Subcellular fractionation, partial purification and characterization of neutral triacylglycerol lipase from pig liver

132

Biochimica et Biophysics Acta, 398 (1975) 132-148 0 Elsevier Scientific Publishing Company, Amsterdam

- Printed

in The Netherlands

BBA 56621

SUBCELLULAR FRACTIONATION, PARTIAL PURIFICATION AND CHARACTERIZATION OF NEUTRAL TRIACYLGLYCEROL LIPASE FROM PIG LIVER

JOANNA

H. LEDFORD*

and PETAR

ALAUPOVIC

Lipoprotein Laboratory, Oklahoma Medical Research Foundation and Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Okla. 73104 (U.S.A.) (Received

March 17th,

1975)

Summary

The subcellular distributions of acidic (pW 4.5) and neutral (pH 7.5) longchain t~acylglycerol lipases (glycerol ester hydrolase, EC 3.1.1.3) of pig liver have been determined. The distribution of the acidic lipase closely paralleled that of the lysosomal marker enzyme, cathepsin D. Approx. 60% of the neutral lipolytic activity resided in the soluble fraction; the distribution of this activity failed to parallel that of marker enzymes for mitochondria, lysosomes, microsomes, or plasma membranes. A method has been developed for purification of the neutral lipase from the soluble fraction by ultracentrifugation. An approximate go-fold purification was achieved, with recovery of 16% of the initial activity. The partially purified neutral lipase exhibited a pH optimum between 7.25 and 7.5. It required 30 m&I emulsified triolein for optimal activity and ceased to liberate fatty acids after 30 min of incubation. The enzymatic activity was destroyed by heating at 60°C. Neutral lipase was inhibited by sodium deoxycholate, Triton X-100 and iodoacetamide. The activity was not inhibited by sodium taurocholate, EDTA, heparin and diethyl-p-nitrophenyl phosphate. Neutral lipase failed to exhibit activity in assay systems specific for lipoprotein lipase, monoolein hydrolase, tributyrinase, and methyl butyrate esterase and showed little or no capacity to hydrolyze chyle chylomicrons or plasma very low density lipoproteins. It is suggested that the function of neutral lipase may be to supply the liver with fatty acids liberated from endogenously synthesized or stored triacylglycerols.

* Present address: Courtauld Institute London WlP 5PB. U.K.

of Biochemistry,

The Middlesex Hospital Medical School.

133

Introduction The ability of mammalian liver to hydrolyze simple organic esters was recognized at the beginning of the century [1,2] , However, the existence of a liver enzyme capable of hydrolyzing long-chain triacylglycerols was not postulated until 1965 [3-71. It has now been established that rat liver contains at least two triacylglycerol lipases. One of these enzymes possesses an acidic pH optimum and is localized in lysosomes [ 8-16 ] , The second has a neutral or alkaline pH optimum and its localization has been variously reported as soluble 161, microsomal [11,17,18] or mitochondrial [19J. Recent evidence also suggests the possib~ity of a hep~n-~nsitive lipase in plasma membranes [ll,lS] . In 1970 Multer and Alaupovic [20] noted that pig liver also contained acidic [pH 4.51 and neutral (pH 7.5) triacylglycerol lipases. The acidic enzyme was clearly lysosomal in origin while the neutral lipase showed highest specific activity in microsomes. The present communication presents in more detail the subcellular distributions of pig liver triacylglycerol lipases, describes a procedure for partial purification of the neutral lipase and reports the characteristics of the partially purified enzyme.

Materials and Methods

Substrates employed in various enzymatic de~rminations included olive oil (Sargent-Welch, Dallas, Texas, U.S.A.), triolein, sodium succinate, adenosine 5’-monophosphate and glucose 6-phosphate (Sigma, St. Louis, MO., U.S.A.), monoolein, tributyrin and methyl butyrate (Analabs, North Haven, Conn., U.S.A.), iodonitrotetrazolium violet and denatured hemoglobin (Nutritional Biochemicals, Cleveland, Ohio, U.S.A.). Standards for analysis of protein content and reaction products were human albumin (Hoechst-Behringwerke, Marburg/Lahn, G.F.R.), palmitic acid (Sigma), iodonitrotetrazolium violet formazan (Nutritional Biochemicals), L-leucine (Mann, New York, N.Y., U.S.A.) and potassium dihydrogen phosphate (J.T. Baker, Phillipsburg, NJ., U.S.A.). Other compounds utilized during the isolation or assay of liver lipases included sodium taurocholate (Maybridge Research Chemicals, Launceston, Cornwall, U.K.), Triton X-100 (Calbiochem, LaJolla, Calif., U.S.A.), and thymol blue and Nile blue A indicators (Allied Chemical, Morristown, N.J., U.S.A.). Chemicals tested for inhibitory action on lipolysis were sodium heparin (as solution, 10 000 U/ml, Upjohn, Kalamazoo, Mich., U.S.A.), EDTA (Calbiochem), sodium deoxycholate (Mann, New York, N.Y., U.S.A.), diethyl-pnitrophenyl phosphate or E-600 (K & K, Plainview, N.Y., U.S.A.), and iodoacetamide (Sigma). Purificationof oliueoil

