294
Biochimica et Biophysics Acta, 488 (1977) 294-304 0 Elsevier/North-Holland Biomedical Press
BBA 57033
~~RACTERIZATION AND PARTIAL FROM H~AN LEUKOCYTES
R. RINDLER-LUDWIG, Department
PURIFICATION
OF ACID LIPASE
W. PATSCH, S. SAILER and H. BRAUNSTEINER
of Rledicine, University of Innsbruck,
A-6020 Znnsbruck {Austria)
{Received March Znd, 1977)
Summary Hydrolysis of glycerol trioleate by human leucocytes was characterized and the enzymes responsible for this activity were obtained in a purified form by means of gel chromatography on Sephadex G-100 as well as by zanal ultracenThe activity is localized in the trifugation followed by gel chromatography. granule fraction of leucocytes (15 000 X g, 20 min) and shows a sharp pH optimum at pH 5.25. As judged from the elution profile obtained by gel chwomatography, two proteins are likely to contribute to the hydrolysis of glycerol trioleate. The approximate molecular weights of the two enzymes are 74 100 and 60 300, respectively. The activity is reduced in the presence of NaCl, KCl, CaClz as well as of p-hydroxymercuribenzoate. The enzymes are stable at -25°C but loose about 50% of their activity within 48 h at 4°C.
Introduction In many metabolic disorders, the lysosomal defect of the target tissue is reflected in leucocytes. In these cases, leucocytes offer the advantage of beeing readily and repeatedly available both for metabolic investigations as well as for the diagnosis of disorders of metabolism. Defects of lysosomal acid lipase have been observed in various inborn disorders of metabolism which are characterized by lipid accumulation in several organs. Severe deficiency of liver and spleen cells in acid lysosomal lipase [l] has been described in Wolman’s disease [2]. In patients with cholesterol ester storage disease [3] also a deficiency of acid lipase has been found in liver cells [ 4] as well as in cultured fibroblasts [ 51. Evidence for the presence of acid lipolytic activity in human leucocytes was presented by Braunsteiner et al. [6,7]. So far, however, detailed investigation was carried out only on the lipolytic activity in rabbit polymorphonuclear leucocytes [S,Q].
295
The present work reports the partial purification and ch~acte~zation lipolytic activity of human leucocytes.
of acid
Materials The following reagents were used: glycerol t.ri[l-14C]oleate, spec. act. 55 mCi/mmol (The Radiochemical Centre, Amersham, England); bovine serum albumin, crystallized and lyophilized, used as a standard for protein determination, azocasein, DL-dithiothreitol (Sigma, St. Louis, U.S.A.); bovine serum albumin, powder, fraction V, used in the lipase assay (Armour Pharmaceutical Company Ltd., ~astbou~e, England); friton X-100, trypsin, cytochrome c from horse heart, glycerol trioleate, Amber&e IRA-400 and ~-hydroxymercuribenzoate (Serva, Heidelberg, G.F.R.); Sephadex G-100, Dextran T 500, Blue Dextran 2000 (Pharmacia, Uppsala, Sweden); NCS-tissue solubilizer (Amersham/ Searle Corporation, Arlington Heights, Ill., U.S.A.). Methods Isolation of ieucocy tes Blood was obtained from patients with a myeloid reaction and processed as described earlier [lo]. The isolated white blood cells consisted of approximately 80% of neutroph~ polymo~honucle~ leucocytes or earlier stages of neutrophil cells, S-12% of monocytes and S---12% of lymphocytes. This was determined by differential counts either of smears stained for 10 min for naphto1 AS-D chloroacetate esterase [ll] or of May-Grtinwald-Giemsa smears. Homogenization of the cells was carried out in 0.34 M sucrose [lo]. After sedimentation of nuclei and agglutinated granules at 600 X g for 15 min the granules were collected at 15 000 X g for 20 min. To determine lipolytic activity in subcellular fractions of human leucocytes, the nuclear as well as the granule pellet were resuspended in 0.34 M sucrose containing 0.013% (v/v) Triton X-100. To the postgranular supernatant 0.5% (v/v) Triton X-100 in 0.34 M sucrose was added to give a final concentration of 0.013%.
