(6), we think that the methylation of DNA and RNA probably. are not involved in the chemotactic response. Because of the

Transcription

1 Pro. Natl. Aad. Si. USA Vol. 76, No. 6, pp , June 1979 Immunology Phospholipid methylation in marophages is inhibited by hemotati fators (SadenosylLmeth ionine/arboxyaomethylation/phagoytosis /phosphatidylholine/phosphat idylethanolam ine) MARILYN. PIK*t, NIHOLAS M. KRDIHM11, AND RALPH SNYDRMAN*t tfi *The Laboratory of Immune ffetor Funtion of the Howard Hughes Medial Institute, in the tdivision of Rheumati and Geneti Diseases and the Departments of Mediine, tmirobiology and Immunology, and Biohemistry, Duke University Medial enter, Durham, North arolina 2771 ommuniated by James B. Wyngaarden, Marh 19, 1979 ABSTRAT hemotaxis by human monoytes has been shown to require methylation mediated by SadenosylLmethionine(AdoMet), but the speifi transmethylation reation neessary for this funtion was not eluidated. In an attempt to define the methylation requirement for hemotaxis, we examined the effet of hemotati agonists and antagonists on protein arboxyomethylation of protein and methylation of phospholipid in guinea pig marophages. hemotati agents tested over a wide dose and time range produed no alteration in arboxyomethylation. However, these agents did produe an effet on the methylation of phosphatidylethanolamine by marophages. AdoMetmediated phospholipid methylation was inhibited by as muh as 73% by hemotati fators, and there was exellent orrelation (r =.99) between their onentrations for produing halfmaximal hemotati responses and for inhibiting phospholipid methylation. The inhibition of methylation by hemotati fators was observed at all inubation times and ould not be explained by an inreased turnover of membrane phospholipid. Neither the hemotaxis antagonist fphemet nor the nonhemotati tripeptide MetMetMet signifiantly depressed phospholipid methylation. Immune phagoytosis by marophages similarly did not alter p ho ipid methylation. The hemotati fators produed no alteration in total marophage phospholipid synthesis or in the phospholipid methylation in a nonhemotati ell type. The formation of newly methylated derivatives of phosphatidylethanolamine in marophages was dereased by abiologially ative dose of hemotati fator. These findings indiate that hemotati fators are apable of altering the methylation of phosphatidylethanolamine in hemotatially responsive ells. The inhibition of phospholipid methylation by hemotati fators may be neessary for the translation of a hemotati signal on the surfae of the ell into diretional ell movement. Transmethylation reations mediated by SadenosylLmethionine (AdoMet) are required for many funtions of both eukaryoti and prokaryoti ells (1, 2). In bateria, hemotaxis requires the methylation of ertain membraneassoiated proteins by protein arboxymethyltransferase (35). Reently, we demonstrated that transmethylation was neessary for the direted migration of at least one type of eukaryoti ell, the human monoyte (6). Inubation of monoytes with a speifi inhibitor of adenosine deaminase, erythro9[3(2hydroxynonyl)]adenine(hna), plus adenosine and Lhomoysteine thiolatone produed marked inreases in intraellular onentrations of SadenosylLhomoysteine (AdoHy), a ompetitive inhibitor of AdoMetmediated methylation reations (7). onomitant with suh inreases in mdnoyte AdoHy was a profound depression of monoyte hemotati responsiveness and inhibition of protein arboxyomethylation (6). Although these studies learly demonstrated the requirement of AdoMetmediated transmethylation for monoyte hemotaxis, they did not define the speifi methylation reations(s) involved in ell movement. Beause ell division and protein The publiation osts of this artile were defrayed in part by page harge payment. This artile must therefore be hereby marked "advertisement" in aordane with 18 U. S solely to indiate this fat. synthesis appear not to be required for monoyte hemotaxis (6), we think that the methylation of DNA