Thrombin- and nucleotide-activated phosphatidylinositol 4,5-bisphosphate phospholipase C in human platelet membranes

Thrombin, nucleotides, and chelators elicited a phosphatidylinositol 4,5-bisphosphate (PtdIns-P2) phospholipase C activity that was associated with human platelet membranes. Both alpha- and gamma-thrombin enhanced phospholipase C activity, whereas active site-inhibited alpha-thrombin did not stimulate PtdIns-P2 hydrolysis. PtdIns-P2 phospholipase C was also activated by nucleoside triphosphates, citrate, EDTA, and NaF. Magnesium was an inhibitor of PtdIns-P2 hydrolysis stimulated by nucleotides and chelators. Only PtdIns-P2 was degraded by the phospholipase C activated by alpha-thrombin, nucleotides, and chelators. The soluble fraction phospholipase C activity was also stimulated at low protein concentrations by nucleotides; however, soluble fraction phospholipase C activity cleaved both PtdIns-P2 and phosphatidylinositol 4-phosphate and was inhibited by chelators, suggesting the presence of a different enzyme in this compartment. The pH optimum for the membrane-associated phospholipase C in the presence of alpha-thrombin or nucleotides was 6.0, and the PtdIns-P2 phospholipase C was inhibited by neomycin and high detergent concentrations. Guanine nucleotides did not synergistically activate phospholipase C in the presence of alpha-thrombin. The characteristics of the membrane-associated PtdIns-P2 phospholipase C suggest that this enzyme is involved in platelet activation by the low-affinity alpha- or gamma-thrombin-dependent pathway.     

Stimulation of phosphatidylinositol 4,5-bisphosphate phospholipase C activity by phosphatidic acid

Phosphatidic acid was a potent activator of the phosphatidylinositol 4,5-bisphosphate (PtdIns-P2) phospholipase C activity associated with human platelet membranes. Lysophosphatidic acid was half as active as phosphatidic acid, and shortening the fatty acid chain reduced the effectiveness of the corresponding phosphatidic acid. Compounds lacking either the phosphate group (diacylglycerol or phorbol ester) or the fatty acid (glycerol phosphate) were not activators. When the negative charge was contributed by a carboxyl group (fatty acid or phosphatidylserine), stimulation of phospholipase C was weak but detectable. Structural analogs of phosphatidic acid (lipopolysaccharide, lipid A, and 2,3-diacylglucosamine 1-phosphate) were less effective but also enhanced PtdIns-P2 hydrolysis. Phosphatidic acid potentiated the activation of phospholipase C by alpha-thrombin, chelators, and guanine nucleotides. Phosphatidylinositol 4-phosphate and PtdIns-P2 were also effective activators of PtdIns-P2 degradation. Other phospholipids were without effect. The production of inositol 1,4,5-trisphosphate and diacylglycerol via the activation of phospholipase C provides a rationale for the cellular responses evoked by phosphatidic acid and the ability of this phospholipid to potentiate and initiate hormonal responses.     

Partial purification and properties of a phosphatidylinositol 4,5-bisphosphate hydrolyzing phospholipase C from the soluble fraction of soybean sprouts

Three soluble enzyme fractions (F-I, F-II, and F-III) that hydrolyze phophoinositides were separated from soybean sprouts by using Matrex green gel column chromatography. Among the three phosphatidylinositol (PI)-specific phopholipsase C (PLC) enzymes, only the third fraction (F-III) was able to hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2) as well as phosphatidylinositol (PI) and phosphatidylinositol phosphate (PIP) as substrates. The F-I and F-II fractions only showed enzymatic activities for PI and PIP. The PIP2-hydrolyzing PLC protein, F-III, was partially purified using the chromatographic steps of the Matrex green gel, phenyl Toyopearl, Matrex orange gel, Mono S cation exchange, and superose 6 gel filtration columns. The molecular weight of the F-III protein was estimated to be about 64 kDa on SDS-PAGE. The protein showed immunocross-reactivity with a polyclonal antibody that was prepared against the X and Y motifs of animal PLC enzymes, the conserved catalytic domains. Ca2+ ion critically affected the PIP2-hydrolyzing PLC activity of the F-III protein, representing maximal activity at 10 microM Ca2+ concentration. The PIP2-hydrolyzing PLC activity of the protein was also significantly increased by sodium deoxycholate (SDC) from 0.05 to 0.08%. However, the activity was greatly reduced above the concentration, and no activity was detected at 0.3% SDC. In addition, the protein exhibited maximal PIP2-hydrolyzing PLC activity at pH, in the range of 6.5-7.5.     

