Characterization of phospholipase C-mediated phosphatidylinositol degradation in rat heart ventricle

Phosphoinositide-specific phospholipase C (PI-PLC) activity was investigated in the rat heart ventricle. Incubation of ventricle homogenate or 100,000g supernatant fraction with [3H]myoinositol or [3H]arachidonate-labeled phosphatidylinositol in the presence of Ca2+ resulted in a decrease in phosphatidylinositol with a concomitant increase in water-soluble [3H]inositol phosphate or [3H]diglyceride, respectively. Total overt homogenate PI-PLC activity could be accounted for in the supernatant fraction. Neutral, zwitterionic, cationic, or anionic detergents did not unmask membrane-associated activity. While cytosolic phospholipase C was active against a pure phosphatidylinositol substrate in the presence of Ca2+, no hydrolytic activity was detected when phosphatidylinositol was presented as a component (4-5%) of a mixture of phospholipids. However, addition of deoxycholate to the incubation mixture (pH 6.5, Ca2+ 10(-3) M) containing mixed phospholipids resulted in the exclusive hydrolysis of inositol phospholipids. Ventricular supernatant phospholipase C-mediated phosphatidylinositol degradation has a sharp pH optimum at 5.5 and a specific requirement for Ca2+. Activity is maximal at 1 to 2 X 10(-3) M Ca2+, with inhibition occurring at higher levels. Under optimized conditions phosphatidylinositol is hydrolyzed at a rate of 20-25 nmol/min/mg protein. Multivalent cations inhibit Ca2+-dependent PI-PLC activity while monovalent cations and anions have no effect. There is no apparent selectivity for specific fatty acid moieties on phosphatidylinositol. Soluble PI-PLC is inhibited by sulfhydryl reagents, neomycin, mepacrine, trifluoperazine, and propranolol. Chlorpromazine, dibucaine, and tetracaine exert a biphasic influence, stimulating at lower and inhibiting at higher concentrations.     

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.     

Studies on the properties of a soluble phosphatidylinositol-phosphodiesterase of rabbit iris smooth muscle

Some properties of the soluble phosphatidylinositol phosphodiesterase (monophosphatidylinositol inositolphosphohydrolase, EC 3.1.4.10) of rabbit iris smooth muscle are described. Studies on its subcellular distribution showed that in this tissue the phosphodiesterase is not exclusively cytosolic. Thus, under our experimental conditions about 58% of the enzyme activity was found in the soluble fraction and the remainder was particulate. When the latter was treated with deoxycholate about 59% of the enzyme activity, compared to 86% of that of ATPase, was still bound to the particulate fraction. The kinetic properties of the enzyme (30--50% (NH4)2SO4 fraction) were examined. Maximum breakdown was 7.7 mumol/h per mg protein and occurred at pH 5.6. The products of [14C]arachidonic acid-labelled phosphatidylinositol were 1,2-diacylglycerol and a mixture of 86% myoinositol 1-phosphate and 14% myoinositol 1,2-(cyclic)phosphate. The enzyme has an absolute requirement for Ca2+. Addition of Ba2+, La3+, Mg2+, Mn2+, EGTA or EDTA at 0.05--5 mM concentrations; Sr2+ at higher concentrations (greater than 0.25 mM) markedly inhibited the phosphodiesterase activity and this inhibition was completely reversed by Ca2+. The enzyme is specific for the phosphoinositides.     

Phosphatidylinositol-specific phospholipase C of murine lymphocytes

Phosphatidylinositol-specific phospholipase C (PI-phospholipase C) was found primarily in the cytosolic fraction of murine splenic lymphocytes. However, small but significant amounts of the activity of the enzyme were detected in the microsome and plasma membrane fractions. Both the cytosolic and membrane-bound phospholipases C specifically hydrolyzed inositol phospholipids, phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate. PI-Phospholipase C activity was detected in the cytosolic and microsome fractions from both T-cell-enriched and B-cell-enriched spleen cells. The membrane-bound enzyme was distinguishable from the cytosolic enzyme in the following properties. The cytosolic PI-phospholipase C showed optimal activity at pH 6.0 while the membrane-bound enzyme had two pH optima between pH 5.0 and 7.0. The activity of the cytosolic enzyme was first detected at 1 microM Ca2+, and maximum activity was observed at 100 microM Ca2+, while the membrane-bound PI-phospholipase C required higher Ca2+ concentrations, of millimolar order. The membrane-bound enzyme could hardly be extracted with 1 M NaCl but was extracted with 0.4% cholate.A portion of the membrane-bound PI-phospholipase C activity in the cholate extract was absorbed by concanavalin A-Sepharose and specifically eluted with an alpha-methylmannoside solution. The cytosolic enzyme, which was water soluble, did not bind to concanavalin A-Sepharose. Trypsinization of lymphocytes before subcellular fractionation caused a significant decrease in the PI-phospholipase C activity in the microsome fraction but almost no loss at all of the cytosolic enzyme activity.     

