©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Partial Purification and Characterization of Arf-sensitive Phospholipase D from Porcine Brain (*)

H. Alex Brown (1), Stephen Gutowski (1), Richard A. Kahn (2), Paul C. Sternweis (1)(§)

From the (1)Department of Pharmacology, University of Texas, Southwestern Medical Center, Dallas, Texas 75235-9041 and the (2)Laboratory of Biological Chemistry, Developmental Therapeutics Program, Division of Cancer Treatment, NCI, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Phospholipase D (PLD) activity from membranes of cultured cells can be activated by guanosine 5`-O-(3-thiotriphosphate) and the small GTP-dependent protein, Arf. While this activity was readily apparent in membranes from HL60 cells, it was much lower or not observable in membranes from various mammalian tissues. However, extraction of porcine brain membranes with detergent and subsequent chromatography with SP-Sepharose revealed a large peak of Arf-sensitive PLD activity. This activity has been enriched through several steps of chromatography and characterized with respect to size, nucleotide specificity, and sensitivity to different Arf and Arf-like proteins. Hydrodynamic analysis indicated that the enriched PLD had an s of 5.1 and a Stokes radius of 4.3 nm. These parameters indicate that the enzyme has an apparent molecular mass of 95,000 Da. Effective stimulation of the enriched enzyme was achieved with GTP as well as nonhydrolyzable analogs. All of the Arf subtypes tested were effective activators of PLD activity. Arf derived from yeast could activate mammalian PLD but with lower potency. The Arf-related Arl proteins were ineffective.

PLD that has been highly enriched retained a requirement for phosphatidylinositol 4,5-bisphosphate for efficient expression of activity. Additionally, the ability of recombinant or purified porcine brain Arf to stimulate PLD activity was reduced relative to impure fractions of Arf activity. Thus, porcine PLD that has been purified about 5,000-10,000-fold is synergistically activated by Arf in combination with other cytosolic components that are described in the accompanying paper (Singer, W. D., Brown, H. A., Bokoch, G. M., and Sternweis, P. C.(1995) J. Biol. Chem. 270, 14944-14950). Taken together, these data suggest that physiological regulation of Arf-sensitive PLD may involve the coordinate assembly of several interacting regulatory subunits.


INTRODUCTION

Lipids can serve as the source of several second messengers important in signal transduction. Inositol 1,4,5-trisphosphate and diacylglycerol are derived from a single phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP),()by the action of a phosphatidylinositol-specific phospholipase C. Stimulation of various isozymes of phosphatidylinositol-specific phospholipase C by heterotrimeric G proteins or receptor tyrosine kinases can thus account for regulation of intracellular Ca and protein kinase C-dependent pathways by a variety of hormones(1) . A cytoplasmic phospholipase A has also been extensively characterized and serves as a mechanism for the production of arachidonic acid and its metabolites(2) .

Progress in understanding the regulation and function of phospholipase D activity has been slower (for reviews, see Refs. 3-5). This enzymatic activity hydrolyzes phospholipids into phosphatidic acid (PA) and their respective polar head groups and is distinct from the better characterized PLD activity that hydrolyzes phosphatidylinositol-glycans (6). Signaling via this reaction is accomplished through the production of PA, which may act directly as a second messenger or serve as a precursor to diacylglycerol or lysophosphatidic acid. As a direct messenger, PA may act to modulate the activity of a variety of intracellular enzymes. Alternatively, the production of PA may serve to alter the local structural characteristics of membranes and subserve fusigenic events. Stimulation of PLD activity via hormones that act through either G protein-dependent or tyrosine kinase-linked mechanisms has been reported (for review, see Ref. 5). However, the mechanisms for this regulation have not been determined. Some experiments have implicated protein kinase C as one potential regulatory influence(7, 8) .

Recently, two groups have demonstrated that PLD activity can be stimulated by the small monomeric G protein, Arf(9, 10) . Arf was originally identified as a factor required for efficient ADP-ribosylation of the heterotrimeric G protein by cholera toxin(11, 12) . Subsequently, a family of Arf proteins has been described that contains six mammalian isoforms that have this activity and are each greater than 60% identical in amino acid sequence. The family has been extended to include a group of Arf-like (Arl) proteins, which typically share 40-55% identity to any of the Arf proteins but which are functionally diverse(13, 14, 15) . The Arf proteins have been found to have a role in intracellular protein traffic (see Ref. 16 for discussion). The essential nature of the Arf proteins was demonstrated by the lethality of coincident deletion of both known Arf proteins in yeast(17) .

PLD activity was first described in higher plants, and the enzymes from these organisms and some procaryotes have been extensively characterized. In contrast, a mammalian counterpart was not described until later(18) , and its isolation has just been reported(19) . A second PLD activity that is stimulated by Arf (9, 10) is the subject of this report. Herein, we report the partial purification of Arf-dependent PLD from porcine brain and its initial characterization with respect to substrate specificity, sensitivity to Arf proteins, and additional cytosolic factors that will be described in more detail in the accompanying manuscript(20) .


EXPERIMENTAL PROCEDURES

General Reagents and Methods

All reagents were analytical grade unless otherwise indicated. Bovine brain phosphatidylethanolamine (PE) and PIP as well as phospholipase D (Type II) from peanut were obtained from Sigma. Chemically defined phospholipids, such as dipalmitoylphosphatidylcholine (PC), were obtained from Avanti. H- and C-labeled dipalmitoyl-PC and other labeled phospholipids were obtained from DuPont NEN. Dioctylphosphatidylinositol 3,4,5-trisphosphate was synthesized through great efforts (21) and was the generous gift of J. R. Falck (University of Texas, Southwestern Medical Center).

