Enzymes of phospholipid synthesis in bacillus Calmette-Guerin induced rabbit alveolar macrophage

311

Biochimica

et Biophysics

@ Elsevier/North-Holland

Acta,

450 (1976) Biomedical Press

311-321

BBA 56900

ENZYMES OF PHOSPHOLIPID SYNTHESIS IN BACILLUS CALMETTE-GUERIN INDUCED RABBIT ALVEOLAR

MACROPHAGE

CHARACTERIZATION AND LOCALIZATION OF CYTIDINE DIPHOSPHOCHOLINE PHOSPHOTRANSFERASE AND MONOACYLPHOSPHOLIPID ACYLTRANSFERASE

PATSY WANG *, LAWRENCE The Department of Biochemistry, N.C. 27103 (U.S.A.)

(Received

June 17th,

R. DECHATELET The Bowman

** and MOSELEY Gray School

WAITE

of Medicine,

Winston-Salem,

1976).

Summary The rabbit alveolar macrophage is capable of renewing its plasma membrane by at least two metabolic pathways. It contains (1) a monoacylphospholipid acyltransferase, which catalyzes the synthesis of diacylphospholipids by recycling monoacylphospholipids produced by the action of phospholipases and (2) a cytidine diphosphocholine phosphotransferase (CDPcholine phosphotransferase), which catalyzes the last step in the synthesis de novo of diacylglycerophosphocholine. These activities have been characterized in the cell homogenate with respect to time, protein, pH optimum (for CDPcholine phosphotransferase), substrate specificity (for monoacylphospholipid acyltransferase) and cation requirement (for CDPcholine phosphotransferase). Monoacylphospholipid acyltransferase activity is localized solely in the endoplasmic reticulum. On the other hand, the CDPcholine phosphotransferase activity can be measured in the endoplasmic reticulum and in the plasma membrane, characterized by both differential and gradient sedimentation techniques. In addition to the normal route of phospholipid synthesis in the endoplasmic reticulum, the rabbit alveolar macrophage may thus possess the capacity for in situ synthesis of phospholipids of plasma membrane as a mechanism for membrane renewal following phagocytosis.

* Present address: Department of Bacteriology and Immunology University of Medicine Chapel Hill, N.C. 27514. ** To whom reprint requests should be addressed. Abbreviations: CDPcholinr phosphotransferase, cytidinc diphosphocholine

of North

Carolina

phosphotransferase.

School

312

Introduction As a long-lived phagocyte that plays an active role in maintaining a sterile environment in the lung, the rabbit alveolar macrophage must be capable of renewing its plasma membrane in order to continue to carry out its defensive, phagocytic function [l]. A number of enzymes that are instrumental in the restructuring of the cellular phospholipid composition have been measured in this cell. The macrophage is known to contain phospholipases A, and Az activities that are localized within lysosomes [ 2- 41, phospholipases A, and A2 in cellular membranes [ 21 and monoacylphospholipid acyltransferase activity which has not been specifically localized [ 51. First identified by Lands in 1960 [6], the acyltransferase in rat liver was found to be located in the microsomal fraction [ 71, as well as in the mitochondrial outer membrane [8]. Elsbach [9] first described this activity in homogenates of rabbit neutrophils and alveolar macrophages and reported that a stimulation of activity was observed upon phagocytosis. Monoacylglycerophosphocholine was thought to be derived from exogenous sources: this process therefore could lead to net accumulation of membranous lipid. The acyltransferasc also was reported to be activated in lymphocytes stimulated by phytohemagglutinin [lo]. Another enzyme which contributes to the synthesis of diacylphospholipids is CDPcholine phosphotransferase which catalyzes the last step in the synthesis de novo of phosphatidylcholine. The enzyme, initially described by Kennedy and Weiss [ll] in rat liver, can utilize the 1,2-diacyl-gl.ycerol which originates from triglyceride or from phosphatidic acid. This activity is located in the microsomal fraction both in rat liver [12---141 and in Acenthamoeba castellanii [151. Since a considerable amount of plasma membrane is internalized during phagocytosis, the macrophage must be capable of carrying out the synthesis of diacylphospholipids in conjunction with the restructuring of these plasma membrane components in order to continue its phagocytic function. The purpose of this study was to determine if this cell possesses the ability to synthesize diacylphospholipids by the de novo and reacylation pathways and to determine the cellular localization of these phospholipid synthesizing activities with a view towards determining their possible contribution in the renewal of plasma membrane. Materials and Methods Vaccination and cell collection An ultrasonic suspension of sterile heat-killed bacillus Calmette-Guerin in Bayol F (100 pg in 0.10 ml) was injected into the marginal ear veins of female New Zealand white rabbits (1.5~-2.0 kg) approximately one month prior to killing. The animals were routinely maintained on tetracycline, obtained from Rochelle Lab., Inc., Long Beach, Calif., which was administered in the drinking water at a concentration of 1.2 g/gal. Animals were killed by air embolism, and the alveolar macrophages were collected by the lung lavage technique of Myrvik et al. [16]. Lungs were

