scholarly journals Isolation and enzymic assay of choline and phosphocholine present in cell extracts with picomole sensitivity

1990 ◽  
Vol 270 (1) ◽  
pp. 63-68 ◽  
Author(s):  
J J Murray ◽  
T T Dinh ◽  
A P Truett ◽  
D A Kennerly
Keyword(s):  
Phospholipase C ◽  
Phospholipase D ◽  
Tissue Sample ◽  
Water Soluble ◽  
Choline Kinase ◽  
Simple Analysis ◽  
Cell Extracts ◽  
Sodium Tetraphenylboron ◽  
Hydrolysis Of ◽  
Enzymic Assay

Increasing interest in receptor-regulated phospholipase C and phospholipase D hydrolysis of cellular phosphatidylcholine motivates the development of a sensitive and simple assay for the water-soluble hydrolytic products of these reactions, phosphocholine and choline respectively. Choline was partially purified from the methanol/water upper phase of a Bligh & Dyer extract by ion-pair extraction using sodium tetraphenylboron, and the mass of choline was determined by a radioenzymic assay using choline kinase and [32P]ATP. After removal of choline from the upper phase, the mass of residual phosphocholine was determined by converting it into choline by using alkaline phosphatase, followed by radioactive phosphorylation. In addition to excellent sensitivity (5 pmol for choline and 10 pmol for phosphocholine), these assays demonstrated little mutual interference (phosphocholine----choline = 0%; choline----phosphocholine = 5%), were extremely reproducible (average S.E.M. of 3.5% for choline and 2.9% for phosphocholine), and were simple to perform with instrumentation typically available in most laboratories. In addition, the ability to apply the extraction technique to the upper phase of Bligh & Dyer extracts permitted simple analysis not only of choline and phosphocholine, but also of phosphatidylcholine and lipid products of phospholipase C and phospholipase D activity (1,2-diacylglycerol and phosphatidic acid respectively) from the same cell or tissue sample.

scholarly journals Assessment of receptor-dependent activation of phosphatidylcholine hydrolysis by both phospholipase D and phospholipase C.

1991 ◽  
Vol 2 (4) ◽  
pp. 299-309 ◽  
Author(s):  
T T Dinh ◽  
D A Kennerly
Keyword(s):  
Mast Cells ◽  
Phospholipase C ◽  
Phospholipase D ◽  
Cell Activation ◽  
Immunoglobulin E ◽  
Water Soluble ◽  
Extracellular Medium ◽  
Acid Accumulation ◽  
The Masses ◽  
Hydrolysis Of

Enhancement of cellular phospholipase D (PLD)-1 and phospholipase C (PLC)-mediated hydrolysis of endogenous phosphatidylcholine (PC) during receptor-mediated cell activation has received increasing attention inasmuch as both enzymes can result in the formation of 1,2-diacylglycerol (DAG). The activities of PLD and PLC were examined in purified mast cells by quantitating the mass of the water-soluble hydrolysis products choline and phosphorylcholine, respectively. Using an assay based on choline kinase-mediated phosphorylation of choline that is capable of measuring choline and phosphorylcholine in the low picomole range, we quantitated the masses of both cell-associated and extracellular choline and phosphorylcholine. Activating mast cells by crosslinking its immunoglobulin E receptor (Fc epsilon-RI) resulted in an increase in cellular choline from 13.1 +/- 1.2 pmol/10(6) mast cells (mean +/- SE in unstimulated cells) to levels 5- to 10-fold higher, peaking 20 s after stimulation and rapidly returning toward baseline. The increase in cellular choline mass paralleled the increase in labeled phosphatidic acid accumulation detected in stimulated cells prelabeled with [3H]palmitic acid and preceded the increase in labeled DAG. Although intracellular phosphorylcholine levels were approximately 15-fold greater than choline in unstimulated cells (182 +/- 19 pmol/10(6) mast cells), stimulation resulted in a significant fall in phosphorylcholine levels shortly after stimulation. Pulse chase experiments demonstrated that the receptor-dependent increase in intracellular choline and the fall in phosphorylcholine were not due to hydrolysis of intracellular phosphorylcholine and suggested a receptor-dependent increase in PC resynthesis. When the extracellular medium was examined for the presence of water-soluble products of PC hydrolysis, receptor-dependent increases in the mass of both choline and phosphorylcholine were observed. Labeling studies demonstrated that these extracellular increases were not the result of leakage of these compounds from the cytosol. Taken together, these data lend support for a quantitatively greater role for receptor-mediated PC-PLD compared with PC-PLC during activation of mast cells.