Protein concentrations were determined by staining with Amido Black (22) using bovine serum albumin as a standard. Protease inhibitors were included in solutions when indicated in the following concentrations: 2.5 µg/ml leupeptin, 0.1 unit of trypsin inhibitor/ml aprotinin, 21 µg/ml N-p-tosyl-L-arginine methyl ester, 1 µg/ml pepstatin A, 10 µg/ml soybean trypsin inhibitor, 21 µg/ml phenylmethylsulfonyl fluoride, 21 µg/ml tosylphenylalanyl chloromethyl ketone (TPCK), and 21 µg/ml N-p-tosyl-L-lysine chloromethyl ketone (TLCK).

Preparation of Arf Proteins

Two preparations of native Arf were utilized for the assay of PLD activity. A cruder preparation of porcine brain Arf, which was obtained after resolution through anion exchange and gel filtration (9, 23) was utilized to survey columns for PLD activity. As described in detail in the accompanying paper(20) , this preparation contains other cytosolic activators of PLD. A highly enriched preparation of Arf, obtained after a fourth chromatographic step through Sephadex G-75 as described previously (9, 23) was utilized to stimulate PLD activity for analytical experiments. This preparation is at least 25% pure Arf, based on estimation by silver stain.

Recombinant Arf proteins were made in Escherichia coli as described previously(24) ; myristoylated proteins were made by co-expression with a yeast N-myristoyltransferase. The recombinant Arf fractions were then isolated by anion exchange on DEAE-Sepharose gel filtration with Ultrogel AcA44 and adsorption to hydroxylapatite (Bio-Rad) as described(9, 24) .

Preparation of Brain Membranes and Cytosol

Porcine brains were obtained from Pel Freez Biologicals (Rogers, AR). All steps were performed at 4 °C. Five brains (about 500 g) were thawed in 4 volumes (2000 ml) of Solution A (10 mM NaHepes, pH 7.5, 5 mM EDTA, 1 mM EGTA, 10% sucrose, and 0.25 mM phenylmethylsulfonyl fluoride). The tissue was minced and disrupted with a Polytron homogenizer for 20 s at a setting of 7. The homogenate was filtered through two layers of cheese cloth, and membranes were separated by centrifugation at 30,000 g for 60 min. Membranes were washed once by suspension in 10 mM NaHepes, pH 7.5, 2 mM EDTA, 10% sucrose, and phenylmethylsulfonyl fluoride/TPCK/TLCK and repeated centrifugation. Both membranes and cytosol were frozen in liquid nitrogen and stored at -80 °C.

Extraction and Chromatography of PLD Activity

PLD was prepared from brain membranes essentially as described previously(23) . Membranes (200 ml, 3 g of protein) from porcine brain were thawed in 200 ml of Solution B (20 mM NaHepes, pH 7.5, 1 mM EDTA, 1 mM dithiothreitol (DTT) and protease inhibitors). After the addition of 60 ml of 4 M NaCl and 25 ml of 20% (w/v) sodium cholate, the mixture was incubated for 1 h at 4 °C while stirring. Insoluble material was removed by centrifugation at 100,000 g for 90 min.

The supernatant was collected and concentrated by pressure filtration through Amicon PM30 filters to approximately 175 ml. This was applied to a 500-ml column of Sephadex G-50 and eluted with Solution C (20 mM NaHepes, pH 7.5, 1 mM EDTA, 1 mM DTT, 1% n-octyl--D-glucopyranoside, and protease inhibitors) containing 200 mM NaCl to lower the ionic strength and exchange detergent. Fractions containing protein were clarified by centrifugation at 100,000 g for 60 min. The supernatant (175 ml, 1750 mg of protein) was applied to a 125-ml column of SP-Sepharose. After washing with 50 ml of Solution C containing 200 mM NaCl, activity was eluted with a 300-ml linear gradient of 100-1000 mM NaCl in Solution C followed by a 150-ml wash of Solution C containing 2 M NaCl. Fractions of 6.8 ml were collected and analyzed for PLD activity in the presence of GTPS and crude Arf, which contains other cytosolic activators of PLD (see ``Preparation of Arf Proteins'' above and the accompanying paper(20) ).

Chromatography with Hydroxylapatite

The activity peaks from two SP-Sepharose columns were combined (150-200 ml) and loaded onto a 15-ml column of Macro-Prep ceramic hydroxylapatite (Bio-Rad), which had been equilibrated with Solution D (20 mM NaHepes, pH 7.5, 1 mM DTT, 1% n-octyl--D-glucopyranoside, and protease inhibitors). The column was eluted with a 60-ml linear gradient of 0-500 mM potassium phosphate, pH 7.5, in solution D followed by a 20-ml wash of Solution D containing 750 mM potassium phosphate, pH 7.5. Fractions of 2.2 ml were collected and assessed for PLD activity.

Anion-exchange Chromatography with Mono Q

The pool (40-45 ml) from the hydroxylapatite column was concentrated to 4-5 ml (with an Amicon PM30 filter), diluted 6-fold, and applied to a Mono Q HR 10/10 anion-exchange column (Pharmacia Biotech Inc.) that had been equilibrated with Solution E (20 mM Tris-Cl, pH 7.5, 1 mM EDTA, 1 mM DTT, 1% n-octyl--D-glucopyranoside, and protease inhibitors). The column was eluted at 0.5 ml/min with a 50-ml linear gradient of 200-800 mM NaCl in Solution E followed by a 10-ml gradient of 0.8-2.0 M NaCl in Solution E. The applied material was collected as fractions 1-4; eluate from the gradients was collected in fractions of 1.25 ml. The peaks of activity were stable at 0-4 °C for several days or were frozen and stored at -80 °C. This material was utilized to characterize several properties of the enzyme as more purified preparations were unstable.

