Specificity of lysophospholipase D

58

Biochimico et Biophysics 0 El sevier/North-Holland

Acta,

619

Biomedical

(1980)

58-67

Press

BBA 57597

SPECIFICITY OF LYSOPHOSPHOLIPASE

ROBERT

L. WYKLE,

WILLIAM

F. KRAEMER

From the Medical and Health Sciences Oak Ridge, TN 3 7830 (U.S.A.)

(Received

Key

words:

November

Division,

D *

and JACALYN Oak Ridge

M. SCHREMMER

Associated

Universities,

lst, 1979)

Lysophospholipase

D; Plasmalogen;

Glycerophospholipid;

(Rat liver microsome)

Summary The specificity of lysophospholipase D (l-alkyl-sn-glycero-3-phosphoethanolamine ethanolaminehydrolase, EC 3.1.4.39; also works on choline analogs) for l-alkyl- and l-acyl-linked substrates was examined using rat liver microsomes. The microsomes were treated with diisopropylphosphorofluoridate to inhibit the hydrolysis of acyl chains from the acyl-linked compounds (l-palmitoyl-snglycero-3-phosphocholine and l-palmitoyl-sn-glycero-3-phosphoethanolamine) and were treated with p-bromophenacyl bromide to block acylation of the compounds tested. In the presence of the inhibitors, 1-alkyl-sn-glycero-3-phosphocholine and l-alkylsn-glycero-3-phosphoethanolamine were hydrolyzed extensively by lysophospholipase D but the corresponding 1-acyl-linked analogs were only negligibly hydrolyzed. Lysophospholipase D therefore appears to be specific for the ether-linked compounds. 1-Alk-1-‘enyl-sn-glycero-3-phosphoethanolamine (lyso plasmalogen) was also tested as a substrate, but a plasmalogenase in the rat liver microsomes rapidly hydrolyzed the compound and we were unable to determine whether it is a substrate for lysophospholipase D. Alkyl-linked substrates containing long-chain acyl groups at the 2-position are not hydrolyzed by the enzymes. We tested l-alkyl-2-acetoyl-sn-glycero-3-phosphocholine and l-alkyl-2-acetoyl-sn-glycero-3-phosphoethanolamine to determine if the less bulky, more hydrophilic acetate group would permit hydrolysis by lysophospholipase D; the derivatives did not appear to be attacked, except after hydrolysis of the acetate group. However, in the absence of inhibitors, the acetate groups were rapidly hydrolyzed by microsomal preparations. --__* This work has been authored by a contractor of the U.S. Government under contract number DEAC05-760R00033. Accordingly. the U.S. Government retains a nonexclusive, royalty-free license to publish or reproduce the published form of this contribution, or allow others to do so. for U.S. Govemment purposes.

