Modulation of Phosphatidylinositol-specific Phospholipase C Activity by Phospholipid Interactions, Diglycerides, and Calcium Ions*

We have investigated the effects of different lipids on the activity of a phosphatidylinositol (PI)-specific phos- pholipase C isolated from sheep seminal vesicular glands. Dispersions of PI in the absence of detergent are hydrolyzed at 1-3 pmol/min/mg of protein, a rate only 3-10% of that obtained when optimal concentra- tions of sodium deoxycholate are present. When hydrolysis of PI in microsomes from mouse L-cells is measured, only 0.01 pmol of PI is hydrolyzed/min/mg of protein. Lipid dispersions prepared from extracts of microsomes are also poor substrates. We added varying amounts of other phospholipids to PI dispersions to explore the effect on PI hydrolysis. Phosphatidylserine (PS) and phosphatidylethanolamine (PE) had only mod- est effects on PI hydrolysis, but phosphatidylcholine (PC) was markedly inhibitory. Diglycerides stimulated PI hydrolysis in dispersions by about 10-fold. Using small unilamellar vesicles containing PI and PE (1:0.4), we found 50 pmol of PI hydrolyzed/min/mg of protein. Incorporation of PC in increasing amounts into these vesicles inhibited PI hydrolysis by 75% at 1 PC/PI, and at 4 PC/PI, no hydrolysis occurred. PC incorporated into vesicles separate from those containing PI was not inhibitory. Incorporation of PS into vesicles containing PI and PC overcame the inhibition of PI hydrolysis in a calcium ion- and PS-dependent

We have investigated the effects of different lipids on the activity of a phosphatidylinositol (PI)-specific phospholipase C isolated from sheep seminal vesicular glands. Dispersions of PI in the absence of detergent are hydrolyzed at 1-3 pmol/min/mg of protein, a rate only 3-10% of that obtained when optimal concentrations of sodium deoxycholate are present. When hydrolysis of PI in microsomes from mouse L-cells is measured, only 0.01 pmol of PI is hydrolyzed/min/mg of protein. Lipid dispersions prepared from extracts of microsomes are also poor substrates. We added varying amounts of other phospholipids to PI dispersions to explore the effect on PI hydrolysis. Phosphatidylserine (PS) and phosphatidylethanolamine (PE) had only modest effects on PI hydrolysis, but phosphatidylcholine (PC) was markedly inhibitory. Diglycerides stimulated PI hydrolysis in dispersions by about 10-fold. Using small unilamellar vesicles containing PI and PE (1:0.4), we found 50 pmol of PI hydrolyzed/min/mg of protein.
Incorporation of PC in increasing amounts into these vesicles inhibited PI hydrolysis by 75% at 1 PC/PI, and at 4 PC/PI, no hydrolysis occurred. PC incorporated into vesicles separate from those containing PI was not inhibitory. Incorporation of PS into vesicles containing PI and PC overcame the inhibition of PI hydrolysis in a calcium ion-and PS-dependent manner. PI hydrolysis at 3 m M Ca2' using vesicles containing PI/PC/PS/PE in proportions of 1:4:2:0.4 was 2.5 pmol/min/mg of protein, compared to undetectable hydrolysis in similar vesicles without PS. Addition of diglyceride further increased the rate of hydroIysis by 2.5-fold. We conclude that PC inhibits PI hydrolysis by interacting with the substrate. The inhibition is overcome by negatively charged lipids and high concentrations of calcium ions. Diglyceride, a product of PI hydrolysis, further stimulates PI breakdown. Since negatively charged lipids are concentrated on the inner leaflet of most cell membranes, we postulate that calcium fluxes resulting from stimulation of cell surface receptors may initiate PI breakdown which is further accelerated as diglyceride accumulates.
The physiological significance of agonist-stimulated PI' breakdown has not been fully elucidated. Recent experiments in platelets (1) and other tissues (2, 3) indicate that 1,2diglyceride, which is produced from PI by the action of a PIspecific phospholipase C following stimulation, is further degraded to release arachidonic acid for conversion to prostaglandins, thromboxanes, leukotrienes, and other arachidonate metabolites that are collectively designated as eicosanoids (4). In addition, diglyceride itself has been shown to stimulate a CAMP-independent protein kinase present in membranes (5).