Chromatography with Heparin-Sepharose

Fractions containing PLD activity (10-15 ml) from the Mono Q step were diluted 3-fold with Solution C and applied to a 10-ml column of heparin-Sepharose CL-6B (Pharmacia). Activity was eluted with a 50-ml linear gradient of 300-1500 mM NaCl in Solution C followed by a 10-ml gradient of 1.5-2.0 M NaCl in Solution C.

Chromatography with Phenyl-Superose

Fractions of activity from heparin-Sepharose were diluted with a 50% volume of 2 M ammonium sulfate (pH 7.5, final concentration 1 M) in Solution C and applied to a fast protein liquid chromatography phenyl-Superose HR 5/5 column, which had been equilibrated with the same solution. Protein was eluted in fractions of 0.5 ml with a 16-ml linear gradient of Solution C containing descending concentrations of ammonium sulfate (1000-0 mM) and ascending concentrations of isopropyl alcohol (0-10%); the flow rate was 0.25 ml/min.

Gel Filtration with AcA34

Fractions containing PLD activity were pooled (12 ml) and concentrated to 3 ml on an Amicon PM30 filter. The preparation was filtered through a 200-ml column of AcA34 Ultrogel (Sepracor) with Solution C containing 900 mM NaCl at a flow rate of 20 ml/h. Fractions of 2.6 ml were collected.

Chromatography with Hi-Trap Heparin-Sepharose

The peak of activity from gel filtration (12 ml) was diluted 3-fold with Solution C and concentrated to a final volume of 3 ml to reduce the concentration of NaCl to 300 mM. This was loaded onto a 5-ml column of Hi-Trap heparin-Sepharose and eluted with a 20-ml linear gradient of 300-1500 mM NaCl in Solution C followed by a 10-ml gradient of 1.5-2.0 M NaCl in Solution C. Fractions of 0.5 ml were collected at a flow rate of 0.5 ml/min.

General Assay for Phospholipase D Activity

PLD activity was measured essentially as described previously(9, 23) . Fractions containing PLD activity, activators (e.g. Arf) from cytosol, and any regulatory ligands (e.g. GTPS) were mixed on ice in 50 µl of Solution F (50 mM NaHepes, pH 7.5, 3 mM EGTA, 80 mM KCl, 1 mM DTT, 3.0 mM MgCl, and 2.0 mM CaCl). The free calcium in the final assay is estimated to be about 300 nM. Unless otherwise indicated, the final concentration of n-octyl--D-glucopyranoside contributed by the PLD preparation was 1.7 mM (0.05% (w/v)) in the assay. Substrate (10 µl) was added in the form of phospholipid vesicles composed of 600 µM PE, 30 µM PIP, 60 µM dipalmitoyl-PC, and L--[choline-methyl-H]dipalmitoyl-PC to give about 50,000 cpm per assay. Lipid vesicles were made as described previously (9) in solution F without divalent cations and diluted 6-fold into the assay. Reactions were initiated by the addition of substrate vesicles and incubated at 37 °C for the indicated times. Reactions were stopped by the addition of 200 µl of 10% trichloroacetic acid and 100 µl of 10 mg/ml bovine serum albumin. Precipitated lipids and protein were removed by centrifugation at 3000 g for 10 min at 4 °C. An aliquot of the supernatant (0.3 ml containing released [H]choline) was removed and analyzed by liquid scintillation spectroscopy. Reported activities have been corrected by subtraction of a blank (1-2% of the total counts), which is obtained by incubation of vesicles in the absence of fractions containing PLD activity, but with 20 µg of fatty acid-free bovine serum albumin. All assays were conducted in duplicate (except for profiles of column fractions); the variance between samples was less than 5%.

Production of PA or Phosphatidylethanol

For analysis of the lipid products of PLD activity, assays were conducted with phosphatidylcholine, L--1-palmitoyl-2-arachidonyl-[arachidonyl-1-C], essentially as described previously(9) . Reactions were halted by the addition of 0.3 ml of cold chloroform/methanol/water (2:1:0.8), mixing, and centrifugation(25) . The aqueous phase was removed, and the organic phase was dried under a stream of nitrogen. Lipids were resuspended in chloroform, applied to the thin-layer chromatography plates (Whatman PE Sil G thin-layer chromatography plates, 20 20 cm), and developed with CHCl/MeOH/NHOH/HO (65:25:2:2). The migration of phospholipids was visualized by staining with iodine, and C-labeled lipids were quantitated with an Ambis 4000 radioanalytic imaging detector (San Diego, CA).

Measurement of GTPS Binding to Arf and Related Proteins

The functional activation of native Arf and recombinant monomeric G proteins was assessed by measuring their extent of association with GTPS under conditions similar to those used for the assay of PLD. Samples were combined with PE/PIP/PC lipid vesicles (as described above) and solutions to give final concentrations of 25 mM NaHepes, pH 7.5, 3 mM EGTA, 1 mM EDTA, 1 mM DTT, 0.5 mM MgCl, 2 mM CaCl, 50 mM NaCl, 0.05% n-octyl--D-glucopyranoside, 5 µM GTPS, and [S]GTPS (700 cpm/pmol) in a 60-µl assay. The binding reaction was incubated for 60 min at 30 °C and terminated by dilution and filtration through BA85 nitrocellulose filters (Scheicher & Schuell) with 6 2-ml washes of ice-cold 20 mM Tris-Cl, pH 8.0, 100 mM NaCl, and 40 mM MgCl as described previously(26) . The amount of GTPS bound was quantitated by liquid scintillation counting.

