(Received for publication, May 11, 1995; and in revised form, July 31, 1995)
From the
Anandamide (arachidonylethanolamide) is known as an endogenous
agonist for cannabinoid receptors. An amidohydrolase, which hydrolyzed
anandamide, was solubilized from the microsomal fraction of porcine
brain with 1% Triton X-100. The enzyme was partially purified by
Phenyl-5PW hydrophobic chromatography to a specific activity of
approximately 0.37 µmol/min/mg of protein at 37 °C. As assayed
with C-labeled substrates, the apparent K
value for anandamide was 60
µM, and anandamide was more active than ethanolamides of
linoleic, oleic, and palmitic acids. Ceramidase and protease activities
were not detected in our enzyme preparation. The purified enzyme also
synthesized anandamide from free arachidonic acid in the presence of a
high concentration of ethanolamine with a specific activity of about
0.16 µmol/min/mg of protein at 37 °C. On the basis of
cochromatographies, pH dependence, heat inactivation, and effects of
inhibitors such as arachidonyl trifluoromethyl ketone, p-chloromercuribenzoic acid, diisopropyl fluorophosphate, and
phenylmethylsulfonyl fluoride, it was suggested that the anandamide
amidohydrolase and synthase activities were attributable to a single
enzyme protein.
An endogenous agonist for cannabinoid receptor was isolated from porcine brain, and this compound referred to as anandamide was identified to be arachidonylethanolamide(1) . It inhibited the specific binding of radiolabeled ligands to cannabinoid receptors, reduced cAMP production, and caused the inhibition of N-type calcium currents and calcium channel antagonist binding(1, 2, 3, 4, 5) . Anandamide also inhibited electrically evoked contraction of vas deferens isolated from mice (1) and mimicked in vivo effects of cannabinoids such as antinociception, hypothermia, hypoactivity, and catalepsy in mice (6, 7, 8) .
It was shown that anandamide
was rapidly degraded by an amidase (amidohydrolase) activity which was
found in the membrane fraction of cultured neuroblastoma and glioma
cells and homogenates of rat tissues (9) . In fact, the
addition of PMSF, ()a serine protease inhibitor(9) ,
increased an apparent affinity of anandamide for cannabinoid receptors,
probably due to protecting the compound from
hydrolysis(10, 11) . Recently, some properties of the
enzyme were reported with a microsomal preparation of rat
brain(12) . On the other hand, the synthesis of anandamide from
free arachidonic acid and ethanolamine was shown with rat(9) ,
bovine(13) , and rabbit (14) brain and was reported to
be independent of ATP and coenzyme A (14) . However, the
enzyme(s) hydrolyzing and synthesizing anandamide has not yet been
purified and well characterized, and it is still unknown whether the
two enzyme activities are attributed to a single enzyme protein or two
enzymes.
DEAE-ion exchange column chromatography was
performed as follows. The partially purified enzyme was concentrated 7
times by ultrafiltration using an Amicon XM-50 membrane. During this
procedure, the concentration of ammonium sulfate was reduced to about
20 mM. This material (1.3 mg of protein in 5 ml) was loaded
onto a Tosoh DEAE-5PW column (7.5 mm inside diameter 7.5 cm)
which had been equilibrated with 20 mM citrate-sodium
phosphate buffer (pH 6.0) containing 0.05% Triton X-100 (solution B).
The flow rate was 1.0 ml/min during the entire procedure. After loading
the sample, the column was washed with 15 ml of solution B, and
adsorbed proteins were eluted in 2.5-ml fractions with a 50-ml linear
gradient of NaCl (0-1 M) and then with 20 ml of solution
B containing 1 M NaCl.