59

Introduction Evidence for the presence of lysophospholipase D (EC 3.1.4.39) in microsomal preparations of brain and liver was presented and discussed earlier [ 1,2] ; the enzyme catalyzes the hydrolysis of base groups from long-chain l-alkyl-snglycero-3-phosphocholine and 1-alkyl-sn-glycero-3-phosphoethanolamine. Lysophospholipase D of the liver is tightly bound to the microsomal membrane fraction and requires Mg2+ for activity [Z]. The initial product of the reaction is l-alkyl-sn-glycero-3-phosphate, which is subsequently converted to l-alkyl-snglycerol by a phosphohydrolase present in the microsomes. If the phosphohydrolase is inhibited by NaF, the major product recovered is l-alkyl-snglycero-3-phosphate [ 1,2]; however, no NaF was added in the present study. Earlier evidence indicated that ‘phospholipase C’ is not responsible for the accumulation of 1-alkylglycerol in the system from brain [l] or liver [ 21. The combined action of lysophospholipase D and the phosphohydrolase provides a pathway for removing ether-linked lysoglycerophospholipid which are potent cell lysing agents that cannot be broken down by acyl hydrolases as are the analogous acyl-linked lipids. The present study was carried out primarily to determine if lysophospholipase D hydrolyzes l-acyl-sn-glycero-3-phosphocholine and l-acyl-sn-glycero3-phosphoethanolamine as it does the alkyl-linked analogs. Initially we found that the microsomal preparations contain an acyl hydrolase (lysophospholipase, EC 3.1.1.5) that rapidly breaks down the l-acyl-linked substrates, releasing fatty acids. However, by inhibiting the acyl hydrolase with diisopropylphosphorofluoridate [3,4], we were able to compare the 1-alkyl- and 1-acyl-linked compounds as substrates for lysophospholipase D. We found earlier that lysophospholipase D does not act on 1-alkyl-2-acyl-snglycero-3-phosphoethanolamine or l-alkyl-2-acyl-sn-glycero-3-phosphocholine containing long-chain acyl groups at the sn-2 position [1,2]. In the present study, l-hexadecyl-2-acetoyl-sn-glycero-3-phosphocholine and l-hexadecyl-2acetoyl-sn-glycero-3-phosphoethanolamine were tested as substrates in order to determine if the enzyme can act on derivatives containing a less bulky, more hydrophilic group at the 2-position, or if the free hydroxyl group is necessary for activity. Materials and Methods Labeled substrates. l-[ 1-14C]Palmitoyl-sn-glycero-3-phosphocholine (55 Ci/ mol) and unlabeled l-palmitoyl-sn-glycero-3-phosphocholine were obtained Inc., State College, PA and mixed in from Applied Science Laboratories, CHC13/CH30H (2 : 1, v/v) to give an appropriate specific radioactivity. l-Palmitoyl-sn-glycero-3-phosphoethanolamine (11 Ci/mol) was prepared from 1,2di[ 1-‘4C]palmitoyl-sn-glycero-3-phosphoethanolamine (Applied Science Inc.) by treatment with phospholipase A2 (Ophiophugus hannah venom); it was purified by preparative thin-layer chromatography (TLC) (silica gel HR) in CHC13/CH30H/CH3COOH/H20 (50 : 25 : 6 : 2, v/v). l-[l-‘VIl-[ 1-14C]Hexadecyl-sn-glycero-3-phosphoethanolamine and hexadecyl-sn-glycero-3-phosphocholine were prepared by the following proce-

60

dures: as described earlier [5], the ether-linked lipids of Ehrlich ascites cells were labeled in vivo with [l-14C]hexadecanol (55 Ci/mol) for 24 h, extracted from the cells, and treated with HCl gas to remove plasmalogens. The lipids were next loaded on an activated silicic acid column (unisil, 100-200 mesh, 40 g; 2 X 40 cm; equilibrated with CH&); the less polar lipids were eluted with 500 ml CHCl,/CH,OH (20 : 1, v/v). Next the ethanolamine-containing fraction was eluted with 500 ml of CHC1JCH30H (5 : 1, v/v). Finally, the cholinecontaining fraction was eluted with CHJOH. Analysis of the ethanolamine- and choline-containing fractions by TLC demonstrates the labeled products migrated as single peaks at the same RF as authentic l-alkyl-2-acyl-s~-glycero-3-phosphoeth~o~~~e and I-alkyl-2-acylso-glycero-3-phosphocholine, respectively. The X-acyl groups and diacyl components were removed from each of the fractions by a mild saponification procedure [6] in which the fractions were dissolved in 1.2 ml CHC&, and 0.6 ml 0.33 M KOH in CH,OH was added at 0°C. After the mixtures were shaken at 25°C for 15 min, 0.5 ml 6 M HCl was added and the products extracted [ 71. The l-[PC]1-r 1-i4C]hexadecyl-sn-glycero-3-phosphocholine and hexadecyl-sn-glycero-3-phosphoethanolamine from the saponification procedure were purified by preparative TLC and were respectively 97 and 95% pure based. on TLC analysis of radioactivity. Only 1-alkyl-glycerol (no fatty alcohol) was found after reduction of the compounds with V&ride [ 81, which indicates that no acyl groups were present in the preparations. The specific radioactivities, based on phosphorus analysis [9], were 1.9 Ci/mol for l-~~-14C]hexadecyls~~lycero-3-phosphocholine and 2.4 Ci/mol for l-[l-‘4C]hexadecyl-snglycero-3-phosphoethanolamine. The l-[ 1-‘4C]hexadecyl-sra-glycero-3-phosphocholine, dissolved in CHClJCH,OH (2 : 1, v/v), and the l-[l-14C]hexadecyl-sn-glycero-3-phosphoethanolamine in CHCl, were stored at -20°C. A similar preparation containing 37% l-[ l-14C]alk-l’-enyl-sn-glycero-3phosphoethanolamine and 63% l-[ l-14C]alkyl-sn-glycero-3-phosphoethanolamine was obtained by the same procedures except the lipids of the Ehrlich ascites cells were labeled with [l-‘4CJhexadecanol for 48 h and were not treated with HCl gas. At the end of the saponification reaction, the mixture was acidified with CH,COOH instead of HCI. The aIk-1-enyl content of the mixture was measured independently by reducing the mixture with Vitride and measuring the label in l-alkyd-so-glycerol and 1-ok-l’~nyl-so-glycerol 181, and by exposing the mixture to HCl fumes and measuring the labeled aldehyde released from the l-alk-1’-enyl-sn-glycero-3-phosphoethanolamine [ 81. The procedure used to prepare l-[ 1-14C]hexadecyl-2-acetoyl-sn-glycero-3phosphoethanolamine (2.4 Ci/mol) has been described [lo] ; the l-[ l-“Clhexadecyl-sn-glycero-3-phosphoethanolamine used in the procedure was the same preparation as described above. l-[ l-14C]Hexadecyl-2-acetoyl-sn-glycero3-phosphocholine (1.9 Ci/mol) was prepared by a procedure similar to that described by Gupta et al. [ll], except J_-]1-14C]hexadecyl-sn-glycero-3-phosphocholine, prepared as described above, was substituted for l-palmitoyl-snglycero-3-phosphocholine. Other mu~er~uls.Diisopropylphosphoro~uo~date, phospholip~e AZ (lyophilized venom from Ophi~phu~s Hannah) and p-bromophenacyl bromide were obtained from Sigma Chemical Co.

