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A purified triglyceride lipase, lipoprotein in nature, from rat adipose tissue
295
PRELIMINARY NOTES
BBA 91293
A purified triglyceride lipase, lipoprotein in noture, from rot odipose tissue We have recently shown that a soluble and stable triglyceride lipase preparation is obtained when adipose tissue is homogenized in 0.25 M sucrose containing I. l O - 3 M EDTA z-3. Lipase activity in this soluble fraction was further separated into two subfractions by preparative ultracentrifugation at a medium density of 1.12. Previous exposure of the intact fat pads to epinephrine caused, preferentially, an enhancement of lipase activity in the floating (d < 1.12) fraction. The present communication describes further purification yielding a fraction homogeneous by analytical ultracentrifugation. Epididymal fat pads from fifty Sprague-Dawley rats (200-300 g) previously fasted for 24 h were incubated for 15 rain in Krebs-Ringer bicarbonate, pH 7.4, containing 5" lO-5 M epinephrine. Homogenization of the tissue and preparation of the supernatant fraction were carried out as described earlier 3. Triglyceride lipase was assayed using a [l*Cltriolein emulsion substrate stabilized with 5 % gum arabic 3. The released radioactive fatty acids were isolated and counted by a modification of the resin method described by KELLEY4. Protein 5, inorganic phosphorus 6, cholesterol7 and triglyceride s were assayed with micromodifications of the published techniques. Sedimentation velocity was measured in a Spinco Model E analytical ultracentrifuge using an An-H rotor. Sedimentation constants were calculated from the ultraviolet scanner patterns with appropriate corrections for viscosity and density. Molecular weights were determined by sedimentation equilibrium technique °. Rotor speed used was 4609 rev./min and pzotein concentrations of the solutions were 0.05-0.2 mg/ml.
TABLE I PURIFICATION OF TRIGLYCERIDE LIPASE FROM RAT ADIPOSE TISSUE F F A s t a n d s for free f a t t y acids.
Fraction
Total Total activity protein (lzEq FFA/h) (rag)
Specific activity (l~Eq FFA/h/ mg protein)
Relative specific activity
Yield (%)
78 ooo × g supernatant
25o
435
o.58
i
p H 5.2 precipitate
235
91
2.6
4-5
94
d < 1.12 fraction
ioo
I o5
15.7
6.7
I 1.5
42
d 1.o6-1.12 fraction
58
8.3
7.o
12. I
23
Agarose fraction
48
3.0
16.o
27.6
19
8 7 ooo × g supernatant
38
o.61
62.0
lO6
15
Biochim. Biophys. Acta, 224 (197 o) 295-298
296
PRELIMINARY NOTES
The purification procedure is summarized in Table I. 9 ° °/o of the total triglyceride lipase activity in the 78 ooo X g supernatant fraction was precipitated at pH 5.2. The precipitate was taken up in 0.02 M tris, pH 7.4, containing lO _3 M EDTA and lO -3 M dithiothreitol, and the density of the solution was adjusted to 1.12 with sucrose. After 48 h ultracentrifugation in a 40.3 rotor (4° ooo rev./min, 5°), 45 % of the lipase activity was recovered in the floating (d < 1.12) fraction. This fraction was adjusted to d = 1.o6 and centrifuged at 40 ooo rev./min for 36 h. More than 5 ° ~o of the activity was found in the sedimenting (d 1.o6-1.12) fraction with only a slight increase in the specific activity. Large amounts of opalescent, lipid-containing material were, however, removed with the floating top fraction. The d 1.o6-1.12 fraction was further purified by chromatography on a 6 °/o agarose gel column. Lipase activity emerged from the column in the void volume, well separated from a second protein peak. Further purification was obtained by centrifugation at 35 ooo rev./min (40.3 rotor) for 30 rain. The lipase in the clear supernatant was used for subsequent characterization of the enzyme. A io6-fold purification over that in the original 78 ooo ×g supernatant fraction was obtained with 15-2o °/o overall recovery of triglyceride lipase activity. Equilibrium density gradient ultracentrifugation of the material in sucrose showed one peak of lipase activity in the d 1.o8-1.o 9 region with constant specific activity across the peak. The relative amounts of protein (bovine serum albumin standard), phospholipid and cholesterol in the purified material were 48 %, 45 %, and 6 °/o respectively. High speed sedimentation equilibrium analysis by the method of YPHANTIS9 was carried out on two independent preparations. The plot of logarithm of fringe displacement against r 2 yielded a single straight line within experimental error, as expected for a homogeneous preparation. Assuming a partial specific volume of 0.89 (the value for rat serum high-density lipoprotein, which also is approximately 50 9/o lipid by weight1°), the molecular weight was calculated to be 7.2. lO6. Additionally, three preparations were examined by the sedimentation velocity method using ultraviolet scanning. Symmetrical curves were observed on all scans (taken at 4 min intervals during runs of 3o to 4 ° min) and the calculated s20,~ values were 33.4, 35.2 and 34.o. Using tissue from fed rats, we have shown (unpublished results) that the bulk of lipoprotein lipase activity is removed during the first density ultracentrifugation step. Fig. I illustrates the pH-activity curve and the effect of dialysed serum on the activity of the purified lipase. At lower pH values a slight inhibition was observed, whereas at higher pH values there was activation. The effect of serum additions at pH 6.8 and 8.2 was followed during the purification of lipase from rats fasted for 36 hr. The shape of the pH-activity curves was similar throughout the purification. This suggests that the shift in the pH-activity curve in the presence of serum is characteristic for the isolated lipase and is not due to contamination by lipoprotein lipase. The lipase-stimulating effect of prior exposure of the fat pad to epinephrine has been demonstrated previously in the d < 1.12 fraction 3. In the present studies, yield of pure lipase from this fraction was about 4o %. Consequently, it seems most likely that the purified material represents the hormone-sensitive lipase of adipose tissue. The chemical composition of the pure enzyme and its behavior on sucrose-density centrifugation indicated that the enzyme is (or is associated with) a high-molecular Biochim. Biophys. Acta, 224 (197o) 295-298
297
PRELIMINARY NOTES
• t
II
_
SERUM
+
SERUM
1.o<
C-- o.5(.J
J
I
i
5.0
6.0
7.0
8.0
9.0
pH
Fig. i. The p H - a c t i v i t y curve for purified lipase in the presence and absence of dialysed serum. Triolein emulsion was incubated with s e r u m or equivalent a m o u n t of buffer for 3 ° min at 37 ° before the assay. Final concentration of s e r u m in the assay m i x t u r e was i o %. Circles indicate the values obtained with p h o s p h a t e buffer and squares the values obtained with Tris buffer, respectively. F F A s t a n d s for free f a t t y acids.