A homogenate of porcine brain was subjected to differential
centrifugation. Each fraction was allowed to react with
[1-C]anandamide, and free
[
C]arachidonic acid as a hydrolytic product was
separated from the remaining [
C]anandamide by
TLC. Approximately 50%, 35%, 13%, and 7% of the total activity of the
whole homogenate were recovered in the 2,000
g pellet,
20,000
g pellet, 105,000
g pellet
(microsomal fraction), and 105,000
g supernatant
(cytosol), respectively. The specific enzyme activities in these
fractions were 5.4, 5.5, 7.5, and 1.7 nmol/min/mg of protein. The yield
of the activity in the 105,000
g pellet was low, but
its specific enzyme activity was the highest. Therefore, the pellet
referred to as the microsomal fraction was chosen as a starting
material for purification of the anandamide amidohydrolase.
The
microsomal fraction was treated with 1% Triton X-100. As shown in Fig. 1, the solubilized protein hydrolyzed
[C]anandamide to free
[
C]arachidonic acid (lane 1). The same
preparation also synthesized [
C]anandamide from
free [
C]arachidonic acid in the presence of a
high concentration of ethanolamine (lane 5), but not in its
absence (lane 6). For identification of the product as
anandamide, it was purified by TLC and reverse-phase high performance
liquid chromatography with a solvent mixture of methanol/water/acetic
acid (85:15:0.01) and analyzed by gas chromatography/mass spectrometry.
Significant ion peaks were observed at m/z 329 (M - 18,
dehydration of the parent ion), 315, 301, 287, 273, 259, 245, 232, 218,
204, 192, 178, 164, 139, 125, 124, 98 (product of a
-cleavage
fragment), and 85 (base peak, McLafferty rearrangement ion).
Essentially the same spectra were obtained with synthetic anandamide.
Either the amidohydrolase or synthase activity was not observed with
heat-treated solubilized protein (lanes 2 and 7 of Fig. 1).
Figure 1:
Anandamide amidohydrolase and synthase
activities as examined by TLC. The solubilized protein (31 µg of
protein, lanes 1, 5, and 6), the
heat-treated solubilized protein (31 µg of protein, lanes 2 and 7), the partially purified enzyme (0.94 µg of
protein, lanes 3 and 8), or the protein-free buffer (lanes 4 and 9) was incubated with
[1-C]anandamide (lanes 1-4) or
with [1-
C]arachidonic acid in the presence (lanes 5 and 7-9) or absence (lane 6)
of ethanolamine under the standard conditions. The enzyme was denatured
at 90 °C for 10 min before the enzyme assay. AA,
arachidonic acid; AE, anandamide.
For enzyme purification, the solubilized protein was directly loaded on a Tosoh Phenyl-5PW column. As shown in Fig. 2, when the proteins were separated by decreasing the concentration of ammonium sulfate, two peaks with the amidohydrolase activity were observed. The amidohydrolase in the peak 2 fractions was purified about 22-fold in a yield of 31% and showed an average specific enzyme activity of 368 (240-540) nmol/min/mg of protein at 37 °C. When the peak 2 fractions were subjected to 8.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, staining with silver showed several major bands. The intensity of a protein band corresponding to about 60 kDa changed in parallel with the amidohydrolase activity from fraction to fraction. Peak 1 also contained anandamide amidohydrolase in a bulk of protein. When the peak 1 fractions were pooled and loaded again on the Phenyl-5PW, the enzyme activity was detected only in the original position. It is unclear whether the enzyme found in peak 1 is an isozyme or a tight aggregate of the enzyme and other proteins.
Figure 2: Hydrophobic chromatography of anandamide amidohydrolase and synthase. The solubilized protein of porcine brain microsome (8.8 mg of protein) was applied onto a Phenyl-5PW column, and 2.5-ml fractions were collected as described under ``Experimental Procedures.'' Closed circles, amidohydrolase activity; open circles, synthase activity; closed triangles, protein concentration; broken line, ammonium sulfate concentration.
Anandamide synthase activity was also found in peaks 1 and 2 (Fig. 2). The synthase in the peak 2 fractions showed an average specific activity of 160 nmol/min/mg of protein at 37 °C in a yield of 29%. Hydrolysis and synthesis of anandamide by the partially purified enzyme are shown in lanes 3 and 8 on thin layer chromatograms (Fig. 1). When the partially purified enzyme (the peak 2 fractions in Fig. 2) was applied onto a DEAE-5PW column and the adsorbed protein was eluted by increasing NaCl concentration, both the amidohydrolase and synthase activities cochromatographed in one major peak (Fig. 3).
