Strain L fibroblast upases. Purification and properties

Biochimica et Biaphysica Acta, 296 (1973) 411-425 6 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 56200

STRAIN L FIBROBLAST

LIPASES. PURIFICATION

AND PROPERTIES

ERNEST LENGLE and ROBERT P. GEYER Department of Nutrition, Harvard School of Public Health, 665 Huntington Avenue, Boston, Mass. 02115 (U.S.A.) (Received September 4th,

1972)

SUMMARY

Triglyceride hydrolysis by sonicated Littlefield strain L fibroblasts was measured by determining the amount of [I-i4C]oleic acid liberated during a r-h incubation at 37 “C with [r-14C]triolein. Three optimal pH values could be observed. Ap proximately 80 % of the total lipolytic activity was associated with the pH 6.5 lipase. The enzyme was inhibited by PO43 - and sodium taurocholate, but was unaffected by NaCl, heparin, eserine, or cyclic AMP. Treatment of sonicated L cells with neutral solvents or gum arabic caused an approximate 3-fold increase in lipolytic activity. The enzyme from sonicated L cells remained with the 105000 xg supernatant liquid and was purified approximately 45-fold by DEAE-cellulose chromatography. When the purified enzyme was treated with diethyl ether and fractionated by column chromatography with DEAE-cellulose or Sephadex G-200, lipolytic activity was now found in a number of fractions. The potential total enzyme activity of the pH 6.5 lipase on a unit cell basis would be sufficient to account for the rate of removal of intracellular lipid droplets in growing L cells, and offers an explanation for the rapid exchange of acyl groups in such droplets during the time of their formation.

INTRODUCTION

Studies in vitro on mammalian cells have shown that under normal growth conditions most of the cellular lipids were derived from the culture mediumip2. In previous investigations strain L mouse fibroblasts were found to oxidize a portion of exogenous fatty acids to C02, but most of these acids were incorporated into triglycerides and phospholipids. When the concentration of fatty acids exceeded the cellular requirements for a source of carbon and energy, the excess fatty acids were esterified and incorporated into lipid-rich cytoplasmic droplets. These particles contained approximately 90 % lipids, of which 90-92 % was triglyceride; small amounts of polar lipids, cholesterol esters, mono- and diglycerides were present also. No free fatty acids were detected3. Electron micrographs showed that each droplet appeared to be separated Abbreviation:

HEPES, N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic

acid.

412

E. LENGLE, R. P. GEYER

from the surrounding cytoplasm by a thin layer of osmiophilic material which lacked the bilaminar appearance of the usual unit membrane4. With the removal of exogenous fatty acids, the droplets disappeared, but at a rate much faster than could be accounted for as dilution due to cell division. Throughout the disappearance phase, the droplets were randomly distributed in the cell and were not associated with lysosomal structures4. The rapidity of droplet disappearance suggested the presence of intracellular lipases. Their presence was further suggested by the fact that the droplets formed from one substrate fatty acid rapidly equilibrated with other fatty acids when the latter sequentially replaced the initial substrate4. The present paper describes the characterization of a partially purified cytoplasmic lipase found in strain L fibroblasts. These studies utilized [r-‘4C]triolein emulsions, cell sonicates, and enzyme fractions derived from such sonicates. Triolein was used because the predominant cellular fatty acid normally present in these cells in either the neutral or phospholipid fractions was oleic acid5. MATERIALS

The following chemicals were purchased from Eastman Organic Chemicals, Rochester, N.Y. : N,N,N’,N’-tetramethylethylenediamine, N,N’-methylenebisacrylamide, and acrylamide. Bromophenol blue, Florisil, and heparin were obtained from Fisher Scientific Corporation, Fair Lawn, N. J. Phenyhnethylsulfonylfluoride and eserine were purchased from Sigma Chemical Company, St. Louis, MO. Paraoxon was generously supplied by Dr Sheldon Murphy, Harvard University, Boston, Mass. Fatty acid-free bovine albumin was purchased from Nutritional Biochemicals Corporation, Cleveland, Ohio. Hyamine hydroxide and Omniflur were obtained from New England Nuclear, Boston, Mass. METHODS