3 13

lavaged with 160 ml of 0.9% NaCl; the cells were collected by centrifugation at 150 X g for 10 min, and washed twice in 0.9% NaCl by repeated suspension and centrifugation. Contaminating red blood cells were lysed for 20 s with cold deionized water, and the suspension was brought to isotonicity with 3.5% NaCl. Differential and total cell counts were performed in a white cell counting chamber. Only preparations containing more than 80% macrophages were used for these studies. The lungs from a single bacillus Calmette-Guerin treated animal typically yielded from 1.0 to 5.0 ml of macrophages (packed cell volume). Normal rabbit alveolar macrophages were obtained by the same method, but the cell yield for uninduced animals ranged from 0.1 to 0.3 ml. Neutrophil migration into the peritoneal cavity of female New Zealand white rabbits was stimulated by the intraperitoneal injection of 50 ml of sterile 1.2% sodium caseinate in saline [17]. Approximately 15 h after the injection, the animals were killed, and the neutrophils were harvested from the peritoneal cavity by repeated washes with 0.9% NaCl. Human peripheral neutrophils were isolated from the venous blood of apparently healthy, volunteer subjects. Red cells were sedimented with plasmagel (HTI Corporation, Buffalo, N.Y.), and the white cells were prepared as described previously [ 181. Enzyme

assays

Acid phosphatase activity (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.2) was assayed using p-nitrophenyl phosphate as substrate, as described by DeChatelet et al. [ 191. /3-Glucuronidase activity (/3-D-glucuronide glucuronosohydrolase, EC 3.2.1.3) was measured by Canonico and Bird’s modification [ 201 of the method of Talalay et al. [ 211. Phenolphthalein glucuronic acid was used as the substrate, and reaction was terminated by the addition of 3 ml of 0.34 M glycine, pH 10.4. Cytochrome oxidase (ferrocytochrome c : oxygen oxidoreductase, EC 1.9.3.1) and NADPH cytochdrome c reductase (NADPH : (acceptor) oxidoreductase, EC 1.6.9.9.1) activities were determined by the method of Sottocasa et al. [22]. For the assay of cytochrome oxidase activity, cytochrome c (Sigma, type VI) was reduced by the addition of sodium borohydride, and the solution was neutralized with 0.5 M HCl. Alkaline phosphodiesterase I activity (oligonucleate 5’-nucelotidohydrolase, EC 3.1.4.1) was assayed by a modification of the method of Touster et al. [ 231, using thymidine-5’-monophospho-p-nitrophenyl ester as substrate. The enzyme was assayed at pH 10.6 with 50 mM cyclohexylaminopropane sulfonic acid as the buffer. The monoacylphospholipid acyltransferase activity was measured by the method described by Elsbach [ 51. [ 14C]-l-Monoacyl-sn-3-glycerophosphocholine (0.05 pmol) or [‘4C]-l-monoacyl-sn-3-glycerophosphoethanolamine (0.05 pmol) in 2% defatted albumin was incubated at 37°C with 5 pmol ATP, 5 pmol pH 7.0, and varying amounts of Mg’+, 0.1 pmol CoA, 50 pmol Tris-maleate, protein in a total volume of 0.5 ml. Exogenous fatty acid did not increase the activity in the whole cell homogenate, but 0.03 mM linoleic acid were added to assay for the activity in the gradient fractions to ensure that there was a sufficient amount of fatty acid for the acylation reaction. Subcellular fractionation probably removes the endogenous fatty acid present in the homogenate. The marcrophage enzyme exhibited no activity in the absence of ATP and CoA.

