Diacylglycerol kinase activity in purified basolateral membranes of kidney tubules. I. Evidence for coupling with phospholipase C.
The diacylglycerol kinase (DGK) catalyzes the phosphorylation of diacylglycerol (DAG) yielding phosphatidic acid (PA) signaling molecules which are involved in the modulation of different cell responses. The aim of this work was to characterize the DGK activity associated to the basolateral membranes (BLM) of kidney proximal tubules, in a native preparation that preserves the membrane microenvironment. The Arrhenius plot of DGK activity was non-linear, indicating a complex influence of the lipid environment of the native membrane. The formation of PA was strongly impaired by U73122, an inhibitor of PLC, whereas remained unmodified when exogenous DAG or PLC were added. The Mg.ATP2- complex is the true phosphoryl-donor substrate, and the very narrow peak of activation at pH 7.0 suggests that amino acids that dissociate at this pH, i.e. hystidine residues, play a role by acting in the coordination of the Mg2+ atoms. The renal DGK is almost completely blocked by 0.1 mM sphingosine, but it is insensitive to micromolar free Ca2+ concentrations and to R59499, the most potent inhibitor of the classical DGKs. Taken as a whole, these data suggest that the DGK isoform present in BLM of proximal tubules is different from those included in the type I family, and that membranous PLC could be the main source of DAG for DGK catalysis.
Nogaroli
Luciana
L
Laboratório de Fisico-Química Biológica Aída Hassón-Voloch, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Ilha do Fundão, 21949-900, Rio de Janerio, Brazil.
Silva
Osman F
OF
Bonilha
Thaís A
TA
Moreno
Pilar A M
PA
Bernardo
Róbson R
RR
Vieyra
Adalberto
A
Einicker-Lamas
Marcelo
M
D000255:Adenosine Triphosphate / Q000378:metabolism; D001485:Basement Membrane / Q000201:enzymology; D002384:Catalysis / Q000187:drug effects; D002462:Cell Membrane / Q000201:enzymology*; D019852:Diacylglycerol Kinase / Q000737:chemistry* / Q000378:metabolism*; D004075:Diglycerides / Q000378:metabolism; D004963:Estrenes / Q000494:pharmacology; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D007687:Kidney Tubules, Proximal / Q000201:enzymology*; D008274:Magnesium / Q000378:metabolism; D010712:Phosphatidic Acids / Q000378:metabolism; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D011760:Pyrrolidinones / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D010738:Type C Phospholipases / Q000378:metabolism*
D004075:Diglycerides; D004963:Estrenes; D007527:Isoenzymes; D010712:Phosphatidic Acids; D010726:Phosphodiesterase Inhibitors; D011760:Pyrrolidinones; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D000255:Adenosine Triphosphate; D019852:Diacylglycerol Kinase; D010738:Type C Phospholipases; D008274:Magnesium