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Lipolytic activity of adipose tissue IV. The diacylglycerol lipase activity of human adipose tissue
25
Biochimica et Biophysics Acta, 369 (1974) 25-33 0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
BBA 56494
LIPOLYTIC
ACTIVITY
OF ADIPOSE TISSUE
IV. THE DIACYLGLYCEROL TISSUE
LIPASE ACTIVITY
OF HUMAN ADIPOSE
H. GIUDICELLI, N. COMBES-PASTRY? and J. BOYER
Unite' Me’tabolique, Centre Hospitalier Rtfgional de Marseille, H6pital de la Conception, 13385 Marseille, Ckdex 4 (France) (Received April 22th, 1974)
Summary Diacylglycerol lipase activity of human adipose tissue was studied by using emulsified glycerol di[9,10-3 H] oleate as substrate. A partially purified preparation of this lipase activity was obtained through stabilization of the enzyme in glycerol-water mixtures. Enzymic hydrolysis was maximum in 4 mM diolein and 1% serum albumin, at pH 7.2 and 37°C. With all preparations tested, the rate for triolein was found to be l/10-1/30 of that of triolein, depending on the experimental conditions. It was shown that a number of intrinsic properties are shared by di- and triacylglycerol lipase activities. Thermal stability (in the presence and absence of glycerol), susceptibility to bile salts and binding affinity for specific lipid interfaces are similar for both activities. This, along with a uniform behaviour upon various purification procedures, support the contention that di- and triacylclycerol lipase activities of human adipose tissue are referable to a single catalytic protein.
Introduction Detailed information concerning the number of lipases in adipose tissue is very fragmentary. As a prerequisite of a project designed to study the physiological mechanism of lipolysis, we had to investigate the lipase distribution in human fat. In this line, we have recently partially purified and characterized a triacylglycerol lipase activity [ 11, the catalytic properties of which are consonant with those of “hormone-sensitive” lipase, essentially described in the rat [2,3]. Human fat also contains a monoester lipase [4-S], active in vitro towards monoacylglycerol and esters of fatty acids and short-chain normal primary alcohols. Tri- and monoacylglycerol lipase activities are very likely to
26
be due to different enzymes. An acylcholesterol lipase activity has also been recently identified [7] in the same tissue. This sterol monoester lipase seems itself to be distinct from monoacylglycerol lipase. So far none of the human adipose tissue preparations tested in our laboratory was found to contain detectable levels of a triacylglycerol lipase activity identifiable as lipoprotein lipase .
This paper is concerned with the characterization of diacylglycerol acylhydrolase activity of human adipose tissue. The data presented below support the contention that, in this tissue, di- and triacylglycerol are hydrolyzed by the same enzyme. Materials and Methods Glycerol di[9,10-3 H] oleate (0.6 @i/mole) was purchased from the Institut des Corps Gras de Marseille, and has been purified by chromatography on Florisil [8]. The final product, used as substrate for the lipolytic assays, was over 98% radiochemically pure and had a composition of about 40% 1,2-diolein and 60% 1,3diolein. Glycerol tri[9,10-3 H] oleate (0.5 Ci/mmole) (The Radiochemical Centre, Amersham) was purified by thin-layer chromatography on silica gel using a system previously published [4] . Unlabeled triolein and diolein (Sigma Chemical Co.) were obtained over 98% pure by chromatography on Florisil [8]. In all substrate purification procedures, artifacts caused by Florisil-catalyzed transesterification reactions were carefully avoided [ 91. Bovine serum albumin B grade (fatty acid-poor) was obtained from Calbiochem (San Diego, Calif.). (NH,), SO,, was recrystallized from EDTA [lo]. Radioactivity of 3 H and ’ 4 C was measured as reported earlier [ 51. Experimental
procedures
Specimens of human subcutaneous adipose tissues were obtained during surgery. Lipase activity was extracted and partially purified as recently described [ 11. With this procedure, lo-30% of the total activity are usually found to be associated with material of low density collected after serial (NH4 )2 SO4 fractionation of a pH 5.4-precipitated fraction. This 20-40-foldpurified preparation is active towards diolein and triolein. It has no tributyrinase activity but still retains high levels of activity towards long-chain monoacylglycerol. It usually contains, by weight, at least twice as much lipids (mainly neutral lipids) as proteins. All the purification procedures were carried out in 25% glycerol, which protects the enzyme(s) against denaturation [ 111. Protein concentrations were determined as reported [l] . The assay of triacylglycerol lipase activity, using [ 3 H] triolein as substrate, has been described [ 11. Diacylglycerol lipase activity was assayed employing the same procedure but substituting 5 pmoles of [3 H] diolein as substrate, in a final volume of 5 ml containing; 0.01 M sodium phosphate buffer, 1% serum albumin and the enzyme, at the final pH 7.2. It has been verified that the substitution of [3 H] diolein for [3 H] triolein in the assay medium does not affect, to any significant degree, the isolation and the counting of the [ 3 H] oleic acid liberated during hydrolysis.