T~a~ylglycerols were isolated from olive oil by column ~hromato~phy over silica gel G according to a modification of &he method of Crider et al. 1211. The purified preparation was a clear, nearly colorless liquid, containing only triacylglycerol by thin-layer chromatography.

134

~amoge~~~~~o~. Pig livers, from animals of either sex, were obtained fresh from a local meat-packing firm and were held on ice for less than 1 h before use. Fresh livers were either processed imm~iately or divided into pieces of approximately 100 g each and frozen at -12°C. Frozen tissue was used within a l-month period. Fresh or thawed tissue, always obtained from several different lobes of the same liver, was chopped into pieces of about 1 cm3 size. 25 g of chopped tissue were mixed with 200 ml of ice-cold 0.25 M sucrose containing 0.001 M d&odium-EDTA, pH 7.2. The mixture was homogenized in a Sorvail Omnimixer (Ivan Sorvall Co., Newton, Corm., U.S.A.) for 30 s at setting 3 (gentle bomogeniz~tion) or for 30 s at setting 3 and 1 min at setting 10 (ha&r homo~e~~~ation}. Homo~ena~s were filtered through 8 layers of coarse cheesecloth into a chilled confiner, and the pH was adjusted to 7.2 with 5 M KOH. S~~ce~~~~~~~r~e~~o~~~~o~.~~rnoge~a~$ prepared from 50 g of fresh tissue by gentle homogenization were fractionated essentially according to the method of de Duve et al. [ 221 and Appelmans et al, [23]. The procedure is summarized in Fig. 1. Centrifugations were carried out near 0°C in, either a refrigerated Servall cen~ifuge (Ivan Sorvall Co,) with GSA rotor or a Spinco Model L 2-653 ultracentrifuge (Beckman Co., Palo Alto, Calif., U.S.A.) with Type 42 rotor. Final pellets were suspended in approximately 50 ml of sucrose/EDTA solution, and the suspensions were frozen immediately in small aliquots until anatyzed. F~rifi~~~o~ of ~e~~~~~~~~~~~~~yce~o~ lipme. The isolation procedure is summ~ized in Fig. 2. For each experiment a homogenate was prepared from 50 g of fresh or frozen pig liver by harsh homogenization.

Fig.

I,

Subcellular

fractionatwn

of Pig liver

homo$enate.

135 Homo

enate

7-

Centrifugatlo”

30

min,

113,000 x g,

0-c

Fig. 2. Purification of pig liver neutral lipase.

An artificial lipid emulsion was prepared by sonication of a mixture of 3.32 g of unpurified olive oil and 21.3 ml of 12 mM sodium taurocholate solution for two 1-min periods at setting 5 on a Bronwill Biosonik II (Bronwill Scientific, Rochester, N.Y ., U.S.A.) equipped with catenoidal probe. Assuming a molecular weight of triolein for the olive oil, the final concentration of triacylglycerol in the prepared emulsions was 150 E.cmol/ml. This volume of emulsion was mixed with 205 ml of soluble fraction, and centrifuged as described in Fig. 2 in a Spinco L 2-65B ultracentrifuge with Type SW-27 swinging bucket rotor. The isolated lipid cakes were suspended in 230 ml of sucroseEDTA solution for washing by ultracentrifugation. Bound lipase was separated from the washed lipid cakes by density gradient centrifugation in media containing detergent. The media were prepared by dissolving sucrose in 0.01 M phosphate buffer, pH 7.5, to give solutions of density 1.25, 1.15, 1.05, and 1.00 g/ml. Triton X-100 at a concentration of 3 mg/ml was added to each solution. The washed lipid cakes were suspended by aspiration in 17 ml of the solution of density 1.25 g/ml. 8-ml aliquots of this mixture were layered beneath discontinuous density gradients formed by sequential layering of 10 ml of each of the three lighter solutions in centrifuge tubes (1 X 3.5 inch). Centrifugation was carried out in the Type SW-27 rotor. A number of discrete bands could be visualized by their opacity. The triacylglycerol-free fraction of density 1.25 g/ml, which contained the purified activity, was collected through a hole punctured in the bottom of the tube. The 1.25 g/ml fraction was washed and concentrated by ultrafiltration in a 150 ml Amicon apparatus (Amicon Corp., Lexington, Mass., U.S.A.) equipped with a Diaflo PM-10 filter. The sample was placed in the ultrafiltration appara-