314

: 1,2-diacylglycerol choline phosCDPcholine phosphotransferase (CDPcholine photransferase, EC 2.7.8.2) activity was assayed by the radioactive procedure of Goldfine [ 241 as modified by Sarzala et al. [ 121. The final incubation contained 3.2 pmol 1,2-diolein in 0.03% Tween-20, 4 E.tmol GSH, 0.2 pmol CDP[‘4C]choline (0.08 pC!i), 10 pmol Mg’+, and 50 pmol 2-amino-2-methyl-l-propanol buffer, pH 8.5, in a total volume of 1.0 ml. Aliquots (0.1 ml) were removed from the reaction mixture after incubation for the specified period of time at 37°C and pipetted onto Whatman 3A filter paper discs, 2 cm in diameter, for the determination of product formation. Reaction was terminated by the immersion of the filter paper discs in 10% trichloroacetic acid and washing for 20 min. The discs were then transferred sequentially to 5% trichloroacetic acid, 1% trichloroacetic acid, and deionized H,O, each with wash periods of 20 min. The trichloroacetic acid precipitates the protein and the reaction product, [ 14C] diacylglycerophosphocholine, onto the filter paper. The subsequent rinses wash off all unreacted starting material. The radioactivity that was associated with the filter disc was identified as diacylglycerophosphocholine by thin-layer chromatography in preliminary studies. After washing, the filter paper discs were dried and placed in scintillation vials containing Triton-omnifluor scintillation fluid and counted in a Nuclear Chicago Corp. Isocap/300 liquid scintillation counter. Omnifluor was dissolved in toluene at a concentration of 4 g/l. The scintillation fluid was composed of omnifluor in toluene/triton/water in a ratio of 2 : 1 : 0.2. Protein assays on the various samples were performed by the method of Lowry et al. [ 251. Bovine serum albumin and lysozyme standards were run with the unknown samples, and the results obtained with the two proteins were averaged for all calculations.

Lipid analysis The radioactive products of the reactions catalyzed by monoacylphospholipid acyltransferase and CDPcholine phosphotransferase were identified by thin-layer chromatography in some experiments in order to verify the validity of the assay. Reaction was terminated by the addition of two volumes of methanol, and the products were extracted by the method of Bligh and Dyer [ 261. Radioactive lipids were separated by thin-layer chromatography on Silica Gel G plates by development in chloroform/methanol/acetic acid/water (50 : 30 : 8 : 4, by vol.) (271. The fractions were visualized by brief exposure to iodine vapor, and the silicic acid containing [ 14C]-1,2-diacyl-sn-3-glycerophosphocholine or [ 14C]-1,2-diacyl-sn-3-glycerophosphoethanolamine and the respective monoacyl compounds were scraped from the plate, placed in scintillation vials, and counted in a scintillation mixture of Triton X-100 and toluene (1 : 2) with omnifluor. Linear sucrose gradient centrifugation The whole homogenate of bacillus Calmette-Guerin induced rabbit alveolar macrophages was fractionated to determine the localization of monoacylphospholipid acyltransferase and CDPcholine phosphotransferase activities. The cells were suspended in 0.25 M sucrose (1 : 8, v/v) and homogenized (30 strokes) in a Dounce homogenizer with a tight pestle. The homogenate was

