The biosynthesis of ribonuclease and its accumulation in protein bodies in the cotyledons of mung bean seedlings

Germination and seedling growth of mung bean are accompanied by a 7- to 10-fold increase in the ribonuclease content of the cotyledons. The increase occurs during the first 4 days of seedling growth and precedes the senescence of the cotyledons. Separation of the RNases in the cotyledons by polyacrylamide gel electrophoresis indicates the presence of several minor bands in seeds imbibed for 24 hr. On the second day of seedling growth a new major band with an R_f of 0.76 is present. In 4- to 5-day old seedlings this major band accounts for nearly all the RNase activity in the tissue. The characteristics of this RNase show that it is a plant ribonuclease I (pH optimum of 5.0; MW 16,000; activity preferentially inhibited by purine nucleotides; no activity toward DNA; no phosphodiesterase activity). When the seedlings are grown in 66% D₂O the RNase activity undergoes a density shift of 0.61% indicating that the increase in enzyme activity is due to the de novo synthesis of the enzyme molecules. A method is described for the isolation of protein bodies from protoplasts of storage parenchyma cells. Fractionation of protoplast lysates on Ficoll gradients results in the recovery of a high proportion (75%) of intact protein bodies. On these gradients RNase activity comigrates with α-mannosidase, a protein body marker enzyme indicating that the newly synthesized RNase accumulates in the protein bodies. We suggest that the synthesis of RNase in the cotyledons and its accumulation in the protein bodies indicates that protein bodies may function in the degradation of cellular macromolecules other than the reserves stored within them.     

Rapid degradation and limited synthesis of phospholipids in the cotyledons of mung bean seedlings

Seedling growth of mung bean is accompanied by the rapid catabolism of the three major phospholipids in the cotyledons (phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol). The decline starts 24 hours after the beginning of imbibition and by the 4th day of growth more than 50% of the phospholipids have been catabolized. Extracts of cotyledons of 24-hour-imbibed beans contain enzymes capable of degrading membrane-associated phospholipids in vitro. This degradation involves phospholipase D and phosphatase activity.     

Formation of diacylglycerol by a phospholipase D-phosphatidate phosphatase pathway specific for phosphatidylcholine in endothelial cells

The conversion of phosphatidylcholine (PC) to diacylglycerol (DAG) was studied in sonicated endothelial cells and in subcellular fractions in the presence of 0.05% Triton X-100 and 2 mM EDTA. DAG formation occurred predominantly in an organelle fraction that sedimented at 15,000 x g. In parallel reactions with exogenous 1-oleoyl-2-[3H]oleoyl-PC (sn-2-[3H]DOPC) and phosphatidyl[3H]choline ([choline-3H]PC), [3H]DAG was formed by a reaction pathway in which [3H]choline was the only product derived from [choline-3H]PC. [3H]Choline was not formed secondarily from [3H]glycerophosphocholine or [3H]phosphocholine. Small amounts of [3H]phosphatidate ([3H]PA) were isolated from reactions with sn-2-[3H]DOPC at short incubation times, and substantial PA phosphatase activity was demonstrated. These data, taken together, supported a phospholipase D-PA phosphatase pathway of DAG formation. Kinetic data established that the low ratio of [3H]PA/[3H]DAG formed in reactions with sn-2-[3H]DOPC was due to a 15-fold higher Vmax and 7-fold lower apparent Km of the PA phosphatase. The [3H]PA/[3H]DAG product ratio was increased by addition of unlabeled PA or by selective extraction of phospholipase D with Triton X-100. The characteristics of the phospholipase D indicated a unique enzyme. Activity was optimal in the presence of EDTA and was almost totally dependent upon Triton X-100. The pH profile displayed a peak at 7.0. Of particular significance was the stringent substrate specificity. Phosphatidylinositol was not hydrolyzed, and activities towards phosphatidylethanolamine and sphingomyelin were at most 30- to 50-fold lower than those towards PC. Phospholipase D and PA phosphatase were identified in a number of rat tissues and other cells. The highest activities of phospholipase D were present in lung and endothelial cells. Phospholipase D was partially purified from rat lung by Triton X-100 extraction and anion exchange chromatography. When linked with PA phosphatase, the phospholipase D could initiate a pathway of DAG formation that is highly specific for PC.     