136

tus along with 3 vols ice-cold distilled water. Pressure was applied by a flow of 5-7 ljmin N, , and filtration was continued until the solution was reduced to less than 10 ml in volume. The preparation was washed 3 times with l.O-ml vols of water. The final preparation, designated purified neutral lipase, was frozen at -12°C in small aliquots until analyzed. Isolation of lipoproteins and upolipoproteins Very low density lipoproteins were isolated from a fasting hypertriacylglyceremic subject according to the method of Gustafson et al. [24]. Chyle chylomicrons were isolated from the abdominal fluid of a patient suffering from a chylous fistufa. Chylous fluid was centrifuged at 93 000 X g in a Type SW-27 swinging bucket rotor for 1.5 h at 0°C. The packed lipid cake was washed twice by ultra~e~~ifugation in 0.15 M NaCl containing 0.001 M sodium-EDTA at pH 7.0. A-I and A-II polypeptides of apolipoprotein A were isolated according to the procedure of Kostner and Alaupovic [25]. Enzyme assays Lipase. Acidic (pH 4.5) triacylglycerol lipase was measured according to Miiller and Alaupovic [ 201. Neutral lipase activity (pH 7.5) was also determined by the method of these authors [ZO] in the subcellular fractionation studies. The optimal system for assay of purified neutral fipase was comprised of 0.5 ml of 0.02 M phosphate buffer (pH 7.25) containing 2 mg denatured hemoglobin, 0.5 ml of substrate emulsion (45 pm01 of triofein emulsified in 12 mM sodium taurocholate [ 33 ), and 0.5 ml of enzyme preparation. After incubation at 37°C [3] f reactions were stopped and fatty acids were extracted by the method of Dole [ 261. 1 ,umol palmitic acid in n-heptane was added to each assay tube, and standard solutions of palmitic acid in n-heptane were extracted and analyzed in identical fashion. Two methods were used for measurement of fatty acids. In the first procedure, 3-ml aliquots of heptane phase were titrated according to Schnatz [ 27 ] utilizing a Radiometer TTT-lc titrator equipped with TTA-31 titration assembly and SBR-2c recorder (Radiometer, Copenhagen, Denmark). Standard samples of palmitic acid contained 1-3 gmof of fatty acid. For the alternative procedure, 1.5 ml of a freshly prepared mixture of eth~ol~~.~2~ aqueous Nile Blue A sulfate~~.~2 M NaUH (9 : 1 : 0.2, v : v : v) were added to each 3 ml aliquot of heptane phase. Individual tubes were shaken rapidly by hand for 10 s and the phases were permitted to separate. The absorbance of the lower ethanolic phase was determined spectrophotometrically at 640 nm against a solvent blank. Standard solutions of palmitic acid in n-heptane containing 1.0-1.6 pmol fatty acid were used as references. Stock solutions of 0.02% aqueous Nile Blue A sulfate were washed with several volumes of n-heptane to remove extractable impurities and stored at room temperature. Marker enzymes. Succinic dehydrogenase, a marker for the presence of mitochondria, was determined according to the method of Pennington [ZS] . The lysosomal protease cathepsin D was assayed by the method of Beck et al. f29]. Released amino acids were quantized according to Rosen [30] with the modification of Grant f31]. ~lu~os~6*phospha~se~ a microsomal marker, was determined by the method of Hubscher and West 1321. The plasma membrane

