Communication

Platelet-Activating Factor

EVIDENCE FOR l-0-ALKYL-2-ACETYL-sn- GLYCERYL-3-PHOSPHORYLCHOLINE AS THE ACTIVE COMPONENT (A NEW CLASS OF LIPID CHEMICAL MEDIATORS) *

(Received for publication, June 22, 1979, and in revised form, July 30, 1979)

Constantinos A. Demopoulos,#j R. Neal Pinckard,g and Donald J. Hanahan$ll From the $ Departments of Biochemistry and aPathology, The University of Texas Health Science Center, San Antonio, Texas 78284

A glyceryl ether containing phosphoglyceride, I-O- alkyl-2-acetyl-sn- glyceryl- 3 -phosphorylcholine (Ac- GEPC), has been shown to have a biological activity indistinguishable from that of naturally generated (rabbit) platelet activating factor (PAF). Its biochemi- cal and biological properties so closely parallel those of naturally occurring PAF that we propose they are one and the same compound. Both PAF and AcGEPC could be converted to an inactive form through base- catalyzed methanolysis and restored to 100% functional activity by reaction with acetic anhydride. The syn- thetic lipid, AcGEPC, elicited 50% secretion of serotonin from rabbit platelets at a level of 10-l” M (based on phosphorus). A propionyl derivative had somewhat comparable activity towards platelets, whereas the bu- tyryl homologue was some ‘I-fold less active and the stearoyl derivative was inactive. These short chain acylglyceryl ether phosphoglycerides represent an en- tirely new, potent and unique class of lipid chemical mediators.

1 - Acyl- 2 - acetyl- sn - glyceryl- 3 -phosphorylcholine (AcLL) also exhibited activity towards platelets but was some 200-fold less active than AcGEPC. The pro- pionyl lysolecithin behaved quite similarly to AcLL, but butyryl and stearoyl lysolecithins showed no activ- ity.

Platelet activating factor (PAF), a most potent chemical mediator released from antigen-stimulated, IgE-sensitized ba- sophils and presumably mast cells, interacts with rabbit plate- lets inducing aggregation and secretion of granular constitu- ents (1). Recently, Pinckard et al. (2) presented data which showed that PAF’ produced, under in vivo as well as in vitro

* This investigation was supported by Grant HL-22555-01 from the National Heart, Blood and Lung Institute and by Grant R-A-54 from the Morrison Trust, 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 USC. Section 1734 solely to indicate this fact.

1 Permanent address, National University of Athens, Department of Food Chemistry, Athens, Greece.

/I To whom all inquiries should be addressed. I The abbreviations used are: PAF, platelet activating factor,

AcGEPC, 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine; AcLL, 1-acyl-2-acetyl-sn-glyceryl-3-phosphorylcholine; GEPC, l-O-alkyl- sn-glyceryl-3-phosphorylcholine; albumin, bovine serum albumin; IgE, immunoglobulin E.

conditions, from antigen-challenged blood obtained from IgE- sensitized rabbits had comparable physicochemical behavior. Although the available amounts of PAF precluded any rigor- ous chemical structure proof, it was still possible to associate certain changes in the biological activity of PAF with specific chemical treatments (2). The evidence clearly supported the conclusion that PAF was a lipid with properties similar to those expected for a neutral phospholipid which contained at least one carboxylic acid ester group.

Our strategy was to utilize the base sensitivity of PAF to investigate its possible resynthesis from the degradation prod- ucts and to attempt chemical synthesis of compounds with analogous biological and biochemical characteristics. During these experiments, we discovered that acetylation of the prod- ucts derived from base treatment of PAF produced a com- pound with high biological activity and an RF indistinguisha- ble from that of the native PAF. Also, we found that acetyla- tion of l-acyl-sn-glyceryl-3-phosphorylcholine (lysolecithin) gave rise to a derivative with platelet-activating behavior. While these lysolecithin derivatives functionally mimicked PAF, certain physicochemical properties differentiated and excluded them as being the native PAF molecule. On the other hand, acetylation of the glyceryl ether phosphoglycer- ide, l-0-alkyl-sn-glyceryl-3-phosphorylcholine (GEPC) pro- duced an exquisitely high activity platelet activating compo- nent with biochemical properties identical with PAF. Thus, we propose that native PAF is very similar if not identical with this compound. The details of these experiments are summarized here and provide an insight into a new, potent group of biologically active lipids.

