A Specific Acetylhydrolase for l-Alkyl-2-acetyl-sn-glycero-3-phosphocholine (a Hypotensive and Platelet-activating Lipid)*

l-A~yl-2-acetyl-sn-glycero-3-phosphocholine, a phospholipid with platelet activating and hypotensive properties, has an extremely labile acetate grouping. The acetate group is obviously important in the expression of the biological properties of this unique deriva- tive of plasmanic acid since once it is hydrolyzed from the parent compound to form the lyso product, all biological activity is lost. Our studies show that the enzyme responsible for the hydrolysis of the acetate moiety, 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine: acetylhydrolase, occurs in the cytosolic fraction of a variety of tissues and has a pH optimum of 7.5 to 8.5. Effects of on the enzyme and acetylhydrolase activity normally A2 utilizes with two long acyl groups. highest kidney; brain

clearly shown that a variety of rat tissues can actively synthesize alkylacetyl-GPC by direct acetylation of alkyllyso-GPC (4,5) or by a selective cholinephosphotransferase that utilizes 1-alkyl-2-acetyl-sn-glycerol as the substrate (4). 3 Both of these synthetic enzymes are of microsomal origin.
The relatively short term antihypertensive effect in rats (lasting 4 0 min) after administration of 1 pg or less of alkylacetyl-GPC (1) suggests that the active phospholipid is rapidly degraded in the body. Our earlier experiments have demonstrated that an active alkylacetyl-GPC:acetylhydrolase exists in the cytosolic fraction of rat liver (4). 3 The product of this reaction, alkyllyso-GPC, does not affect blood pressure (1) or platelet aggregation ( 2 ) and, therefore, emphasizes the important role of the acetylhydrolase in the metabolism of alkylacetyl-GPC. Perhaps the transfer of the acetate group to other molecules, such as proteins, is the crucial step for expression of the biological properties of alkylacetyl-GPC.
In view of the importance of the removal of acetate from alkylacetyl-GPC, we have investigated the details of the tissue distribution and kinetic characteristics of the acetylhydrolase responsible for the removal of the acetate group. Until now, acetylhydrolases that utilize phospholipids as substrates have not been described.
Livers from adult, male CDF rats were homogenized in 0.25 M sucrose by using 25 strokes of a glass pestle in a loose fitting Dounce homogenizer tube; various other tissues were homogenized with five strokes of a motor-driven Potter-Elvehjem homogenizer. We prepared subcellular fractions by conventional centrifugation methods. For tissues other than liver, only the postnuclear (600 X g X 10 min supernatant) and cytosolic fractions ( l O 0 , O O O X g X GO min supernatant) were used. Protein was determined by the method of Lowry et al. (13).
Incubations were done in screw-capped polypropylene tubes containing protein and l-hexade~y1-2-[~H]acetyl-GPC in a final volume of 0.5 ml of 0.1 M phosphate buffer (pH 8.0) at 37°C for 5 min. Substrate and protein concentrations are stated in the legends of the tables and figure. Incubations were terminated by adding 1 ml of chloroform and 0.5 ml of a 10% sodium bicarbonate solution. This mixture was shaken and then centrifuged at 1500 rpm; the upper layer was washed three times with I-ml aliquots of fresh chloroform.
["]Acetate in the aqueous layer (0.5-ml aliquot) was radioassayed by liquid scintillation spectrometry. Results obtained with the boiled enzyme preparations containing added internal standards of [l-"C]acetic acid and l-hexade~y1-2-[~H]acetyl-GPC revealed that >90% of the I4C and only t l % of 3H were extracted into the aqueous layer under these conditions. These percentages were used to correct the acetate calculations for the amount of 'H-labeled 1-hexadecyl-2-acetyl-GPC found in the aqueous layer after each incubation. Incubations using 1-[1,2-'H]alkyl-2-acetyl-GPC were terminated by the addition of I ml of methanol containing 2% acetic acid. The entire contents were evaporated to dryness with N2, redissolved in 1 ml of chloroform/ methanol (l:l, v/v), and the products analyzed for radioactivity after separation by thin layer chromatography (1).
In an experiment to verify that the tritium released by the acetylhydrolase was in the form of acetate, we incubated 40 pg of protein from the soluble fraction of kidney cortex with 10 nmol of l-hexadecyl-2-["H]acetyl-GPC for 10 min and then terminated the reaction by adding 50 pl of glacial acetic acid. The samples were immediately frozen and then lyophilized. Volatile components were collected in a trap submersed in liquid nitrogen. Acetic acid was converted to the ammonium salt by exposing the trapped solution to an excess of ammonia vapor generated from NHrOH. An aliquot of the ammonium acetate solution was then chromatographed on Silica Gel G layers (250 pm thick) in chloroform/methanol/ammonium hydroxide (G5:35: 8, v/v). After staining the developed plates with iodine vapor to locate the ammonium acetate band (identified by co-chromatography with a standard having an RF of 0.2 to 0.3). various areas of the plate were scraped into vials and the tritium assayed in a liquid scintillation spectrophotometer. When sodium [l-I4C]acetate was subjected to this same procedure, the overall recovery of radioactivity was 72%.

