Identification of type-2 phosphatidic acid phosphohydrolase (PAPH-2) in neutrophil plasma membranes

Identification of type-2 phosphatidic acid phosphohydrolase (PAPH-2) in neutrophil plasma membranes

Cellular Signalling Vol. 6, No. 8, pp. 933-941, 1994. Elsevier Science Ltd Printed in Great Britain Pergamon 0898-6568(94)00051-4 IDENTIFICATION OF...

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Cellular Signalling Vol. 6, No. 8, pp. 933-941, 1994. Elsevier Science Ltd Printed in Great Britain

Pergamon 0898-6568(94)00051-4







ERIC BODER,t~ G R E G TAYLOR,§ LUKE AKARD, JAN JANSEN and DENIS ENGLISH~ SBone Marrow Transplant Laboratory, Methodist Hospital of Indiana, IN, U.S.A. and Department of Physiology and Biophysics, Indiana University School of Medicine, Indianapolis, IN, U.S.A. (Received 2 June 1994; and accepted 14 June 1994)

Abstract--Plasma membrane phosphatidic acid phosphohydrolase (PAPH) plays an important role in signal transduction by converting phosphatidic acid to diacylglycerol. PAPH-2, a Mg2+-independent, detergent-dependent enzyme involved in cellular signal transduction, is reportedly absent from the plasma membranes of neutrophilic leukocytes, a cell that responds to metabolic stimulation with abundant phospholipase D-dependent diacylglycerol generation. The present study was designed to resolve this discrepancy, focusing on the influence of cellular disruption techniques, detergent availability and cation sensitivity on the apparent distribution of PAPH in neutrophil subcellular fractions. The results clearly indicate the presence of two distinct types of PAPH within the particulate and cytosolic fractions of disrupted cells. Unlike the cytosolic enzyme, the particulate enzyme was not potentiated by magnesium and was strongly detergent-dependent. The soluble and particulate enzymes displayed dissimilar pH profiles. Separation of neutrophil particulate material into fractions rich in plasma membranes, specific granules and azurophilic granules by high speed discontinuous density gradient centrifugation revealed that the majority of the particulate activity was confined to plasma membranes. This activity was not inhibited by pretreatment with n-ethylmaleimide in concentrations as high as 25 mM. PAPH activity recovered in the cytosolic fraction of disrupted neutrophils was almost completely inhibited by 5.0 mM n-ethylmaleimide. We conclude that resting neutrophils possess n-ethylmaleimide-resistant PAPH (type 2) within their plasma membranes. This enzyme may markedly influence the kinetics of cell activation by metabolizing second messengers generated as a result of activation of plasma membrane phospholipase D. Key words: Phosphatidic acid, second messengers, phospholipase D.


vates protein kinase C [8], an enzyme which is a constituent of several signal transduction pathw a y s and w h i c h p l a y s a r o l e in n e u t r o p h i l N A D P H oxidase activation [1-3]. PA has recently been implicated as a stimulator of several different cellular responses [9-13]. Previous studies have revealed that the kinetics of PA formation in neutrophils closely parallel the kinetics of cellular activation, leading to the hypothesis that PA is required for neutrophil metabolic stimulation [14-16]. PA has been demonstrated to activate the N A D P H oxidase in disrupted cell preparations [17-19] and to induce superoxide release of intact neutrophils [3, 20]. In stimulated neutrophils, PA may be generated directly--when phospholipids are hydrolysed by phospholipase D - - o r indirectly

A m o n g the second messengers involved in neutrophil activation, d i a c y l g l y c e r o l ( D A G ) and phosphatidic acid (PA) have both been implicated as important stimulatory lipids [1-7]. D A G acti*This work was supported by an NIH Shannon Award and by a grant from the Phi Beta Psi Sorority. tPresent address: Department of Chemical Engineering, University of Illinois, Urbana, IL. §Present address: Department of Biological Chemistry, Purdue University, Lafayette, IN. ~To whom correspondence should be addressed at: Bone Marrow Transplantation Laboratory, Methodist Hospital of Indiana, Indianapolis, IN 4602, U.S.A. Abbreviations: DAG--~iacylglycerol; NEM--n-ethylmaleimide; PA--phosphatidic acid; PAPH--phosphatidic acid phosphohydrolase. 933