Ca2+-sensitive phosphatidylinositol 4-phosphate metabolism in a rat beta-cell tumour.

Subcellular fractions were isolated from a rat beta-cell tumour by centrifugation of homogenates on Percoll and Urografin density gradients. Fractions were incubated with [gamma-32P]ATP, and labelling of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate was used to measure phosphatidylinositol kinase and phosphatidylinositol 4-phosphate kinase activities, respectively. The distribution of enzyme markers in density gradients indicated that phosphatidylinositol kinase was located in both the plasma membrane and the secretory-granule membrane. Phosphatidylinositol 4-phosphate kinase activity was low in all fractions. Phosphatidylinositol kinase activity of secretory granules and plasma membranes was decreased to 10-20% of its initial value by raising the free [Ca2+] from 1 microM to 5 microM. The enzyme had a Km (apparent) for ATP of 110 microM (secretory granule) or 120 microM (plasma membrane) and a Ka for Mg2+ of 7 mM (secretory granule) or 6 mM (plasma membrane). Ca2+-sensitivity of phosphatidylinositol kinase in calmodulin-depleted secretory granules and plasma membranes was not affected by addition of exogenous calmodulin, although activity was stimulated by trifluoperazine in the presence of 0.1 microM or 40 microM-Ca2+. Trifluoperazine oxide had no effect on the enzyme activity of secretory granules. Plasma membranes had a phosphatidylinositol 4-phosphate phosphatase activity which was stimulated by raising the free [Ca2+] from 0.1 to 40 microM. The secretory granule showed no phosphatidylinositol 4-phosphate-degrading activity. These results suggest the presence in the tumour beta-cell of Ca2+-sensitive mechanisms responsible for the metabolism of polyphosphoinositides in the secretory granule and plasma membrane.     

Purification and characterization of a phosphoinositide-specific phospholipase C from guinea pig uterus. Phosphorylation by protein kinase C in vivo

Two peaks of phosphoinositide-specific phospholipase C (PI-PLC) activity were resolved when guinea pig uterus cytosolic proteins were chromatographed on a DEAE-Sepharose column. The first peak of enzyme activity eluting from the DEAE-Sepharose column (PI-PLC I) was further purified to homogeneity, whereas the second peak of enzyme activity was enriched 300-fold. PI-PLC I migrated as a 62-kDa protein on sodium dodecyl sulfate-polyacrylamide gels. Antibodies prepared against PI-PLC I failed to react with PI-PLC II. PI-PLC I hydrolyzed all three phosphoinositides, exhibiting a greater Vmax for phosphatidylinositol 4,5-bisphosphate greater than phosphatidylinositol 4-phosphate greater than phosphatidylinositol. Hydrolysis of phosphatidylinositol was calcium-dependent, whereas significant hydrolysis of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate occurred in the presence of 2.5 mM EGTA. At physiological concentrations of calcium, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate were the preferred substrates. Antibodies specific for PI-PLC I reacted with a 62-kDa protein in both the cytosol and membrane fractions from guinea pig uterus. Quantitation of the immunoblots revealed that 25% of the 62-kDa protein was membrane-associated, whereas only 5% of the total enzyme activity was membrane-associated. Approximately 20% of the membrane-bound phospholipase C activity and immunoreactive material were loosely bound, whereas the remainder required detergent extraction for complete solubilization. The 62-kDa protein associated with the membrane fractions did not bind lectin affinity columns, suggesting that it was not glycosylated. PI-PLC I was identified as a phosphoprotein in [32P]orthophosphate-labeled rat basophilic leukemia (RBL-1) cells by two-dimensional gel electrophoresis followed by immunoblotting. In untreated cells, 32P-labeled PI-PLC I was found in the cytosolic fraction. Treatment of RBL-1 cells with those phorbol esters which are known to activate the Ca2+/phospholipid-dependent enzyme protein kinase C, resulted in a time-dependent increase in the phosphorylation of both membrane-bound and cytosolic PI-PLC I. Thus, in RBL-1 cells, protein kinase C may play an important role in the regulation of phospholipase C through protein phosphorylation.     

