Study of the hydrolysis of 4-methylumbelliferyl oleate by acid lipase and cholesteryl oleate by acid cholesteryl esterase in human leucocytes, fibroblasts and liver

98

Biochimico et Biophysics @ Elsevier/North-Holland

Acta, 618 (1980) Biomedical Press

98-105

BBA 57541

STUDY OF THE HYDROLYSIS OF 4-~ETHYL~B~LLIFERYL OLEATE BY ACID LIPASE AND ~HOLESTERYL OLEATE BY ACID CHOLESTERYL ESTERASE IN HUMAN LEUCOCYTES, FIBROBLASTS AND LIVER

JOHAN

F. KOSTER

*, HEDWIG

VAANDRAGER

and THEO J.C. VAN BERKEL

Department of Biochemistry I, Medical Faculty, Erasmus University P.0. Box 1738, 3000 DR Rotterdam (The Netherlands) (Received April llth, 1979) (Revised manusc~pt received

Key words: Methylumbelliferyl (Human)

November

Rotterdam,

8th, 1979)

oleate; Acid lipase; Cholesteryl

e&erase; Cholesteryl

oleate;

Summary 1. The characteristics of acid lipase with 4-methylum~llife~l oleate as substrate and acid cholesteryl e&erase with cholesteryl [l-%]oleate as substrate were investigated in homogenates of human leueocytes, fibroblasts and liver. The substrates were encapsulated in egg yolk lecithin vesicles and assays were developed which were linear with amount of protein and with time. 2. With 4-methylumbelliferyl oleate as substrate, a pH optimum of 4.0 was found in all preparations. Half-maximal activity was obtained in leucocytes at 7 ,uM, fibroblasts at 4 j&l and liver at 14 fl. Similar inhibition of the enzyme from the three sources was observed with various inhibitory compounds. It is concluded that the hydrolysis of 4-methylumbellife~l oleate is catalyzed by one enzyme common to the three cell types. 3. With cholesteryl [ l-14C Joleate as substrate, the pH optima for leucocytes, fibroblasts and liver were 4.5, 4.0 and 3.5, respectively. Furthermore, the concentration necessary for half-maximal activity for leucocytes, fibroblasts and liver was 23, 25 and 20 @I, respectively. The inhibition percentages with the different substances were different for the three cell types. It is suggested that these differences between the three cell types might be due to the different environments of the enzyme. 4. Comparing the results of the hydrolysis of 4-methylumbelliferyl oleate with those of the hydrolysis of cholesteryl [l-‘4C]oleate, we concluded that * To whom correspondence should be addressed.

99

two different proteins (or one protein sites for both substrates) are involved.

which possesses two distinct

catalytic

Introduction Lysosomal acid lipase is considered to be important in the catabolism of lipids and/or cholesteryl esters. Deficiency of this enzyme leads to Wolman’s disease and cholesteryl ester storage disease [ 11. With both diseases, the hydrolysis of 4-methylumbelliferyl oleate by acid lipase, as well the hydrolysis of cholesteryl oleate by acid cholesteryl esterase, are greatly impaired. However, the clinical course for both diseases is quite different. Wolman’s disease occurs in infancy and is almost fatal before the age of one, whereas cholesteryl ester storage disease is more benign and cannot be detected until the adult. Furthermore, with Wolman’s disease, there is an accumulation of triacylglycerols and cholesteryl esters, whereas with cholesteryl ester storage disease there is much more storage of cholesteryl esters and much less storage of triacylglycerols [2]. ~thou~ the defect of hydrolyses for both ~a~ylglycerols and cholesteryl esters in one cell strongly suggest that one enzyme [3] is responsible for these hydrolyses, the striking differences in the expression of these diseases initiated us to investigate the relationship between these hydrolyses. The acid lipase (with 4-methylumbelliferyl oleate as substrate) and acid cholesteryl e&erase (with cholesteryl oleate as substrate) were studied in leucocytes, fibroblasts and liver. These three cell types were chosen because they are normally analysed in the diagnosis of these diseases. The data suggest that acid lipase and acid cholesteryl esterase are different enzymes, or, perhaps, one protein with two different active centres for both substrates. Methods and ~a~~a~ Leucocytes were obtained from heparinized blood and isolated according to the method of Wyss et al, [4]. Polymorphonuclear cells and lymphocytes were isolated from heparinized blood according to the method of Boyum [5]. Human fibroblasts were grown in HAM F10 medium supplemented with fetal calf serum and harvested with trypsin at confluency. Human liver was obtained at autopsy (within 4 h after death), and immediately frozen in liquid Nz and stored at -70°C until use. The homogenates of the various cell types were prepared by sonication twice for 30 s at 21 kHz just before the assays. The substrates, 4-methylumbelliferyl oleate and cholesterol oleate, were incorporated into vesicles [ 63. With the substrates in this form, more reliable and reproducible results were obtained than with the assays in which the substrates are dispersed into Triton + albumin, or in acetone. The vesicles were prepared as follows: 1 ml 10 mM 4-methylumbelliferyl oleate in hexane and 1 ml 16 mM L+phosphatidylcholine in hexane were evaporated together to dryness under a stream of nitrogen. The residue was resuspended in 25 ml 2.4 mM sodium taurocholate and sonicated in an ice-bath twice 1 min at 25 kHz. The resulting preparations were stable for four days. The desired substrate condensation