61

Enzyme preparation, incubations, and analysis of products. Microsomal preparations, obtained as described earlier [2] from livers of adult CD rats, were prepared and washed in 0.25 M sucrose, 10 mM EDTA [ 21. Lysophospholipase D activity was assayed as described earlier [ 1,2] ; details are given in the legends of tables and the figure. Procedures used to extract products from the incubation mixtures and the methods used for chromatographic analysis and radioassay were the same as before [ 1,2]. In addition, phospholipids were analyzed by two-dimensional TLC. Products were chromatographed in the first dimension along the edge of a 20-cm plate by developing the layers in CHClJ CH30H/NH40H (65 : 35 : 8, v/v); then solvents were removed by placing the plate in a desiccator under vacuum for 1 h. Next, the plate was rotated and developed in the second dimension in CHCl,/CH,OH/glacial CH&!OOH/H,O (50 : 25 : 6 : 2: v/v).

Results Two problems were encountered in determining if lysophospholipase D acts on l-acyl-sn-glycero-3-phosphocholine and 1-acyl-sn-glycero-3-phosphoethanolamine. First, as discussed earlier, the acyl groups were rapidly hydrolyzed by an acyl hydrolase present in the microsomal preparations; after incubating l-[ 1-14C]palmitoyl-sn-glycero-3-phosphocholine (17 nmol) for 10 min in the absence of inhibitors, 30% of the label was recovered as the fatty acid. Second, a fraction of the lyso substrates was acylated by the microsomal preparations during the incubation period, and thus was rendered inactive as a substrate for lysophospholipase D. However, by using inhibitors, it was possible to block both reactions without abolishing lysophospholipase D activity. Treatment of the microsomal preparations (5 mg protein per ml) with 10 mM diisopropylphosphorofluoridate at 0°C for 10 min inhibited >98% of the acyl hydrolase activity and >90% of the acylation activity. We found that addition of 0.1 mM

