Purification and characterization of a lysophospholipase from human amnionic membranes.

We have identified the presence of a lysophospholipase in human placental tissues and have purified this enzyme from the amnion. The specific activity was highest in the amnion and decreased across adjacent tissues. The purification involved the use of DEAE-Sephadex, phenyl-Sepharose, hydroxylapatite, and sulfylpropyl Sephadex chromatography. The activity of the purified enzyme toward palmitoyl lysophospha- tidylcholine is 2.5 &mol min” mg-I and the pH optimum is 7.0. The enzyme is not inhibited by EDTA and does not appear to have a metal ion requirement. The en- zyme may be of membrane origin; the purified enzyme requires the presence of detergent during storage. The effects of substrate composition and physical state on enzymatic activity were explored. The enzyme was not active toward mono-, di-, or triglycerides, nor toward diacyl phospholipid. The enzyme was active toward myristoyl and palmitoyl lysophosphatidylcholine at concentrations where these substrates spontaneously form micelles or where Triton X-100 was used to in-duce co-micellization of the substrate at low concentrations with detergent. A role for this enzyme in process- ing the lysophospholipid product of phospholipase A action must be considered in evaluating arachidonic acid production in human fetal membranes and placental tissue, particularly during the initiation of labor.

We have identified the presence of a lysophospholipase in human placental tissues and have purified this enzyme from the amnion. The specific activity was highest in the amnion and decreased across adjacent tissues. The purification involved the use of DEAE-Sephadex, phenyl-Sepharose, hydroxylapatite, and sulfylpropyl Sephadex chromatography. The activity of the purified enzyme toward palmitoyl lysophosphatidylcholine is 2.5 &mol min" mg-I and the pH optimum is 7.0. The enzyme is not inhibited by EDTA and does not appear to have a metal ion requirement. The enzyme may be of membrane origin; the purified enzyme requires the presence of detergent during storage. The effects of substrate composition and physical state on enzymatic activity were explored. The enzyme was not active toward mono-, di-, or triglycerides, nor toward diacyl phospholipid. The enzyme was active toward myristoyl and palmitoyl lysophosphatidylcholine at concentrations where these substrates spontaneously form micelles or where Triton X-100 was used to induce co-micellization of the substrate at low concentrations with detergent. A role for this enzyme in processing the lysophospholipid product of phospholipase A action must be considered in evaluating arachidonic acid production in human fetal membranes and placental tissue, particularly during the initiation of labor.
Recent attention in the field of lipid metabolism has focused on the role of an important group of metabolites having arachidonic acid as their common precursor, including the thromboxanes, prostacyclins, leukotrienes, and prostaglandins (1)(2)(3). Arachidonic acid is not only the necessary precursor for these metabolites, but its availability appears to be the limiting factor controlling the synthesis of prostaglandins and the other biologically active lipids (1,4). Therefore, we have initiated studies on the role of lipolytic enzymes in the release of this fatty acid, as these enzymes appear to be an important control point in prostaglandin biosynthesis.
Prostaglandin biosynthesis in the human placenta and fetal membranes is of particular interest because of the involvement of prostaglandins in the initiation of labor (5)(6)(7)(8)(9). TWO pathways have been proposed for the release of arachidonate in the human amnion (7,(10)(11)(12)(13), one involves phospholipase AI and one involves phospholipase C coupled with diglyceride lipase. Study of these pathways is complicated by the presence * This work was supported by National Institutes of Health Grant GM-20501. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ Guggenheim Fellow, 1983-84. To whom correspondence and requests for reprints should be addressed. of enzymes such as the lysophospholipases and acyl lipases which further degrade the products produced by the initial enzyme action on phospholipids (4). This is illustrated for the phospholipase Aflysophospholipase A pathways in Fig. 1.
We have now identified a very active lysophospholipase in human amnion. The specific activity of this enzyme is highest in the fetal tissues associated with the placenta: the amnion and the chorion. Because of the magnitude of the lysophospholipase activity, which was much greater than the phospholipase A activity in broken cell preparations, the lysophospholipid product of phospholipase A action does not accumulate.