Propagation of cell line

Suspension cultures of Littlefield strain L mouse fibroblasts were grown in 125 ml screw-capped conical flasks containing 50 ml of a modified Waymouth MB752/1 medium. Each liter of medium contained D-sorbitol (0.1 I g), thymidine (0.01 g), deoxycytidine (0.01 I g), m-inositol (0.04 g), polyvinylpyrrolidone (I .o g) (Antara Chemicals, New York, N. Y .), Methocel, I 5 centipoise (0.25 g) (Dow Chemical Co., Midland, Mich.), and heat-inactivated horse serum (xoo ml) (Grand Island Biological Company, Grand Island, N. Y .). The flasks were agitated on a rotary shaker (Model G27, New Brunswick, N. J.) having a r-inch radial stroke at 120 rev./min. The incubation temperature was 36 “C. A Model B Coulter Counter and size distribution plotter (Coulter Electronics, Hialeah, Fla.) were used to monitor changes in cell number and size. At various time intervals, cell viability was checked employing the trypan blue exclusion method described by Merchant et aL6. The cells were monitored at frequent intervals for possible contamination with pleuropneumonia-like organisms2 and other microorganisms. Substrate

A mixture of 5 &i of [ r-‘4C]triolein (145 mCi per mmole) (Applied Science Laboratories, State College, Pa.) and I g of triolein (Applied Science Laboratories) was

STRAIN L FIBROBLAST LIPASES

413

dissolved in heptane and purified by chromatography on Florisil’, stored in heptane, and repurified whenever necessary. No partial glycerides or free fatty acids were detected in the purified material by thin-layer chromatography. After an aliquot of the triolein mixture containing 330 mg of lipid was evap orated to dryness under a stream of Nz, 15 mg of Pluronic F-68 (a polyoxyethylenepolyoxypropylene block polymer) (Wyandotte Chemicals Corporation, Wyandotte, Mich.) and 2 ml of distilled water were added. The mixture was placed in an ice-bath and dispersed with a Biosonik Sonicator (Bronwill Scientific, Rochester, N. Y.) using a microprobe for a total of 15 min at 5-min intervals just prior to use. Dark phase microscopy (Unitron Instrument Co., Newton Highland, Mass.) revealed a homogeneous, stable emulsion. Most of the lipid particles were approximately 0.5-1.0 pm in diameter, being as large as many of the naturally occurring fatty acid induced cytoplasmic, lipid-rich particles 4. Like the naturally occurring particles, most of the visible emulsion particles exhibited Brownian movement. Lipase assay

For a single assay, o. I ml of the above substrate emulsion was placed in a 3-ml screw-cap reaction vessel containing a mixture of 5 mg fatty acid-free albumin and o. I ml enzyme preparation. The reaction mixture was diluted to a total volume of 1.5 ml with 15 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES) buffer (pH 6.5) (Calbiochem, Los Angeles, Calif.) and agitated with an Aliquot Mixer (Lab-Line Instruments, Inc., Melrose Park, Ill.) at 37 “C for 6o min. Lipolysis was terminated by adding the reaction mixture along with heptane rinses of the reaction vial to 5 ml of Dole’s extraction medium’. Fatty acids were isolated from neutral lipids by one of two methods. The first was by thin-layer chromatography on o.4-mm-thick Silica Gel G (Brinkman Instrument, Inc., Westbury, N. Y.) coated thin-layer chromatographic plates (20 cm x 20 cm) using ascending chromatography in hexan-thy1 ether-glacial acetic acid (60 : 39 : I, v/v/v). The second utilized the ion-exchange chromatograph paper method of Marsh and Fitzgerald’ modified as follows: After the sample had been chromatographed and RF values determined, the lipid bands were removed with scissors, cut into small pieces, and dropped into a counting vial containing I ml Hyamine. A blank was prepared by cutting a piece of treated paper of approximately the same size as the radioactive bands from each chromatogram. Radioactivity was determined in a Nuclear Chicago Liquid Scintillation Counter (Des Plaines, Ill.) with a r5-ml scintillation mixture (8 g Omniflur in I liter toluene-absolute ethanol (2 : I, v/v)). The results were corrected for quenching by the channels ratio method”. Purification of enzyme

Approximately 4 - IO* Littlefield L strain mouse fibroblasts were pelleted by centrifugation, and then resuspended and recentrifuged twice with 0.9 % saline containing 0.025 % Methocel and once with glass distilled water. The final pellet was suspended in 2 ml of ice-cold 15 mM HEPES buffer (pH 6.5) containing 0.88 M sucrose and I mM EDTA. This mixture was sonicated at 20 kHz with a Biosonik sonicator for a total of 60 s in periods of IO-S intervals; each interval followed by a cooling period of about 15 s. The completeness of cellular disruption was determined with a phase microscope.