The availability of free arachidonate controls the rate of synthesis of arachidonate metabolites. PI-specific phospholipase C is thought to regulate arachidonate release from PI, partly because agents that elevate platelet CAMP levels inhibit both PI breakdown and arachidonate release (6) and because the enzyme requires calcium ions for activity. Calcium fluxes are important in the activation of platelets and other cells. The release of free calcium ions from platelet intracellular storage granules following thrombin stimulation (7) may allow the PI-specific phospholipase C to hydrolyze PI in the membrane and thus trigger arachidonate release.
PI-specific phospholipase C activity has been described in crude extracts from many mammalian tissues (8). We have recently identified two immunologically distinct PI-specific phospholipase C enzymes in sheep seminal vesicles and have purified one of them to homogeneity. While the enzyme readily metabolizes pure dispersions of PI, the PI in cellular membranes is a poor substrate. These findings contrast with studies of bacterial PI-specific phospholipac C enzymes, which do not require calcium ions and readily hydrolyze PI from cell membranes. Imine et al. (9) have studied the ability of the mammalian enzyme in crude brain homogenate to hydrolyze PI in various lipid dispersions and have concluded that PC inhibits the enzyme and that acidic phosphoIipids and negatively charged amphiphiles overcome the inhibition. In the current report, we have investigated the effects of various lipids on homogeneous PI-specific phospholipase C using SUV of defined lipid composition. We find that PC inhibits enzyme activity by "masking" substrate PI. The inhibition is overcome by increasing the concentration of PS in the vesicles in a manner which is dependent on calcium ions. Diglycerides further enhance enzyme activity, suggesting that this product of PI-specific phospholipase C may have a physiological role in enhancing the rate of PI breakdown.
PI-specific Phospholipase C I from Sheep Seminal Vesicles-Purification was carried out as previously described (13). Purification steps included a sulfated dextran batch separation, aminohexyl Sepharose chromatography, gel filtration using Sephadex G-160, and heparin-Sepharose chromatography. The enzyme preparation was homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Assay of PI-specific Phospholipase C Actiuity-When PI hydrolysis was measured in sonicated dispersions of PI with other lipids, the lipids were first mixed in chloroform/methanol solution (or ether in the case of diglycerides to avoid isomerization) and then dried under a stream of ND. Buffer was added and the samples were sonicated for 15 s X 50 watts using a Branson Sonifier with a small probe just prior to use. Reaction mixtures contained 250 p~ soybean PI, 60,000-100, OOO dpm of [3H]PI (specific activity, 600-1,000 dpm/pmol), 1 mM CaC12, 0.5 mg/ml of bovine serum albumin, 100 mM NaC1, 100 mM Tris-maleate buffer, pH 6.7, appropriate amounts of enzyme, and other lipids as described in the figure legends in a total volume of 200 pl. After 10 min at 37 "C, 1 ml of chloroform/methanol/HCl (100:1000.6) was added, followed by 0.3 mi of 1 N HCI containing 5 mM EGTA. The aqueous and organic phases were separated by centrifugation, and a 400-pI portion of the upper aqueous phase was removed for liquid scintillation counting. This assay was linear with respect to enzyme concentration when less than 15% of the substrate was hydrolyzed during the reaction. We define 1 unit of enzyme activity to be that amount which cleaves 1 pmol of PI/min. Assays involving SUV were performed in a manner similar to those described above, except that reaction mixtures contained enzyme and SUV in a buffer containing 50 mM Hepes, pH 7.0, 100 mM NaC1, 0.5 mg/ml of bovine serum albumin, and CaCL as indicated. The CaC12 was added to the reaction mixture at 37 "C 2 min prior to the addition of enzyme. These assays were performed for 2 min unless otherwise noted. Assays performed with very low concentrations of enzyme (0.1-0.2 ng/mI) were done in polystyrene tubes to minimize the slow absorption of enzyme to glass tubes.
Preparation of Single Bilayer Vesicles (SUV)-Stock solutions of individual lipids were mixed in the desired molar proportions in glass vials and dried for 30 min under a stream of nitrogen. One or two milliliters of 50 mM Hepes, pH 7.0, 100 mM NaCI were added, and the mixture was vortexed for several minutes. The final concentration of phospholipid was 0.3-2 mM. Nitrogen gas was bubbled through the solution, and the vials were flushed with nitrogen and tightly stoppered. The samples were sonicated using an ultrasonic cleaner (Cole Parmer, Model 8849) for 1 h. The temperature did not exceed 42 "C during sonication. Samples were then centrifuged at 134,000 X g,, for 90 min, and the optically clear supernatant was carefully removed. Usually 185% of the phospholipid was recovered in the supernatant. This method of preparation yields small unilamellar vesicles that are mostly homogeneous in size (14). Samples were stored at room temperature and used within 24-48 h.