Purification of a phospholipase C from rat liver cytosol that acts on phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate

A soluble phospholipase C from rat liver was purified to homogeneity using phosphatidylinositol 4,5-bisphosphate (PIP2) as substrate. After ammonium sulfate fractionation, the purification involved chromatography on phosphocellulose, DEAE-Sepharose CL-6B, hydroxylapatite, Reactive Blue 2 dye-linked agarose, and Mono S cation exchanger. Under the conditions of the assay, the pure enzyme had a specific activity of 407 mumol/mg protein/min. It migrated as a single band with a molecular mass of 87 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The water-soluble product formed during the hydrolysis of PIP2 by the purified enzyme was inositol 1,4,5-trisphosphate. The enzyme shows one-half of maximum velocity at 2 microM Ca2+ with PIP2 as substrate. Between 0 and 100 microM Ca2+, the enzyme shows approximately the same activity with phosphatidylinositol 4-phosphate (PIP) as it does with PIP2, and very low activity with phosphatidylinositol. The enzyme is activated by low concentrations of basic proteins; for example, with PIP2 as substrate, 1 microgram/ml histone activates the enzyme 3.6-fold. The enzyme shows an almost absolute requirement for monovalent salts which can be met by different alkali metal halides. A second, minor peak of PIP2-hydrolyzing phospholipase C activity was resolved during chromatography of the enzyme on hydroxylapatite. The substrate specificity suggests that PIP and PIP2 are normal substrates of this enzyme. Under physiological conditions of activation, the enzyme may therefore generate inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate in amounts determined by the ratio of PIP and PIP2 present in the cellular membranes.     

Prolonged ischemia differently affects phospholipase C acting against phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate in brain subsynaptosomal fraction

The effect of 10 min ischemia on the activity of phospholipase C acting against [3H]inositol-phosphatidylinositol (PI) and [3H]inositol-phosphatidylinositol 4,5-bisphosphate (PIP2) in the brain subsynaptosomal fractions was investigated. In the presence of endogenous CaCl2, specific activity of phospholipase C acting on phosphatidylinositol was as follows: synaptic cytosol (SC) greater than synaptic vesicles (SV) greater than synaptic plasma membrane SPM). Brain ischemia activated phospholipase C acting on PI by about 60% and 40% in SV and SPM, respectively. The enzyme of synaptic cytosol was not affected by ischemic insult. Phospholipase C acting against PIP2 in the presence of endogenous calcium expressed the specific activity in the following order: SV greater than SPM greater than SC. After 10 min of brain ischemia, activity of phospholipase C acting on PIP2 was significantly suppressed in all subsynaptosomal fractions by about 50-60%. These results indicate that prolonged ischemia produced activation exclusively of phospholipase C acting against phosphatidylinositol.     

Rapid and transient thrombin stimulation of phosphatidylinositol 4,5-bisphosphate synthesis but not of phosphatidylinositol 3,4-bisphosphate independent of phospholipase C activation in platelets

When platelets are stimulated by thrombin they immediately undergo inositol lipid hydrolysis via phospholipase C activation. However, subsequently an increased production of phosphatidylinositol 4,5-bisphosphate is observed. Phospholipases C were inhibited by lowering the cytoplasmic free calcium concentration by preincubation with Quin-2-tetra(acetoxymethyl) ester. Aggregation and secretion were also totally suppressed. Under these conditions we observed an increased labeling of phosphatidylinositol 4,5-bisphosphate, indicating a stimulation of inositol lipid kinases, independent of lipid hydrolysis by phospholipase C. Conversely the production of phosphatidylinositol 3,4-bisphosphate was totally abolished. These results suggest a different regulation of the kinases/phosphatases responsible for the production of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4-bisphosphate.     