Hydrodynamic Analysis

Samples of PLD were mixed with calibrating proteins and layered on a 4-ml linear gradient of sucrose (5-20%) in Solution G (20 mM Tris-Cl, pH 7.5, 1 mM EDTA, 5 mM DTT, 1 mM MgCl, 500 mM NaCl, and protease inhibitors) with or without 1% n-octyl--D-glucopyranoside, as indicated. Samples were sedimented at 4 °C for 14 h at 170,000 g in a SW60 rotor (Beckman). The Stokes radius of porcine PLD was measured by filtration through AcA34 Ultrogel (Sepracor) in Solution G containing 1 M NaCl and 1% n-octyl--D-glucopyranoside. Sedimentation coefficients and Stokes radii were determined by comparison with calibrating proteins included in the analysis. The calibrating enzymes (, s) used were -galactosidase (6.9 nm), catalase (5.2 nm, 11.3 S), lactate dehydrogenase (4.75 nm, 7.3 S), malate dehydrogenase (3.7 nm, 4.3 S), horseradish peroxidase (3.5 S), myoglobin (1.9 nm, 2.0 S); these were assayed as described previously(27, 28, 29) .


RESULTS

Resolution of Arf-sensitive PLD in Mammalian Tissues and Initial Steps of Chromatography

Previous studies have demonstrated that exogenous lipid could be utilized to measure a PLD activity from membranes of HL60 cells that was stimulated by a cytosolic protein identified as Arf(9) . Membranes isolated from several cultured cell lines show a similar response to added Arf, although the PLD activities observed varied widely and were not readily observed in membranes from all cell lines examined (data not shown). The Arf utilized in these experiments was prepared from porcine brain cytosol. Curiously, membranes from mammalian brain and several other tissues (e.g. rat lung or bovine kidney) show very little activity either in the presence or absence of Arf and GTPS. The low activities observed in tissues and some cell lines are probably due to factors that interfere with the assay of PLD activity. This was suggested in part by the observation that the Arf in cytosol from brain (as opposed to cytosol from HL60 cells) was not effective in stimulating PLD activity in HL60 membranes until it was partially resolved by a step of chromatography(9) .

Fig. 1demonstrates that PLD activity from porcine brain membranes can be readily observed if it is extracted and resolved by chromatography. In this case, the activity was extracted with a mixture of 25 mM sodium cholate and 0.4 M NaCl. Like the membranes, the extract had little or no activity (not shown). After gel filtration (to reduce salt and switch the detergent into n-octyl--D-glucopyranoside (see ``Experimental Procedures'')) and chromatography through SP-Sepharose, Arf-dependent PLD activity was easily detected. This activity was most apparent when assayed with the addition of preparations of Arf and GTPS (about 10-25 times the activity observed in their absence). The activity resolved by SP-Sepharose is fairly stable and can be maintained for several weeks on ice or frozen in liquid nitrogen and stored at -80 °C for months. Less than 5% of the starting protein was recovered in the pool of activity (Fig. 1). If 50% of the original activity in membranes is recovered by this procedure, it then represents a 10-fold or greater enrichment of the PLD. PLD activity, which had been extracted from HL60 membranes with salt and enriched by two steps of chromatography (9) had a specific activity of 1.2 nmol/min/mg. This is comparable with the specific activity of the brain PLD (about 450 pmol/min/mg) after SP-Sepharose.


Figure 1: Chromatography of PLD activity through SP-Sepharose. Membranes from porcine brains were prepared, extracted, and processed as described under ``Experimental Procedures.'' Eluted fractions (6.8 ml) were assayed for protein () and PLD activity () in the presence of 10 µM GTPS and a cruder preparation of Arf that contained other cytosolic activators (see ``Experimental Procedures''). Fractions 24-42 were pooled for subsequent chromatography.



The pool of PLD activity from SP-Sepharose was further resolved by chromatography on hydroxylapatite and Mono Q anion-exchange resin (Fig. 2). These two steps resulted in a further enrichment of PLD activity of 6-10-fold ( Fig. 2and see ); the pool of activity from the last step could be stored on ice for several days and utilized for subsequent characterization or could be frozen at -80 °C for several months. The three steps gave an overall enrichment of about 100-fold if one assumes a 50% recovery on the first step. It should be noted that the apparent behavior of PLD activity on different resins varied between preparations. Thus, a single peak of activity, rather than two peaks (Fig. 1), was often observed with SP-Sepharose, and a sharp peak of activity, which was resolved from the bulk of protein (see ) could be obtained with hydroxylapatite rather than the broad peak observed in Fig. 2. The reasons for this variability are not clear.


Figure 2: Chromatography of PLD with hydroxylapatite and Mono Q anion-exchange resins. Top, the fractions containing PLD activity from two SP-Sepharose columns were combined and loaded onto a 15-ml Macro-Prep ceramic hydroxylapatite column and chromatographed as described under ``Experimental Procedures.'' Fractions (2.2 ml) were assayed for protein () and PLD activity (): fractions 11-24 were pooled, diluted, and concentrated as described under ``Experimental Procedures.'' Bottom, the pool from hydroxylapatite was loaded onto a Mono Q HR 10/10 anion-exchange column and chromatographed as described under ``Experimental Procedures.'' Fractions were assayed for protein () and PLD activity (). Typically two peaks of PLD activity were observed. In this example, Peak I (fractions 5-10) was very small, and peak II (fractions 20-25) was dominant. Typically, the peaks were combined into a single pool for storage or further chromatography.