enzyme 5’-nucleotidase was assayed according to Emmelot et al. [33]. Inorganic phosphate resulting from glucose-6-phosphatase and 5 ‘nucleotidase assays was quantitated by the method of Fiske and Subbarow [ 343. Monoolein hydroluse. Be&age’s method [ 351 for the measurement of this activity in pig liver preparations was followed except that the substrate used was unlabeled. Sodium taurodeoxycholate served as solubilizing agent for the monoacylglycerol. The reaction was quantitated by measurement of the released fatty acid as in lipase assays. Tributyrinase. The hydrolysis of tributyrin was assayed in a system adapted from Egelrud and Olivecrona [ 361. Small volumes (0.025-0.2 ml) of enzyme preparations were mixed with 0.1 M NaCl, pH 8.25, to a total volume of 1.9 ml in the sample cup of the Radiometer automatic titrating apparatus. The flow of 0.02 M NaOH required to maintain a pH of 8.25 was recorded for approximately 5 min. The reaction was started by injection of 0.1 ml of tributyrin and the flow of titrant recorded for another 5 min; reactions were adequately linear to determine rates. Results were expressed as E.tmol titrant/ min/mg protein, and specific activity was the difference between values in the presence and absence of substrate. Corrections were also made for non-enzymic hydrolysis. The tributyrin was maintained in dispersed form by rapid stirring; assays were carried out in duplicate at ambient temperature (approx. 23°C). Methyl butyrute esterase. The activity of methyl butyrate esterase was measured according to the procedure described by Arndt and Krisch [37] except that the temperature was 23°C. Results of this titrimetric procedure were expressed in the same manner as those for the hydrolysis of tributyrin. Protein determination Protein was determined by the method necessary, lipids were removed from samples analysis of protein content.

of Lowry by solvent

et al. [38]. Where extraction prior to

Results Calorimetric assay for long-chain fatty acids Attempts to quantitate long-chain fatty acids by manual titration in a two-phase system employing Nile Blue indicator [39] were not considered satisfactory due to difficulties in visual detection of the end point. However, the fact that a color change occurred in this reaction suggested the possibility that the assay might be adapted to spectrophotometric quantitation. In the presence of alkali, ethanolic Nile Blue indicator exhibited a pink color which failed to display an absorption maximum within the wavelength range 220-800 nm. In the absence of base, however, the blue indicator solution absorbed maximally at 640 nm. It was reasoned that the mixing of samples of fatty acid with indicator solution previously taken to its alkaline end point should produce blue color by downward titration of the indicator. Experiments were set up to determine conditions necessary for the reaction and to establish whether the color change could be quantitated spectrophotometrically. Known amounts of fatty acid, dissolved in 4 ml n-heptane, were assayed as described in Methods, utilizing color reagents in which the volume ratios of ethanol, 0.02%

1600

0600

0

1

2

3

4

5

pmoles PALMITIC ACID Fig. 3. Relationship of color yield to fatty acid concentration with several experimental color Color reagent mixtures contained absolute ethanol: 0.02% Nile Blue: 0.02 M NaOH in volume 9.0 : 1.0 : 0.5 (.---. ) and 9.0 : 1.0 : 0.2 (“- - - - - -0).

reagents. ratios of

aqueous Nile Blue, and 0.02 M NaOH were varied. The results of two representative assays are shown in Fig. 3, Development of blue color was sigmoidal with respect to fatty acid concentration. A range of linearity occurred in each plot; this range was determined primarily by the ratio of NaOH to Nile Blue present in the color reagent. On the basis of these results, the method was considered to be quite satisfactory for quantitation of long-chain fatty acids released during lipolysis and was adopted for use under the following specific conditions. Standard solutions of palmitic acid in n-heptane contained from 1.0 to 1.6 pmol of fatty acid; all assay samples contained 1.0 pmole fatty acid dissolved in n-heptane and added during the extraction procedure, and the enzyme concentration in assay samples was adjusted to maintain fatty acid release at less than 0.6 pmol.

Subcellular fractionation Subcellular fractionation of fresh pig liver resulted in quantitative isolation of five subcellular fractions: the nuclear (N), the mitochondrial (M), the light mitochondrial (L), the microsomal (P), and the soluble (S). The homogenate and each of the isolated fractions were assayed for their protein content and activities of six different enzymes: succinic dehydrogenase, cathepsin D, glucose-6-phosphatase, 5’-nucleotidase, triacylglycerol lipase at pH 4.5 and triacylglycerol lipase at pH 7.5. The data reported are based on the means of three separate experiments. The recovery of protein in the five subcellular fractions averaged 92% of that in the homogenate. Likewise, recoveries of the enzymatic activities cathepsin D (89%), glucose-6-phosphatase (91%) and lipase at pH 7.5 (95%) were nearly complete. The recoveries of succinic dehydrogenase (79%), lipase at pH 4.5 (80%) and 5’nucleotidase (68%) activities were somewhat less satisfactory. The latter enzyme, especially, appeared rather labile under the experimental

139

Cathcpsm

D

. % TOTAL PROTEIN Fig. 4. Distribution profiles of enwmes in pig liver subcellular fractions. N. nuclear fraction: dzial fraction; L. tight mitochondrial fraction; P, microsomal fraction: S. soluble fraction.