Phorbol ester and the actions of phosphatidylinositol 4,5-bisphosphate specific phospholipase C and protein kinase C in microsomes prepared from cultured cardiomyocytes

Microsomes were prepared from cultured neonatal rat cardiomyocytes. Incubation of microsomes in buffer containing 5µM CaCl₂, 5 mM cholate and 100 nM [3H-]Phosphatidylinosito14,5-bisphosphate (PtdIns(4,5) P₂) resulted in the formation of [³H-]InsP ₃. GTP-gamma-S (125 µM) stimulated the production of [³H-]InsP ₃. Microsomes prepared from phorbol ester-treated (100 nM phorbol 12-myristate 13-acetate, PMA) cardiomyocytes showed decreased activities of basal as well as GTP-gamma-S-stimulated [³H-]Ptdlns(4,5)P ₂ hydrolysis. In the microsomes a 15 kD protein was demonstrated to be the major substrate phosphorylated by intrinsic protein kinase C, which was activated by 0.5 mM Ca²⁺. Addition of phorbol ester (100 nM PMA) enhanced the ³²P-incorporation into the 15 kD protein. Protein kinase C, purified from rat brain, in the presence of Ca²⁺, diglyceride, and phosphatidylserine did not change the phosphorylation pattern any further. In conclusion, it was shown that phorbol ester pretreatment of neonatal rat cardiomyocytes reduces microsomel GTP-gamma-S-stimulated Ptdlns(4,5)P ₂-specific phospholipase C activity, as estimated with exogenous substrate, and that in cardiomyocyte microsomes phorbol ester activates protein kinase C-induced 15 kD protein phosphorylation. The results indicate that phorbol ester may down-regulate -adrenoceptor mediated Ptdlns(4,5)P ₂ hydrolysis by activation of protein kinase C-induced 15 kD protein phosphorylation.List of abbreviations ATP Adenosine 5-Trphosphate - CSU Catalytic Subunit of cyclic AMP-dependent protein kinase - DG Diacylglycerol - DMSO Dimethylsulfoxide - DTT DL-dithiothreitol - EDTA Ethylenedinitrilotetraacetic Acid - EGTA Ethyleneglycol-0,0-bis(aminoethyl)-N,N,N,N,-tetraacetic acid - GTP-gamma-S Guanosine 5-O-(3-thiotriphosphate) - HPTLC High Performance Thin Layer Chromatography - InsP ₃ Inositol monophosphate - InsP ₂ Inositol bisphosphate - InsP ₃ Inositol trisphosphate - MES 2-Morpholinoethanesulfonic acid - MOPS 3-[N-Morpholino]Propanesulfonic acid - PAGE Polyacrylamide-gel Electrophoresis - PKC Protein Kinase C - PLase C Phospholipase C - PMA Phorbol 12-Myristate 13-Acetate - PMSF Phenylmethylsulfonyl Fluoride - PtdSer Phosphatidylserine - PtdIns Phosphatidyl inositol - PT Pertussis Toxin - Ptdlns(4)P Phosphatidylinositol 4-monophosphate - Ptdlns (4,5)PZ-Phosphatidylinositol4,5-bisphosphate - SDS-Sodium Dodecyl Sulfate Tris-Tris(hydroxymethyl) aminomethane     

Effects of phosphatidylinositol 4,5-bisphosphate and neomycin on phospholipase D: Kinetic studies