21
Throughout this work, lipase activities refer to initial reaction rates determined by plotting the amounts of [” H] oleic acid released 0,lO and 20 min after the addition of enzyme. Enzyme kinetics were of zero order over the 20 min duration of the assay. For each value of activity, duplicate assays were reproducible within 10% of [3 H] oleic acid liberated. One unit of activity corresponds to the release of one E.tmole of acid per min, at 37” C. Results Characterization of diacylglycerol lipase activity The influence of pH on the hydrolyzing activity of human adipose tissue towards diolein is shown in Fig. 1. This activity has a broad maximum between pH 6.8 and pH 7.2, i.e. at a pH range slightly lower than with triolein as substate. Fig. 2 shows the increase of the reaction rates measured at pH 7.2 as a function of increasing amounts of substrate. Maximum velocity was attained in 4 mM diolein. In 1 mM, hydrolysis occured at a rate amounting to about 75% of the maximum velocity. The values plotted according to Lineweaver and Burk (Fig. 2, inset) give an apparent K, of 0.5 mM. The measured catalytic activity of the extracts towards diolein was dependent on the concentration of serum albumin in the assay medium (Table I). The hydrolysis rate showed a 4-fold increase when albumin concentration was raised from 0.01% to 2.5%. At higher concentration, the hydrolysis rate tended to fall off. Under similar conditions of assay (Table I), serum albumin inhibited the hydrolysis of triolein. The lipolytic activity towards diolein was consistently found to be lo-40 times greater than that towards triolein. The magnitude of the difference was clearly dependent on the experimental conditions. Table I shows that the ratio of di- to triacylglycerol lipase activity measured in the same enzymatic preparation may be as different as 3 and 28 in 0.01% and 2.5% albumin, respectively.
PH
Fig. 1. Effect of pH on lipase activity. Initial adjustment at the indicated pH values. and pH assays, have been carried out under pH-stat control. They necessitated the addition of minimum amounts of HCl or NaOH. Enzymatic assays were as indicated under Materials and Methods.
28
~-3--?-&
6
0
Diolein
Fig. 2. Hydrolysis Lineweaver-Burk
(mM)
of [3H]diolein plot.
as a function
Comparative determination enzyma tie preparations
of substrate
concentration,
at pH 7.2 and 37OC. Inset:
of di- and triacylglycerol lipase activities in various
Hydrolyzing activities towards di- and triolein have the same pattern of distribution upon serial (NH4 )Z SO4 fractionation (Fig. 3). In this experiment, 5500 munits of activity towards diolein (versus 310 munits towards triolein) were found to be bound to the floating fraction, with a specific activity of 150 munits/mg of proteins. This latter value indicated a 22fold purification over the crude extract. As for triacylglycerol lipase activity [l] , the pellets sedi-
TABLE EFFECT
I OF ALBUMIN
ON DI- AND TRIACYLGLYCEROL
LIPASE
ACTIVITIES
Activities were measured against di- and triolein as indicated under Materials and Methods. The indicated amounts of serum albumin were added to the assay medium before sonic emulsification of the substrate. In all cases. enzyme kinetics were of zero order over the 20-min duration of the assays. --.-. Albumin
Lipase activity
(%)
0.01 0.05 0.10 0.50 1.0 2.5 5.0
--_-
~____
--
(munitslml of extract) _____-__
Diolein
Triolein
16 21 31 59 68 65 53
5.1 4.8 4.3 4.0 3.8 2.3 2.0
~towards
29
Saturation
of(NH,),SO,
Fig. 3. Distribution pattern of di- and triacylglycerol lipase activities upon serial (NH4)2SO4 fractionation in 5% increment from 0.15 to 0.80 of saturation. Fractions are those obtained from the experiment presented in ref. 1 (Fig. 1). in which a pH 5.4 precipitate served as enzyme source. Prior to assay. each fraction was dialyzed for 24 h at 4OC against four 500~ml changes of 10 mM sodium phosphate (PH 7.4) containing 25% glycerol and lo4 M EDTA. Di- and trioleinase activities were concomitantly assayed in each dialyzed fraction. 1 to 3 assays contributed to each value. The dotted column represents dioleinase activity in the floating layer collected after centrifugation of the 0.40 (NH4)2SO4 fraction.
mented above 0.40 of saturation of (NH4 )n SO4 contained enzyme(s) of lesser specific activity, with a much lower ratio, by weight, of lipids to proteins. Likewise, di- and triacylglycerol lipase activities were distributed evenly in four fractions taken at various stages of purification, with a ratio of di- to triacylglycerol lipase activities averaging 15 (Table II). A comparable uniformity was noted when both activities were concomitantly assayed in fractions obtained by successive differential centrifugation (Table III). TABLE
II
RATIO OF DI- TO TRIACYLGLYCEROL CATION
LIPASE
ACTIVITY
AT SUCCESSIVE
STAGES
IN PURIFI-
Various enzymatic fractions were concomitantly assayed towards di- and triolein as substrate. The dioleinase specific activity of the fractions ranged from 6.8 to 150 munits/mg of proteins. Comparison between fractions does not hold, since all did not derive from a single purification procedure. Fraction
Total homogenate 12 000 X * supernatant pH 5.4 precipitate Fraction floating in 40% (NH&S04
Lipase activity
(munits/ml)
Diolein
Triolein
25 16 120 550
1.9 1.1 9.4 31
towards
Ratio
13 15 13 18
c_i_..2_
0
.A
.A.
