Analysis of Human Synovial Fluid Phospholipase A, on Short Chain Phosphatidylcholine-Mixed Micelles: Development of a Spectrophotometric Assay Suitable for a Microtiterplate Reader’ Laure
at San Diego, La Jolla,
Phospholipase A, (PLA,; EC 188.8.131.52)2 catalyzes the hydrolysis of fatty acids from the sn-2 position of phos1 Financial support was provided by Lilly Research Laboratories and NIH Grant GM 20,501. ’ Abbreviations used: PLAx, phospholipase A,; HSF, human synovial fluid, DTP, 4,4’-dithiodipyridine; DTNB, 5,5’-dithiobis(:!-nitrobenzoic acid); BSA, bovine serum albumin; racemic diheptanoyl thio-PC, 1,2-bis(heptanoylthio)-1,2-dideoxy-rac-glycero-3-phosphorylcholine; racemic didecanoyl thio-PC, 1,2-bis(decanoylthio)-1,2-di190
The development of a reliable assay for human synovial fluid phospholipase A, (HSF PLA,) is important for the kinetic characterization of the enzyme and for the identification of enzyme inhibitors. This enzyme behaves differently from other extracellular PLA,s in many standard phospholipase assays and is generally assayed using radiolabeled, autoclaved Escherichia coli as a substrate. We have now developed a nonradioactive, continuous, spectrophotometric assay for this enzyme that is adaptable for use with a microtiterplate reader and is suitable for screening enzyme inhibitors. The assay uses a thioester derivative of diheptanoyl phosphatidylcholine as a substrate, with which the enzyme displays a specific activity of about 25 pmol min-’ The substrate concentration curve fits a Hill mg-l. equation with an apparent K, of 500 pM and a Hill coefficient of two. The enzyme has a pH optimum of 7.5 in this assay and requires about 10 mM Ca2+ for maximal activity. The presence of 0.3 mM Triton X-100 was necessary to solubilize the substrate; however, higher concentrations of the detergent inhibited enzyme activity. Using this spectrophotometric assay, inhibition of HSF PLA2 by a thioether phosphonate phosphatidylethanolamine analog was observed with an IC,, of 18 pM. 0 1992 Academic
pholipids, yielding a free fatty acid and a lysophospholipid as products (1). The release of arachidonic acid from membrane phospholipids by this enzyme is believed to be a key step in the control of eicosanoid production within the cell (2). PLA, has been purified from the synovial fluid of arthritic patients and from a variety of mammalian cell types (3-5). This enzyme has not only been found in high levels at sites of inflammation, but has also been shown to induce inflammation in experimental animals (6). Thus, there is considerable interest in identifying inhibitors of this enzyme as potential antiinflammatory agents. The amino acid sequence of the human synovial fluid (HSF) PLA, reveals a high degree of homology to PLA,s obtained from pancreatic juice and from various snake venoms (7). The three dimensional structures of these enzymes are also very similar (8). Despite the physical similarity, however, this enzyme behaves very differently than other extracellular enzymes and displays a lower activity in many standard assays. Currently, the most popular assay for the detection of HSF PLA, utilizes radiolabeled Escherichia coli membranes (3,9-12). Other radioactive phospholipid substrates have also been used (4,11-14), but the activity in these assays is usually less than that observed in the E. coli assay. These methods can detect very small amounts of enzyme and have thus been useful for enzyme purification. deoxy-rat-glycero-3-phosphorylcholine; chiral diheptanoyl thio-PC, 1,2-bis(heptanoylthio)-1,2-dideoxy-sn-glycero-3-phosphorylcholine; dihexanoyl thio-PC, 1,2-bis(hexanoylthio)-1,2-dideoxy-sn-glycero-3phosphorylcholine; SPPE, I-hexadecylthio-l-deoxy-2-hexadecylphosphono -an- glycero -3 -phosphorylethanolamine; SPPC, 1 - hexa decylthio-l-deoxy-2-hexadecylphosphono-s~-glycero-3-phosphorylcholine; CMC, critical micelle concentration; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; PE, phosphatidylethanolamine; PC, phosphatidylcholine; I&, concentration giving 50% inhibition. 0003-2697192
Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
However, these radioactive assays are discontinuous, laborious, and costly (15). Further characterization of this enzyme, as well as the identification of inhibitors, would be facilitated by the availability of a convenient, reliable assay. Now that the protein has been cloned (S), larger quantities of the enzyme are available making the development of a continuous, nonradioactive assay for this enzyme feasible. We have previously developed a thiolbased spectrophotometric assay for the kinetic analysis of cobra venom (N. nclja nczja) PLA, (16-19). In this report, we describe a similar assay for human synovial fluid PLA, that is suitable for detection with a microtiterplate reader. MATERIALS
1,2 - Bisjheptanoylthio) - 12 - dideoxy - sn - glycero - 3 phosphorylcholine (chiral diheptanoyl thio-PC) was prepared as described previously (19) except for the final purification. A Bond Elut NH, aminopropyl solid phase extraction column was employed instead of the first silica column (chloroform/methanol/ammonia, 50/100/5), which was used with short chain acylthiophospholipids, or the Rexyl ion-exchange column, which was used with long chain fatty acylthiophospholipids. This solid phase extraction column was purchased from Analytichem International (Harbor City, CA). The prepacked column contains 10 g of solid phase, which can be used to purify the crude product containing about 250 mg of phospholipid. The column was washed with 50 ml of chloroform prior to use. The sample was dissolved in 2-3 ml of chloroform and then applied to the column. The column was eluted in a stepwise manner with 50-ml volumes of chloroform/ methanol which contained 0 to 60% methanol in 10% increments. After this column, the product is about 85% pure. Further purification was performed on a silica column using chloroform/methanol/water as described in the original method. 1,2 - Bis(heptanoylthio) - 1,2 - dideoxy - rut- glycero - 3 phosphorylcholine (racemic diheptanoyl thio-PC) and 1,2-bis(decanoylthio)-1,2-dideoxy-rac-glycero-3-phosphorylcholine (racemic didecanoyl thio-PC) were synthesized according to the procedure of Hendrickson et al. (20) using racemic tritylglycidol as the starting material. 1,2 - Bis(hexanoylthio) - 1,2 - dideoxy - srz - glycero-phosphorylcholine (dihexanoyl thio-PC) was synthesized using a modification (18) of the Hendrickson procedure. 1-Hexadecylthio-l-deoxy-Z-hexadecylphosphono-sn-glycero-3-phosphorylethanolamine (SPPE) and 1-hexadecylthio-1-deoxy-2-hexadecylphosphonoso-glycero-3-phosphorylcholine (SPPC) were synthesized as described elsewhere (L. Yu and E. A, Dennis, manuscript in preparation). Pure recombinant human synovial fluid PLA, was kindly provided by Drs. Lee Bobbit, Don McClure, and John Sharp of Lilly Research
Laboratories (Indianapolis, IN). Cobra venom (N. naja @a) PLA, was purified as described previously (21,22). Porcine pancreatic PLA, and fatty acid free bovine serum albumin were purchased from Sigma Chemical Co. (St. Louis, MO). The thiol sensitive reagents 4,4’dithiodipyridine (DTP) and 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB, Ellman’s reagent) were purchased from Aldrich Chemical Co. (Milwaukee, WI). Triton X-100 was from Calbiochem (San Diego, CA).
