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W F Boss

Publications and source records attributed to W F Boss.

At least 37 records · Page 2Linked to original sources
PubMed1992

Release of carrot plasma membrane-associated phosphatidylinositol kinase by phospholipase A2 and activation by a 70 kDa protein.

Plasma membranes were isolated from carrot (Daucus carota L.) cells grown in suspension culture and treated with phospholipase A2 from snake or bee venom for 10 min. As a result of this treatment, phosphatidylinositol kinase activity was recovered in the soluble fraction. There was no detectable diacylglycerol kinase or phosphatidylinositol monophosphate kinase activity released from the membranes after the phospholipase A2 treatment. Treating the plasma membranes with phospholipase C or D did not release PI kinase activity. The phospholipase A2-released PI kinase was activated over 2-fold by a heat stable, soluble 70 kDa protein. The partially purified 70 kDa activator increases the Vmax but does not affect the Km of the phospholipase A2-released PI kinase.

1-Phosphatidylinositol 4-Kinase↗
PubMed1992

Association of Phosphatidylinositol Kinase, Phosphatidylinositol Monophosphate Kinase, and Diacylglycerol Kinase with the Cytoskeleton and F-Actin Fractions of Carrot (Daucus carota L.) Cells Grown in Suspension Culture : Response to Cell Wall-Degrading Enzymes.

Phosphatidylinositol kinase (PI), phosphatidylinositol monophosphate (PIP) kinase, and diacylglycerol (DAG) kinase activities were detected in the cytoskeletal fraction isolated from microsomes and plasma membranes of carrot (Daucus carota L.) cells grown in suspension culture. The lipid kinase activities were associated with the actin filament fraction (F-actin fraction) isolated from the cytoskeleton. The PI and PIP kinase activity in the F-actin fraction significantly increased after cells were treated with Driselase, a mixture of cell wall-degrading enzymes; however, the DAG kinase activity in the F-actin fraction was unaffected by the Driselase treatment. These data indicate that at least one form of PI, PIP, and DAG kinase preferentially associates with actin filaments and/or actin binding proteins and that cytoskeletal-associated PI and PIP kinase activities can change in response to external stimulation.

Journal Article↗
PubMed1991

Phosphorylation of lysophosphatidylinositol by carrot membranes.

sn-1 Palmitoyl lysophosphatidylinositol is found in carrot suspension culture cells and can be phosphorylated to [32P]lysophosphatidylinositol monophosphate (LPIP) when [gamma 32P]ATP is added to isolated membranes. Based on in vivo labeling studies, [3H]inositol sn-1 palmitoyl LPIP was found predominantly in the plasma membrane-rich fraction or upper phase isolated by aqueous two-phase partitioning and LPI was found in the intracellular membrane-rich fraction or lower phase (Wheeler and Boss, Plant Physiol. 85, 389-392, 1987). While both membrane fractions phosphorylated LPI in vitro, the apparent Km for LPI in the intracellular membrane fraction was 180 microM and for the plasma membrane was 580 microM. When cells were treated with the ionophore, monensin, the percentage of [3H]inositol LPIP increased in the whole cell lipid extract. However, the monensin treatment decreased the amount of [3H]inositol LPIP and PIP recovered in the plasma membrane fraction relative to the sum of the individual lipid, [3H]inositol LPIP or PIP, respectively, recovered in both membrane fractions.

Cell Membrane↗
PubMed1991

Neomycin inhibits the phosphatidylinositol monophosphate and phosphatidylinositol bisphosphate stimulation of plasma membrane ATPase activity.