Polyphosphoinositide phospholipase C in wheat root plasma membranes. Partial purification and characterization.

The effect of various detergents on polyphosphoinositide-specific phospholipase C activity in highly purified wheat root plasma membrane vesicles was examined. The plasma membrane-bound enzyme was solubilized in octylglucoside and purified 25-fold by hydroxylapatite and ion-exchange chromatography. The purified enzyme catalyzed the hydrolysis of phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) with specific activities of 5 and 10 mumol/min per mg protein, respectively. Phosphatidylinositol (PI) was not a substrate. Optimum activity was between pH 6-7 (PIP) and pH 6-6.5 (PIP2). The enzyme was dependent on micromolar concentrations of Ca2+ for activity, and millimolar Mg2+ further increased the activity. Other divalent cations (4 mM Ca2+, Mn2+ and Co2+) inhibited (PIP2 as substrate) or enhanced (PIP as substrate) phospholipase C activity.     

The metabolism of phosphatidylinositol in the thyroid gland of the pig

1. The metabolism of phosphatidylinositol in pig thyroid has been investigated as a basis for understanding the specific stimulation of the synthesis of this phospholipid in the gland by thyrotropin. 2. The gland contained an active Ca(2+)-dependent phosphatidylinositol-splitting enzyme with an optimum pH of 5.3-5.5. 3. The major water-soluble product (65%) formed by this catabolic enzyme was not phosphorylinositol but a related compound, which may be a cyclic phosphorylinositol. Both this and phosphorylinositol (35%) were released simultaneously from the phosphatidylinositol substrate. 4. The phosphatidylinositol-splitting enzyme was found almost exclusively in the supernatant fraction obtained by homogenization of the gland. It was not present in the acid-phosphatase-containing particulate fraction. 5. The incorporation of [2-(3)H(1)]inositol into phosphatidylinositol in the presence of either CDP-diglyceride or CTP+ATP was most active in the microsomal fraction. 6. When thyroidal microsomes were labelled with [(3)H]inositol and (32)P, and then incubated with unlabelled inositol, there was a dramatic loss of (3)H labelling from the phosphatidylinositol, which was not accompanied by an equivalent loss of (32)P from the phosphate moiety. This turnover of the inositol moiety required nucleotide coenzymes. It is postulated that the phosphatidylinositol is split into inositol and a phosphorus-containing lipid precursor of the phospholipid that remains on the microsomal membrane and is recycled. 7. Isolated thyroidal mitochondria synthesized phosphatidylinositol from [2-(3)H(1)]inositol only because of their contaminating microsomal component. 8. Some evidence has been obtained of a rapid transfer of phosphatidylinositol molecules from thyroidal microsomes to mitochondria when these were incubated together in the presence of a supernatant fraction. 9. Both phosphatidylinositol breakdown by the supernatant fraction of the gland and synthesis by the microsomes were totally inhibited by 1mm-chlorpromazine. This drug is known to suppress thyrotrophin-induced stimulation of activity in thyroid slices.     