-L
-
24
12 lncubotton
i..i
36
48
at 50”C(min:
Fig. 4. Comparative behaviour of di- and triacylglycerol fipase activities upon incubation at 50°C. Aliquots were taken at the indicated times from the incubated extracts and concomitantly assayed at 37” C towards diolein (black symbols) and triolein (open symbols) as substrate. A pH 5.4 precipitate served as enzyme source. Assays were performed on samples incubated in the absence (circles) or presence (squares) of 25% glycerol. Activity is expressed as percent of control values measured in the extracts kept at 4’C, immediately prior to incubation.
Fig. 4 shows that di- and triacylglycerol lipase activities elicited an identical pattern of inactivation upon incubation of a purified extract at 50°C. Moreover, both activities were equally and fully protected by inclusion of 25% glycerol in the extract. Hydrolyzing activities towards di- and triolein were consistently found to be inhibited by about 80% and 90%, respectively, in the presence of 10 m&l sodium taurocholate in the assay medium. Glycerol had only a slight and variable protective effect against this inactivation.
TABLE
III
RATIO TION
OF DI- TO TRIACYLGLYCEROL
The enzymatic Methods.
preparations
derived
from
-. Enzymatic fraction
Total homogenate 600 X g supernate 12 000 X g supernate 100000 X g supernate
LIPASE
ACTIVITY
UPON DIFFERENTIAL
a single tissue sample.
Assays as indicated
-~-
CENTRIFUGAunder Materials and -
Total lipase activity --.
(munits) _l____-
Diolein
Trio&n
2460 1890 1450 1310
77 63 43 38
towards
Ratio
32 30 34 35
31 TABLE
IV
COMPARATIVE DIOLEIN
ADSORPTION
AND TRIOLEIN
OF
DI-
AND
TRIACYLGLYCEROL
LIPASE
ACTIVITY
ONTO
INTERFACES
The enzymatic extract (12000 X g supernatant fluid) was prepared in 10 mM sodium phosphate (PH 7.4) containing 104M EDTA and 25% glycerol. A 20 ml-aliquot fraction was dialyzed for 2 h at 4’C against l-l of a solution made of 10 mM sodium phosphate and 10”M EDTA (final pH 7.4). without glycerol. 7.7 ml of the dialyzed preparation were gently stirred for 90 min at 4’C with either 1 ml of a diolein emulsion or 1 ml of a triolein emulsion. Each emulsion was prepared by sonicating with a Branson sonifier B-12 (Setting 1 for 10 s at 4’C) 1 g of acylglycerol with 1 ml of 10 mM sodium phosphate buffer (pH 7.4). Both mixtures were then centrifuged at 100000 X g for 1 h at 4OC and the clear supernatant fluids were concomitantly assayed for di- and trioleinase activity. Similar samples containing no awlglycerol emulsion were run in parallel: the lipase activity measured in the corresponding 100000 X g supernatant fluids (240 munits and 15 munits towards di- and triolein, respectively) were taken as control values (100%). Activity
(%) in the 100000
Added acylglycerol emulsion
Diolein
Triolein
None Diolein Triolein
100 7 34
100 11 45
X g supernate
towards
Behaviour of di- and triacylglycerol lipase activities in the presence of a lipid interface Data presented in Table IV show the comparative behaviour of di- and trioleinase activities in the presence of diolein or triolein emulsions at 4°C. The disappearance from the supernatant fluid of di- and trioleinase activities are quantitatively similar with both emulsions. However, diolein retained both activities with an efficiency greater than triolein, presumably indicating a lower affinity of the enzyme(s) for triacylglycerol. This difference of affinity is not reflected by the apparent Km values, which have been found to be identical (0.5 mM) for both substrates (see ref. 1). However, it should be recalled that dealing with water insoluble esters, Km values expressed in molar concentration cannot be accurate, and may fluctuate from one to another experiment. Interestingly, inclusion of 2% serum albumin in the mixture decreased by 40-60% the retention of di- and trioleinase activities by both emulsions. Discussion It is commonly admitted that, in adipose tissue from most animal species, triacylglycerol lipase differs in a number of properties from monoacylglycerol lipase, and that each activity is referable to a distinct catalytic protein [ 12181. We have indicated earlier [6] that under optimal experimental conditions, that is, in the presence of 10 mM sodium taurocholate in the assay medium, a 39-fold purified preparation of human adipose tissue monoacylglycerol lipase had no activity towards long-chain diacylglycerol. It is therefore most likely that in the present work, the monoacylglycerol hydrolyzing activity retained in the purified extract does not contribute to diolein hydrolysis. However, it is of interest to note that, so far, human as well as animal adipose tissue mono- and
32
diacylglycerol lipases on one hand, mono- and triacylglycerol lipases on the other hand, have not been separated by physical methods, and seem actually tightly associated in a multienzyme complex (Verine, A. and Boyer, J., unpublished). In view of our data, it is our present working hypothesis that diolein and triolein are hydrolyzed in human adipose tissue by the same enzymatic entity. Several lines of evidence support this interpretation, and especially those which essentially reflect identical intrinsic molecular properties: thermal stability, protection by glycerol (see ref. ll), susceptibility to bile salts and binding affinity for lipid interfaces. Failure to separate di- and triacylglycerol lipase activities by various purification procedures only represents tentative clues, since the degree of purification attained is still very partial. In contradiction with these common properties Fig. 1 shows that the pH curves for the two substrates are slightly different (by a factor of about 0.5 pH unit with respect to the optimum pH value). However, it should be recalled that similarity of pH optimum for two substrates is not necessarily to be expected, even when both are acted upon by the same enzyme. We have already reported such slight differences with human adipose tissue monoester lipase [ 61. Also, di- and triacylglycerol lipase activities are apparently inversely influenced by the addition of serum albumin to the incubation medium. Such a