Characterization of the Partially Purified PLD

The hydrodynamic parameters of the PLD obtained from Mono Q chromatography were determined in the presence of calibrating enzymes (). The activity sedimented with an s of 5.1 Svedberg units and had a Stokes radius of 4.3 nm. If a partial specific volume of 0.74 cm/g is assumed, these parameters indicate that Arf-dependent PLD has a molecular mass of about 95,000 Da. It is important to note that the same values were obtained when the PLD was sedimented through sucrose gradients either in the presence or absence of detergent. This indicates that bound detergent probably does not contribute significantly to the mass of the protein. In spite of this, detergent was required for effective purification; it is clearly important for controlling the behavior of other membrane proteins, and a small amount of detergent may be critically important for uniform behavior of PLD itself.

Most PLD enzymes can catalyze a transphosphatidylation reaction (for review, see Ref. 30). In this reaction, a primary alcohol substitutes for water in the hydrolytic step to yield a phosphatidyl alcohol. This is thought to be a characteristic reaction of PLD enzymes and was used to detect stimulation of this activity by Arf in permeabilized HL60 cells(10) . In the presence of increasing concentrations of ethanol, production of PA by the partially purified enzyme was progressively reduced in favor of the production of phosphatidylethanol. By 500 mM ethanol, this shift in product formation was greater than 80% (). The shift in product formation had little effect on the overall rate of reaction of the enzyme.

Three phospholipids were tested as substrates for the enzyme. While no hydrolysis of phosphatidylinositol was observed, phosphatidylethanolamine was hydrolyzed, albeit to a much lower extent than phosphatidylcholine (data not shown). This coincides with the substrate selectivity indicated for other PLDs (30) and explains the viability of the current assay that uses PC in a background of PE and PIP.

Both Ca and Mg enhanced the activity of Arf-dependent PLD, although about 40-50% of maximal activity is observed in the absence of either metal ion. Half-maximal stimulation by Mg is obtained by 1 mM, while calcium is effective in the submicromolar range but inhibitory at higher concentrations. The stimulatory effect of Ca is negated by the presence of mM Mg. The apparent lack of dependence on Ca clearly contrasts with the apparent large effect of Ca on the activity of Arf-dependent PLD measured in permeabilized HL60 cells (10) or the requirements observed for the plant enzymes(30, 31) . Thus, maximal activities for PLD from a variety of plant species have been obtained with 10-100 mM Ca; in contrast, PLD obtained from red algae had no absolute cation requirements, but its catalytic rate could be stimulated by Ca(32) .

Porcine PLD activity shows a narrow dependence on pH. Maximal activity was obtained at pH 7.5; this declined by about 50% at pH 7 or 8 and was reduced to less than 20% at pHs 6.5 and 8.5. This contrasted markedly with a PLD activity derived from peanut, which showed high activity at relatively low pH and a marked decline between pH 6.5 and 8 to almost nothing (data not shown). The narrow pH profile of the brain enzyme could be due to intrinsic properties of the catalyst itself or to a combination of pH requirements for activation of Arf proteins, interaction of the regulatory proteins, or association with PIP.

Partially purified PLD retained its requirement for PIP as a component of vesicles containing substrate. Substitution with other acidic phospholipids such as phosphatidylserine, phosphatidylinositol and phosphatidylinositol 4-phosphate were largely ineffective (0, 0, and 13% of activity obtained with PIP, respectively). It was of some interest to test for the efficacy of phosphatidylinositol 3,4,5-trisphosphate (PIP). Lipid vesicles made with either 15 µM PIP or dioctyl-PIP had equivalent activities when incubated with PLD and Arf. Thus dioctyl-PIP does not appear to be a more effective cofactor or regulator of this enzyme.

Nucleotide Dependence of Arf-dependent PLD

The original observations demonstrating stimulation of PLD activity by Arf used GTPS as the activating agent(9) . In crude preparations of PLD, such as extracts of HL60 membranes, GTP was less effective (data not shown). As the preparations of PLD have been purified, GTP has become a more effective stimulator of this complex (Fig. 3). The nonhydrolyzable analog, Gpp(NH)p was less potent than GTPS but had a similar efficacy. GTP was an effective stimulator but of lower potency and efficacy than either of the nonhydrolyzable analogs. In contrast, both GDP and GDPS were ineffective at stimulating the reaction (data not shown); either nucleotide could inhibit the action of the nucleotide triphosphates at relatively high concentrations (>1000-fold above that for the stimulating nucleotide). This relatively low potency of GDPS and GDP to inhibit GTPS activation contrasts with more potent blocks of activation observed with other effectors.


Figure 3: Dependence of Arf-stimulated PLD activity on nucleotide concentration. Assays of PLD activity were conducted at 37 °C for 30 min with 2 µg of Mono Q PLD and 1.2 µM native Arf from a Sephadex G-75 column as described under ``Experimental Procedures.'' Nucleotides were included in the assays as indicated.



Adenine nucleotides could also stimulate PLD under these conditions, although with much lower potency than guanine nucleotides. Upon further analysis, at least part of this effect could be attributed to the action of contaminating nucleoside-diphosphate kinase activity.

Specificity of PLD for Arf Proteins

Several isoforms of Arf have been described in mammals(13) . The actions of three recombinant Arf proteins on porcine PLD activity are compared with native Arf in Fig. 4(upperpanel). All three recombinant Arf proteins stimulate PLD activity albeit with apparent differences in potency. Two reasons for this apparent difference could be differential activation by guanine nucleotides and potential different extents of N-myristoylation. The importance of the latter modification was noted previously (9) and is repeated here in the form of the low potency of nonmyristoylated hArf1 (Fig. 4). Potential differential activation was tested directly by measurement of binding of GTPS to added Arf under conditions similar to those used for the assay of PLD activity. If activity is then expressed as a function of the activated GTPS-bound Arf, the result obtained is shown in the lowerpanel of Fig. 4. Thus, when the Arfs are compared by their specific activities, the recombinant and native proteins act with similar potencies. The apparent higher potency of Arf5 is due to a much higher efficiency of guanine nucleotide exchange. The extent of myristoylation of the recombinant Arf proteins has not been assessed for these specific preparations. Analysis of the extent of myristoylation in other preparations of recombinant proteins has indicated similar extents of modification (estimated at 10-20%) among the expressed human Arf proteins.()A remaining distinction is the apparent lower efficacy of Arf5 relative to Arf1 and Arf3. Such a variance in extent is not explained by a differential extent of GTPS activation or myristoylation.