and RNA probably are not involved in the hemotati response. Beause of the known or suspeted importane of protein arboxyomethylation (8, 9) and phospholipid methylation (1, 11) in ertain speialized membratie funtions, we measured the effets of hemotati fators on these two types of methyltransferase reations in guinea pig marophages. MATRIALS AND MTHODS hemials. NFormylLmethionylLmethionylLmethionine (fmetmetmet), fphemet, MetMetMet, and fmetleu were purhased from Andrulis Researh (Bethesda, MD); fmetleuphe and fnorleuleuphe were obtained from Peninsula Laboratories (San arlos, A). Adenosine and Hepes were produts of albiohem; Lhomoysteine thiolatone, phosphatidylholine (Ptdho), and phosphatidyln,ndimethylethanolamine (Ptdtn) were obtained from Sigma, and HNA was from Burroughs Wellome (Researh Triangle Park, N). PhosphatidylNmonomethylethanolamine was purhased from GIBO. L[methyl3HJMethionine (8 i/ mmol; 1 i = bequerels) and 32P1 as orthophosphori aid were purhased from New ngland Nulear. The hemotati stimulants ativated guinea pig serum (AS) and the hemotati fragment derived from the fifth omponent of omplement (5a) were prepared as desribed (12). ell Preparations. Mononulear leukoytes were obtained from the peritoneal avities of male Hartley guinea pigs (56 g) 3 days after an intraperitoneal injetion of 25 ml of.5% shellfish glyogen (Sigma). Mononulear leukoytes were removed by lavage of the peritoneal avities with Gey's balaned salt solution ontaining 2% (wt/vol) bovalbumin (Flow Laboratories, Rokville, MD),.1 M Hepes at ph 7., and 1 units of heparin per ml. Guinea pig lymphoytes were obtained by mining reseted spleens and passing the resultant ell suspension through gauze to remove large partiulate matter. After entrifugation at 4 X g for 1 min, the ells were resuspended to 2 X 16 ells per ml in RPMI 164 (GIBO) ontaining 1% (wt/vol) heatinativated (3 min X 37) fetal alf serum (GIBO). Twelve milliliters of this suspension was plaed into eah of a series of 1nimdiameter Falon tissue ulture plates. The plates were then inubated for 1 hour at 37 in humidified air ontaining 5% O2 to allow the marophages to adhere to the plasti. The nonadherent ells were then pooled and standardized to ontain 46 X 16 ells per ml in Gey's solution. Abbreviations: AdoMet, SadenosylLmethionine; HNA, erythro 9[3(2hydroxynonyl)ladenine; AdoHy, SadenosylLhomoysteine; AS, ativated guinea pig serum; 5a, hemotati fragment derived from the fifth omponent of omplement; Ptdho, phosphatidylholine; Ptdtn, phosphatidylethanolamine; TL, thinlayer hromatography. 11 To whom reprint requests should be addressed. 2922

2 Immunology: Pike et al. hemotaxis Assay. hemotaxis was measured after a 2hr inubation in modified Boyden hambers using.5.iinmjpol arbonate filters (13). hemotati ativity of the various stimulants was determined as the mean number of migrating ells per oilimmersion field in tripliate samples. Assay of ellular Phospholipid Methylation. The methylation of both membrane and total intraellular Ptdho preursors was assayed in intat marophages and lymphoytes by measuring the inorporation of the methyl group from L Imethyl3Hlmethionine into phospholipids. Aliquots (1 ml) of ell suspensions ontaining 46 X 16 ells per ml in Gey's solution were preinubated at 37 for 15 min, after whih 1 gi of L[nethyl3H]methionine (.125 AM) was added. Where indiated, hemotati agonists, antagonists, or medium alone was added simultaneously with the labeled methionine. After inubation at 37 for various times, the ells were pelleted, and washed one in isotoni saline, and the ell pellet was resuspended in.1 ml of saline. Then,.5ml of hloroform/ methanol, 2:1 (vol/vol), was added to eah tube. The tubes were vortexed vigorously for 3 se and then entrifuged for 5 min at 2 X g. The aqueous phase was aspirated and.1 ml of the organi phase was applied to a ellulose thinlayer hromatography (TL) plate ontaining a fluoresent indiator (astman hromogram) along with 1,g of Ptdho. Separation of labeled and authenti