Purification and Characterization of Membrane-Bound Inositol Phospholipid-Specific Phospholipase C from Suspension-Cultured Rice (Oryza sativa L.) Cells (Identification of a Regulatory Factor)
Abstract:A membrane-bound inositol phospholipid-specific phospholipase C was solubilized from rice (Oryza sativa L.) microsomal membranes and purified to apparent homogeneity using a series of chromatographic separations. l h e apparent molecular mass of the enzyme was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 42,000 D, and the isoelectric point was 5
“…With respect to biochemical characterizations of PIP 2 -PLC, it has been reported that Ca 2+ concentration, pH and protein concentration are among the most important affectors Pfaffmann et al, 1987;Pical et al, 1992;Yotsushima et al, 1993). The properties of PLC toward PIP 2 from pea leaves examined in this study (Fig.…”
“…With respect to biochemical characterizations of PIP 2 -PLC, it has been reported that Ca 2+ concentration, pH and protein concentration are among the most important affectors Pfaffmann et al, 1987;Pical et al, 1992;Yotsushima et al, 1993). The properties of PLC toward PIP 2 from pea leaves examined in this study (Fig.…”
“…We report here a kinetic study of the partially purified, membrane-associated enzyme using different approaches. Few reports regarding the biochemical characterization of plant PLC are available (Tate et al, 1989;Melin et al, 1992;Pical et al, 1992;Yotsushima et al, 1992Yotsushima et al, , 1993Hirayama et al, 1995;Huang et al, 1995;Shi et al, 1995;Kopka et al, 1998). To our knowledge, this is the first report in which different assays were used to characterize this enzyme in plants.…”
Section: Discussionmentioning
confidence: 99%
“…PLC is a family of isoenzymes that has been classified into three groups: , ␥, and ␦ (Rhee and Bae, 1997). In plants several studies have reported the biochemical presence of this enzyme (McMurray and Irvine, 1988;Tate et al, 1989;Melin et al, 1992;Pical et al, 1992;Yotsushima et al, 1992Yotsushima et al, , 1993Huang et al, 1995;De Los Santos-Briones et al, 1997). Different genes for PLC have also been cloned (Hirayama et al, 1995(Hirayama et al, , 1997Shi et al, 1995;Yamamoto et al, 1995;Kopka et al, 1998), all of them resembling the ␦ type.…”
The properties of phospholipase C (PLC) partially purified from Catharanthus roseus transformed roots were analyzed using substrate lipids dispersed in phospholipid vesicles, phospholipiddetergent mixed micelles, and phospholipid monolayers spread at an air-water interface. Using [ 33 P]phosphatidylinositol 4,5-bisphosphate (PIP 2 ) of high specific radioactivity, PLC activity was monitored directly by measuring the loss of radioactivity from monolayers as a result of the release of inositol phosphate and its subsequent dissolution on quenching in the subphase. PLC activity was markedly affected by the surface pressure of the monolayer, with reduced activity at extremes of initial pressure. The optimum surface pressure for PIP 2 hydrolysis was 20 mN/m. Depletion of PLC from solution by incubation with sucrose-loaded PIP 2 vesicles followed by ultracentrifugation demonstrated stable attachment of PLC to the vesicles. A mixed micellar system was established to assay PLC activity using deoxycholate. Kinetic analyses were performed to determine whether PLC activity was dependent on both bulk PIP 2 and PIP 2 surface concentrations in the micelles. The interfacial Michaelis constant was calculated to be 0.0518 mol fraction, and the equilibrium dissociation constant of PLC for the lipid was 45.5 M. These findings will add to our understanding of the mechanisms of regulation of plant PLC.
“…As seen in Fig. 4, NPC4 showed very low activity for PIP 2 (Ͻ10% of PC-hydrolyzing activity), suggesting that NPC4 has different substrate selectivity from PI-PLC (32,33). Next, we analyzed the effect of divalent cations on the enzyme activity, since many plant phospholipases are characterized as Ca 2ϩ -dependent enzymes.…”
Section: Npc4 Encoded a Functional Pc-plc That Prefers Pc For The Submentioning
During phosphate starvation, it is known that phospholipids are degraded, and conversely, a nonphosphorus galactolipid digalactosyldiacylglycerol accumulates in the root plasma membrane of plants. We report a novel phospholipase C that hydrolyzes phosphatidylcholine and is greatly induced in response to phosphate deprivation in Arabidopsis. Since phosphatidylcholinehydrolyzing activity by phospholipase C was highly upregulated in phosphate-deprived plants, gene expression of some phospholipase C was expected to be induced during phosphate starvation. Based on amino acid sequence similarity to a bacterial phosphatidylcholine-hydrolyzing phospholipase C, six putative phospholipase Cs were identified in the Arabidopsis genome, one of which, NPC4, showed significant transcriptional activation upon phosphate limitation. Molecular cloning and functional expression of NPC4 confirmed that the NPC4 gene encoded a functional phosphatidylcholinehydrolyzing phospholipase C that did not require Ca 2؉ for its activity. Subcellular localization analysis showed that NPC4 protein was highly enriched in the plasma membrane. Analyses of transferred DNA-tagged npc4 mutants revealed that disruption of NPC4 severely reduces the phosphatidylcholine-hydrolyzing phospholipase C activity in response to phosphate starvation. These results suggest that NPC4 plays an important role in the supply of both inorganic phosphate and diacylglycerol from membrane-localized phospholipids that would be used for phosphate supplementation and the replacement of polar lipids in the root plasma membrane during phosphate deprivation.Phosphorus is an essential element for plant growth, development, and reproduction. It plays decisive roles not only in regulation of various enzymes but also in constitutive components such as membrane phospholipids and nucleic acids. In most soils, despite its abundance, phosphorus is not freely available for assimilation by roots (1). Therefore, plants have developed distinct systems to cope with phosphate deficiency.When plants suffer from phosphate limitation, highly integrated systems are activated both for assimilation of P i and supplementation of P i from innermost phosphorus storage. The former action is represented by a morphological modification of root architecture upon P i starvation, which presumably facilitates P i uptake by enlargement of absorptive root surface areas (2). On the other hand, the latter has been described by the dynamic evolution of metabolism that is altered toward the supply of free P i . During P i starvation, overall phospholipid content that corresponds to 30% of total P i storage is decreased, and conversely, contents of nonphosphorus galactolipid, digalactosyldiacylglycerol (DGDG), 1 increase significantly (3). Galactolipids such as monogalactosyldiacylglycerol (MGDG) and DGDG are ubiquitous in plants, but are typically found only in plastids, especially in photosynthetic membranes (4). These galactolipids are synthesized by the galactosylation of diacylglycerol (DAG) by MGDG synthase an...
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