The kinetics of phosphatidylcholine-specific phospholipase D activated by phosphatidylinositol 4,5-bisphosphate (PIP₂) and inhibition by neomycin were studied in an enzyme preparation partially purified from human hepatocarcinoma cell line. It was found that phospholipase D was marginally activated by phosphatidyl-4-phosphate (PIP) and phosphatidylethanolamine (PE). In contrast, it was considerably activated by PIP₂ in different concentration of phosphatidylcholine (PC). Sphingomyelin (SM), lysophosphatidylcholine (LPC) and phosphatidylserine (PS) were neither substrates nor inhibitors of the phospholipase D. PIP₂ induced an allosteric effect on phospholipase D and a negative cooperative effect with respect to phosphatidylcholine as indicated in the Lineweaver-Burk plot. In the absence of PIP₂, a straight line was obtained, whereas a downward concave curve was observed in the presence of 25 M of PIP₂. The Hill coefficient and the apparent K_m of phosphatidylcholine in the presence of 25 M PIP₂ were calculated to be 0.631 and 10.79 mM, respectively. PIP₂ also increased the maximal velocity (V_max) of the phospholipase D reaction, suggesting that the affinity of substrate to enzyme was decreased, and the turnover number of the enzyme (kcat) was increased by PIP₂. The activation of phospholipase D by PIP₂ was dose dependent up to 50 M of PIP₂. The Ka of PIP₂ was 15.8 mM. Neomycin, a polycationic glycoside, was shown to be an uncompetitive inhibitor of phospholipase D, and revealed the formation of a neomycin-PIP₂ complex. The Ki of neomycin was estimated to be 8.7 mM.     

Subcellular localization and some properties of the enzymes hydrolysing inositol polyphosphates in rat liver

The hydrolysis of inositol [³²P]trisphosphate (IP₃) and inositol [³²P]bisphosphate (IP₂) has been examined in subcellular fractions of rat liver. IP₃ was degraded by an enzyme located in the plasma membrane which did not degrade IP₂. Cytosolic fractions were found to degrade both IP₃ and IP₃. The IP₃ phosphatase activity of liver plasma membranes displayed a neutral pH optimum, was Mg²⁺ dependent and was not inhibited by high concentrations of Li⁺. Half-maximal activity of the enzymes hydrolysing IP₃ and IP₂ was obtained with substrate concentrations in the range 1–2μM. The significance of these observations to the proposed Ca²⁺ -mobilizing role of IP₃ is discussed.     

In vitro synthesis of 32P-labelled phosphatidylinositol 4,5-bisphosphate and its hydrolysis by smooth muscle membrane-bound phospholipase C

For studies of phospholipase C (PLC) activity in cell-free systems, 32P-labelled phosphatidylinositol 4,5-bisphosphate (PIP2) was prepared enzymatically by phosphorylating phosphatidylinositol 4-phosphate (PIP) in the presence of [gamma-32P]ATP using a PIP kinase partially purified from bovine retinae. PLC activity was determined by incubating membranes of DDT1 MF-2 cells with 32P-PIP2 and measuring remaining non-hydrolyzed substrate as well as accumulation of the hydrolysis product, inositol trisphosphate (IP3). Guanine nucleotides stimulated PIP2 hydrolysis and IP3 release. Additional increase in IP3 accumulation was observed with adrenaline plus guanine nucleotides.     

Proteome profiling of peripheral mononuclear cells from human blood

Peripheral blood mononuclear cells (MNCs) are accessible through blood collection and represent a useful source for investigations on disease mechanisms and treatment response. Aiming to build a reference proteome database, we generated three proteome data sets from MNCs using a combination of SDS‐PAGE and nanoflow LC‐MS. Experiments were performed in triplicates and 514 unique proteins were identified by at least two non‐redundant peptides with 95% confidence for all replicates. Identified proteins are associated with a range of dermatologic, inflammatory and neurological conditions as well as molecular processes, such as free radical scavenging and cellular growth and proliferation. Mapping the MNC proteome provides a valuable resource for studies on disease pathogenesis and the identification of therapeutic targets.     

Interaction of cerebral-cortical membranes with exogenously added phosphatidylinositol 4,5-bisphosphate. Effects on measured phospholipase C activity.

