Stimulation of phospholipase D in rabbit platelet membranes by nucleoside triphosphates and by phosphocreatine: roles of membrane-bound GDP, nucleoside diphosphate kinase and creatine kinase

Abstract

Previous work has shown that guanosine 5'-[gamma-thio]triphosphate (GTP[S]) and GTP stimulate phospholipase D (PLD) in rabbit platelet membranes and that these effects are greatly enhanced by pretreatment of platelets with phorbol esters that activate protein kinase C [Van der Meulen and Haslam (1990), Biochem. J. 271, 693-700]. In the present study, the effects of Mg2+, various nucleoside triphosphates and phosphocreatine (PCr) were investigated. Platelet membranes containing phospholipids labelled with [3H]glycerol were assayed for PLD in the presence of an optimal Mg2+ concentration (10 mM) by measuring [3H]phosphatidylethanol formation in incubations that included 300 mM ethanol. In membranes from phorbolester-treated platelets, the same maximal increases in PLD activity (5-fold) were seen with 1 microM GTP[S]), and 100 microM GTP. Addition of adenosine 5'-[gamma-thio]triphosphate (ATP[S]), ITP, XTP, UTP and CTP had similar stimulatory effects, but only at > or = 1 mM. In contrast, ATP had a biphasic action, causing a maximal (2-fold) stimulation at 10 microM and smaller effects at higher concentrations; the inhibitory component of the action of ATP was blocked by 2 microM staurosporine. Guanosine 5'-[beta-thio]diphosphate decreased the stimulatory effects of ATP and ATP[S]. UDP, which can inhibit nucleoside diphosphate kinase (NDPK), decreased the activation of PLD by ATP[S], ATP, XTP, CTP and to a lesser extent ITP, but had no effect on the actions of GTP[S] and GTP. Rabbit platelet membranes contained NDPK and addition of [gamma-32P]ATP led to the formation of [32P]GTP in amounts sufficient to explain most or all of the activation of PLD; UDP prevented GTP formation. PCr (0.04-1 mM) also stimulated membrane PLD activity, an effect that was dependent on endogenous membrane-bound creatine kinase (CK). UDP and guanosine 5'-[beta-thio]diphosphate each inhibited this effect of PCr. The results show that in rabbit platelet membranes, CK, NDPK and the GTP-binding protein that activates PLD can be functionally coupled. However, assay of membrane preparations at increasing dilutions showed that stimulation of PLD by the compounds studied, with the partial exception of ATP[S], involved diffusible rather than protein-bound intermediates.

Association of nucleoside diphosphate kinase with pancreatic zymogen granules: effects of local GTP generation on granule membrane characteristics

“…Since the mechanism of action of NDP kinase involves transphosphorylation of nucleoside diphosphates, it has been proposed that competitive inhibition can be produced by the addition of UDP at high concentrations [27]. As shown in Figure 4, UDP decreased phosphorylation of p21 in a concentration-dependent manner, a result which supports the suggestion that p21 is NDP kinase.…”

Phosphocreatine-dependent Glutamate Uptake by Synaptic Vesicles

“…Creatine Kinase Activity Is Not Responsible for PCr-dependent Glutamate Uptake Activity-PCr can be considered a shortterm energy buffer. In the reaction catalyzed by creatine kinase, one molecule of PCr and one molecule of ADP are converted to one molecule of creatine and one molecule of ATP (23). Therefore, it is important to determine if the stimulatory effect of PCr on glutamate uptake activity is simply due to ATP formation, the additional ATP being the actual species responsible for the increased uptake activity.…”

“…Assay of Vesicular [ 3 H]Glutamate Uptake-The ATP-dependent uptake of glutamate into synaptic vesicles was assayed according to a published procedure (14). The standard incubation mixture contained 50 g of protein from synaptic vesicles, 5 mM Tris maleate (pH 7.4), 4 mM MgSO 4 , 0.25 M sucrose, 4 mM KCl, either 2 mM Tris-ATP neutralized with Tris base or 30 mM PCr, either 5 or 10 Ci of [ 3 H]glutamate (specific activity, 46 Ci/mmol) and either 50 M or 1 mM glutamate as indicated in the figure legends in a final volume of 100 l. Although the K m for glutamate uptake is near 1 mM, most previous studies of ATPdependent vesicular glutamate uptake were performed using 50 M glutamate with high specific activity to increase the signal to noise ratio (2,5,11,23). In order to compare our results with these studies, we use both 50 M and 1 mM glutamate in the assay system.…”