differential effect is observed whether or not Ca” (10 mM) is present in the assay system. It has been recognized [18] that albumin, at physiological pH value, binds the fatty acids released during lipolysis, thereby preventing inhibition of the reaction. However, current findings from our laboratory (Jubelin, J., and Boyer, J., unpublished) indicate that albumin exerts this stimulating effect within an optimal range of concentration, variable with the experimental conditions. Beyond this range, albumin has been consistently found to inhibit lipolysis. These observations might explain the differential effect of albumin on diolein and triolein hydrolysis (Table I) attributable, at least in part, to the basic difference in the reaction rates. Within a given range, the increase of albumin concentration stimulates the rapid hydrolysis of diolein (generating relatively large amounts of fatty acids), whereas it inhibits that, much slower, of triolein. The reason for the inhibition is not known. But it is remarkable that the presence of 2% albumin decreases the binding affinity of the enzyme for diolein and triolein interfaces. In practice, it is therefore important to carefully determine, on an experimental basis, the amount of albumin optimal for each assay system. In any case, it should be emphasized that the in vitro hydrolysis of diolein and triolein are virtually null in the absence of added albumin. If indeed diolein and triolein are attacked in human adipose tissue by a single enzyme, the difference between their respective hydrolysis rates should be noted. It is assumed that lipases (and esterases in general), attack the carbonyl group of fatty esters as a nucleophile. Pancreatic lipase, in accordance with a nucleophilic mechanism, hydrolyzes more rapidly triolein than diolein [ 191. Since the reverse order is observed in vitro with human adipose tissue lipase, it is probable that steric factors, in this latter case, counteract the inductive effects and hamper the formation of the enzyme-ester complex. If, as suggested [20], an essential requirement for the lipase attack is that the aliphatic chains
33
can conveniently bend away from the enzyme, it is understandable that a greater mobility of the chains in diolein, as compared to that in triolein, may facilitate the approach of the carbonyle group by the enzyme. As a consequence, the measured rates of hydrolysis of the first primary ester bond of triacylglycerol, termed in this (and other) paper “triacylglycerol lipase” activity, might be theoretically overestimated, due to the concomitant attack of the di- and eventually monoacylglycerol produced. In practice, the very low reaction rates dealt with in adipose tissue preparations, i.e. the very low concentrations of partial acylglycerol formed, as well as the permanent reference in the present work to initial reaction rates, should render minimal these difficulties. Acknowledgements We wish to thank Professor G. Michotey for his courtesy in making available human adipose tissue. This work was supported by Grant No. 732.084.7 from the Institut National de la Sante et de la Recherche Medicale and by the Fondation pour la Recherche Medicale Francaise. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Giudicelh, H., Pastre, N. and Bayer, J. (1974) Biochim. Biophys. Acta 348. 221-231 Hollenberg, C.H.. Raben, M.S. and Astwood, E.B. (1961) Endocrinology 68, 589-598 Rizack, M.A. (1961) J. Biol. Chem. 236,657-662 Bayer, J., Amaud-Le Petit, J. and Charbonnier, M. (1971) Biochim. Biophys. Acta 239. 353-356 Charbonnier. M.. Amaud, J. and Bayer, J. (1973) Biochim. Biophys. Acta 296,471-480 Arnaud, J., Charbonnier, M. and Boyer, J. (1973) Biochii. Biophys. Acta 316, 162-172 Arnaud, J. and Bayer, J. (1974) Biochim. Biophys. Acta 337, 165-168 Carroll, K.K. (1961) J. Lipid Res. 2,135-141 Amaud. J. and Bayer. J. (1972) Biochim. Biophys. Acta 270, 189-196 Beireriherz. G.. Boltze. H.J. Bucher, T., Czok, R., Garbade. K.H.. Meyer-Arendt, E. and Pfleiderer, G. (1953) Z. Naturforsch. 8. 555-564 Giudicelli, H. and Bayer, J. (1973) J. Lipid. Res. 14, 592-593 Lynn, W.S. and Perryman, N.C. (1962) J. Biol. Chem. 235, 1912-1916 Kupiecki, F.P. (1966) 7, 230-235 Mann, J.T. and Tow, S.B. (1966) J. Biol. Chem. 241, 3595-3599 Vaughan, M., Berger. J.E. and Steinberg, D. (1964) J. Biol. Chem. 239, 401-409 Gorin, E. and Shafrir, E. (1964) Biochim. Biophys. Acta 84,24-34 Katocs, AS., Calvert, D.N. and Lech, J.J. (1970) Biochim. Biophys. Acta 229. 608417 Gordon, R.S., Bayle. E., Brown, R.K., Cherkes, A. and Anfinsen. C.B. (1953) Proc. Sot. EXP. Biol. Med. 84.168-170 DesnueBe, P. (1961) Adv. Enzymol. 23.129-161 Brockerhoff, H. (1970) Biochim. Biophys. Acta 212.92-101
Biochimica et Biophysics Acta, 369 (1974) 25-33 0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
BBA 56494
LIPOLYTIC
ACTIVITY
OF ADIPOSE TISSUE
IV. THE DIACYLGLYCEROL TISSUE
LIPASE ACTIVITY
OF HUMAN ADIPOSE
H. GIUDICELLI, N. COMBES-PASTRY? and J. BOYER
Unite' Me’tabolique, Centre Hospitalier Rtfgional de Marseille, H6pital de la Conception, 13385 Marseille, Ckdex 4 (France) (Received April 22th, 1974)
Summary Diacylglycerol lipase activity of human adipose tissue was studied by using emulsified glycerol di[9,10-3 H] oleate as substrate. A partially purified preparation of this lipase activity was obtained through stabilization of the enzyme in glycerol-water mixtures. Enzymic hydrolysis was maximum in 4 mM diolein and 1% serum albumin, at pH 7.2 and 37°C. With all preparations tested, the rate for triolein was found to be l/10-1/30 of that of triolein, depending on the experimental conditions. It was shown that a number of intrinsic properties are shared by di- and triacylglycerol lipase activities. Thermal stability (in the presence and absence of glycerol), susceptibility to bile salts and binding affinity for specific lipid interfaces are similar for both activities. This, along with a uniform behaviour upon various purification procedures, support the contention that di- and triacylclycerol lipase activities of human adipose tissue are referable to a single catalytic protein.