EXPERIMENTAL PROCEDURES

Preparation and Assay for PAF-PAF was prepared by antigen stimulation of washed rabbit buffy coat containing 8 to 10% IgE- sensitized basophils as described by Pinckard et al. (2). The crude PAF samples in Tris/Tyrode’s buffer containing bovine serum albu- min were extracted into 70% methyl alcohol and the PAF was phased into chloroform by the addition of chloroform and water and purified as described below.

PAF and the various test lipid compounds were assessed for platelet stimulatory activity as recently described (2). Briefly, rabbit platelets, internally labeled with [‘Hlserotonin (New England Nuclear; 28.2 Ci/ mmol) were washed on Ficoll-Paque cushions and adjusted to 2.5 x lo* platelets/ml of Tyrode’s buffer, pH 7.2. Appropriate dilutions of PAF or the test lipids were prepared in pyrogen-free 0.15 M NaCl containing 2.5 mg/ml of crystallized bovine serum albumin; the al- bumin was required for dispersion of PAF and the test lipids. Four microliters of the various dilutions of PAF and test lipids were added to 200 ~1 of prewarmed (37’C) [‘HI serotonin-labeled platelets in plastic test tubes and the reaction mixture was incubated for 60 s at which time 20 ~1 of cold 1.5 M formaldehyde were added to stop the reactions. The tubes were immediately cooled to O”C, centrifuged at 2200 x g for 10 min and the supernatants were assayed for percentage of [.‘H]serotonin secretion relative to 100% controls prepared by the addition of Triton X-106 to 200 ~1 of the starting platelet suspension. The data were plotted linearly and 1 unit of activity was defined as the amount of PAF or test lipids required to effect 50% serotonin release.

Purification of PAF-PAF was purified by chromatography on silicic acid (SilicAR CC-7, 100 to 200 mesh) using a sequential solvent system of chloroform, acetone, acetone/methanol (9:1, v/v), acetone/ methanol (l:l, v/v), chloroform/methanol (1:4, v/v), and chloroform/ methanol/water (1:2:0.8, v/v). Over 80% of the PAF was recovered in the chloroform/methanol (1:4, v/v) eluate with the remainder in the chloroform/methanol/water (1:2:0.8, v/v) fraction. Both eluates con-

9355

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9356 PAF and Acetylglyceryl Ether Phosphoglyceride

tamed PAF with identical thin layer chromatographic behavior and usually had as a contaminant either sphingomyelin or lysolecithin, or both. Preparative thin layer chromatography on Analtech precoated Silica Gel plates, 1000 I*, using a solvent system of chloroform/meth- anol/water (65:35:6, v/v), showed PAF migrating as a very sharp band (RF, 0.21) very close to sphingomyelin (RF, 0.23) but well away from the lysolecithin (RF, 0.15). The PAF could be eluted from these plates with chloroform/methanol/water (1:2:0.8, v/v) and further purified by rechromatography using a solvent system of chloroform/metha- nol/water (65:35:4., v/v) with PAF (RF, 0.10) migrating coincident with lysolecithin (RF, 0.09) and with good separation from sphingo- myelin (RF, 0.18).

Preparation and Reactions of Specific Phospholipids-Lysoleci- thin (I-acyl-sn-glyceryl-3-phosphorylcholine) was prepared through action of phospholipase AS on purified egg lecithin (3) and recovered as a single band (RF, 0.15) by thin layer chromatography using a solvent system of chloroform/methanol/water (65:35:6, v/v). It con- tained 5.52% phosphorus and had an infrared spectrum with the following bands: 2925 cm-‘, 2855 cm-‘, 1458 cm-‘, -CH:j and -CH,; 1730 cm-‘, ester C=O, 1082 cm-‘, P-O ; 1055 cm-‘, P-O-C; 965 cm-‘, P-O-choline. Its fatty acid composition (%) as determined by gas- liquid chromatography was: 16:0, 68.0; 18:0, 27, and 18:1, 4.

