Activation by saturated and monounsaturated fatty acids of the O2- -generating system in a cell-free preparation from neutrophils.

Saturated and monounsaturated fatty acids with appropriate chain length such as laurate and oleate activated an O2- -generating enzyme system in a cell-free preparation from porcine neutrophils. The activated preparation catalyzed a stoichiometric conversion of O2 to O2- by utilizing NADPH as the electron donor. The preparation contained both membrane and soluble fractions and, upon separation into subfractions, the O2- -generating activity resided exclusively in the membrane fraction. Polyunsaturated fatty acids including arachidonate also activated the system, but they concurrently stimulated NADPH-independent O2 consuming reactions which yield neither O2- nor H2O2. The amount of such a non-O2- -producing O2 consumption often reached twice as much as that of O2- production. For the activation of the O2- -generating system in the membrane, the presence of the soluble fraction was essential. However, the soluble fraction was no longer effective when once used for the activation, suggesting that the effective component(s) in the fraction was consumed or translocated to the membrane during the activation. When the activated membrane was incubated with delipidated albumin, the activity was lost with concomitant decreases in the amount of membrane-associated fatty acids. The lost activity was restored by the replenishment of the fatty acid in the presence of a fresh soluble fraction. We also found that Ca2+ augmented a non-O2- -producing O2 consumption in the cell-free preparation by unsaturated fatty acids and interfered with the activation of the O2- -generating system, especially that by saturated fatty acids.

Saturated and monounsaturated fatty acids with appropriate chain length such as laurate and oleate activated an 0;-generating enzyme system in a cell-free preparation from porcine neutrophils. The activated preparation catalyzed a stoichiometric conversion of 0 2 to 0, by utilizing NADPH as the electron donor. The preparation contained both membrane and soluble fractions and, upon separation into subfractions, the 0;-generating activity resided exclusively in the membrane fraction. Polyunsaturated fatty acids including arachidonate also activated the system, but they concurrently stimulated NADPH-independent O2 consuming reactions which yield neither 0; nor H202. The amount of such a non-0,"producing O2 consumption often reached twice as much as that of 0; production.
For the activation of the 0;-generating system in the membrane, the presence of the soluble fraction was essential. However, the soluble fraction was no longer effective when once used for the activation, suggesting that the effective component(s) in the fraction was consumed or translocated to the membrane during the activation. When the activated membrane was incubated with delipidated albumin, the activity was lost with concomitant decreases in the amount of membrane-associated fatty acids. The lost activity was restored by the replenishment of the fatty acid in the presence of a fresh soluble fraction. We also found that Ca2+ augmented a non-0;-producing 0 2 consumption in the cell-free preparation by unsaturated fatty acids and interfered with the activation of the 02-generating system, especially that by saturated fatty acids. &-Unsaturated fatty acids such as arachidonate' and oleate * This investigation was supported in part by grants from Keio University, by a grant from Takeda Science Foundation, and by grants from the Ministry of Education, Science and Culture, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
have been known to stimulate the release of superoxide anion (0;)' from intact neutrophils and macrophages (1)(2)(3)(4). The stimulation was reversed by the removal of fatty acids from the cells with delipidated albumin and was restored by the replenishment of the fatty acids (2). It was also known that the stimulation was accompanied by morphological changes of the cells (2). Saturated fatty acids such as myristate and palmitate and trans-unsaturated fatty acids including linolelaidate were described as inert both in stimulating the 0; release and in provoking the morphological changes. The effects of fatty acids were thus interpreted to be due to the perturbation of plasma membrane caused by the intercalated fatty acids with the double bond(s) in the &-configuration (1,2). Recently, an NADPH-dependent 0;-generating system in cell-free preparations from neutrophils or macrophages was shown to be activated by unsaturated fatty acids (5-10) and sodium dodecyl sulfate (ll), but again not by saturated fatty acids such as stearate and palmitate (6,9,10).