RESULTS AND DISCUSSION
The activity of alky1acetyLGPC:acetylhydrolase in the soluble fraction of rat liver was linear with protein concentrations up to at least 50 pg and with the time of incubation through at least 10 min. A broad pH optimum existed between 7.5 and 8.5.
Results obtained for the subcellular distribution of alkylacetyl-GPC:acetylhydrolase in rat liver (Table I) demonstrate that the hydrolase activity is highest in the cytosolic fraction. Therefore, it seems likely that once alkylacetyl-GPC enters the cell, it is first hydrolyzed to the 2-lyso-compound by the cytosolic acetylhydrolase. The product produced by the hydrolase, l-hexadecyl-2-lyso-GPC, could then be subsequently metabolized in microsomes by several enzymes, e.g. alkyllysophospholipase D (15) and alkyllysoglycerophosphocholine monooxygenase,4 and/or resynthesized to alkylacetyl-GPC by an acetyl-coA transferase (4, 5).
In order to determine whether the alkylacetyl-GPC:acetylhydrolase activity had properties similar to the phospholipase A2 that utilizes phospholipids containing two long chain acyl groups, we added various compounds to the incubations that have been reported to influence the activity of the typically encountered phospholipase Az. Results obtained with the liver cytosolic fraction (Table 11) show that the acetylhydrolase activity was not greatly affected by additions of Ca2+ or Mg", and that EDTA and dithiothreitol had slight stimulating effects. However, the reaction was severely inhibited by diisopropylfluorophosphate (0.11 t o 11 mM) and, to some extent, by deoxycholate. The lack of stimulation by Ca2+ or Mg", and the slight stimulation by EDTA indicate that the acetylhydrolase activity in the cytosolic fraction of rat liver does not behave like the typical phospholipase A2 activity of rat liver (16) since the latter exhibits completely different properties. Moreover, the severe inhibition of the acetylhydrolase by diisopropylfluorophosphate, which does not inhibit phospholipase Az (17), suggests that the acetylhydrolase may be similar t o the short chain acylhydrolase activities encountered by Wykle and Schremmer (10) in their studies of plasmalogen biosynthesis with microsomal fractions of Fischer sarcomas. Table 111 presents the acetylhydrolase activities found in the soluble fraction of several rat tissues. Specific activities of acetylhydrolase were also measured in the postnuclear fractions (600 X g X 10 min supernatant) from each tissue and were found to be two-thirds to one-half less than that in the cytosolic fraction (100,000 X g X 60 min supernatant). The soluble fraction from kidney had the highest acetylhydrolase activity of all tissues examined, although the lung and brain also had relatively high activities.
T h e specific activity of acetylhydrolase found in the soluble fraction of the kidney medulla was two-thirds to one-half that of the kidney cortex. In view of the high activities present, the cytosolic fraction of the kidney cortex was used to obtain the kinetic data depicted in the double reciprocal plot shown in Fig. 1. Acetylhydrolase had apparent K , and V,,, values of 3.1 p M and 17.8 nmol/ min/mg of protein, respectively.
After formation of the ammonium salt, the tritium released from l-hexadecyl-2-["H]acetyl-GPC by the kidney cortex soluble fraction was shown to co-chromatograph on thin layer chromatograms with an acetate standard. The overall yield was equal to that found using the collection technique for sodium     Only a slight inhibition of alky1acetyLGPC:acetylhydrolase activity was observed when egg phosphatidylcholine (long chain diacyl type) was added to the incubations containing the cytosolic enzyme from rat kidney cortex (Table IV). In contrast to the data obtained with egg phosphatidylcholine, the phosphatides having sn-2 acetyl groups significantly in- hibited the hydrolysis of the alkylacetyl-GPC (Table IV). However, l-alkyl-2,3-diacetyl-sn-glycerol caused only a slight decrease in acetylhydrolase activity. These findings are consistent with our results obtained with the inhibitor studies (Table 11) and further support the premise that the acetylhydrolase for alkylacetyl-GPC differs from the phospholipase A2 that utilizes long chain diacylphospholipids as substrates.
Our results might also be explained on the basis of phospholipase AS having a higher specificity for the acetyl group; however, this does not seem likely since phosphatidylcholines containing two short chain acyl groups are known to be poor substrates for phospholipase AZ (17). Apparently, the substrate specificity for the enzyme is not dependent on the type of aliphatic moiety at the sn-1 position since the acyl analog also inhibits the activity of the acetylhydrolase. However, only the alkyl derivative exhibits the platelet activating and hypotensive properties; therefore, the alkyl moiety must be a critical determinant of its profound biological activity. Perhaps it is the alkyl portion of the A Phospholipid Acetylhydrolase molecule that determines the specificity of the transfer of the acetate to other compounds that modulate the biological responses involved. Regardless, the acetylhydrolase that removes the acetate moiety of alkylacetyl-GPC appears to be a very important enzyme in the metabolism of this biologically active phospholipid.