100

was obtained as follows: 1 vol. substrate was added to several ~01s. of 0.2 M buffer (acetate/Tris-HCl) of the appropriate pH. For the assay, 2 ml substrate suspension was added to a cuvette and equilibrated for 2 min at 37°C. The cell homogenate (usually 25 or 50 111)was added and the activity measured after a further 2 min incubation. The change in relative fluorescence was recorded against time at 37°C (excitation, 335 nm; emission, 445 nm). The reaction was linear with time up to 30 min. The non-enzymic hydrolysis (measured by mixing substrate and buffer alone or substrate with enzyme heated for 10 min at 8O’C) was negligible. Reference was made to a standard curve for 4-methylumbelliferone fluorescence. For studying the effect of pH, standard curves were also run at the various pH values. The activities were expressed as nmol substrate hydrolysed/min per mg protein. Cholesteryl [1-14C]oleate was used as substrate also encapsulated into vesicles as described for 4-methylumbelliferyl oleate. The incubations were performed in a total volume of 0.3 ml for 90 min (the reaction was linear for 3 h). The reaction was stopped with 3.0 ml benzene/CHC13/CH30H (1.0 : 0.5 : 1.2, v/v), containing 0.1 mM oleate as carrier. 0.6 ml 0.3 M NaOH was added the mixture was shaken for 45 s and subsequently centrifuged for 10 min at 1000 X g. 0.5 ml of the upper layer was removed and counted in 10 ml Lumagel, after addition of acetic acid to minimize quenching. The activities were expressed as pmol substrate hydrolyzed/min per mg protein. Egg yolk lecithin was obtained from Sigma, 4-methylumbelliferyl oleate from Koch-Light Laboratories and cholesteryl [1-‘4C]oleate from the Radiochemical Centre (Amersham, U.K.). All other reagents were of the highest purity commercially available. Results Fig. 1 shows the effect of leucocyte protein concentration upon the hydrolysis of 4-methylumbelliferyl oleate and cholesteryl oleate. With 4-methylumbelliferyl oleate, the reaction was linear up to 15 1.18protein, whereas with cholesteryl oleate there is a continued linearity between reaction rate and protein concentration. For fibroblasts and liver, the reaction was linear in time and

60

MU-&ate

3c

rg protein

Chokoleate

added

Fig. 1. Protein dependency of the hydrolyses of 4-methylumbelliferyl &ate in leucocytes. Concentration of both substrates 50 JLM.

(MU) oleate and cholesteryl

(Chol.)