Only free fatty acid and glycerophosphorylcholine are observed when crude homogenates are studied with exogenous phosphatidylcholine as substrate. Identification of specific phospholipase and lipase activities is rendered difficult by the possibility that a very active lysophospholipase could also exhibit phospholipase AI, A2, or various glycerol lipase activities. Because the amnion is the membrane most heavily implicated in the initiation of labor through the release of arachidonic acid from that membrane, we have undertaken the purification of the lysophospholipase from this human fetal membrane; a preliminary report has appeared (14). We have also examined the enzyme specificity and activity toward various lysophospholipids in monomer and micellar form (below and above the CMC') as well as the effects of the nonionic detergent Triton X-100 on its activity; a preliminary report of these results has also appeared (15).

RESULTS
Enzyme Pwification-The pH profile obtained from an isoelectric focusing column of the crude lysophospholipase activity indicated the enzyme should have a net negative charge above pH 5.0. Thus, an anion exchange column, DEAE-Sephadex, was used for the first column in the purification scheme. The column profile is shown in Fig. 2A. When this purification step was attempted using buffer not containing urea, the second peak was very broad, eluting between approximately 0.2 and 1.5 M NaCl. Urea eliminated this problem, possibly by disrupting aggregated forms of the lyso-The abbreviations used are: CMC, critical micelle concentration; lyso-PC, palmitoyllysophosphatidylcholine or l-palmitoyl-sn-glycerol-3-phosphorylcholine; dipalmitoyl-PC, 1,2-dipalmitoyl-sn-glycerol-3-phosphorylcholine.
Portions of this paper (including "Experimental Procedures") are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 84111-821, cite the authors, and include a check or money order for $1.60 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press. 1. Products of phospholipases AI and Az and lysophospholipases AI and Az (also called phospholipases L1 and Lz, respectively). Possible nonenzymatic migration between the 1-acyland 2-acyllysophospholipids is shown.
phospholipase. The enzyme tolerates 1.5 M urea for several hours, but urea will eventually inactivate the enzyme.
Enzyme which had been partially purified by anion exchange chromatography on DEAE-Sephadex was further purified on the basis of hydrophobicity by a phenyl-Sepharose column with a linear ethylene glycol gradient, as shown in Fig. 2B. This column gave a 5-fold purification. When the phenyl-Sepharose-purified enzyme was first applied to hydroxylapatite columns, broad double peaks of lysophospholipase activity were observed, with little resulting purification. It was suspected there were problems with protein aggregation or with lipids in the partially purified protein preparation. A number of column purification techniques were tried without success. The enzyme activity matched the protein profile in each case. It should be possible to remove lipid from the preparation by treatment with the nonionic detergent Triton X-100. However, when Triton X-100 was added to the protein preparation, no detectable enzymatic activity remained. Fortunately, this effect is reversible and removal of Triton X-100 restores activity. Triton X-100 does not adhere to hydroxylapatite and the lysophospholipase does, so that phenyl-Sepharose-purified enzyme was brought to 3 mM Triton before being applied to a hydroxylapatite column. This procedure eliminated the broad double peak previously observed, presumably by removal of lipids. The enzyme now eluted in a single peak (Fig. 2C). The hydroxylapatite column gave a 2fold purification. The final column used in this purification procedure contained a cation exchange resin, sulfopropyl (SP)-Sephadex. This column was used at pH 4.7; this pH is very close to the pI of the enzyme as demonstrated by isoelectric focusing. The enzyme may not tolerate a pH of 4.7 for long periods of time, so every effort was made to limit the amount of time the enzyme spent exposed to this low pH. In order to raise the pH of the effluent as soon as possible, 0.6 ml of 1 M NaH2P04 buffer was placed in each fraction collection tube before the column was begun.
