Inhibition of lysosomal phospholipase A and phospholipase C by

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The lipid extract was applied to an 0.25-mm layer of Silica Gel H prepared with .... cific activity, 20 mCi/mmol; 50 m~ sodium acetate buffer, pH 4.4 or. 5.4. Varying ...
Inhibition of Lysosomal Phospholipase A and Phospholipase Chloroquine and 4,4‘-Bis(diethylaminoethoxy)c@ diethyldiphenylethan~* (Received for publication,

Yuji Matsusawa$

C by

December 7, 1979)

and Karl Y. Hostetler

From the Department of Medicine, Division af Metabolic Disease, Veterans Administration University of California, San Diego, California 9.2161

The administration of 4,4’-bis(~e~yl~noethoxy)e,@liethyldiphenylethane to mau or animals causes phospho~pid storage in liver, spleen, and other body tissues. Chloroquine has also been shown to cause phospho~pid storage in animal tissues. In a previous study of drug-induced lipidosis in rat liver, we found that the lysosomal fraction is the intracelhrlar site of the stored phospholipid and that it contains most of tbe ehloroquine or 4,4’-bis(diethylaminoethoxyh@diethyldiphenylethane (Matsuzawa, Y., aud Hostetler, K. Y. (1980) 4 Lipid Res. 21,202-214). In this paper, we have examined the effect of these cationic ampbiphilic agents on a soluble delipidated preparation from rat liver lysosomes which contains phospholipase A and C activity. Our data show that &loroquine and 4,4’-bis(~ethyl~inoetho~)a,~-~ethyldiphenylethane are potent inhibitors of lysosomal phospbo~pase A and C activities. However, these agents do not cause substantial iuhibition of the lysosomal phospholipase A wbieh catalyzes the trausaeylation step in the synthesis of bis(monoacylglycero)phosphate, a lysosomal marker lipid which is greatly increased in this drug-induced phosphoiipidosis. The inhibition of lysosomal phospholipases A and C is reversible siuce full activity can be restored by dialysis or desalting. The mechanism of iuhibition of lysosomal phospholipases A and C by these drugs is as yet unknown and will require purification of the respective enzymes. However, our experiments clearly demonstrate direct inhibition of iysosomal phospholipases A and C chloroquine and 4,4’-bis(~ethyl~noethoxy)a,#&diethyldiphenylethane and suggest that it may be a major factor in the biochemical pathogenesis of drug-induced phospholipidosis.

Certain cationic amphiphilic drugs have been shown to cause tissue phospholipid accumulation when administered chronically to man or animals (1). For example, 4,4-bis(diethylaminoethoxy)a$-diethyldiphenylethane was discovered to be the cause of an iatrogenic lipid storage disease in patients who had been treated with this agent (2). In rats, chronic arbitration of c~oroquine or 4,4-bis(diethyl* These studies were supported by Nationsi Institutes of He&h Grsnt GM-24979 and by the Research Service of the Veterans Administration Medical Center, San Diego, Calif. The costs of publication of this srticle were defrayed in psrt by the payment of page charges. This srticle must therefore be hereby marked “advertisement” in accordance with 18 USC. Section Ii’34 solely to indicate this fact. $ Present address, Second Department of Internal Medicine, Osaka University Medical School, Fukushima-ku, Osaka 553, Japan. 5190