In contrast to above observations, Kakinuma and her associates (12)(13)(14)(15) have repeatedly reported that saturated fatty acids with appropriate chain length were also good stimuli for the 0;-generating system in intact neutrophils from various sources. Recently, we were also able to show that, besides unsaturated fatty acids, saturated fatty acids were effective in stimulating the 0 2 release in human and porcine neutrophils (16). In these papers, evidence was provided that the discrepant results on the effects of saturated fatty acids are due at least in part to the different Ca2' concentrations in the medium employed in each study. It has been known that Ca'+ interacts with fatty acids affecting their solubilities (17) or altering the physicochemical states of fatty acids which were inserted in the biological membrane (18).
In the present study, we examined the activation of the 0;-generating system in a cell-free preparation from porcine neutrophils by various fatty acids with special emphasis on the stoichiometry between 0; generation and 0 2 consumption in the activated reaction. The stoichiometry was determined by using a heme-substituted horseradish peroxidase as a trapping reagent for both 02 and Hz02 (19). The results show that all kinds of straight-chain fatty acids with appropriate carbon numbers (Clz-C20) are capable of activating the 0 2generating system in the cell-free preparation irrespective of their structures. The reported inertness of saturated and trans-monounsaturated fatty acids in the activation of 0 2generating system was presumably due to the way of adding them to the reaction mixture. A difference was noted among the effects of various kinds of fatty acids. The activation by saturated and monounsaturated fatty acids was specific to the 0;-generating enzyme system in the sense that they did not stimulate other 02-consuming reactions, while polyunsaturated fatty acids stimulated both 02-generating and non-02generating O2 consumptions. We also found that the OFgenerating system in the membrane fraction could recurrently be activated and deactivated in vitro by the addition and removal of a fatty acid, respectively. The presence of fresh soluble fraction was always required for the activation, but i t was not necessary to maintain the activity. The significance of these results is discussed. A preliminary report pertinent t o a portion of this work has appeared (16).

MATERIALS AND METHODS
Isolation of Neutrophils-Porcine neutrophils were isolated as described previously (20) with minor modifications. The whole blood (1 liter) anticoagulated with heparin was mixed with an equal volume of phosphate-buffered saline at pH 7.4, followed by sedimentation of erythrocytes in 0.9% dextran T-500 (Pharmacia LKB Biotechnology Inc.) at 1 X g, 25 "C. The granulocyte-rich supernatant was collected and centrifuged at 200 X g for 10 min. The pellets were suspended in 280 ml of a Ca2+-free HEPES-buffered Hanks' balanced salt solution containing 137 mM NaC1, 5.4 mM KC1, 0.81 mM MgSO4, 0.44 mM KH2P04, 0.34 mM NazHP04, 5.6 mM glucose, and 25 mM HEPES-NaOH at pH 7.3, which is hereafter called as Ca2+-free HBSS. The suspension (10 ml) was centrifuged at 400 X g for 30 min over 12 ml of Ficoll/Conray solution (IBL, Japan). Each precipitate was suspended in 30 ml of distilled water for 30 s to lyse the remaining erythrocytes, and the isotonicity of the medium was restored by the addition of 3 ml of 10% NaCI. The isotonicity was further assured by the addition of an appropriate amount of physiological saline. The granulocytes were obtained by centrifugation at 200 X g and washed at least twice with Ca2+-free HBSS. The cell preparation thus obtained consisted of 88-97% neutrophils, and the yield was 2-10 X loB cells from 1 liter of the blood. The cell suspension (5 X lo7 cells in 0.5 ml of Ca2+-free HBSS) in a glass culture tube (12 X 75 mm, Corning Glass Works) was frozen in liquid N2 and stored at -70 "C. After the storage for 2 months, loss of activity was less than 10% as judged by the fatty acid-stimulated 0; generation.