scholarly journals Hydrolysis of membrane phospholipids by phospholipases of rat liver lysosomes

1979 ◽  
Vol 182 (2) ◽  
pp. 599-606 ◽  
Author(s):  
Donald E. Richards ◽  
Robin F. Irvine ◽  
Rex M. C. Dawson
Keyword(s):  
Phospholipase C ◽  
Rat Liver ◽  
Lysosomal Enzymes ◽  
Microsomal Fraction ◽  
Water Soluble ◽  
Membrane Phospholipids ◽  
Liver Lysosomes ◽  
Rat Liver Lysosomes ◽  
Hydrolysis Of

(1) The hydrolysis of 32P- or myo-[2-3H]inositol-labelled rat liver microsomal phospholipids by rat liver lysosomal enzymes has been studied. (2) The relative rates of hydrolysis of phospholipids at pH4.5 are: sphingomyelin>phosphatidylethanolamine>phosphatidylcholine> phosphatidylinositol. (3) The predominant products of phosphatidylcholine and phosphatidylethanolamine hydrolysis are their corresponding lyso-compounds, indicating a slow rate of total deacylation. (4) Ca2+ inhibits the hydrolysis of all phospholipids, though only appreciably at high (>5mm) concentration. The hydrolysis of sphingomyelin is considerably less sensitive to Ca2+ than that of glycerophospholipids. (5) Analysis of the water-soluble products of phosphatidylinositol hydrolysis (by using myo-[3H]inositol-labelled microsomal fraction as a substrate) produced evidence that more than 95% of the product is phosphoinositol, which was derived by direct cleavage from phosphatidylinositol, rather than by hydrolysis of glycerophosphoinositol. (6) This production of phosphoinositol, allied with negligible lysophosphatidylinositol formation and a detectable accumulation of diacylglycerol, indicates that lysosomes hydrolyse membrane phosphatidylinositol almost exclusively in a phospholipase C-like manner. (7) Comparisons are drawn between the hydrolysis by lysosomal enzymes of membrane substrates and that of pure phospholipid substrates, and also the possible role of phosphatidylinositol-specific lysosomal phospholipase C in cellular phosphatidylinositol catabolism is discussed.

Elevated phosphocholine concentration in ras-transformed NIH 3T3 cells arises from increased choline kinase activity, not from phosphatidylcholine breakdown

1989 ◽  
Vol 9 (1) ◽  
pp. 325-328
Author(s):  
I G Macara
Keyword(s):  
Phospholipase C ◽  
Kinase Activity ◽  
Transformed Cells ◽  
Choline Kinase ◽  
3T3 Cells ◽  
Cellular Concentration ◽  
Nih 3T3 ◽  
Hydrolysis Of ◽  
Nih 3T3 Cells

The cellular concentration of phosphocholine has been reported to be significantly elevated in Ha-ras-transformed NIH 3T3 cells, but not in v-sis transformants (J. C. Lacal, J. Moscat, and S. A. Aaronson, Nature [London] 330:269-271, 1987). It was suggested that the phosphocholine arises from constitutive hydrolysis of phosphatidylcholine by phospholipase C, an activity that would also account for the elevated 1,2-diacylglycerol found in ras-transformed cells. I have demonstrated that the increased phosphocholine arises through the induction of choline kinase activity. No increased breakdown of phosphatidylcholine was observed in ras-transformed cells. The elevation in diacylglycerol is therefore unlikely to be a consequence of phosphatidylinositol or phosphatidylcholine turnover.