When this last purification step was carried out without the presence of Triton in the buffer solutions, the column purified the protein successfully but the enzyme lost activity upon storage. A low level (200 PM) of detergent prevented this loss of activity. The enzyme is very dilute at this point and detergent may stabilize a dilute protein. In addition, Triton may substitute for lipid in stabilizing membrane-associated enzymes. The effects of Triton X-100 on lysophospholipase activity are complicated and will be considered in the "Discussion." The SP-Sephadex column profile is shown in Fig.  2D, a 7-fold purification was achieved the bulk of the protein did not elute from the column. The results of a typical purification of the lysophospholipase are summarized in Table  I.
Tissue Distribution of Lysophospholipase Activity-  activity in the various tissues associated with the placenta and in whole blood. The specific activity in terms of protein was highest in the amnion and there appeared to be a gradient of activity across adjacent tissues, from amnion to chorion, to decidua, to the main placental mass, and finally very little activity in associated blood. When centrifuged, the amnion homogenate lost more activity to the pellet than did the other tissues. This may reflect the difficulty of homogenizing this tissue or it may indicate that this protein is membraneassociated. Although the amnion exhibits the highest specific activity, it has a lower activity when expressed per g of whole tissue (wet weight) reflecting the low percentage of soluble protein in this tissue compared to the others studied. Lowry protein assays showed only 8 mg of protein g" of amnion, 25 mg g" of chorion, 37 mg g" of decidua, 90 mg g" of placental lobe, and 205 mg g" of blood.
Lysophospholipase Assay-The addition of EDTA or Ca2' to the purified enzyme did not affect the activity of the lysophospholipase within experimental error. Thus, no attempt was made to restrict or include any particular metal ion in the purification or assay of the lysophospholipase. Hydrolysis of lyso-PC by the purified enzyme was linear with added enzyme up to at least 0.3 gg/assay and the assay was linear for at least 50 min even with 30% hydrolysis. Standard assay conditions used 0.3 gg of protein or less and an incubation time of 30 min.
This time course sheds some light on the acyl chain preference of this enzyme. The lipid used in the assay can undergo acyl migration as discussed by Pliickthun and Dennis (20) and illustrated in Fig. 1. Although the lyso-PC was generated by phospholipase Az action on dipalmitoyl-PC and it should be 1-acyllyso-PC, it could contain as much as 10% 2-palmitoyllyso-PC. The pseudo-first order rate constant for the conversion of the 2-acyllyso-PC at pH 7.0 is 1.5 X s-' (20). This rate constant gives a half-life for 2-acyllyso-PC of 13 h. It is safe to assume that very little migration takes place during a 50-min time course in which 30% hydrolysis OCcurred. Since 30% of the lipid was hydrolyzed and only about 10% of the lipid could be the 2-acyllyso-PC, the linear time course eliminates the possibility that the enzyme is solely an sn-2 lysophospholipase. While the enzyme is clearly a 1- conditions were used with the exception of the buffer. Sodium acetate was used between pH 3.5 and pH 5.6 (0); Tris/malate was used between pH 5.8 and 8.6 (0); glycine was used between pH 9.3 and 10.5 (A). The final buffer concentration was 167 p~. The assay mixtures contained 0.38 pg of lysophospholipase. The pH of each assay tube was measured and recorded at 37 "C. acylhydrolase, the possibility that it also acts on 2-acyllyso-PC at an equivalent rate has not been ruled out.
The activity of the lysophospholipase between pH 3.5 and 10.5 is shown in Fig. 3. The peak of activity was at pH 7.0. All points were corrected for background hydrolysis which was quite low a t acid pH but higher in the basic range, as expected. Three different buffers were used to cover this pH range. There was no obvious "buffer effect" with this enzyme and there was remarkably good overlap between the different buffers used. De Jong and co-workers (21) found large buffer effects on a lysophospholipase isolated from pancreas where the activity in Tris-HC1 buffer was half of that observed in phosphate buffer.
