Effect of phospholipase D on micellar phospholipids and the role of calcium ions]

The rates of the reaction products formation under simultaneous phospholipase D effect on phosphatidyl ethanolamine and phosphatidyl choline were studied. The hydrolysis of cephalin, unlike the phospholipase D effect on lecithin, does not require Ca2+ ions. Ca2+ does not affect the enzymatic degradation of lecithin and inhibits the reaction with cephalin in "inorganized" phospholipid emulsions. The hydrolysis of micellar phospholipids by phospholipase D (in the presence of the anionic detergent sodium dodecyl sulfate) is accelerated by Ca2+ ions for both substrates. The apparent Km value is equal to 1.5 mM and does not depend on the phospholipid type. In contrast, the value of kcat for lecithin is twice as high as that for cephalin. It was demonstrated that the phase state of the phospholipids and the chemical nature of the alcohol residue in the phospholipid molecule are essential for the substrate specificity of phospholipase D.     

Action of phospholipase C (Bacillus cereus) on isolated myelin sheath preparations

The action of phospholipase C (Bacillus cereus) on the phospholipids of myelin sheath preparations has been investigated. With freshly isolated bovine brain myelin about 40% of the total phospholipid could be hydrolyzed by this enzyme. With bovine spinal cord myelin the phospholipid seemed more resistant to attack whereas the opposite was the case with myelin from guinea-pig brain or rat brain. With fresh bovine brain myelin, phosphatidylcholine and the ethanolamine-containing phospholipids were the main targets for the enzyme with lesser extents of hydrolysis occurring with phosphatidylserine and sphingomyelin. The effect of exposing bovine brain myelin to structural perturbants prior to enzyme digestion indicated that trypsin pretreatment had no significant effect, whereas marked enhancement of the extent of phospholipid hydrolysis occurred following lyophilization + rehydration, or pretreatment of myelin with HCl, Triton TX-100/ammonium acetate or deoxycholate. The effect of myelin pretreatment on the degradation of the individual phospholipid classes was also studied.     

Evidence for a catalytic role of phospholipase A in phorbol diester- and zymosan-induced mobilization of arachidonic acid in mouse peritoneal macrophages

Inositol phospholipid degradation and release of phospholipid-bound arachidonic acid was induced in intact peritoneal macrophages by exposure to phorbol myristate acetate (PMA) or zymosan particles. PMA, known to activate protein kinase C, selectively enhanced the deacylation of phosphatidylinositol (i.e., degradation by phospholipase A), while zymosan particles enhanced degradation via both phospholipase A and inositol lipid phosphodiesterase (phospholipase C). The release of arachidonic acid was found to correlate with the degradation of phosphatidylinositol by the phospholipase A pathway and could be dissociated from the phospholipase C-catalyzed cleavage of inositol phospholipids in several experimental situations: (i) when PMA was the stimulus, (ii) by the difference in Ca2+ dependence between the two enzymatic processes when zymosan was the stimulus and (iii) by the parallel inhibition by chlorpromazine of the phospholipase A pathway and arachidonic acid release, but not inositol phospholipid phosphodiesterase. In addition, phloretin, a reported inhibitor of protein kinase C, was found to inhibit arachidonic acid release and the deacylation of phosphatidylinositol. The results are consistent with a model in which arachidonic acid release is mediated by phospholipase(s) A and in which PMA or the phosphodiesterase-catalyzed degradation of phosphoinositides causes activation of the phospholipase A pathway via protein kinase C.     