Standard assay conditions were adapted from the method of Hendrickson and Dennis (16). The desired amount of diheptanoyl thio-PC (final concentration 2 mM) in chloroform solution was dried under a stream of nitrogen. Triton X-100 (0.3 mM) in assay buffer (10 mM CaCl,, 0.1 M KCl, 1 mg/ml BSA, 25 mM Tris-HCl, pH 7.5) was added. The mixture was warmed to 40°C and vortexed until a clear solution was obtained. Substrate solution (0.3 ml) was placed in a cuvette (2 X 10 mm) followed by 5 ~1 of thiol reagent (50 mM DTNB in either H,O, pH 7.5, or 0.5 M Tris, pH 8.0, or 50 mM DTP in ethanol) and carefully mixed with a sealed capillary tube. This solution was placed in a water-jacketed, temperature controlled cell holder of a Perkin-Elmer 552 uv-vis spectrophotometer and allowed to equilibrate for 3 min at 40°C. The background absorbance was recorded for at least 1 min and later subtracted from the observed enzymatic rate. The reaction was initiated by the addition of 5 ~1 of enzyme solution (containing about 25 ng of PLA, in assay buffer) and the reaction rate recorded for 1-2 min at 405 nm [for DTNB, t = 12,800 M-’ cm-” (19)] or 324 nm [for DTP, t = 19,800 M-l
Inhibition Substrate mixed micelles composed of 2 mM chiral diheptanoyl thio-PC and 0.3 mM Triton X-100 were prepared in 25 mM Tris-HCl (pH 7.5) buffer with 0.1 M KCl, 10 mM CaCl,, and 1 mg/ml BSA as described above. Substrate/inhibitor mixed micelles were formed by the addition of this substrate solution to a quantity of dried phosphonate PE (SPPE final concentration 100 pM) and sonicated until the SPPE became solubilized. Aliquots of this solution were mixed with substrate micelles to obtain the desired concentration of inhibitor in the assay. Assays were run at 40°C with DTNB as the thiol reagent and initiated by addition of 100 ng HSF PLA,.
Microtiterplate assays were performed with a THERMOmax plate reader from Molecular Devices that was equipped with a thermostated reading chamber and an
automixing option. Racemic diheptanoyl thio-PC substrate was prepared as described for the standard assay above (final concentration 2 mM thio-PC and 0.3 mM Triton X-100). Substrate solution (0.2 ml) and 5 ~1 of DTNB (33 mM in H,O, pH 7.5) per well were placed in a 96-well polystyrene plate and equilibrated at 40°C for 3 min. After equilibration, 10 ~1 of HSF PLA, containing 10 ng of enzyme, in standard assay buffer, was added. The solution was mixed, and the absorbance at 405 nm was read every minute for lo-30 min. RESULTS
Previous studies with the human synovial fluid phospholipase A,, and other mammalian nonpancreatic PLA,s, have indicated that the enzyme hydrolyzes phosphatidylcholine (PC) substrates poorly (12,14,24). Most of these studies have been performed on PC substrates with long fatty acid chains in vesicle form. We have now observed that HSF PLA, acts well on short chain PC substrates in micelle form and have utilized this information to develop a spectrophotometric assay for the enzyme. This assay utilizes a thioester phospholipid analog as a substrate and measures the amount of free thiol that is produced upon enzymatic hydrolysis by observing its reaction with a thiol sensitive reagent. Optimization
of the Assay
In initial studies, using the conditions employed for the assay of cobra venom PLA, (16,19), negligible activity was observed toward racemic didecanoyl thio-PC (1 mM) either in mixed micelles with Triton X-100 or in sonicated vesicles. Activity was observed, however, using micellar substrates with shorter chain lengths (six and seven carbons). These short chain thiophospholipids have critical micelle concentrations (CMCs) around 1.0 and 0.17 mM (for dihexanoyl and diheptanoyl thio-PC, respectively) (20,25) and form micelles at assay concentrations. The conditions of the assay were adjusted to optimize enzyme activity and allow for adaptation to an automated microtiterplate assay. With 2 InM diheptanoyl thio-PC, a small amount of Triton X-100 (0.3 mM) was added to the substrate in order to create an optically clear solution, which is essential for a spectrophotometric assay. In contrast, the dihexanoyl thio-PC, at 2 mM, was optically clear and did not require the addition of detergent. The enzyme displayed a greater activity toward the diheptanoyl substrate than toward the dihexanoyl substrate. Thus, diheptanoyl thio-PC was employed in standard assays. With both substrates, large fluctuations in specific activity were observed which were alleviated by the addition of 1 mg/ml of BSA. BSA is included in many of the E. coli assays for this PLA, (3,9,11,14) and may minimize the amount of enzyme adhering to the curvette and other surfaces. When BSA
PH FIG. 1. pH rate profile of human synovial fluid PLA,. Assays were performed with DTNB (in H,O, pH 7.5) as the thiol reagent and 25 ng PLA, in either 25 mM acetate (A), 25 mM Tris (m), or 25 mM glycine (0) buffers. The activities shown reflect corrections for the change in extinction coefficient of the thiol reagent with pH (19). The data presented are the average of duplicates.