Preparation of Labeled S~bstrates-[~H]InositoI-labeled PI was prepared from ['H]inositol-fed LM-cells and purified by high performance liquid chromatography as described (13). Microsomes were prepared from [3H]inositol-labeled LM-cells as follows. LM-cells were grown to a density of 2-3 X lo6 cells/ml in inositol-free Higuchi (15) medium containing 0.6 mCi of [3H]inositol and 5 pmol of supplemental arachidonic acid in a total volume of 180 ml. Incorporation of label into the cells was 90%. The cells were pelleted at low speed (320 X g X 8 min), washed three times in 50 mM Hepes, pH 7.0, 100 mM NaCI, and resuspended in 16 mi of this buffer. Following sonication for 3 X 20 s at 50 watts on ice, the resulting solution was centrifuged for 5 min at 10,000 X g to remove unbroken cells and nuclei, and the pellet was discarded. The supernatant was centrifuged at 100,ooO X g X 45 min, and the resulting pellet was washed with 3 X 16 mi of cold buffer. The pellet at each stage of the washing was resuspended by brief sonication. The final pellet was resuspended in 1.5 ml of buffer and about 7.5 pmol of phospholipid and 8 mg of protein and 2.5 X IO8 dpm stored at -80 "C. This preparation yielded microsomes containing of [3H]inositol, 97.0% of which are in PI. Phospholipids were extracted from a portion of these microsomes using chloroform/methanol/KCl/ EDTA as described by Cohen et al. (16) for use in some experiments.

RESULTS
We initially investigated the rate of PI hydrolysis using our homogeneous seminal vesicle PI-specific phospholipase C with sonicated dispersions of PI with or without other phospholipids. When PI is dispersed by brief sonication in the absence of detergent at pH 7.0,l-3 pmol of PI are hydrolyzed/ min/mg of protein as shown in Table 11. The rate of hydrolysis is stimulated 10-fold by the addition of the anionic detergent sodium deoxycholate, which presumably forms mixed micelles with the substrate (Table 11 and Ref. 13). When P I is dispersed in the presence of arachidonate or other fatty acids (9), hydrolysis is similar to that obtained using deoxycholate (Table 11). Addition of varying proportions of P E or PS had minimal effects on PI hydrolysis (data not shown). PC markedly inhibited PI hydrolysis as illustrated below. An unexpected result was that a diglyceride (1,2-diolein) enhanced the rate of PI hydrolysis, as shown in Table 11. This observation is of potential physiological significance since diglyceride is a product of the PI-specific phospholipase C.
Both 1,2and 1,3-diglyceride stimulated PI hydrolysis as shown in Fig. 1. The inhibition of PI hydrolysis observed at high 1,2-diolein concentrations was associated with the formation of a precipitate in reaction mixtures. Mixtures of 1,2diarachidonin also stimulated hydrolysis but, did not inhibit (or precipitate) a t higher concentrations. Other neutral lipids including 1,2-distearin, triolein, and 1-monoolein only slightly stimulated the rate of PI hydrolysis. The ability of diglyceride   to enhance PI hydrolysis is due to an effect on the substrate rather than the enzyme since the optimal activation of hydrolysis varies with substrate concentration at constant amounts of enzyme, as shown in Table 111. Approximately 1 diglyceride molecule/7 PI molecules gives half-maximal activation over a wide range of substrate concentrations. Diglyceride is more potent than deoxycholate under these conditions since approximately 3 deoxycholate molecules/PI give similar stimulation of hydrolysis.

Hydrolysis of PI in Microsomal Membranes and Lipids-
PI was hydrolyzed poorly when microsomes derived from LMcells were used as substrate, as shown in Fig. 2 A . In this case, hydrolysis was stimulated by calcium ions at much higher concentrations than in dispersions of P I where calcium concentrations above 1.0 mM were inhibitory. The initial rate of PI hydrolysis at 50 mM calcium ions was approximately 0.01 pmol of PI hydrolyzed/min/mg of protein. The slow PI hydrolysis was caused by lipid interactions rather than by masking of P I by proteins because dispersions of extracted microsomal lipids give results similar to intact microsomes, as shown in Fig. 2B. In this case, high concentrations of calcium ions were also stimulatory, with an initial rate of hydrolysis of approximately 0.02 pmol of PI hydrolyzed/min/mg of protein at 50 mM calcium ions. The effect of e a 2 + is not merely due to increased ionic strength because increasing NaCl concentrations up to 0.75 M had little effect on PI hydrolysis (data not shown). When diolein was added to intact microsomes, there was a 2-fold enhancement in the rate of PI hydrolysis (Fig.   3A) even though the access of diglyceride to microsomal phospholipids under these conditions is probably minimal. When 30 p~ diolein was added to microsomal lipids, there was a 3-fold stimulation of PI hydrolysis (Fig. 3B) which was not increased by higher diolein concentrations.