The phospholipid-dependence of uridine diphosphate glucuronyltransferase. Reactivation of phospholipase A-inactivated enzyme by phospholipids and detergents

Specific degradation of the phospholipid membrane of guinea-pig liver microsomal fraction with phospholipase A inactivated glucuronyltransferase. The inactivation was reversed by phosphatidylcholine and mixed microsomal phospholipid micelles at concentrations similar to those present in intact microsomal preparations. The other commonly occurring phospholipids did not reactivate phospholipase A-treated enzyme. Since the mixed microsomal phospholipids consisted mainly of phosphatidylcholine, it is concluded that the reactivation by phospholipids is phosphatidylcholine-specific. Reactivation was also achieved by low concentrations of the cationic detergents cetylpyridinium chloride and cetyltrimethylammonium bromide. Higher concentrations of these detergents inactivated the glucuronyltransferase activity of intact and phospholipase A-treated microsomal fractions. Anionic detergents were potent inactivators of the glucuronyltransferase activity of untreated and phospholipase A-treated microsomal fractions, whereas non-ionic detergents had little effect on the activity of either preparation. Measurements of the zeta-potentials of the micellar species used in this study showed that no obvious relationship existed between the zeta-potentials and the ability to reactivate glucuronyltransferase. However, high positive or negative zeta-potentials were correlated with the ability of the amphipathic compound to inactivate glucuronyltransferase.     

Studies on the enzymatic pathways of calcium ionophore-induced phospholipid degradation and arachidonic acid mobilization in peritoneal macrophages

Exposure of mouse peritoneal macrophages to ionophore A23187 caused a rapid and extensive Ca2+-dependent phospholipid degradation and mobilization of arachidonic acid. Phosphatidylinositol, phosphatidylcholine and phosphatidylethanolamine all contributed to the arachidonic acid release, although the ethanolamine phospholipids incorporated [3H]arachidonic acid more slowly during the prelabeling period, particularly the plasmalogen form. Several enzymatic pathways could be positively identified as contributing to the ionophore-induced phospholipid degradation by the use of several different radiolabeled phospholipid precursors: (i) a phospholipase A-mediated deacylation, (ii) a phosphodiesterase (phospholipase C) reaction, rapidly generating diacylglycerol units from inositol phospholipids, and (iii) enzymatic processes generating diacylglycerol and CDP- and phosphocholine/ethanolamine from phosphatidylcholine/ethanolamine. The diacylglycerol formed was in part phosphorylated and in part hydrolyzed to monoacylglycerol, with retention of its arachidonic acid. These, and other, results indicate that the Ca2+-ionophore activates several apparently distinct phospholipid-degrading processes, in contrast to stimuli acting via cellular receptors.     

Binding of Crotoxin, a Presynaptic Phospholipase A2Neurotoxin, to Negatively Charged Phospholipid Vesicles

Crotoxin, isolated from the venom of Crotalus durissus terrificus, is a potent neurotoxin consisting of a basic and weakly toxic phospholipase A2 subunit (component B) and an acidic nonenzymatic subunit (component A). The nontoxic component A enhances the toxicity of the phospholipase subunit by preventing its nonspecific adsorption. The binding of crotoxin and of its subunits to small unilamellar phospholipid vesicles was examined under experimental conditions that prevented any phospholipid hydrolysis. Isolated component B rapidly bound with a low affinity (Kapp in the millimolar range) to zwitterionic phospholipid vesicles and with a high affinity (Kapp of less than 1 microM) to negatively charged phospholipid vesicles. On the other hand, the crotoxin complex did not interact with zwitterionic phospholipid vesicles but dissociated in the presence of negatively charged phospholipid vesicles; the noncatalytic component A was released into solution, whereas component B remained tightly bound to lipid vesicles, with apparent affinity constants from 100 to less than 1 microM, according to the chemical composition of the phospholipids. On binding, crotoxin or its component B caused the leakage of a dye entrapped in vesicles of negatively charged but not of zwitterionic phospholipids. The selective binding of crotoxin suggests that negatively charged phospholipids may constitute a component of the acceptor site of crotoxin on the presynaptic plasma membrane.     