was included, the assay was stable and reproducible. Assays with HSF PLA, were run at 4O”C, whereas previous assays with cobra venom PLA, were run at 30°C. This increase in temperature resulted in an approximately 40% increase in activity with little or no increase in the background rate of hydrolysis. A pH rate profile, determined using DTNB as the thiol reagent, is shown in Fig. 1. The optimum pH for HSF PLA, in this assay was observed to be about 7.5. A broad pH optimum in the same region was observed when DTP was the thiol reagent. At pH 7.5, 25 mM Tris-HCl was found to have adequate buffering capacity. Hepes buffer was also satisfactory; however, high background rates were observed with imidazole buffers. HSF PLA, exhibits an absolute requirement for the presence of Ca ‘+ for activity, since no activity was observed in the presence of EGTA. Enzyme activity was examined as a function of Ca2+ concentration and optimal activity was observed at about 10 mM or higher CaCl, (Fig. 2). The Ca2+ concentration curve can be fit to a simple Michaelis-Menton equation and the line drawn in Fig. 2 represents a theoretical curve with a V,,, of 28 pmol min-’ mg-’ and an apparent K, for Ca2+ of 1.5 mM (where the K, for Ca2+ is the Ca2+ concentration at half-maximal velocity). The addition of M$+ to the PLA, assay has been reported to replace a part of the Ca2+ requirement in some PLA, systems, allowing the activation of PLA, at lower concentrations of Ca2+ (26,27). However, in this system, Mg2+ had the opposite effect and appears to be competing with Ca2+. In the presence of 10 mM Mg2+, the apparent K, for Ca2+ increased to about 5 mM.
tCa2+l (mM) FXG. 2. Ca” dependence of HSF PLA,. Assays were run with DTNB (in 0.5 M Tris, pH 8.0) as the thiol reagent and 25-50 ng PLA,. PLA, activities were measured with variable CaCl, concentrations in the absence (A,) or presence (e) of 10 mM MgC&. The background rate of hydrolysis, in the absence of added CaCl,, was 0.7 pmol min-’ from each point. The lines drawn are mg-’ and has been subtracted theoretical Michaelis-Menton curves as described in the text. The data represent the average of duplicate assays.
There are two thiol-sensitive reagents which have been used in PLA, assays, DTP, whose product has an absorption maximum at 324 nm fc = 19,800 M-’ cm-’ (19,23)] and DTNB, whose dianion has an absorption maximum at 412 nm [r = 12,800 M-’ cm-’ (19,28)]. For the cobra venom PLA,, DTP was chosen as the thiol reagent because of its higher extinction coefficient. However, there are several other factors that affect the choice of reagent, including pH and solubility. The extinction coefficients of both DTP and DTNB are dependent on the pH of the assay solution (I9,23). The absorption of DTP is stable at acidic pH, but drops off at basic pHs above about 7.5. The absorption of DTNB, however, is stable at basic pH but drops off at acidic pHs below about 6.5. Thus, the pH of the assay is a consideration in the choice of reagent. An additional considerat.ion is the solubility of the reagents, since DTNB is water soluble at assay pH, while DTP requires an organic solvent, such as ethanol. Preliminary experiments showed that the assay of HSF PLA, with DTNB (in aqueous solution) gave a higher activity than with DTP (in ethanol solution). In a closer comparison of the two reagents, both in ethanol solution, the rate with DTNB was equal to that observed with DTP. As described below, further investigation using the aqueous DTNB reagent indicated that the addition of ethanol to the assay inhibited enzyme activity. Thus, the lower activity originally observed with DTP was most likely due to the addition of ethanol.