The inositol phospholipids, phosphatidylinositol monophosphate (PIP) and phosphatidylinositol bisphosphate (PIP(2)), have been shown to increase the vanadate-sensitive ATPase activity of plant plasma membranes (AR Memon, Q Chen, WF Boss [1989] Biochem Biophys Res Commun 162: 1295-1301). In this paper, we show the effect of various concentrations of phosphatidyinositol, PIP, and PIP(2) on the plasma membrane vanadate-sensitive ATPase activity. PIP and PIP(2) at concentrations of 10 nanomoles per 30 microgram membrane protein per milliliter of reaction mixture caused a twofold and 1.8-fold increase in the ATPase activity, respectively. The effect of these negatively charged phospholipids on the ATPase activity was inhibited by adding the positively charged aminoglycoside, neomycin. Neomycin did not affect the endogenous plasma membrane ATPase activity in the absence of exogenous lipids.

Journal Article↗
PubMed1991

Inositol phospholipids as plant second messengers.

Two plasma membrane lipids, phosphatidylinositol monophosphate (PtdIns4P) and phosphatidylinositol bisphosphate (PtdIns(4,5)P2), have been shown to be key intermediates in stimulus response pathways in many animal cells. PtdIns4P and PtdIns(4,5)P2 act as sources of second messengers and they directly alter the activity of membrane enzymes. These lipids and the enzymes involved in their metabolism are found in the plasma membranes of plant cells. A clear role for the inositol phospholipids in plant signal transduction, however, has not emerged. In this chapter we present some of the questions raised by current work in the area and propose an alternative focus for phosphoinositide metabolism in plants: a role for PtdIns4P and PtdIns(4,5)P2 as direct effectors of membrane structure and function.

Animals↗
PubMed1990

Rapid light-induced changes in phosphoinositide kinases and H(+)-ATPase in plasma membrane of sunflower hypocotyls.

Irradiation of sunflower (Helianthus annuus L. cv. Russian Mammoth) hypocotyls with white light resulted in a 51% decrease in plasma membrane phosphatidylinositol monophosphate (PIP) kinase activity. As little as 10 s of white light irradiation was sufficient to lower the phosphatidylinositol bisphosphate (PIP2) produced in the in vitro phosphorylation assay. This decrease was not caused by an increase in phospholipase C activity since analysis of the water-soluble products indicated no increase in inositol bisphosphate or inositol trisphosphate. Treatment of the plasma membrane with 200 microM vanadate prior to phosphorylation enhanced the PIP kinase and appeared to overcome the light inhibition. In addition to decreasing the PIP kinase activity, light irradiation resulted in a corresponding decrease in the H(+)-ATPase activity to 53% of the dark control values. The plasma membrane ATPase activity increased approximately 2-fold when PIP or PIP2 was added to the isolated membranes. Thus, effects of external stimuli on the level of plasma membrane PIP or PIP2 could affect plasma membrane ATPase activity directly and thereby provide an alternative mechanism for control of cell growth.

1-Phosphatidylinositol 4-Kinase↗
PubMed1990

Short-term treatment with cell wall degrading enzymes increases the activity of the inositol phospholipid kinases and the vanadate-sensitive ATPase of carrot cells.

Treating carrot (Daucus carota L.) suspension culture cells with a mixture of cell wall degrading enzymes, Driselase, resulted in an increase in the percentage of [(3)H]phosphatidylinositol bisphosphate. Analysis of the lipid kinase activities in the isolated plasma membranes after whole cell treatment indicated that treatment with Driselase (2% weight/volume; the equivalent of 340 units per milliliter of hemicellulase and 400 units per milliliter of cellulase activity) or treatment with hemicellulase (31.7% weight/volume, 20.7 units per milliliter) resulted in an increase in the inositol phospholipid kinase activity. However, treatment with cellulase alone had no effect at 0.5% (weight/volume, 17.2 units per milliliter) or inhibited the kinase activity at 1% (weight/volume, 34.4 units per milliliter). The active stimulus in Driselase was heat sensitive. The plasma membrane vanadate-sensitive ATPase activity also increased when the cells were treated with Driselase. A time course study indicated that both the inositol phospholipid kinases and the plasma membrane vanadate-sensitive ATPase responded to as little as 5 seconds of treatment with 2% Driselase. However, at the lowest concentration of Driselase (0.04%, weight/volume) that resulted in an increase in inositol phospholipid kinase activity, the ATPase activity was not affected. Because inositol phospholipids have been shown to activate the vanadate-sensitive ATPase from plants (AR Memon, Q Chen, WF Boss [1989] Biochem Biophys Res Commun 162: 1295-1301), a stimulus-response pathway involving both the inositol phospholipid kinases and the plasma membrane vanadate-sensitive ATPase activity is discussed.