414

E. LENGLE, R. P. GEYER

After centrifuging the sonicate at 103 x g at 4 “C for 15 min, the supernatant fluid was removed and recentrifuged at ro5 ooo x g at o “C in a Beckman L2-65 ultracentrifuge using SW 50 L swinging bucket rotors. The supematant fraction (S,, s fraction) contained 10-15 mg protein/ml. The particulate fraction was discarded. Column procedure Anion-exchange chromatography on DEAE-cellulose (H. Reeve Angel and Co., Clifton, N. J.) and gel filtration on Sephadex G-200 (Pharmacia Fine Chemicals, Inc., Piscataway, N. J.) were used for additional purification and characterization of the enzyme. The SIOs fraction was separated on a I cm x 25 cm DEAE-cellulose column previously equilibrated with 15 mM HEPES buffer (pH 8.2). The column was eluted by a continuous decrease in PH. Fractions of I .5 ml were collected at a velocity of 0.3 ml/min at 4 “C. Chromatographic separation according to molecular weight of the S, O5fraction was accomplished on a 2.5 cm x 50 cm Sephadex G-200 column. Elution was performed with 0.9 % NaCl at a velocity of 0.3 ml/min at 4 “C. Fractions of 1.5 ml were collected. Blue Dextran 2000 and reference calf serum were used as standards to determine both void volume of the gel and the approximate molecular weights of the eluted cellular fractions. Absorbance of the collected fractions from either anion-exchange chromatography or gel filtration was read at 280 nm. The corresponding protein peaks were pooled and dialyzed overnight against distilled water at 4 “C. The lyophilized protein peaks were reconstituted in I ml of distilled water and assayed for lipolytic activity. Protein analysis Total protein was determined by the method of Lowry et al.” with bovine serum albumin as a standard. Analytical electrophoresis on 7 % acrylamide gel was performed according to the technique of Davis” using bromophenol blue as a marker. RESULTS

Efict of detergent:substrate ratio on enzyme activity A series of substrate emulsions were prepared having different ratios of Pluronit Fd8/triolein, but with the same concentration of the triglyceride in each. At the lowest concentration of Pluronic F-68 used, sonication for longer than 15 min served no useful purpose; therefore, this time interval was chosen in preparing all of the emulsions. Replicate batches of an emulsion of a given composition appeared similar when viewed by means of phase microscopy. Fig. I shows that enzymatic activity was linear when the ratio of Pluronic F-68 to triolein was from 0.023 to 0.046. Within this range, the emulsions were milky-white in color with many lipid particles exhibiting Brownian movement. As the emulsions became opalescent, i.e. as the ratio of detergent to substrate increased to 0.648, there was a decrease in enzymatic activity. E&ct of pH on activity Three pH optima could be consistently observed for the lipolytic activity of Littlefield L cells (Fig. 2). Approximately 80 % of the total lipolytic activity was associated with the pH 6.5 lipase. The addition of 50 I.U. heparin or 1.5 hM cyclic AMP

STRAIN

L FIBROBLAST

‘0

LIPASES

41.5

0.04 0.08 0.12 0.16 0.20 0.24 0.28 0.32 Ratio (mg pluronic F-68 Img triolein 1

Fig. I. Effect of detergent: substrate ratio on lipase activity. Except for the Pluronic F-68 concentration, the standard lipase assay using the Slos fraction was done.

alone or in combination with 3 mM ATP and IO mM MgCl, to the incubation medium did not stimulate lipolysis at any pH. All further studies were done at pH 6.5 (the pH 6.5 lipase). Eflect of

medium composition on lipolytic activity In view of the fact that Huttunen et al.” reported that when rat epididymal fat pads were homogenized in 0.25 M sucrose instead of 0.15 M KC1 there was a significantly higher yield of hormone-activated lipase in the S,,s fraction, it was of TABLE I EFFECT OF MEDIUM

