Differentiation of microsomal from lysosomal triacylglycerol lipase activities in rat liver

230

BBA 5 1359

DIFFERENTIATION OF MICROSOMAL ACTIVITIES IN RAT LIVER ROSALIND Departmenr

A. COLEMAN of Pediatrics.

and ELAINE

FROM LYSOSOMAL

TRIACYLGLYCEROL

LIPASE

B. HAYNES

Duke Universrty Medical Center, Durham. NC 27710 (U.S.A.)

(Received June 14th. 1982) (Revised manuscript received

December

Key words: Glycerolipid metabolrsm;

14th. 1982)

Triacylglycerol

lipuse; Cholestqvl

ester; Wolmon ‘s drsease; (Rat hoer)

Non-lysosomal triacylglycerol lipase (EC 3.1.1.3) and cholesteryl esterase (EC 3.1.1.13) activities from rat liver were distinguished from lysosomal activities using assays based on the release of oleic acid from glycerol-[ ‘H]oleate or cholesteryl-ll-‘4C)oleate. Dispe rsion of the lipid substrates in acetone and the addition of a 1: 1 (w/w) mixture of sonicated phosphatidylcholine/phosphatidylserine to the incubation mixture permitted the development of sensitive assays that allowed comparison under identical conditions. MnCl 2 stimulated the non-lysosomal triacylglycerol lipase and cholesteryl esterase activities 2-fold but inhibited the lysosomal activities 85%. The lysosomal activities could be solubilized by osmotic disruption with water, whereas the non-lysosomal activities repelleted after centrifugation at 100000 X g for 1 h. pH optima of the lysosomal and non-lysosomal activities were 5.5 and 6.0, respectively, under the conditions employed. Cobalt acetate, CaCl, and MgCl, inhibited the lysosomal triacylglycerol lipase and cholesteryl esterase activities greater than 60% but had no effect on the non-lysosomal activities. Upon subcellular fractionation, the Mn*+ -stimulated triacylglycerol lipase and cholesteryl esterase activities partitioned with microsomal marker enzymes. Pronase inactivated the Mn*+- stimulated activities 7542% under conditions in which mannose-6-phosphatase latency and activity remained unchanged, suggesting that the Mn*+-stimulated activities contain protease-sensitive sites exposed on the cytoplasmic surface of microsomal vesicles. The triacylglycerol lipase and cholesteryl esterase activities in fibroblasts from patients with Wolman’s disease and cholesteryl ester storage disease were stimulated by MnCl,, had pH optima of 6.0-6.5, and were not solubilized by osmotic disruption, suggesting that human fibroblasts may also contain both the microsomal and the lysosomal activities. Microsomal triacylglycerol lipase and cholesteryl esterase activities may function to hydrolyze endogenously synthesized triacylglycerol and cholesteryl esters.

Introduction Triacylglycerol is synthesized in hepatocytes by a series of microsomal enzymes whose active sites face the cytoplasmic surface of the endoplasmic reticulum [I]. During the biosynthesis of verylow-density lipoprotein (VLDL), endogenously synthesized triacylglycerol must be transported into Abbreviation:

VLDL,

very-low-density

OOOS-2760/83/0000-0000/$03.00

lipoprotein(s).

0 1983 Elsevier Science Publishers

the lumen of the endoplasmic reticulum. Triacylglycerol may also accumulate in droplets within liver cells during several pathological states and may disappear upon recovery [2]. In chick hepatocytes in culture, some triacylglycerol in lipid droplets appears to be hydrolyzed and then reesterified prior to secretion as VLDL [3]. The subcellular site of hydrolysis is unknown, as is the mode of regulation of triacylglycerol degradation. Lysosomal triacylglycerol lipase (EC 3.1.1.3) is

231

believed to hydrolyze triacylglycerol that enters hepatocyte lysosomes via receptor-mediated endocytosis of serum lipoproteins [4]. The lysosomal activity, which has an acid pH optimum, may be identical to lysosomal cholesteryl esterase (EC 3.1.1.13) [5-71. Both the lysosomal triacylglycerol lipase and the lysosomal cholesteryl esterase activities are deficient in fibroblasts from patients with Wolman’s disease and cholesteryl ester storage disease [8,9]. Although investigators have measured a non-lysosomal triacylglycerol lipase activity with a neutral pH optimum in hepatocytes, this activity may not be distinct from the hepatic lipoprotein lipase that appears to be secreted by hepatocytes and ultimately located on the outer surface of liver endothelial cells [ IO,1 I]. DeBeer et al. [ 121 have presented evidence that hepatic membrane-bound lipase activity measured at neutral and alkaline pH values is lipoprotein lipase on plasma membranes that contaminated microsomal preparations. These workers suggest that the lysosomal triacylglycerol lipase is the only intracellular hepatic lipase and that endogenously synthesized triacylglycerol would thus require transport into lysosomes for degradation. We have identified a novel microsomal triacylglycerol lipase with a pH optimum well below that of hepatic lipoprotein lipase. The activity differs substantially from the lysosomal lipase activity which has a similar pH profile. Because the microsomal cholesteryl esterase activity has many characteristics similar to the novel microsomal triacylglycerol lipase, and because hepatic microsomal cholesteryl esterase has not been well characterized, we have examined both the microsomal triacylglycerol lipase and the microsomal cholesteryl esterase activities in rat liver and have localized these activities within the transverse plane of the endoplasmic reticulum. Materials Cholesteryl-[ I4 Cloleate was obtained from Amersham. Glycerol [ 3H]triolein, [ “C]oleic acid and Aquasolwere from New England Nuclear. [ 3H]Palmitoyl-CoA was synthesized as previously described [ 131. [ 32P]Mannose 6-phosphate was the generous gift of Dr. L.M. Ballas (North Carolina State University School of Veterinary Medicine).