Lipolytic activity was determined by the method of Krauss et al. [12]. The assay mixture contained in a final volume of 1 ml: 10.4 nmol glycerol tri[l-14C]oleate (rechromatographied before use to a purity of more than 99%); 8.5 pmol unlabeled glycerol trioleate; 15 mg bovine serum albumin; 0.40 ~1 Triton X-100. These constituents were sonicated at 4°C in 0.1 M sodium acetate buffer (pH 5.25) by four bursts of 60 s each in conical tubes using an ultrasonic disintegrator (Megason Ultrasonic Instruments International, Farmingdale, N.Y., U.S.A.). In the standard assay, 0.2 ml of enzyme sample were added to 0.8 ml of substrate emulsion and incubated for 60 min at 37°C in a shaking water bath. The reaction was terminated by lipid extraction [13]. Free fatty acids were isolated by the method of Kelley 1141. Counting was performed in a Packard Tricarb liquid scint~lation spectrometer. All assays were performed in duplicate; duplicate values differed by less than 5%. Blanks were constituted by incubating the substrate emulsion with the appropriate buffer
without
liberated
enzyme
sample.
Lipdytic
either per h per mg protein?
activity was expressed as nmol of fatty acid or per h per ml enzyme sample. as described by Bretz and Baggialini [ 1 S].
Myeloperoxidase was measured Unless otherwise stated, 0.1 ml enzyme sample were added to I ml of substrate solution and incubated for 1 min. Neutral protecrlytic activity was measured by hydrolyzing 1% (w/v) azocasein dissolved in 0.05 M phosphate buffer of indicated pH for 60 min at 37°C
f16J. Protein was determined by the microbiuret method of Goa [ 171 modified by Winkler et al. [18]. 2on~I uttrrxcentrifugab~u~~* This was carried out in a Beckman L2-65B ultracentrifuge using a Ti-44 zonal rotur and a piston gradient pump (Beckman, model 141). The stock solutions to form the gradient (5%, w/v, and 40% sucrose) were made by dissolving the appropriate amount of sucrose in 30 mM phosphate buffer (pH 6.0) containing 0.5 M MaCl. 20 ml of sample 137 mg protein) containing 2% (w/v) sucrose were layered over 450 ml gradient extending from 5 to 40% sucrose which was linear with the radius. The sample was overlayed with 130 ml phosphate buffer (pH 6.0) containing 0.5 M NaCl. Ultracentrifugation was carried out at 46 000 rev,/min for 26 h at 4°C. Loading and unloading of the rotor was performed according to Patsch et al. [19]. After centrifugation, the rotor effluent was monitored continuously at 280 nm and IO-ml fractions were collected and assayed for enzyme activities. Fractions containing lipolytic activity were pooled and concentrated by uItrafil~ratio~ using Sartsrius coflodion bags SM 13 200. Gel ~~~o~~~ogr~~~~. This was performed at 4°C on Sephadex G-IOQ column (2.5 X 90 cm) which was equilibrated and eluted with 10 mM phosphate buffer (pH 6.0) containing 0,013% (v/v) Triton X-la0 and 0.5 M NaCl. Fractions of 4 ml were collected at a flow rate of 23 ml/h and assayed for enzyme activities. The column was calibrated by the separate chromatography of blue dextran, bovine serum albumin (69 OOO), trypsin (24 000) and cytochrome c (12 500), The moleculrur weight of the proteins showing lipolytic activity was estimated according to the method of Whitaker [ 201. Results
Prelimirrary experiments were carried out to establish optimal. assay conditions for the hydrolysis of glycerol trioleate, Evidence had been obtained that albumin is needed in the assay mixture as a fatty acid acceptor for optimal lipolytic activity, but that the activity is independent from the albumin concentration in the range from 12 to 18 mg/ml assay mixture. In addition, the effect of increasing amounts of Triton X-100 in the substrate emulsion was investigated since the availability of substrate molecules for the enzyme at the oil-water interface depends on the amount of detergent present in the emulsion [21,221f. Fig. la shows that acid lipolytic activity of human leucocyte granules is optimal in the presence of 0.3 to 0.5 ~1 of Triton X-100 per mi of assay mixture, Subsequently, the effect of increasing amounts of glycerol trioleate at a constant T&on X-100 concentration of 0.4 @/ml of assay mixture was determined. Hydrolysis of trioleate reached a plateau at about 8 gmol of trioleate