The vinyl ether-containing phospholipids of fresh beef heart were isolated and purified as outlined by Pugh et at. (4). The choline- containing fraction was further purified on thin layer chromatography and a portion was subjected to catalytic hydrogenation using PtO, as catalyst at a hydrogen pressure of 60 p.s.i. for a period of 3 hours at room temperature. The resulting mixture gave no reaction to Schiff base reagent (5) and was subjected to a short term base-catalyzed methanolysis (6). The resulting phospholipid, LO-alkyl-sn-glyceryl- 3-phosphorylcholine, was isolated by thin layer chromatography (RF., 0.15) using chloroform/methanol/water (65:35:6, v/v). It contained 5.1% phosphorus and exhibited the following infrared pattern: 2930 cm-‘, 2860 cm-‘, 1460 cm-‘, 1375 cm-‘, -CHz and -CHa; 1090 cm-‘, P-O; 1060 cm-‘, P-O-C; 968 cm-‘, P-O-choline. Its glyceryl ether composition (mole per cent) was determined by gas-liquid chroma- tography (6) and was found to be 16:0, 78; 18:0, 22.

Reacylation of lysolecithin and the ether analogues was accom- plished by two procedures: 1) long chain fatty acids were reinserted by the technique of Cubero-Robles and van den Berg (7); 2) short chain fatty acids (C,, C:l, Cd) were incorporated by dissolving the sample in chloroform and adding the specific anhydride in a ratio of one part substrate and five parts anhydride, with warming at 60°C for 45 min. Each reaction mixture was mixed with chloroform, meth- anol, and water to allow phase separation to occur. The chloroform- rich layer was washed with methanol/water (10:9, v/v) until it was acid-free, and then purified by thin layer chromatography using chloroform/methanol/water (65:35:6, v/v). In addition, each deriva- tive was further characterized by phosphorus assay and infrared spectral pattern.

Materials and Other Procedures-All solvents were ACS reagent grade or of the highest purity available. Acetic anhydride (99.7%) was a product of Fisher Scientific Co.; propionic anhydride (97%) and butyric anhydride (99%) were purchased from Aldrich Chemical Co. Total weights of samples were obtained by drying in a tared flask in Uacao over P20, for 6 h. Organic phosphorus was determined by the method of Bartlett (8). Gas-liquid chromatography was conducted on a Varian model 3700 gas chromatograph equipped with a 2 m x ‘/H inch outer diameter stainless steel column containing 15% DEGS on Chromosorb AW 80/100. The temperature conditions were: column, 18O”C, flame ionization detector, 230°C; inlet 220°C. Infrared spectra were obtained on a Beckman IR 4230 recording spectrophotometer, using 1 mm NaCl cells.

RESULTS

Chemical Studies on PAF

Native PAF was exposed separately to 0.10 M periodic acid in methanol (15 min to 2 h in the dark), lithium aluminum hydride (9), 6 N HCl at 100°C for 3 h, or to 0.5 N NaOH for 3 h at 100°C. Except for the periodic acid treatment, all these treatments led to a total loss of functional activity and it was not possible to recover activity by acetylation of the products. The periodic acid experiments showed that there were no free vicinal glycol groups or any amino alcohol function. The

results with lithium aluminum hydride and acid and base hydrolysis were supportive (but not proof) for a compound with characteristics similar to a phosphoglyceride.