Introduction Detailed information concerning the number of lipases in adipose tissue is very fragmentary. As a prerequisite of a project designed to study the physiological mechanism of lipolysis, we had to investigate the lipase distribution in human fat. In this line, we have recently partially purified and characterized a triacylglycerol lipase activity [ 11, the catalytic properties of which are consonant with those of “hormone-sensitive” lipase, essentially described in the rat [2,3]. Human fat also contains a monoester lipase [4-S], active in vitro towards monoacylglycerol and esters of fatty acids and short-chain normal primary alcohols. Tri- and monoacylglycerol lipase activities are very likely to
26
be due to different enzymes. An acylcholesterol lipase activity has also been recently identified [7] in the same tissue. This sterol monoester lipase seems itself to be distinct from monoacylglycerol lipase. So far none of the human adipose tissue preparations tested in our laboratory was found to contain detectable levels of a triacylglycerol lipase activity identifiable as lipoprotein lipase .
This paper is concerned with the characterization of diacylglycerol acylhydrolase activity of human adipose tissue. The data presented below support the contention that, in this tissue, di- and triacylglycerol are hydrolyzed by the same enzyme. Materials and Methods Glycerol di[9,10-3 H] oleate (0.6 @i/mole) was purchased from the Institut des Corps Gras de Marseille, and has been purified by chromatography on Florisil [8]. The final product, used as substrate for the lipolytic assays, was over 98% radiochemically pure and had a composition of about 40% 1,2-diolein and 60% 1,3diolein. Glycerol tri[9,10-3 H] oleate (0.5 Ci/mmole) (The Radiochemical Centre, Amersham) was purified by thin-layer chromatography on silica gel using a system previously published [4] . Unlabeled triolein and diolein (Sigma Chemical Co.) were obtained over 98% pure by chromatography on Florisil [8]. In all substrate purification procedures, artifacts caused by Florisil-catalyzed transesterification reactions were carefully avoided [ 91. Bovine serum albumin B grade (fatty acid-poor) was obtained from Calbiochem (San Diego, Calif.). (NH,), SO,, was recrystallized from EDTA [lo]. Radioactivity of 3 H and ’ 4 C was measured as reported earlier [ 51. Experimental
procedures
Specimens of human subcutaneous adipose tissues were obtained during surgery. Lipase activity was extracted and partially purified as recently described [ 11. With this procedure, lo-30% of the total activity are usually found to be associated with material of low density collected after serial (NH4 )2 SO4 fractionation of a pH 5.4-precipitated fraction. This 20-40-foldpurified preparation is active towards diolein and triolein. It has no tributyrinase activity but still retains high levels of activity towards long-chain monoacylglycerol. It usually contains, by weight, at least twice as much lipids (mainly neutral lipids) as proteins. All the purification procedures were carried out in 25% glycerol, which protects the enzyme(s) against denaturation [ 111. Protein concentrations were determined as reported [l] . The assay of triacylglycerol lipase activity, using [ 3 H] triolein as substrate, has been described [ 11. Diacylglycerol lipase activity was assayed employing the same procedure but substituting 5 pmoles of [3 H] diolein as substrate, in a final volume of 5 ml containing; 0.01 M sodium phosphate buffer, 1% serum albumin and the enzyme, at the final pH 7.2. It has been verified that the substitution of [3 H] diolein for [3 H] triolein in the assay medium does not affect, to any significant degree, the isolation and the counting of the [ 3 H] oleic acid liberated during hydrolysis.