297
a
04 0.2
0
Tntm
0.4
X - 100
$~llml
0.6 assay
0.8
10
mIxtore>
0 0
10
5 trioleate
(pmol/ml
15
20
assay mixture1
Fig. 1. Assay conditions for zlycerol trioleate hydrolysis by human leucocyte granules. Assay mixture consisted of 0.2 ml granule suspension (400 fig protein) in 0.34 M sucrose containing 0.013% (v/v) Triton X-100 and 0.3 ml of substrate emulsion. Incubation was carried out for I h at 37OC. (a) Lipolytic activity in the presence of increasing amounts of T&on X-100 and a constant substrate eoncentretion of 8.5 pmd per ml of assay mixture. (b> Lipolytic activity in the presence of increasing amounts of 8;t~ceroI triolellte and a constant Triton X-100 e~n&e~tratiu~ of 0.4 $.alper ml of assay mixture. The two sxnbok represent vaiues of two separate experiments.
per ml of assay mixture and was then found to be independent from the substrate ~on~ent~~ti~n up to 20.0 ~mol~rnl (Fig, lb). Triton X-100 had also to be present in the suspension af leucocytes or granules, respectively, to get optimal rates of hydrolysis. A concentration of 0.01% (v/v) in the enzyme sample was sufficient to reach the plateau of optimal activity. In subsequent experiments, the concentration of Triton X-100 in the enzyme samples was 0.013% (v/v>. At standard concentrations of 8.5 ymol glycerol trioleate, 0.4 ~1 Triton X-100 and 15 mg albumin per ml of assay mixture, the amount of liberated fatty acid was Iinear with the time of incubation up to 75 min as we11as with the amount of added granule protein in the studied range from 200 to 950 pg protein, Dependence of EiJxdytic activity on pH Hydrolysis of glycerol trioleate by homogenates of leucocytes as well as by
of these cells was measured at pH values ranging from 4.0 Fig, 2 shows a sharp optimum uf lipofytic activity in homogenates of
the $igrmule fra&iun
to
9.0,
leucoeytes at pH 5.25-5.30. Constantly, a broad shoulder between pH 5.8 and 7.0 was found. Essentially the same dependence on pH was determined with the granule fraction of human leucocytes.
Acid lipolytic activity in various fractions of human leucocy tes The distribution of acid lipolytic activity in various fractions of human leucoeytes has been compared to that of the typical granule bound enzyme myefoperoxidase, The results are shown in Table I_ A similar distribution pattern has been observed for both the enzymes, thus ~d~~at~ng that the e~ymes res~o~s~b~e for the hydru~ys~s of h-k&-&e are ~~~~~e~ in the granules of human feucocytes. The relatively high amount of both enzymes in the nuc‘iear fraction might be due to agglutinated granules which sedimented with the nuclei. The specific activity of the granule fraction (170.4 rt 38.9 nmol * h-” * wz-‘, n = 7) was increased about 3-fold over that of the homogenate (59.6 rt. 21.6 nmol - h-” * mg-‘, n = 12).
Properties of lipoly tic activity Hydrolytic activity on trioleate in human leucocytes proved to be very labile at temperatures above 0°C. When ahquots of a granufe suspension in 0.34 M sucrose ~o~t~~~~g U.~~3~ Triton X-100 were stared at 4”6, the hydrolytic activity on trioleate dropped to about 50% of the InSal vahze within two days and to 20% within nme days, respectively. Qn the other hand, when granules were stored at --25”C, after nine days, 95% of the original activity could still be measured. Thus it was possible to pool and store granules for purification
299 TABLE
I
ACID LIPOLYTIC
ACTIVITY
IN VARIOUS
The results are expressed as percentages, the activities in the fractions was taken
FRACTIONS
OF HUMAN
Protein (n = 5)
Nuclei
(600
LEUCOCYTES
mean + S.D.; n = number of separate experiments. The sum of to be loo%, and the percentage in each fraction was calculated.