Exogenously added phosphatidylinositol 4,5-bisphosphate (PtdInsP2) is rapidly associated with cerebral-cortical membranes. Substrate association with membranes was promoted by Mg2+, but inhibited by bivalent chelators. Once associated with the membrane, the PtdInsP2 was resistant to displacement by EDTA. The apparent phospholipase C activity was dependent on the degree of association of substrate with membranes. After preincubation of membranes with substrate, PtdInsP2 hydrolysis was independent of the incubation volume, indicating that substrate and membrane-associated phospholipase C were not independently diluted. Hydrolysis of the membrane-associated substrate was stimulated by Ca2+, guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG), guanosine 5'[gamma-thio]triphosphate and carbachol in the presence of p[NH]ppG. Carbachol in the absence of guanine nucleotides, GDP, GTP, ATP and pyrophosphate was ineffective. These results demonstrate that exogenously added PtdInsP2 substrate is rapidly associated with membranes and hydrolysed by a phospholipase C whose activity is regulated by guanine nucleotides and agonist in the presence of guanine nucleotides. Use of exogenously added substrate for studies on the regulation of membrane phospholipase C requires consideration as to possible effects of incubation conditions on the partitioning of substrate into membranes.     

Plasma membrane associated phospholipase C from human platelets: synergistic stimulation of phosphatidylinositol 4,5-bisphosphate hydrolysis by thrombin and guanosine 5'-O-(3-thiotriphosphate)

The effects of thrombin and GTP gamma S on the hydrolysis of phosphoinositides by membrane-associated phospholipase C (PLC) from human platelets were examined with endogenous [3H]inositol-labeled membranes or with lipid vesicles containing either [3H]phosphatidylinositol or [3H]phosphatidylinositol 4,5-bisphosphate. GTP gamma S (1 microM) or thrombin (1 unit/mL) did not stimulate release of inositol trisphosphate (IP3), inositol bisphosphate (IP2), or inositol phosphate (IP) from [3H]inositol-labeled membranes. IP2 and IP3, but not IP, from [3H]inositol-labeled membranes were, however, stimulated 3-fold by GTP gamma S (1 microM) plus thrombin (1 unit/mL). A higher concentration of GTP gamma S (100 microM) alone also stimulated IP2 and IP3, but not IP, release. In the presence of 1 mM calcium, release of IP2 and IP3 was increased 6-fold over basal levels; however, formation of IP was not observed. At submicromolar calcium concentration, hydrolysis of exogenous phosphatidylinositol 4,5-bisphosphate (PIP2) by platelet membrane associated PLC was also markedly enhanced by GTP gamma S (100 microM) or GTP gamma S (1 microM) plus thrombin (1 unit/mL). Under identical conditions, exogenous phosphatidylinositol (PI) was not hydrolyzed. The same substrate specificity was observed when the membrane-associated PLC was activated with 1 mM calcium. Thrombin-induced hydrolysis of PIP2 was inhibited by treatment of the membranes with pertussis toxin or pretreatment of intact platelets with 12-O-tetradecanoyl-13-acetate (TPA) prior to preparation of membranes. Pertussis toxin did not inhibit GTP gamma S (100 microM) or calcium (1 mM) dependent PIP2 breakdown, while TPA inhibited GTP gamma S-dependent but not calcium-dependent phospholipase C activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

The efflux of biotin from human peripheral blood mononuclear cells

Peripheral blood mononuclear cells (PMBCs) are readily available for sampling and are a useful model for studying biotin metabolism in human cells. To better understand biotin handling by PMBCs, we investigated the mechanism(s) and kinetics of biotin efflux from PMBCs. Human PMBCs were incubated with [(3)H]biotin at 475 pmol/L to load the cells. The [(3)H]biotin-loaded cells were then harvested and incubated in [(3)H]biotin-free media for up to 20 hours. At various intervals, aliquots of the PMBC suspensions were collected and analyzed for intracellular [(3)H]biotin. [(3)H]Biotin efflux from cells at 37 degrees C was fast and triphasic; the half-lives for the three elimination phases were 0.2 +/- 0.02 hours, 1.2 +/- 0.1 hours, and 21.9 +/- 13.6 hours. Such a triphasic [(3)H]biotin efflux could reflect (1) rapid efflux of free biotin, (2) slower release of biotin bound to intracellular molecules, and (3) even slower release from carboxylases in cellular organelles. Incubation at 4 degrees C rather than 37 degrees C increased the [(3)H]biotin retained at 20 hours from 27% to 85%. This observation is consistent with transporter-mediated efflux. When cellular glucose utilization was reduced by 2-deoxy-d-glucose and sodium fluoride, [(3)H]biotin efflux was similar to controls, suggesting that biotin efflux does not directly require metabolic energy. When [(3)H]biotin-loaded cells were incubated in external medium containing unlabeled biotin analogs, [(3)H]biotin efflux was accelerated approximately two times compared with incubation in a biotin-free medium. This observation suggests that biotin efflux is mediated by the same transporter that mediates biotin uptake from the extracellular medium (i.e., classic countertransport).     