21
Throughout this work, lipase activities refer to initial reaction rates determined by plotting the amounts of [” H] oleic acid released 0,lO and 20 min after the addition of enzyme. Enzyme kinetics were of zero order over the 20 min duration of the assay. For each value of activity, duplicate assays were reproducible within 10% of [3 H] oleic acid liberated. One unit of activity corresponds to the release of one E.tmole of acid per min, at 37” C. Results Characterization of diacylglycerol lipase activity The influence of pH on the hydrolyzing activity of human adipose tissue towards diolein is shown in Fig. 1. This activity has a broad maximum between pH 6.8 and pH 7.2, i.e. at a pH range slightly lower than with triolein as substate. Fig. 2 shows the increase of the reaction rates measured at pH 7.2 as a function of increasing amounts of substrate. Maximum velocity was attained in 4 mM diolein. In 1 mM, hydrolysis occured at a rate amounting to about 75% of the maximum velocity. The values plotted according to Lineweaver and Burk (Fig. 2, inset) give an apparent K, of 0.5 mM. The measured catalytic activity of the extracts towards diolein was dependent on the concentration of serum albumin in the assay medium (Table I). The hydrolysis rate showed a 4-fold increase when albumin concentration was raised from 0.01% to 2.5%. At higher concentration, the hydrolysis rate tended to fall off. Under similar conditions of assay (Table I), serum albumin inhibited the hydrolysis of triolein. The lipolytic activity towards diolein was consistently found to be lo-40 times greater than that towards triolein. The magnitude of the difference was clearly dependent on the experimental conditions. Table I shows that the ratio of di- to triacylglycerol lipase activity measured in the same enzymatic preparation may be as different as 3 and 28 in 0.01% and 2.5% albumin, respectively.
PH
Fig. 1. Effect of pH on lipase activity. Initial adjustment at the indicated pH values. and pH assays, have been carried out under pH-stat control. They necessitated the addition of minimum amounts of HCl or NaOH. Enzymatic assays were as indicated under Materials and Methods.
28
~-3--?-&
6
0
Diolein
Fig. 2. Hydrolysis Lineweaver-Burk
(mM)
of [3H]diolein plot.
as a function
Comparative determination enzyma tie preparations
of substrate
concentration,
at pH 7.2 and 37OC. Inset:
of di- and triacylglycerol lipase activities in various
Hydrolyzing activities towards di- and triolein have the same pattern of distribution upon serial (NH4 )Z SO4 fractionation (Fig. 3). In this experiment, 5500 munits of activity towards diolein (versus 310 munits towards triolein) were found to be bound to the floating fraction, with a specific activity of 150 munits/mg of proteins. This latter value indicated a 22fold purification over the crude extract. As for triacylglycerol lipase activity [l] , the pellets sedi-
TABLE EFFECT
I OF ALBUMIN
ON DI- AND TRIACYLGLYCEROL
LIPASE
ACTIVITIES
Activities were measured against di- and triolein as indicated under Materials and Methods. The indicated amounts of serum albumin were added to the assay medium before sonic emulsification of the substrate. In all cases. enzyme kinetics were of zero order over the 20-min duration of the assays. --.-. Albumin
Lipase activity
(%)
0.01 0.05 0.10 0.50 1.0 2.5 5.0
--_-
~____
--
(munitslml of extract) _____-__
Diolein
Triolein
16 21 31 59 68 65 53
5.1 4.8 4.3 4.0 3.8 2.3 2.0
~towards
29
Saturation
of(NH,),SO,
Fig. 3. Distribution pattern of di- and triacylglycerol lipase activities upon serial (NH4)2SO4 fractionation in 5% increment from 0.15 to 0.80 of saturation. Fractions are those obtained from the experiment presented in ref. 1 (Fig. 1). in which a pH 5.4 precipitate served as enzyme source. Prior to assay. each fraction was dialyzed for 24 h at 4OC against four 500~ml changes of 10 mM sodium phosphate (PH 7.4) containing 25% glycerol and lo4 M EDTA. Di- and trioleinase activities were concomitantly assayed in each dialyzed fraction. 1 to 3 assays contributed to each value. The dotted column represents dioleinase activity in the floating layer collected after centrifugation of the 0.40 (NH4)2SO4 fraction.
mented above 0.40 of saturation of (NH4 )n SO4 contained enzyme(s) of lesser specific activity, with a much lower ratio, by weight, of lipids to proteins. Likewise, di- and triacylglycerol lipase activities were distributed evenly in four fractions taken at various stages of purification, with a ratio of di- to triacylglycerol lipase activities averaging 15 (Table II). A comparable uniformity was noted when both activities were concomitantly assayed in fractions obtained by successive differential centrifugation (Table III). TABLE
II
RATIO OF DI- TO TRIACYLGLYCEROL CATION
LIPASE
ACTIVITY
AT SUCCESSIVE
STAGES
IN PURIFI-
Various enzymatic fractions were concomitantly assayed towards di- and triolein as substrate. The dioleinase specific activity of the fractions ranged from 6.8 to 150 munits/mg of proteins. Comparison between fractions does not hold, since all did not derive from a single purification procedure. Fraction
Total homogenate 12 000 X * supernatant pH 5.4 precipitate Fraction floating in 40% (NH&S04
Lipase activity
(munits/ml)
Diolein
Triolein
25 16 120 550
1.9 1.1 9.4 31
towards
Ratio
13 15 13 18
c_i_..2_
0
.A
.A.