Determination of phosphatidylcholine in a flow injection system using immobilized enzyme reactors

Two alternative procedures are described for the quantitative determination of phosphatidylcholine in a flow-injection system utilizing immobilized enzymes. Phospholipase C from Bacillus cereus and phospholipase D from cabbage were covalently bound to the surface of controlled-pore glass beads and the enzyme-derivatized beads were packed in small columns. In the first procedure, the phospholipase C column was connected with a second column containing coimmobilized alkaline phosphatase and choline oxidase. In the alternative procedure, the column packed with immobilized phospholipase D was connected with a column packed with immobilized choline oxidase. The hydrogen peroxide produced through the action of choline oxidase in both flow-injection systems was detected amperometrically. Both procedures are suitable for an accurate and rapid quantitation of phosphatidylcholine. The sensitivity of the method based on phospholipase C and alkaline phosphatase is higher than that using phospholipase D. Quantitation of phosphatidylcholine at the nanomole level can be easily obtained using the first method.     

Calcium- and calmodulin-regulated breakdown of phospholipid by microsomal membranes from bean cotyledons

Evidence for the involvement of Ca²⁺ and calmodulin in the regulation of phospholipid breakdown by microsomal membranes from bean cotyledons has been obtained by following the formation of radiolabeled degradation products from [U-¹⁴C]phosphatidylcholine. Three membrane-associated enzymes were found to mediate the breakdown of [U-¹⁴C] phosphatidylcholine, viz. phospholipase D (EC 3.1.4.4), phosphatidic acid phosphatase (EC 3.1.3.4), and lipolytic acyl hydrolase. Phospholipase D and phosphatidic acid phosphatase were both stimulated by physiological levels of free Ca²⁺, whereas lipolytic acyl hydrolase proved to be insensitive to Ca²⁺. Phospholipase D was unaffected by calmodulin, but the activity of phosphatidic acid phosphatase was additionally stimulated by nanomolar levels of calmodulin in the presence of 15 micromolar free Ca²⁺. Calmidazolium, a calmodulin antagonist, inhibited phosphatidic acid phosphatase activity at IC₅₀ values ranging from 10 to 15 micromolar. Thus the Ca²⁺-induced stimulation of phosphatidic acid phosphatase appears to be mediated through calmodulin, whereas the effect of Ca²⁺ on phospholipase D is independent of calmodulin. The role of Ca²⁺ as a second messenger in the initiation of membrane lipid degradation is discussed.     

DIFFERENTIAL ACTIVITIES OF ACID PHOSPHATASE FROM ADAXIAL AND ABAXIAL REGIONS OF LUPINUS LUTEUS (FABACEAE) COTYLEDONS

Acid phosphatases of abaxial and adaxial regions in the cotyledons of the Lupinus luteus which possess structurally distinct protein bodies were examined. Acid phosphatase activity was investigated by enzyme assays and by gel electrophoresis and was localized by cytochemical methods in the cotyledons of Lupinus luteus L. during germination and seedling development. Acid phosphatase activity was significantly higher in the adaxial (heterogeneous protein body) region as compared to the abaxial (homogeneous protein body) region of the cotyledon. The pH optimum of acid phosphatase from the abaxial region and from the adaxial region was 4.5 and 5.0, respectively. There were significant differences in substrate specificity and isoenzymic composition of the enzyme between the two regions. Isoenzymic composition changed during the course of germination and seedling development. Acid phosphatase was localized in the matrix of the homogeneous protein bodies and in the globoids of the heterogeneous protein bodies at imbibition. After germination (d 3, d 4, d 7) acid phosphatase was localized primarily in the inner cell walls and intercellular spaces of both regions. These results show that different isoenzymes of acid phosphatase show differential localization and the rate of acid phosphatase activation or synthesis differs in cells from the two regions of the cotyledon.     