X g, 15 min)
Lipolytic activity (n = 6)
16.9 f 1.1 36.1 + 12.2 46.9 f 10.8
Granule fraction (15 000 X g, 20 min) Postgranular supernatant
16.8 +
Myeloperoxidase (n = 5) 8.8
20.1 * 11.3
65.4 + 14.6 17.8 * 8.7
68.7 ? 10.8 11.2 * 8.8
experiments at -25°C without significant loss of activity. The influence of various substances on lipolytic activity are summarized in Table II. The presence of NaCl, KCl, CaCl, and p-hydroxymercuribenzoate, respectively, considerably reduced the lipolytic activity, whereas the presence of EDTA was of no great influence. Dithiothreitol as well as sodium iodoacetate slightly inhibited the lipolytic activity. Purification In order TABLE
of lipolytic activity to purify lipolytic activity
by chromatographic
or ultracentrifugal
II
INFLUENCE GRANULES
OF
VARIOUS
SUBSTANCES
ON LIPOLYTIC
ACTIVITY
OF HUMAN
LEUCOCYTE
Granules were suspended in 0.34 M sucrose containing 0.013% (v/v) Triton X-100 and the appropriate concentration of the substance to be tested. The assay mixture consisted of 0.2 ml of granule suspension and 0.8 ml of substrate emulsion. The enzyme sample containing dithiothreitol was preincubated for 30 min at 37’C; all the other samples were used without preincubation. Lipolytic activity was measured as described in Methods. Added
substance
Concentration in the assay
Activity
(%)
Number of experiments
(mM) None NaCl
KC1
CaCl2
EDTA
Dithiothreitol Sodium iodoacetate p-Hydroxymercuribenzoate
30 100 200 300 400 30 100 200 2.5 5 10 20 1 5 10 1 2 0.5 2.5
100 88 70 60 51 41 72 41 35 76 75 68 47 101 101 102 92 93 42
Biochimica et Biophysics Acta, 488 (1977) 294-304 0 Elsevier/North-Holland Biomedical Press
BBA 57033
~~RACTERIZATION AND PARTIAL FROM H~AN LEUKOCYTES
R. RINDLER-LUDWIG, Department
PURIFICATION
OF ACID LIPASE
W. PATSCH, S. SAILER and H. BRAUNSTEINER
of Rledicine, University of Innsbruck,
A-6020 Znnsbruck {Austria)
{Received March Znd, 1977)
Summary Hydrolysis of glycerol trioleate by human leucocytes was characterized and the enzymes responsible for this activity were obtained in a purified form by means of gel chromatography on Sephadex G-100 as well as by zanal ultracenThe activity is localized in the trifugation followed by gel chromatography. granule fraction of leucocytes (15 000 X g, 20 min) and shows a sharp pH optimum at pH 5.25. As judged from the elution profile obtained by gel chwomatography, two proteins are likely to contribute to the hydrolysis of glycerol trioleate. The approximate molecular weights of the two enzymes are 74 100 and 60 300, respectively. The activity is reduced in the presence of NaCl, KCl, CaClz as well as of p-hydroxymercuribenzoate. The enzymes are stable at -25°C but loose about 50% of their activity within 48 h at 4°C.
Introduction In many metabolic disorders, the lysosomal defect of the target tissue is reflected in leucocytes. In these cases, leucocytes offer the advantage of beeing readily and repeatedly available both for metabolic investigations as well as for the diagnosis of disorders of metabolism. Defects of lysosomal acid lipase have been observed in various inborn disorders of metabolism which are characterized by lipid accumulation in several organs. Severe deficiency of liver and spleen cells in acid lysosomal lipase [l] has been described in Wolman’s disease [2]. In patients with cholesterol ester storage disease [3] also a deficiency of acid lipase has been found in liver cells [ 4] as well as in cultured fibroblasts [ 51. Evidence for the presence of acid lipolytic activity in human leucocytes was presented by Braunsteiner et al. [6,7]. So far, however, detailed investigation was carried out only on the lipolytic activity in rabbit polymorphonuclear leucocytes [S,Q].