MedicaL Center, and The

~~oethoxy)~,~-diethyldiphenylethane has also been found to cause phosphohpid storage in tissues (3). Recently, efforts to elucidate the biochemical basis for lipid storage induced by cationic amphiphilic drugs have concentrated on hpidosis in rat. In a previous publication from this laboratory, the intracellular distribution of phospholipid, free and ester cholesterol, and the respective drugs was examined in the liver of control and drug-treated rats. We found that the excess phospholipid was present iu lysosomes, which also contained most of the intracelhdar chloroquine and 4,4bis(diethyhuninoethoxy)~,/?-diethyldiphenylethane (4). The size of other ~trace~~~ pools of phospholipid, such as microsomes and mitochondria, was not increased. The liver of rats treated with cbloroquine or 4,4-bis(diethyIaminoethoxy)a&diethyldiphenylethane also contained a greatly increased ester cholesterol content in microsomes, lysosomes, and in the cell supernatant fractious (4). However, with regard to tissue phospholipid storage, it is clear that the lysosome is the principal site to be considered when seeking the biochemical basis for thii condition. Liver lysosomes have been shown to contain several phospholipases A (5, 6). Recently, we demonstrated the presence of a phospholipase C activity in rat liver lysosomes which is active against all phosphoglycerides tested (7). (&her workers have isolated a lysosomal phospholipase C which is specific for ph~phatidyl~ositol (8, 9). All of these phospholipases participate in the catabolism of phospholipids in lysosomes. Our previous work showed that both c~oroqu~e and 4,4bis(diethylaminoethoxy)~,~-diethyldiphenylethane concentrate greatly in lysosomes (4). Since the phospholipid storage in liver was exclusively lysosomal, we considered the possibility that these drugs might be inhibitors of phospholipid catabolism. We have examined their effects on lysosomsl phospholipase A and C activity and this paper reports results which show that both chloroquine and 4,4-bis(diethy~~noethoxy)a&diethyldiphenylethane are potent inhibitors of lysosomal phosphohpases A and C. MATERIALS

AND

MET?IODS

Preparation

of Z,ysoeomes-Lysosomes were prepared from rats injected with Triton WR-1339 as described by Trouet (10). Delipidated soluble protein fractions were obtained from lysosomes aa previously reported and stored frozen at -60% until use (Xl). Protein was determined by the method of Lowry et aZ. (12).

Assaw of Lvsosomal Phosvholivase ‘Y!]Di&eo&p~osphatidylchoiine~The

A attd C: Hydrolysis

of fl-

standard incubation mixture contained 4.5 x IO-’ M [l-‘4C]dioleoylphosphatidylcholine, specific activity, 20 mCi/mmol, 50 rnM sodium acetate buffer, pH 4.4 and 5.4, or 50 mu ~s(hy~ox~ethyl)am~omethane/m~eate buffer at pH 6.4, and 50 ag of lysosomal protein. Vsrying amounte of chloroquine or 4,~-bis(~ethyl~~oethoxy)~,~-diethyl~phenyleth~e (0.0125 to 50 mr+i) were added to incubation mixtures; the total volume was

InhibitionPhospholipases of Lysosomal 0.200 ml. After incubation for 30 min in a shaking water bath at 37”c, the reaction was stopped by adding 4 ml of chloroform/methanol (2: 1 v/v). The lipids were extracted by the method of Folch et al. (13). The lipid extract was applied to an 0.25-mm layer of Silica Gel H prepared with 0.01 M magnesium acetate. A stock solution containing 15 or 20 nmol each of unlabeled reference phosphatidylcholine, lysophosphatidylcholine, I-monoolein, 1,2-diolein,and oleic acid in chloroform/methanol (21v/v), was also applied to theorigin of the plate. The thin layer plates were developed to a height of 7 cm above the origin with chloroform/methanol/water (6535:5 v/v, dried for 20 min in a nitrogen atmosphere, and developed to the topof the plates (20 cm) with heptane/diethylether/formic acid ( W W 4 v/v). The respective lipids were located by scanning for radioactivity with a Panax thin layer chromatography scanner (PanaxInstruments, Redhill, Surrey, England) and by staining with iodine vapors. Theareas representing the respective lipids were scraped into liquid scintillation vials and counted in a Searle Mark 111 liquid scintillation counter with 15 ml of 0.5%, 2,5-diphenyloxazole and 0.04%p-bis[2-(5-phenyloxazole)]benzene in toluene/TritonX-I00/water (2:1:0.2 v/v).as counting fluid. Lysosomal phospholipase A activity was assessed by the formation of lysophosphatidylcholine;phospholipase C activity was measured as the sum of diglyceride and monoglycerideformation. In making the calculations, a specific activity of 10 mCi/mmol was used for monoglyceride and lysophosphatidylcholine,while a specific activity of 20 mCi/mmol was used to calculate the amount of diglyceride formed. Assay of Bis(monoacylg1ycero)phosphate Synthetase-The formation of bis(monoacylg1ycero)phosphate from phosphatidyl[l’,3’14Clglycerol or lysopho~phatidyl[l’,3”’~C]glycerol was assayed as described previously (15).Briefly, the reaction mixture contained 50 mM sodium acetate buffer, pH 4.4, 80 pgof lysosomal protein (intact lysosomes), and 4 X M ph0sphatidyl[l’,3”’~C]plycerol or lysopho~phatidyI[l’,3’-’~C ]glycerol, specific activity, 63 mCi/mmol, and varying amounts of chloroquine or 4,4’-bis(diethylaminoethoxy)a,Pdiethyldiphenylethane, in a final volume of 0.200 ml. The mixture was incubated for 60 min in a shaking water bath a t 37°C. stopped by the addition of 4.0 ml of chloroform/methanol (2:l v/v). The lipids were extracted by the method of Folch et al. (13) and bis(monoacylg1ycero)phosphateformation was determined by thin layer chromatography and liquid scintillation counting as previously described (11). Chemicals-Triton WR-1339 was obtained from Supelco, Bellefonte, PA. [l-’4C]Dioleoylphosphatidylcholinewas purchased from Applied Science Laboratories, College Park, PA. 2-Monoolein and 1,2-dioleinwere obtained from Serdary Research Laboratories, London, Ontario, Canada. sn-[1’,3”’4C]Glycerol-3-Pw a s purchased from New England Nuclear Corp., Boston, MA. Phosphatidyl[l’,3”’‘C]glycerolwas prepared biosynthetically and purified as previously described (16, 17). l-Acyllysophosphatidyl[l’,3’-’4C]glycerolwas prepared by the action of phospholipase A2 on phosphatidyl[l’,3’14C]glycerolas described by van Golde and van Deenen (18). Silica Gel H was purchased from EM Laboratories, Elmsford, NY. Phospholipase A2 was purchased from Boehringer Mannheim, Indianapolis, IN. Chloroform and methanol was redistilled before use; other chemicals were of analytical reagent grade.