The role of choline on the activity-temperature relationship of brush-border alkaline phosphatase

We have studied the effect of choline on the activity and temperature dependency of the brush-border alkaline phosphatase isoenzymes from rat intestine (tissue-specific type), and from kidney and placenta (tissue-nonspecific type). The removal of choline with phospholipase D resulted in the loss of enzyme activity in all the membranes, whereas in situ loss in the discontinuity of Arrhenius plots occurred in the kidney and the placental membranes, but not in the intestinal membranes. The lost activity was restored either by addition of free choline or phosphatidylcholine or by the removal of the enzyme from the membrane surface. Intestinal enzyme was removed by papain, while the tissue-nonspecific enzyme was released by subtilisin and by phosphatidylinositol-specific phospholipase C. The enzyme from kidney and placental membranes aggregated (rho = 1.13) upon removal of choline, and addition of choline resulted in disaggregation (rho = 1.03). Conversion of discontinuous to continuous linear plots of alkaline phosphatase in the kidney and placental membranes paralleled the increase in membrane phosphatidic acid content, and the decrease in total phosphatidylcholines. The intestinal enzyme produced plots with break points at all phosphatidic acid/phosphatidylcholine ratios. The change brought about by treatment with phospholipidase D was not due to changes in the half-saturation kinetics (Km) for the substrate. Based on these studies we conclude that the active site of the tissue-nonspecific phosphatase is approximated to exterior membrane cholines, as in the case of the intestinal isoenzyme; that despite similar effects on the membrane content of phospholipids, phospholipase D treatment caused much greater effects on the tissue-nonspecific enzyme, as assessed by Arrhenius plots and density centrifugation; that these effects are due to different protein structures rather than to a lipid milieu unique to each brush-border membrane.     

A simplified assay for phospholipase C.

A new assay for phospholipase C activity that uses alkaline phosphatase to convert phosphorylcholine to inorganic phosphate is described. The determination of inorganic phosphate is performed in the presence of phosphatidylcholine and protein after the addition of sodium dodecyl sulfate. Phospholipase C activity determined by this coupled enzyme assay agrees well with data obtained by extracting and measuring phosphoryl[¹⁴C]choline produced from phosphatidyl[methyl-¹⁴C]choline. The assay is sensitive to 1 nmol of phosphate, requires no removal of protein or phospholipid, and will work with a variety of phospholipid substrates. The assay is faster and more sensitive than previously published procedures. Stimulation of phospholipase C from Clostridium perfringens by ammonium sulfate is also reported.     

Purification, Characterization and Cloning of Phospholipase D from Peanut Seeds

We purified phospholipase D (PLD) enzyme from peanut seeds, and the PLD enzyme eluted as two distinct peak fractions on Mono-Q chromatography, the first of which was characterized. N-terminal sequencing indicated that the N-terminus was blocked. The molecular mass of the purified enzyme was estimated to be 92 kDa by SDS-PAGE. The pH optimum of the enzyme was 5.0, and the K _m value against its substrate phosphatidylcholine (PC), in the presence of 10 mM CaCl₂ and 4 mM deoxycholate, was estimated to be 0.072 mM. The enzyme catalyzed two reactions, i.e., hydrolysis of PC generating phosphatidic acid (PA) and choline, and transphosphatidylation of the PA-moiety in the PC molecule to the acceptor glycerol, generating phosphatidylglycerol. Furthermore, we cloned two types of full-length cDNA, Ahpld1 and Ahpld2, each encoding distinct PLD molecules having 794 and 807 residues, respectively. The partial amino acid sequence of the purified PLD was consistent with the deduced sequence of AhPLD2.     

v-Src increases diacylglycerol levels via a type D phospholipase-mediated hydrolysis of phosphatidylcholine.

Activating the protein-tyrosine kinase of v-Src in BALB/c 3T3 cells results in rapid increases in the intracellular second messenger, diacylglycerol (DAG). v-Src-induced increases in radiolabeled DAG were most readily detected when phospholipids were prelabeled with myristic acid, which is incorporated predominantly into phosphatidylcholine. Consistent with this observation, v-Src increased the level of intracellular choline. No increase in DAG was observed when cells were prelabeled with arachidonic acid, which is incorporated predominantly into phosphatidylinositol. Inhibiting phosphatidic acid (PA) phosphatase, which hydrolyzes PA to DAG, blocked v-Src-induced DAG production and enhanced PA production, implicating a type D phospholipase. Consistent with the involvement of a type D phospholipase, v-Src increased transphosphatidylation activity, which is characteristic of type D phospholipases. Thus, v-Src-induced increases in DAG most likely result from the activation of a type D phospholipase/PA phosphatase-mediated signaling pathway.     