COMPOSITION

Except for the addition of the compounds Slos fraction was done. Bugler (pH 6.5)

0.5 M KH2P04-Na2HP04 o. I M Tri-HCl 0.015 M HEPES

ON LIPOLYTIC ACTIVITY indicated in the table, the standard lipase assay using the

Oleic acid released (pgjassay) Sucrose Buffer only 0.1 M 0.88 M

KC1 ‘0.1 M

0.88 M

113.9

105.3

109.9

113.7

154.6 170.9

152.3 172.3

151.1

153.4

113.9

151.9

170.3

171.0

171.0

interest to see what effect the polarity of the incubation medium had on enzymatic activity. As shown in Table I, the activity in the crude homogenate was not significantly higher when either sucrose or KC1 was used. Lipolytic activity of the pH 6.5 lipase, however, was dependent on the type of buffer used (Table I). Greatest enzyme activity was observed with HEPES buffer, an organic buffer commonly used in tissue culture media.

E. LENGLE, R. P. GEYER

416

C

8 16C 14c

120

100 m x Et30 z n ‘p 60 ._v P 040

\

A 3

9 20

1 Oo

10

2

30

np. (“C) Fig. 2. Effect of pH on lipase activity. The standard lipase assay using the Slos fraction was done. pH was adjusted to values noted in figure. A, 0.2 M acetic acid-NaOH buffer; B, 0.2 M KH2P04-Na2HP04 buffer; C, 0.2 M Tris-HCl buffer. Fig. 3. Effect of incubation temperature on lipase. Except for the incubation temperature, the standard lipase assay using the Slos fraction was done.

Effect of temperature on activity

The optimum temperature for the triolein pH 6.5 lipase was 37 “C (Fig. 3). This lipase was heat labile. If the enzyme preparation was heated to 50 “C in a water bath for 5 min, shock cooled in an ice-bath, and then assayed at 37 “C in the usual manner, over 25 % of the lipolytic activity was lost. E&ct of enzyme concentration on activity

Prior to purification a study was made of the effect of enzyme concentration on activity. Fig. 4 shows that enzyme activity was directly proportional to the number of cells added to the incubation medium in the form of the sonicate. Time course of hydrolysis

The rate of hydrolysis of a triolein emulsion by a crude enzyme preparation was linear with time for 60 min (Fig. 5). Maximum release was achieved within 120 min. Enzyme purajication

The pH 6.5 lipase from L cells, like other lipases, was easily adsorbed on the enzyme from Ca,(PO,), gels l4 . Once adsorbed, however, it was not pos&& to free the gels. An alternative procedure of preincubating the enayme with a triglyceride emulsion for 15 min at 37 “C. ultracentrifuging the resulting enzyme-lipid oomplex, and removing the enzyme from the corr~plex~~also proved to be unworkable. The difficulty seemed to be in the initial formation of a stable enzyme-lipid complex.

STRAIN L FIBROBLAST

180

LIPASES

417

I

120 n % ; 100 L

200

0 : 80 .u 4J -d ?y 60

40

20

40

60

80

loo

*o

~41S,05 fraction

20

40 60 80 Incubation time (min

loo

120

1

Fig. 4. Effect of enzyme concentration on lipase activity. Except for the enzyme concentration, standard lipase assay using the Slos fraction was done. The S i,,s fraction was derived from z cells/ml.

the * IO*

Fig. 8. Time course of hydrolysis of triolein emulsion. Except for the length of incubation, the standard lipase assay using the SIoL fraction was done.

A summary of a typical pH 6.5 lipase purification procedure finally adopted is presented in Table II. At the end of the three steps of the procedure, the specific activity of the enzyme had increased approximately 46 times. However, only 21 % of the total activity in the original homogenate was recovered. Further purification of this material has not thus far been attempted, although the disc gel’electrophoresis data presented in Fig. 6 show that additional purification is possible. During the early attempts to purify the lipase, it was observed that the lipolytic activity in the S,,, fraction was low if sonication and subsequent steps were performed in the absence of I mM EDTA. Phase microscopy revealed the formation of a gelatinous precipitate shortly after sonication of the cell pellet. However, in the presence of EDTA no precipitate was observed and the lipase activity increased. Data from TABLE II PURIFICATION

OF L CELL pH 6.5 LIPASE

Fraction

mg protein

Total activity (mg oleic acidlh)

mg oleic acid/ mg protein

Crude enzyme fraction &es fraction DBAE-cellulose fraction

141.0 92.2 0.654

163.38

0.1159

-_

-

0.1486 5.321

83.9 21.3

1.28 45.9

137.02

34.80

0/eRecovery Puri$cation factor

418

Fig. 6. Acrylamide gel electrophoresis bottom.