PI Hydrolysis in Small Unilamellar
Vesicles-The highest rates of PI hydrolysis were observed using SUV (Table 11). PI with PE in a ratio of 1:0.4 is rapidly hydrolyzed (13-30 pmol/ min/mg at 60 p~ PI) until 60-70% of the total PI is hydrolyzed. Although 60 p~ PI is less than saturating, this concentration was used in all subsequent experiments in order to conserve material. When the SUV concentration was raised to 250 PM PI, approximately 2-fold greater rates of hydrolysis were observed. Vesicles prepared using only PI were equally rapidly hydrolyzed but were unstable. Therefore, PE was included in all vesicle preparations. Inclusion of PC in PI/PE vesicles inhibited PI hydrolysis by 75% in SUV containing 1 PC/PI; PI hydrolysis was completely inhibited in SUV containing 4 PC/PI, as shown in Fig. 4. When PC-containing vesicles were added to PI/PE vesicles, there was almost no effect on PI hydrolysis, indicating that PC and PI must be in the same

FIG. 2.
Time course of PI cleavage in intact L"cel1 microsomes (A) or in extracted microsomal lipids (B) and effect of added calcium. The enzyme concentration was 4.6 pg/ml, and the total phospholipid concentration was 0.15 mM. We determined that PI accounts for 5.3 +-1.2% of the total phospholipid in these microsomal preparations by high performance liquid chromatography as described under "Experimental Procedures." Therefore, the PI concentration is approximately 8 p~ (compare to PI/PE vesicles at this concentration, 1.5 units/mg at 1 mM Ca"). The buffer contained 50 mrd Hepes, pH 7.0, 100 m M NaCl, and 0.5 mg/ml of bovine serum albumin. A free Ca2' concentration of approximately 4 PM was attained using a CaClz/EGTA buffer ([CaCL]/[EGTA] = 0.9) as previously described (13). The other components of the assay mixture were then added and the mixture was sonicated for 15 s X 50 watts. In B. the microsomal lipids and 1,2-diolein (D.O.) were mixed first in organic solvent and the assays were performed as described above.  (1:0.4). The PI concentration in each assay was 50 VM, the enzyme concentration was 0.115 pg/ml, and the total calcium concentration was 1 mM. Assays were conducted for 5 min at 37 "C. vesicle in order for PC to be inhibitory. It appears that PC inhibits PI hydrolysis by masking substrate PI rather than by affecting the enzyme. Calcium ion concentrations above 1 mM did not stimulate PI hydrolysis in PC/PI/PE vesicles. In contrast, rates of PI hydrolysis using SUV made from LMcell microsomal lipids were 0.03, 0.05, and 0.06 pmol of PI/ min/mg of protein at 1, 10, and 50 mM CaC12, respectively. PC/PE vesicles do not inhibit hydrolysis of ['HIPI in PI/ P E vesicles, as shown in Fig. 5. In fact, PC/PI/PE vesicles also failed to inhibit, indicating that the enzyme has no apparent affinity for the unlabeled PI in the PC-containing vesicles, as compared to unlabeled PI/PE vesicles which were inhibitory (Fig. 5). PC/PS/PE-containing vesicles were only slightly inhibitory, indicating that the enzyme does not have a high affinity for the acidic phospholipid PS in PC-containing vesicles. However, when PS was incorporated into vesicles containing PC/PI/PE, there was a marked increase in PI hydrolysis from undetectable to 2 pmol of PI hydrolyzed/min/ mg of protein at 0.5 PS/PI, as shown in Fig. 6. The rate of PI  ., 2 PS and 1 DG; A, 2 PS and 2 DG. The PI concentration in all assays was 60 p~. Enzyme concentration was 0.46 Fg/ml, and assays were conducted for 2 min at 37 "C.

Modulation of PI-specific Phospholipase C Activity
hydrolysis was further stimulated in vesicles containing additional PS, and the reaction was dependent on high calcium ion concentrations a t higher proportions of PS. Above 3 mM calcium ions, the vesicles aggregated and precipitated. Similar results were seen using SUV a t 250 p~ PI (data not shown). The ability of PS to overcome inhibition by PC is not due to a direct effect of PS on either enzyme or PI since PS in vesicles without PC (Le. PI/PE/PS) did not stimulate PI hydrolysis, but was actually inhibitory (data not shown). 1,2-Diolein did not stimulate PI hydrolysis unless the vesicles contained PS, as shown in Fig. 7. When PS was present, there was a further 2.5-fold stimulation of PI hydrolysis by diolein.