Phospholipase D/phosphatidic acid signal transduction: Role and physiological significance in lung

Phospholipase D (PLD), a phospholipid phosphohydrolase, catalyzes the hydrolysis of phosphatidylcholine and other membrane phospholipids to phosphatidic acid (PA) and choline. PLD, ubiquitous in mammals, is a critical enzyme in intracellular signal transduction. PA generated by agonist- or reactive oxygen species (ROS)-mediated activation of the PLD1 and PLD2 isoforms can be subsequently converted to lysoPA (LPA) or diacylglycerol (DAG) by phospholipase A₁/A₂ or lipid phosphate phosphatases. In pulmonary epithelial and vascular endothelial cells, a wide variety of agonists stimulate PLD and involve Src kinases, p-38 mitogen activated protein kinase, calcium and small G proteins. PA derived from the PLD pathway has second messenger functions. In endothelial cells, PA regulates NAD[P]H oxidase activity and barrier function. In airway epithelial cells, sphingosine-1-phosphate and PA-induced IL-8 secretion and ERK1/2 phosphorylation is regulated by PA. PA can be metabolized to LPA and DAG, which function as first- and second-messengers, respectively. Signaling enzymes such as Raf 1, protein kinase C and type I phosphatidylinositol-4-phosphate 5-kinase are also regulated by PA in mammalian cells. Thus, PA and its metabolic products play a central role in modulating endothelial and epithelial cell functions.     

Phospholipase D from Soybean (Glycine max L.) Suspension-Cultured Cells: Purification, Structural and Enzymatic Properties

Phospholipase D (phosphatidylcholine phosphatidohydrolase EC3.1.4.4 [EC] ) from soybean (Glycine max L.) suspension-cultured cellwas purified around 1,200-fold to homogeneity by acetone precipitation,Macro-Prep High Q anion exchange, and octyl-Sepharose CL-4Baffinity chromatography. The purified enzyme released 1,600µmol of choline per min per mg of protein. The enzymeis monomeric with a molecular mass of 92 kDa, as estimated bySDS-PAGE. One of the most interesting characteristics of thepurified soybean phospholipase D was the dependence of the pHoptimum on the Ca²⁺ ion concentration in the assay. With 10mM, 20 mM and 40 mM Ca²⁺ ions, the optima were at pH 7.5, 6and 5.5, respectively. The specific adsorption of phospholipaseD onto octyl-Sepharose gel suggests that the molecule becomesmore hydrophobic in the presence of Ca²⁺ ions. The amino acidsequence of the first 18 N-terminal residues of soybean phospholipaseD revealed a high degree of homology with those previously publishedfor cabbage leaf and castor bean endosperm enzymes. Westernblots of the soybean phospholipase D showed an immunoreactivitywith antibodies raised against a synthetic peptide correspondingto the 15 N-terminal aminoacid residues of phospholipase D fromcabbage leaves. (Received March 13, 1995; Accepted May 29, 1995)     

Phospholipase D/phosphatidic acid signal transduction: role and physiological significance in lung

Phospholipase D (PLD), a phospholipid phosphohydrolase, catalyzes the hydrolysis of phosphatidylcholine and other membrane phospholipids to phosphatidic acid (PA) and choline. PLD, ubiquitous in mammals, is a critical enzyme in intracellular signal transduction. PA generated by agonist- or reactive oxygen species (ROS)-mediated activation of the PLDI and PLD2 isoforms can be subsequently converted to lysoPA (LPA) or diacylglycerol (DAG) by phospholipase A1/A2 or lipid phosphate phosphatases. In pulmonary epithelial and vascular endothelial cells, a wide variety of agonists stimulate PLD and involve Src kinases, p-38 mitogen activated protein kinase, calcium and small G proteins. PA derived from the PLD pathway has second-messenger functions. In endothelial cells, PA regulates NAD[P]H oxidase activity and barrier function. In airway epithelial cells, sphingosine-1-phosphate and PA-induced IL-8 secretion and ERKI/2 phosphorylation is regulated by PA. PA can be metabolized to LPA and DAG, which function as first- and second-messengers, respectively. Signaling enzymes such as Raf 1, protein kinase Czeta and type I phosphatidylinositol-4-phosphate 5-kinase are also regulated by PA in mammalian cells. Thus, PA and its metabolic products play a central role in modulating endothelial and epithelial cell functions.     