E. LENGLE, R. P. GEYER

of partially purified pH 6.5 lipase. Mobility was from top to

later experiments shown in Table IV suggest that the primary effect of the EDTA is as a nonspecific stabilizer of solubilized cellular protein, not as an activator of the lipase. Effect of lipid extraction on enzyme activity About 25 - 10~ cells were distributed to each 3 ml screw-cap reaction vessel. After centrifugation at 150 xg for IO min and removal of the supernate, the cells were sonicated as previously described. The sonicate was mixed thoroughly with either 3 ml of the various organic solvents or I ml of the detergents (Table III) at either room temperature or in an icebath. After 20 min the vials were centrifuged and the organic solvent removed. The particulate matter was placed in a stream of N, to remove the last traces of solvent. The standard lipase assay at pH 6.5 was then carried out, and the results are presented in Table III. Pretreatment with the neutral solvents, diethyl ether and heptane, caused a very large increase in lipolytic activity. These solvents removed chiefly triglycerides and free and esterified sterols with only small amounts of partial glycerides and free fatty acids. Extraction with the more polar solvents greatly decreased the enzymatic activity, and this was accompanied by removal of phospholipid as well as the neutral lipids. The treatment with neutral detergents gave

STRAIN L FIBROBLAST LIPASES

419

TABLE III EFFECT OF LIPID EXTRACTION ON LIPASE ACTIVITY Conditions assay

of lipid extraction described in text. The reaction m&Ire

Solvents

Control Diethyl ether Heptane Benzene Ethanol-diethyl ether (3:1, v/v) Ethanol-diethyl ether (3 :2, v/v) Acetone then diethyl ether Chloroform-methanol (2 : I, v/v) Chloroform-methanol (2: I, v/v)* 5 % Gum arabic 5 % Pluronic F-68 I % Triton WB-1339 5 % Tween 80

was that of the standard lipase

pg oleic acid released Extracted at room temperature

Extracted ice bath

134.3 313.9 240.7 21.7 IO.5

138.0 453.5 314.8 120.5

124.4

4.9

31.0

32.9

46.0

0.0 0.0 338.0 96.4 13.7 0.0

in

10.0 27.0 488.3 239.0 14.2 II.2

* Sonicate lyophilized before addition of organic solvents.

varying effects on the lipase activity. Triton WR-1339 and Tween 80 caused almost complete loss of activity, while the effect of a high concentration of Pluronic F-68 was temperature dependent. Gum arabic had as great an enhancing effect as the neutral solvents. E$ect of lipase activators and inhibitors The effect of a number of compounds which are known to inhibit or activate lipases from different sources were tested (Table IV). Preliminary experiments demonstrated that free fatty acids formed from exogenous triglyceride greatly inhibited the lipolytic activity of the pH 6.5 lipase. As little as 5 mg fatty acid-free bovine albumin per reaction vial could prevent this inhibition. EDTA, Ca’+, and Mg2+ had little effect on this L cell lipase. Pancreatic lipase, however, was reported to be greatly stimulated by Ca’+ (ref. 16) whereas Ca2+ and Mg2+ inhibited the lipolytic activity of rat liver lipase ” . NaCl did not inhibit significantly the pH 6.5 lipase activity in concentrations up to IO M. Similar effects were observed for rat liver plasma membrane’*, liver microsomal lipase I7 , but not for lipoprotein lipaselg and lipases from rat adipose tissue20921.In the present study, heparin or cyclic AMP alone or in combination with ATP and Mg 2+ failed to enhance the lipase activity under the assay conditions. This is in contrast to the hormone-activated lipase described for rat epididymal adipose cellist. The pH 6.5 lipase was inhibited by protamine sulfate. Protamine sulfate has been shown to inhibit liver lipoprotein lipase23, to activate rat liver plasma membrane lipa&*, and to have no effect on rat liver microsomal’lipase”, rat adipose tissue lipase20*2’ and rabbit polymorphonuclear leukocyte acid lipase24. The pH 6.5 lipase of L cells was not inhibited by I mM NaF, thus being less sensitive to this ion-dependent inactivation than rat liver and kidney lysosomal lipase2’, pancreatic lipa.@, rat adipose tissue lipase20*2’, and hormone-activated lipase26. Iodoacetate and Hg2 +