Action of Phospholipase A2 and Phospholipase C on Bacillus subtilis Protoplasts

Protoplasts prepared from Bacillus subtilis by lysozyme digestion lysed in the presence of pure pancreatic phospholipase A(2). The phospholipids cardiolipin, phosphatidylethanolamine, phosphatidylglycerol and lysylphosphatidylglycerol, which are present in the membrane, are degraded by phospholipase A(2) only after removal of the cell wall, giving free fatty acids and lyso derivatives. The four phospholipids are hydrolyzed equally well at a given enzyme concentration. Differences in the phospholipid composition of the protoplasts were obtained by variations in the growth medium, time of harvesting, and preincubation time with lysozyme. The extent of hydrolysis appeared to depend on the initial phospholipid composition. A relative increase in acidic phospholipids in the membrane facilitated the action of phospholipase A(2), whereas the rate of hydrolysis was diminished when protoplasts were tested which contained a relatively high amount of positively charged phospholipid. Pure phospholipase C from B. cereus preferentially hydrolyzed phosphatidyl-ethanolamine in the B. subtilis membrane. More than 80% of this phospholipid was converted into diglyceride, whereas only 30% of the cardiolipin was hydrolyzed. Such a loss of phospholipids, however, was not followed by lysis of the protoplasts. Liposomes were prepared from the lipid extracts of B. subtilis and incubated with both phospholipases. The hydrolysis pattern of the phospholipids in these model membrane systems was identical to the hydrolysis pattern of the phospholipids in the protoplast membrane. Phospholipase A(2) hydrolyzed all the phospholipids in the liposomes equally well, whereas phospholipase C preferentially degraded phosphatidylethanolamine.     

Effect of membrane sterol content on the susceptibility of phospholipids to phospholipase A2

The effects of membrane sterol level on the susceptibility of LM cell plasma membranes to exogenous phospholipases A2 has been investigated. Isolated plasma membranes, containing normal or decreased sterol content, were prepared from mutant LM cell sterol auxotrophs. beta-Bungarotoxin-catalyzed hydrolysis of both endogenous phospholipids and phospholipids introduced into the membranes with beef liver phospholipid exchange proteins was monitored. In both cases, phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were degraded at similar rates in normal membranes, while PC hydrolysis was specifically accelerated in sterol-depleted membranes. Additional data suggest that this preferential hydrolysis of PC is not a consequence of the phospholipid head group specificity of the phospholipase, nor of a difference in the accessibility of PC versus PE to the enzyme. Analysis of the reaction products formed during treatment of isolated membranes with phospholipase A2 showed almost no accumulation of lysophospholipids. This was found to be due to highly active lysophospholipase(s), present in LM cell plasma membranes, acting on the lysophospholipids formed by phospholipase A2 action. A soluble phospholipase A2 was partially purified from LM cells and found to behave as beta-bungarotoxin with regard to membrane sterol content. These results demonstrate that the nature of phospholipid hydrolysis, catalyzed by phospholipase A2, can be significantly affected by membrane lipid composition.     

The role of lipid-protein interactions in NADH-cytochrome c reductase (rotenone-insensitive) of rat liver mitochondria

The phospholipid depletion of rat liver mitochondria, induced by acetone-extraction or by digestion with phospholipase A₂ or phospholipase C, greatly inhibited the activity of NADH-cytochrome c reductase (rotenone-insensitive). A great decrease of the reductase activity also occurred in isolated outer mitochondrial membranes after incubation with phospholipase A₂. The enzyme activity was almost completely restored by the addition of a mixture of mitochondrial phospholipids to either lipid-deficient mitochondria, or lipid-deficient outer membranes. The individual phospholipids present in the outer mitochondrial membrane induced little or no stimulation of the reductase activity. Egg phosphatidylcholine was the most active phospholipid, but dipalmitoyl phosphatidylcholine was almost ineffective. The lipid depletion of mitochondria resulted in the disappearance of the non-linear Arrhenius plot which characterized the native reductase activity. A non-linear plot almost identical to that of the native enzyme was shown by the enzyme reconstituted with mitochondrial phospholipids. Triton X-100, Tween 80 or sodium deoxycholate induced only a small activation of NADH-cytochrome c reductase (rotenone-insensitive) in lipiddeficient mitochondria. The addition of cholesterol to extracted mitochondrial phospholipids at a 1 : 1 molar ratio inhibited the reactivation of NADH-cytochrome c reductase (rotenone-insensitive) but not the binding of phospholipids to lipid-deficient mitochondria or lipid-deficient outer membranes.These results show that NADH-cytochrome c reductase (rotenone-insensitive) of the outer mitochondrial membrane requires phospholipids for its activity. A mixture of phospholipids accomplishes this requirement better than individual phospholipids or detergents. It also seems that the membrane fluidity may influence the reductase activity.     