Although both reagents were used successfully in the assay of HSF PLA,, aqueous DTNB was chosen as the thiol reagent in the standard assay because of its higher activity compared to ethanolic DTP. One further advantage of using DTNB is that this reagent is more compatible with microtiterplate readers than DTP. The microtiterplate reaction must be observed in the visible light range since assays are run in polystyrene plates. The absorption maximum of 412 nm is also close to the 405-nm filter that is available for most microtiterplate readers. The absorption at 405 nm is nearly the same as that observed at 412 nm (&~i~ = 0.994), thus, the standard assay reported here utilizes 405 nm as the detection wavelength. When employing this assay, one should be aware that any free thiols present in the enzyme or inhibitor solutions will react with the thiol reagent. The resulting chromophore, or any compound present that absorbs at 405 nm, would add to the apparent activity observed in the enzymatic assay. This type of interference can easily be tested for by checking for an increase in the absorbance of t.he thiol reagent, in the absence of substrate, upon the addition of enzyme or inhibitor. Standard
On the basis of these results and observations, a standard assay was established for HSF PLA, that contained 2 mM diheptanoyl t.hio-PC, 0.3 IXIM Triton X-100, 10 InM CaCl,, 0.1 M KC!& 1 mg/ml BSA, 0.8 m&f DTNB, and 25 rnM Tris-WC1 at pH 7.5. Assays were run at 40°C and detected at 405 nm. The standard assay and the experiments presented here were determined with chiral diheptanoyl thio-PC as a substrate, unless otherwise stated. However, racemic diheptanoyl thio-PC has also been used as a substrate and behaves similarly to the chiral compound. Under the standard assay conditions, the enzymatic reaction rate was observed to be linear with time. The reaction was also linear with protein concentration between 2.5 and 100 ng. An examination of enzyme activity as a function of substrate concentration is shown in Fig. 3. The data was fit to a Hill equation with a V,,, of 24.7 pmol min-’ mg-“, an apparent &, of 0.53 mM, and a Hill coefficient of 1.8. This result indicates a positive cooperativity of phospholipid binding to HSF PLA, with at least two phospholipid binding events. Using the standard assay, the activity of two other extracellular PLA,s from cobra venom and porcine pancreatic fluid were compared to that of the HSF PLA,. The activities of these three enzymes were determined using both the diheptanoyl thio-PC/Triton X-100 mixed micelles and pure dihexanoyl thio-PC micelles (Table I). All three enzymes were more active (3- to 6-fold) against the seven carbon substrate than against the six carbon substrate. With both substrates, the activ-
Dependence of HSF PLA, activity on the concentration of substrate. Assays were run with DTNB (in 0.5 M Tris, pH 8.0) as the thiol reagent with 40 to 100 ng HSF PLA,. The diheptanoyl thio-PC: Triton ratio was kept constant at 1:0.15. The line drawn is that calculated for a Hill equation, as described in the text. The data represent the average of two experiments, each performed in duplicate. Error bars represent the standard deviation.
ity of the pancreatic enzyme was only 2- to 3-fold faster than that of the HSF PLA,. The cobra enzyme, however, had much higher activities against these substrates and was 50-70 times faster than the HSF PLA,. The activity of HSF PLA, was also determined toward racemic didecanoyl thio-PC (2 mM) in vesicle form and was 10% of that seen vs diheptanoyl thio-PC-mixed micelles. Effect of Triton
The activity of HSF PLA, was examined in the presence of increasing concentrations of the nonionic detergent, Triton X-100. As described above, the standard
Effect of Triton X-100 on HSF PLA, activity. Assays were run with DTNB (in 0.5 M Tris, pH 8.0) as the thiol reagent and 25 to 30 ng HSF PLAz. Enzymatic activity was measured in the presence of increasing concentration of Triton X-100. The data represent the average of duplicate assays.