Enzymatic determination of phospholipase D activity with choline oxidase

A new enzymatic method was developed for the assay of phospholipase D [phosphatidylcholine phosphatidohydrolase EC 3.1.4.4] from cabbage leaves using choline oxidase from Arthrobacter globiformis cells. The method was based on the estimation of choline by the following series of enzymatic reactions after ending the phospholipase D reaction: Choline + 202 + h2o Choline oxidase Betaine + 2H2O2 2H2O2 + Phenol + 4-Aminoantipyrine Peroxidase Quinoneimine dye + 4H2O The amount of choline was proportional to the amount of resulting quinoneimine dye with an absorbance maximum at 500 nm. The phospholipase D reaction (choline liberation) was carried out at pH 5.5 in the presence of Ca2+ ions and ended by adding EDTA in conc. Tris-HCl buffer, pH 8, to give a final pH of around 8. The initial rate of the phospholipase D reaction was proportional to the enzyme concentration over the absorbance change range of 0 to 0.25 (equivalent to 0-21 micron of choline) under the optimal reaction conditions.     

Evidence for a protein kinase C-directed mechanism in the phorbol diester-induced phospholipase D pathway of diacylglycerol generation from phosphatidylcholine

In this study we provide evidence for the involvement of protein kinase C (PKC) in phorbol diester-induced phosphatidylcholine (PC) hydrolysis by the phospholipase D pathway. Rat embryo fibroblasts (REF52) were prelabeled with either tritiated choline or myristic acid; these compounds are preferentially incorporated into cellular PC. Phorbol diester-induced PC degradation was determined by measuring the release of [3H]choline, and the formation of [3H]myristoyl-containing phosphatidate (PA), diacylglycerol (DG), and phosphatidylethanol (PE). Staurosporine, a PKC inhibitor, blocked from 73 to 90% of the phorbol diester-induced PC hydrolysis. The inhibition of phorbol diester-induced choline release by staurosporine was dose dependent with an approximate ED50 of 150 nM. Pretreatment of cells with phorbol diester inhibited subsequent phorbol diester-induced PC degradation by 78-92%. A close correlation between the ED50 for phorbol diester-stimulated choline release and the Kd for phorbol diester binding was demonstrated. Neither forskolin nor dibutyryl cAMP elicited cellular PC degradation. In vitro experiments using phospholipase D from Streptomyces chromofuscus showed that staurosporine did not inhibit and TPA did not stimulate enzyme activity.     

Partial purification and properties of an acid nucleotidase from the postmicrosomal supernatant of rat spleen

1. The dephosphorylation of 3′-AMP, 3′-dAMP, 3′-CMP and 3′-dCMP was studied in the postmicrosomal supernatant of rat spleen and liver. In both organs 3′-AMP and 3′-dAMP were dephosphorylated at an appreciable rate, in both the presence and the absence of Mg²⁺. The pH optimum for this dephosphorylation was in the range 4.5–5.0. 3′-CMP and 3′-dCMP were very slowly degraded, though the activity towards 3′-dCMP increased somewhat in the presence of Mg²⁺. The optimum pH for this Mg²⁺-dependent dephosphorylation was 5.5–6.0. 2. The rate of dephosphorylation of 3′-AMP and 3′-dAMP per mg of protein was about 5 times as high in spleen as in liver. 3. The dephosphorylation of 3′-AMP could be ascribed to a single enzyme with pH optimum about 4.5. The activity towards 3′-dAMP could be resolved into one component coinciding with the 3′-dAMP-degrading enzyme, and one Mg²⁺-requiring component probably identical with the soluble deoxyinosine-activated nucleotidase. The dephosphorylation of 3′-dCMP seemed to be performed only by the latter enzyme. 4. The enzyme dephosphorylating 3′-AMP was purified 200-fold from the postmicrosomal supernatant and its physical and catalytic properties were compared with those of acid nucleotidase (EC 3.1.3.31) purified from rat liver lysosomes. The two enzymes were identical in all properties tested (substrate specificity, K_m, molecular weight, response to phosphatase inhibitors), but some of the data differed from earlier reports on the acid nucleotidase. 5. The subcellular localization of the acid nucleotidase, its relationship to the acid phosphatase(s) and its role in the breakdown of nucleic acid constituents are discussed.     