Phospholipid metabolism and prostacyclin synthesis in hypoxic myocytes.

We observed that in hypoxic myocardial cells prostacyclin and arachidonic acid release increased and that during hypoxia phospholipid degradation also occurred. In order to clarify the mechanism of phospholipid degradation, we determined the activity of phospholipases A2 and C. We found that phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were markedly decreased and that lysophosphatidylcholine and lysophosphatidylethanolamine were increased. In contrast, there was only slight phosphatidylinositol degradation and no lysophosphatidylinositol elevation was observed. These results show that phospholipase A2 was activated in hypoxic myocytes and had substrate specificity towards PC and PE. To study phospholipase C activity, membrane phospholipids were labeled with [3H]choline, [3H]inositol or [3H]ethanolamine. The release of inositol was observed, but neither choline nor ethanolamine was released. In hypoxia, myocardial-cell phospholipase C has high substrate specificity towards phosphatidylinositol. The activation of phospholipases is closely related to the intracellular Ca2+ concentration; it is though that inositol polyphosphatides may regulate intracellular Ca2+. We determined how Ca2+ influx occurs in hypoxia. beta-Adrenergic blockade and Ca2+ antagonists markedly suppressed Ca2+ influx, phospholipase A2 activity, phospholipase C activity and cell death. However, the alpha 1-adrenergic blockade was less effective in suppressing these phenomena. These results suggest that in hypoxic myocardial cells Ca2+ influx mediated by beta-adrenergic stimulation activates phospholipases A2 and C, and that phospholipid degradation and prostacyclin release then occur.     

Regulatory aspects of mitochondrial phospholipase A2 from rat liver: effects of proteins, phospholipids and calcium ions

This paper deals with the search for specific inhibitors or activators of the mitochondrial phospholipase A2. Convincing evidence for the existence of proteins in the mitochondrial or cytosolic fraction that function as specific regulators of this enzyme was not obtained. The enzymatic activity appeared to be inhibited at low substrate concentrations by lipocortin isolated from human monocytes. However, at higher substrate concentrations, the inhibition disappeared, suggesting either that lipocortin sequestered the phospholipid substrate or that the putative inactive complex of enzyme and lipocortin dissociated in the presence of excess phospholipids. The hydrolysis of the neutral phospholipid phosphatidylethanolamine was stimulated by the presence of cardiolipin and phosphatidylglycerol. It is unlikely that this is caused merely by the negative charge of these phospholipids, since other negatively charged phospholipids did not show this effect. Using a phospholipid extract from mitochondria as substrate, the enzymatic activity as a function of the Ca2+ concentration was determined. Only one enzyme activity plateau was observed. The calculated KCa2+ value of 0.05 mM suggests that the mitochondrial phospholipase A2 could be regulated strictly by the modulation of the free Ca2+ concentration in vivo. The two activity plateaus observed previously upon variation of the Ca2+ concentration using phosphatidylethanolamine as substrate could be explained by a Ca2+-induced transition of the phospholipid structure.     

