Elektron transzfer rendszerek élettani szerepe = The physiological role of electron transfer systems

Fagocitákban leírtuk a NADPH oxidázt szabályozó két különböző GTPáz aktiváló fehérje szabályozását és a kísérő K+ transzport baktérium ölő hatását. Agyi mitokondriumokban (mito) a légzési lánc I. komplexének szubsztrátjai membránpotenciál (Em) függően reaktív oxigénszármazékot (ROS) képeznek. Az alfa-glicerofoszfát (aGP) ROS-t képez az I. komplexen és az aGP-dehidrogenáz enzimen, utóbbit a Ca2+ aktivája. Idegvégződésekben a mito ROS képzését az Em nem befolyásolja. A mito-k elektromos szincíciumot képeznek, de a Ca2+ diffúziója korlátozott. Alacsony O2.- szint a Ca2+ -mobilizáló agonista Ca2+ jel képző hatását glomerulóza sejtben gátolja. A ROS támadáspontja a belső raktárból történő Ca2+ felszabadulás. UV hatására a mito Ca2+ felvétele is csökkent. Angiotenzin II -vel ingerelt H295R sejtben a mito Ca2+ jel képzés sebessége a mito és az endoplazmás retikulum (ER) közelségével korrelál. A p38 MAPK és az újtípusú PKC izoformák egyidejű gátlása a Ca2+ jelnek a citoszolból a mito-ba történő áttevődését gátolja és a fenti korrelációt megszünteti. Az ER lumenében a tiol/diszulfid rendszertől elkülönülő NAD(P)+/NAD(P)H rendszer működik. Redox állapotát a glukóz-6-foszfát transzporter és az intraluminális oxidoreduktázok határozzák meg. A redukált állapot fenntartása szükséges a glukokortikoidok prereceptoriális aktiválásához, s egyes sejtekben antiapoptotikus hatású. Jellemeztük az ER szulfát transzporterét, valamint a transzlokon peptid csatorna anion permeabilitását. | We described in phagocytes the regulation of two GTPase activating proteins, terminating the activity of plasmalemmal NADPH oxidase and the role of K+ movements in bacterial killing. In brain mitochondria complex I dependent substrates show a membrane potential (Em) dependent reactive oxygen species (ROS) formation. ROS production by alpha-glycerophosphate (aGP) occured at complex I and on the aGP-dehydrogenase enzyme. The latter is activited by Ca2+. Mitochondria form an electric syntitium but the diffusion of Ca2+ is limited. In glomerulosa cells, at low [O2.-] angiotensin-induced Ca2+ signalling is attenuated, the site of ROS action is Ca2+ release from the internal stores. The rate of mitochondrial Ca2+ uptake in angiotensin-stimulated cells correlates with the vicinity of the mitochondrion and the endoplasmic reticulum (ER). Simultaneous activation of p38 MAPK and the novel isoforms of PKC attenuates the transfer of cytosolic Ca2+ signal into the mitochondria and abolishes this correlation. In the ER we observed a novel NAD(P)+/NAD(P)H system different from the thiol/disulphide system. Its reduced state is tuned by the glucose-6-phosphate transporter and the luminal oxidoreductases and is required for the prereceptorial activation of glucocorticoids. We have characterized the sulphate transport in the ER, and the contribution of the translocon peptide channel to the membrane permeation of small anions

Comparative analysis of involvement of UGT1 and UGT2 splice variants of UDP-galactose transporter in glycosylation of macromolecules in MDCK and CHO cell lines

Nucleotide sugar transporters deliver nucleotide sugars into the Golgi apparatus and endoplasmic reticulum. This study aimed to further characterize mammalian UDP-galactose transporter (UGT) in MDCK and CHO cell lines. MDCK-RCAr and CHO-Lec8 mutant cell lines are defective in UGT transporter, although they exhibit some level of galactosylation. Previously, only single forms of UGT were identified in both cell lines, UGT1 in MDCK cells and UGT2 in CHO cells. We have identified the second UGT splice variants in CHO (UGT1) and MDCK (UGT2) cells. Compared to UGT1, UGT2 is more abundant in nearly all examined mammalian tissues and cell lines, but MDCK cells exhibit different relative distribution of both splice variants. Complementation analysis demonstrated that both UGT splice variants are necessary for N- and O-glycosylation of proteins. Both mutant cell lines produce chondroitin-4-sulfate at only a slightly lower level compared to wild-type cells. This defect is corrected by overexpression of both UGT splice variants. MDCK-RCAr mutant cells do not produce keratan sulfate and this effect is not corrected by either UGT splice variant, overexpressed either singly or in combination. Here we demonstrate that both UGT splice variants are important for glycosylation of proteins. In contrast to MDCK cells, MDCK-RCAr mutant cells may possess an additional defect within the keratan sulfate biosynthesis pathway

The glucose-6-phosphate transporter-hexose-6-phosphate dehydrogenase-11 beta-hydroxysteroid dehydrogenase type 1 system of the adipose tissue

11 beta-Hydroxysteroid dehydrogenase type 1, expressed mainly in the endoplasmic reticulum of adipocytes and hepatocytes, plays an important role in the prereceptorial activation of glucocorticoids. In liver endoplasmic reticulum-derived microsomal vesicles, nicotinamide adenine dinucleotide phosphate reduced supply to the enzyme is guaranteed by a tight functional connection with hexose-6-phosphate dehydrogenase and the glucose-6-phosphate transporter ( G6PT). In adipose tissue, the proteins and their activities supporting the action of 11 beta-hydroxysteroid dehydrogenase type 1 have not been explored yet. Here we report the occurrence of the hexose-6-phosphate dehydrogenase in rat epididymal fat, as detected at the level of mRNA, protein, and activity. In the isolated microsomes, the activity was evident only on the permeabilization of the membrane because of the poor permeability to the cofactor nicotinamide adenine dineucleotide phosphate ( NADP(+)), which is consistent with the intralumenal compartmentation of both the enzyme and a pool of pyridine nucleotides. In fat cells, the access of the substrate, glucose-6-phosphate to the intralumenal hexose-6-phosphate dehydrogenase appeared to be mediated by the liver-type G6PT. In fact, the G6PT expression was revealed at the level of mRNA and protein. Accordingly, the transport of glucose-6-phosphate was demonstrated in microsomal vesicles, and it was inhibited by S3483, a prototypic inhibitor of G6PT. Furthermore, isolated adipocytes produced cortisol on addition of cortisone, and the production was markedly inhibited by S3483. The results show that adipocytes are equipped with a functional G6PT-hexose-6-phosphate dehydrogenase-11 beta-hydroxysteroid dehydrogenase type 1 system and indicate that all three components are potential pharmacological targets for modulating local glucocorticoid activation