5191

bis(diethylaminoethoxy)o,P-diethyldiphenylethane on the hydrolysis of [l-’4C]dioleoylphosphatidylcholine by the lysosomal soluble, delipidated protein. Phospholipase A was measured as theformation of lysophosphatidylcholine while phospholipase C was determined by the formation of mono- and diglyceride. Fig.2 shows that both drugs inhibit phospholipase

-

F2

FA 1,2 DG 1,3 DG MG -FI

PC LPC

-0 ~~~

FIG. 1. Thin layer chromatogram of reference lipids as described under “Materials and Methods”. The abbreviations used are: FA, oleic acid; DG, diolein, MG, monoolein, PC, phosphatidylcholine; LPC, lysophosphatidylcholine; FI, first front; F2, second front; 0, origin.

0

RESULTS

In a previous publication, we reported the presence of phospholipase C activity in a lysosomal soluble protein preparation; with phosphatidylcholine as substrate, monoglyceride and diglyceride were the reaction products (7). However, the two-dimensional thin layer chromatography system used in these earlier studies did not separate monoglyceridefrom diglyceride. In order to simplify the complete analysis of the hydrolysis products of [l-’4C]dioleoylphosphatidylcholine,we have developed a one-dimensional thin layer chromatography system which is shown in Fig. 1. This system employs two successive developments; the first is a partial development with a polar solvent and is followed by a full development with a nonpolar solvent as described under ‘‘Materials and Methods.” All of the lysosomal degradation products of phosphatidylcholine are well separated as shown in Fig. 1. The method also allows large numbers of samples to be assayed more rapidly. We examined the effect of chloroquine and 44’-

Y

0

# 0

0

25t

-d DRUG CONCENTRATION. rnM

CONCENTRATION, DRUG

25

10

mM

FIG. 2. Effect of chloroquineor 4,4-bis(diethylaminoethoxy)a,/?-diethyldiphenylethane on the conversion of [l-“C]dioleoylphosphatidylcholine to [*4C]lysophosphatidylcholine (LYSO P C ) by lysosomal phospholipase A at pH 4.4 and 5.4. The incubation mixture contained: 50 pl of lysosomal soluble delipidated protein; 4.5 X M [l-14C]dioleoylphosphatidyicholie, specific activity, 20 mCi/mmol; 50 m~ sodium acetate buffer, pH 4.4 or 5.4. Varying amounts of 4,4’-bis(diethylaminoethoxy)a,fi-diethyldiphenylethane or chloroquine were added as indicated. The total volume was 0.200 ml; the mixture was incubated at 37°C for 30 min. Symbols: 0, 4,4’-bis(diethylaminoethoxy)a,~-diethyldiphenylethane at pH 4.4, or a,pH 5.4; A, chloroquine a t pH 4.4, or A, pH 5.4; left panel, chloroquine (CLQ); right panel, 4,4”bis(diethylaminoethoxy)a,P-diethyldiphenylethane(OH).