-L
-
24
12 lncubotton
i..i
36
48
at 50”C(min:
Fig. 4. Comparative behaviour of di- and triacylglycerol fipase activities upon incubation at 50°C. Aliquots were taken at the indicated times from the incubated extracts and concomitantly assayed at 37” C towards diolein (black symbols) and triolein (open symbols) as substrate. A pH 5.4 precipitate served as enzyme source. Assays were performed on samples incubated in the absence (circles) or presence (squares) of 25% glycerol. Activity is expressed as percent of control values measured in the extracts kept at 4’C, immediately prior to incubation.
Fig. 4 shows that di- and triacylglycerol lipase activities elicited an identical pattern of inactivation upon incubation of a purified extract at 50°C. Moreover, both activities were equally and fully protected by inclusion of 25% glycerol in the extract. Hydrolyzing activities towards di- and triolein were consistently found to be inhibited by about 80% and 90%, respectively, in the presence of 10 m&l sodium taurocholate in the assay medium. Glycerol had only a slight and variable protective effect against this inactivation.
TABLE
III
RATIO TION
OF DI- TO TRIACYLGLYCEROL
The enzymatic Methods.
preparations
derived
from
-. Enzymatic fraction
Total homogenate 600 X g supernate 12 000 X g supernate 100000 X g supernate
LIPASE
ACTIVITY
UPON DIFFERENTIAL
a single tissue sample.
Assays as indicated
-~-
CENTRIFUGAunder Materials and -
Total lipase activity --.
(munits) _l____-
Diolein
Trio&n
2460 1890 1450 1310
77 63 43 38
towards
Ratio
32 30 34 35
31 TABLE
IV
COMPARATIVE DIOLEIN
ADSORPTION
AND TRIOLEIN
OF
DI-
AND
TRIACYLGLYCEROL
LIPASE
ACTIVITY
ONTO
INTERFACES
The enzymatic extract (12000 X g supernatant fluid) was prepared in 10 mM sodium phosphate (PH 7.4) containing 104M EDTA and 25% glycerol. A 20 ml-aliquot fraction was dialyzed for 2 h at 4’C against l-l of a solution made of 10 mM sodium phosphate and 10”M EDTA (final pH 7.4). without glycerol. 7.7 ml of the dialyzed preparation were gently stirred for 90 min at 4’C with either 1 ml of a diolein emulsion or 1 ml of a triolein emulsion. Each emulsion was prepared by sonicating with a Branson sonifier B-12 (Setting 1 for 10 s at 4’C) 1 g of acylglycerol with 1 ml of 10 mM sodium phosphate buffer (pH 7.4). Both mixtures were then centrifuged at 100000 X g for 1 h at 4OC and the clear supernatant fluids were concomitantly assayed for di- and trioleinase activity. Similar samples containing no awlglycerol emulsion were run in parallel: the lipase activity measured in the corresponding 100000 X g supernatant fluids (240 munits and 15 munits towards di- and triolein, respectively) were taken as control values (100%). Activity
(%) in the 100000
Added acylglycerol emulsion
Diolein
Triolein
None Diolein Triolein
100 7 34
100 11 45
X g supernate
towards
Behaviour of di- and triacylglycerol lipase activities in the presence of a lipid interface Data presented in Table IV show the comparative behaviour of di- and trioleinase activities in the presence of diolein or triolein emulsions at 4°C. The disappearance from the supernatant fluid of di- and trioleinase activities are quantitatively similar with both emulsions. However, diolein retained both activities with an efficiency greater than triolein, presumably indicating a lower affinity of the enzyme(s) for triacylglycerol. This difference of affinity is not reflected by the apparent Km values, which have been found to be identical (0.5 mM) for both substrates (see ref. 1). However, it should be recalled that dealing with water insoluble esters, Km values expressed in molar concentration cannot be accurate, and may fluctuate from one to another experiment. Interestingly, inclusion of 2% serum albumin in the mixture decreased by 40-60% the retention of di- and trioleinase activities by both emulsions. Discussion It is commonly admitted that, in adipose tissue from most animal species, triacylglycerol lipase differs in a number of properties from monoacylglycerol lipase, and that each activity is referable to a distinct catalytic protein [ 12181. We have indicated earlier [6] that under optimal experimental conditions, that is, in the presence of 10 mM sodium taurocholate in the assay medium, a 39-fold purified preparation of human adipose tissue monoacylglycerol lipase had no activity towards long-chain diacylglycerol. It is therefore most likely that in the present work, the monoacylglycerol hydrolyzing activity retained in the purified extract does not contribute to diolein hydrolysis. However, it is of interest to note that, so far, human as well as animal adipose tissue mono- and
32