420

E. LENGLE, R. P. GEYER

TABLE IV EFFECT OF LIPASE ACTIVATORS

AND INHIBITORS

Except for the addition of the compounds Sio5 fraction was done.

indicated in the table, the standard lipase assay using the

Compounds 5 10

I IO

0.01 0.1 1.0 0.1 1.0 10.0 0.1 1.0 10.0 0.5

1.0 10.0 I5 75 1.50 1.5 0.15 I .50 15.0 0.5 1.0 10.0 0.1 1.0 I IO 0.1 I .o 0.01 0.10 1.0 0.01 0.1 1.0 0.5 5.0 0.5 5.0 0.5 5.0 0.5 5.0

mg mg PM pM mM mM mM mM mM mM mM mM mM M M M I.U. I.U. I.U. PM mg mg mg mM mM mM mM mM ,uM PM mM mM mM mM mM mM mM mM mM mM mM mM mM mM mM mM

% Control

albumin albumin oleic acid* oleic acid* EDTA EDTA EDTA CaCl,* CaCl,* CaCl,* MgC12* MgCl2* MgCl,*

NaCl NaCl NaCl heparin heparin heparin cyclic AMP+3 mM ATP+Io protamine sulfate protamine sulfate protamine sulfate NaF NaF NaF iodoacetate iodoacetate HgClz HgClz mercaptoethanol mercaptoethanol eserine eserine eserine paraoxon paraoxon paraoxon deoxycholate** deoxycholate** sodium cholate** sodium cholate** sodium glycocholate* sodium glycocholate* sodium taurocholate** sodium taurocholate**

mM MgC12

100.0 103.4 90.3 I.7 101.1 86.6 73.9 72.5 77.9 63.0 99.3 90.7 77.5 90.2 86.6 89. I 99.0 85.2 86.4 101.1 89.7 63.7 35.5 101.6 100.5 43.5 78.7 59.6 88.2 79.9 85.4 114.7 86.3 91.8 88.2 88.4 47.2 15.0 63. I 30.7 80.8 20.7 66.0 33.3 77.3 23.8

* No albumin present. fir Emulsion made with stated bile salt in the absence of Pluronic F-68.

STRAIN L FIBROBLAST LIPASES

421

inhibited the lipolytic activity of the L cell lipase. This is in agreement with rat liver microsomal lipasel’, rat adipose tissue lipase2’p21 and rabbit polymorphonuclear leukocyte acid lipase 24. Eserine, a powerful potential inhibitor of esterases2’, had very little effect on the pH 6.5 lipase. However, I mM paraoxon, an irreversible organophosphorus inhibitor of esterases2*, inhibited 85 % of the lipolytic activity of the pH 6.5 lipase. A decrease in lipolytic activity of the pH 6.5 lipase was observed when bile salts were substituted for Pluronic F-68 in preparing the substrate. Depending on the concentration chosen, either deoxycholate or taurocholate either has no effect or decreases the reaction rate of lipoprotein lipase. The inhibition by high concentrations of these bile salts has been used to distinguish lipoprotein lipase from pancreatic lipase29. E#ect of lipid extraction on enzyme configuration

As shown in Table III, partial removal of the neutral lipids increased enzymatic activity. A study of this phenomenon was made. When the Slos fraction was further fractionated on a Sephadex G-200 column, all of the lipolytic activity was associated with protein peak A, which corresponds to the macroglobulin peak of calf serum. After treatment of this active Fraction A with diethyl ether as described previously, refractionation by means of Sephadex G-200 yielded lipase activity in three additional fractions. Since the lipolytically active protein fragments appear at the 0.5 0.4 0.3 0.2 0.1

0.6 $0.5

t

II

0.4 0.3 0.2 0.1 0 Tube

No.

Fig. 7. Chromatography on Sephadex G-200 column. T’he column was equilibrated and eluted with 0.9% NaCl. (I) Heat inactivated calf serum. (II) Slos fraction. (III) Fraction A of II treated with diethyl ether. Enzymatic activity was found in Peak A of II and Peaks 1-4 of 111.