The potential for regeneration of PAF activity from base- catalyzed methanolysates was investigated. PAF was sub- jected to base-catalyzed methanolysis, with complete loss of functional activity. The mixture was neutralized and subjected to reacylation using stearic acid anhydride and tetraethyl ammonium stearate at 60°C for 60 h (7). The reaction mixture was extracted with chloroform/methanol/water (1:2:0.8, v/v), cNoroform and water were added, and the chloroform phase was washed several times with methanol/water (10:9, v/v). In repeat experiments, there was consistently less than 25% recovery of functional PAF activity. This finding suggested that the reaction was either quite inefficient or that some component specific for PAF activity was being lost during the above extractions. Consequently, a neutralized methanolysate of PAF was phased into chloroform, which was washed re- peatedly with methanol/water (10:9, v/v) and this fraction (A) subjected to acylation with stearic acid anhydride as above. There was no recovery of functional PAF activity, and this suggested that a component(s) was being lost in the extraction procedures, most likely in the methanol/water washes. Consequently, a series of low molecular weight fatty acid anhydrides, i.e. acetic, propionic, and butyric, were re- acted with the functionally inactive chloroform-soluble frac- tion (A). This led to recovery of biological activity. Most revealing was the fact that the acetylated PAF component had an RF identical with that of native PAF, while the starting material (A) was located in a region comparable to lysolecithin on thin layer chromatograms.

Synthesis of PAF “Analogues”

Lysolecithin Deriuatiues-The above results precluded ly- solecithin as a precursor molecule, since lysolecithin would have been completely deacylated and the key product for reacylation, sn-glyceryl-3-phosphorylcholine, would be water- soluble. Nevertheless, it seemed reasonable to assess the pos- sibility that lysolecithin could be converted to a biologically active derivative by reaction with the short chain fatty acid anhydride. Accordingly, lysolecithin was subjected to treat- ment with the short chain anhydride as outlined above. Their behavior on thin layer chromatography is shown in Fig. 1. The acetylated lysolecithin migrated with the same R,v as native PAF. Each of the other derivatives had increasing RF values and also had similar infrared absorption bands to those noted for lysolecithin alone. The phosphorus content of the acetyl, propionyl, and butyryl derivatives ranged from 5.50 to 5.10%.

Reacylation of lysolecithin was accomplished with stearic acid anhydride, as described above, and yielded a product which migrated with pure (egg) phosphatidylcholine. How- ever, neither lysolecithin nor its stearoyl derivative exhibited any PAF-like activity.

Glyceryl Ether Phospholipid Derivatives-Since only the chloroform-soluble fraction of a base-catalyzed methanolysate of native PAF could be acetylated to full (biological) recovery and to identical thin layer chromatographic behavior as the starting PAF preparation, it seemed very likely that PAF was a glyceryl ether phospholipid or a compound of comparable structure. Consequently, the choline containing glyceryl ether phosphoglyceride, 1-0-alkyl-sn-glyceryl-3-phosphorylcholine, was reacted with short chain anhydrides and the correspond- ing acylated derivatives were isolated and characterized (see “Experimental Procedures”). On thin layer chromatography, only the acetylglyceryl ether phosphoglyceride migrated co-

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PAF and Acetylglyceryl Ether Phosphoglyceride 9357

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FIG. 1. Thin layer chromatography of short chain-subeti- tuted phosphoglycerides. These samples were run on a precoated Silica Gel G plate (25Op)(Analtech) in a solvent system of chloroform/ methanol/water (65356, v/v). Lanes 1 to 3 contained, respectively, the following derivatives of I-acyl-sn-glycero-3-phosphorylcholine (lysolecithin): I, acetyl; 2, propionyl; 3, butyryl. Lanes 5 to 7 con- tained, respectively, the following derivatives of 1-O-alkyl-sn-glycero- 3-phosphorylcholine (glyceryl ether phosphoglyceride): 5,2-acetyl; 6, 2-propionyl; 7,2-butyryl. Lane 4 represented phospholipid standards in ascending order from the origin: lysolecithin, sphingomyelin, and phosphatidylcholine. The spots were visualized by spraying the plate with concentrated sulfuric acid and charring.

incidentally with the acetylated lysolecithin and the native PAF (Fig. 1). As with the lysolecithin derivatives, the pro- pionyl and butyryl derivatives had increased RF values. All of these compounds had the same absorption bands as reported under “Experimental Procedures” and, in addition, the (ex- pected) carboxylic acid ester band at 1733 cm-‘. The phos- phorus content of the acetyl, propionyl, and butyryl com- pounds ranged from 5.60 to 5.40.