phospholipids from free L tmethyl3hlmethionine was ahieved by developing the plates in hloroform/methanol/water, 75:18:2 (vol/vol); authenti phospholipids were visualized under UV light. The spots ontaining labeled phospholipids were sraped into glass sintillation vials and the total amount of radioativity in dupliate samples was determined by liquid sintillation spetrophotometry after the addition of 12 ml of Aquasol (New ngland Nulear). In some instanes, the speifi ativity of Sadenosyl[methyl3H]methionine in aidsoluble ellular extrats was determined in parallel experiments using highperformane liquid hromatography as desribed (6). haraterization of Reation Produts. A hloroform/ methanol extrat derived from approximately 3 X 16 ells was spotted on ellulose TL plates and developed as desribed above. The [3HImethylated phospholipids ontained in the TL srapings were eluted with 1 ml of hloroform/methanol (2:1) whih was then evaporated under a stream of nitrogen gas. After the addition of 1 Ml of hloroform/methanol (2:1), samples were applied to a silia gel G plate (Uniplate, Analteh, Newark, D) and developed in eah of two solvent systems: (i) hloroform/propioni aid/1propanol/water, 2:2:3:1 (vol/vol); and (ii) hloroform/methanol/water, 65:25:4 (vol/vol) (1). Authenti Ptdho and Ptdtn and its mono and dimethylated intermediates were hromatographed and visualized by spraying with.6% rhodamine 6G (astman). Assay of ellular Phospholipid Synthesis. Total phopholipid synthesis in marophages was assayed by measuring the inorporation of 32P; into organi solventextratable material. Four to 6 X 16 ells ontained in 1 ml of phosphatefree balaned salt solution (.135 M NaI/4.5 mm KI/.1% dextrose/1.5 mm Mgl2/.15 mm a12, ph 7.) were inubated for 15 min at P1 (5 Mi) was then added, and the ell suspension was inubated for an additional 1 hr at 37. Phospholipids were extrated as outlined above for studies of phospholipid methylation. The aqueous phase was aspirated and the organi phase was washed one with.2 ml of phosphatebuffered saline. Onetenth milliliter of the organi phase was transferred to a glass sintillation vial and the radioativity was assayed by liquid sintillation spetrophotometry after the addition of 12 ml of Aquasol. ellular Protein arboxymethylase Assay. arboxyomethylation of proteins was assayed as desribed (6, 9). Pro. Natl. Aad. Si. USA 76 (1979) 2923 Preparation of Opsonized rythroytes for Phagoytosis. Opwnizvd sheep erythroytes for use in phagoytosis were prepaired as desribed (14). RSULTS A role for AdoMetmediated transmethylation in human monoyte hemotaxis has already been established (6). A methylation requirement also exists for guinea pig marophage hemotati responsiveness, beause inubation of these ells with HNA, adenosine, and Lhomoysteine thiolatone also inhibits their hemotaxis (data not shown). In an attempt to define the speifi methyltransferase reation(s) neessary for marophage hemotaxis, we investigated the effets of various hemotati agonists and antagonists on ellular phospholipid and protein methylation. ffets of hemotati Fators on Marophage Phospholipid Methylation. Guinea pig marophage phospholipid methylation was measured during a 6min period in the presenop of a series of hemotati formylated tripeptides and 5a (12). fmetmetmet, fnorleuleuphe, fmetleuphe, and 5a inhibited marophage phospholipid methylation in a dosedependent fashion, with maximal inhibition ranging from 5 to 73% with the various hemotati agonists (Fig. 1A). The onentrations neessary to inhibit phospholipid methylation were in the same range (.11 nm) that gave a hemotati response (Fig. 1B). omparison of the effetive onentrations of these different substanes required for 5% of a maximal response shows exellent agreement (orrelation oeffiient,.999) between their effets on hemotaxis and phospholipid methylation (Fig. 2). ff4et peptides at 1 nm and less did not signifiantly alter the speifi ativity of intraellular AdoMet when measured at 5, 3 4nd 6 min during the