Mannose 6-phosphate, sodium taurocholate, triolein, cholesteryl oleate, bovine serum albumin (essentially fatty acid-free), oleic acid, type 3 cytochrome c (horse heart), sodium deoxycholate, phenazine methosulfate and 3-(4,5_dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide were from Sigma. Pronase was from Calbiochem. Phosphatidylcholine (egg), phosphatidylserine (beef brain), triolein and diolein were from Serdary. Silica gel G plates were from Analtech, Inc. Silicic acid was from Mallinkrodt. NADPH was from PL Biochemicals. Fetal bovine serum, penicillin, streptomycin and minimal essential medium (Eagle) with Earle’s salts were from Grand Island Biological Company. Wolman’s disease fibroblasts (GM 1606) and cholesteryl ester storage disease fibroblasts (GM 3347) were from the American Type Culture Collection. Triton WR-1339 was obtained from Ruger Chemical Co. A highly purified rat liver peroxisomal fraction was the generous gift of Dr. P.B. Lazarow (Rockefeller University). Methods Isolation of rut liver lysosomes and microsomes.

Livers from fasted 250-g Charles River CD strain female rats were homogenized by 10 up-and-down strokes in a motor-driven, Teflon-glass homogenizer in 5 vol. of cold medium I (0.25 M sucrose/l mM EDTA/lO mM Tris-HCl, pH 7.4). The homogenate was centrifuged at 1900 X g for 10 min and the pellet was discarded. To obtain a lysosomal fraction, the supernatant was centrifuged at 27 000 X g for 10 min. The resulting pellet was resuspended in medium I and centrifuged at 1600 x g for 10 min. The pellet was discarded and the supernatant was centrifuged at 20000 X g for 10 min to obtain the lysosomal pellet. To obtain a microsomal fraction, the 1900 x g, 10 min, supernatant was centrifuged at 22000 X g for 15 min. The pellet was discarded and the supernatant was centrifuged at 100000 X g for 1 h. Microsomal and lysosomal pellets were resuspended in medium I and stored in aliquots at -2O’C. Once thawed, specimens were discarded. Protein was determined by the method of Lowry et al. [14] using bovine serum albumin as the standard. Microsomal fractions from other rat tissues were prepared similarly.

232

Release of soluble lysosomal triaqlglycerol lipase and cholesteryl esterase. A 1 : 5 total liver homogenate was centrifuged at 100000 X g for 1 h. The pellet was homogenized with 10 up-and-down strokes as described above in deionized water at 1.5 X the original volume, and recentrifuged at 100000 X g for 1 h. Approximately 54% of the lysosomal triacylglycerol lipase and cholesteryl esterase activities were released into the supernatant which was employed for studies on the soluble lysosomal triacylglycerol lipase and cholesteryl esterase activities. A second solubilization treatment released another 25% of each activity into the supernatant. The pellet obtained after the second osmotic disruption was employed for studies on the Mn*+-stimulated triacylglycerol lipase and cholesteryl esterase activities as indicated in the text. Subcellular fractionation. Female Charles River CD rats (250 g) were injected intraperitoneally with 1 ml of 20% (w/v) Triton WR-1339 in 0.9% NaCl [15]. 5 days later the rats were decapitated and the livers were minced and homogenized in 3 vol. of 0.25 M sucrose in 0.1% ethanol (medium II) with three gentle up-and-down strokes of a motor-driven Teflon-glass homogenizer. Subcellular fractions were obtained by differential centrifugation as described previously [ 15,161 and are summarized in the legend to Fig. 4. Aliquots of each fraction were resuspended in medium II and stored at -20°C. Pronase treatment of microsomes. Intact untreated microsomes and microsomes that had been disrupted by a 10 min exposure at 0°C to 0.05% deoxycholate and 50 mM KC1 in medium I without EDTA were treated for 20 min at 30°C with pronase, 68.6 pg/mg microsomal protein, as described by Nilsson and Dallner [ 171. Control intact and disrupted microsomes were treated similarly except that pronase was omitted. The four initial samples each contained 15 mg of microsomal protein. After the incubation, the preparations were cooled in ice water and centrifuged at 100 000 X g for 2 h. The recovered microsomes were suspended in medium I. Enzyme assays were performed within 3 days on samples stored at - 20°C. CeN culture and preparation of total particulate cell fractions. Normal human fibroblasts and