Comparison of Functional Activity of PAF with Synthetic Phosphoglycerides

Table I lists the relative biological activities of the various synthetic phosphoglycerides with respect to their ability to

induce platelet shape change without aggregation, platelet aggregation but without serotonin secretion, and irreversible platelet aggregation with 50% secretion of serotonin (the latter measurement being defined as 1 unit of PAF-like activity). It can be seen that both the acetyl and propionyl derivatives of the GEPC were the most highly active compounds studied (1 unit being equal to lo-*’ M). The acetylated derivative was significantly more active than the propionyl derivative of GEPC (paired Students t test, P < 0.001).

Several other experiments were performed to compare the functional activities of the native PAF with phosphoglycer- ides. Native PAF and most of the synthetic phosphoglycerides listed in Table I induced a calcium-independent platelet shape change with no aggregation or serotonin release. However, in the presence of added calcium (1.3 X 10V3 M), 1 unit of most of the compounds induced irreversible platelet aggregation and released 50% of the serotonin. These latter reactions were not affected by the presence of indomethacin (10.0 pM, final concentration), which completely inhibited the ability of ara- chidonic acid (2.0 p, final concentration) to induce platelet aggregation and secretion of serotonin. PAF- or AcGEPC- induced serotonin secretion, as high as 80%, was not accom- panied by any detectable platelet lactic dehydrogenase re- lease, thus documenting a noncytolytic mechanism for these two substances. The sensitivity of the lactic dehydrogenase assay was such that release of 1% of platelet enzyme by PAF or AcGEPC could have been detected.

Finally, native PAF and AcGEPC were utilized in cross- desensitization experiments to evaluate functional similarities. These experiments were conducted as previously described (IO) and were based upon the observations that exposure of platelets to PAF under nonsecreting conditions (i.e. in absence of extracellular calcium) desensitized the platelets to a second exposure to PAF in the presence of calcium. The desensitixa- tion was stimulus-specific since serotonin secretion induced by other platelet stimulators not related to PAF, e.g. collagen or thrombin, was not decreased. Thus, platelets were desen- sitized to native PAF, AcLL, or to AcGEPC, and secretion profiles were determined upon control and desensitized plate- lets utilizing the native PAF, the synthetic phosphoglycerides,

TABLE I

Effect of variousphosphoglycerides on functional activity of rabbit platelets

Derivative RpO [‘H]Serotonin Aggrsga- release. 5mh

Shape tion’ cbanae’

Lysolecithin Acetyl Propionyl Butyryl

M x 10-10

0.21 24Ozt 50 80 40 0.25 3OOf60 70 30 0.26

Glyceryl ether phos- phoglycerided

Acetyl 0.21 1.0 f 0.3 0.3 0.1 Propionyl 0.24 1.4 * 0.4 0.3 0.1 Butyryl 0.30 7.0 f 2.0 10 6

n Measured on Silica Gel G thin layer plates with chloroform/ methanol/water (6535~6, v/v) as solvent. Native PAF had a value of 0.21 while lysolecithin and GEPC each had one of 0.15 in this system.

h Final molar concentration (mean -f standard deviation) equiva- lent to 1 functional unit of native PAF activity. One unit activity in every case induced irreversible platelet aggregation.

V Platelet shape change with no aggregation and reversible platelet aggregation (50% increase in light transmission) were determined as described (2) utilizing a Chronolog aggregometer at 37’C with con- stant stirring (1200 rpm) of the washed rabbit platelets (500 fl, 250,006 platelets /jd).

d 1-0-Alkyl-sn-glyceryl-3-phosphorylcholine exhibited no activity towards rabbit platelets even at a level of 1 x lo-” M.

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9358 PAF and Acetylglyceryl Ether Phosphoglyceride

TABLE II Cross-desensitization ofplatelets with native PAF and AcGEPC

[‘H]Serotonin-labeled platelets were prepared as described under “Experimental Procedures” and were resuspended in Tyrode’s buffer, pH 7.2, containing 100 FM EGTA (ethylene glycol his@-aminoethyl ether)N,N,N’,N’-tetraacetic acid) and no calcium. The platelets were then divided into three portions to which was added either 10 units/ ml of PAF, 10 units/ml of AcGEPC, or albumin-saline as a control. Following incubation at 37°C for 20 min, the platelets were washed twice prior to resuspension in Tyrode’s buffer, pH 7.2, containing 1.3 x lo-,’ M calcium, The desensitized and control platelets then were tested for their respective reactivity to 1.0 unit of native PAF, Ac- GEPC, AcLL, calf skin collagen (25 pg/ml, Worthington Biochemical Co.), or thrombin (purified o-thrombin, 0.25 units/ml, kindly provided by Dr. John W. Fenton, II).