scholarly journals Quantification of inositol phospholipid breakdown in isolated rat hepatocytes

1993 ◽  
Vol 290 (3) ◽  
pp. 865-872 ◽  
Author(s):  
C J Allan ◽  
J H Exton
Keyword(s):  
Phospholipase C ◽  
Phospholipase D ◽  
Inositol Phosphates ◽  
Rat Hepatocytes ◽  
Isolated Rat Hepatocytes ◽  
Phospholipid Hydrolysis ◽  
Inositol Phospholipids ◽  
Inositol Phospholipid ◽  
Inositol Phospholipid Breakdown ◽  
Hydrolysis Of

The hydrolysis of inositol phospholipids induced by vasopressin in hepatocytes during 60 min was quantified chemically. There was a large release of myo-inositol which was abolished by Li+, indicating that it was derived from inositol phosphates and not from phospholipase D action on PtdIns. There was also a large release of inositol phosphates which was increased approx. 2-fold by Li+ at 30 min, but then remained constant, suggesting that inositol phospholipid breakdown declined substantially beyond this time. In cells prelabelled with myo-[3H]inositol and treated with Li+, [3H]PtdIns(4,5)P2 decreased maximally (50%) at 15 s and then recovered to a level at 5 min that was maintained at 25% below control for 40 min. [3H]PtdIns4P and [3H]PtdIns showed slower decreases to approx. 30% below control at 15 min, but with no further changes. Labelled Ins(1,4,5)P3 and Ins(1,3,4)P3 showed 2-4-fold increases within 30 s and then declined to values that were maintained at a constant level above the control, except for [3H]Ins(1,3,4)P3, which showed a second increase. [3H]Ins(1,4)P2 showed a very large increase over 10 min, whereas [3H]Ins4P and [3H]Ins1P showed little change before 6 and 15 min respectively. The total [3H]inositol phosphates showed little further increase after 20 min. These data are consistent with a rapid, but not sustained, hydrolysis of PtdIns-(4,5)P2, but not of PtdIns, by phospholipase C, but do not exclude PtdIns4P as a substrate. Phosphatidate was rapidly increased by vasopressin, whereas diacylglycerol was increased after a 1-2 min lag. Both were maintained at levels 2-3-fold above control for 60 min. The vasopressin-induced increase in inositol phosphates plus myo-inositol (approx. 120 nmol/100 mg) was greater than the increase in diacylglycerol plus phosphatidate (approx. 60 nmol/100 mg) between 10 and 40 min. This indicates that there was substantial further metabolism of these lipids. Addition of 75 mM ethanol resulted in rapid production of phosphatidylethanol in response to vasopressin and a 35% reduction in phosphatidate, but no decrease in diacylglycerol. In summary, the results indicate that inositol phospholipid hydrolysis by phospholipase C can account for most of the diacylglycerol and phosphatidate that accumulate during 60 min of vasopressin action, but that these phospholipids are probably not the major source of the phosphatidate that is formed during the first 2 min by phospholipase D, or of the diacylglycerol and phosphatidate that are formed beyond 30 min.

scholarly journals Potentiated bradykinin-induced increase of 1,2-diacylglycerol generation and phospholipase D activity in human senescent fibroblasts

1995 ◽  
Vol 312 (3) ◽  
pp. 799-803 ◽  
Author(s):  
E Meacci ◽  
V Vasta ◽  
P Faraoni ◽  
M Farnararo ◽  
P Bruni
Keyword(s):  
Phospholipase D ◽  
Human Fibroblasts ◽  
Term Treatment ◽  
Water Soluble ◽  
Short Term Treatment ◽  
Kinase C ◽  
The Comparative Study ◽  
Hydrolysis Of ◽  
Pkc Activation