Journal Article↗
PubMed1989

Inositol phospholipids activate plasma membrane ATPase in plants.

Phosphatidylinositol-4-monophosphate and phosphatidylinositol-4,5-bisphosphate increased the activity of the vanadate-sensitive ATPase associated with plasma membranes isolated from both sunflower hypocotyls and carrot suspension culture cells. The response was not due to the metabolism of the polyphosphoinositides since diacylglycerol, inositol-1,4-bisphosphate, inositol-1,4,5-trisphosphate, glycerophosphoinositol monophosphate and glycerophosphoinositol bisphosphate had no effect. These data suggest that activation of the inositol phospholipid kinases could be a critical step in signal transduction in plants.

1-Phosphatidylinositol 4-Kinase↗
PubMed1989

Characterization of Inositol Phosphates in Carrot (Daucus carota L.) Cells.

We have shown previously that inositol-1,4,5-trisphosphate (IP(3)) stimulates an efflux of (45)Ca(2+) from fusogenic carrot protoplasts (M Rincón, WF Boss [1987] Plant Physiol 83: 395-398). In light of these results, we suggested that IP(3) might serve as a second messenger for the mobilization of intracellular Ca(2+) in higher plant cells. To determine whether or not IP(3) and other inositol phosphates were present in the carrot cells, the cells were labeled with myo-[2-(3)H]inositol for 18 hours and extracted with ice-cold 10% trichloroacetic acid. The inositol metabolites were separated by anion exchange chromatography and by paper electrophoresis. We found that [(3)H]inositol metabolites coeluted with inositol bisphosphate (IP(2)) and IP(3) when separated by anion exchange chromatography. However, we could not detect IP(2) or IP(3) when the inositol metabolites were analyzed by paper electrophoresis even though the polyphosphoinositides, which are the source of IP(2) and IP(3), were present in these cells. Thus, [(3)H] inositol metabolites other than IP(2) and IP(3) had coeluted on the anion exchange columns. The data indicate that either IP(3) is rapidly metabolized or that it is not present at a detectable level in the carrot cells.

Journal Article↗
PubMed1989

Inositol Trisphosphate Metabolism in Carrot (Daucus carota L.) Cells.

The metabolism of exogenously added d-myo-[1-(3)H]inositol 1,4,5-trisphosphate (IP(3)) has been examined in microsomal membrane and soluble fractions of carrot (Daucus carota L.) cells grown in suspension culture. When [(3)H]IP(3) was added to a microsomal membrane fraction, [(3)H]IP(2) was the primary metabolite consisting of approximately 83% of the total recovered [(3)H] by paper electrophoresis. [(3)H]IP was only 6% of the [(3)H] recovered, and 10% of the [(3)H]IP(3) was not further metabolized. In contrast, when [(3)H]IP(3) was added to the soluble fraction, approximately equal amounts of [(3)H]IP(2) and [(3)H]IP were recovered. Ca(2+) (100 micromolar) tended to enhance IP(3) dephosphorylation but inhibited the IP(2) dephosphorylation in the soluble fraction by about 20%. MoO(4) (2-) (1 millimolar) inhibited the dephosphorylation of IP(3) by the microsomal fraction and the dephosphorylation of IP(2) by the soluble fraction. MoO(4) (2-), however, did not inhibit the dephosphorylation of IP(3) by the soluble fraction. Li(+) (10 and 50 millimolar) had no effect on IP(3) metabolism in either the soluble or membrane fraction; however, Li(+) (50 millimolar) inhibited IP(2) dephosphorylation in the soluble fraction about 25%.