fibroblasts derived from patients with Wolman’s disease or cholesteryl ester storage disease were grown to confluence in lOO-mm dishes in Eagle’s minimal essential medium with Earle’s salts and 10% fetal bovine serum. At confluence the dishes were washed three times with ice-cold medium I. The cells were scraped from the dishes with a rubber policeman and homogenized in medium I as described for the liver microsomes. A total particulate pellet was obtained by centrifuging the homogenate at 100000 X g for 1 h. No acid or Mn*+-stimulated triacylglycerol lipase or cholesteryl esterase activities were detected in the supernatant. The pellet was resuspended in medium I and aliquots were stored at -2O’C. Assays of triacylgbcerol lipase and cholesteryl triacylglycerol esterase activities. Mn2+ -stimulated lipase activity was determined in 13 x 100 mm screw-capped culture tubes in a final volume of 200 ~1. The reaction mixture contained 175 mM 2-( N-morpholino)ethanesulfonic acid, pH 6.0. 100 pg of bovine serum albumin, 15 pg of a 1 : 1 (w/w) mixture of phosphatidylcholine and phosphatidylserine sonicated for 2 min in 10 mM TrisHCl, pH 7.0, 10 mM MnCl,, and 250 PM [“Hltriolein (5 pCi/pmol) that had been dissolved in acetone. The final acetone concentration in the assay was 2.5%. Triolein was purified prior to assay [ 181. The reaction was initiated by adding l-10 pg of protein and was terminated after 10 min at 37°C by adding 3.25 ml methanol/chloroform/heptane (1.41 : 1.25 : 1.0, v/v) followed by 1.05 ml of 0.1 M potassium carbonate/sodium borate, pH 10.5, and 10 pg of oleic acid [ 181. After vortexing vigorously, the capped tubes were centrifuged for 15 min. An aliquot of the aqueous phase was counted in 4.0 ml of Aquasolusing a Beckman LS 8000 liquid scintillation counter. A known amount of [‘4C]oleic acid was routinely carried through the extraction procedure to verify the efficiency of extraction. 80-85% of the standard [ “C]oleic acid was extracted under all conditions tested, and specific activities were corrected for recovery. Unless otherwise indicated, each specific activity was obtained by assaying a sample at three different protein concentrations. Mn*‘-stimulated cholesteryl esterase activity was determined by a method analogous to that described above for triacylglycerol lipase activity

233

except that 250 PM cholesteryl [‘4C]oleate (0.4 pCi/pmol) replaced the [3H]triolein. For triacylglycerol lipase and cholesteryl esterase assays, greater than 93 and 98%, respectively, of the extracted labelled material co-migrated with authentic oleic acid on thin-layer chromatography using silica gel G 250-pm plates developed in ether/diethyl ether/acetic petroleum acid, (80 : 20 : 1, v/v). Lysosomal triacylglycerol lipase and cholesteryl esterase activities were determined at pH 5.5 by methods similar to those described for the Mn”stimulated activities except that MnCl, was omitted. Other enzyme assuys. Diacylglycerol acyltransferase activity was determined by a previously described radiochemical method except that the 1,2-sn-dioleoylglycerol was added in acetone so that the final incubation mixture contained 10% acetone and 200 PM dioleoylglycerol [19]. The activity of mannose-6-phosphatase was determined by a radiochemical method [20]. Succinate dehydrogenase [21] and NADPH cytochrome c reductase [22] were determined spectrophotometritally. Hexosaminidase was measured fluorometritally [23]. All assays were proportional to the time and protein employed. Results Dependencies cholesteryl

esterase

of

triacylglycerol

activities.