A activity at pH 4.4 and 5.4. In the left panel, it can be seen that chloroquine inhibition of lysophosphatidylcholine formation is less pronounced at pH 4.4 than at pH 5.4. A similar effect of pH is observed with 4,4’-b~(diethyl~inoethoxy)~,~diethyldiphenylethane (right panel), but the degree of inhibition produced by the latter agent is much greater than that found with chloroquine. At pH 4.4, 50% inhibition is noted at about 10 mM for chloroquine uersus 0.23 mM for 4,4’b~(diethylaminoethoxy)~,~-diethylphenylethane. At pH 5.4, 50% inhibition occurs at 0.3 mM for c~oroquine compared with 0.04 mu for 4,4’-bis(diethylaminoethoxy)a,/?-diethyldiphenylethane (Table I). Lysosomal phospholipase C is also inhibited by both of these agents as shown in Fig. 3. In contrast to their effects on phospholipase A activity, both agents are about equally effective as inhibitors of phospholipase C activity at pH 4.4 with a 50% reduction of activity at 0.33 mu for chloroquine (right panel) and 0.20 mM for 4,4’-bis(diethylaminoethoxy)cu,P-diethyldiphenylethane (left panel). However, at pH 5.4 the inhibition is less marked; 10 mu chloroquine and 6.0 mM 4,4’bis(diethylam~oethoxy)ff,~-diethyldiphenylethane were required to produce a 50% reduction of activity. The reduction in phospholipase C inhibition with an increased pH is opposite to the effect of pH on inhibition of lysosomal phospholipase A (Fig. 2 and Table I). Finally, it should be noted that these reaction rates represent minimum values since lysophosphatidylcholine and monoglyceride can be degraded by the action of other lysosomal acid hydrolases present in the preparation. Fig. 4 shows the effect of pH on lysosomal phospholipases A and C in the presence and absence of 10 mu chloroquine or 10 XXIM 4,4’-bis(diethylaminoethoxy)tu,P-diethyldiphenylethane. At pH 4.4, which is optimal for both lysosomal phospholipases, the rate of lysophosphatidylcholine generation in the absence of inhibitors is 74 nmol mg-’ h-’ versus a rate of mono- and diglyceride formation of 80 nmol mg-’ h-‘. Thus, Effect

--.I_

TABLE I ofpH on the inhibition of lysosomalphospholipase A and by chloroquine and 4,4’-bis(diethylaminoethaxy)a,f3diethyldiphenylethane Phospholipase c” Phospholipase A” ~__^ pH -DH DH CLQ CLQ mhf ?nM

4.4 5.4_

>lO 0.30

0.23 0.04

0.33 >lO

C

0.20 6.00

” Concentration of chloroquine (CLQ) or 4,4’-bis(diethylaminoethoxy)a&diethyldiphenylethane (DH) required to produce 50% inhibition of the respective phospholipase activities. The incubation conditions are described in the legend to Fig. 2; the data in this table have been calculated from the data shown in Figs. 2 and 3.

DRUG CONCENTRATION.mM

FIG. 3. Effect

DRUG CONCENTRATION.mM

of chloroquine and 4,4’-bis(diethylaminoethoxy)a&diethyldiphenylethane on the conversion of [l“C]phosphatidylcholine to [‘“C]monoglyceride and [‘%]diglyceride at pH 4.4 and 5.4. Incubations and symbols as noted in Fig. 2. Lefi panel, c~o~qu~e (CLQ); right panel, 4,4’-bis(diethyl~inoethoxy)a,~-diethyl~phenylethane WI). MG, monoolein; DG, diolein.