1. The comparative study of the effect of bradykinin (BK) in young and old IMR-90 human fibroblasts shows that old cells are characterized by a reduced increase in 1,2-diacylglycerol (1,2-DAG) generation upon stimulation after short-term treatment and a significant higher increase after long-term agonist treatment. BK-induced activation of phospholipase D (PLD), the major enzyme involved in sustained 1,2-DAG generation, was 2.5-fold higher in old cells, strongly suggesting that it is involved in the potentiated increase of 1,2-DAG formation. The increased activation of PLD by BK in old cells was specific, since in parallel experiments the effect of thrombin was not significantly different in young and old cells. PLD activity in old cells was only reduced by down-regulation of protein kinase C (PKC) activity, in contrast to what was observed in young cells where it was completely abolished. This indicates that the enzyme activity in old cells was partially PKC-independent. BK was also able to increase the release of [14C]ethanolamine, a water-soluble product of hydrolysis of phosphatidylethanolamine (PtdEtn), through PLD activation in young and old cells. The BK effect was significantly higher in old cells and, very likely, PKC-independent, since phorbol 12-myristate 13-acetate failed to induce PtdEtn hydrolysis. 2. The present results indicate that the PLD/1,2-DAG pathway is specifically potentiated by BK in old fibroblasts, demonstrating that the formation of positive effectors of PKC activation is not necessarily decreased in cellular senescence. It remains to be established whether the increased generation of DAG upon BK stimulation plays any role in the altered PKC signalling pathway which characterizes old fibroblasts.

scholarly journals Epidermal growth factor stimulates distinct diradylglycerol species generation in Swiss 3T3 fibroblasts: evidence for a potential phosphatidylcholine-specific phospholipase C-catalysed pathway

1994 ◽  
Vol 298 (3) ◽  
pp. 655-660 ◽  
Author(s):  
T R Pettitt ◽  
M Zaqqa ◽  
M J Wakelam
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Phospholipase C ◽  
Phospholipase D ◽  
Kinase Inhibitor ◽  
Inositol Phosphates ◽  
Egf Receptors ◽  
3T3 Fibroblasts ◽  
Epidermal Growth ◽  
Hydrolysis Of

Stimulation of 3T3 fibroblasts with epidermal growth factor (EGF) results in an increase in 1,2-diacylglycerol (DAG) mass which is maximal at 25 s, declining at 1 min and returning to basal levels by 30 min. No changes in alkylacylglycerol or alkenylacylglycerol were detected. Three species account for most of this mass increase: 18:0/20:5,n-3, 18:0/20:4,n-6 and 18:0/20:3,n-9. These species are characteristic of the phosphoinositides; however, previous work failed to detect any EGF-stimulated rise in inositol phosphates in these cells [Cook and Wakelam (1992) Biochem. J. 285, 247-253]. This ruled out phosphoinositide hydrolysis by phospholipase C, but raised the possibility of phospholipase D/phosphatidate phosphohydrolase-catalysed hydrolysis of phosphatidylinositol. The inclusion of butanol in the incubation medium failed to block the diacylglycerol changes, indicating that the phospholipase D pathway is not involved and that DAG must be derived from another source, probably via phospholipase C-catalysed hydrolysis of a phosphatidylcholine pool that is particularly rich in these species. The tyrosine kinase inhibitor ST-271 almost abolished the elevation in 18:0/20:5,n-3, 18:0/20:4, n-6 and 18:0/20:3,n-9 at 25 s, but only reduced the rise in total DAG mass by about 50%. The protein kinase C (PKC) inhibitor Ro-31-8220 increased DAG levels at all time points but had no effect on the species profiles. This provides additional evidence for PKC-mediated regulation of cell-surface EGF receptors, since the inhibition of PKC would increase the availability and/or ligand binding affinity of receptors at the plasma membrane and hence increase and prolong the response to EGF.

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