Preparation of Cell-free System, Its Fractionation and Reconstitution-To obtain a cell-free preparation, frozen suspensions of porcine neutrophils (5 X IO7 cells/0.5 ml) were quickly thawed in a water bath at 39 "C with vigorous shaking. The cells were found to be disrupted by this procedure and showed no 0;-generating activity upon stimulation by phorbol 12-myristate 13-acetate. In most experiments, the freeze-thawed preparation was further disrupted by sonic oscillation (for conditions see below) and was used as the cell-free preparation. To obtain unstimulated membrane and soluble fractions, the freezethawed preparation before sonic oscillation was fractionated into precipitate and supernatant by centrifugation at 6,000 X g for 5 s with a Beckman Microfuge B. The precipitate, which contained 80-90% of the total alkaline phosphatase activity and less than 3% of the total lactate dehydrogenase activity, was used as the membrane fraction. The soluble fraction was obtained by centrifuging further the supernatant fraction at 400,000 x g for 5 min at 4 "C by a microultracentrifuge (Hitachi CP100H). In reconstitution studies, the 6,000 X g precipitate was used in combination with the 400,000 X g supernatant. When membrane and soluble fractions were prepared from a previously sonicated preparation (see text), they were obtained by 400,000 X g centrifugation of the preparation. After mixing, the preparations were dispersed evenly by sonic oscillation at 0 "C for 30 times with 0.2-s pulses using a microprobe of the Branson sonifier at 20% of the full power.
Determination of 0; and H202 Generation and O2 Consumptwn- The generation of both 0; and H202 were determined spectrophotometrically by using diacetyldeuteroheme-substituted horseradish peroxidase as the trapping reagent (19). The heme-substituted horseradish peroxidase reacts stoichiometrically with 0; and H202 to form compounds I11 and 11, respectively, which are stable enough for our measurements and are spectrally distinguishable from each other (19). 0 2 consumption was measured with a Clark-type oxygen electrode (YSI Inc., 5331 probe) with a high sensitivity membrane (YSI Inc., 5776 membrane). 0; and H202 generation and 0 2 consumption were measured simultaneously in a single cuvette which was set in a Hitachi 557 dual wavelength-double beam spectrophotometer under constant stirring (19). The standard reaction mixture (0.4 ml) contained the cell-free preparation obtained from 4 X lo7 neutrophils, 5 mM EGTA, 0.16-0.25 mM diacetyldeuteroheme-substituted horseradish peroxidase and other ingredients in Ca2+-free HBSS at pH 7.3. For the activation of the 07-generating system, a desired amount of a fatty acid was added slowly to the reaction mixture with vigorous stirring at a rate of 5-10 nmol/s, which usually took about 2 min. If the fatty acid, especially a saturated fatty acid, was added more rapidly, activation of the 0;-generating system could hardly be observed. After further incubation for 5 min at 25 "C, NADPH (0.1 mM) was added to initiate the reaction.
Binding of "C-Fatty Acids to the Membrane Fraction-The cellfree preparation (4 X lo7 cells/0.4 ml) was incubated with 2 mM 1-14C-labeled laurate (0.04 mCi/mmol, Du Pont-New England Nuclear) or 1.5 mM [l-'4C]oleate (0.04 mCi/mmol, Amersham Corp.) for 5 min at 25 "C. The mixture was centrifuged at 200,000 X g for 30 min at 4 "C, and the surface of the resulting pellet was rinsed with Ca2+-free HBSS. Radioactivities in both pellet and supernatant fractions were determined in a liquid scintillation counter (Beckman Model LS 3800) using a Triton X-100-toluene-based scintillation fluid (21). Gas Chromatographic Analysis of Metabolites from Fatty Acids-Metabolism of laurate or oleate, if any, during the activation process of the cell-free system was evaluated by gas chromatography with a Shimadzu GC-14A gas-chromatograph equipped with a fused silica capillary column (Supelco Inc., SP-2330,300 cm). The cell-free preparation (4 X lo7 cells/0.4 ml) was incubated with 2.0 mM laurate or 1.5 mM oleate for 5 min at 25 'C. Then 1/19 volume of delipidated albumin solution (6.0 mM) in Ca2+-free HBSS was added to the mixture followed by further incubation for 10 min. The mixture was cooled and centrifuged at 400,000 X g for 5 min at 4 "C. The lipids in the resultant supernatant fraction were extracted by chloroform/ methanol (107, v/v) as described previously (22). Fatty acids thus extracted were methylated by diazomethane and quantitated with myristic acid or capric acid as the standard.