diacylglycerol lipases on one hand, mono- and triacylglycerol lipases on the other hand, have not been separated by physical methods, and seem actually tightly associated in a multienzyme complex (Verine, A. and Boyer, J., unpublished). In view of our data, it is our present working hypothesis that diolein and triolein are hydrolyzed in human adipose tissue by the same enzymatic entity. Several lines of evidence support this interpretation, and especially those which essentially reflect identical intrinsic molecular properties: thermal stability, protection by glycerol (see ref. ll), susceptibility to bile salts and binding affinity for lipid interfaces. Failure to separate di- and triacylglycerol lipase activities by various purification procedures only represents tentative clues, since the degree of purification attained is still very partial. In contradiction with these common properties Fig. 1 shows that the pH curves for the two substrates are slightly different (by a factor of about 0.5 pH unit with respect to the optimum pH value). However, it should be recalled that similarity of pH optimum for two substrates is not necessarily to be expected, even when both are acted upon by the same enzyme. We have already reported such slight differences with human adipose tissue monoester lipase [ 61. Also, di- and triacylglycerol lipase activities are apparently inversely influenced by the addition of serum albumin to the incubation medium. Such a differential effect is observed whether or not Ca” (10 mM) is present in the assay system. It has been recognized [18] that albumin, at physiological pH value, binds the fatty acids released during lipolysis, thereby preventing inhibition of the reaction. However, current findings from our laboratory (Jubelin, J., and Boyer, J., unpublished) indicate that albumin exerts this stimulating effect within an optimal range of concentration, variable with the experimental conditions. Beyond this range, albumin has been consistently found to inhibit lipolysis. These observations might explain the differential effect of albumin on diolein and triolein hydrolysis (Table I) attributable, at least in part, to the basic difference in the reaction rates. Within a given range, the increase of albumin concentration stimulates the rapid hydrolysis of diolein (generating relatively large amounts of fatty acids), whereas it inhibits that, much slower, of triolein. The reason for the inhibition is not known. But it is remarkable that the presence of 2% albumin decreases the binding affinity of the enzyme for diolein and triolein interfaces. In practice, it is therefore important to carefully determine, on an experimental basis, the amount of albumin optimal for each assay system. In any case, it should be emphasized that the in vitro hydrolysis of diolein and triolein are virtually null in the absence of added albumin. If indeed diolein and triolein are attacked in human adipose tissue by a single enzyme, the difference between their respective hydrolysis rates should be noted. It is assumed that lipases (and esterases in general), attack the carbonyl group of fatty esters as a nucleophile. Pancreatic lipase, in accordance with a nucleophilic mechanism, hydrolyzes more rapidly triolein than diolein [ 191. Since the reverse order is observed in vitro with human adipose tissue lipase, it is probable that steric factors, in this latter case, counteract the inductive effects and hamper the formation of the enzyme-ester complex. If, as suggested [20], an essential requirement for the lipase attack is that the aliphatic chains
33
can conveniently bend away from the enzyme, it is understandable that a greater mobility of the chains in diolein, as compared to that in triolein, may facilitate the approach of the carbonyle group by the enzyme. As a consequence, the measured rates of hydrolysis of the first primary ester bond of triacylglycerol, termed in this (and other) paper “triacylglycerol lipase” activity, might be theoretically overestimated, due to the concomitant attack of the di- and eventually monoacylglycerol produced. In practice, the very low reaction rates dealt with in adipose tissue preparations, i.e. the very low concentrations of partial acylglycerol formed, as well as the permanent reference in the present work to initial reaction rates, should render minimal these difficulties. Acknowledgements We wish to thank Professor G. Michotey for his courtesy in making available human adipose tissue. This work was supported by Grant No. 732.084.7 from the Institut National de la Sante et de la Recherche Medicale and by the Fondation pour la Recherche Medicale Francaise. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Giudicelh, H., Pastre, N. and Bayer, J. (1974) Biochim. Biophys. Acta 348. 221-231 Hollenberg, C.H.. Raben, M.S. and Astwood, E.B. (1961) Endocrinology 68, 589-598 Rizack, M.A. (1961) J. Biol. Chem. 236,657-662 Bayer, J., Amaud-Le Petit, J. and Charbonnier, M. (1971) Biochim. Biophys. Acta 239. 353-356 Charbonnier. M.. Amaud, J. and Bayer, J. (1973) Biochim. Biophys. Acta 296,471-480 Arnaud, J., Charbonnier, M. and Boyer, J. (1973) Biochii. Biophys. Acta 316, 162-172 Arnaud, J. and Bayer, J. (1974) Biochim. Biophys. Acta 337, 165-168 Carroll, K.K. (1961) J. Lipid Res. 2,135-141 Amaud. J. and Bayer. J. (1972) Biochim. Biophys. Acta 270, 189-196 Beireriherz. G.. Boltze. H.J. Bucher, T., Czok, R., Garbade. K.H.. Meyer-Arendt, E. and Pfleiderer, G. (1953) Z. Naturforsch. 8. 555-564 Giudicelli, H. and Bayer, J. (1973) J. Lipid. Res. 14, 592-593 Lynn, W.S. and Perryman, N.C. (1962) J. Biol. Chem. 235, 1912-1916 Kupiecki, F.P. (1966) 7, 230-235 Mann, J.T. and Tow, S.B. (1966) J. Biol. Chem. 241, 3595-3599 Vaughan, M., Berger. J.E. and Steinberg, D. (1964) J. Biol. Chem. 239, 401-409 Gorin, E. and Shafrir, E. (1964) Biochim. Biophys. Acta 84,24-34 Katocs, AS., Calvert, D.N. and Lech, J.J. (1970) Biochim. Biophys. Acta 229. 608417 Gordon, R.S., Bayle. E., Brown, R.K., Cherkes, A. and Anfinsen. C.B. (1953) Proc. Sot. EXP. Biol. Med. 84.168-170 DesnueBe, P. (1961) Adv. Enzymol. 23.129-161 Brockerhoff, H. (1970) Biochim. Biophys. Acta 212.92-101