M. mitochon-

conditions. To correct for this variability, the amounts of protein and activities actually recovered in the five subcellular fractions have been taken as 100% in the summary of the data presented in Fig. 4. Purificationof neutraltriacylglycerollipase Determinations of the pH optimum of the neutral lipolytic activity in several preparations of soluble fractions (S) yielded curves similar to that presented by MtiIler and Alaupovic [20] for the pig liver microsomal enzyme. In one instance, the optima of the soluble fraction and the particulate fraction

140 TABLE

I

PURIFICATION S, soluble fraction; Number

OF PIG LIVER EBL-3,

of experiments

NEUTRAL

emulsion-bound

LIPASE bpase fraction

Fraction

8

S EBL-3

6

Purifwd

Protein

neutral

lipase

* Defined as specific activity of fraction/specific ** Numbers in parentheses give range of values.

3. (%)

100 37 ( 22-49) 16 ( 12-26)

100 1 ( 0.4-1.3j** 0.2 ( 0.1-0.2) activity

Lipase activity

(%)

Purification

’

1 41 (2656) 92 (84-111)

of S.

isolated by centrifugation at 113 000 X g for 30 min from a single homogenate were determined. The shapes of the curves were quite similar with peak activity occurring at pH 7.5 in both fractions. Purification of neutral triacylglycerol lipase was carried out according to the scheme presented in Fig. 2. The soluble fraction was chosen as starting material because of its high percentage of total lipase activity and its lack of membranous particles. Results of the purification procedure are shown in Table I. Successful isolation of emulsion-bound lipase fractions depended on maintenance of a smooth, unbroken emulsion. The sodium taurocholate-stabilized emulsions, however, tended to coagulate under certain conditions, including the presence of ions in the starting material or variation of the temperature during centrifugation by more than one degree from 0°C. The bound lipase was separated from the emulsion by density gradient

Fig. 5. PH dependence of neutral triacylglycerol lipase. Enzyme concentration was 14 fig protein/test. Substrate was purified olive oil at concentration of 60 I.cmol/test based on molecular weight of triolein. The buffer system was 0.02 M phosphate/O.1 M glycyl-glycine. Incubations were carried out for 15 min.

0

15


co

45

TIME

15

tn rmnutes

Fig. 6. Time dependence of neutral trlacylglycerol 7.25; substrate was 60 firno trioleinltest. Enzyme Fig. 7, Substrate-concentration for 30 min in 0.02 M phosphate

90

0

15

30 pmoles

45

60

75

TRIOLEIN

lipase reaction. The buffer was 0.02 M phosphate concentration was 14 fig protein/test.

dependence of neutral trlacylglycerol lipase. Incubation buffer at pH 7.25. Enzyme concentration was 23 fig/test.

at pH

was carried

out

centrifugation through media containing Triton X-100. After centrifugation, a number of discrete bands of varying density could be visualized. In initial experiments, the neutral lipolytic activity occurring in each major band was measured. Some lipolytic activity was found in all fractions, but approx. 50% of the total recovered activity occurred in the triacylglycerol-free fraction of density 1.25 g/ml. Attempts to separate bound lipase from the emulsion by several other methods, including treatment with sodium taurocholate and sodium deoxycholate, or extraction with organic solvents, were unsuccessful. Chamcteristics of purified neutral lipase The pH dependence of neutral lipase is shown in Fig. 5. The release of fatty acids as a function of time is plotted in Fig. 6, and Fig. 7 shows the activity of the enzyme as a function of substrate concentration. The lipolytic response was linear over a 4-fold range in enzyme protein concentration (4-l 6 ,ug protein). Pretreatment of purified neutral lipase at 60” or 100°C for short time intervals caused complete loss of lipolytic activity towards triolein. The enzyme was quite stable, however, to preincubation at 37°C for up to 1 h. Effects of proteins included in assay system During the course of purification of neutral lipase, it had been observed that the sodium taurocholate-stabilized triacylglycerol emulsions used as assay substrates coagulated during incubation at 37°C when the enzyme source contained less than 1 mg total protein per assay tube. The addition of denatured hemoglobin to the assay buffer at a concentration of 2 mg/test stabilized the emulsion and permitted determination of the activities of emulsion-bound lipase preparations of low protein content. Albumin at a similar concentration was less effective as a stabilizer. This phenomenon was re-investigated with purified lipase as enzyme source. In contrast to the earlier results, elimination