phospholipid methylation assays. However, at peptide onentrations of 1,uM or greater, AdoMet speifi ativities were appreiably lowered, presumably by hydrolyti release of nonradiolabeled methionine from these ompounds. The data given here have been orreted for suh effets. The nonhemotati peptide MetMetMet (15) produed no inhibition of phospholipid methylation at a onentration of 1 nm. The hemotati agonist fphemet (16) inhibited phospholipid methylation by only 13% at.1 mm. ffet of Phagoytosis on Phospholipid Methylation. The effet of a phagoyti stimulus on phospholipid methylation was also investigated in order to determine whether the inhibition of phospholipid methylation was a general onsequene of stimulated metaboli funtions within the ell or more speifially related to hemotaxis. Marophages were inubated for 15 min at 37 with sheep erythroytes or with sheep erythroytes treated with antisheeperythroyte hemolysin; then L[methyl3Hlmethionine was added and, 1 hr later, the ells were proessed for phospholipid methylation. A ratio of opsonized erythroytes to marophages as high as 5:1 did not signifiantly alter the amount of marophage phospholipid rnethylation (Table 1). Unopsonized erythroytes, whih are not ingested, also did not alter the methylating ability of the ells. The phospholipid methyltransferase ativity of the opsonized and unopsonized erythroytes themselves did not signifiantly ontribute to these reations beause, at the onentrations of erythroytes used, their phospholipid methylation was undetetable. Kinetis of Inhibition of Phospholipid Methylation by hemotati Fator. Marophages were inubated at 37 with LImethyl3Hlmethionine in the presene and absene of 1 nm fmetmetmet and harvested at various times for the measurement of phospholipid methylation. Inhibition of the rate of aumulation of radiolabel in methylated phospholipid

3 2924 Immunology: Pike etal. e._ T w 41U in. ~. x At an.a rd L. an m.. 12 A 1 % '6 4 2 I I FIG. 1. (A) ffets of hemotati fator on inorporation of [3H]methyl groups into phospholipids in guinea pig marophages. % of normal = (/) X 1 in whih is inorporation in the presene of hemotati agent and is inorporation in the presene of buffer alone. alulations were orreted for the alteration of speifi ativity of the [3H]AdoMet formed in the presene of fmetleu., 5a; A, fmetmetmet; *, fnorleuleuphe; *, fmetleuphe;, fmetleu. (B) hemotati ativity of hemotati agents for guinea pig marophages. % of maximal response = (SIM) X 1 in whih M is the maximal response to a hemotati agent and S is response to the same fator at a submaximal onentration. Maximal responses for the individual hemoattratants (expressed as ells per oilimmersion field) were: 5a, 114.2; fmetmetmet, 29.7; fmetleuphe, 12.5; fnorleuleuphe, 28.7; fmetleu, Symbols as in A. by hemotati fator was evident within 5 min of inubation and persisted for as long as 6 min (Fig. 3). At no time did phospholipid methylation appear to be stimulated by fmet MetMet. Table 1. Pro. Natl. Aad. Si. USA 76 (1979) ffet of immune phagoytosis on marophage phospholipid methylation [3H]Methyl inorporation, Marophages inubated into phospholipids, % of with* pmol/19 ells normalt Buffer alone fmetmetmet(1 nm) ShA (5:1) ShA (1:1) Sh (5:1) Sh (1:1) * Marophages (4 X 16) were inubated under the indiated onditions at 37 for 6 min with 1,gi of [methyl3h]methionine, and then total phospholipid methylation was quantified. ShA, sheep erythroytes treated with antisheeperythroyte hemolysin; Sh, sheep erythroytes. t % of normal = (/) X 1 in whih is the [3H]methyl inorporation determined in the presene of the indiated substanes and is the [3H]methyl inorporation in marophages inubated with buffer alone. We also asked whether hemotati fator aelerates the turnover of preformed methylated derivatives of Ptdtn in marophages. Marophages were labeled with L[methyl 3H]methionine' for 1 hr at 37, olleted by entrifugation, and resuspended in Gey's solution ontaining.1 mm unlabeled methionine. These ells were then inubated at 37 