Detergent Dependence of Lysophospholipase-Since Triton X-100 could not be totally excluded from the assay system without inactivating the enzyme, the activity of the lysophospholipase was always investigated in the presence of at least 3.3 ~L M Triton X-100. When the time course of the reaction was determined below the CMC of the lyso-PC substrate (5.3 p~) in the presence either of 3.3 p~ Triton or 15 pM Triton, the results were linear for both plots and the hydrolysis rate a t 15 p~ Triton was significantly higher (Fig. 4). This experiment was designed to achieve a reasonable amount of hydrolysis a t low Triton concentrations and short time periods, while keeping the amount of lysophospholipase in each assay constant. Under these conditions, the 40-min time point with Lysophospholipase activity uet-sus Triton X-100 concentration is shown. All assays were 100 p~ in lyso-PC and 3.2 pg of protein was used per assay.
higher Triton (15 p~) proceeded to 39% hydrolysis. This is probably the reason this graph curves over slightly at the longer time points, and the least squares line which is shown does not intersect zero. Under standard assay conditions with a large excess of substrate (typically 100 p~) above its CMC (initial rate conditions), the time course passes through zero and appears linear as indicated above. The CMC of palmitoyllyso-PC is reported to be 7 X M (22). Fig. 5 shows the inhibition of lysophospholipase activity by detergent at higher substrate levels (above the CMC of lyso-PC), while Fig. 6 demonstrates that if the substrate to Triton ratio is held constant, no inactivation is seen. Fig. 7 shows the effect of the same Triton concentrations used in Fig. 6 when the substrate concentration is held constant. Above the CMC of the lipid, only inhibition is seen. Below the CMC, an initial activation followed by an inhibition is observed.
The dependence of the lysophospholipase activity on substrate concentration was determined in the presence of 3.3 p~ and 15 p~ Triton X-100. The Lineweaver-Burk plots generated from these data are shown in Fig. 8. These low levels of Triton have no effect at high concentrations of substrate, but do effect the activity below the CMC of the lysophospholipase substrate.
Specificity of the Lysophospholipase-The enzyme was tested against several different lipid substrates and the results are shown in Table 111. The phosphate assay results with nonradiolabeled palmitoyllyso-PC agree within experimental error with the radioactive lipid assays using the same substrate.
The CMC of myristoyllyso-PC has not been reported, but can be estimated to be about an order of magnitude higher than palmitoyllyso-PC (23). The myristoyllyso-PC was a very good substrate above the CMC and was hydrolyzed 50% better than the longer chain palmitoyllyso-PC. When the concentration of myristoyllyso-PC was dropped from 187 p~ to 14 p~, the specific activity decreased by 80%. As the concentration of palmitoyllyso-PC was dropped from 188 p M to 16 pM, the specific activity decreased much less (by 30%). If myristoyllyso-PC has a CMC of about 0.1 mM, then 14 pM is well below the CMC and monomer lipid is present. This is consistent with the idea that the lysophospholipase works poorly on monomeric lipid as opposed to micellar lipid.
In addition to the different lysophospholipids, this enzyme was tested for esterase activity against several other lipids ( Table 111). The lysophospholipase was not active against mono-, di-, or trioleoylglyceride nor was it active toward dipalmitoyl-PC. Thus, this lysophospholipase does not exhibit acylglyceride lipase activity or phospholipase A, or Az activity toward the substrates tested. Apparently, the enzyme requires both a phosphoryl head group and a single acyl tail for activity. Furthermore, in experiments with lysophospholipids as substrates, phospholipid formation was never observed in the TLC plates ruling out transacylase activity. Lysophospholipases have been isolated from a number of sources (21, 27, 40, 48-56). These enzymes are remarkably variable as to their size, specificity, and pH optima. Lysophospholipase activities have been reported in many systems with pH optima ranging from pH 4 (57, 58) to as high as pH 10 (53, 59). Many of these optima were measured on crude systems and it is possible that some of these enzymes are not primarily lysophospholipases, but are enzymes which hydrolyze other lipid esters at a higher rate at physiological pH (29).