315

centrifuged at 1000 X g for 15 min to sediment nuclei, cell debris, and the remaining intact cells. 4 ml of the supernatant solution were layered over a linear sucrose gradient (0.73 to 1.61 M in 24 ml), which was cushioned with 6 ml of 1.93 M sucrose. The samples were placed in a Beckman SW 25.1 swinging bucket rotor and centrifuged at 24000 rev./min (64000 X g) for 2 h at 4°C in a Spinco Model L ultracentrifuge. At the end of the centrifugation, the bottom of the centrifuge tube was punctured, and fractions were collected from the top of the gradient by pumping 1.93 M sucrose from below. The volume layered over the gradient (4.0 ml) was pumped off; and nine fractions of 3.0 ml each were collected. The density of the gradient fractions was measured in each experiment with a refractometer, and the fractions were assayed for various enzymatic activities. Discontinuous sucrose gradient centrifugation Plasma membranes were isolated from bacillus Calmette-Guerin induced rabbit alveolar macrophages by a method described in detail elsewhere [28]. Briefly, cells were lysed for 1 h under hypotonic conditions in 1 mM NaHC03 at 4°C. Large plasma membrane fragments, nuclei, and cell debris were sedimented by centrifugation at 2000 X g for 20 min. The 2000 X g pellet was resuspended in 55% sucrose (w/v) containing 5 mM Mg’+, and 10 ml of the sample were placed at the bottom of a SW 27 centrifuge tube. This was then overlaid with 10 ml 45% sucrose, 10 ml 40% sucrose and finally with 8 ml 30% sucrose, all containing 5 mM Mg*+. The tube was centrifuged at 4°C for 2 h at 90000 X g. At the end of the centrifugation, plasma membrane fragments were collected from the 30/40% and 40/45% interfaces. Plasma membrane purification was monitored by the plasma membrane marker, alkaline phosphodiesterase I [ 23,28,29]. Substrate synthesis [‘4C]-l-Monoacyl-sn-3-glycerophosphorylcholine (spec. act. 4.0 . 10” cpm/ pmol) and [‘4C]-l-monoacyl-sn-3-glycerophosphorylethanoine (spec. act. 1.2 . lo6 cpm/pmol) were synthesized by hydrolysis of the diacyl compound, using the phospholipase A, of the venom of Crotalus adamenteus [8]. The radiolabelled precursor was obtained by the intraperitoneal injection of either [ “C]choline or [ “C]ethanolamine into rats and, after 30-60 min, removing the liver and extracting and purifying the labeled lipid [30]. Materials New Zealand white rabbits were purchased from Franklin Rabbitry, Wake Forest, N.C. The heat-killed bacillus Calmette-Guerin was the generous gift of Mrs. Eva Leake and Dr. Quentin Myrvik (Department of Microbiology, Bowman Gray School of Medicine, Winston-Salem, N.C.). The designated chemicals were obtained from the following sources: trypan blue, Allied Chemical, Morristown, N.J.; lipid standards and 1,2-dioleylglycerol, Serdary Research Laboratories, London, Ontario, Canada; Silica Gel type G, Brinkman Instruwas synthements, Inc., Westbury, N.Y. [Me-14C] Cytidine diphosphocholine sized by New England Nuclear Corp., Boston, Mass. [1,2-14C]Choline chloride (61 Ci/M) was obtained from Amersham Searle, Arlington Heights, Ill., while

316

[ “C]ethanolamine (UL) (78 Ci/M) came from ICN, Irvine, Calif. Sigma Chemical Co., St. Louis, MO., supplied all reagent grade substrates. All other chemicals were purchased from Fisher Scientific Co., Fair Lawn, N.J.