101

the protein dependency for both substrates was similar to that of leucocytes. Table I shows the distribution of the hydrolysis of 4-methylumbelliferyl oleate and cholesteryl oleate in lymphocytes and polymorphonuclear cells. In agreement with Coates et al. [ 81, the hydrolysis of 4-methylumbelliferyl oleate was much higher in lymphocytes than in polymorphonuclear cells. Table I shows that this also holds for the hydrolysis of cholesteryl oleate. Fig. 2 shows the effect of different pH buffer on the hydrolysis of $-methylumbelliferyl oleate and cholesteryl oleate for leucocytes. The hydrolysis of 4-methylumbelliferyl oleate had a pH maximum of 4.0, whereas the hydrolysis of cholesteryl oleate was maximal at pH 4.4. Furthermore, at alkaline pH, there was some hydrolysis of 4-methylumbelliferyl oleate (much less than the activity at acid pH), ivhereas there was no hydrolysis of cholesteryl oleate at these pH values. For fibroblasts, hydrolysis of both 4-methylumbelliferyl oleate and cholesteryl oleate were maximal at pH 4.0; at alkaline pH there was no (or little) hydrolysis of 4-methylumbelliferyl oleate. The hydrolysis of 4-methylumbelliferyl oleate by liver had the same pH maximum as for leucocytes and fibroblasts, but the pH optimum for the hydrolysis of cholesteryl oleate was at pH 3.5. Furthermore, there was a substantial hydrolytic activity towards 4-methylumbelliferyl oleate at neutral pH. The effect of substrate concentration on the reaction velocity is shown in Fig. 3, for leucocytes. The half-maximal velocity for the hydrolysis of 4-methylumbelliferyl oleate was at 7 I/M, whereas for cholesteryl oleate this half-maximal activity was observed at 23 a. The half-maximal hydrolyses of 4-methylumbelliferyl oleate and cholesteryl oleate for fibroblasts were 4 w and 25 @I, respectively. For liver, these figures are 14 @I for 4-methylumbelliferyl oleate and 20 fl for cholesteryl oleate. To study the possibility that the hydrolysis of these two substrates are catalyzed by two different enzymes, we investigated the effects of a number of agents which are generally used to identify and to discriminate between different classes of lipases [ 71. The effect of each substance depends on the nature of the substrate (high-salt, protamine sulphate, Triton X-100) or upon the enzyme itself (iodoacetate, N-ethylmaleimide, p-chloromercuribenzoate). All agents which modify the nature of the substrate have a similar inhibition with both substrates for leucocytes, whereas for liver and fibroblasts, the effect of NH&l and EDTA were not similar with both substrates. In addition, for fibroblasts (but not for leucocytes or liver), Triton X-100 had a different effect on the hydrolTABLE I DISTRIBUTION CHOLESTERYL

OF THE HYDROLYSIS RATE OLEATE IN LEUCOCYTES

OF

4-METHYLUMBELLIFERYL

The activities were measured at 50 pM substrate. With 4-methylumbelliferyol parations were used. 4-Methylumbelliferyl (nmol/min per ma) Total leucocytes Polymorphonuclear Lymphocytes

cells

4.98; 6.00 0.93: 1.06 8.60: 6.49

oleate

OLEATE

oleate two different cell pre-

Cholesteryl oleate (pmol/min per mg) 1.99 0.83 4.90

AND

Fig. 2. The pH dependency of the hydrolyses of 4-mrthylumbelliferyl (MU) o&ate and cholesteryl oleate in leucocytes. Substrate concentration is 50pM; lA, I-methylumbelliferyl oleate; A------+ cholesteryl oleate.

yses of both substrates (Table Ii). ‘With the effecters which influence the enzyme itself, there were some differences in inhibition between these two substrates. For leucocytes, it was found that, with 4-methylumbelliferyl oleate both p-chloromercuribenzoate and N-methylmaleimide inhibited to the same extent (about 31%), whereas with cholesteryl oleate, p-chloromercuribenzoate

I

20

rM

1

40 MU-/Cholroleate

Fig. 3. The saturation cytes at pH 4.0. .A,

I

00

plot for 4-methylumbelliferyl (MU) oleate 4-methylumbelliferyl oleate: A-----+

and cholesteryl cholesteryl

(Chol.)

oleate.

oleate in leuco-

103 TABLE II THE EFFECT OLEATE AND

OF VARIOUS CHOI,ESTERYL

AGENTS ON THE HYDROLYSIS OF I-METHYLUMBELLIFERYL OLEATE IN LEUCOCYTES, FIBROBLASTS AND LIVER AT ACID

PH (4.0) The activity is expressed as percentage of the activity at 50 PM substrate and at PH 4.0. The given value is the mean of three different measurements with three different preparations (-tS.E.) of each cell type. Cells