Phospholipase C from human sperm specific for phosphoinositides

Human sperm lysates were incubated in the presence of 1-[14C]stearoyl-2-acyl-sn-glycero-3-phosphocholine, 1-[14C]stearoyl-2-acyl-sn-glycero-3-phosphoethanolamine or 1-[14C]stearoyl-2-acyl-sn-glycero-3-phosphoinositol. Only the latter substrate was hydrolyzed to a significant extent, with a concomitant formation of 1-[14C]stearoyl-2-acyl-sn-glycerol. Furthermore, incubation of phosphatidyl[3H]inositol under the same conditions was accompanied by the formation, in roughly equal amounts, of [3H]inositol 1-phosphate and [3H]inositol 1:2-cyclic monophosphate. Finally [32P]phosphatidylinositol 4-phosphate and [32P]phosphatidylinositol 4,5-bisphosphate were degraded into [32P]inositol 1,4-bisphosphate and [32P]inositol 1,4,5-trisphosphate, respectively. The phosphoinositide-specific phospholipase C was activated by calcium (optimal concentration 5-10 mM) and inhibited by EGTA, although endogenous calcium supported a half-maximal activity. The enzyme displayed an optimal pH of 6.0 and an apparent Km of 0.08 mM. Its specific activity was around 10 nmol/min per mg protein, which is approximately the same as that found in human blood platelets. Subcellular fractionation revealed that 55% of the enzyme was solubilized under conditions where 80% of acrosin appeared in the supernatants. The majority of the particulate phospholipase C activity (37% of total) was found in the 1000 X g pellet, which contained only 8% of total acrosin activity. Further fractionation of spermatozoa into heads and tails indicated no specific enrichment of phospholipase C activity in any of these two fractions. However, owing to a 4-fold higher protein content in the head compared to the tail fraction, it is concluded that about 80% of particulate phospholipase C activity is located in sperm head. The physiological significance of this enzyme is discussed in relation to a possible role in acrosome reaction and (or) in egg fertilization.     

Glycerol-3-phospho-D-myo-inositol 4-phosphate (Gro-PIP) is an inhibitor of phosphoinositide-specific phospholipase C

The deacylated forms of the phosphoinositides were used to determine whether the guinea pig uterus phosphoinositide-specific phospholipase C (PI-PLC I, Mr 60,000) required fatty acids at the sn-1 and sn-2 positions for the hydrolysis of the sn-3 phosphodiester bond. L-alpha-Glycerophospho-D-myo-inositol 4-phosphate (Gro-PIP), but not glycerol 3-phosphate (Gro-3-P), L-alpha-glycerophospho-D-myo-inositol (Gro-PI), or L-alpha-glycerophospho-D-myo-inositol 4,5-bisphosphate (Gro-PIP2), inhibited PI-PLC I in a concentration-dependent manner. Assays performed with 10 microM [3H]phosphatidylinositol ([3H]PI), 10 microM [3H]phosphatidylinositol 4-phosphate ([3H]PIP) or 10 microM [3H]phosphatidylinositol 4,5-bisphosphate ([3H]PIP2) as substrates, with increasing [Gro-PIP] revealed an IC50 = 380 microM. Kinetic studies with increasing [3H]PI substrate concentrations in the presence of 100 microM and 300 microM Gro-PIP demonstrated that Gro-PIP exhibited competitive inhibition; Kis = 40 microM. Ca2+ concentrations over the range 1.1 microM to 1 mM did not effect inhibition, suggesting that Gro-PIP inhibition of [3H]PI hydrolysis was calcium-independent. To determine whether Gro-PIP was a substrate, 20 microM and 500 microM [3H]Gro-PIP were incubated with PI-PLC I. Anion-exchange HPLC analysis revealed no [3H]IP2 product formation, indicating that [3H]Gro-PIP was not hydrolyzed. Assays performed with [3H]PI and [3H]PIP substrates in the presence of 500 microM [3H]Gro-PIP revealed approx. 75% less [3H]inositol 1-phosphate ([3H]IP1) and [3H]inositol 1,4-bisphosphate ([3H]IP2) product formation, respectively, indicating that [3H]Gro-PIP inhibited the hydrolysis of the substrates by PI-PLC I. These data suggest that Gro-PIP does not serve as a substrate, and that it inhibits PI-PLC I by competitive inhibition in a Ca2(+)-independent fashion.     