295
The present work reports the partial purification and ch~acte~zation lipolytic activity of human leucocytes.
of acid
Materials The following reagents were used: glycerol t.ri[l-14C]oleate, spec. act. 55 mCi/mmol (The Radiochemical Centre, Amersham, England); bovine serum albumin, crystallized and lyophilized, used as a standard for protein determination, azocasein, DL-dithiothreitol (Sigma, St. Louis, U.S.A.); bovine serum albumin, powder, fraction V, used in the lipase assay (Armour Pharmaceutical Company Ltd., ~astbou~e, England); friton X-100, trypsin, cytochrome c from horse heart, glycerol trioleate, Amber&e IRA-400 and ~-hydroxymercuribenzoate (Serva, Heidelberg, G.F.R.); Sephadex G-100, Dextran T 500, Blue Dextran 2000 (Pharmacia, Uppsala, Sweden); NCS-tissue solubilizer (Amersham/ Searle Corporation, Arlington Heights, Ill., U.S.A.). Methods Isolation of ieucocy tes Blood was obtained from patients with a myeloid reaction and processed as described earlier [lo]. The isolated white blood cells consisted of approximately 80% of neutroph~ polymo~honucle~ leucocytes or earlier stages of neutrophil cells, S-12% of monocytes and S---12% of lymphocytes. This was determined by differential counts either of smears stained for 10 min for naphto1 AS-D chloroacetate esterase [ll] or of May-Grtinwald-Giemsa smears. Homogenization of the cells was carried out in 0.34 M sucrose [lo]. After sedimentation of nuclei and agglutinated granules at 600 X g for 15 min the granules were collected at 15 000 X g for 20 min. To determine lipolytic activity in subcellular fractions of human leucocytes, the nuclear as well as the granule pellet were resuspended in 0.34 M sucrose containing 0.013% (v/v) Triton X-100. To the postgranular supernatant 0.5% (v/v) Triton X-100 in 0.34 M sucrose was added to give a final concentration of 0.013%.
Lipolytic activity was determined by the method of Krauss et al. [12]. The assay mixture contained in a final volume of 1 ml: 10.4 nmol glycerol tri[l-14C]oleate (rechromatographied before use to a purity of more than 99%); 8.5 pmol unlabeled glycerol trioleate; 15 mg bovine serum albumin; 0.40 ~1 Triton X-100. These constituents were sonicated at 4°C in 0.1 M sodium acetate buffer (pH 5.25) by four bursts of 60 s each in conical tubes using an ultrasonic disintegrator (Megason Ultrasonic Instruments International, Farmingdale, N.Y., U.S.A.). In the standard assay, 0.2 ml of enzyme sample were added to 0.8 ml of substrate emulsion and incubated for 60 min at 37°C in a shaking water bath. The reaction was terminated by lipid extraction [13]. Free fatty acids were isolated by the method of Kelley 1141. Counting was performed in a Packard Tricarb liquid scint~lation spectrometer. All assays were performed in duplicate; duplicate values differed by less than 5%. Blanks were constituted by incubating the substrate emulsion with the appropriate buffer
without
liberated
enzyme
sample.
Lipdytic
either per h per mg protein?
activity was expressed as nmol of fatty acid or per h per ml enzyme sample. as described by Bretz and Baggialini [ 1 S].