TABLE I EFFECTS OF DIISOPROPYLPHOSPHOROFLUORIDATE LYSOPHOSPHOLIPASE D

AND p-BROMOPHENACYL

BROMIDE

ON

The incubation mixture contained MgClZ (5 mM), Tris-HCl buffer (0.1 M, pH 7.1). treated or untreated microsomal preparation (0.5 mg protein) and l-[l-14Clhexadecyl-sn-glycero-3-phosphocholine (74 000 dpm; 17 nmol), added in 20 pl ethanol. The mixture in a final volume of 3 ml was shaken at 37’C for 10 min; the reaction was stopped by extracting the lipids. Treatment with diisopropylphosphorofluoridate was carried out by incubating the microsomal preparation (5 mg protein per ml water) with the inhibitor (10 mM) for 10 min at O’C before the preparation was added to the incubation mixture. The final concentration of diisopropylphosphorofluoridate in the incubation mixture was 0.33 mM and of protein 0.5 mg per 3 ml. The p-bromophenacyl bromide (0.13 mM) was added directly to the incubation mixture in 20 ~1 acetone. The major hydrolysis product recovered in all samples was l-[l-‘4Clhexadecyl-sn-glycerol. but there were also smsll amounts (
Percentage of l-[l-14C1hexadecyl-snglycero-3-phosphocholine hydrolyzed

None p-Bromophenacyl bromide Diisopropylphosphorofluoridate p-Bromophenacyl bromide + diisopropylphosphorofluoridate

32: 15; 19; 13;

31 16 19 14

62

p-bromophenacyl bromide to the incubation mixture blocked >99% of the acylation activity. We next examined lysophospholipase D activity after treatment with the inhibitors. When l-[ 1-14C]hexadecyl-sn-glycero-3-phosphocholine, which is known to be a substrate, was used to measure lysophospholipase D activity, the treatments with diisopropylphosphorofluoridate and p-bromophenacyl bromide inhibited the rate of hydrolysis by lysophospholipase D approx. 50% (Table I). In the presence of the inhibitors the reaction rate was linear for 15 min. No inhibition of the phosphohydrolase was observed under these conditions and >95% of the hydrolyzed products was recovered as l-[ 1-14C]hexadecyl-sn-glycero1. The results demonstrate that even though

I-Alkyl-GPC

I-Alkyl-GPC

- Mg2’

t l-Acyl-GPC+Mg”

I1 Zone

/

1213

I

number

Fig. 1. Distribution of label in products after incubation of l-[l- “C]hexadecyl-sn-$lycero-3-phosphocholine (l-aIkyl-GPC) and l-[l-14Clhexadecanoyl-sn-gIycero-3-phosphochoIine (1-acyl-GPC) in the lysophosphollpase D assay system. Substrates were tested in an incubation mixture that contained microsomal preparations (0.5 mg protein) treated with diisopropylphosphorofluoridate as described in Table I, p-bromophenacyl bromide (0.13 mM), Tris-HCl buffer (0.1 M, pH 7.1). MgClZ (5 mM) as indicated. and the labeled substrates in a final volume of 3 ml. AII samples were incubated for 1 h at 37’C before the products were extracted. The products were separated on silica gel G developed in tanks (without a wick) containing hexane/diethyl ether/methanol (80 : 20 : 5, v/v). Area 1 (zone 1 to 3; 5-mm zones) contained the unhydrolyzed substrate; area 2 (usuaIIy zone 13-15) contained 1-acylgIycero1 and area 3 (usualIy zone 16-20) contained 1-alkylglycerol. Panels A and B: l-[1-*4ClHexadecyl-sn-glycero-3-phosphocholine (17 nmol; 74 000 dpm; added in 20 ~1 ethanol) was incubated in the system in the absence of Mg*+ (A) or the presence of Mg2* (B); in the presence of Mg 2+, 59% of the label was in l-alkylglycerol. Panel C: this shows the products isolated after a mixture of l-[l-l4 C]hexadecyl+n-glycero-3-phosphocholine (17 nmol; 74 000 dpm) and 1-[1-14Clhexadecanoyl-sn-glycero-3-phosphocholne (17 nmol. 86 000 dpm) were incubated in the system in the presence of Mg 2+. The two substrates were mixed first and added to the incubation mixture together in 20 ~1 ethanol. No label (
63