Desensitizing agent Test stimulus ___

Control PAF AcGEPC ---

PAF 50.4 le_ 4.2” 16.8 f 9.6 16.8 + 11.4 AcGEPC 53.4 -c 0.3 15.7 * 1.5 15.4 + 6.1 AcLL 46.1 f 5.4 12.1 * 6.6 11.2 -t 6.6 Collagen 55.8 k 4.3 57.5 k 1.8 60.9 f 1.1 Thrombin 64.3 f 10.4 54.7 f. 9.3 66.6 f 11.1

n Mean -c standard deviation. 3% [,‘H]serotonin release.

collagen, and purified thrombin (Table II). It can be seen that desensitization of platelets to native PAF also desensitized these cells to AcGEPC and AcLL, but not to collagen or thrombin. In a similar fashion, desensitization of the platelets to AcGEPC desensitized the platelets to AcLL and most importantly to native PAF, but did not desensitize with re- spect to collagen or thrombin.

DISCUSSION

The evidence presented here clearly shows that the inser- tion of a short chain fatty acid into glycerylether phospho- glyceride produces a compound with extremely potent biolog- ical activity towards washed rabbit platelets. The most active compound was l-0-alkyl-2-acetyl-sn-glyceryl-3-phosphoryl- choline (AcGEPC). The propionyl derivative was nearly as potent, but the butyryl and long chain fatty acid derivatives had relatively little or no activity. Comparable derivatives of the lysolecithin series, while having activity toward platelets, had significantly lower potency than the GEPC series.

Though the AcGEPC was a synthetic compound, there was strong support for its existence in nature as platelet activating factor (PAF). Several lines of evidence support this conclu- sion. First, both PAF and AcGEPC had exactly the same RF values on thin layer chromatography, and treatment with base, which yielded a biologically inactive product in each case, gave rise to a chloroform-soluble component which migrated with the same RF value. The latter upon treatment with acetic anhydride resulted in complete recovery of the initial biological activity with products identical in thin layer chromatographic behavior with each other and to native PAF. Thus, on the basis of these data, we conclude that naturally derived PAF and 1-0-alkyl-2-acetyl-sn-glyceryl-3-phospho- rylcholine are likely one and the same compound. The fact that the propionyl derivative consistently had a slightly in- creased RF value (over that of AcGEPC and native PAF) and a slightly lower biological activity than the acetyl derivative would argue against it being PAF.

The impressive fact about the AcGEPC described in this communication is its biological potency, perhaps being un- matched by any other known platelet stimulator. As shown in Table I, AcGEPC elicited 50% secretion of [“Hlserotonin from

rabbit platelets at a concentration of 1.1 X lo-“’ M, induced significant platelet aggregation at 3 X lo-” M, and promoted a platelet shape change at 1 x lo-“1 M. All the other com- pounds listed in Table I were of lower activity. The dose- response characteristics of secretion profiles for AcGEPC and PAF were identical. Moreover, the functional and cross-de- sensitization experiments also demonstrated identical biolog- ical characteristics between PAF and AcGEPC. Thus, one can conclude, at least tentatively at this time, that for optimum PAF activity, there must be an ether linkage on position 1 of a sn-glyceryl-3-phosphorylcholine backbone and that a short chain fatty acid occupies position 2.

The occurrence of a biologically active 0-acetyl- or O- propionyl-substituted phosphoglyceride in mammalian tissue has not been reported to date. This can be attributed to the very small amounts in tissues and to the fact that the short chain fatty acids would have been lost or not detected in the usual analytical procedures. However, it is not without prec- edent that short chain fatty acids are found in naturally occurring lipids. An acetyl moiety has been reported in the seed oil triglycerides of Euonymus alatus (1 l), butyric acid in bovine butterfat triglycerides (12), and a mixture of short chain fatty acids in a bacterial lipopolysaccharide (13).