in the presene or absene of 1nM fmetmetmet and aliquots were removed at times ranging between 1 min and 5 hr for measurements of residual phospholipid radioativity. There was no appreiable loss of newly formed methylated phospholipids in the presene or absene of hemotati fator during the ourse of the 5hour inubation. Thus, fmetmetmet did not aelerate the degradation of newly formed methylated phospholipid derivatives assoiated with the marophages. Speifiity of Inhibition of Phospholipid Methylation by hemotati Fators. Beause the derease in phospholipid methylation produed by hemotati fators ould be the result of inhibiting total phospholipid synthesis, the inorporation of 2P1 into lipidextratable radioativity after inubation for 1 hr at 17' was measured in the presene and absene of various onentrations of the hemotati fators fmetmet Met, fmetleuphe, and 5a. The results of these experiments (Fig. 4) indiate that the hemotati fators produed no inhibition of phospholipid synthesis. Indeed, slight but signifiant 9. ^ & " () 9 w log (5 for inhibition of methylation) (M) FIG. 2. orrelation of hemotati poteny of the hemotati agonists and their ability to inhibit phospholipid methylation. The onentrations of eah hemoattratant ausing half maximal responses (ro) were omputed from data in Fig. 1. Linear regression analysis was performed to obtain a best fit line: slope, 3.6; orrelation oeffiient,.999; standard error of estimate, ± Time, man FIG. 3. ffet of fmetmetmet on the kinetis of inorporation of [3H]methyl into guinea pig marophage phosopholipids. Marophages were inubated with [methyl3hlmethionine in the presene or absene of 1 nm fmetmetmet, and total phospholipid methylation was determined after inubation for various times at 37.@*, Buffer alone;, fmetmetmet.

4 , i > Immunology: Pike et al log [attratant] (M) FIG. 4. ffets of hem~a~ti fators on marophage total phospholipid synthesis. Marophages were inubated with 32Pi in the presene or absene of hemotati fators for 6 min at 37; then, total phospholipid synthesis was measured. ; 5a; A, fmet MetMet; *, fmetleuphe. (P <.25) enhanement of synthesis (152%) was onsistently observed with 5a. ffet of hemotati Fators on Marophage Protein arboxyomethylation. To determine whether the inhibition of phospholipid methylation produed by hemotati fators were speifi or simply refleted a general inhibition of all AdoMetmediated methylation reations, the effets of various onentrations of fmetmetmet, fmetleuphe, and 5a on protein arboxyomethylation in marophages were determined. There was no signifiant effet on marophage protein arboxyomethyltransferase ativity produed by inubation for 1 hr at 37 with the three different hemotati fators (Fig. 5A). Beause other laboratories (17) have reported a rapid and transient (1 min) stimulation of protein arboxymethylase ativity in rabbit neutrophils inubated with a fmet hemotati peptide, we investigated the effets of 1 nm fmetmetmet on the kinetis of marophage arboxyomethylation (Fig. 5B). No alteration in protein arboxymethylase ativity was observed in the presene of the hemoattratant even as early as 1 min after introdution of labeled methionine. ellular Speifiity of Inhibition of Phospholipid Methylation by hemotati Fators. The effets of three different hemotati fators (fmetmetmet, fmetleuphe, and 5a) on the phosphatide methyltransferase ativity of guinea pig spleni lymphoytes were examined. These ells did not respond hemotatially to the above ompounds (data not shown). No inhibitory effets on lymphoyte phospholipid methylation were observed with any of the hemotati fators tested (Fig. 6). haraterization of Methylated Phospholipids. Radiolabeled phospholipids extrated from marophages inubated with L[methy1:3HImethionine were frationated into mono, di, and trimethyl(holine) derivatives by TL using two separate solvent systems (1). Fig. 7A illustrates the profile of._ Pro. Natl. Aad. Si. USA 76 (1979) 2925 X o 14 O, 12 U....b s >. 