In vitro synthesis of phosphatidylinositol and phosphatidylcholine by phospholipase D

Phosphatidylinositol (PI) was prepared from egg lecithin by a one-step transphosphatidylation reaction catalysed by phospholipase D in the presence of myo-inositol. Similarly phosphatidylcholine (PC) has been synthesized by the same technique from egg phosphatidylethanolamine using phospholipase D and choline chloride.The yield of PI was ca 25 % and that of PC ca 28 %. The transphosphatidylase function of phospholipase D offers a useful route for the synthesis of different classes of phospholipids.     

Biochemical characterization of phospholipase D activity from human neutrophils

We have found a phospholipase D activity in the postnuclear fraction of human neutrophils, employing phosphatidylinositol as exogenous substrate. This phospholipase D activity was assessed by both phosphatidate formation and by free inositol release in the presence of 15 mM LiCl in the reaction mixture and in the absence of Mg2+ ions to prevent inositol-1-phosphate phosphatase activity. To assess further the phospholipase D activity, we studied its capacity to catalyze a transphosphatidylation reaction, as a unique feature of the enzyme. It was detected as [14C]phosphatidylethanol formation when the postnuclear fraction was incubated with [14C]phosphatidylinositol in the presence of ethanol. The phospholipase D showed a major optimum pH at 7.5 and a minor one at pH 5.0. Neutral and acid phospholipase D activities were differentially located in subcellular fractionation studies of resting neutrophils, namely in the cytosol and in the azurophilic granules, respectively. Neutral phospholipase D required Ca2+ ions to the active, whereas the acid enzyme activity was Ca2(+)-independent. The neutral phospholipase D activity showed a certain specificity for phosphatidylinositol, as it was able to hydrolyze phosphatidylinositol at a much higher rate than phosphatidylcholine, in the absence and in the presence of different detergents. This neutral phospholipase D activity behaved as a protein of high molecular mass (350-400 kDa) by gel filtration chromatography. Moreover, neutral phospholipase D activity was detected in the postnuclear fraction of human monocytes, by measuring free inositol release from phosphatidylinositol as exogenous substrate, under the same experimental conditions as those used with neutrophils. The enzyme displayed similar specific activities in both cell types as well as the same degree of activation after cell stimulation with the calcium ionophore A23187. These results demonstrate the existence of two phospholipase D activities with different pH optima and intracellular location in human neutrophils. Furthermore, these results suggest that this phospholipase D can play a role in signal-transducing processes during cell stimulation in human phagocytes.     

Two uncommon phospholipase D isoenzymes from poppy seedlings (Papaver somniferum L.)

Phospholipase D (PLD) has been detected in seedlings of Papaver somniferum L. cv. Lazúr (Papaveraceae). Purification of the enzyme revealed the existence of two forms of PLD (named as PLD-A and PLD-B). The two enzymes strongly differ in their catalytic properties. The pH optima were found at pH 8.0 for PLD-A and at pH 5.5 for PLD-B. While both enzymes show hydrolytic activity toward phosphatidylcholine (PC) and phosphatidyl-p-nitrophenol (PpNP), PLD-B only was able to catalyze the exchange of choline in PC by glycerol. Both enzymes were activated by Ca²⁺ ions with an optimum concentration of 10 mM. In contrast to PLDs from other plants, PLD-B was still more activated by Zn²⁺ ions with an optimum concentration of 5 mM. The apparent molecular masses of PLD-A and PLD-B, derived from sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), were estimated to be 116.4 and 114.1 kDa. N-terminal protein sequencing indicated N-terminal blockage in both cases. The isoelectric points were found to be 8.7 for PLD-A and 6.7 for PLD-B. Both enzymes were shown to be N-linked glycoproteins. This paper is the first report on PLD in poppy and indicates some important differences of the two enzyme forms to other PLDs known so far.     

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

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.