lipase

and

Mn2+-stimulated triacylglycerol lipase and Mn2+-stimulated cholesteryl esterase activities in a total particulate rat liver pellet recollected after osmotic disruption were proportional to the amount of protein added up to 12 and 10 pg, respectively. When 10 pg of protein was employed, both assays were proportional with time to at least 14 min (Fig. 1). Similar results were obtained with liver microsomal fractions and with total particulate fractions from human fibroblasts. Both activities were completely dependent on the addition of enzyme protein. Phospholipid and bovine serum albumin stimulated triacylglycerol lipase activity 250 and 15%, respectively, and were routinely included in both incubation mixtures although neither compound affected cholesteryl esterase activity. NaCl at 50 mM or FeSO, at 5

pug protein

ti

6

12 18 Minutes

24

Fig. I. Dependence of hepatic Mn *+-stimulated triacylglycerol lipase (0, n) and cholesteryl esterase (0, 0) activities with the amount of protein (circles) and time (squares) employed. Assays were performed as described in Methods. The time dependencies employed 10 pg of protein that was repelleted at 100000 X g for 1 h after osmotic disruption of a total particulate pellet (see Methods). The protein dependencies employed a 10 min incubation.

mM inhibited both activities about 50%. N-Ethylmaleimide at 2.5 mM inhibited triacylglycerol lipase 17% but had no effect on cholesteryl esterase. The addition of EDTA (10 mM) had no effect on triacylglycerol lipase but inhibited cholesteryl esterase 58%. Effect of MnCl,. Two triacylglycerol lipase activities and two cholesteryl esterase activities in rat liver subcellular fractions were initially differentiated by their responses to MnCl, (Fig. 2). To facilitate comparison, all activities were assayed at pH 6.0. Lysosomal activities were about 70% of maximal at this pH (Fig. 3). 10 mM MnCl, inhibited triacylglycerol lipase activity 79% in lysosomes and 85% in solubilized homogenate. In contrast, the triacylglycerol lipase activity that pelleted at 100000 X g for 1 h after two osmotic disruptions of a total liver homogenate was inhibited only 22% by MnCl,. Similarly, 10 mM MnCl, inhibited the lysosomal and the solubilized cholesteryl esterase activities 64 and 84%, respectively. In contrast, the cholesteryl esterase activity

234

A

Triacyiglycerol

lipase

B

Cholesteryl

esterase

35(

30(

25(

I

I

I

I

5

IO

15

20

Fig. 2. Dependencies of triacylglycerol lipase (A) and cholesteryl esterase (B) activities on MnCl,. Assays were performed on rat liver and human fibroblast preparations as described in Methods except that MnCI, was added to the incubation mixture as indicated. The total particulate pellets from Wolman’s disease fibroblasts, cholesteryl ester storage disease fibroblasts, normal human fibroblasts, and rat liver were recollected by centrifugation at 100000 X g for 1 h after osmotic disruption (see Methods). Rat liver lysosomes were prepared as described in Methods. Without added MnCl,. the respective specific activities for the triacylglycerol lipase and cholesteryl esterase activities were as follows: cholesteryl ester storage disease pellet (A), under 0.20 and under 0.1 nmol/min per mg; Wolman’s disease pellet (0) 0.58 and 0.24 nmol/min per mg; normal fibroblast pellet (0) I. 11and 0.50 nmol/min per mg; residual liver pellet (0) 1.64 and 0.27 nmol/min per mg; liver lysosomal pellet (A), 13.50 and 2.15 nmol/min per mg; soluble fraction from an osmotically-disrupted liver lysosomal pellet (m), 14.62 and 0.75 nmol/min per mg.

remaining in the pellet recollected after osmotic disruption was stimulated 2.7-fold by MnCl,. These findings suggested the presence of membrane-bound triacylglycerol lipase and cholesteryl esterase activities that were distinct from the soluble lysosomal activities. This hypothesis was tested in two human fibroblast mutants that lack the lysosomal activities [8,9]. After osmotic disruption and recollection of the total particulate pellet,

MnCl, stimulated the cholesteryl esterase and triacylglycerol lipase activities 3.5 and 2.9-fold, respectively, in fibroblasts derived from a patient with cholesteryl ester storage disease. In a similar preparation of fibroblasts derived from a patient with Wolman’s disease, MnCl, stimulated the cholesteryl esterase and triacylglycerol lipase activities 2.7- and 1.7-fold, respectively. Thus, both types of mutant cells contained residual mem-

235

A

Triocylglycerol

brane-bound activities that were enhanced by Mn’ +. Like liver, normal human fibroblasts contain a lysosomal activity that can be solubilized by osmotic disruption and is inhibited over 90% by 10 mM Mn2+. MnCl, appeared to stimulate tri-