A calcium-dependent mechanism for associating a soluble arachidonoyl-hydrolyzing phospholipase A2 with membrane in the macrophage cell line RAW 264.7

Arachidonoyl-hydrolyzing phospholipase A2 plays a central role in providing substrate for the synthesis of the potent lipid mediators of inflammation, the eicosanoids, and platelet-activating factor. Although Ca2+ is required for arachidonic acid release in vivo and most phospholipase A2 enzymes require Ca2+ for activity in vitro, the role of Ca2+ in phospholipase A2 activation is not understood. We have found that an arachidonoyl-hydrolyzing phospholipase A2 from the macrophage-like cell line, RAW 264.7, exhibits Ca2(+)-dependent association with membrane. The intracellular distribution of the enzyme was studied as a function of the Ca2+ concentration present in homogenization buffer. The enzyme was found almost completely in the 100,000 x g soluble fraction when cells were homogenized in the presence of Ca2+ chelators and there was a slight decrease in soluble fraction activity when cells were homogenized at the level of Ca2+ in an unstimulated cell (80 nM). When cells were homogenized at Ca2+ concentrations expected in stimulated cells (230-450 nM), 60-70% of the phospholipase A2 activity was lost from the soluble fraction and became associated with the particulate fraction in a manner that was partly reversible with EGTA. Membrane-associated phospholipase A2 activity was demonstrated by [3H]arachidonic acid release both from exogenous liposomes and from radiolabeled membranes. With radiolabeled particulate fraction as substrate, this enzyme hydrolyzed arachidonic acid but not oleic acid from membrane phospholipid, and [3H]arachidonic acid was derived from phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol/phosphatidylserine. We suggest a mechanism in which the activity of phospholipase A2 is regulated by Ca2+: in an unstimulated cell phospholipase A2 is found in the cytosol; upon receptor ligation the cytosolic Ca2+ concentration increases, and the enzyme becomes membrane-associated which facilitates arachidonic acid hydrolysis.     

Characterization of phospholipase C-mediated polyphosphoinositide hydrolysis in rat heart ventricles

Phospholipase C (PLC)-mediated degradation of polyphosphoinositides (phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 4-phosphate (PIP] was found to be present in rat heart ventricular soluble and total membrane fractions (100,000g supernatant and pellet). Distribution of polyphosphoinositide-specific phospholipase C activity between the membrane and soluble fraction was approximately 63 and 33% of total activity, respectively, whereas, phosphatidylinositol (PI) degradation could be detected only in the soluble fraction. Optimal PIP2-PLC activity occurred at a pCa2+ of 4.5. A similar peak in PIP-PLC activity could be demonstrated in soluble and membrane preparations; however, the rate of PIP degradation in the soluble fraction continued to increase at the highest calcium level tested (pCa2+ 3). With the exception of Sr2+, other noncalcium polycations did not support homogenate PIP2-PLC activity. In the presence of Ca2+, addition of Mg2+, La3+, or Sr2+ (10(-3) M) inhibited PIP2-PLC while Mn2+ and Gd3+ stimulated activity. In both the total membrane and soluble fractions, maximal polyphosphoinositide degradation occurs at pH 5.5 and 6.8. The detergents deoxycholate, cholate, and saponin exert a biphasic effect on PIP2-PLC activity (stimulating at lower concentrations and inhibiting at higher concentrations). The deoxycholate effect is observed in both the cytosolic and membrane fractions. Neutral and cationic detergents inhibit PIP2-PLC activity in a concentration-dependent manner. Similar to cytosolic PI-PLC activity, PIP2-PLC appears to depend on intact sulfhydryl groups. In the presence of a mixture of all three inositol phospholipids or the three phosphoinositides plus noninositol phospholipids, polyphosphoinositides are preferentially degraded.     

Soluble phosphatidylinositol phosphodiesterase in normal and denervated fast and slow muscles of the rat.

Phosphatidylinositol phosphodiesterase activity was determined in cytosol prepared from rat slow (soleus) and fast (extensor digitorum longus) muscles. The substrate was prepared by incubation of sarcoplasmic reticulum with myo-[2-3H]inositol. The enzyme hydrolysed both membrane-bound and extracted phosphatidylinositol. The activity determined with the isolated phospholipid exhibited an optimum at pH 5.5. Ca2+ ions stimulated the activity. The enzyme specific activity was higher in cytosol prepared from soleus muscle than in that from extensor digitorum longus muscle. After section of the motor nerve, the activity of the enzyme increased in both muscles up to 36 h and then declined. A function for this enzyme in the control of acetylcholine sensitivity in muscle is discussed.     