1 ) m D F FIG. 6. ffets of hemotati fators on [3Hlmethyl inorporation into phospholipids of guinea pig spleni lymphoytes. Lymphoytes were inubated with or without hemotati fators in the presene of [methyl3h]methionine for 6 min at 37. Then, total phospholipid methylation was measured; % of normal was alulated as in Fig. 1A., 5a; A, fmetmetmet; *, fmetleuphe. [3H]methylated phospholipids found in marophages inubated in the absene of hemotati fator. Most of the radioativity migrated with the three methylated derivatives of Ptdtn, partiularly with Ptdho and phosphatidylnndimethylethanolamine. Another peak of radioativity, the nature of whih has yet to be determined, was onsistently found to migrate with the solvent front. The profile of [3H]methylated phospholipids from marophages that had been treated with 1 nm fmetmetmet showed a uniform depression of all three methylated derivatives ompared to ells inubated in the absene of hemotati fator (Fig. 7B). DISUSSION The aumulation of phagoyti ells at sites of antigeni penetration or neoplasti transformation is a ritial event in immunologially mediated host defense. There is substantial evidene indiating that leukoytes are apable of sensing ertain hemial gradients by means of speifi ellular reeptors (16, 18) and of migrating diretionally along suh onentration gradients. Although the biohemial mehanism by whih oupany of a hemotati reeptor leads to a hemotati response in eukaryoti ells is as yet poorly understood, we have reently shown a requirement for AdoMetmediated methylation in this phenomenon (6). In bateria, hemotaxis requires the binding of a hemoattratant to speifi surfae reeptors followed by AdoMet mediated arboxyomethylation of ertain membrane proteins (35, 19). In the studies reported here, we did not observe any signifiant alteration of 9 (12 >= 12 io. ' 1 x 8 U A.1_ log[attratantl (M) L u 'a 6. I'U 2) Time, min FIG. 5. ffets of hemotati fators on marophage protein arboxymethylation. (A) Various doses of hemotati fators were inubated with the marophages in the presene of [methyl3hlmethionine for 6 min at 37. Then, protein arboxy methylation was determined; % of normal was alulated as in the legend to Fig. 1A., 5a; A, fmetmet Met; *, fmetleuphe. (B) Kinetis of marophage protein arboxymethylation in the presene () and absene () of fmetmetmet. Marophages were in 6 ubated for various times at 37 with [methyl3h1 6 methionine in the presene or absene of fmetmetmet (1 nm); then, arboxymethylation was measured.

5 2926 Immunology: Pike et al. 1 m IIl] A FIG. 7. hromatographi pattern of the [3H]methylated 8 phospholipid reation produts in the presene of medium 6 alone (A) and of 1 nm fmet MetMet. (B) Reation prod 4 uts were isolated, applied to a 2 W silia gel G plate, and hroma tographed with a solvent. sys Itemof h hloroform/propioni B aid/npropyl alohol/water, 8 2:2:3:1 (vol/vol). Similar results were obtained with hloro 6 form/methanol/water, 65:25:4 (vol/vol). Region identifiation: 1, lysophosphatidylholine; 2, 2 Ptdho; 3, phosphatidyl N,N. dimethylethanolamine; 4, phosphatidylnmono methylethanolamine; 5, Ptdtn. RF arboxyomethylation over a wide dose range of hemoattratants, at any inubation time, even as early as 1. min after exposure of guinea pig marophages to these agents. These findings ontrast with the results of O'Dea et al. (17) who reported a rapid stimulation of arboxyomethyltransferase ativity in rabbit peritoneal polymorphonulear leukoytes by fmetleuphe. Whether the differenes in our data reflet differenes in speies or ell type has not yet been determined. In ontrast to their lak of effet on protein arboxyomethylation, hemotati fators inhibited phospholipid methylation in intat guinea pig marophages by as muh as 573% when measured after 56 min of inubation. A physiologi signifiane for this phenomenon is suggested by the fat that, for eah ompound tested, hemotati ativity and inhibition of phospholipid methylation ourred over the same onentration range. Furthermore, neither the hemotati antagonist fphemet, whih binds to the formylated