142 TABLE ir EFFECTS

OF PROTEINS ON HYDROLYSIS

OF TRICLEIN

BY PURIFiED

NEUTRAL

LIPASE

Assays were carried out at pH 7.25 with 45 .umoi triolein/test as substrate. Incubations were for 30 mm. Proteins were added in assay buffer: all protein amounts represent an approximate concentration of 2 . 10M5M in the final mixture. Protein --

Protein added (m&f)

None Denatured hemoglobin Bovine serum albumin Human plasma A-i potypeptide Human plasma A-H pofypeptide

2 2 0.84 0.54

Specific activity ~_..-_13.778 56.667 22.444 1.333 11.556

of all extraneous protein from the assay system no longer caused coagulation of the substrate emulsion. However, as shown in Table II, the activity of purified neutral lipase was stimulated 4-fold by denatured hemoglobin, but not by albumin or the polypeptides of human apolipoprotein A. None of the proteins tested exhibited intrinsic lipolytic activity.

Effects of add ittues The effects of severat compounds on the activity of purified neutral fipase are shown in Table III ~~fo~unately~ it was not possible to test the effects of highly ionic substances such as NaCl and protamine sulfate in this assay system, as they caused severe coagulation of the substrate. Relationship of neutral lipase to other triacylglycerol lipmes

The soluble fraction of pig liver homogenates, starting material for the isolation of neutral lipase, also contained a large percentage of the acidic or lysosomal triacylglycerol lipase. It was considered important to determine the behavior of the acidic enzyme during the purification procedure. In four experiments, the acidic and neutral lipolytic activities were measured on both the TABLE III EFFECTS

OF ADDFPIVES ON HYDROLYSIS

OF TRIDLEIN

BY PURIFIED

NEUTRAL

LlPASE

Assays were performed in optimal system as described in Methods. The concentrations of sodium taurocholate iisted here were the amounts present above the basal Ievei of 4. l& M. Thus, tot& sodium taurocholate concentrations were 5 . 10T3 M in the first case and 8 * 10m3 M in the second. Compound -.-

Concentration

Sodium taurocholate Sodium taurocholate Sodium deoxycholate Sodium deoxycholate Triton X-100 T&on x-100 Iodoacetamide EDTA Hepa&l ~iethyl-p.~trup~e~yl

1 . 1O-3 M 4 110-3 M 1 . 1O-3 M 4. 1Q3M 0.13 mgfmi 1.33 m&ml 5 * 10-Z M 1 1lo-+ M 1 Uhnl 1.165M

Percent of control activity

-

phosphate

106 107 72 6 36 3 27 99 91 94

143 TABLE

IV

COMPARATIVE

PURIFICATION

OF NEUTRAL

AND ACIDIC

Purifications are defined as the ratios of the specific activity of the soluble fraction at the appropriate pH values. Experiment

number

Purification Neutral

1 2 3 4

TRIACYLGLYCEROL

of the purified

enzyme

LIPASES to the specific

activity

of

lipase

59 17 II 103

Acidic lipase 20 7 41 93

soluble fraction and the final purified enzyme. As shown in Table IV, the purification of the acidic enzyme was variable and independent of the purification of the neutral lipase. The soluble fraction and purified lipase failed to release fatty acids from triolein when measured in an assay system specific for the detection of lipoprotein lipase [40] . The converse was also true: a preparation of human postheparin plasma lipoprotein lipase, purified according to Ganesan and Bradford [41], did not release fatty acids from triolein in the liver lipase assay system. Attempts were made under a number of experimental conditions to demonstrate hydrolysis of human chyle chylomicrons and plasma very low density lipoproteins by purified neutral lipase. The basic system consisting of phosphate buffer (pH 7.25), lipoprotein preparation and enzyme preparation was varied by the addition of albumin, Ca2’, Mg2+ (alone or in combination), denatured hemoglobin and sodium taurocholate. In all instances, release of fatty acids was minimal or absent. Relationship of neutral lipase to other esterolytic activities

In a set of three experiments, the soluble and particulate fractions were isolated from liver homogenates, and neutral lipase was then purified from the soluble fractions. Quantitative measurements were made of the methyl butyTABLE

V

DISTRIBUTIONS

OF ESTEROLYTIC

Enzyme

ACTIVITIES

ISOLATION

OF NEUTRAL

LIPASE

Fraction Particulate Specific activity relative to homogenate

Methyl butyrate esterase Tributyrinase Monoolein hydrolase Neutral triolein lipase