Recently, Benveniste et al. (14) proposed 2-acyl-sn-glyceryl- 3-phosphorylcholine (a lysolecithin) as the structure for PAF. Although we could prepare a similar compound by his pro- cedure, it had no activity in our assay system. In addition, the physicochemical behavior of our PAF preparations eliminated the 2-acyl-sn-glyceryl-3-phosphorylcholine as the active con- stituent.

Though we feel confident that native PAF is indeed similar to, if not the same as, 1-0-alkyl-2-acetyl-an-glyceryl-3-phos- phorylcholine, the very limited quantitites of PAF available for chemical study mandate that final structure proof await mass spectrographic analysis.

Acknowledgments-We are grateful to Mary Berthier and Cynthia Morley for their very skilled assistance in these studies. The thought- ful and penetrating comments of Dr. Linda McManus are appreciated. We are indebted to Dr. Merle S. Olson for the LDH assays.

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Cubero-Robles, E., and van den Berg, D. (1969) Biochim Rio- phys. Acta 187, 520-526

Bartlett. G. (1959) J. Biol. Chem. 234. 466-468 Thompson, G. A., Jr. (1965) J. B~ol. &em. 240,1912-1918 Henson, P. M., and Pinckard, R. N. (1977) J. Zmmunol. 119,

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C A Demopoulos, R N Pinckard and D J Hanahannew class of lipid chemical mediators).

1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine as the active component (a Platelet-activating factor. Evidence for

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Additions and Corrections

Vol. 254 (1979) 8594-8604 Vol. 254 (1979) 9355-9358

Electron and proton transport in the ubiquinone cyto- Platelet-activating factor. Evidence for l-O-alkyl-2- chrome b-cz oxidoreductase of Rhodopseudomonas acetyl-sn-glyceryl-3-phosphorylcholine as the active sphaemides. Patterns of binding and inhibition by an- component (a new class of lipid chemical mediators). timycin.

Constantinos A. Demopoulos, R. Neal Pinchard, and Willem H. van den Berg, Roger C. Prince, C. Lindsay Donald J. Hanahan

Bashford, Ken-ichiro Takamiya, Walter D. Bonner, Jr., and P. Leslie Dutton Page 9358, Table 11, Footnote a:

Page 8597, Fig. 5, Part C Delete 3%. It should read:

The vertical scale is too large; to get the correct AA, divide by 8.

a Mean & standard deviation, % [''Hlserotonin release.

Page 9358, 2nd column, Line 3:

Vol. 254 (1979) 4220-4226 1 X lo-" 1 M should read 1 X 10"' M.

Cell-free synthesis of fructose diphosphate aldolases A, B, and C.

Janis E. Shackelford and Herbert G. Lebherz Vol. 254 (1979) 9237-9246

Page 4220, "Experimental Procedures," Line 6: The topological orientation of N,N'-diacetylchitobio- sylpyrophosphoryldolichol in artificial and natural

Line 6 should read 0.01% heparin (not 1%) and 0.005% sperm- ine (not 0.5%) John A. Hanover and William J. Lennarz

membranes.

The correct line is as follows: Page 9237, Right-hand column, 2nd paragraph:

7.5, which contained 0.01% heparin (Sigma) and 0.005% spermine (Sigma) The equation should read:

Page 4221, Left column, Line 7:

Line 7 should read 25 mM KC1 (not 25 mM NaCl)

The correct line is as follows:

of 25 mM Tris-HC1, 25 mM KCl, 1 mM MgCL, pH 7.5, and these

UDP-Gal + (G1cNAc)s-P-P-dolichol - Gal-(GlcNAch-P-P-dolichol + UDP. Mn2+

I I We suggest that subscribers photocopy these corrections and insert the photocopies at the appropriate places where the article to bc corrected originally appeared. Authors are urged to introduce these corrections into any reprints they distribute. Secondary (abstract) services are urged to carry notice of these corrections as prominently as they carried the original abstracts.

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