Control of arachidonic acid accumulation in bone marrow-derived macrophages by acyltransferases

The turnover of phospholipid fatty acid moieties of bone marrow-derived macrophages was analyzed by separate determination of degrading and acylating activities. Acylating activities were followed in intact cells by incubation with excess arachidonic acid and degradation of phospholipids was followed in cells prelabeled with fatty acids. Significant phospholipase A2 activity was detectable only if the reutilization of liberated fatty acid was inhibited , e.g. by p-chloromercuribenzoate. It was of interest that the divalent cation ionophore A 23187 and various antiphlogistic drugs like indomethacin, diclofenac, and acetylsalicylic acid were found to inhibit the acylation reaction. These compounds led to increased levels of free arachidonic acid in stimulated, as well as in unstimulated cells. Increased activities of phospholipase A2 were achieved by treatment with the bivalent cation ionophore A 23187 and with zymosan. The effect of zymosan obtained from various sources was found to be exclusively due to contamination of tee zymosan particles with phospholipase A2 activity. Even when the cellular phospholipase activity was increased by the addition of exogenous phospholipase activity contained in the zymosan particles, degradation of cellular phospholipids was not measurable unless the reacylation was inhibited. These results suggest that in the cells studied, the level of free arachidonic acid is mainly controlled by the activity of the lysophosphatide acyltransferase.     

Role of phospholipases in myocardial ischemia: effect of cardioprotective agents on the phospholipases A of heart cytosol and sarcoplasmic reticulum in vitro

Increased breakdown of myocardial phospholipids to fatty acids and lysophosphoglycerides is an early feature of myocardial ischemic injury and many investigators believe that enhanced phospholipase action is an important factor in the process. Several recent reports indicate that inhibitors of phospholipase A, such as mepacrine, chloroquine and chlorpromazine, can prevent heart phosphoglyceride breakdown in vivo. We isolated the phospholipases A from rat heart cytosol and sarcoplasmic reticulum and examined the effects of various cardioprotective substances on their activity. Most of the cardioprotective agents studied inhibited the heart phospholipases in vitro, providing further evidence that phospholipid degradation in ischemic myocardial injury may be modulated by pharmacologic agents.     

Gonadotropin receptors in plasma membranes of bovine corpus luteum. II. Role of membrane phospholipids.

The role of phospholipids in the binding of 125I-choriogonadotropin to bovine corpus luteum plasma membranes has been investigated with the use of purified phospholipase A and phospholipase C to alter membrane phospholipids. The phospholipase C-digested plasma membrane preparation showed 85 to 90% inhibition of 125I-choriogonadotropin binding activity when 70% of the membrane phospholipid was hydrolyzed. Similarly treatment of plasma membranes with phospholipase A resulted in 45 to 55% hydrolysis of membrane phospholipid and almost 75% inhibition of receptor activity. Both these enzymes hydrolyzed membrane-associated phosphatidylcholine to a greater extent than phosphatidylethanolamine and phosphatidylserine. Phosphorylaminoalcohols of phospholiphase C end products were completely released into the medium, while phospholipase A by-products remained associated with plasma membranes. Addition of a phospholipids suspension or liposomes to plasma membranes pretreated with phospholipase A and C did not restore gonadotropin binding activity. Soluble phosphorylcholine, phosphorylethanolamine, and phosphorylserine and insoluble diglyceride products of phospholipase C action had no effect on receptor activity. In contrast, end products of the phospholipase A action, such as lysophosphatides and fatty acids, inhibited both on the membrane-associated and solubilized receptor activity. Lysophosphatidylcholine was the most effective end product inhibiting the binding of gonadotropin to the receptor, followed by lysophosphatidylethanolamine and lysophosphatidylserine. The inhibitory effects of phospholipase A or lysophosphatides were completely reversed upon removal of membrane-bound phospholipid end products by washing the membranes with defatted bovine serum albumin. However, phospholipase C inhibition could not be overcome by defatted albumin washings. Solubilization of plasma membranes with detergents which had been pretreated with phospholipase C partially restored the inhibited activity. It is concluded that the phospholipase-mediated inhibition of gonadotropin binding activity was due to hydrolysis and alterations of the phospholipid environment in the case of phospholipase C and by direct inhibition by end products in the case of phospholipase A.     