E. LENGLE, R. P. GEYER

422

5.0 60 7.2* 8.2a

0.3

5.0

0.2

6.0

0.1

7.0

0

8.2 Tube NO.

Fig. 8. Chromatography on DEAE-cellulose column. The column was equilibrated with 15 mM HEPES buffer (pH 8.2) and eluted with decreasing pH gradient. (I) S 105fraction. (II) Sephadex G-ZOOenzymatically active fraction (Peak A of II, Fig. 7) treated with diethyl ether. Enzymatic activity was found in Peak A of I and Peaks 1-7 of II.

extreme lower range of separation for this column and since adequate standards were not available, the molecular weight of these fractions cannot be estimated. If the ether-extracted material from Fraction A was separated on DEAE-cellulose, six additional anionic peaks were obtained (Fig. 8). Stability

of the enzyme

The crude enzyme was stable at room temperature for a few hours. It could be stored at - 20 “C for over six months, and at 4 “C for at least a week, without loss of activity. The enzyme fraction eluted from Sephadex G-zoo could be stored at - 20 “C for over a month and repeatedly frozen and thawed without appreciable inactivation. However, the DEAE-cellulose fraction could be stored at - 20 “C for only a few days before inactivation occurred. Both the Sephadex G-200 and DEAEcellulose fractions lost approximately IO % of their activity when stored overnight at 4 “C. DISCUSSION

The enzyme described in this paper should be termed a lipase rather than an esterase since it is active on water-insoluble triolein and is not inhibited by eserine. Benzonana and Desnuelle have reported that pancreatic lipase activity depends primarily on two phenomena: An oil-water interface and the number of enzyme molecules adsorbed at the interface 3o. Thus, the rate of hydrolysis is a function of the total surface area of the interface rather than the substrate concentrationf6. Increased Pluronic F-68 concentrations gave finer dispersions, but the increased total surface area was associated with less lipolysis. This loss of enzymatic activity was not due solely to the concentration of the Pluronic F-68. No significant inhibition was detected when a sonicated emulsion having a detergentltriolein ratio of 0.04 was sup-

STRAIN L FIBROBLAST

LIPASES

423

plemented with enough Pluronic F-68 to bring the ratio to 0.32. However, if this mixture was sonicated inhibition occurred. Other effects such as molecular organization30 may come into play as the detergent: substrate ratio increases beyond some point. The lipolytic activity of strain L cells had three different pH optima (Fig. 2). Guder ei aZ.31have reported that three distinct lipases, which differ in localization and substrate specifity, were present in rat liver. An acid lipase (optimum activity at pH 5.0) was shown to be lysosomal in origin; an alkaline lipase (pH 8.5) was found to sediment with the microsomes; and a heparin-activated lipase (pH 7.5) was located in the plasma membrane. The three L cell lipases may be compartmentalized similarly. TABLE V COMPARISON OF PANCREATIC LIPASE, LIPOPROTEIN ACTIVATED LIPASE, AND L CELL pH 6.5 LIPASE

LIPASE, HORMONE-

Efector agent or property

Enzyme studied Puncreatic lipase* Lipoprotein lipase* Hormone-activated lipase*

L cell pH 6.5 lipase

Albumin

No effect or

Taurocholate

stimulation Stimulation

Protamine sulfate

Stimulatiou

-

Stimulation

-

Inhibition

No effect

Noeffector inhibition Strong inhibition

Inhibition

NaCl

No effect

Strong inhibition

po4sF-

-

PH Molecular weight

8.0-9.0 38 ooo**

Inhibition No effect or slight inhibition 8.0-8.5 73 ooo***

No effect or slight stimulation No effect or slight stimulation Stimulation Strong inhibition 6.o-7.5 5ooooot