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as a consequence of phosphorylation of DAG generated by the activity of phospholipase C. In the light of the proposed roles of both PA and DAG in signal transduction, phosphatidic acid phosphohydrolase (PAPH) has been implicated as an important neutrophil regulatory enzyme [3, 21-22]. This hypothesis is supported by results with PAPH inhibitors which markedly affect neutrophil function [3, 14, 21, 23-25]. Several reports have d e m o n s t r a t e d that inhibitors of P A P H enhance neutrophil functional responses to the chemotactic agonist, FMLP, presumably as a result of enhanced accumulation of PA [21, 23, 25]. On the other hand, decreased accumulation of DAG in cells treated with PAPH inhibitors leads to inhibition of responses triggered by PMA and other metabolic agonist [21, 24]. Previous studies have confirmed the existence of PAPH in human neutrophils but these studies have yielded vastly conflicting results regarding the subcellular distribution of the enzyme [21, 22]. In other cells, two predominant forms of PAPH have been identified by Brindley and colleagues; PAPH-1, a soluble Mg2+-dependent enzyme which is involved in glycerolipid synthesis, and PAPH-2, which is located in the plasma membrane and regulates signal transduction [26, 27]. Activities of these enzymes are measured by differential sensitivity to N-ethylmaleimide. In a recent study, Truett et al. reported an absence of type 2 activity in the plasma membranes of resting neutrophils, a result which would limit the involvement of the enzyme in regulation of signal transduction [22]. Since definition of the subcellular distribution of PAPH is critical for a full appreciation of the potential role of this enzyme in regulation of neutrophil activation, the present study was designed to resolve the discrepancy regarding the existence of PAPH in neutrophil plasma membranes. The results demonstrate that the apparent distribution of PAPH activity in neutrophil subcellular fractions is markedly influenced by the conditions employed for assay of enzyme activity. The data indicate that relatively high levels of PAPH activity are present in neutrophil plasma membranes. Several characteristics of this activity differentiate it from its cytosolic

counterpart, including pH optima, cation dependence and detergent requirements. Based on resistance to inhibition with n-ethylmaleimide, the neutrophil plasma membrane PAPH activity is identified as the type 2 PAPH activity implicated as an important component of the signal transduction apparatus in other systems. MATERIALS AND METHODS Reagents

[7-32p]ATP (6000 Ci/mmol) was purchased from DuPont New England Nuclear (Boston, MA). DAG kinase was from Lipidex (Westfield, NJ), and Hanks' Balanced Salt Solution (HBSS) was from GIBCO (Grand Island, NY). All other chemicals and reagents were obtained from Sigma Chemical (St Louis, MO). Cel/s Human neutrophils were isolated from heparinized whole blood of healthy donors by enhanced sedimentation followed by centrifugation on ficoll-Hypaque to remove mononuclear leukocytes. Residual erythrocytes were lysed by suspension in 0.85% ammonium chloride containing 0.1% KH2CO3. Finally, the neutrophils were washed with HBSS and resuspended in sonication buffer (10 mM HEPES, 154 mM NaCI, 1 mM EGTA, pH 7.2) at a concentration of approximately 108 cells/ml and chilled on melting ice. Subcellular fractionation