lipose

A

NADPH

cytochrome

C

6

succ4note dehydrogenose

0

Hexosominidose

reductaze d

e r

4

5

6

7

8

9

C

acyltmnsferase

t

B Cholesteryl

1

Diacylglycerol

t

esterase

E

Mn2+

c

-stunuloted

triocylglycerol

F

lipose

31

3

I

I

I

I

t

I

4

5

6

7

8

9

PH

Fig 3. pH dependencies of lysosomal and Mn2+-stimulated triacylglycerol lipase (A) and cholesteryl esterase (B) activities in various preparations. Rat liver lysosomes and post-osmotically-disrupted particulate fractions were prepared from rat liver (II), cholesteryl ester storage disease fibroblasts (A), and Wolman’s disease fibroblasts (0) as described in Methods. Assays were performed as described in Methods except that the pH was varied. MnC12 was not included in assays of the lysosomal(0) activities. Buffers employed were sodium acetate (pH 4.5-5.0); 2-( N-morpholino)ethanesulfonic acid (pH 5.5-6.5); and Tris-HCl (pH 7.0-9.0). Specific activities at pH 6 for the triacylglycerol lipase and cholesteryl esterase activities, respectively, were: Wolman’s, 1.5 1 and 0.80 nmol/min per mg; cholesteryl ester storage disease, 0.85 and 0.62 nmol/min per mg; residual liver, 1.53 and 0.43 nmol/min per mg. At pH 5.5 lysosomal triacylglycerol lipase and cholesteryl esterase specific activities were 12.19 and 3.29 nmol/min per mg, respectively.

cholesteryl

LySOSOmoI esterase

esterase

50

Lysosomal triacylglycerol

GO Cumulative

50 ‘10

lipose

cholesteryl

160

protein

Fig. 4. Distribution pattern of different enzymes in liver from a rat that had been treated with Triton WR-1339 (see Methods). Relative specific activity with respect to that of homogenate is plotted as a function of percent protein according to the method of DeDuve et al. (16). The fractions are: a, nuclear fraction (O-6 000 X g. min); b, mitochondrial fraction (6000-33000~ g’min); c, lysosomal fraction (33000-250000 x g.min); d, microsomal fraction (250000-6000000X g.min; e, cytosol (6000000x g.min supematant). The specific activities in the homogenate were: A, NADPH cytochrome c reductase, 23.0 nmol/min per mg; B, succinate dehydrogenase, 3.14 nmol/min per mg; C, diacylglycerol acyltransferase, 1.52 nmol/min per mg; D, hexosaminidase, 23.8 nmol/min per mg; E, Mn2+-stimulated triacylglycerol lipase, 1.62 nmol/min per mg; F, lysosomal triacylgfycerol lipase, 8.19 nmol/min per mg; G, Mn2+-stimulated cholesteryl esterase, 1.53 nmol/min per mg; H, lysosomal cholesteryl esterase, 2.25 nmol/min per mg.

236

acylglycerol lipase activity less in osmotically disrupted, recollected pellets from normal fibroblasts than in pellets derived from the mutant cells, probably reflecting the combined measurement of the Mn2~~stimulat~d activity and residual Mn2+-inhibited lysosomal activity that had not been fully released. pH ~~~~~~e~~j~~* ~ysusomal tria~yl~y~~rol lipase and choiesteryl esterase activities had sharp pH optima of 5.5. Little activity was observed at pH values greater than 6.0 (Fig. 3). The Mn’+-stimulated residual activities were optimal at pH 6 with broader curves, and substantial activity was present at pH 7. A second peak of tsiacylglycerol lipase at pH 8 in the liver preparation may reflect the presence of residual hepatic lipoprotein lipase. As has been demonstrated by other workers [24], the activities present in Wolman’s disease and cholesteryl ester storage disease f~broblasts have higher pH optima and broader pH profiles (Fig. 3) than we observed in human fibroblasts which contain lysos~mal activities with sharp pH optima at 5.5. ~ub~@~~u~a~focalization. Fig. 4 shows the relative specific activities of different enzymes in subcellular fractions of liver obtained from rats treated with Triton WR-1339. Relative specific activity is the specific activity of the sub~ellular fraction compared to that of the homogenate [l6]. The results of the experiment depicted in Fig. 4 are similar to those obtained in two other experiments. The recoveries of the marker enzymes varied between 62 and 90% The recoveries of the lysosomal t~a~ylglycerol Iipase and cholesteryl esterase activities varied between 75 and 88% The recoveries of the Mn2 “-stimulated tria~ylgl~~ero1 lipase and cholesteryf esterase activities varied between 74 and 77%. The Mn~‘-stimulated activities had distribution patterns similar to those of two microsomal activities, diacylglycerol acyltransferase [ 791; and NADPH cytochrome c reduetase [25], differing substa~ti~ly from the distribution pattern of the fysosomal triacylglycerol lipase and cholesteryl esterase activities *.

* No t~ac~Ig~ycero$ Iipase or choiesteryl

esterase activities at pfi 5-6 with or without Mn*” present were detected in a highiy purified pernxisomal fraction from rat liver.