assay with diheptanoyl thio-PC required the presence of 0.3 mM Triton X-100 in order to create an optically clear solution. This concentration corresponds approximately to the CMC of Triton X-100. At concentrations of Triton X-100 below 0.3 mM, the solutions were not optically clear and anomalous activities were observed. Above 0.3 mM, however, the activity of HSF PLA, decreased with increasing Triton concentration (Fig. 4). Similar results were also observed using dihexanoyl thio-PC as a substrate. The inhibition of HSF PLA, activity by high concentrations of detergents has also been observed with other assays (11,12,14). This decrease in enzymatic activity with increasing detergent concentration could result from surface dilution kinetics, as was observed previously with cobra venom PLA, (29,30). Effect of Solvents
Comparison of Extracellular PLA, Activities on Short Chain Thio-PC Substrates Specific activity (pm01 min-’ mg-I) Substrate
Pancreatic 10 61
Cobra 370 1080
Note. Assays were performed with 2 mM dihexanoyl thio-PC (diC, PC) or 2 mM diheptanoyl thio-PC/O.3 mM Triton X-100 mixed micelles (diC, PC) in the standard assay using DTNB (50 mM in 0.5 M Tris, pH 8.0) as the thiol reagent. Assays were initiated with 25 ng HSF, porcine pancreatic, or cobra venom PLA,. The data represent the average of two experiments, each performed in duplicate.
As mentioned above, a lower enzymatic activity was observed when the thiol reagents were added in ethanol solution than in aqueous solution. This sensitivity of HSF PLA, activity to the presence of solvents in the assay was examined further using DTNB in aqueous solution as the thiol reagent (Fig. 5). A dramatic decrease in enzymatic activity was observed with increasing concentrations of ethanol in this assay. The addition of methanol to the assay also affected enzymatic activity, but to a lesser extent than was observed with ethanol. The addition of dimethyl sulfoxide, however, had little effect on enzyme activity. In the standard assay, the thiol reagent is typically added in a 5-~1 volume. When the reagent is in ethanol solution, this results in the addition of 1.6% ethanol to the assay. In this experi-
FIG. 5. The effect of solvents on HSF PLA, activity. HSF PLA, activity was determined under standard assay conditions using DTNB (in 0.5 M Tris, pH 8.0) as the thiol reagent with 25 ng PLA, and increasing concentrations of ethanol (A), methanol (o), or dimethyl sulfoxide (@. The data represent the average of two experiments, each performed in duplicate, and are expressed relative to a control, with no solvent added.
ment, after the addition of 1.6% ethanol to the assay, the activity of the HSF PLA, was only 67% of the control activity observed in the absence of solvent. On addition of methanol, at the same concentration, the enzymatic activity was 84% of the control while the activity in the presence of dimethyl sulfoxide was about 90% of the control. The lower activity observed in the presence of ethanol was not due to a denaturation of the HSF PLA, by the solvent since no loss of enzyme activity was detected upon preincubation of the enzyme at 40°C for 45 min in the presence of 1.6% ethanol, followed by dilution of the enzyme into the assay cuvette. The lower activity observed in the presence of alcohols could be due to an effect of the solvent on the phospholipid interface or to an effect on the thiol reagent or chromophore. The sensitivity of the enzymatic rate to the presence of ethanol and methanol suggests that, in future studies, any inhibitors added to the assay should be in dimethyl sulfoxide solution rather than in alcohol solution. Inhibition of HSF PLA, Analogs
heptanoyl thio-PC, and SPPE. In Tris buffer (pH 7.5), with DTNB as the thiol reagent, this long chain phosphonate PE analog was found to inhibit the synovial fluid enzyme with an IC,, of 18 PM (Fig. 6), which is over loo-fold less than the concentration of substrate in the assay. A similar experiment, run in Hepes buffer at pH 6.5 with DTP as the thiol reagent, gave a comparable IC,, value of 14 prvi. In comparison to SPPE, the phosphatidylcholine analog, SPPC, was at least lo-fold less potent as an inhibitor with only 20% inhibition occurring at 100 PM. This difference in inhibition potency between the two long chain phosphonates suggests that PE binds better to the HSF PLA, than