Cells were disrupted by sonication at 35% power on a Fisher Sonic Dismembrator as described below. In some experiments, cells were disrupted by pressurization to 300 psi under N2 followed by rapid depressurization. Microscopic examination revealed greater than 90% disruption by both techniques. Between all steps following disruption, lysates were kept on melting ice. To remove intact cells and nuclei, lysates were centrifuged at 1500 g for 5 min. Particulate and cytosolic fractions were then isolated after centrifugation at 40,000 g for 60 min (4°C) as indicated below. After removal of cytosol, pellets were resuspended in sonication buffer at a volume equal to that of the original sample, centrifuged and fractions were stored frozen (-20°C) until use. Protein concentrations were determined using a Bio-Rad Protein Assay Kit. For some experiments, cell lysates were layered over discontinuous sucrose gradients (40, 50 and 60% sucrose) and centrifuged for 40 min at 100,000 g to separate azurophilic granules, specific granules and plasma membranes as previously described [28]. Each fraction

PAPH-2 in neutrophil plasma membranes was carefully aspirated from the gradient through a blunted 26 gauge needle and diluted with sonication buffer to a volume equivalent to that of the original fractionated sample. Assays of the plasma membrane marker alkaline phosphatase within each subcellular fraction were performed with p-nitrophenol phosphate at pH 10 as previously described [29].

(approximately 100-500 ~tg cellular protein) and 10 p,l PA solution (66-250 ktM final concentration) unless indicated otherwise. Assays were incubated for 30 min at 37°C and reactions were then stopped by addition of 2 ml of chloroform/methanol/HC1 (1:2:0.03). Water and chloroform (0.5 ml) were then added to separate phases. Vials were thoroughly vortexed and then centrifuged at 1500 g for 5 min. The entire aqueous phase was collected, washed twice with chloroform and assayed for radioactivity by Cherekov counting. Previous studies have confirmed that release of radioactivity in this system is due to release of labelled inorganic phosphate resulting from the action of PAPH on [32p]PA [21, 22].

Preparation of radiolabelled phosphatidic acid [32p]PA was synthesized by incubating DAG and [~/32p]ATP at 37°C in the presence of DAG kinase, as previously described [30]. Lipids were then extracted from the reaction mixture with chloroform/methanol/concentrated HC1 (1:2:0.03). Thin-layer chromatographic analysis of the extracted lipids under previously described conditions [16] revealed that p2P]PA accounted for virtually all the recovered radioactivity. Radiolabelled PA was mixed with cold PA suspended in chloroform (final activity 250-850 IxCi/mmol) and the mixture was dried under nitrogen and stored at 4°C until use.


Divergent characteristics of particulate and soluble neutrophil PAPH W e first compared the effects of detergent on P A P H activity in the particulate and cytosolic fractions of sonicated neutrophils. The divergent effects of NP-40 on these activities are clearly evident in Fig. 1. Plasma membrane fractions required 0.08% (w/v) NP-40 for optimal activity, while the optimal concentration for elaboration of cytosolic activity was much lower (0.01%). Although under optimal assay conditions the total

Assay of PAPH Radiolabelled PA was sonicated into solution in an assay buffer containing 50 mM HEPES (pH 7.0), 40 mM NaC1 and 2 mM EGTA and detergent (Triton X100 or NP-40) at indicated concentrations. Assays consisted of 190 p,l assay buffer, 50 I11 cell fraction 400





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i 0.04

i 0.12




j 0.16


Fig. 1. Detergent dependence of particulate and soluble neutrophil PAPH. Cytosol and membranes were obtained after high speed centrifugation of sonicated cells and assayed for PAPH activity in the presence of indicated concentrations of the detergent, NP40. Assays were carded out in the absence of added Mg 2÷. Values plotted represent the means of triplicate data from two separate experiments. The absence of error bars indicates that the standard deviation was smaller than the respective data point.


E. Boder et al.



E rE ..... "o Q) N






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3x Sonicati0n

4x Cycles

Fig. 2. Effect of sonication on membrane and cytosolic PAPH activities. The specific activities of PAPH in the particulate and cytosolic fraction of sonicated cells were measured by incubation with [32p]PA at 37°C in the presence of 0.08% (w/v) NP-40 and the absence of added Mg 2÷. Values are the result of a single experiment performed in triplicate (mean ± S.D.). Cells were sonicated by discrete 15 s, bursts at a setting of 35% on a Fisher Sonic Dismembrator. 200


E e-


0 ~





Membranes Cytosol Fig. 3. Comparison of PAPH activity in membranes and cytosol of neutrophils lysed by sonication and N2 cavitation. Membrane and cytosol fraction were incubated with [32p]PA (66 IJ.M) at 37°C in assay buffer containing 0.08% NP40. Results are from one experiment conducted in triplicate.