the lysophospholipase D activity is depressed by the inhibitors at the levels required to block the acyl hydrolase and aeylation reactions, sufficient activity remains to test whether lysophospholipase D acts on 1-a~yl-so-glycero-3-phosphocholine and l-acyl-so-glycero-3-phosphoeth~olamine. l-Acyl-sn-glycero-3-phosphocholine and I-alkyl-sn-glycero-3-phosphocholine were tested as substrates both separately and as a mixture (Fig. lA--D). The incubation period was extended to 1 h in this experiment in order to demonstrate the negligible activity obtained with the l-acyl-linked substrate. Zonal scans of the products separated by TLC are shown; the chromato~aphy system used resolves 1-acylglycerol (RF 0.45) and l-alkylglycerol (RF 0.58). When l-alkyl-sn-glycero-3-phosphocholine alone was tested in the complete system (Fig. lB), 10 nmol of 1-alkylglycerol was released; a similar amount of l-alkylglycerol (9.9 nmol; the percentage of [14C]alkylglycerol is decreased with the mixture, but the total dpm as percentage of initial 1-alkyl-sn-glycero-3phosphocholine are not) was released when 1-acyl-so-glycero-3-phosphoeholine was mixed with the l-alkyl-sn-glycero-3-phosphocholine at an equal concentration (Fig. 1C). No significant hydrolysis of 1-acyl-sn-glycero-3-phosphocholine was observed when it was tested alone (Fig. 1D) or when mixed with 1-alkyl-sn-glycero-3-phosphocholine (Fig. 1C). The phospholipids obtained from each of the experiments described in Fig. 1 were analyzed by two-dimensional TLC. A maximum of 0.2% of the label was in 1-alkylsn-glycero-3phosphate or l-acyl-sn-glycero-3-phosphate in any of the experiments and
64 TABLE II HYDROLYSIS OF l-ALKYL-sn-GLYCERO-3-PHOSPHOETHANOLAMINE 3-PHOSPHOETHANOLAMINE BY LYSOPHOSPHOLIPASE D

AND l-ACYL-sn-GLYCERO-

The incubation mixture, time. and other conditions were the same as described in Table I for the diisopropylphosphorofluoridate-treated sample. 1-[1-14C]Hexadecyl-sn-glycero-3-phosphoethanolamine (60000 dpm; 11 nmol), 1-[1-14C]hexadecanoyl-sn-~ycero-3-phosphoethanolamine (60 000 dpm; 2.5 nmol), or a mixture of the two (60 000 dpm of each were added in 20 ~1 diethyl ether/ethanol (2: 1, v/v). Values are based on the total 14C-labeled lipids recovered in the incubation products; the distribution of label was determined by TLC. Compounds tested

Percentage of label in products

MgClz (5 mM)

l-Alkylglycerol

l-Awlglycerol

I.

1-[1-14C]Hexadecyl-sn-glycero-3-phosphoethanolamine

+

39 +2* 3.4 i 1

_

II.

1-[1-‘4C]Hexadecanoyl-sn-glycero-3-phosphoethanola~nine

+

_

1.6 t 0.1 0.5 + 0.4

III.

l-[l-14C]Hexadecyl-sn-glycero-3-phosphoethanol~ine 1-[1-14C]Hexadecanoyl-sn-glycero-3-phosphoethanol~ine

+

24

* In sample I. 39% represents 4.3 nmol t 1“C]alkylglycerol,

plus il


while in sample III, 24% represents 5.3 nmol.

mine with certainty whether lysophospholipase D hydrolyzes l-alk-l’-enyl-snglycero-3-phosphoethanolamine. Treatment of the microsomes with diisopropylphosphorofluoridate, p-bromophenacyl bromide, or 3 mM imidazole, which was earlier found to inhibit the hydrolysis of 1-alk-1’-enyl-sn-glycero-3phosphocholine by a plasmalogenase of rat liver microsomes [ 131, failed to block hydrolysis of the l-alk-l’-enyl-sn-glycero-3-phosphoethanolamine by the plasmalogenase. Derivatives of l-alkyl-sn-glycero-3-phosphoethanolamine and l-alkyl-snglycero-3-phosphocholine containing acetate at the Z-position were tested as TABLE III HYDROLYSIS OF l-[l-‘4C]HEXADECYL-2-ACETOYL-sn-GLYCERO-3-PHOSPHOETHANOLA~~INE AND l-[1-14C]HEXADECYL-2-ACETOYL-sn-GLYCERO-3-PHOSPHOCHOLINE BY LYSOPHOSPHOLIPASE D The incubation mixture and conditions were the same as described in Table I for the diisopropylphosphorofluoridate-treated sample. 1+1-l 4ClHexadecyl-sn-glycero-3-phosphoethanolamine (11 nmol; 58 000 dpm) and l-[l-14C]hexadecyl-2-acetoyl-sfl-~ycero-3-phoaphoethanolamine (13 nmol: 71 000 dpm) were added to the incubation mixture in 20 ~1 of diethyl ether/ethanol (2:l. v/v). l-[l-‘“ClHexadecyl-snglycero-3-phosphocholine (14 nmol; 60 000 dpm) and l-[l-14C]hexadecyl-2-acetoyl-sn-glycero-3-phosphocholine (15 nmol; 65 000 dpm) were added in 20 ~1 ethanol. The hydrolyzed product recovered from each of the compounds tested was 1-alkylglycerol. Compound tested