Location of Nn’ f-stim.41ated activities

~i~~jn th2

t~a~~~~~~~ pbne of the ~~d~~~~~~~~ ~~r~~u~~33~. Since the Mn’*-stimulated activities appeared to be microsomal, their location was determined within the transverse plane of the microsomal membrane. The latency of mannose-6-phosphatase, a lumenal enzyme, was used as a quantitative index of microsomal integrity ]26], Assay components employed to determine the t~ia~yIgIy~~ro1 lipase and cholesteryl esterase activities did not affect mannosy-6-phosphatase latency (data not shown). Latency of triacylglycerol lipase and cholesteryl esterase could not be estimated since concentrations of sodium taurocholate or sodium deoxycholate that allowed full expression of mannosephosphatase activity ]26,2?] inhibited one or both of the lipase activities. Microsomes that were greater than 94% intact, as determined by manlose-6-pbosphat~s~ latency, were incubated with pronase and recollected by centrifugation (see Methods), In the non-detergent-treated preparation, mannose-6-phosphatase was not inhibited by pronase, indicating that the microsomes had remained sealed during the exposure to pronase (Table I). Since maunose-6-phosphatas~ activity was determined after microsomes were rendered permeable by exposure to taurochoate [28], full recovery of activity also indicates that residual pronase was not present after the 2 h centrifugation ]29]. Pronase substantially inactivated the Mn2 +-stimulated tria~ylg~ycerol lipase and cholesteryl esterase activities in intact microsomal vesicles (Table I). When microsomes that had been disrupted with deoxycholate were then incubated with pronase, the mannose-6-phosphatase activity was inactivated 87% inhibition b,v salts and detergents. To distinguish further between the lysosomal and the Mn”stimulated triacy~gly~ero~ lipase and cholesteryf esterase activities, dependencies on various salts and detergents were investigated. Cobalt acetate bad little effect on the microsomal activities but inhibited the solubilized lysosomal activities 80% at 7.5 mM (Fig. 5A). SimiIar results were obtained using MgCl 2 and CaCl z. The Mn~~-sti~~ulated cholesteryl esterase and triacylglycerol lipase activities were unaffected by incubation with 20 mM MgCl, or 20 mM CaCl,, but the lysosomal activities were inhibited about 604%(Figs. SB, C). Sodium

237

I

I

I

0.25

0.50

0.75

I

I

0.25

I 0.50

I 0.75

% Taurocholate

I

I

I

2.5

5.0

7.5

[Co acetate]

mM

e \‘\;_ B

1oc

Fig. 6. Dependencies of solubilized lysosomal (0, 0) and Mn2+-stimulated microsomal (0, n) cholesteryl esterase (A) (0, @)and triacylglycerol lipase (B), (0, n) activities on sodium taurocholate. Lysosomal or microsomal fractions were exposed to the indicated concentration of taurocholate for 10 min at 0°C. Aliquots were then taken for assay at pH 5.5 (lysosomal) or pH 6.0 (Mn2+-stimulated) as described in Methods. The maximal concentration of sodium taurocholate in the assay was 0.075%. Specific activities for the untreated triacylglycerol lipase and cholesteryl esterase activities, respectively, were 5.7 and 2.35 nmol/min per mg for the lysosomal and 1.11 and 0.31 nmol/min per mg for the Mn2+-stimulated activities in microsomes.

0

5(

-

0

n

0

0

-

I

I

I

I

10 [QC12]

20 mM

C

5 > E z z f t

I

I

I

I 20

~a~l~~ Fig. 5. Dependencies Mn2+-stimulated (0,

of

mM

solubilized

l) cholesteryl

lysosomal (0, n) and esterase (0, W) and tri-

taurocholate (0.5%) had little effect on lysosomal or Mn*+-stimulated cholesteryl esterase activities (Fig. 6A). However, taurocholate enhanced the lysosomal triacylglycerol lipase activity at 0.3% and inhibited less than 25% at 0.75%. The Mn*‘stimulated triacylglycerol lipase was inhibited 50 and 84% at 0.3 and 0.5% sodium taurocholate concentrations, respectively (Fig. 6B). Tissue distribution. The distribution of Mn2+stimulated triacylglycerol lipase and cholesteryl esterase activities was investigated in microsomal fractions from various rat tissues (Table II). Lung and brain contained the highest specific activities. Liver, skeletal muscle, and heart had relatively low

acylglycerol lipase (0, 0) activities on A, cobalt acetate; B, Assays were performed using 5 pg of M&I,; C, CaCI,. solubilized lysosomal protein or 10 gg of microsomal protein at pH 6.0 as described in Methods except that the salt concentrations were varied. Specific activities for the untreated solubilized lysosomal triacylglycerol lipase and cholesteryl esterase were 18.57 and 0.80 nmol/min per mg, respectively. Untreated Mn2+-stimulated triacylglycerol lipase and cholesteryl esterase specific activities were, respectively, 2.75 and 1.24 nmol/min per mg. Data are the mean of four separate experiments.