PAPH-2 in neutrophil plasma membranes

particulate activity exceeded that recovered in the cytosol, in the absence of detergent very little particulate activity was evident but cytosolic activity was only slightly impeded. At concentrations between 0.02% and 0.08%, NP-40 potentiated membrane activity but strongly inhibited soluble activity. As previously reported for triton X-100 [21, 22], NP-40 inhibited both forms of the enzyme at high concentrations. We next examined the influence of the cell disruption method on neutrophil PAPH activity, since sonication has previously been implicated in altering apparent enzyme levels in disrupted cells [22]. As shown in Fig. 2, increasing the duration of sonication did decrease levels of activity in the cytosolic fraction, as 'reported by Truett et al. [22]. However, this decrease was not associated with increased levels of membrane activity as would be expected if sonication caused cytosolic activity to incorporate in membrane particles. Most importantly, levels of activity recovered in the particulate fraction of cells disrupted by nitrogen cavitation were equivalent to those recovered in membranes of sonicated cells (Fig. 3), confirming that sonication was not the cause of the particulate activity.

Effects of cations

Walton and Possmayer observed differential effects of Mg 2÷on rat lung PAPH [31 ], and Jamal et al. reported similar results in rat liver cells [26]. Similarly, magnesium effected a dramatic enhancement of neutrophil cytosolic PAPH activity but did not potentiate particulate activity (Fig. 4). At a concentration of 0.25 raM, Mg 2÷ approximately doubled cytosolic PAPH activity but had no discernible effect on particulate activity. Higher levels of Mg 2÷ did not further potentiate cytosolic activity and caused an inhibition of particulate activity. Both cytosolic and particulate activity were inhibited by Ca 2÷in approximately the same manner. The above experiments were performed in the presence of detergent, but the enhancing effects of Mg 2. on cytosolic PAPH activity were not detergent-dependent. As shown in Table 1, the divergent effects of Mg 2÷ on cytosolic and particulate neutrophil PAPH were observed in assays performed both in the presence and absence of detergent. The conditions of assay thereby strongly influenced the apparent distribution of recovered PAPH activity. For example, when analyses were Co 2 + MQ 2 +


Plasmo Membranes




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Fig. 4. Effects of Mg2÷ (open symbols) and Ca 2÷ (closed symbols) on membrane (solid lines) and cytosol (dotted lines) PAPH activity. Subcellular fractions were prepared from sonicated cells. PAPH activity was measured by incubating cellular fractions with [32P]PA(66 [aM) at 37°C in assay buffer containing 0.05% Triton X-100 and the indicated levels of cations.

E. Boder et al.


Table 1. Distributionof neutrophilPAPH Assay components* Mg2÷ Detergent

PAPH activity(pmol/min/mg) Membranes Cytosol

0 0 + +

18.6 ± 7.6 331.6 ± 25.6 22.0± 1.1 155.4 ± 1.9

0 + 0 +

% Total activity Membranes Cytosol

146.5 ± 5.6 49.2 ± 2.4 273.1 ±5.0 98.3 ± 2.1

11.2 87.1 7.5 61.3

88.7 12.9 92.5 38.7

*Subcellularfractions were incubatedwith 132 laM [32p]PAat 37°C in the presence of absence of 3 mM MgCI2 and 0.08% NP-40 as indicated.Results are means + S.D. of triplicate determinations.

p H optima

performed in the presence of Mg 2. and absence of detergent, over 90% of the recovered activity was localized in the cytosolic fraction. Conversely, in the presence of detergent and absence of Mg 2÷, 87.1% of the total activity resided in the particulate fraction. The results confirm the existence of membrane-bound PAPH in neutrophilic leukocytes and underscore the importance of assay conditions in characterizing this activity.