Using SUV at 250 p~ PI, the same degree of stimulation was seen (data not shown).
We next constructed vesicles that mimic the composition of the cytoplasmic leaflet of the platelet (or other cell) membrane bilayer (17). Using vesicles containing PC/PS/PI/PE (4:2:1:3), we found 4 pmol of P I hydrolyzed/min/mg of protein at 3 mM calcium ions, as shown in Fig. 8. Cholesterol had no effect on the rate of P I hydrolysis. This rate of P I hydrolysis is 100-fold higher than the rate observed when SUV are made from phospholipids extracted from LM-cells or platelets. The high concentration of acidic lipids on the inner face of membranes may be important for PI-specific phospholipase C activity.

DISCUSSION
PI breakdown occurs rapidly when platelets are activated by thrombin. The PI-specific phospholipase C appears to be equally active when assayed in crude extracts from thrombinstimulated uersus unstimulated platelets.' Studies of the distribution of phospholipids of the platelet membrane indicate that PI is contained almost exclusively on the inner leaflet (17,18). In unstimulated cells, the cytoplasmic PI-specific phospholipase C is prevented from hydrolyzing PI. In the current work, we have investigated the effects of various lipids on PI hydrolysis. The most informative experiments are those using SUV, where the lipids are available in a fairly uniform state. We find that PI is rapidly hydrolyzed in such vesicles unless they contain PC. When the PC content approaches that seen in platelets and other cells, PI hydrolysis is completely inhibited. PC inhibits by preventing the enzyme from interacting with P I because PC in separate SUV has no effect on PI hydrolysis. It is unlikely that the effect of PC is due to the sequestration of PI inside the SUV because PC is also observed to inhibit PI hydrolysis in monolayers of PI at an air-water interface (19). Addition of PS to vesicles in concentrations that are found in the inner membrane leaflet of platelets and other cells overcomes the inhibition by PC. The addition of diglyceride, a product of P I hydrolysis, further enhances activity. PS may be effective by increasing the negative charge at the vesicle surface and increasing the binding of calcium ions. PS may also promote calcium-de-' S. L. Hofmann and P. W. Majerus, unpublished observations pendent lateral phase separation of PC and the acidic lipids PI and PS, thereby relieving inhibition. Jacobson and Papahadjopoulos (20) and others (21,22) have demonstrated such phase separations in mixtures of PC and PS. Diglycerides may also enhance such phase separations, thereby increasing activity (23). Thus, we postulate that PI breakdown may be triggered in cells by the agonist-induced release of calcium ions. The rise in cytoplasmic calcium may cause a redistribution of lipids, thereby transforming PI to a state where it is accessible for hydrolysis. As PI is hydrolyzed, local 1,2-diglyceride accumulation may further enhance the rate of hydrolysis. Hydrolysis then ceases when all available PI is hydrolyzed. The failure to find significant rates of PI hydrolysis using microsomes or total membrane lipid extracts may result from the fact that the total concentration of acidic lipids in such mixtures is insufficient to allow hydrolysis. Irvine et al. (9,24) and Dawson et al. (25) have also studied the effects of various lipids on PI hydrolysis by PI-specific phospholipase C present in crude extracts of brain. They also found inhibition by PC similar to that reported here. The effects that they observed with other lipids are quantitatively different from our results. However, it is difficult to compare the studies since initial rates of PI hydrolysis are not measured, the methods for preparation of lipids are different, and other proteins present in the crude brain extract used may modify the results.
Another factor that might affect the rate of PI hydrolysis is the fatty acid composition of various lipids. The lipids used in our experiments were not of defined fatty acid composition, although they contained similar proportions of saturated and unsaturated fatty acids which would probably result in relatively uniform dispersions of the different lipids in vesicles. It is possible that particular phospholipids of a specific fatty acid composition will prove to be better activators or inhibitors. The fatty acid composition of PI itself is unlikely to affect the rate of hydrolysis. In thrombin-stimulated platelets, all species of PI appear to be hydrolyzed equally (11). The seminal vesicle enzyme used in the current study hydrolyzes lyso-PI a t nearly the same rate as PI, indicating that the 2-position fatty acid is unimportant (data not shown).