Myeloperoxidase was measured Unless otherwise stated, 0.1 ml enzyme sample were added to I ml of substrate solution and incubated for 1 min. Neutral protecrlytic activity was measured by hydrolyzing 1% (w/v) azocasein dissolved in 0.05 M phosphate buffer of indicated pH for 60 min at 37°C
f16J. Protein was determined by the microbiuret method of Goa [ 171 modified by Winkler et al. [18]. 2on~I uttrrxcentrifugab~u~~* This was carried out in a Beckman L2-65B ultracentrifuge using a Ti-44 zonal rotur and a piston gradient pump (Beckman, model 141). The stock solutions to form the gradient (5%, w/v, and 40% sucrose) were made by dissolving the appropriate amount of sucrose in 30 mM phosphate buffer (pH 6.0) containing 0.5 M MaCl. 20 ml of sample 137 mg protein) containing 2% (w/v) sucrose were layered over 450 ml gradient extending from 5 to 40% sucrose which was linear with the radius. The sample was overlayed with 130 ml phosphate buffer (pH 6.0) containing 0.5 M NaCl. Ultracentrifugation was carried out at 46 000 rev,/min for 26 h at 4°C. Loading and unloading of the rotor was performed according to Patsch et al. [19]. After centrifugation, the rotor effluent was monitored continuously at 280 nm and IO-ml fractions were collected and assayed for enzyme activities. Fractions containing lipolytic activity were pooled and concentrated by uItrafil~ratio~ using Sartsrius coflodion bags SM 13 200. Gel ~~~o~~~ogr~~~~. This was performed at 4°C on Sephadex G-IOQ column (2.5 X 90 cm) which was equilibrated and eluted with 10 mM phosphate buffer (pH 6.0) containing 0,013% (v/v) Triton X-la0 and 0.5 M NaCl. Fractions of 4 ml were collected at a flow rate of 23 ml/h and assayed for enzyme activities. The column was calibrated by the separate chromatography of blue dextran, bovine serum albumin (69 OOO), trypsin (24 000) and cytochrome c (12 500), The moleculrur weight of the proteins showing lipolytic activity was estimated according to the method of Whitaker [ 201. Results
Prelimirrary experiments were carried out to establish optimal. assay conditions for the hydrolysis of glycerol trioleate, Evidence had been obtained that albumin is needed in the assay mixture as a fatty acid acceptor for optimal lipolytic activity, but that the activity is independent from the albumin concentration in the range from 12 to 18 mg/ml assay mixture. In addition, the effect of increasing amounts of Triton X-100 in the substrate emulsion was investigated since the availability of substrate molecules for the enzyme at the oil-water interface depends on the amount of detergent present in the emulsion [21,221f. Fig. la shows that acid lipolytic activity of human leucocyte granules is optimal in the presence of 0.3 to 0.5 ~1 of Triton X-100 per mi of assay mixture, Subsequently, the effect of increasing amounts of glycerol trioleate at a constant T&on X-100 concentration of 0.4 @/ml of assay mixture was determined. Hydrolysis of trioleate reached a plateau at about 8 gmol of trioleate
297
a
04 0.2
0
Tntm
0.4
X - 100
$~llml
0.6 assay
0.8
10
mIxtore>
0 0
10
5 trioleate
(pmol/ml
15
20
assay mixture1
Fig. 1. Assay conditions for zlycerol trioleate hydrolysis by human leucocyte granules. Assay mixture consisted of 0.2 ml granule suspension (400 fig protein) in 0.34 M sucrose containing 0.013% (v/v) Triton X-100 and 0.3 ml of substrate emulsion. Incubation was carried out for I h at 37OC. (a) Lipolytic activity in the presence of increasing amounts of T&on X-100 and a constant substrate eoncentretion of 8.5 pmd per ml of assay mixture. (b> Lipolytic activity in the presence of increasing amounts of 8;t~ceroI triolellte and a constant Triton X-100 e~n&e~tratiu~ of 0.4 $.alper ml of assay mixture. The two sxnbok represent vaiues of two separate experiments.
per ml of assay mixture and was then found to be independent from the substrate ~on~ent~~ti~n up to 20.0 ~mol~rnl (Fig, lb). Triton X-100 had also to be present in the suspension af leucocytes or granules, respectively, to get optimal rates of hydrolysis. A concentration of 0.01% (v/v) in the enzyme sample was sufficient to reach the plateau of optimal activity. In subsequent experiments, the concentration of Triton X-100 in the enzyme samples was 0.013% (v/v>. At standard concentrations of 8.5 ymol glycerol trioleate, 0.4 ~1 Triton X-100 and 15 mg albumin per ml of assay mixture, the amount of liberated fatty acid was Iinear with the time of incubation up to 75 min as we11as with the amount of added granule protein in the studied range from 200 to 950 pg protein, Dependence of EiJxdytic activity on pH Hydrolysis of glycerol trioleate by homogenates of leucocytes as well as by
of these cells was measured at pH values ranging from 4.0 Fig, 2 shows a sharp optimum uf lipofytic activity in homogenates of
the $igrmule fra&iun
to
9.0,
leucoeytes at pH 5.25-5.30. Constantly, a broad shoulder between pH 5.8 and 7.0 was found. Essentially the same dependence on pH was determined with the granule fraction of human leucocytes.