Journal Article↗
PubMed1988

Rapid Changes in Plasma Membrane Protein Phosphorylation during Initiation of Cell Wall Digestion.

Plasma membrane vesicles from wild carrot cells grown in suspension culture were isolated by aqueous two-phase partitioning, and ATP-dependent phosphorylation was measured with [gamma-(32)P]ATP in the presence and absence of calcium. Treatment of the carrot cells with the cell wall digestion enzymes, driselase, in a sorbitol osmoticum for 1.5 min altered the protein phosphorylation pattern compared to that of cells treated with sorbitol alone. Driselase treatment resulted in decreased phosphorylation of a band of M(r) 80,000 which showed almost complete calcium dependence in the osmoticum treated cells; decreased phosphorylation of a band of M(r) 15,000 which showed little calcium activation, and appearance of a new band of calcium-dependent phosphorylation at M(r) 22,000. These effects appeared not to be due to nonspecific protease activity and neither in vivo nor in vitro exposure to driselase caused a significant loss of Coomassie blue-staining bands on the gels of the isolated plasma membranes. However, protein phosphorylation was decreased. Adding driselase to the in vitro reaction mixture caused a general decrease in the membrane protein phosphorylation either in the presence or absence of calcium which did not mimic the in vivo response. Cells labeled in vivo with inorganic (32)P also showed a response to the Driselase treatment. An enzymically active driselase preparation was required for the observed responses.

Journal Article↗
PubMed1987

myo-Inositol Trisphosphate Mobilizes Calcium from Fusogenic Carrot (Daucus carota L.) Protoplasts.

To determine whether or not inositol trisphosphate (IP(3)) mobilizes calcium in higher plant cells, we investigated the effect of IP(3) on Ca(2+) fluxes in fusogenic carrot (Daucus carota L.) protoplasts. The protoplasts were incubated in (45)Ca(2+)-containing medium and the (45)Ca(2+) associated with the protoplasts was monitored with time. Addition of IP(3) (20 micromolar) caused a 17% net loss of the accumulated (45)Ca(2+) within 4 minutes. There was a reuptake of (45)Ca(2+) and the protoplasts recovered to their initial value by 10 minutes. Phytic acid (IP(6)), also stimulated (45)Ca(2+) efflux from the protoplasts. Both the IP(3(-) ) and the IP(6(-) )induced (45)Ca(2+) efflux were inhibited by the calmodulin antagonist, trifluoperazine.

Journal Article↗
PubMed1987

Polyphosphoinositides are present in plasma membranes isolated from fusogenic carrot cells.

Fusogenic carrot cells grown in suspension culture were labeled 12 hours with myo-[2-(3)H]inositol. Plasma membranes were isolated from the prelabeled fusogenic carrot cells by both aqueous polymer two-phase partitioning and Renografin density gradients. With both methods, the plasma membrane-enriched fractions, as identified by marker enzymes, were enriched in [(3)H]inositol-labeled phosphatidylinositol monophosphate (PIP) and phosphatidylinositol bisphosphate (PIP(2)). An additional [(3)H]inositol-labeled lipid, lysophosphatidylinositol monophosphate, which migrated between PIP and PIP(2) on thin layer plates, was found primarily in the plasma membrane-rich fraction of the fusogenic cells. This was in contrast to lysophosphatidylinositol which is found primarily in the lower phase, microsomal/mitochrondrial-rich fraction.

Journal Article↗
PubMed1985

Polyphosphoinositides are present in plant tissue culture cells.

Polyphosphoinositides have been isolated from wild carrot cells grown in suspension culture. This is the first report of polyphosphoinositides in plant cells. The phospholipids were identified by comigration with known standards on thin-layer plates. After overnight labeling of the cells with myo-[2-3H] inositol, the phosphoinositides as percent recovered inositol were 93% phosphatidylinositol., 3.7% lysophosphatidylinositol, 1.7% phosphatidylinositol monophosphate, 0.8% phosphatidylinositol bisphosphate.