T • . . . / I '",.. T


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We investigated the pH dependence of neutrophil cytosolic and membrane PAPH in an attempt to determine whether these activities resulted from the same or different enzymes, The results demonstrate strikingly different pH profiles for the soluble and insoluble activities (Fig. 5). The cytosolic enzyme showed a clear optimum at pH 6.9; activity rapidly fell in assays conducted





Plasma Membranes



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60.0 5.7 6.0 6..5 6.6 6.9 7.2 7.5 7.8 8.1 r






pH Fig. 5. Influence of pH on cytosolic and particulate PAPH activities. Assays were conducted in the presence of 0.5% Triton X-100. Results reflect mean _+S.D. of triplicate determinations.

PAPH-2 in neutrophil plasma membranes



Particulate Fraction

Disrupted Cell Preparation ~



% Recovered PAP Activity

Cytosol Plasma Membranes

~' 66.26_+ 0.85

Specific Granules

~' 20.04_+ 0.55

40% Sucrose

40% Sucrose 100,000 x g 40 minutes

50% Sucrose

50% Sucrose

--t, Azurophillic Granules


"i3.70 + 0.82

60% Sucrose



Fig. 6. Distribution of particulate neutrophil PAPH. Particles were isolated by density gradient centrifugation of crude sonicates as described in Materials and Methods above. Fractions were incubated with 66 I.tM [32P]PA in assay buffer containing 0.05% Triton X-100 and no Mg2÷. Results shown are means _ S.D. (n = 3) of the percentage of total particulate activity recovered in each fraction. at higher or lower pH values. In contrast, the membrane-associated enzyme displayed a much lower and broader pH optimum with maximal reaction rates observed at pH 6.0. These results indicate that the molecules contributing to the particulate and soluble activities are not identical.

Subcellular distribution of particulate PAPH activity We employed a well-characterized discontinu-

Table 2. Effect of NEM on plasma membrane and cytosolic PAPH

Preincubationt Untreated NEM, 5.0 mM NEM, 25.0 mM

PAPH activity* (pmol/min/mg) Cytosol Plasma membrane 353.4 ± 40.8 39.7 ± 12.4 9.9 ± 1.6

246.3 ± 25.3 268.2 ± 9.6 221.1 ± 5.2

*Cytosolic PAPH activity was determined in the presence of 3.0 mM MgC12 and no added detergent. For assays of plasma membrane activity, MgCI2 was omitted and NP-40 was added at a concentration of 0.08%. Results are means ± S.D. of triplicate determinations. tFractions were preincubated with the indicated concentrations of n-ethylmaleimide for 10 rain at 37°C prior to dilution in assay buffer and analysis of PAPH activity.

ous density gradient technique to separate plasma membranes from two major granule populations (specific granules and azurophilic granules) in the particulate fraction of sonicated cells. Each fraction was recovered and diluted to a volume equivalent to that of the original unseparated material in order to estimate its relative contribution to total neutrophil particulate PAPH activity. The activity of each fraction as a percent of the total is shown in Fig. 6. Most of the total particulate activity was localized in the plasma membrane fraction, with lower but significant levels of activity recovered in the specific and azurophilic granule fractions of resting cells. At least a portion of the activity recovered within each granule fraction could be attributed to contamination with plasma membranes as revealed by analysis of the plasma membrane marker alkaline phosphatase in each fraction. In three separations, 7.1 _+ 0.5% of the total amount of this enzyme within the unseparated particulate fraction was recovered in the specific granule fraction and 2.6 +_ 0.2% was recovered in the azurophilic granule fraction. Thus, just under 10% of the plasma membranes sedimented with granules under the conditions used. Since 33% of the particulate PAPH activity was recovered in granules, however, it is evident that the