Temporal profiling of changes in phosphatidylinositol 4,5-bisphosphate, inositol 1,4,5-trisphosphate and diacylglycerol allows comprehensive analysis of phospholipase C-initiated signalling in single neurons

Phosphatidylinositol 4,5-bisphosphate (PIP(2)) fulfils vital signalling roles in an array of cellular processes, yet until recently it has not been possible selectively to visualize real-time changes in PIP(2) levels within living cells. Green fluorescent protein (GFP)-labelled Tubby protein (GFP-Tubby) enriches to the plasma membrane at rest and translocates to the cytosol following activation of endogenous Galpha(q/11)-coupled muscarinic acetylcholine receptors in both SH-SY5Y human neuroblastoma cells and primary rat hippocampal neurons. GFP-Tubby translocation is independent of changes in cytosolic inositol 1,4,5-trisphosphate and instead reports dynamic changes in levels of plasma membrane PIP(2). In contrast, enhanced GFP (eGFP)-tagged pleckstrin homology domain of phospholipase C (PLCdelta1) (eGFP-PH) translocation reports increases in cytosolic inositol 1,4,5-trisphosphate. Comparison of GFP-Tubby, eGFP-PH and the eGFP-tagged C1(2) domain of protein kinase C-gamma [eGFP-C1(2); to detect diacylglycerol] allowed a selective and comprehensive analysis of PLC-initiated signalling in living cells. Manipulating intracellular Ca(2+) concentrations in the nanomolar range established that GFP-Tubby responses to a muscarinic agonist were sensitive to intracellular Ca(2+) up to 100-200 nM in SH-SY5Y cells, demonstrating the exquisite sensitivity of agonist-mediated PLC activity within the range of physiological resting Ca(2+) concentrations. We have also exploited GFP-Tubby selectively to visualize, for the first time, real-time changes in PIP(2) in hippocampal neurons.     

Regulation of apoptosis by phosphatidylinositol 4,5-bisphosphate inhibition of caspases, and caspase inactivation of phosphatidylinositol phosphate 5-kinases

Phosphoinositides such as phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate promote cell survival and protect against apoptosis by activating Akt/PKB, which phosphorylates components of the apoptotic machinery. We now report that another phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PIP2) is a direct inhibitor of initiator caspases 8 and 9, and their common effector caspase 3. PIP2 inhibited procaspase 9 processing in cell extracts and in a reconstituted procaspase 9/Apaf1 apoptosome system. It inhibited purified caspase 3 and 8 activity, at physiologically attainable PIP2 levels in mixed lipid vesicles. Caspase 3 binding to PIP2 was confirmed by cosedimentation with mixed lipid vesicles. Overexpression of phosphatidylinositol phosphate 5-kinase alpha (PIP5KIalpha), which synthesizes PIP2, suppressed apoptosis, whereas a kinase-deficient mutant did not. Protection by the wild-type PIP5KIalpha was accompanied by decreases in the generation of activated caspases and of caspase 3-cleaved PARP. Protection was not mediated through PIP3 or Akt activation. An anti-apoptotic role for PIP(2) is further substantiated by our finding that PIP5KIalpha was cleaved by caspase 3 during apoptosis, and cleavage inactivated PIP5KIalpha in vitro. Mutation of the P(4) position (D279A) of the PIP5KIalpha caspase 3 cleavage consensus prevented cleavage in vitro, and during apoptosis in vivo. Significantly, the caspase 3-resistant PIP5KIalpha mutant was more effective in suppressing apoptosis than the wild-type kinase. These results show that PIP2 is a direct regulator of apical and effector caspases in the death receptor and mitochondrial pathways, and that PIP5KIalpha inactivation contributes to the progression of apoptosis. This novel feedforward amplification mechanism for maintaining the balance between life and death of a cell works through phosphoinositide regulation of caspases and caspase regulation of phosphoinositide synthesis.     