Results The homogenate from bacillus Calmette-Guerin induced rabbit alveolar macrophages was examined for CDPcholine phosphotransferase activity under a variety of conditions. When assayed over a pH range of 7.5-~10.0 using Tris * HCl and 2amino-2methyl-l-propanol as buffers, the enzyme exhibited a sharp peak of activity at pH 8.5. Since higher activity was observed with 2-amino-Zmethyl-l-propanol, this buffer was used in all subsequent studies. With 1.8 mg of homogenate protein in the incubation medium, the reaction was linear with time for up to 20 min; linearity was observed with protein concentrations up to 2.0 mg for an incubation time of 15 min (Fig. 1). Optimal cor~centrations of the substrates, 1,2-diacylglycerol and CDPcholine, were determined (Fig. 2). Tween-20 was required to disperse the diacylglycerol during sonication and for the expression of enzymatic activity, but the concentration was found to be critical. Final concentrations of Tween-20 greater than 0.03% were inhibitory to the enzyme; 0.1% of the detergent abolished 88% of the activity. Inhibition was also observed at high concentrations of CDPcholine (Fig. 2). The enzyme showed an absolute requirement for Mg” for activity with maximal activity observed with 5 mM Mg’+. In contrast, 0.2 mM Ca*’ inhibited the enzymatic activity by 90% in the presence of 10 mM Mg’+. Although the enzyme is active without the addition of reduced glutatione to the incubation medium the presence of 4 E.tmol of the compound increased the activity by 55% and so this concentration was included routinely in the incubation mixture. Homogenates of normal alveolar macrophages, rabbit peritoneal neutrophils and human peripheral neutrophils were examined for CDPcholine phosphotransferase activity

t

1

0

I

15

I

I

30 45 Time (mln)

Fig. 1. CDPcholine phosphotransferasr activity as a function of (A) protein concrntration tion period of 15 min and (B) timt* of incubation using 2.0 mg homogenate protein.

I

60

for an incuba-

317

flfl

I

I

IO 20 30 1.2.d~ole~n (iimOlS)

L--l.._-..l.-05

CDP-

cholbne

IO

i,umOiS

1

Fig. 2. Effect of substrate concentration on CDPcholine phosphotransferase activity in the homogenate of bacillus Calmctte-Guerin induced rabbit alveolar macrophages: (A) 1,2-dioleoylglycerol (1.2-diacylglycerol) *emulsified with 0.03% Tween-20; (B) CDPcholine. Activity is expressed as nmol [14Cldiacylglycerophosphucholine formed/min per mg protein. Incubation time was 15 min. Reaction was initiated with 1.8 mg homogenate protein.

under the assay conditions defined for the bacillus Calmette-Guerin induced macrophage (Table I). The specific activity of the rabbit peritoneai neutrophil and the normal macrophage homogenate was 2-fold less than that of the macrophage homogenate obtained from the bacillus Calmette-Guerin induced animals, whereas no measurable activity was found in the human neutrophil. The ability of the bacillus C~mette-Gue~n induced rabbit alveolar macrophage to synthesize diacylphospholipids by Land’s reacylation pathway [6] was examined using the assay procedure described by Elsbach [ 51. The activity with [ 14C]-l-monoacyl-sn-3-glycerophosphocholine as substrate increased with substrate concentration up to 60 PM. The macrophage homogenate was capable of reacylating both l-monoacyl-so-3-glycerophosphocholine and l-monoacylso-3-glycerophosphoethanolamine in the presence of exogenous ATP and CoA, but monoacylglycerophosphocholine served as a better substrate, with greater conversion of the monoacyl derivative to diacylglycerophosphocholine as a function of protein. Activity was linear up to 0.9 mg homogenate protein. TABLE

I

SPECIFIC ACTIVITY OF CDPCHOLINE TION FROM VARIOUS CELL TYPES

PHOSPHOTRANSFERASE

IN THE HOMOGENATE

The specific activity is expressed as nmol [ 14CI-diacylglycerophosphocholine The values represent the mean of the specific activities t S.E. The number parentheses. Cell

type

Specific -~

activity

Bacillus CalmetteGuerin induced rabbit alveolar macrophage 0.90

* 0.11 ~..