Chemicals-Extra pure preparations of lauric acid (99.8% pure), oleic acid (99.9% pure), myristic acid (99.7% pure), palmitic acid (99.5% pure), stearic acid (99.9% pure), linoleic acid (99.3% pure) were the gifts of Nippon Oil & Fats Co. Ltd. (Hyogo, Japan). Other fatty acids were of the highest grade available from Sigma (98-100% pure). These fatty acids were dissolved in absolute ethanol and neutralized with a minimal amount of 1 N NaOH except for arachidic acid. Arachidic acid was used without neutralization. The concentrations of ethanol in the stock solution were adjusted to 50 or 70% (v/ v). Usually 10-12.5 p1 of these fatty acid solutions were added to 0.4 ml of the reaction mixture slowly as described already. Ethanol up to the final concentration of 4% (v/v) in the reaction mixture had no detectable effect on the 0; generation activity. Horseradish peroxidase was purchased from Toyobo Co. Ltd. (Osaka). Diacetyldeuteroheme-substituted horseradish peroxidase was prepared as described previously (23). Delipidated albumin (bovine, prepared from fraction V) was from Sigma. BW-755C was the gift of Wellcome Research Laboratories (England). Other chemicals were of the highest grade commercially available.

Stimulation of 0;-generating System in a Cell-free Preparation by Fatty
Acids- Fig. 1 shows effects of various concentrations of fatty acids with t h e chain length from Clo t o C18 on NADPH-dependent 0; generation in a cell-free preparation from porcine neutrophils. As seen, saturated fatty acids exerted their maximal effects at concentrations over 2 mM, while unsaturated fatty acids had t h e maximal effect at around 1.5 mM and thereafter became inhibitory. In these experiments, fatty acids were added slowly under vigorous stirring to the reaction mixture which contained the cell-free preparation derived from 4 X lo7 cells (approximately 2 mg as protein) in 0.4 ml of Ca2+-free HBSS. The use of such a high concentration of the cell-free preparation was to obtain an accurate stoichiometry between O2 consumption and 0; generation. Under comparable conditions, the concentration of arachidonate which induced the maximal 0; generation was also around 1.5 mM. Saturated fatty acids with more longer or shorter chain length such as arachidate (Czo) and caprylate (C,) were not effective at similar concentrations.  tion. Upon addition of 0.1 mM NADPH, it immediately began to consume 0 2 and produced 0 2 , where the initial rates of 0 2 consumption and 0; generation were both 0.21 mM/min. Formation of Hz02 was not detected throughout the reaction. When the reaction was carried out in the absence of laurate, both 0 2 consumption and 0; production were negligible in the presence of the same concentration of NADPH. The reaction practically ceased at 2 min after the addition of NADPH, and the total amounts of 0 2 consumed and 0; generated during the reaction were 57.6 and 60.0 nmol, respectively. From these results, it is evident that the cell-free preparation activated by laurate converts molecular O2 stoichiometrically to 0;. Table I summarizes the initial rates of O2 consumption and 0; generation upon stimulation by various fatty acids. When the cell-free preparation was stimulated by saturated, and cisand trans-monounsaturated fatty acids, the ratios of the two reaction rates were all approximately 1:l. The ratios of total O2 consumption to total 0; production were also about 1:l.
On the other hand, the ratios of 0; production to O2 consumption were as low as 0.27, 0.53, and 0.49, when the preparation was stimulated with cis-polyunsaturated fatty acids such as arachidonate, linolenate, and linoleate, respectively. Furthermore, a massive O2 consumption was observed with these cis-polyunsaturated fatty acids, when they were added to the cell-free preparation in the absence of NADPH (data not shown). During such NADPH-independent 0 2 consumption, no formation of 0; was detected, and the amount of O2 consumed was roughly equal to that of the non-0;producing O2 consumption observed in the presence of NADPH. Thus both saturated and unsaturated fatty acids are capable of activating the 0;-generating system, but the latter fatty acids also induced a large amount of non-02producing O2 consumption. The NADPH-independent 0 2 consumption induced by 0.25 mM linoleate was inhibited by nordihydroxyguaiaretic acid and BW755C lipoxygenase inhibitors, with apparent Ki values of 8 and 9 pM, respectively.