4.4

5.4

PH

6.4

4.4

5.4

6.4

PH

FIG. 4. Effect

of pH on the activities of phospholipase A and C with chloroquine or 4,4’-bis(diethylaminoethoxy)a,@liethyldiphenylethane and without. The incubation mixture contained: 50 pg of Iysosomai soluble delipida~d protein, 4.5 x lo-” M [l“C]dioleoylphosphatidylcholine, specific activity, 20 mCi/mmol; 50 mM sodium acetate buffer at pH 4.4 and 5.4, or 50 mu, tris(hydroxymethylaminomethane)/maleate buffer at pH 6.4, and 10 mM 4,4’-bis(diethylaminoethoxy)a,P-diethyldiphenylethane or chlo-

roquine. The totai volume was 0.200 mI and the mixture was incubated at 37‘Y for 30 mm. Symbols: El, no drug; 0, 4,4’-~~(diethylaminoethoxy)ff,~-~ethyl~phenyleth~e; A, chloroquine; lefi panel, phospholipaae A, right panel, phospholipase C. LysoPC, lysophosphatidylcholine; MG, monoolein; DC, diolein.

lysosomal phospholipase C may represent a quantitatively ~po~ant mechanism for phosphatidylcho~ne degradation. These experiments clearly demonstrate the direct inhibitory effect of chloroquine and 4,4’-bis(diethylaminoethoxy)ol,P-diethyldiphenylethane on lysosomal phospholipases A and C. This inhibition is most pronounced at lower pH values as shown in Figs. 2 to 4. Increased pH greatly reduces the rates of phosphatidylcholine degradation by both phospholipase A and C. Chloroquine is known to raise the intralysosomal pH (19,20). We have shown that 4,4’-bis(diethylaminoethoxy)a$diethyldiphenylethane concentrates in liver lysosomes (4), but its effects on intralysosomal pH have not been measured. However, as a weak base, it would also be expected to raise the intralysosomal PH. Further experiments were done to elucidate the nature of the drug inhibition. Substrate-velocity studies were carried out at five fixed concentrations of the respective inhibitors. However, when the data were plotted as the double reciprocals, the results did not fit the classical models for competitive, noncompetitive, or uncompetitive inhibition. This is due, in all probability, to the complexity of the soluble lysosomal hydrolase preparation which contains at least two phospholipases A (5, 6), phospholipase C (7), and lipase (21) and a monoglyce~de lipase (22). Covalent modification of the enzyme is probably not involved since the inhibition of lysosomal phospholipase A and phospholipase C by chloroquine and 4,4’-bis(diethylaminoethoxy)a,P-diethyldiphenylethane is reversible. After incubation of these inhibitors with lysosomal soluble protein, essentially full activity can be restored upon gel Ntration desalting with Sephadex G-25 or by dialysis (data not shown). Further studies of the mechanism of inhibition by cationic amphiphilic drugs will require purification of lysosomal phospholipase A and C. Tissue levels of bis(monoacylglycero)phosphate are greatly increased in rats or humans who have chronically ingested 4,4’-bis(diethylaminoethoxy)a,P-diethyldiphenyleth~e (1). In rats, chloroquine also caused marked increases in the level