peptide hemotati reeptor but produes no hemotati response (16, 18), nor the nonhemotati peptide MetMetMet (15) produed any inhibition of phospholipid methylation. The lak of effet of fphemet indiates that hemotati fator reeptor oupany per se is not suffiient to trigger inhibition of methylation. This point is further emphasized by the finding that immune phagoytosis by marophages did not inhibit phospholipid methylation. Phagoytosis and hemotaxis bear ertain similarities in that both proesses are initiated by oupany of membrane reeptors followed by ellular movement (2). However, in hemotaxis there is polarized ell movement whereas in phagoytosis there is membrane and ytoplasmi movement around the partile to be ingested but no aompanying ellular polarization. Our data thus suggest that inhibition of phospholipid methylation by hemotati fators may be important for polarized ell movement. The failure of hemotati agonists to inhibit phospholipid methylation in erythroytes and lymphoytes also argues in favor of a relationship between alterations of phospholipid transmethylation reations and hemotaxis. In the presene of hemotati fators, marophages failed to aumulate all three methylated derivatives of Ptdtn. Whether hemotati fators affet these transmethylation reations diretly or indiretly remains to be determined. The present study learly shows that hemotati fators inhibit a speifi AdoMetmediated methylation reation in hemotatially responsive ells. We have previously demonstrated, however, that AdoMetmediated reations are indeed neessary for hemotaxis to our, in that inhibition of methylation resulted in inhibition of hemotaxis (6). One explanation Pro. Natl. Aad. Si. USA 76 (1979) of this paradox is that methylation might be requisite for some aspet of hemotaxis, and inhibition of phospholipid methylation is neessary for another. Another possibility is that a gradient of membrane phospholipid methylation or loal inhibition of this proess is neessary for a direted migratory response. During nondireted migration or at rest, phospholipid methylation might be a generalized membrane property. By binding to responsive ells and by altering the methylation of membrane phospholipids in a nonuniform manner, hemotati fators ould effet loal hanges in the biophysial properties of the membrane. This would our as a onsequene of the inhibition of the formation of Ptdho without affeting total phospholipid synthesis. It an be expeted that, under these onditions, there would be a loal aumulation of Ptdtn, a moleule whose head group is both smaller than that of Ptdho and apable of forming more hydrogen bonds. Global inhibition of phospholipid methylation by HNA, adenosine, and L homoysteine thiolatone would depress hemotaxis by preventing a ell from establishing a gradient of the ratio of Ptdtn to Ptdho in newly synthesized membrane. Many onstruts of the possible relevane of the alteration of phospholipid omposition to hemotaxis an be made. Several that deserve mention are that these hanges ould be aompanied by loal alterations in membrane mirovisosity, with the ability of the membrane to interat with external surfaes, or with the anhoring of the membrane to internal ytoskeletal elements. Determining the exat role of the inhibition of phospholipid methylation in hemotaxis will have a major impat on our understanding of ellular motility as well as the mehanism by whih membrane phospholipid omposition is ontrolled. 1. Franesa, S., Borek,., Zappia, V., WilliamsAshman, H. G. & Shlenk, F. (1977) The Biohemistry of Adenosylmethionine (olumbia Univ. Press, New York). 2. antoni, G. L. (1975) Annu. Rev. Biohem. 44, Kort,. N., Goy, M. F., Larsen, S. H. & Adler, J. (1975) Pro. Natl. Aad. Si. USA 64, Springer, W. R. & Koshland, D.., Jr. (1977) Pro. Natl. Aad. Si. USA 74, Springer, M. S., Goy, M. F. & Adler, J. (1977) Pro. Natl. Aad. Si. USA 74, Pike, M.., Kredih, N. M. & Snyderman, R. (1978) Pro. Natl. Aad. Si. USA 75, Kredih, N. 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