Percentage of compound hydrolyzed by lysophospholipase D 19.6 3.2 16.3 4.4

? f ? f

0.5 0.1 0.2 0.1

65

substrates of lysophospholipase D. The acetylated compounds were hydrolyzed at a much slower rate than the analogous lyso compounds (Table III). Microsomes were treated with diisopropylphosphorofluoridate to prevent hydrolysis of acetate from the sn-2-position of the substrates. With untreated microsomes under the same conditions, acetate was hydrolyzed from 60% of the 1-alkyl-2acetoyl-sn-glycero-3-phosphoethanolamine and from 25% of the l-alkyl-2acetoyl-sn-glycero-3-phosphocholine after 15 min. In the experiment shown in Table III, l-alkyl-glycerol was found in the products, but neither 1-alkyl-2acetoylglycerol nor 1-alkyl-2-acetoylglycero-3-phosphate was detected. A small amount of label was also found in the respective lyso compounds. These findings indicate the acetate groups were hydrolyzed to a small extent, even though the microsomes were treated with diisopropylphosphorofluoridate. Thus, the labeled 1-alkyl-glycerol was most likely formed by hydrolysis of the lyso compounds after the acetate groups were removed. Discussion The results of the present study provide evidence that lysophospholipase D does not act on either l-acyl-sn-glycero-3-phosphocholine or l-acyl-sn-glycero3-phosphoethanolamine. Results were equivocal for the l-alk-l’enyl-snglycero-3-phosphoethanolamine because it was rapidly hydrolyzed by a plasmalogenase present in the liver microsomes. The l-alkyl- and 1-acyl-linked substrates were tested separately and as mixtures of the two. Several conclusions can be drawn from the mixing experiment. The mixing experiment described in Fig. 1C demonstrates that the lack of hydrolysis of the 1-acyl-linked compound is not due to the presence of an inhibitor in the l-acyl-sn-glycero-3-phosphocholine preparation nor due to inhibition of lysophospholipase D by l-acylsn-glycero-3-phosphocholine itself. Furthermore, the lack of activity toward l-acyl-sn-glycero-3-phosphocholine cannot be attributed to differences in detergent action of the two compounds. If the l-alkyl-sn-glycero-3-phosphocholine preparation contained an activator or activated the enzyme by detergent action, the 1-acyl-sn-glycero-3-phosphocholine should be hydrolyzed in the presence of the l-alkyl-sn-glycero-3phosphocholine. However, only the 1-alkyl-linked compound was hydrolyzed. Analysis of the products also demonstrates that the lack of hydrolysis was not due to selective acylation at the sn-2-position of the 1-acyl-linked substrate. Similar results were observed when the ethanolamine-linked compounds were compared. Derivatives of the alkyl-linked substrates (1-alkyl-2-acetoyl-sn-glycero-3phosphocholine and l-a.lkyl-2-acetoyl-sn-glycero-3-phosphoethanolamine) containing short-chain acyl groups at the 2-position were tested in the system to determine if the more hydrophilic, smaller chain would render the substrates inactive as do fatty acyl chains. Neither of the substrates containing an acetate group appeared to be hydrolyzed directly by lysophospholipase D. However, the microsomes contained an acyl hydrolase, similar to one observed in tumor microsomes [lo], that removed the acetate groups to yield the lyso substrates. Treatment of the microsomes with diisopropylphosphorofluoridate strongly inhibited the hydrolysis of the acetate groups, but a low level of the activity