Inhibition of Lysosomal Phospholipases

5193

microsomes, nuclear fraction, and cell supernatant) were essentially unchanged (4).The respective lysosomal fractions also contained most of the intracellular drug content; the lysosomal drug content relative to protein was 13.5- and 10.9BMP synthetase" fold greater than that of the tissue homogenates for chloroquineand 4,4'-bis(diethylaminoethoxy)a,P-diethyldiphenyl["C]Phosphatidyl[l'C]LysophosphaInhibitor Concentration tidylglycerol glycerol ethane, respectively (4).These findings suggested the possibility that these drugs might inhibit the degradation of phosSpecific Q conSpecific Q control trol Activitv Activity pholipids by lysosomes. rnM Our present results demonstratetwo probable mechanisms 2.01' 100 100 3.85b None for the drug-induced phospholipid storage. Both drugs are directinhibitors of lysosomalphospholipaseA and C. In n.dd n.d 102 2.04 0.025 Chloroaddition, lysosomal accumulation of phospholipids may also 1.92 95 96 quine 0.25 3.67 be due to the tendency of these agents to concentrate in 103 2.02 101 0.5 3.98 lysosomes, raising the intralysosomal pH to levels which are n.d n.d 127 1.25 4.89 unfavorable for phospholipid catabolism. The levels of 4,4'2.29 114 117 2.5 4.50 10 137 133 2.67 5.27 bis(diethylan~inoethoxy)a,P-diethyldiphenylethane have pre141 n.d n.d 25 5.43 viously been determined in liver homogenates of rats treated with 100 mg/kg for 2 weeks and approximate 5 to 10 mM, 2.20 110 n.d DH n.d 0.025 assuming free distribution in cellular water (1). In another 0.25 100 2.56 127 3.84 study, rats treated with 100 mg/kg of chloroquine or 4,4'90 2.33 116 0.5 3.48 bis(diethylaminoethoxy)a,P-diethyldiphenylethanefor 7 days 1.25 90 n.d n.d 3.45 2.5 2.30 114 82 3.14 had liver homogenate levels of drug estimated at 2 to 3 mM. 10 1.10 1.24 29 62 However, the drugs are highly concentrated in lysosomes 25 8.9 n.d n.d 0.34 which are 10.9- to 13.6-fold enriched relative to thehomogeThe abbreviationsused are: BMP, bis(monoacylg1ycero)-P; APG, nate (4).Thus, the true concentrationof chloroquine or 4.4'acylphosphatidylglycerol; DH, 4,4'-bis(diethylaminoethoxy)a,P-di- bis(diethylaminoethoxy)a,P-diethyldiphenylethaneinside the ethyldiphenylethane. lysosome may be presumed to be much higher, perhaps apNanomoles BMP + APG formed mg" protein h-I. proaching or even exceeding 50 to 100 m ~Under . our expere Nanomoles BMP formed mg" protein h". imental conditions, 50%inhibition of phospholipase Ais found n.d, not determined. at 0.04 to 0.23 m~ and 0.2 to 6.0 m~ for phospholipase C, of bis(monoacylg1ycero)phosphate in liver and other tissues respectively (Table I). Concentrations of thesedrugsap(3,4).The tissue increases inbis(monoacylg1ycero)phosphate proaching 50 mM inside the lysosome would be expected to in these cases is substantially greater than the increases in produce profound inhibition of both phospholipases A and C, other phospholipids (3,4).Bis(monoacylg1ycero)phosphateis therefore representing a major mechanism for the phosphoformed by the transfer of an acyl group from phosphatidyli- lipid storage reported in thiscondition (1, 3,4). Our findings are in general agreement with the work of nositol to the free glycerol moiety of lysophosphatidylglycerol (11).We have previously presented evidence which indicates Seydel and Wasserman who showed that cationic amphilic phospholipids and suggested that the drug. that bis(monoacylg1ycero)phosphate synthetase is not identi- drugs interact with cal withlysosomal phospholipase A basedon differential heat phospholipid complex might be resistant to degradation by stability and differing effects of certain inhibitors (10).How- phospholipases (23).Although they did not test this hypothever, since it removes a fatty acid from phosphatidylinositol, esis with lysosomal phospholipases, our data show concluthis enzyme may also be classified as a specialized phospho- sively thatthese twocationic amphiphilicdrugsstrongly lipase A. We examined the effects of chloroquine and 4,4'- inhibit lysosomal phospholipases A and C. At present, the bis(diethylaminoethoxy)a,P-diethyldiphenylethane on the mechanism of the inhibition of these phospholipases is unsynthesis of bis(monoacylg1ycero)phosphatefrom radioactive known. Kasama et al. haveshown in rat liver that acid phosphatidylglycerol or lysophosphatidylglycerol by intact triglyceridelipase and acid esterase are inhibited by 4,4'lysosomes and the results are shown in Table 11. Interestingly, bis(diethylaminoethoxy)a,P-diethyldiphenylethane(24).The chloroquine, over a wide range of concentrations, did not drug appears tobe a noncompetitive inhibitor of the lysosomal inhibit the synthesis of bis(monoacylg1ycero)phosphate.At acid esterase; detailed studies of the drug inhibition of acid 1.25 to 25 mM chloroquine, stimulation of bis(monoacy1- lipase have not been done (24). g1ycero)phosphate synthetase activitywas found with bothof the radioactive substrates. 4,4"Bis(diethylaminoethoxy)a,/lREFERENCES diethyldiphenylethaneinhibited bis(monoacylg1ycero)phos1. Matsuzawa, Y., Yamamoto, A., Adachi, S., and Nishikawa, M. phatesynthetase only a t concentrations in excess of 2.5 (1977) J. Biochem. (Tokyo) 82, 1369-1377 mM. This series of experiments suggests that the synthesis 2. Yamamoto, A., Adachi, S., Ishikawa, K., Yokomura, T., Kitani, T., Nasu, T., Imoto, T., and Nishikawa, M. (1971) J. Biochem. of bis(monoacylg1ycero)phosphate is partially preserved even (Tokyo)70,775-584 at concentrations of these drugs which cause profound inhi3. Yamamoto, A., Adachi, S., Matsuzawa, Y., Kitani, T., Hiraoka, bition of lysosomal phospholipases A and C. A., and Seki, K. (1976) Lipids 11,616-622 4. Matsuzawa, Y., and Hostetler, K. Y.(1980) J. Lipid Res. 21,202DISCUSSION TABLE I1 Effect of chloroquine and 4,4'- bis(diethylaminoethoxy)a,Pdiethyldiphenylethane on bis(monoacylg1ycero)phosphate synthesis from [14C]phosphatidylglycerolor ["C]lysophosphatidylglycerol