Porcine neutrophils have been known to contain a significant amount of 12-lipoxygenase which metabolizes cis-polyunsat- System-It should be noted that all of the foregoing experiments were carried out under Ca2+-free conditions. The reason for such experimental conditions was due to the previous findings that Ca2+ interfered with the activation of the 0; generating system by fatty acids both in intact cells and in the cell-free systems (5, 6, 15, 16). Then we examined here the effects of Ca2+ ion on the activation by various fatty acids of 0;-generating system in the present cell-free system. As seen in Fig. 3, the laurate-induced 0; generation was completely inhibited by the presence of 1.0 mM Ca2+, while 0; generation with linolenate or arachidonate was scarcely affected by the same concentration of Ca2+. Intermediate degree of inhibition was observed with oleate-and linoleate-induced systems. In these experiments, 0 2 generation was initiated by the addition of NADPH immediately after the addition of a fatty acid. When NADPH was added at 5 min after the addition of a fatty acid in the presence of Ca2+, however, even linoleate-or arachidonate-induced 0; generation was inhibited (see below).
In the next experiments, we examined the effects of Ca2+ on the 0;-generating system which had been activated by the incubation with a fatty acid for 5 min. At 1 min after the addition of Ca2+, none of the 0;-generating activity induced by both saturated and unsaturated fatty acid was affected. After 5 min of incubation with Ca2+, however, the activity was found to be decreased significantly; the laurate-and linolenate-induced 0; generation decreased to 30 and 60% of their original activities, respectively. It should be noted that the activity induced by saturated fatty acids was more affected than that by unsaturated fatty acids. The reason(s) for this phenomenon will be discussed in detail under "Discussion." Ca2+ had another effect on the oxygen metabolism in the present system. When the cell-free preparation was incubated with cis-or trans-unsaturated fatty acid in the presence of Ca2+, a significant amount of 0 2 consumption was induced in 0 0.5 3. Effect of Ca" on the 0;-generating activity induced by various fatty acids in the cell-free system. The reaction mixture was the same as described under Fig. 1 except that EGTA was omitted from the system. The mixture, which received an indicated concentration of CaC12, was incubated at 25 "C for 3 min and then received a fatty acid (1.5 mM). The reaction was initiated by the addition of 0.1 mM NADPH immediately after the addition of fatty acid. 0; generation was measured by using diacetyldeuterohemesubstituted horseradish peroxidase and was expressed as percent control activity obtained under Ca2+-free conditions. The control activities under experimental conditions were 40 the absence of NADPH. For example, the rate of 0 2 consumption in the absence of exogenous NADPH was 82 k 13 kM/min (mean f S.E. in 10 experiments), when the system was stimulated with 0.5-1.0 mM oleate in the presence of 2 mM Ca2+. The concentration of Ca2+ required for a halfmaximal activation was 0.03 mM. The 0 2 consumption was not affected by up to 2 mM of NaN3 and KCN, and formation of 0 2 or Hz02 was not detected during the process. No similar 0 2 consumption in the absence of NADPH was observed with saturated fatty acids nor with the sonicates of porcine lymphocytes and platelets. We also found that at a pH lower than 6.5, all kinds of fatty acids including saturated fatty acids elicited an NADPH-independent O2 consumption even in the absence of Ca2+.