66

was observed even after treatment. Thus although some hydrolysis by lysophospholipase D was observed with the 2-acetoyl compounds, the hydrolysis appeared to result via attack on the lyso compounds released. The specificity of lysophospholipase D for the ether-linked substrates is novel since the specificity is conferred at a position of the molecule distal to the site of hydrolysis. In addition, phospholipases are generally less active toward substrates containing ether-linked chains, e.g., Lands and Hart [ 141 and Waku and Nakazawa [ 151. The lack of hydrolysis of the 2-acetoyl derivatives and those containing long-chain acyl groups suggests a free hydroxyl group may be required for activity. In this respect the enzyme is similar to the phospholipase D-type isolated from Corynebacterium pseudotuberculosis by Soucke et al. [ 161; it hydrolyzes 1-acyl-sn-glycero-3-phosphocholine and sphingomyelin but not diacyl-sn-glycero-3-phosphocholine. Lysophospholipase D differs from the phospholipase D solubilized from rat brain by Saito and Kanfer [17] in that hydrolyzes diacyl-sn-glycero-3-phosphocholine and is stimulated by Ca’+. Acyl-sn-glycero-3-phosphocholine has been shown to influence the activity of certain enzymes such as guanylate and adenylate cyclase [ 18,191 and phosphorylcholine cytidyltransferase [20] ; these effects were attributed in part to the detergent action of l-acyl-sn-glycero-3-phosphocholine. If the alkyl-linked analogs have similar effects, control of their levels by lysophospholipase D, and possibly other enzymes, could influence these enzyme activities. Lysophospholipase D might also be involved in metabolism of the synthetic analogs of lysophosphatidylcholine that have been reported to have antitumor activity [21, 221. In addition, the enzyme could play a role in the metabolism of dietary sources of alkyl-linked glycerophospholipids. The acylation of substrates observed in the absence of inhibitors was promoted by an endogenous acyl source. The acyl donor was not identified but is possibly acyl-CoA. In other studies, inhibition of acyl CoA:lysophosphatidylcholine acyltransferase by diisopropylphosphorofluoridate was observed in microsomal preparations of rabbit lung [23] and of mouse pulmonary adenomas and liver [ 241. However, Lands and Hart [25] earlier reported that the acyltransferase of liver is not inhibited by diisopropylphosphorofluoridate. The discrepant results are possibly due to differences in the concentrations of the inhibitor or substrates employed. Based on the results of the present study, lysophospholipase D appears to be mainly important for the catabolism of the alkyl-linked lysoglycerophospholipids, which, if allowed to accumulate, could result in cell lysis. Since the ether chains cannot be hydrolyzed by phospholipases, the lysophospholipase D-dependent pathway may be essential for the catabolism of the alkyl-linked phosphoglycerides. However, there are other pathways known to catabolize the alkyl-linked glycerophospholipids. The alkyl chains of 1-alkyl-2-acyl-sn-glycero3-phosphoethanolamine can be converted to 1-alk-l’-enyl chains [26], which can be hydrolyzed by plasmalogenase [ 121. Furthermore, the tetrahydropteridine-dependent cleavage enzyme has been shown to cleave the aIky1 moiety from 1-alkyl-sn-glycero-3-phosphoethanolamine [ 271. We have determined, using EDTA-washed microsomal preparations, that the alkyl group of l-alkylsn-glycero-3-phosphoethanolamine is cleaved directly by the cleavage enzyme,

67

releasing an aldehyde, without the action of lysophospholipase D. Lysophospholipase D appears to be the first ester hydrolase known that is specific for the ether-linked lipids. We are currently attempting to purify lysophospholipase D from the microsomal membrane in order to compare the kinetics of the isolated enzyme and the membrane-bound system. Our findings that the derivatives containing acetate at the sn-Z-position are very poor substrates for lysophospholipase D, but that acetate groups can be rapidly hydrolyzed by the enzyme system, were highlighted by recent evidence that platelet activating factor is l-alkyl-2-acetoyl-sn-glycero-3-phosphocholine [ 281. 1-Alkyl-2-acetoylsn-glycero-3-phosphocholine was also recently found to have powerful antihypertensive properties in one-kidney, one-clip hypertensive rats when given either intravenously or orally [ 291. Acknowledgements We thank Mr. Neil F. Martin for helping on this project as a research trainee in the Student Research Participation Program operated by University Programs of the Oak Ridge Associated Universities. The work was supported by the U.S. Department of Energy (Contract No. DE-AC05-760R00033), the National Cancer Institute (Grant CA-11949-lo), and the American Cancer Society (Grant BC-70J). References 1

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