Characterization of rat kidney proximal tubule brush border membrane-associated phosphatidylinositol phosphodiesterase

Isolated rat kidney proximal tubule brush border membrane vesicles exhibit an increase in diacylglycerol levels (20- to 30-fold) and a concomitant decrease in phosphatidylinositol when incubated with [3H]arachidonate-labeled lipids, Ca2+, and deoxycholate. Levels of free arachidonate, triglyceride, and noninositol phospholipids are not altered. These results suggest phosphatidylinositol phosphodiesterase activity is associated with rat proximal tubule brush border membrane. Presence of both deoxycholate and certain divalent cations was necessary to demonstrate enzyme activity. Optimum pH ranged from 7.0 to 8.5. Ca2+, Mg2+, and Mn2+ stimulated diglyceride production while Ba2+, Zn2+, Hg2+, and K+ were ineffective. HgCl2 inhibited Ca2+-stimulated phosphatidylinositol phosphodiesterase. Mg2+ and deoxycholate-dependent enzyme activity was shown to be phosphatidylinositol specific. Sodium lauryl sulfate, tetradecyltrimethylammonium bromide, and Triton X-100 did not activate phosphatidylinositol phosphodiesterase in the presence of Ca2+. In combination with deoxycholate, diglyceride formation was not affected by sodium lauryl sulfate, partially inhibited by Triton X-100, and completely abolished by tetradecyltrimethylammonium bromide. Diglyceride kinase activity was not found associated with brush border membrane phosphatidylinositol phosphodiesterase. ATP (1-5 mM) inhibited Ca2+- or Mg2+-stimulated, deoxycholate-dependent phosphatidylinositol hydrolysis by chelating the required divalent cation.     

Gonadotropin-releasing hormone activates a rapid Ca2+-independent phosphodiester hydrolysis of polyphosphoinositides in pituitary gonadotrophs

Addition of gonadotropin releasing hormone (GnRH) to pituitary cells prelabeled with [32P]Pi or with myo-[2-3H]inositol, resulted in a rapid decrease in the level of [32P]phosphatidylinositol 4,5-bisphosphate (approximately 10 s), and in [32P]phosphatidylinositol 4-phosphate (approximately 1 min), followed by increased labeling of [32P]phosphatidylinositol and [32P]phosphatidic acid (1 min). GnRH stimulated the appearance of [3H]myo-inositol 1,4,5-trisphosphate (10 s), [3H]myo-inositol 1,4-bisphosphate (15 s), and [3H]myo-inositol 1-phosphate (1 min) in the presence of Li+ (10 mM). Li+ alone stimulated the accumulation of [3H]myo-inositol 1-phosphate and [3H]myo-inositol 1,4-bisphosphate but not [3H]myo-inositol 1,4,5-trisphosphate, but had no effect on luteinizing hormone release. The effect of GnRH on inositol phosphates (Ins-P) production was dose-related (ED50 = 1-5 nM), and was blocked by a potent antagonist [D-pGlu,pClPhe,D-Trp]GnRH. Elevation of cytosolic free Ca2+ levels ([Ca2+]i), by ionomycin and A23187 from intracellular or extracellular Ca2+ pools, respectively, had no significant effect on [3H]Ins-P production. GnRH-induced [3H]Ins-P production was not dependent on extracellular Ca2+ and was noticed also after extracellular or intracellular Ca2+ mobilization by A23187 or ionomycin, respectively. The effect of GnRH on [3H]Ins-P accumulation was not affected by prior treatment of the cells with the tumor promoter phorbol ester 12-O-tetradecanoylphorbol-13-acetate or with islet-activating protein pertussis toxin. These results indicate that GnRH stimulates a rapid phosphodiester hydrolysis of polyphosphoinositides. The stimulatory effect is not mediated via an islet-activating protein-substrate, is not dependent on elevation of [Ca2+]i, neither is it negatively regulated by 12-O-tetradecanoylphorbol-13-acetate which activates Ca2+/phospholipid-dependent protein C kinase. The results are consistent with the hypothesis that GnRH-induced phosphoinositide turnover is responsible for Ca2+ mobilization followed by gonadotropin release.     