Acid lipolytic activity in various fractions of human leucocy tes The distribution of acid lipolytic activity in various fractions of human leucoeytes has been compared to that of the typical granule bound enzyme myefoperoxidase, The results are shown in Table I_ A similar distribution pattern has been observed for both the enzymes, thus ~d~~at~ng that the e~ymes res~o~s~b~e for the hydru~ys~s of h-k&-&e are ~~~~~e~ in the granules of human feucocytes. The relatively high amount of both enzymes in the nuc‘iear fraction might be due to agglutinated granules which sedimented with the nuclei. The specific activity of the granule fraction (170.4 rt 38.9 nmol * h-” * wz-‘, n = 7) was increased about 3-fold over that of the homogenate (59.6 rt. 21.6 nmol - h-” * mg-‘, n = 12).
Properties of lipoly tic activity Hydrolytic activity on trioleate in human leucocytes proved to be very labile at temperatures above 0°C. When ahquots of a granufe suspension in 0.34 M sucrose ~o~t~~~~g U.~~3~ Triton X-100 were stared at 4”6, the hydrolytic activity on trioleate dropped to about 50% of the InSal vahze within two days and to 20% within nme days, respectively. Qn the other hand, when granules were stored at --25”C, after nine days, 95% of the original activity could still be measured. Thus it was possible to pool and store granules for purification
299 TABLE
I
ACID LIPOLYTIC
ACTIVITY
IN VARIOUS
The results are expressed as percentages, the activities in the fractions was taken
FRACTIONS
OF HUMAN
Protein (n = 5)
Nuclei
(600
LEUCOCYTES
mean + S.D.; n = number of separate experiments. The sum of to be loo%, and the percentage in each fraction was calculated.
X g, 15 min)
Lipolytic activity (n = 6)
16.9 f 1.1 36.1 + 12.2 46.9 f 10.8
Granule fraction (15 000 X g, 20 min) Postgranular supernatant
16.8 +
Myeloperoxidase (n = 5) 8.8
20.1 * 11.3
65.4 + 14.6 17.8 * 8.7
68.7 ? 10.8 11.2 * 8.8
experiments at -25°C without significant loss of activity. The influence of various substances on lipolytic activity are summarized in Table II. The presence of NaCl, KCl, CaCl, and p-hydroxymercuribenzoate, respectively, considerably reduced the lipolytic activity, whereas the presence of EDTA was of no great influence. Dithiothreitol as well as sodium iodoacetate slightly inhibited the lipolytic activity. Purification In order TABLE
of lipolytic activity to purify lipolytic activity
by chromatographic
or ultracentrifugal
II
INFLUENCE GRANULES
OF
VARIOUS
SUBSTANCES
ON LIPOLYTIC
ACTIVITY
OF HUMAN
LEUCOCYTE
Granules were suspended in 0.34 M sucrose containing 0.013% (v/v) Triton X-100 and the appropriate concentration of the substance to be tested. The assay mixture consisted of 0.2 ml of granule suspension and 0.8 ml of substrate emulsion. The enzyme sample containing dithiothreitol was preincubated for 30 min at 37’C; all the other samples were used without preincubation. Lipolytic activity was measured as described in Methods. Added
substance
Concentration in the assay
Activity
(%)
Number of experiments
(mM) None NaCl
KC1
CaCl2
EDTA
Dithiothreitol Sodium iodoacetate p-Hydroxymercuribenzoate
30 100 200 300 400 30 100 200 2.5 5 10 20 1 5 10 1 2 0.5 2.5
100 88 70 60 51 41 72 41 35 76 75 68 47 101 101 102 92 93 42