DURING

1.8 2.0 1.5 1.4

Soluble

Purified

neutral

lipase

Percent of particulate + soluble activities

Specific activity relative to homogenate

Percent of particulate + soluble activities

Specific activity relative to homogenate

Percent soluble activity

75

0.4 0.5 0.4 0.9

25 24 24 46

0.03 0.2 0.5 100

0.01 0.06 0.24 19

76 76 54

of

144

rate esterase, tributyrinase, monoolein hydrolase, and neutral lipase activities each of the 3 fractions. The averaged results are presented in Table V.

in

Discussion The spectrophotometric assay for long-chain fatty acids developed in the present study offers the simplicity common to titrimetric methods. It is superior to automatic titration [2?] in speed and eliminates the human bias of manual endpoint titration [26,39] . The principal drawback to this method is the relatively narrow range (1.0-1.6 ymol fatty acid) of linear color response. However, for qu~titation of the fatty acids released by lipolytic enzymes this represents little problem since fatty acid release can be maintained within the necessary limits by simple adjustment of the amount of enzyme protein incubated. Results of the marker enzyme studies indicated that subcellular fractionation of pig liver homogenates achieved a partial separation of mitochondria, lysosomes, and microsomes. The distribution of the acidic lipase, measured at pH 4.5, followed very closely that of the lysosomal marker cathepsin D. This confirms the lysosomal origin of this lipase. The occurrence of a rather large percentage of the lysosomal activities in the soluble fraction was probably due to the use of a blade-type homogenizer. The fragility of lysosomes is well recognized, and treatment in a bung Blendor is a known method of releasing their enzymic activities into soluble form [ 221. A precise subcellular localization could not be assigned to the neutral triacylglycerol lipase activity on the basis of these experiments. The activity failed to show a distinct increase in specific activity in any subcellular fraction or to parallel the distribution of any marker enzyme. The high percentage of total activity occurring in the soluble fraction was striking. There are several possible explanations for this unusual distribution. The observed pattern might reflect the presence of two or more enzymes, one of which is localized in the cytoplasm and the other(s) in particulate organelles. Guder et al. [ll] observed a similar distribution of the triolein-hydrolyzing activity at pH 8.5 in rat liver. The activity displayed some tendency to localize in the microsomal fraction, but failed to parallel the distribution of the microsomal marker enzyme, glulose-6-phospha~se. Approx. 40% of the total activity resided in the soluble fraction. These authors suggested the presence of two particulate lipases with alkaline pH optima, one specific to microsomes and the other to plasma membranes. The soluble activity was considered to represent either a third enzyme or activity washed off the microsomes during isolation. Likewise, a grossly similar distribution may be inferred from the data presented by Assmann et al. [15] for the hydrolysis of triolein at pH 9.5 by rat liver subcellular fractions. Again, the implication is made that several lipases are probably contributing to the overall distribution of activity. In neither of these studies, however, was the existence of more than one alkaline triacylglycerol lipase proven. The observed distribution of the neutral lipolytic activity in pig liver could also be explained by assuming the presence of only one enzyme, lipophilic in nature, and lacking a specific subcellular comp~mentation. A free lipophilic enzyme might be expected to adhere to the lipoprotein membranes of all