Figure 3: Cochromatography of the anandamide amidohydrolase and synthase activities on a DEAE-5PW column. The partially purified enzyme (1.3 mg) was applied onto a DEAE-5PW column as described under ``Experimental Procedures.'' Closed circles, amidohydrolase activity; open circles, synthase activity; broken line, NaCl concentration.
The
amidohydrolase and synthase activities of the partially purified enzyme
increased depending on the amount of protein almost in a linear fashion (Fig. 4A). The amidohydrolase reaction proceeded
linearly up to 30 min while the rate of the anandamide synthase
decreased gradually (Fig. 4B). The amidohydrolase
activity increased depending on the concentrations of anandamide with
an apparent K value of about 60 µM (Fig. 5A). The synthase activity depended on the
concentrations of arachidonic acid (Fig. 5B) and
ethanolamine (Fig. 5C). Their apparent K
values were approximately 100 µM and 50 mM,
respectively. V
values of the hydrolase and
synthase reactions were 0.48 µmol/min/mg of protein and 0.11
µmol/min/mg of protein. With the same amount of enzyme, the
amidohydrolase activity was 3-4 times higher than the synthase
activity. The two enzymes were active between pH 7 and 9 (Fig. 6). Furthermore, when the enzyme was preincubated at
various temperatures for 5 min, the amidohydrolase and synthase
activities were lost almost in parallel as the temperature was raised (Fig. 7).
Figure 4: Dependence on protein amount and time course of the anandamide amidohydrolase and synthase reactions. A, different amounts of the partially purified enzyme were assayed for amidohydrolase activity (closed circles) and synthase activity (open circles) under the standard conditions. B, the partially purified enzyme (0.48 µg of protein) was allowed to react for the indicated time periods for amidohydrolase activity (closed circles) and synthase activity (open circles).
Figure 5:
Substrate specificity of the enzyme
reactions. The partially purified enzyme (0.94 µg of protein) was
allowed to react under the standard conditions with various
concentrations of substrates as follows. A, ethanolamides of
various C-labeled fatty acids (closed circles,
arachidonylethanolamide; open circles, linoleylethanolamide; closed triangles, oleoylethanolamide; open triangles,
palmitoylethanolamide); B, various
C-labeled free
fatty acids in the presence of 250 mM ethanolamine (closed
circles, arachidonic acid; open circles, linoleic acid; closed triangles, oleic acid; open triangles,
palmitic acid); and C, ethanolamine in the presence of 250
µM arachidonic acid.
Figure 6:
pH dependence of the anandamide
amidohydrolase and synthase reactions. The partially purified enzyme
(0.94 µg of protein) was allowed to react at various pH values for
amidohydrolase activity (solid line) and synthase activity (broken line) under the standard conditions. pH was adjusted
with the following buffers: closed circles,
citrate-NaHPO
; open circles, Tris-HCl; closed triangles,
Na
CO
-NaHCO
; open
triangles, NaHCO
-NaOH.
Figure 7: Heat inactivation of anandamide amidohydrolase and synthase. The partially purified enzyme was kept at various temperatures for 5 min. An aliquot (0.94 µg of protein) was removed for the standard assays of amidohydrolase (closed circles) and synthase (open circles). The activity of untreated enzyme was expressed as 100%; amidohydrolase, 0.29 µmol/min/mg of protein; and synthase, 0.13 µmol/min/mg of protein.
Substrate specificity of the amidohydrolase reaction was examined with different fatty acyl ethanolamides (Fig. 5A). At 300 µM concentration, the relative amidohydrolase activity was 44% with linoleylethanolamide, 27% with oleoylethanolamide, and 19% with palmitoylethanolamide as compared with arachidonylethanolamide (anandamide). On the other hand, the rate of ethanolamide synthesis was not very different with palmitic, oleic, linoleic, and arachidonic acids (Fig. 5B).