Requirement of Soluble Factor(s) for the Activation-As described earlier, saturated and monounsaturated fatty acids activated specifically the 0; production in the cell-free preparation from the neutrophils. We therefore tried to characterize further the system by fractionating it into the membrane and soluble fractions (Table 11). When the cell-free preparation was first activated by laurate (C,,,,) or oleate (C,,,,) and then separated into membrane and soluble fractions by centrifugation, the membrane fraction was found to be fully active in producing 0; upon addition of NADPH. In contrast, the soluble fraction had neither 0; producing activity nor any effect on the activity of the activated membrane fraction (Table 11, Experiment A). On the other hand, when the system was first separated into membrane and soluble fractions and each of them was incubated separately with laurate or oleate, 0;-generating activity was found in neither fractions. When the two fractions were combined and incubated with the fatty acid, a full activation of the 0;-generating system was obtained (Experiment B). Similar results were obtained upon activation of the system with other fatty acids such as myristate and a trans-monounsaturated fatty acid, elaidate. These results are in agreement with those of others who demonstrated the requirement of a soluble fraction in the activation of 0;-generating system in cell-free preparations from neu- Requirement of fresh solubk fraction for the activation of 0;generating system and localization of the activated system In Experiment A, the membrane and soluble fractions were obtained from a cell-free preparation which had been activated with 2.5 mM laurate or 1.5 mM oleate, and were assayed for 0 2 generation either separately or together. In Experiment B, the membrane, soluble or their combined fraction from an unstimulated cell-free preparation, was incubated with 2.5 mM laurate or 1.5 mM oleate for 5 min and was assayed for 02 generation. In Experiment C, a membrane fraction from unstimulated cell-free preparation was combined with fresh or used soluble fractions, and their activities after incubation with 2.5 mM laurate for 1.5 mM oleate for 5 min were determined. The amounts of cell-free preparations, soluble and membrane fractions, used were all that amount derived from 4 X lo7 cells. The reaction mixture (0.4 ml) was the same as that in Fig. 2. Reaction and assay conditions were also as in Fig. 2. Control activities were those of nonfractionated cell-free preparations activated by the same amount of respective fattv acids.

Experiment
Fraction Obtained from a cell-free preparation activated by laurate as in Experiment A.
dObtained from a cell-free preparation activated by oleate as in Experiment A. trophils or macrophages by unsaturated fatty acids and sodium dodecyl sulfate (5-11).
We found, however, that a soluble fraction obtained from the cell-free preparations previously activated by fatty acids was no longer effective even when it was combined with a fresh membrane fraction and incubated with the same or different fatty acids used for the prior activation (Table 11, Experiment C). Thus the effective component for the activation was lost from the soluble fraction during the activation. The loss of activity was also observed by using other fatty acids. Additions of ATP (0.01-1.0 mM) or GTP (0.01-1.0 mM) to the used soluble fraction had no effect on the lost activity. A similar loss of the active component(s) seems to take place in vivo, since a soluble fraction obtained from intact neutrophils which were activated with laurate (0.45 mM) or phorbol 12-myristate 13-acetate (200 ng/ml) for 5 min was less effective than that from untreated cells by 40-60% in stimulating the membrane-bound 09-generating system. The viability of these activated neutrophils was more than 95% as judged by trypan blue dye exclusion test.
Reversible Activation of 09-generating System-When the cell-free preparation was activated by a fatty acid and then incubated with a sufficient amount of delipidated albumin, the activity decreased with increasing time of incubation resulting in an almost complete deactivation within 10 min (Fig. 4  The cell-free preparation was incubated with "C-labeled laurate (2.0 mM) or oleate (1.5 mM) for 5 min at 25 "C and then incubated with 0.3 mM delipidated albumin for 10 min at 25 "C. Distribution of radiolabeled fatty acids was determined before and after the albumin treatment, and the values were expressed as percent recovery. The membrane and soluble fractions were isolated by centrifugation at 200,000 X g for 30 min at 4 "C (Experiment A) or at 25 "C (Experiment B). Each value is the mean from dudicate emeriments.  The cell-free preparation obtained from 4 X lo7 cells which had been activated by laurate, myristate, oleate, or elaidate was deactivated by the incubation with delipidated albumin. The albumin concentrations used were 0.38 mM for laurate-and myristate-activated preparations and 0.3 mM for oleateand elaidate-activated preparations. Then the albumin-treated membrane fraction was obtained by centrifugation at 400,000 X g and reactivated by fatty acids in the presence or absence of a fresh soluble fraction (derived from 4 X lo7 cells). Fatty acid concentrations used for both preactivation and reactivation were 2.5 mM for laurate and myristate or 1.5 mM for oleate and elaidate. Other conditions and assay methods were the same as those in Fig. 1 radioactivity associated with the membrane was removed and recovered in the supernatant fraction containing albumin (Table 111). The total recovery of radioactive fatty acids (laurate and oleate) was 98-100% after the albumin treatment. No metabolite of these fatty acids was detected in a significant quantity upon examination by gas chromatographic analyses.