Lysophospholipases-Lysophospholipases
Amnionic Lysophospholipase-The amniotic enzyme was purified almost lZOO-fold, with a final specific activity of 2.5 @mol min" mg". The recovery of enzyme activity was about 12%; some of the loss was due to the separation of two lysophospholipase activities on the DEAE-Sephadex column. The minor activity peak, consisting of 30% of the applied activity, was not further purified after separation. All four purification columns required conditions and buffers which  phosphatidylglycerol. * A , radioactive lyso-PC assay, detection limit 2 nmol min" mg"; B, radioactive PC assay, detection limit 20 nmol min" mg-'; C radioactive acylglyceride assay, detection limit 2 nmol min" mg"; D, phosphate assay, detection limit 200 nmol min" mg". ND, not detectable. might be considered unusual or extreme for a monomeric soluble enzyme. The DEAE-column required the presence of urea to achieve a good separation. Enzymes are typically loaded onto phenyl-Sepharose in high salt and eluted with low salt, but this lysophospholipase required 30% ethylene glycol in the eluting buffer. The hydroxylapatite step involved the use of Triton X-100 in the loading buffer and the SP-Sephadex step also involved detergent in the buffer. These findings, coupled with the apparent stabilization of the purified enzyme by detergent, suggests a membrane origin for this enzyme, although this has not been shown directly.
The results presented in Table I1 show the distribution of lysophospholipase across several placental tissues. It is not known a t this time what functional significance the high proportion of lysophospholipase activity in the amnion may have. However, it does prevent the accumulation of lysophosphatidylcholine at least in broken cell preparations. The subcellular distribution of the enzyme may be difficult to determine. Methods which efficiently homogenize the tough amnionic membrane also disrupt the subcellular organelles. However, because of the high pH optima of the enzyme it can be assumed the lysophospholipase is at least not a lysosomal enzyme.
A lysophospholipase may in principle exhibit some low level of phospholipase activity toward diacylphosphatides in some substrate configurations or physical states. In the placental tissues studied, this lysophospholipase appears to be present at concentrations which exhibit orders of magnitude more activity than other phospholipases. The presence of the active lysophospholipase activities in these and other tissues makes the study of the phospholipases acting on the diacylphosphatides a difficult and challenging task.
Triton Protection-Because the amnionic lysophospholipase requires the presence of Triton X-100 to maintain activity, total exclusion of Triton X-100 from the assay is not practical. The lysophospholipase is stored in 200 p~ Triton X-100, a concentration such that 3.33 p~ Triton is the lowest level one can, in practice, achieve in the assay system. When Triton is included with membrane-associated enzymes acting on water-soluble substrates, it is generally assumed that the Triton is solely associated with the enzyme and the only effect is to stabilize the enzyme's tertiary structure in the absence of the normal membrane. In the lysophospholipase assay system, the substrate itself forms micelles. For the substrate used in the studies reported herein, palmitoyllyso-PC, the CMC is approximately 7 p~ (22). The CMC of Triton X-100 is about 250 pM (60).
An ideal mixture of the two compounds would have a CMC between the CMC of lyso-PC and the CMC of Triton X-100. However, studies on mixtures of detergents have demonstrated that such systems often do not behave in an ideal fashion and the CMC observed is often lower than the CMC of either component alone (61-64). The CMC of mixtures of palmitoyllyso-PC and Triton X-100 have not been determined, but one would expect deviation from ideality in this system due to the markedly different structures of the two detergents. Knowing the CMC of the mixed system would still not give a clear picture of the state of the lipid in the actual assay system, since it is clear from the stabilization of the enzyme by Triton X-100 that Triton also associates with the protein to some significant degree.