Figure 4: Comparison of the ability of native Arf and various recombinant Arf subtypes to stimulate porcine PLD activity. Assays containing Mono Q PLD activity (1 µg) and the indicated amounts of various Arf proteins as described were incubated at 37 °C for 30 min as described under ``Experimental Procedures.'' Binding of GTPS to the Arf proteins was measured in separate assays which used identical conditions. The top panel compares activation of PLD by a variety of Arf subtypes as a function of the concentration of Arf protein. The lower panel illustrates PLD activity obtained as a function of activated Arf (as assessed by the extent of GTPS binding to Arf under the same conditions).



Two other human recombinant Arf proteins (myristoylated and nonmyristoylated Arf6 and a nonmyristoylated Arf4) were examined for interaction with earlier preparations of PLD activity derived from HL60 cells (data not shown). These were not characterized for extent of binding or by rigorous titration. The pattern of results was similar to that with rArf1(9) ; myristoylated Arf6 was effective in the 100 nM range (2.4-4-fold stimulations at 170 and 830 nM) while the nonmyristoylated Arf6 was ineffective below 2 µM but yielded modest stimulation of about 60-80% at 4.5 and 9 µM. The nonmyristoylated Arf4 was only tested at submicromolar concentrations where it was essentially without effect (a maximal stimulation of 33% at 700 nM Arf).

Results with a selection of other recombinant members of the Arf family are shown in I. In this analysis, activity was assessed with Arf both in the presence and absence of another cytosolic activity (called CFII) that can enhance PLD activity along with Arf (see accompanying article, Ref. 20). Inclusion of the other cytosolic factors provides a more sensitive assay to detect Arf proteins with low potency. Three observations are worthy of note. First, nonmyristoylated Arf proteins can stimulate PLD activity at high concentrations. By virtue of this activity, the nonmyristoylated Arf1 appears to be significantly more potent than nonmyristoylated Arf5. This may reflect the lower efficacy of Arf5 observed with the myristoylated proteins (Fig. 4). At concentrations examined so far, nonmyristoylated Arf did not compete effectively with the action of the myristoylated forms (data not shown). A simple interpretation of this observation is that the lowered potency of the nonmyristoylated proteins is due to either a lower affinity for the PLD enzyme or reduced association of nonmyristoylated Arf with the substrate vesicles.

A second observation of interest is the ability of two recombinant yeast Arf proteins to stimulate mammalian PLD activity, although at significantly lower potency (I). The significantly lower activity of the nonmyristoylated yeast Arf1 supports the importance previously imparted to this covalent modification.

Finally, the Arl proteins are a recently discovered subgroup of Arf. Two members of this family did not stimulate PLD activity when assayed at the same concentrations where the nonmyristoylated Arfs were active; this is especially emphasized by a lack of synergism with the CFII factors (I). It is notable that the recombinant Arl proteins are not myristoylated when coexpressed in bacteria with N-myristoyltransferase.

Further Resolution of PLD Activity

PLD activity from porcine brain has been subjected to further purification. Four steps of a preparation subsequent to the Mono Q resin are shown in Fig. 5, and the purification table for an entire preparation is given in . Activities determined under two conditions are presented. The first of these is stimulation by Arf and GTPS; the second condition is stimulation in the presence of Arf, GTPS, and CFII, the additional cytosolic factor indicated previously. The overall purification determined by either assay was approximately 5,000-10,000-fold with a yield in activity of about 2%. This assumes a 50% yield and 10-fold purification in the first step. These procedures have been performed several times (n = 7) with similar results. The yields and specific activities achieved have been variable; this is due largely to increasing instability of the highly enriched protein; the half-life at this stage is 1-2 days while stored on ice. This material retains a requirement for PIP as a component of the substrate vesicles used for expression of activity.


Figure 5: Further chromatographic resolution of PLD. PLD activity enriched through the step of Mono Q anion exchange was subjected to further purification as described under ``Experimental Procedures.'' Fractions from each column were assayed for PLD activity in the presence of 10 µM GTPS and a crude AcA44 fraction of Arf containing other cytosolic activators. Pools used for each subsequent step of chromatography were as follows: fractions 28-38 from heparin-Sepharose, fractions 6-20 from phenyl-Superose, fractions 32-36 from AcA34. The resulting enrichments from these steps are further delineated in Table IV.



The highly enriched PLD has been examined for its response to both purified Arf and the CFII fraction (Fig. 6). Either preparation alone stimulates the PLD, but both together provide a synergistic activation. The time course of activity is fairly linear up to the better part of an hour. A short lag (1-3 min) is sometimes observed before full activity is attained. The dependence of the activity on added PLD (Fig. 6) is clearly nonlinear and may be more revealing. At low concentrations of the preparation, little activity is observed, while at higher concentrations, activity is repressed. One possibility for the lack of activity at low concentrations is that a critical concentration of the PLD may be required for expression of activity. The drop in specific activity at high concentrations of the proteins may be due, in part, to faster utilization of the substrate. However, other assays indicate that depletion of substrate does not appear to be a major factor until greater than 200 pmol of the PC is hydrolyzed.