Agents

Leucocytes

50 500 1 20 0.25 1

Fibroblasts

Liver

4-Methylumbelliferyl oleate

Significance

96.6 27.4 90.2 84.5 1.7 96.6

+ 5.8 f 5.8 f 4.9 t 4.8 * 1.1 f 10.0

0.05 < P < 0.1 0.05

1.2 3.5 6.2 5.0 2.7 5.0

51.2 11.5 126.5 66.6 5.1 97.4

* 7.6 f 11.6 f 4.3 * 5.1 f 5.1 + 15.3

P < 0.1 P < P < P < P <

0.01

4.6 3.2 3.5 4.4 4.6 2.2

131.8 64.9 111.3 93.9 2.7 95.5

f 2.2 f 0.6 * 3.3 * 21.4 + 2.7 + 4.2

P < 0.2 P < 0.7 P < P <

0.001 < P < 0.3 0.02 < P < 0.8 0.001 0.005

76.6 45.5 87.2 92.9 68.9 68.6

+ 6.2 + 4.4 f 4.7 * 0.9 f 10.3 f 1.6

NH4Cl NaCl EDTA Triton X-100 p-chloromercuribenzoate N-ethyhnaleimide

87.6 37.6 93.8 97.5 63.2 59.5

f * * * f *

NH4Ci NaCl EDTA Triton X-100 .n-chloromercuribenzoate N-ethylmaleimide

69.8 54.4 93.5 84.9 52.6 63.0

f f f f * f

mM NH4Cl mM NaCl mM EDTA pg/ml Triton X-100 mM p-chloromercuribenzoate mM N-ethylmaleimide

Cholestervl oleate

completely inhibited the enzyme and Nethylmaleimide had hardly any influence. The same pattern was found for fibroblasts and liver. The significance of these differences can be concluded from the last column of Table II. Furthermore, it should be noted that, with 1 mM CaCl,, 1 mM MgClz and 1 mM NaF, no difference in inhibition was detected between the hydrolyses of both substrates in leucocytes, fibroblasts or liver. 50 mM iodoacetate completely inhibited the hydrolyses of both substrates in these three cell types. With the substrate 4-methylumbelliferyl oleate, the reaction was normally followed up to 30 min, while for cholesteryl oleate a reaction of 90 min is used. To exclude reaction time dependence on the inhibition of the various substances, we followed the 4-methylumbelliferyl oleate hydrolysis for 90 min. No difference in the inhibition was found. Discussion Most of the previous studies concerning e&erases were performed either with 4-methylumbelliferyl fatty acid or with cholesteryl fatty acids as substrate. We believe that we have made the first direct comparison of these substrates. Reliable assays were developed, in which both substrates were encapsulated into lipid vesicles and this allowed a direct comparison of the hydrolytic activities to both substrates. The method8 are easy to apply and lead to reproducible results (see tables).

104

The substrates for lipase assays are usually prepared in different ways [8-121 and there is, therefore, a great variation in the reported K, values. We presumed that the affinity of the enzyme for the substrate is greatly influenced by the way the substrate is presented to the enzyme. The incorporation of the substrates into vesicles is more comparable to the way the substrate normally reaches the lysosomes (attached to low density lipoproteins [13]). Also the enzyme(s) environment seems of great importance and it might be argued that reliable enzyme assays in cell homogenates (proportional to protein and time), as presented here, more closely resemble the physiological function of the enzyme(s) in vivo. From our results, we concluded that there is no difference between the enzymes of leucocytes and fibroblasts that catalyze the hydrolysis of 4-methylumbelliferyl oleate. For liver, half-maximal activity is obtained at a higher substrate concentration than for fibroblasts and leucocytes. It might be possible that this higher value is due to the heterogeneity of the liver. Recently, we [14] found that half-maximal activity was obtained at 4 fl 4-methylumbelliferyl oleate for rat liver non-parenchymal cells and 32 fl for rat liver parenchymal cells. The value for human liver (14 fl) is intermediate between these two values and we assume that this value will be the result of the contribution of parenchymal and non-parenchymal cells in human liver preparations. For the three human cell types studied here, the same pH optimum was found and enzyme inhibition by various agents was similar. As suggested earlier, the difference between non-parenchymal and parenchymal cells might be due to a different environment of the enzyme. This can also be suggested for human liver, and it seems likely that the enzyme responsible for the hydrolysis of 4-methylumbelliferyl oleate in leucocytes, fibroblasts and human liver will be the same protein molecule. This conclusion cannot be drawn for the hydrolysis of cholesteryl oleate. First, the pH optimum in the three cell types are different, although the halfmaximal activity concentration for the cell types is almost the same. Besides the difference in pH optimum significant differences (Table III) were found between the action of inhibitor NH&l on the three cell types, whereas no significant differences were observed with Triton X-100. NaCl had a similar effect on leucocytes and fibroblasts but a very different effect on liver cells. EDTA had a different effect on leucyte to that on fibroblast and liver cell preparations.