Since it is not practical to totally exclude Triton from the assay, the effects of low levels of Triton X-100 on the assay system were investigated. At high levels of substrate, variations in the amount of Triton had very little effect if the detergent level was kept below the level of the lysophospholipid substrate. However, at low levels of substrate, the Triton caused a dramatic enhancement of the activity. Triton X-100 protects the lysophospholipase activity during storage of the enzyme and it is possible that this apparent enhancement of activity by Triton is actually due to denaturation of the enzyme that would occur in the absence of or a t lower levels of Triton over the time period of the assay. If this were the case, one would expect that the time course a t low and high Triton concentrations would be coincidental at first and then the low Triton plot would decrease in slope and result in a lower overall activity; this was not observed. Therefore, this increase in activity at higher Triton levels is not due to the Triton protecting the enzyme activity over the typical 30-min assay period.
MonomerjMicelle Transition and Surface Dilution-When the concentration of lyso PC is held constant at 100 p M and the Triton in the assay is increased, the Triton exhibits an inhibiting effect (Fig. 5 ) . However, when the Triton to lipid ratio is held constant at 1 to 1 (Fig. 6), no inhibition of activity was found. These results are suggestive of surface dilution kinetics, but are far from conclusive (4,65,66). One of the basic requirements for surface dilution kinetics to be applicable is for the enzyme to work on micellar lipid as opposed to monomer lipid. Triton has a stimulatory effect at lyso-PC Concentrations which are below the CMC (7 p M ) of that lipid (Fig. 7 ) . This stimulation effect is followed by a drop in activity at higher detergent concentrations. At 26 pM lyso-PC, well above the CMC of the lipid, the Triton exhibits only an inhibitory effect. All of these results can be explained if the detergent and the lyso-PC have a mixed micelle CMC lower than the lyso-PC alone, so that at low lyso-PC concentrations the detergent brings the substrate into a micellar conformation which the enzyme prefers. Once the substrate is micellar, either because of the Triton presence lowering the CMC or by virtue of the lyso-PC concentration alone, additional Triton serves only to surface-dilute the substrate in the micelle surface.
Triton has no significant effect on activity at high substrate concentrations (Fig. 8). A lowering of Triton concentration results in lower activity only a t low substrate concentrations. This result could be explained if the lyso-PC (when it is below 10 p~) is in micellar form at 15 p M Triton and is in monomer form at 3.3 mM Triton. Thus, the Lineweaver-Burk plot is straight and conventional at 15 p~ Triton. With lower detergent, but above the CMC of lyso-PC, the lines coincide. Below the CMC of lyso-PC, and at low Triton levels, the line rises sharply, indicating a greatly reduced activity toward what should be monomer lyso-PC.
It is difficult to explain the results in Fig. 8 as being anything other than an increased activity of the lysophospholipase toward aggregated lipid. This strengthens the hypothesis that the results in Figs. 4 through 7 can be explained by surface dilution kinetics. However, the difference between surface dilution and competitive inhibition is difficult to prove in a system such as this. In a normal soluble substrate/soluble enzyme system, a competitive inhibitor is expected to be chemically related to the substrate and to act by binding to the active site thereby preventing substrate-enzyme binding. In this case, the inhibitor Triton X-100 (a polydisperse preparation of p-t-octylphenoxypolyoxyethols with oxyethylene chain lengths averaging 9-10 oxyethylene units) (60) is not structurally related to the substrate, lyso-PC, and the inhibitor enhances catalysis a t very low substrate concentrations. It would seem more probably that the effects of Triton X-100 are due to co-micellization with the lyso-PC followed by surface dilution of the substrate rather than due to classical competitive inhibition.
Lysophospholipids-Unfortunately, there is a dearth of information available about the physical properties of the lysophospholipids compared with the phospholipids (23). While it has always been assumed that the monoacyl glycerophosphatides form micelles above their CMCs (67, 68), recent evidence suggests that they may also form bilayer structures at temperatures below a critical lamellar/micelle transition (69-71). Furthermore, the CMC has been determined for palmitoyllyso-PC by equilibrium dialysis and density gradient centrifugation (22) and has also been reported for lyso-PC derived from egg yolk PC (72-74) in which the reports vary Human Amnionic Membrane Lysophospholipase between 12 IrM and 450 pM. Unfortunately, a report by H~~-37. Hung, c. c., and Melnykovych, G. (1976)