Figure 6: Activation of enriched PLD by Arf and CFII. PLD from the Hi-Trap heparin-Sepharose (Fig. 5) was measured for activity in the absence or presence of Arf (2 µg of a Sephadex G-75 pool) and/or CFII (4 µg of an AcA44 fraction, see accompanying paper (20)) as indicated. Top panel, a fixed amount of PLD (130 ng, fraction 31) was incubated in the presence of 10 µM GTPS at 37 °C for the indicated times. Bottom panel, the indicated amounts of PLD from Hi-Trap heparin-Sepharose fraction 29 were incubated at 37 °C for 30 min in the presence of 10 µM GTPS and 20 µg of fatty-acid free bovine serum albumin. The amount of detergent in the assay (usually added as part of the PLD preparation) was kept constant at 1.7 mMn-octyl--D-glucopyranoside. The results are the mean values of assays conducted in duplicate.




DISCUSSION

This report delineates progress in the investigation of a mammalian phospholipase D, which is stimulated by the monomeric Arf proteins. This PLD has been extensively purified and characterized at various stages of the preparation. During the enrichment, it became apparent that cytosolic factors (referred to as CFII) other than Arf could regulate this activity. The resolution of these components and initial characterization of them is presented in the accompanying paper (20). The PLD activity stimulated by these various components co-elutes during a variety of chromatographic procedures, which suggests that all of the factors are acting on the same enzyme. This is further supported in that the extent of purification achieved is similar when the activity is assessed with either Arf or a combination of Arf and CFII ().

The original detection of Arf-stimulated PLD activity, assayed with exogenous substrate, required PIP as part of the substrate vesicles(9) . As hydrolysis of the molecule was not required, the PIP apparently acts as a cofactor of the reaction rather than in its well described role as a precursor for second messengers. This stimulatory action of PIP was recently confirmed in an undefined preparation of PLD from brain, along with evidence that PIP could also fulfill this function at similar concentrations(33) . The requirement for PIP (or PIP) by the highly enriched preparations of Arf-dependent PLD reported here suggests that association of PIP may be integral to full functional expression of activity by this enzyme. One potential mechanism involved may be indicated by the stimulation of guanine nucleotide exchange on Arf by PIP(34) .

A major hurdle to proceeding with purification of Arf-sensitive PLD was an inability to detect substantial activity in membranes derived from tissues. This included rat brain where PLD activity has been reported using a different assay dependent on exogenous substrate(35, 36) . PLD activity, measured by this procedure, either in crude membranes and extracts or after resolution on the SP-Sepharose, was not stimulated by Arf (data not shown). This has also been reported recently by others (37). The impediment to measurement of Arf-dependent activity in brain and other tissues was surmounted by extraction of the membranes and resolution of the PLD activity from inhibitory constraints (Ref. 23 and herein). Similar procedures were used to measure Arf-dependent PLD activity from rat brain in a report that appeared during the preparation of this manuscript(37) . Since the detection of PLD activity requires PIP as a component of the substrate vesicles, one potential reason for the masking of activity in tissues could be interference by various PIP binding proteins. Alternatively, the lack of activity in crude preparations could signify the presence of more specific inhibitory proteins.

So far, Arf-dependent PLD activity from tissues has only been efficiently extracted and chromatographed from particulate fractions with detergent. However, modest activities have been detected in cytosol and salt extractions of membranes that have undergone similar chromatographic resolution (data not shown). This contrasts with membranes from HL60 cells where approximately half of the measurable activity in membranes could be extracted with salt(9) . The latter observations and the similar sedimentation of the enriched brain PLD through sucrose gradients either in the absence or presence of detergent () suggests that the enzyme is not an intrinsic membrane protein. Rather, it is likely to be a peripheral protein that is associated with more hydrophobic moieties in the particulate fraction. If this is the case, and the enzyme is activated by cytoplasmic factors (such as Arf), the hydrolytic action of the enzyme should occur exclusively on the cytoplasmic leaflet of membrane bilayers.

The potency of Arf on the porcine brain PLD appears significantly less (about 10-fold) than that previously reported for PLD activity from HL60 cells(9) . The reason for this is not clear. It is possible that these are different isozymes of PLD with intrinsic differences in Arf sensitivity. Alternatively, the preparations of PLD may consist of several components that differ between the two sources. For example, the enriched preparation of salt extracted PLD from HL60 cells may contain a component that facilitates activation by Arf, and this component is lacking in the detergent-extracted enzyme from brain. Evidence for multiple factors, including the monomeric G proteins, RhoA and Cdc42, that interact with PLD and affect stimulation by Arf is presented in the accompanying paper(20) . The stimulatory effects of RhoA have also been implicated by others(38, 39) . Further suggestive evidence that expression of PLD activity requires interaction of multiple components comes from the dependence of PLD activity on protein concentration (Fig. 6). The loss of apparent specific activity of the highly enriched enzyme at low concentrations of protein could be explained by a dissociation of interacting subunits. The existence of PLD as a complex of interacting subunits would resemble the structural and functional characteristics of the neutrophil NADPH oxidase system, which is stimulated by Rac, another monomeric G protein (for review, see Ref. 40).

The chromatographic behavior of PLD sometimes suggested the existence of more than one form of the enzyme. This could indicate that more than one Arf-dependent PLD is present in mammalian tissues. Alternatively, the variable behavior of PLD activity during several chromatographic procedures may offer support for the existence of this enzyme as a multimeric complex. Thus, association of a catalytic unit with different proteins that may assemble into a functional complex could account for elution of the activity as multiple peaks or broad profiles from these resins. The broad pattern of elution could also be due to nonspecific aggregation of the enzyme with other proteins.