Presence of a Ca2+-sensitive CDPdiglyceride-inositol transferase in canine cardiac sarcoplasmic reticulum

Sarcoplasmic reticulum (SR) and plasma membranes from canine left ventricle were used to evaluate the presence of the enzyme CDPdiglyceride-inositol transferase in these membranes. (K+,-Ca2+)-ATPase activity, a marker for SR, was 79.2 +/- 5.0 (SE) and 11.2 +/- 2.0 mumol.mg-1.h-1 in SR and plasma membrane preparations, respectively, and (Na+,K+)-ATPase activity, a marker for plasma membranes, was 5.6 +/- 1.2 and 99.2 +/- 8.0 mumol.mg-1.h-1, respectively. Contamination of SR and plasma membrane preparations by mitochondria was estimated to be 2% and 8%, respectively, and by Golgi membranes, 0.9% and 1.8%, respectively. Transferase activity, measured at pH 6.8, was 1.32 +/- 0.04 (SE) and 0.28 +/- 0.04 nmol of [3H]phosphatidylinositol ([3H]PtdIns).mg-1.min-1 in three SR and plasma membrane preparations, respectively. The transferase activity detected in the plasma membrane preparation could be accounted for largely, but not entirely, by contaminating SR membranes. The pH optimum for the SR transferase activity was between 8.0 and 9.0; little or no activity was detectable at pH 6.3 and 5.5, the lowest pH tested. Ca2+ inhibited the enzyme, half-maximal inhibition occurring at about 10 microM Ca2+; removal of the Ca2+ by addition of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid restored activity. No loss of [3H]PtdIns could be detected when membranes were incubated in the presence or absence of Ca2+. The Ca2+ inhibition of the transferase was noncompetitive with respect to CDP-dipalmitin while that with respect to myo-inositol was slightly noncompetitive at low [Ca2+] and became uncompetitive at higher [Ca2+].(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential regulation by phosphatidylinositol 4,5-bisphosphate of pituitary plasma-membrane and cytosolic phosphoinositide kinases.

Regulation of phosphatidylinositol kinase (EC 2.7.1.67) and phosphatidylinositol 4-phosphate (PtdIns4P) kinase (EC 2.7.1.68) was investigated in highly enriched plasma-membrane and cytosolic fractions derived from cloned rat pituitary (GH3) cells. In plasma membranes, phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] added exogenously enhanced incorporation of [32P]phosphate from [gamma-32P]MgATP2- into PtdIns(4,5)P2 and PtdIns4P to 150% of control; half-maximal effect occurred with 0.03 mM exogenous PtdIns(4,5)P2. Exogenous PtdIns4P and phosphatidylinositol (PtdIns) had no effect. When plasma membranes prepared from cells prelabelled to isotopic steady state with [3H]inositol were used, there was a MgATP2- dependent increase in the content of [3H]PtdIns(4,5)P2 and [3H]PtdIns4P that was enhanced specifically by exogenous PtdIns(4,5)P2 also. Degradation of 32P- and 3H-labelled PtdIns(4,5)P2 and PtdIns4P within the plasma-membrane fraction was not affected by exogenous PtdIns(4,5)P2. Phosphoinositide kinase activities in the cytosolic fraction were assayed by using exogenous substrates. Phosphoinositide kinase activities in cytosol were inhibited by exogenously added PtdIns(4,5)P2. These findings demonstrate that exogenously added PtdIns(4,5)P2 enhances phosphoinositide kinase activities (and formation of polyphosphoinositides) in plasma membranes, but decreases these kinase activities in cytosol derived from GH3 cells. These data suggest that flux of PtdIns to PtdIns4P to PtdIns(4,5)P2 in the plasma membrane cannot be increased simply by release of membrane-associated phosphoinositide kinases from product inhibition as PtdIns(4,5)P2 is hydrolysed.     

Characterization of the forward and reverse reactions catalyzed by CDP-diacylglycerol:inositol transferase in rabbit lung tissue.

CDPdiacylglycerol:inositol transferase activity in rabbit lung tissue has been characterized and the optimum conditions for assaying this enzyme in vitro were determined. Rabbit lung tissue CDPdiacylglycerol:inositol transferase activity was found primarily in the microsomal fraction. The pH optimum of the enzyme activity was between 8.8 and 9.4, and the reaction was dependent on either Mn2+ or Mg2+. Detergents and Ca2+ inhibited the activity of the enzyme. The apparent Km values of the enzyme for CDPdioleoylglycerol and myoinositol were 0.18 mM and 0.10 mM, respectively. The reversibility of the reaction catalyzed by CDPdiacylglycerol:inositol transferase in microsomes prepared from rabbit lung tissue was demonstrated by the synthesis of [3H]CMPdiacylglycerol when [3H]CMP and phosphatidylinositol were present in the incubation mixture. The reverse reaction was characterized and its importance in the regulation of the acidic phospholipid composition of surfactant during lung development is discussed. The pH optimum for the reverse reaction was 6.2, and the reverse reaction was also dependent on Mn2+ or Mg2+. The apparent Km value of CDPdiacylglycerol:inositol transferase for CMP was found to be 2.8 mM.     