PC does. The high activity of this enzyme against E. coli membranes, which contain no phosphatidylcholine, and earlier studies on long chain vesicular substrates have also suggested a preference for the PE headgroup over the PC! headgroup. PE is known to undergo phase transitions in vesicular systems from a bilayer to a hexagonal array (33). It is possible that the HSF PLA, exhibits a preference for a particular substrate form rather than for a particular headgroup. Thus, the preference for the PE inhibitor in this mixed micelle system provides further evidence that the HSF PLA, does bind the PE headgroup better than the PC headgroup. Application
The spectrophotometric assay for HSF PLA, was adapted for use with a microtiterplate reader. As dis-
Analogs of phospholipids that contain a tetrahedral phosphonate instead of the acyl group at the m-2 position have been found to be potent transition state inhibitors of cobra venom PLA, with IC,,s in the low- to submicromolar region (31,32). The inhibition of HSF PLA, by a thioether phosphonate analog, SPPE, was studied by measuring the enzymatic activity toward substrate/ inhibitor mixed micelles composed of Triton X-100, di-
FIG. 6. Inhibition of HSF PLA, by thioether phosphonate PE and PC. Enzyme activity was determined toward substrate/inhibitor mixed micelles composed of 2 mM chiral diheptanoyl thio-PC, 0.3 mM Triton X-100, and variable amounts of SPPE (A) or SPPC (0) in standard Tris--FIG1 (pH 7.5) assay buffer. The assays were performed at 40°C with 100 ng PLA, using DTNB (in ethanol) as the thiol reagent. The data represent the average of duplicates and are expressed relative to the activity of the control, with no inhibitor added.
h 5 ,o :!
o.oo 3 0
also make the assay particularly useful for screening enzyme inhibitors. Using the thio assay, we have detected a cooperativity of phospholipid binding to the enzyme and a sensitivity of enzyme activity to the presence of alcohols. We have also observed an inhibition of the human synovial fluid PLA, by a thioether phosphonate PE analog. The inhibition by this PE compound was at least lo-fold stronger than the inhibition by the corresponding PC analog, which is consistent with the observed preference of this enzyme for PE substrates over PC substrates.
Adaptation of HSF PLA, thiol assay to a reader. Assays were performed in a 96.well polystyrene plate using 2 mM rat-diheptanoyl thio-PC/O.3 mM mixed micelles as a substrate in standard assay buffer H,O, pH 7.5) as a thiol reagent. Assays were initiated amounts of HSF PLA, and allowed to run for 30 min. sent the average of nine assays (three wells on each and the error bars represent the standard deviation.
microtiterplate microtiterTriton X-100 with DTNB (in with variable The data repreof three plates)
ACKNOWLEDGMENTS We are especially grateful to Dr. Lee Bobbit from Lilly Laboratories for purifying, characterizing, and supplying the nant human synovial fluid PLA, preparation employed in periments. We also appreciate the helpful discussions with ward Mihelich, Ruth Kramer, Don McClure, and John Sharp Research Laboratories regarding this enzyme. We thank Lin our laboratory for providing the phosphonate inhibitors and PC substrates and we thank Raymond Deems for constructive We also acknowledge Molecular Devices for lending us the terplate reader.
Research recombithese exDrs. Edat Lilly Yu from the thioadvice. microti-
cussed above, DTNB was employed as the thiol-sensitive reagent since it is compatible with the 405nm filter of standard microtiterplate readers. The assay volume was reduced in order to accomodate a 96-well microtiterplate. The amount of enzyme added for a typical assay was also reduced, from about 25 ng per assay to about 10 ng per assay with data collection occurring for 10 to 30 min instead of 1 to 3 min. The reaction rate was linear over this time period and was also linear with protein concentration (Fig. 7) between 1 and 50 ng. The specific activity observed was about 24 prnol min-’ mg-‘, which is similar to that observed in the standard spectrophotometric assay. Thus, the microtiterplate assay appears to behave the same as the standard spectrophotometric assay and, in addition to performing several assays at once, has the advantage of using less enzyme and substrate per assay.
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