We tested
whether the partially purified enzyme had a certain protease activity
with hydrophobic peptidyl-MCA substrates; t-butoxycarbonyl-Val-Leu-Lys-MCA (a substrate for plasmin),
Leu-MCA (for aminopeptidase), succinyl-Ala-Ala-Ala-MCA (for elastase),
succinyl-Ala-Ala-Pro-Phe-MCA (for chymotrypsin),
succinyl-Ala-Pro-Ala-MCA (for elastase), succinyl-Leu-Leu-Val-Tyr-MCA
(for chymotrypsin), and Met-MCA. These peptidyl-MCA substrates were
inactive with our enzyme preparation. For the assay of ceramidase
activity, [C]ceramide (N-oleoylsphingosine) was incubated with the Triton
X-100-solubilized enzyme and the partially purified enzyme, and the
produced [
C]oleic acid was separated from the
remaining [
C]ceramide by TLC. The cholate
extract of the 27,000
g pellet of 16-day-old rat brain (18) was used as a positive control. The result showed that a
low ceramidase activity (approximately 16 pmol/min/mg of protein) was
detected in the Triton X-100-solubilized enzyme at pH 7.4 and pH 9, but
not at pH 5. However, the partially purified enzyme did not show a
detectable ceramidase activity at pH 5, 7.4, or 9.
We also tested
the effects of various inhibitors on the amidohydrolase and synthase
activities (Fig. 8). PMSF (9) and arachidonyl
trifluoromethyl ketone (a cytosolic phospholipase A inhibitor) (20) have been reported to inhibit the
anandamide hydrolysis with crude enzyme preparation. PMSF also
inhibited the anandamide synthase activity(13) .
Sulfhydryl-reactive agents such as PCMB inhibited rat liver N-acylethanolamine amidohydrolase(21) . In our assays,
arachidonyl trifluoromethyl ketone (Fig. 8A) and PCMB (Fig. 8B) inhibited both the amidohydrolase and
synthase activities almost in parallel. PMSF (Fig. 8C)
also inhibited both the activities although its higher concentration
was required for the inhibition of the synthase. Diisopropyl
fluorophosphate, another serine protease inhibitor, inhibited both of
the activities in a similar manner (Fig. 8D).
Figure 8: Various inhibitors for anandamide amidohydrolase and synthase reactions. The partially purified enzyme (0.94 µg of protein) was assayed for amidohydrolase activity (closed circles) and synthase activity (open circles) under the standard conditions in the presence of different concentrations of arachidonyl trifluoromethyl ketone (A), PCMB (B), PMSF (C), and diisopropyl fluorophosphate (D). The activity in the control run was expressed as 100%; amidohydrolase, 0.27 µmol/min/mg of protein; and synthase, 0.10 µmol/min/mg of protein.
In consideration of potent psychoactivity of cannabinoids, there must be an in vivo mechanism which metabolizes and inactivates anandamide as an endogenous cannabinoid receptor agonist. It was reported that radiolabeled anandamide was hydrolyzed rapidly to free arachidonic acid and ethanolamine in neuroblastoma and glioma cells(9) . The brain and several other tissues of rats also hydrolyzed anandamide(9, 12) . In the present paper, we attempted to isolate and purify the anandamide amidohydrolase from porcine brain, from which anandamide was first isolated(1) . The enzyme was solubilized from the microsomal fraction with 1% Triton X-100, and the solubilized enzyme was partially purified by hydrophobic chromatography of high performance (Fig. 2). We tried several conventional column chromatographies such as ion exchange chromatography, hydrophobic chromatography, and gel filtration. However, probably due to its hydrophobicity, we have so far been unsuccessful in high purification of the enzyme protein by these methods giving only a low yield of the enzyme activity and a low purification of the enzyme. By the method presented here we could prepare the enzyme constantly with a specific activity of 240-540 nmol/min/mg of protein. This is the first report of the preparation of partially purified enzyme which could be used for enzymological studies.