GTP gamma S-stimulated hydrolysis of phosphatidylinositol-4,5-bisphosphate soluble phospholipase C from human platelets requires soluble GTP-binding protein

GTP-binding activity was fractionated into two peaks (GI and GII) by chromatography on heparin-agarose. GTP-dependent PLC activity eluted as a single peak, which co-chromatographed with GTP-binding peak GII. Rechromatography of peak GII on heparin-agarose, in the presence of 0.5% sodium cholate, resulted in separation of PLC and GTP-binding activities, and loss of GTP-dependent PLC activity. Recombining fractions containing PLC and GTP-binding activities restored GTP-dependent PLC activity. A specific GTP-binding protein of 29,000 daltons was identified in peak GII by Western blotting of column fractions with [alpha-32P]GTP. These results demonstrate that the soluble phospholipase C from human platelets is regulated by GTP S-binding protein (G29).     

Rapid accumulation of phosphatidylinositol 4,5-bisphosphate and inositol 1,4,5-trisphosphate correlates with calcium mobilization in salt-stressed arabidopsis

The phosphoinositide phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] is a key signaling molecule in animal cells. It can be hydrolyzed to release 1,2-diacyglycerol and inositol 1,4,5-trisphosphate (IP(3)), which in animal cells lead to protein kinase C activation and cellular calcium mobilization, respectively. In addition to its critical roles in constitutive and regulated secretion of proteins, PtdIns(4,5)P(2) binds to proteins that modify cytoskeletal architecture and phospholipid constituents. Herein, we report that Arabidopsis plants grown in liquid media rapidly increase PtdIns(4,5)P(2) synthesis in response to treatment with sodium chloride, potassium chloride, and sorbitol. These results demonstrate that when challenged with salinity and osmotic stress, terrestrial plants respond differently than algae, yeasts, and animal cells that accumulate different species of phosphoinositides. We also show data demonstrating that whole-plant IP(3) levels increase significantly within 1 min of stress initiation, and that IP(3) levels continue to increase for more than 30 min during stress application. Furthermore, using the calcium indicators Fura-2 and Fluo-3 we show that root intracellular calcium concentrations increase in response to stress treatments. Taken together, these results suggest that in response to salt and osmotic stress, Arabidopsis uses a signaling pathway in which a small but significant portion of PtdIns(4,5)P(2) is hydrolyzed to IP(3). The accumulation of IP(3) occurs during a time frame similar to that observed for stress-induced calcium mobilization. These data also suggest that the majority of the PtdIns(4,5)P(2) synthesized in response to salt and osmotic stress may be utilized for cellular signaling events distinct from the canonical IP(3) signaling pathway.     

Further characterization of four lipocortins from human peripheral blood mononuclear cells

Four calcium and phospholipid binding proteins purified from mononuclear cells were characterized for PKC and EGF phosphorylation, actin binding capacity, and partial tissue distribution. Those named 35K, 32K, and 73K are equivalent, respectively, to lipocortin III, endonexin II and the 67 kDa calelectrin; 36K is a fragment of 73K. After purification, 35K and 73K were phosphorylated by protein kinase C in vitro but 36K nor 32K were not. None were phosphorylated by the epidermal growth factor receptor kinase in vitro; 73K bound F-actin in a calcium-dependent manner, whereas 35K, 36K, and 32K did not. Using Western blotting analysis, 32K and 73K were detected in high amounts in human lymphocytes, monocytes, liver, and placenta and in rat adrenal medulla; but 32K was not detected in polymorphonuclear cells, and 36K and 35K were detected in high amounts only, respectively, in human blood lymphocytes and polymorphonuclear cells. Thus, 32K and 73K appear to have a wide tissue distribution, whereas 35K has a much more restricted distribution.