E. Boderet al.

enzyme was localized both in granule and plasma membranes. However, the majority of the particulate activity resides in the plasma membranes. N-ethylmalelmide sensitivity

In a previous study, we identified n-ethylmaleimide-insensitive PAPH within a relatively crude particulate fraction of human neutrophils that contained plasma membranes as well as other subcellular organelles [21]. Because of the proposed pivotal role of n-ethylmaleimide-resistant plasma membrane PAPH in cellular signal transduction and the reported failure of previous investigators to identify this activity in neutrophil plasma membranes [22], we assessed the n-ethylmaleimide sensitivity of PAPH within neutrophil plasma membranes isolated by differential sucrose discontinuous gradient centrifugation, as described above. Sensitivity of the cytosolic neutrophil enzyme to n-ethylmaleimide was determined for comparison. The results are shown in Table 2. Preincubation of purified plasma membranes for 10 min at 37°C with 25 mM N-ethylmaleimide had little effect on PAPH activity. In contrast, nethylmaleimide almost completely abolished cytosolic PAPH activity when added at concentrations as low as 5 mM. DISCUSSION Brindley and associates have characterized two forms of PAPH based on differential sensitivity to N-ethylmaleimide [26, 27]. The N-ethylmaleimide sensitive enzyme, PAPH-1, is found in the cytosol of many cells and is predominantly involved in glycerolipid synthesis. PAPH-2, a Nethylmaleimide insensitive plasma-membrane enzyme, is involved in cellular signal transduction. In rat liver, the cytosolic enzyme was Mg 2+ dependent whereas activity of the plasma-membrane enzyme was not potentiated by the cation. In a previous analysis of neutrophil PAPH, Truett et al. failed to identify plasma membrane n-ethylmaleimide-resistant (PAPH-2) activity [22]. A small amount of activity was recovered in the particulate fraction of disrupted neutrophils, but like

cytosolic activity, this activity was inhibited by nethylmalemide and, as the authors suggested, may have represented residual cytosolic activity recovered in the particulate fraction after centrifugation. Absence of PAPH in plasma membranes of resting neutrophils would markedly limit the potential involvement of the enzyme in the regulation of signal transduction, since most of the phosphatidic acid generated by the activation of phospholipase D, a membrane bound enzyme, is itself confined to the plasma membrane. In addition, the configuration of phosphatidic acid within the plasma membrane may prevent its hydrophilic polar region from interacting with enzymes confined to the cytosol. Our own previous studies with crude particulate fractions of disrupted neutrophils indicated that the cells did possess a particulate, Mg2*-independent activity that was nethylmaleimide resistant, although the presence of this activity within plasma membranes was not directly confirmed [21]. These considerations led to the present investigation which was designed to determine the presence and subcellular distribution of type 1 and type 2 PAPH activity in neutrophilic leukocytes. The results confirm the existence of type 2 activity in isolated neutrophil plasma membranes and type 1 activity in the cytosol. In addition to their differential sensitivity to magnesium ions and n-ethylmaleimide, these two entities display strikingly different pH optima and detergent requirements. Previous failure to localize PAPH-2 activity in neutrophil plasma membranes most likely result from failure to employ detergent in the assay system. Our results demonstrate that detergent is required for expression of type 2 but not type 1 activity in neutrophil subcellular fractions. Furthermore, the amount of detergent necessary for optimal expression of type 2 activity (0.08% NP-40) strongly inhibited type 1 activity. Thus, within the cellular milieu where membrane-bound enzyme interacts with lipid substrates under physiological conditions, the activity of type 2 PAPH may actually be much higher. Therefore, we conclude that plasma membrane PAPH activity is present in neutrophil plasma membranes in relatively high levels. This enzyme potentially exerts an important regulatory influ-

PAPH-2 in neutrophil plasma membranes ence on second messengers involved in neutrophil signal transduction. Acknowledgements--The authors wish to thank STEPHANIE McGILLEM for her secretarial support with the preparation of this manuscript. E. BODER and G. TAYLOR contributed equally to this work.

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