The membrane fraction thus deactivated by the albumin treatment was not activated by the addition of fatty acid alone, but about 40-80% of the 02-generating activity was restored by the incubation with a fresh soluble fraction followed by the addition of a fatty acid (Table IV). In the absence of soluble fraction, the restoration was less than 15%. Thus the 0;-generating system in the membrane fraction was reversibly and repeatedly activated by fatty acids with the aid of the fresh soluble fraction.

DISCUSSION
We have shown in this study that the saturated and monounsaturated fatty acids are both good stimuli for the activation of the 0;-generating system in a cell-free preparation from porcine neutrophils. As judged by the stoichiometric relationship between O2 consumption and 0 2 formation in the reaction, these fatty acids activate specifically the 0;generating system in the cell-free preparation. The results, together with the previous findings by Kakinuma and coworkers (12-15) with intact phagocytes, have unambiguously established that the activation of the phagocyte 02-generating system by fatty acids is not limited to that by cis-unsaturated fatty acids.
The reason why most of previous investigators have overlooked the effect of saturated fatty acids on the 0;-generating system both i n vivo and i n uitro is not clear, but the following points may be discussed as possibilities. 1 ) Ca", which was often included in the reaction mixtures of the past experiments, interacts more easily with saturated fatty acids than with unsaturated fatty acids to alter their solubilities (17), which in turn affect the effective concentration of fatty acids in the reaction mixture. It has been pointed out that Ca2+ elevates the Kraft point of a fatty acid (15). Alternatively, Ca2+ can interact with saturated fatty acids in the membrane, for example, to form copolymeric lattice structure (18), which may harden the membrane to reduce its permeability. 2) The method of adding fatty acids is critical. As we have mentioned in this paper, a fatty acid, especially a saturated fatty acid, must be added to the reaction mixture slowly at a constant rate under vigorous stirring. If not, much less activation or irreproducible results were obtained, presumably due to the formation of fatty acid micelles or aggregates, which may reduce effective interactions of the fatty acid with the enzyme system in the membrane and/or with the soluble component(s).
The mechanism(s) by which fatty acids activate the 0;generating system has been the subject of considerable investigation. For example, Badwey et al. (2) have suggested physicochemical changes in the plasma membrane induced by the intercalation of a cis-unsaturated fatty acid, while Bromberg and Pick (11) have raised various possibilities primarily ascribable to the detergent action of sodium dodecyl sulfate. The role of guanine nucleotide-binding protein has also been emphasized by many workers (9, 10, 25, 26), who suggested that fatty acids facilitate interactions among membranebound oxidase, soluble component(s), and the guanine nucleotide-binding protein(s). Then the present study provides the following insights into understanding the mechanism(s) of the activation by fatty acids of neutrophil 0;-generating system.
First, the finding that the laurate and oleate, most frequently used as stimuli in this study, were not metabolized in our cell-free system indicates that the fatty acids themselves and not their metabolites are responsible for the activation. Second, the fatty acid must remain in the membrane to maintain the activated state of the 0;-generating system both i n uiuo and i n uitro. Karnovsky and his associates (2) have shown that intact neutrophils activated by arachidonate can be deactivated by the addition of delipidated albumin and reactivated by the replenishment of the fatty acid. We demonstrated here that the removal and the replenishment of the fatty acid from and to the membrane preparation deactivated and reactivated recurrently the 0;-generating system in a cell-free state. Third, a soluble fraction was required also for the activation by saturated fatty acids. Similar requirement of a soluble fraction has been pointed out for the unsaturated