Cells, Cultured↗
PubMed1985

Intracellular calcium and calmodulin involvement in protoplast fusion.

(45)Ca(2+) uptake was compared between fusogenic and nonfusogenic Daucus carota L. protoplasts. Fusogenic protoplasts took 10 minutes to reach calcium equilibrium compared to 5 minutes in the nonfusogenic protoplasts. Intracellular stores of calcium were manipulated by isolating protoplasts in different calcium regimes. Lowering of intracellular calcium lowered fusion potential, while raising intracellular stores of calcium enhanced fusion potential. Regardless of the amount of calcium sequestered in a store, mobilization with A23187 increased fusion levels within 10 minutes. Calmodulin antagonists were potent inhibitors of protoplast fusion. This inhibition was obtained by treating cells with the calmodulin antagonists during protoplast isolation. A23187, however, only allowed a partial recovery from this inhibition, indicating that calcium flux alone was not sufficient for maximum fusion potential. On the basis of the evidence presented, we propose that calcium fluxes during protoplast isolation activate a calmodulin-mediated biochemical process that is necessary for the formation or maintenance of a fusion permissive state.

Journal Article↗
PubMed1984

Monensin-induced swelling of Golgi apparatus cisternae mediated by a proton gradient.

Monensin, a monovalent ionophore, caused swelling of mature cisternae of plant Golgi apparatus. The appearance of swollen cisternae was time-dependent and linear over a period of 1 h with an estimated maximum rate of production of one swollen cisterna every 3 to 4 min. Implicit in these observations was a need for the uptake of osmotically active monovalent cations to have occurred accompanied by a concomitant efflux of H+ and the entry of water. Furthermore, to sustain the H+ efflux, a source of H+ influx also would be required. To test for the latter, cisternal swelling, as visualized by electron microscopy, was monitored by treatment of wild carrot cells in suspension culture with drugs and inhibitors known to interfere with proton gradients. Swelling was inhibited by the protonophore, FCCP, by the inhibitor of lysosomal acidification, quercetin, and by the lysosomotropic amines, chloroquine and ammonia. While antimycin A, an inhibitor of mitochondrial oxidative phosphorylation, was ineffective, cyanide dramatically decreased swelling. The numbers of swollen cisternae produced could be reduced by prolonged treatment with arsenate, such that an ATP requirement is indicated, at least, for cisternal formation. Swelling was promoted by citrate, representative of a permeant organic anion. Reductions in numbers of monensin-induced swollen cisternae in the presence of quercetin, vanadate, and chloroquine could be compensated for by the addition of citrate. We conclude that the monensin-induced swelling of Golgi apparatus cisternae may involve a mechanism generating a proton gradient at or near the mature Golgi apparatus face.

Arsenates↗
PubMed1983

Ozone Degrades into Hydroxyl Radical under Physiological Conditions : A Spin Trapping Study.

Defining the reactants is a critical step towards elucidating the mechanism of ozone toxicity to biomembranes. To document ozone-induced HO.radicals, the spin trap 5,5-dimethyl-1-pyrroline-N-oxide was used and the resulting spin adduct was monitored with electron spin resonance spectroscopy. Chelexed potassium phosphate buffer (10 millimolar and 0.2 molar) at pH 7.2 and 7.8 was exposed to ozone (1-40 microliters per liter) by directing a stream of ozone over the surface for 60 seconds. Under these conditions, no HO. was detected. Using 0.5 x 10(-4) molar caffeic acid in phosphate buffer, strong DMPO.OH electron spin resonance signals were obtained, indicating HO. production. Air controls yielded no signal. High pH (7.8) enhanced signal strength. Furthermore, with sorbitol (0.4 osmolal final concentration), a net HO. signal loss of 28% was observed, while a carbon-centered sorbitol radical adduct appeared. Although HO. radicals were produced, no breakage of Daucus carota protoplast plasma membranes was observed nor were differences in membrane fluidity observed as determined by 5-doxyl stearic acid.

Journal Article↗