitochondria, followed by mass spectrometry. Our results are consistent with the possibility that the ratio of Type A/Type B sites is due to differential VDAC interactions in bovine and rat neuronal cells.
Asunto(s)
Encéfalo/metabolismo , Electroforesis en Gel de Poliacrilamida/métodos , Hexoquinasa/metabolismo , Espectrometría de Masas/métodos , Canales Aniónicos Dependientes del Voltaje/metabolismo , Animales , Sitios de Unión , Western Blotting , Encéfalo/enzimología , Bovinos , RatasRESUMEN
Voltage-dependent anion channels (VDAC) are pore-forming proteins found in the outer mitochondrial membrane of eukaryotes. VDACs are known to play an essential role in cellular metabolism and in early stages of apoptosis. In mammals, three VDAC isoforms have been identified. A proteomic approach was exploited to study the expression of VDAC isoforms in rat, bovine, and chicken brain mitochondria. Given the importance of mitochondrially bound hexokinase in regulation of aerobic glycolysis in brain, we studied the possibility that differences in the relative expression of VDAC isoforms may be a factor in determining the species-dependent ratio of type A/type B hexokinase binding sites on brain mitochondria. The spots were characterized, and the signal intensities among spots were compared. VDAC1 was the most abundantly expressed of the three isoforms. Moreover the expression of VDAC1 plus VDAC2 was significantly higher in bovine than in rat brain. Chicken brain mitochondria showed the highest VDAC1 expression and the lowest of VDAC2. Bovine brain mitochondria had the highest VDAC2 levels. We concluded that the nature of hexokinase binding site is not determined by the expression of a single VDAC isoform.
Asunto(s)
Encéfalo , Hexoquinasa/metabolismo , Mitocondrias/metabolismo , Isoformas de Proteínas/metabolismo , Canal Aniónico 1 Dependiente del Voltaje/metabolismo , Canal Aniónico 2 Dependiente del Voltaje/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Encéfalo/citología , Encéfalo/metabolismo , Bovinos , Pollos , Electroforesis en Gel Bidimensional , Espectrometría de Masas , Proteínas Mitocondriales/metabolismo , Datos de Secuencia Molecular , Isoformas de Proteínas/genética , Ratas , Canal Aniónico 1 Dependiente del Voltaje/genética , Canal Aniónico 2 Dependiente del Voltaje/genéticaRESUMEN
One-day-old chicks were reared using diets differing in their vitamin E and/or selenium content. The purpose of this research was to detect any possible imbalance in the antioxidant defense system, which could be related to development of nutritional pancreatic atrophy. Mitochondrial membranes from animals deficient in both nutrients, or just vitamin E, submitted to peroxidizability 'in vitro' had the production of TBARS greatly enhanced. Measurements of the 2-GSH/GSSG ratio suggested that selenium and vitamin E, the latter in higher magnitude, were responsible for maintenance of the reducing capacity of the cell. Enzymatic defense systems against oxidative stress were also studied. The results indicated that the total antioxidant enzymatic activity of pancreatic cells was not sufficient to scavenge all the ROS generated in the nutritionally deficient animals. The present study suggests that nutritional deficiency of selenium and/or vitamin E generates one imbalance between pro-oxidant and antioxidant systems in chicken pancreas, leading to oxidative stress and pancreatic atrophy.
Asunto(s)
Antioxidantes/metabolismo , Páncreas/patología , Fenómenos Fisiológicos Nutricionales de los Animales , Animales , Atrofia , Catalasa/metabolismo , Pollos , Glutatión/metabolismo , Peroxidación de Lípido , Masculino , Mitocondrias/metabolismo , Estrés Oxidativo , Oxígeno/metabolismo , Selenio/farmacología , Sustancias Reactivas al Ácido Tiobarbitúrico/farmacología , Factores de Tiempo , Vitamina E/farmacologíaRESUMEN
Hexokinase is released from Type A sites of brain mitochondria in the presence of glucose 6-phosphate (Glc-6-P); enzyme bound to Type B sites remains bound. Hexokinase of freshly isolated bovine brain mitochondria (Type A:Type B, approximately 40:60) selectively uses intramitochondrial ATP as substrate and is relatively insensitive to the competitive (vs ATP) inhibitor and Glc-6-P analog, 1,5-anhydroglucitol 6-phosphate (1,5-AnG-6-P). After removal of hexokinase bound at Type A sites, the remaining enzyme, bound at Type B sites, does not show selectivity for intramitochondrial ATP and has increased sensitivity to 1,5-AnG-6-P. Thus, the properties of the enzyme bound at Type B sites are modified by removal of hexokinase bound at Type A sites. It is suggested that mechanisms for regulation of mitochondrial hexokinase activity, and thereby cerebral glycolytic metabolism, may depend on the ratio of Type A:Type B sites, which varies in different species.