TABLE

III

PROBABILITY VARIOUS All

values

(P) AGENTS

are smaller

VALUES ON than

THE

FOR

THE

DIFFERENCES

HYDROLYSIS

the indicated Leucowtes

OF

OF

THE

CHOLESTEROL

MEANS

OF

THE

number.

vs. fibroblasts

Leucocytes

vs. liver

Fibroblasts

NH4Cl

0.01

0.005

0.05

EDTA

0.01

0.025

0.10

NaCl Triton

X-100

Protamine

sulphate

INHIBITIONS

OLEATE

0.40

0.05

0.05

0.1

0.7

0.5

0.005

0.1

0.01

vs. liver

OF

105

Protamine sulphate has the same effect on leucoeytes and liver, but a different effect on fibroblasts. However, it should be emphasized that these differences are not found for the hydrolysis of 4-methylumbelliferyl oleate and that the effect on N-ethylmaleimide and pchloromercuribenzoate in the hydrolysis of 4-methylumbelliferyl oleate is quite different from their action on the hydrolysis of cholesteryl oleate. Together with the differences in pH optimum, these results suggest that the hydrolysis of 4-me~ylumbellife~l oleate and cholesteryl oleate are catalyzed by two different proteins, or, alternatively, by one protein which possesses two different catalytic active centres. These different catalytic centres may be influenced differently by the two thiol reagents. With the hypothesis of two catalytic centres, the absence of hydrolysis of cholesteryl ester and 4-methylumbelhferyl ester in Wolman’s disease and cholesteryl ester storage disease can be explained. The different centres may even be localized on different subunits, in which each subunit has a specific activity towards either 4-methylumbelhferyl oleate or cholesteryl oleate. If this is true, hybridization of fibroblasts from patients with Wolman’s disease with fibroblasts from patients with cholesteryl ester storage disease will be helpful in the elucidation of the problem, as restoration of activity in the hybrid will prove the last possibility. Acknowledgements The Dutch Heart Foundation is acknowledged for partial financial support. Miss A.C. Hanson is thanked for her help in the preparation of the manuscript. References 1 Fredrickson, D.S. and Ferrans, V.J. (1978) in The Metabolic Basis of Inherited Diseases (Stanbury, J.B. and Fredrickson, D.S., eds.), 4th edn.. pp. 670-687, McGraw-Hill, New York 2 Burke, J.A. and Schubert, W.K. (1972) Science 176.309-310 3 Sloan, H.R. and Fredrickson, D.S. (1972) J. Clin. Invest. 51, 1923-1926 4 Wyss, S.R., Koster, J.F. and Hiilsmsnn. W.C. (1971) Clin. Chim. Acta 36, 277-280 5 Bliyum, A. (1968) &and. J. Clin. Lab. Invest., Suppl. 97, 77-89 6 Cortner, J.A., Coates, P.M., Swoboda. E. and Sehnatz. J.D. (1976) Pediatr. Res. 10. 907-932 7 Hayese, K. and Tappel, H.L. (1970) J. Biol. Chem. 245. 169-175 8 Coates, P.M., Cortner, J.A.. Hoffman, G.M. and Brown, S.A. (1979) Biochim. Biophys. Acta 572, 226-234 9 GUY, G.J. and 3utterwo~h. J. (1978) Clin. Chim. Acta 84.361-371 10 Warner, T.G., Tennant. L.L., Veath. M.C. and O’Brien, J.S. (1979) Biochim. Biophys. Acta 572, 201210 11 Stokke. K.T. (1971) Bioehim. Biophys. Acta 270.156-166 12 Stokke, K.T. (1972) Biochim. Biophys. Acta 280. 329-335 13 Brown, M.S.. Dana. S.E. and Goldstein, J.L. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 2925-2929 14 Van Berkel. T.J.C., Vaandrager, H., Kruijt. J.K. and Koster, J.F. (1980) Biochim. Biophys. Acta 617, 446457