Hydrodynamic measurements indicate that the size of the Arf-dependent PLD is about 95,000 Da. This contrasts with a PLD activity that does not require Arf and has been reported to consist of a single polypeptide of 190,000 Da when purified from porcine lung (19). It may be of interest that a PLD from castor bean (41) has recently been cloned, sequenced, and predicted to have a molecular mass of 92 kDa. Is this coincidence or will the Arf-stimulated mammalian PLD also consist of a single polypeptide in this range? So far, a polypeptide of this size has not been observed in the most enriched preparations of the enzyme (about 10,000-fold, , data not shown); it is equally likely that the enzyme is an oligomeric protein composed of smaller polypeptides. Separation of such subunits during purification could help explain some of the low yields and instability of the enzyme, as well as the loss of enzymatic activity at low concentrations of protein.

Recent observations with two heterotrimeric G protein systems, G-phospholipase C (42) and G-phosphodiesterase(43, 44) , indicate that the effector proteins for the pathway can act as GTPase-activating proteins. Likewise, GTPase-activating proteins have been described for several of the monomeric G proteins, although the relationship of these proteins to effectors remains unclear. The effective stimulation of PLD by Arf in the presence of GTP suggests that the lipase does not act as an efficient GTPase activator on Arf or other potential G proteins in this system. This may indicate that specific GTPase-activating proteins will play a prominent role in reducing the activity of PLD.

All of the mammalian Arf proteins tested have been effective in stimulating enriched mammalian PLD, either of porcine or human (from HL60 cells) origin. This agrees with the recent observations of Massenberg et.al.(37) who tested Arfs -1, -5, and -6 on a cruder preparation of activity. Limited attempts to quantitate the relative potency of the different Arf proteins in this report (e.g. measurement of actual extent of activation of each protein) indicate no obvious differences. This suggests that PLD is a potential mechanism for explaining actions of each of the isoforms of Arf. It is apparently not the mechanism of action of the Arl proteins. This coincides with the inability of these proteins to either stimulate ADP-ribosylation of G by cholera toxin or complement Arf deletions in yeast(13, 14, 17) ; this further reinforces the functional distinction between these Arf-like proteins and the classical Arf proteins.

The ability of the recombinant yeast Arf proteins to stimulate mammalian PLD indicates that this is a well conserved function in cells and predicts that this regulatory pathway will be fundamental in cellular regulation. It will be of interest to see whether this pathway is fundamental to regulated events such as hormone stimulation of PA production or to the function of constitutive processes such as protein traffic. Evidence for the latter comes from a demonstration that PLD activity is abundant in Golgi-enriched membranes from a variety of cell lines and that this activity was stimulated by added Arf and GTPS. In addition, this stimulation could be inhibited by brefeldin A, a drug that blocks transport through Golgi, and basal PLD activity is highly elevated in Golgi membranes from PtK1 cells, a cell line that is naturally resistant to brefeldin A(45) .

Finally, the stimulation of PLD activity by multiple cytoplasmic factors indicates that the regulation of this enzyme is complex. The accompanying report (20) examines in more detail the influence of other cytoplasmic factors that can act on PLD and facilitate the action of Arf on this enzyme.

  
Table: 0p4in Frictional ratios were calculated according to the following formula.

On-line formulae not verified for accuracy

This value does not consider solvation of the PLD. If solvation were 20%, the value of f/f would decrease to 1.3.(119)

  
Table: Catalysis of exchange reactions by Arf-sensitive PLD

Production of PA or phosphatidylethanol was conducted as described under ``Experimental Procedures.'' Assays were incubated at 37 °C for 30 min in the presence of 10 µM GTPS. The results are the mean values of assays conducted in duplicate and analyzed on separate PE Sil G thin-layer chromatography plates.


  
Table: Action of various Arf proteins on porcine PLD activity

Assays that contained Mono Q PLD (1 µg), 10 µM GTPS, and the indicated amounts of Arf proteins and CFII were conducted at 37 °C for 30 min as described under ``Experimental Procedures.'' The results are the mean values of assays conducted in duplicate. The following abbreviations are used: nArf is native porcine Arf (Sephadex G-75 pool); H indicates a recombinant, human protein; Y indicates a recombinant, yeast protein; and m indicates that the protein was co-expressed with N-myristoyltransferase as described under ``Experimental Procedures.''


  
Table: Purification of PLD activity from porcine brain

Pools from fractionation steps were assayed as indicated in the presence of Arf (1 µM final concentration from a Sephadex G-75 pool) and CFII (2 µg from an AcA44 pool) as described under ``Experimental Procedures.'' Assays were conducted at 37 °C for 30 min in the presence of 10 µM GTPS.



FOOTNOTES

*
This work was supported in part by National Institutes of Health Research Grant GM31954 (to P. C. S.) and by National Research Fellowship Award GM15817 (to H. A. B.). Support was also obtained from the Robert A. Welch Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed.

The abbreviations used are: PIP, phosphatidylinositol 4,5-bisphosphate; PLD, phospholipase D; G protein, guanine nucleotide regulatory protein; Arf, ADP-ribosylation factor; PA, phosphatidic acid; PE, phosphatidylethanolamine; PC, phosphatidylcholine; TPCK, tosylphenylalanyl chloromethyl ketone; TLCK, N-p-tosyl-L-lysine chloromethyl ketone; DTT, dithiothreitol; GTPS, guanosine 5`-O-(3-thiotriphosphate); GDPS, guanyl-5`-yl thiophosphate; Gpp(NH)p, guanyl-5`-yl imidodiphosphate; PIP, phosphatidylinositol 3,4,5-trisphosphate; CFII, cytoplasmic factor II, cytosolic activators of phospholipase D other than Arf.

R. A. Kahn, unpublished results.


ACKNOWLEDGEMENTS

We thank J. R. Falck for supplying dioctyl-PIP and Kim Edwards for administrative support.


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