145

subcellular particles, producing a smeared distribution during subcellular fractionation. That the soluble enzyme is lipophilic has been adequately proven by the success of the isolation procedure based on the formation of an enzymesubstrate complex. The identity of the pH curves determined for the particulate and soluble fractions from a single liver supports the one-enzyme hypothesis. The use of an enzyme-substrate complex as a means for purifying postheparin lipoprotein lipase was first described by Anfinsen and Quigley [42] in 1953. Fielding [43,44] developed a procedure for removal of the purified enzyme from the complex by detergent treatment. Further modifications were made by Ganesan and Bradford [41]. The present study represents the first attempt to use this approach for purification of a lipase other than lipoprotein lipase . The measured pH optimum of 7.25 for purified neutral lipase is similar to the value of 7.5 reported by Miiller and Alaupovic [ 201 for pig liver microsomal lipase. Several investigators [7,17,19,45] have studied long-chain triacylglycerol lipolytic activity at neutral pH (7.2-7.4) in rat liver, but only in the report of Biale et al. [18] has a neutral pH optimum actually been determined. Most studies on the pH dependency of rat liver lipases suggest more strongly alkaline optima: pH 8.0 according to Vavrinkova and Mosinger [4], pH 8.5 according to Guder et al. [ll] and pH U-9.5 in the report of Assmann et al. [ 151. Hydrolytic activity of beef liver preparations toward micellar dispersions of tripalmitin exhibited a plateau of activity between pH 5.6 and 7.6 according to Kaplan and Teng [46] . However, it is uncertain whether enzymes active on such dispersions are identical to the lipases which hydrolyze emulsified substrates. The release of fatty acids by purified neutral lipase leveled off between 15 and 30 min in the presence of apparently non-limiting amounts of substrate. This feature is a common characteristic of long-chain triacylglycerol lipases. Similar curves have been reported for liver lipases [11,17,20], for adipose tissue lipases [ 47-491, and for lipoprotein lipase [ 50-521. The tendency for inhibition of lipolytic activity to occur above the optimal substrate concentration has been reported for long-chain triacylglycerol lipases of rat liver [9,11] and human aorta [ 531, and may be inferred from data presented for dog lipoprotein lipase [ 511 and human adipose tissue lipase [54]. The phenomenon, which also occurred in the present study, has never been explained. The activating effect of denatured hemoglobin on neutral lipase remains unexplained. It does not appear to be required for stabilization of the emulsion when purified lipase serves, as enzyme source. It probably does not serve as a fatty acid acceptor, since albumin is not able to replace it fully. In fact, the bile salt solution present in this assay system probably serves to solubilize released fatty acids [ 551, making an additional fatty acid acceptor unnecessary. In contrast to the result of Mtiller and Alaupovic [20] for pig liver microsomal lipase, purified neutral lipase was inhibited by iodoacetamide. It is possible, however, that the inhibition resulted from interaction of the reagent with the denatured hemoglobin present in the assay system rather than with the enzyme itself. The concentration of Triton X-100 at which complete inhibition of neu-

146

tral lipase activity occurred (1.33 mg/ml) is the same concentration routinely used in the assay for acidic liver lipase [ 201. This and the disparate behavior of the two enzymes during purification establishes their non-identity. Neutral lipase appears different from the alkaline activity of rat liver studied by Guder et al. [ll] on the basis of pH optimum, although the subcellular distributions of the activities are similar. A lipase postulated by these authors to occur in plasma membranes [ll] of rat liver seems to have a pH optimum close to that of neutral lipase, but no other points of similarity were noted. The very high pH optimum (9.5) of the rat liver plasma membrane activity discussed by Assmann et al. [ 151 makes any functional relationship between it and neutral lipase unlikely. The lack of effect of heparin and EDTA on the activity of purified neutral lipase suggests some difference between this enzyme and various species of lipoprotein lipase [ 51,56-591. The failure of neutral lipase tc release fatty acids either from chylomicrons or from triolein in a lipoprotein lipase assay system proves its basic dissimilarity to the lipoprotein lipase type of activity [ 50,59,60-621. The striking differences in the hydrolytic activities of particulate and soluble fractions of pig liver homogenates toward triolein as compared to their activities toward the esters monoolein, tributyrin and methyl butyrate; and the failure of purified neutral lipase to hydrolyze the ester substrates clearly establish the specificity of the enzyme for long-chain triacylglycerols. This is the first time that the substrate specificity of a liver lipase has been thus clarified. It has been noted on several occasions that pig liver [63] and rat liver [64] microsomal esterases failed to hydrolyze long-chain triacylglycerols. These observations and the results of the present study strongly support the view that esterolytic and lipolytic activities reside on different enzyme proteins in this organ. The physiological significance of the pig liver neutral lipase remains uncertain. In view of its lack of specificity for chylomicrons and plasma very low density lipoproteins and its probable intracellular localization, it appears unlikely that the enzyme could participate directly in the catabolism of circulating lipoproteins. It is generally recognized that the liver satisfies a large part of its energy needs from the oxidation of fatty acids. However, it has been assumed that these fatty acids are supplied to the liver from the adipose tissue depots, following mobilization by hormone-sensitive lipase. It is conceivable that the liver may not be wholly dependent on exogenously-supplied fatty acids, but may also be able to utilize fatty acids supplied by the action of lipases such as the presently described one from endogenously synthesized or stored triacylglycerols. Acknowledgments The technical assistance of Mr P. Suttle, Mr A. Suenram and MS E. Phillips is gratefully acknowledged. Purified human post-heparin lipoprotein lipase was lipase assays were kindly provided by Dr Devaki Ganesan, and lipoprotein performed by MS Helen Bass. We thank Mr R. Bums for assistance in the preparation of figures and MS M. Farmer for typing this manuscript. These studies were supported in part by Grants HE-7005 and HE-10575 from the

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U.S. Public Health Service, and by the resources of the Oklahoma Medical Research Foundation. References

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