Effect of hyper- and hypothyroidism on phospholipid fatty acid composition and phospholipases activity in sarcolemma of rabbit cardiac muscle.

Lipid content and composition of fatty acids esterified to phospholipids of cardiac sarcolemma isolated from hyperthyroid, hypothyroid and control rabbits were analysed. Hyperthyroidism resulted in a significant reduction of the cholesterol to phospholipid molar ratio as compared to control animals, while hypothyroidism exerted the opposite effect. Complex changes in composition of phospholipid fatty acids observed in hyperthyroid state led to an elevation of the fatty acid unsaturation index over the control value. The unsaturation index value was, however, not affected in the hypothyroid state. Thyroxine hormone administration increased phospholipase A1 and decreased phospholipase A2 activity. The opposite effect was observed in thyreodectomized animals. The effect of changes in sarcolemmal bulk phospholipids upon thyroxine administration or deficiency on regulation of activity of membrane-bound enzymes is discussed.     

Studies on ether phospholipids. I. A new method of determination using phospholipase A1 from guinea pig pancreas: application to Krebs II ascites cells

A new method for ether phospholipid analysis has been devised, based on the selective destruction of diacyl phospholipids by guinea pig phospholipase A1 and of plasmalogens by acidolysis. The paper describes optimal conditions allowing a specific degradation of diacyl phospholipids by the enzyme(s). This requires the incubation of a total lipid extract in the presence of 2.4 mM sodium deoxycholate, at pH 8.0, at a temperature of 42 degrees C. As shown with various radioactive markers, all the diacyl phospholipids become degraded, whereas sphingomyelin and ether phospholipids remain refractory to phospholipase A1 attack. Phospholipids are then separated by a bidimensional thin-layer chromatography involving the exposure of the plates to HCl fumes between the two runs, in order to hydrolyse plasmalogens. Selectivity of both hydrolytic procedures is further demonstrated upon analysis of acetyl diacylglycerol derived from phospholipids. Various phospholipids can thus be determined by phosphorus measurement using sphingomyelin as an internal standard. By this way, it is shown that Krebs II cells present a very high content of ether phospholipid species (around 25% of total). Among these, about 50% are alkyl forms in ethanolamine phosphoglycerides, whereas this value reaches 70% in choline phosphoglycerides.     

The formation of phosphatidylglycerol and other phospholipids by the transferase activity of phospholipase D

1. Purified phospholipase D can catalyse the transfer of a `phosphatidyl' unit from lecithin to various aliphatic alcohols such as glycerol, ethanolamine, methanol and ethylene glycol with the formation of the equivalent phospholipid. 2. The transferase reaction occurs simultaneously with hydrolase activity but at high alcohol concentrations the former predominates. 3. The acceptor molecule must contain a primary alcoholic grouping. 4. The chromatographic and ionophoretic mobilities of the deacylation products of many enzymically synthesized phospholipids are reported. 5. Enzymically prepared phosphatidylglycerol has been isolated in good yield. Chemical degradation showed that the `phosphatidyl unit' of lecithin had been transferred predominantly to the α-hydroxyl groups of glycerol. 6. Water-soluble alcohols can markedly stimulate the liberation of choline from ultrasonically treated lecithin by phospholipase D. The stimulation is usually due to an increase in hydrolase activity although often the associated transferase activity contributes.     