Purification of a phospholipase A2 from human monocytic leukemic U937 cells. Calcium-dependent activation and membrane association

The existence of an intracellular phospholipase A2 (PLA2) involved in the production of 1-O-alkyl-sn-glycero-3-phosphocholine and free arachidonic acid has been repeatedly postulated. Using 1-O-hexadecyl-2-[3H]arachidonoyl-sn-glycero-3-phosphocholine as a substrate and a series of conventional and high-pressure liquid chromatographic techniques, we have purified a PLA2 from the soluble fraction of differentiated human monocytic U937 cells. The enzyme has been purified nearly 2000-fold to homogeneity. The purified enzyme has a molecular mass of 56 kDa, under reducing conditions, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The enzyme activity has a pH optimum of 8.0 and is calcium concentration-dependent. The EC50 for the activation of the enzyme activity by calcium is 300 nM. When the cells were homogenized in the presence of the calcium chelator EGTA (0.2 mM), the enzyme was found to be soluble (more than 90% of the activity in the 100,000 x g supernatant). However, when Ca2+ concentration was controlled from 10 nM to 100 microM in Ca2(+)-EGTA buffers, increasing amounts of the activity were found in the particulate fraction (100,000 x g pellet). This suggests that membrane translocation and activation of the soluble PLA2 may be regulated by physiological intracellular levels of Ca2+. The purified enzyme hydrolyzed different phosphatidylcholine substrates presented in either vesicular or Triton X-100 mix micellar forms. In both situations, the enzyme showed a high degree of specificity for arachidonic acid on the sn-2 position of the substrate. Substitution of palmitic or oleic on the sn-2 position substantially reduced the hydrolytic activity of the enzyme. When vesicles of arachidonic acid-containing phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol were presented to the purified enzyme, all of them were hydrolyzed with comparable efficiency. However, only phosphatidylcholine and phosphatidylinositol were hydrolyzed when presented in Triton X-100 mixed micelles.     

A neutral phospholipase D activity from rat brain synaptic plasma membranes. Identification and partial characterization

Rapid activation of phospholipase D (PLD) in response to cell stimulation was recently demonstrated in many systems, raising the hypothesis that PLD participates in transduction of extracellular signals across the plasma membrane. In the present study, we describe the identification of a neutral PLD activity in purified rat brain synaptic plasma membranes, and the in vitro conditions required to assay its catalytic activity with exogenous [3H]phosphatidylcholine as substrate. Production of [3H]phosphatidic acid, the natural lipid product of PLD and of [3H]phosphatidylethanol, catalyzed by PLD in the presence of ethanol via transphosphatidylation, were measured. The synaptic membrane PLD exhibited its highest activity at pH 7.2 and was thus defined as a neutral PLD. Enzyme activity was absolutely dependent on the presence of sodium oleate and was strongly activated by Mg2+ ions (at 1 mM). Ca2+ at concentrations up to 0.25 mM was as stimulatory as Mg2+, but at 2 mM it completely inhibited enzyme activity. Mg2+ extended the linear phase of PLD activity from 2 to 15 min, suggesting that it may stabilize the enzyme under our assay conditions. The production of [3H]phosphatidylethanol was a saturable function of ethanol concentration. Production of [3H] phosphatidic acid was inversely related to the concentration of ethanol and to the accumulation of phosphatidylethanol, indicating that the two phospholipids are indeed produced by the competing hydrolase and transferase activities of the same enzyme. beta,beta-Dimethylglutaric acid, utilized previously as a buffer in studies of rat brain PLD, inhibited enzyme activity at neutral pH but not at acidic pH. The properties of the neutral synaptic membrane PLD and its relationships with other in vitro, acid, and neutral PLD activities, as well as with the signal-dependent PLD detected in intact cells, are discussed.