(8) - .-...

.~

FRAC-

formed/min per mg protein. of experiments is indicated in

Normal rabbit alveolar macrophage macrophage

Rabbit peritoneal neutrophil

Human peripheral neutrophil

0.40

0.42

0.00

t 0.05

(4)

C 0.08

(6)

(3)

[

qJy,/g[;[~_ 15 25

35

45

55

Sucrose (percent, w/v

i._;_ii__i-_i

65

1

-45-55

15 25

35

65

sucrose (fx!rcent, w/v 1

IO5 109 113 I17 I20 1.24 Density igm/cm3) Fig. 3. Monoacylphosphoglyceride acyltransferase activity as a function of time. (A) [ *4Clmonoacylglvccrophosphocholinr as substrate; (B) [ “Clmo~~oacylglycerophoshpethanolamine as substrate. Reaction was initiated by the addition of 2.0 mg homogenate protein. GPC, glycerophosphoeholine; GPE, glycrrophosphoethanolamine. Fig. 4. Distribution of various rwymatic activities after linear sucrose gradient centrifugation of a 1000 X .q supcrnate from the homogenate of bacillus Calmette-Guerin induced rabbit alveolar macrophages: (A) NADPH cytochrome c reductase; (B) cytochrome oxidase; (C) CDPcholine phosphotransferase; (D) alkaline phosphodiesterase I; (E) monoacylphospholipid acyltransferasc; (F) fl-glucuronidase; (G) acid phosphatase; (H) protein.

Fig. 3 shows a concomitant decrease in the monoacyl compound with an increase in the diacyl analog as a function of time for both substrates. Again, it can be observed that monoacylglycerophosphocholine serves a a better substrate. CDP~holine phosphotransfer~e and monoa~ylphos~holipid a~yltr~sfer~e activities were assayed in the gradient fractions obtained from the isopycnic sucrose gradient centrifugation of a 1000 X g supernatant fraction of the homogenate. The acyltransferase activity (Fig. 4E) was localized in the same fractions as NADPH cytochrome c reductase (Fig. 4A), a microsomal marker. CDPcholine phosphotransferase exhibited a bimodal distribution in the gra-

319 TABLE

II

LOCALIZATION Activities

OF CDPCHOLINE

are expressed

as nmoI/min

PHOSPHOTHANSFERASE per mg protein.

ACTIVITY

Each value represents

the mean of three determina-

tions. Th? 100000 X g pellet (approx. microsomal fraction) was obtained as foiiows: the supernatant fraction was centrifuged in a tvpc 30 rotor at 19600 X g (average) for 20 min. The 19600 X g supernate then was further centrifuged at 100000 X g for 60 min. Activity Lysate

of fractions 100 000 x K

30140% and 401454 interface (plasma membrane)

pellet CDPcholine phosphotransferase NADPH cytochrome c reductase (microsomal marker) Alkaline phosphodiesterase I (plasma membrane marker)

0.6

2.7

1.2

3.6

8.5

0

373

480 -.

3208 --..