When the partially purified enzyme was allowed to react with ethanolamides of arachidonic, linoleic, oleic, and palmitic acids as substrates, anandamide (arachidonylethanolamide) was the most active, suggesting that the physiological role of this enzyme was the metabolic inactivation of anandamide. While we were preparing this manuscript, a similar substrate specificity was reported with the rat brain microsome (12) . Moreover, several hydrophobic peptidyl-MCA substrates tested were inactive with our anandamide amidohydrolase, which could not be attributed to a certain protease with wide substrate specificity. Ceramidase, an amidohydrolase to hydrolyze ceramide (N-acylsphingosine) to sphingosine and a fatty acid, was distinguished from our enzyme which was inactive with N-oleoylsphingosine.
Previously, N-acylethanolamine amidohydrolase activity was found in various mammalian tissues(22) , and the enzyme of rat liver membrane was solubilized with sodium taurodeoxycholate(21) . In these works, however, the enzyme was not purified, and its reactivity with anandamide was not described.
Our purified anandamide
amidohydrolase catalyzed the reverse reaction and produced anandamide
from arachidonic acid and ethanolamine. The synthase was also reactive
with other fatty acids (palmitic acid, oleic acid, and linoleic acid)
at similar reaction rates (Fig. 5B). It was reported
that arachidonic acid was a better substrate for the rabbit brain
anandamide synthase than palmitic, oleic, and linoleic acids, but the
reactivity was assayed at a low substrate concentration (5
µM) below K and was not compared in
terms of V
(14) . Another report showed
that with bovine hippocampal P2 membrane arachidonic acid was more
active than palmitic acid at 30 µM-1
mM(13) . It is unknown at the present time whether or
not the discrepancy of substrate specificity between these results and
our finding was attributable to different animal species or different
assay conditions.
Both the amidohydrolase and synthase activities were copurified as peak 2 on a Phenyl-5PW column (Fig. 2) and cochromatographed as one major peak on a DEAE-5PW column (Fig. 3). The two activities were lost essentially in parallel by heat inactivation (Fig. 7) and various inhibitors (Fig. 8). Although our enzyme preparation was not purified to homogeneity, the results suggested that a single enzyme protein catalyzed both the synthesis and hydrolysis of anandamide. A previous report suggested that the anandamide synthesis and hydrolysis were catalyzed by separate enzymes since the synthase activity was not inhibited by PMSF(9) . However, we found that the synthase was also inhibited by PMSF with the partially purified enzyme (Fig. 8C) and the Triton X-100-solubilized protein (data not shown), although PMSF was less effective on the synthase than on the amidohydrolase. It should be noted that other lipid-related amidohydrolases such as N-acylethanolamine amidohydrolase of rat liver mitochondria (21) and ceramidase (23) are thought to be catalytically reversible enzymes. In agreement with this view, several lines of evidence so far available with our partially purified enzyme support the attribution of the hydrolysis and synthesis of anandamide to one enzyme protein. This conclusion must be confirmed by further purification of the enzyme to homogeneity and by expression of cDNA for this enzyme.
As presented in Fig. 5C,
the ethanolamine concentration which gave a half-maximum activity of
the anandamide synthase was as high as 50 mM. Other
investigators also reported a high K value for
ethanolamine (27 ± 4 mM) with bovine hippocampal P2
membrane(13) . Since such a high concentration of ethanolamine
must be supplied for the enzyme to work as anandamide synthase, it is
unlikely that the anandamide synthesis is catalyzed by this enzyme
under physiological conditions. However, we cannot rule out a certain
mechanism to activate the enzyme and to lower the K
for ethanolamine by either covalent modification of or allosteric
effect on the enzyme protein. As an alternative biosynthetic pathway,
it was recently proposed that anandamide was released from N-arachidonyl phosphatidylethanolamine by the catalysis of a
certain phospholipase D(24, 25) .