From the
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
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.
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
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
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) .
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
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) .
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-
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-
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
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
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.
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
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
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
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
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 GTP
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.
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)
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 GTP
Assays that contained Mono Q PLD (1 µg), 10 µM GTP
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 GTP
We thank J. R. Falck for supplying dioctyl-PIP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
),
(
)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) .
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) .
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).
-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.
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.
-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 GTP
S
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/NH
OH/H
O (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 GTP
The functional activation of native Arf and recombinant
monomeric G proteins was assessed by measuring their extent of
association with GTPS Binding to Arf and Related
Proteins
S 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 GTP
S, and [
S]GTP
S (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 GTP
S 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) .
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) .
-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 GTP
S (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 GTP
S 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.
.
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) .
.
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 GTP
S 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 GDP
S 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 GDP
S and
GDP to inhibit GTP
S 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 GTP
S-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 GTP
S 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 GTP
S 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).
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, GTP
S, 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 GTP
S 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.
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) .
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.
-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.
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.
S. 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) .
Table: 0p4in
Frictional ratios were calculated according
to the following formula.
Table: Catalysis of
exchange reactions by Arf-sensitive PLD
S. 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
S, 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
S.
,
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; GTP
S, guanosine
5`-O-(3-thiotriphosphate); GDP
S, 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.
and Kim Edwards for administrative support.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.