6.5 8ooooo

* ** *** t

Slight inhibition

No effect Inhibition Slight inhibition

G. Hubscherz6. L. Sarda et al.“z. C. J. Fieldingis. J. P. Schwartz and R. L. Jungas2*.

Table V summarizes the pH 6.5 lipase’s characteristics and compares them with three classical lipases present in the tissues of higher animals i.e. pancretic lipase, lipoprotein lipase, and hormone-activated lipase. The pH optimum of the hormoneactivated lipase lies between 6.0 and 7.5. This range is lower than the pH optima reported for pancreatic lipase and lipoprotein lipase but is compatible with the pH 6.5 lipase of Littlefield L cells. The pH 6.5 lipase, hormone-activated lipase and pancreatic lipase are not at all or slightly inhibited at concentrations of NaCl which strongly inhibit lipoprotein lipase. The hormone-activated lipase is more sensitive to inhibition by F- than either pancreatic lipase, lipoprotein lipase, or pH 6.5 lipase. Phosphate buffers stimulate the enzymatic activity of the hormone-activated lipase but inhibit lipoprotein lipase and pH 6.5 lipase. Finally, the molecular weight of a purified lipoprotein lipase from rat post heparin plasma was reported to be 73000 (ref. 15). The molecular weights of pancreatic lipase and hormone-activated lipase are 38000 (ref. 32) and 5ooooo (ref. 22), respectively, while that for the pH 6.5 lipase found in strain L cells is estimated to be at least 8ooooo. These data show that the pH 6.5

424

E. LENGLE, R. P. GEYER

lipase does not fit all the criteria usually used to differentiate the three classical lipases. The diethyl ether or heptane stimulation of lipase activity (Table III) may be due in part to removal of endogenous triglycerides which would otherwise decrease the specific activity of the labeled substrate. This is probably not the entire explanation since gum arabic, which is not known to extract lipid material, also caused enzyme activation. It is possible that the increased enzymatic activity observed after treatment with either detergents or organic solvents may be due to an unfolding of the lipase, thus exposing more active sites of the enzyme to substrate molecules. The low pH needed to remove the various protein fragments from the DEAE-cellulose column implies that the native enzyme molecule contains a number of very strong ionizable protein groups. These groups are not readily accessible under normal conditions. They may be buried within the folded enzyme molecule and/or intimately associated with an enzyme-apolipoprotein complex. The drastic reduction in lipolytic activity observed when an attempt was made to remove the phospholipids supports the latter hypothesis. Schneeberger et ~1.~ by means of electron microscopy and chemical analysis, studied the formation and disappearance of cytoplasmic lipid-rich particles in L cells. The intracellular particles obtained by feeding the cells oleic acid were essentially triolein in composition. Their data showed a biphasic curve for removal of these intracellular particles. The cells lost approximately 0.0181 pmole triglyceride or 0.0543 pmole fatty acid per 10~ cells during the first 24 h and 0.0282 pmole triglyceride or 0.0846 pmole fatty acid per 10~ cells during each of the next two subsequent 24-h periods. From the data in the present paper the lipolytic activity per 10~ cells per 24 h could hydrolyze 0.0158 pmole triolein yielding 0.0474 pmole oleic acid. If activation of the enzyme(s) occurred in the cells similar to that observed with the diethyl ether or gum arabic in the cell-free preparation, approximately 0.0474 pmole triolein could be hydrolyzed per 10~ cells per 24 h with the liberation of 0.1422 pmole oleic acid. Thus, the activity of the lipolytic enzymes of the L cell is sufficient to account for the observed rate of intracellular triolein remova14. This activity also offers a mechanism for the rapid exchange of acyl groups of intracellular lipid particles when different substrate fatty acids are given sequentially to the L cells4. In this case constant triglyceride biosynthesis and breakdown makes for a rapid equilibration of the acyl groups in the triglyceride. ACKNOWLEDGMENTS

The authors wish to thank Dr John W. Littlefield (Massachusetts General Hospital, Boston, Mass.) for suspension cultures of strain L mouse fibroblasts, Mrs M. McGagh for her technical assistance, and Mrs Elnora Fairbank for her aid in the preparation of the manuscript. This study was supported in part by U.S. Public Health Service Nutrition Training Grant 5-TOI-GM 333-1 I, National Cystic Fibrosis Research Foundation, and Nutrition Department’s Research and Teaching Fund, Harvard School of Public Health. REFERENCES I Bailey, J. M. (1967) in Lipid Metabolism in Tissue Culture Cells (Rothblat, G. H. and Kritchevksy, D., eds), pp. 85-1 I 3, Philadelphia, Pa.

STRAIN L FIBROBLAST

LIPASES

425

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