Phospholipase A2 sensitivity of uterine smooth muscle membrane phospholipids and adenylate cyclase activity. Effect of temperature on the action of phospholipase present in excess

Basal as well as GTP-dependent adenylate cyclase activity was partially resistant to porcine pancreatic phospholipase A2, although more activity was degraded at 16 than at 2 degrees C. In contrast, isoproterenol-dependent activity was completely destroyed regardless of the temperature. Snake venom phospholipase A2 destroyed approximately 90% of basal and GTP-dependent adenylate cyclase activity at all temperatures. The difference between the lipases is consistent with earlier evidence that elevated temperature facilitates the entry of some forms of phospholipase into the membrane bilayer. The temperature dependence of adenylate cyclase activation by the GTP analog Gpp[NH]p and its pancreatic phospholipase sensitivity were compared. The Arrhenius plots were markedly similar and biphasic with discontinuities at approximately 8 degrees C. The same temperature-dependent phospholipid phase transition might account, therefore, for both adenylate cyclase properties. Only small amounts of membrane phosphatidylethanolamine and phosphatidic acid were hydrolyzed by pancreatic phospholipase in a temperature-dependent manner analogous to adenylate cyclase degradation. These results suggest that specific phospholipids support catalysis and adenylate cyclase activation, but that different phospholipids are required for receptor coupling which may occur in a less viscous part of the membrane.     

The use of a phospholipase A-less Escherichia coli mutant to establish the action of granulocyte phospholipase A on bacterial phospholipids during killing by a highly purified granulocyte fraction.

Phospholipase A2 present in a highly purified, potently bactericidal, fraction from rabbit graulocytes produces net bacterial phospholipid degradation during killing of a phospholipase A-less strain of Escherichia coli. In the wild-type parent strain phospholipid breakdown is caused not only by the action of phospholipase A2 but also by phospholipase A1, indicating activation of the most prominent phospholipase of E. coli. This activation occurs as soon as the bacteria are exposed to the granulocyte fraction. Phospholipid breakdown by both phospholipases A is dose dependent but reaches a plateau after 30-60 min and at higher concentrations of the fraction. Phospholipid degradation is accompanied in both strains by an increase in permeability to actinomycin D that is also dose dependent. Even though net hydrolysis of phospholipids is greater in the parent strain than in the mutant, the increase in permeability is the same in the two strains. The addition of 0.04 M Mg2+, after the effects on phospholipids and permeability have become manifest, initiates in both strains the restoration of insensitivity to actinomycin D, the net resynthesis of phospholipids, and the disappearance of monoacylphosphatides and the partial disappearance of free fatty acids that had accumulated. Loss of ability to multiply is not reversed by Mg2+ in either strain. Less than 5 micrograms of granulocyte fraction causes loss of viability of from 90 to 99% of 1 X 10(8) microorganisms of both strains. However, at lower concentrations the parent strain is considerably more sensitive to the bactericidal effect of the granulocyte fraction than the mutant strain.     

Phorbol ester stimulates the hydrolysis of phosphatidylethanolamine in leukemic HL-60, NIH 3T3, and baby hamster kidney cells

Treatment of leukemic HL-60, NIH 3T3, and baby hamster kidney (BHK-21) cells, prelabeled with [2-14C]ethanolamine, with 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent activator of protein kinase C, resulted in increased degradation of both 14C-labeled phosphatidylethanolamine and its alkenyl (plasmalogen) derivate. A half-maximal and a maximal (approximately 3.4-fold) stimulation of ethanolamine phospholipid degradation required 3 and 10-20 nM TPA, respectively. TPA had a similar concentration-dependent stimulatory effect on the hydrolysis of phosphatidylcholine in cells previously prelabeled with [methyl-14C]choline. Increased phospholipid degradation was not accompanied by the formation of lysophosphatidylethanolamine, indicating that a phospholipase A-type enzyme was not involved. About 80% of total water-soluble degradation products was ethanolamine, suggesting that phospholipid hydrolysis was catalyzed by a phospholipase D-type enzyme. Increased formation of ethanolamine with exposure of cells to TPA was observed only after a 10-min lag period. Mezerein, bryostatin, sn-1-oleoyl-2-acetylglycerol, and polymyxin B, all of which mimic the action of TPA on protein phosphorylation in vivo, also stimulated the hydrolysis of ethanolamine phospholipids in HL-60 cells, suggesting that the TPA effect was mediated by protein kinase C.