238

TABLE

I

EFFECT

OF PRONASE

ON MICROSOMAL

ACTIVITIES

Protein recoveries from intact control and pronase-treated microsomes were 9.5 and 6.2 mg. respectively. Disrupted microsomes were treated with 0.05% deoxycholate/O mM KCI. Protein recoveries from disrupted control and pronase-treated microsomes were 8.7 and 4.4 mg, respectively. Mannose-6-phosphatase was assayed after treatment with 0.5% taurocholate. Final assay mixtures contained under 0.05% taurocholate. Mannose-6-phosphatase latency was 94.4% in the initial microsomal preparation. Control and pronase values are nmol/min. Intact

microsomes

Control

+ Pronase

Disrupted Pronase/ control

Control

microsomes + Pronase

(%) Mannose-6-phosphatase Mn*+-stimulated triacylglycerol lipase Mn2+-stimulated cholesteryl esterase

TABLE

754 19.3 10.9

756 5.1 1.9

II

DISTRIBUTION OF MICROSOMAL Mn2+-STIMULATED TRIACYLGLYCEROL LIPASE AND CHOLESTERYL ESTERASE ACTIVITIES IN RAT TISSUES Specific activities were determined in two independent surveys (A and B). All activities from each independent survey were determined on the same day. Tissue

Lung Brain Kidney Intestinal mucosa Liver Skeletal muscle Heart

Triacylgly cerol lipase (nmol/min

Cholesteryl esterase (nmol/min

per mg)

per mg)

Triacylglycerol lipase/ cholesteryl lipase

A

B

A

B

9.00 7.15 4.56

12.4 6.59 6.20

8.30 4.00 3.14

10.30 4.18 4.61

1.2 1.7 1.4

6.72 2.69

4.59 2.12

2.17 1.57

2.36 1.19

2.5 1.7

2.19

1.57 1.23

2.18 2.35

1.61 I .88

0.6

1.22

tissue tissue

1.o

activities. The ratios of microsomal triacylglycerol lipase to cholesteryl esterase ranged from 2.5 in intestinal mucosa to 0.6 in heart.

Discussion Differentiation acylglycerol lipase

of a novel microsomal activity in rat liver from

trithe

100.0 26.4 17.4

Pronase/ control (%)

692 17.4 8.2

87 2.3 1.3

12.6 13.2 15.9

lysosomal triacylglycerol lipase activity was facilitated by the varying responses of each activity to Mn’+. MnCl, inhibited the lysosomal activity and stimulated the microsomal activity. The similar pH optima of the two lipases and the significantly greater specific activity of the lysosomal activity has probably masked the microsomal activity in previous investigations of rat liver. The lysosomal activity could be solubilized by osmotic disruption, indicating that it is not an intrinsic membrane protein, whereas the Mn2+-stimulated activity repelleted after this procedure. A cholesteryl esterase activity assayed in a manner similar to that employed for the Mn*+-stimulated triacylglycerol lipase activity also repelleted after osmotic disruption which released lysosomal cholesteryl esterase activity into the supernatant. Although a non-lysosomal cholesteryl esterase is believed to hydrolyze endogenously synthesized cholesteryl esters [30], this activity has not been extensively characterized. The pH dependencies of the lysosomal and microsomal triacylglycerol lipases and cholesteryl esterases were similar but not identical. The Mn’+-stimulated triacylglycerol lipase activity had a broader pH profile, retaining 80% of its maximal activity at pH 6.5 and 50% at pH 7.0. At pH 6.5 lysosomal triacylglycerol lipase activity was only 10% that of maximal. A rat brain microsomal triacylglycerol lipase with a pH optimum of 4.8 may be similar to the rat liver microsomal activity

239

[31,32]. The effect of Mn*+ on the brain activity was not reported. Virtually no lysosomal cholesteryl esterase activity was seen at pH values above 7. Previously reported pH profiles of acid lipase activity in fibroblasts from patients with Wolman’s disease or cholesteryl ester storage disease exhibited a broader and less acidic pH optimum than did normal cells [24]. Our studies confirmed these results and also demonstrated that triacylglycerol lipase and cholesteryl esterase activities from the fibroblasts lacking the lysosomal activities were stimulated by Mn*+ and were not solubilized by osmotic disruption. Thus, the acid triacylglycerol lipase and cholesteryl esterase activities in tissues from patients with Wolman’s disease and cholesteryl ester storage disease may be microsomal rather than residual lysosomal activities [24]. The Mn*+-stimulated triacylglycerol lipase and cholesteryl esterase activities in rat liver partitioned with the microsomal marker enzymes, diacylglycerol acyltransferase and NADPH cytochrome c reductase. In contrast, the lysosomal activities partitioned with the lysosomal marker enzyme, hexosaminidase (Fig. 4). It has been suggested that hepatic lipoprotein lipase has contaminated other hepatic microsomal preparations and has been falsely identified as a neutral microsomal triacylglycerol lipase [ 121. The pH optimum of the Mn*+-stimulated triacylglycerol lipase, however, is well below that of hepatic lipoprotein lipase (Fig. 3). The Mn*+-stimulated activities were further localized to the cytosolic side of the endoplasmic reticulum under conditions in which microsomal impermeability to pronase was maintained. Pronase substantially inactivated microsomal triacylglycerol lipase and cholesteryl esterase activities but had no effect on the lumenal enzyme mannose-6-phosphatase. These data indicate that the microsomes had remained sealed during the incubation with pronase and suggest that, unlike trypsin [29], essentially all pronase was removed by centrifugation prior to enzyme assay. When microsomes were rendered permeable to the protease by previous detergent treatment, mannose6-phosphatase was inhibited, confirming that mannose-6-phosphatase is susceptible to pronase inactivation after microsomal integrity has been dis-