.-

dient (Fig. 4C). One peak of activity coincided with that for NADPH cytochrome c reductase, while the second peak was the same as the major peak of alkaline phosphodiesterase I activity (Fig. 4D), a reported plasma membrane marker [28] for rabbit alveolar macrophage. Both appeared before the cytochrome oxidase peak, a marker for mitochondria [31-351. &Glucuronidase (Fig. 4F) and acid phosphatase (Fig. 4G), which are associated with lysosomes, were also assayed in the gradient fractions. Their major peak of activity was observed at a higher density (1.20 g/cm”) than the other marker enzymes. The dual localization of CDPcholine phosphotransferase activity was further subst~tiated by the me~urement of its activity in the microsomal fraction obtained by differential centrifugation and in the plasma membrane fraction obtained by discontinuous sucrose gradient centrifugation. When the specific activity of CDPcholine phosphotransferase was determined in the different subcellular fractions, the specific activity was four-fold greater in the microsomes when compared with the lysate (Table II). Also, there was a two-fold increase in the phosphotr~sferase activity in the plasma membrane fraction (Table II). Assay of NADPH-cytochrome c reductase activity (microsomal marker) demonstrated a more than two-fold increase in the specific activity of the enzyme in the 100000 X g pellet fraction with no measurable activity in the plasma membrane fraction. Thus the CDPcholine phosphotransferase activity measured in the plasma membrane fraction cannot be accounted for by microsomal contamination, and the enzyme is localized both in the plasma membrane and in the endoplasmic reticulum. Discussion Alveolar macrophages play an important role in the elimination of foreign particles that enter the lung. Since the lifespan of the macrophage is measured in months rather than hours, it must possess the capacity for renewing its plasma membrane. This study demonstrates that the macrophage possesses a number of enzymes that can contribute toward the synthesis of additional plasma membrane by the modification of endogenous phospholipids, by their synthesis

320

via the de novo pathway, or by the uptake and acylation of exogenous lipid including monoacylphosphoglycerides. The macrophage contains monoacylphospholipid acyltransferase activity that is located exclusively in the microsomal fraction. This enzyme can contribute toward the synthesis of plasma membrane phospholipids by the acylation of monoacylphospholipids in the microsomes followed by the transport of the diacyl product to the plasma membrane. Such a mechanism has been reported in rat liver, plants, and guinea pig brain 171. The substrate for the acylation reaction can be produced via phospholipase action on endogenous diacylphospholipids. Acid-active and alkaline-active phospholipases A, and A1 have been identified in rabbit alveolar macrophages [2-- 41. For the synthesis of new membrane, the substrate can be derived from medium monoacylphosphoglycerides [ 361, with cellular triglycerides serving as a source of fatty acid [ 37 J . Phagocytized microorganisms may also serve as a source of substrate [ 381. CDPcholine phosphotransferase, also present in the rabbit alveolar macrophage, exhibits a dual localization in the endoplasmic reticulum and the plasma membrane. The fatty acid composition of diacyl phospholipids is determined in part by the route of synthesis [ 7,391. Diacylglycerophosphocholine such as and l-palmitoyl-2-linoleoyl-glycl-palmitoyl-2-oleoyl-glycerophosphocholine erophosphocholine are formed mainly via the CDPcholine pathway, at least in liver [ 71. These phospholipids make up a large percentage of total macrophage phospholipids [40], Further support for the role of CDPcholine phosphotransferase in the synthesis of plasma membrane diacylglycerophosphocholine is provided by the higher specific activity observed in the homogenate from bacillus Calmette-Guerin induced rabbit alveolar macrophage than from the normal macrophage. The bacillus Calmette-Guerin induced cell has altered morphology and is metabolically more active [41,42], and increased phospholipid synthesis may be needed to support the increased membrane activity. At this point it is not known whether the increased activity is due to the plasma membrane or the microsomal enzyme, or to combined activation. It can be inferred from the phospholipid composition of the macrophage that the de novo pathway plays an important role in the synthesis of plasma membrane. Furthermore, the presence of CDPcholine phosphotransferase in the‘plasma membrane may well contribute to the synthesis in situ of additional plasma membrane. Acknowledgments This research was supported by a grant from the Forsyth Cancer Service and by Public Health Service Grants AI-10732 from the National Institute of Allergy and Infectious Disease, CA-12197 from the National Cancer Institute, and AM-11799 from the National Institute of Arthritis and Metabolic Disease. Dr. Waite is the recipient of Research Career Development Award AM-17392. References 1

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2

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2. and

296,365-373

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C.E. (1972)

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