~

~~

Our previous studies on the subcellular distribution of phospholipids in the liver of rats with phospholipidosis induced by chloroquine or 4,4"bis(diethylaminoethoxy)a,~-diethyldiphenylethane showed that the 50% increase in tissue phospholipids was due to an increased lysosomalphospholipid pool while other cellular pools of phospholipid (mitochondria,

214 5. Mellors, A., and Tappel, A. L. (1967) J . Lipid Res. 8,479-484 6. Franson, R.,Waite, M., and La Via, M. (1971) Biochemistry 10, 1942-1946 7. Matsuzawa, y.,and Hostetler, K. Y.(1980) J. Biol. Chem. 255, 646-652 8. Irvine, R. F., Hemington,N.,andDawson,R. M. C. (1977) Biochem. J . 164,277-280

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Inhibition of Lysosomal Phospholipases

9. Irvine, R. F., Hemington, N., and Dawson, R. M. C. (1977) Bwchem. J. 176,475-484 10. Trouet, A. (1964)Arch. Int. Physiol. Biochim. 72, 698-699 11. Matsuzawa, Y., Poorthuis, B. J. H. M., and Hostetler, K. Y. (1977) J. Bwl. Chem. 253,6650-6653 12. Lowry, 0. H., Rosebrough, N. J., Farr,A. L., and Randall, R. J. (1951) J. BWZ. Chem. 193,265-275 13. Folch, J., Lees, M., and Sloane Stanley, G.H. (1957) J. Biol. Chem. 226,497-509 14. Matsuzawa, Y., and Hostetler, K. Y. (1979) J. Biol. Chem. 254, 5997-6001 15. Poorthuis, B. J. H. M., and Hostetler, K. Y. (1976) J. Biol. Chem. 251.45964602 16. Hostetler, K. Y., and van den Bosch, H. (1972) Biochim. Biophys. Acta 260,380-386 17. Poorthuis, B. J. H. M., and Hostetler, K. Y. (1975) J.BZoZ. Chem.

250,3297-3302 18. Van Goide, L. M. G., and van Deenen, L.L. M.(1966) Biochim.

Biophys. Acta 125,496-509 19. De Duve, C., de B m y , T., Trouet, A., Tulkens, P., and van Hoof, F. (1974) Biochem. Pharmacol. 23,2495-2531 20. Poole, B., Ohkuma, S., and Warburton, M. J. (1977) Acta Biol. Med. Ger. 36, 1777-1778 21. Teng, M.H., and Kaplan, A. (1974) J. Biol. Chem. 249, 10641070 22. Colbeau, A., Cualt, F.0.M., Shapiro, D., and Brady, R. 0. (1961) J. Biol. Chem. 241,1081-1084 23. Seydel, J. K., and Wassermann, 0. (1976) Biochem. Pharmacol. 25,2357-2364 24. Kasama, K., Yoshida, K., Takeda, S., Tsujimura, R., and Hasegawa, S. (1976) Lipids 11, 718-721