rupted [ 1,281. These data indicate that triacylglycerol lipase and cholesteryl esterase activities contain pronase-susceptible sites exposed on the cytoplasmic surface of the microsomal membrane and suggest that the Mn*+-stimulated enzymes have active sites that face the cytoplasmic surface of the endoplasmic reticulum. The data are also consistent, however, with protolytic cleavage of a cytoplasmic domain that results in inactivation of a lumenal active site of a transmembrane enzyme. When the protease-treated microsomes were disrupted with taurocholate prior to assay, cholesteryl esterase activity did not increase. Thus, the activity did not appear to rely on the transport of substrate by a protein that was inactivated by pronase. The latency of triacylglycerol lipase could not be determined since detergent treatment inhibited the activity. Although lack of latency would support the concept that the substrate has free access to the enzyme’s active site [l], both trioleoylglycerol and cholesteryl oleate can probably permeate membrane bilayers freely. In this instance, therefore, lack of latency would remain a weak argument for a cytoplasmic active site. Studies with several salts (Fig. 5) show further differences in the susceptibility of the lysosomal and microsomal triacylglycerol lipase and cholesteryl esterase activities to inhibition. The Mn*+-stimulated triacylglycerol lipase and cholesteryl esterase maintained relatively normal activity in the presence of MgCl,, CaCl, and cobalt acetate, whereas the lysosomal activities were inhibited 60-80%. On the other hand, the detergent taurocholate inactivated only the Mn2 +stimulated triacylglycerol lipase (Fig. 6B). The differences in pH optima, the differences in inhibition by various salts, the stimulation by Mn2’, the differential solubilization after osmotic disruption and the different subcellular localizations strongly support the conclusion that at least two intracellular triacylglycerol lipase activities and at least two intracellular cholesteryl esterase activities are present in rat liver. The lysosomal triacylglycerol lipase .and cholesteryl esterase may be dual activities of a single protein [5-71. The microsomal triacylglycerol lipase and cholesteryl esterase activities responded similarly to inhibition by several salts and had similar pH

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dependencies; however, the activities differed in response to phospholipid, taurocholate, N-ethylmaleimide and EDTA, and activities varied independently in several rat tissues. Demonstration that the two microsomal activities are functions of the same or separate enzymes will require purification to homogeneity. The microsomal cholesteryl esterase characterized in this study may hydrolyze endogenously synthesized cholesteryl esters. An active cycle of hydrolysis and reesterification of cholesteryl esters has been demonstrated in mouse macrophages [30]. Location of the cholesteryl esterase activity on the cytosolic surface of the endoplasmic reticulum would provide cholesterol and long-chain fatty acids on the cytosolic side where fatty acid CoA ligase is located [I]. The topography of acyl-CoA : cholesterol acyltransferase has not been determined, but its active site probably faces the cytosolic surface of the endoplasmic reticulum since the long-chain acyl-CoA substrate does not permeate the membrane [33]. Microsomal triacylglycerol lipase may hydrolyze endogenously synthesized triacylglycerol that accumulates in hepatocytes during various pathological states. Location of the activity on the cytosolic surface of the endoplasmic reticulum would result in the diacylglycerol and fatty acid products being available for resynthesis of glycerolipids or for further metabolism by cytosolic or mitochondrial enzymes. Acknowledgements This work was supported by a grant from the National Institutes of Health (HL 25927) and by Basil O’Connor Starter Research Grant No. 5-275 from the March of Dimes Birth Defects Foundation. References Bell, R.M., Ballas, L.M. and Coleman, R.A. (1981) J. Lipid Res. 22, 391-403 Hoyumpa, A.M., Greene, H.L., Dunn, G.D. and Schenker, S. (1975) Am. J. Digest. Dis. 20, 1142-l 170 Mooney, R.A. and Lane, M.D. (1981) J. Biol. Chem. 256. 11724-I 1733

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