weight lipoprotein particle with a density of 1.o8-1.o 9. Whether the particle represents a multienzyme complex with several catalytic and possibly regulatory subunits remains to be established. Hormone-sensitive lipase is converted from an inactive to an active form by exposure of adipose tissue to any of a number of so-called lipolytic hormones and this conversion is related to increases in tissue levels of cyclic-3',5'adenosine monophosphate 11,1~. Recent studies in this laboratory show that activation can be effected in our d < 1.12 fraction using protein kinase from rabbit muscle in the presence of ATP and cyclic-3',5'-adenosine monophosphate TM. Thus, the inactive (or less active) form of the hormone-sensitive lipase fractionates with the active form at least through that stage of purification. Studies are in progress to determine whether the most highly purified lipase preparation also contains both "inactive" and active forms of the enzyme. We are indebted to Mr. Johannes Everse, Department of Chemistry, University of California, San Diego, for carrying out the ultracentrifuge analyses. This work was supported in part by a grant from the National Institutes of Health, USPHS, HE-I2373 and by a Public Health Service International Postdoctoral Research Fellowship, Fo5 TW 01386, to Jussi K. Huttunen.
Division o/Metabolic Disease, Department o~ Medicine, University o/Cali/ornia, San Diego, La Jolla, Call/. 92037 (U.S.A,)
JossI K. HUTTUNEN ALEGRIA A. AOUINO DANIEL STEINBERG
Note added in proo]: Since this m a n u s c r i p t was s u b m i t t e d we h a v e s h o w n t h a t the p u r e p r e p a r a t i o n is a c t i v a t e d b y r a b b i t muscle p r o t e i n kinase. Activation is t o t a l l y d e p e n d e n t on addition of b o t h cyclic 3', 5 '-adenosine m o n o p h o s p h a t e a n d ATP. I n the presence of [ySZp-]ATP the increase in lipase a c t i v i t y as a f u n c t i o n of time is accompanied b y a parallel increase in lipase-bound r a d i o a c t i v i t y 14. Biochim. Biophys. Acta, 224 (197 o) 295-298
298
PRELIMINARY NOTES
I J. K. HUTTUNEN, J. ELLINGBOE, t{. C. PITTMAN AND D. STEINBERG, Federation Proc., 29 (197o) 267, Abs. 2 J. K. HUTTUNEN, J. ELLINGBOE, IR. C. PITTMAN AND D. STEINBERG, Clin. Res., 18 (197 o) 14o. 3 J. K. HUTTUNEN, J. ELLINGBOE, It. C. PITT.MAN AND D. STEINBERG, Biochim. Biophys. Acta, 218 (197 ° ) 333. 4 T. F. KELLEY, J. Lipid Res., 9 11968) 799. 5 0 . H. LOWRY, N. j. ROSEBROUGH, A. L. FARR AND R. j. I{ANDALL, J. Biol. Chem.,I93 (1951) 265 . 6 G. R. BARTLETT, J. Biol. Chem., 234 (1959) 466. 7 B. ZAK, Am. J. Clin. Path., 27 (1957) 1583. 8 E. VAN HANDEL AND D. B. ZILVERSMIT, J . Lab. Clin. Med., 5 ° (1957) 152. 9 D. A. YPHANTIS, Biochemistry, 3 (1964) 297. Io S. I{OGA, D. L. HORWlTZ AND A. M. SCANU, J. Lipid Res., IO (1969) 577. I I D. STEINBERG, in D. KRITCHEVSKY, R. PAOLETTI AND D. STEINBERG, Progress in Biochemical Pharmacology, Karger, Basel a n d N e w York, Vol. 3, 1967, PP. 139-15o. 12 I{. XV. BUTCHER AND E. W. SUTHERLAND, Ann. N . Y . Acad. Sci., 139 (1967) 849. 13 J. K. HUTTtlNEN, D. STEINBERG AND S. E. MAYER, Proc. Natl. Acad. Sci. U.S., 67 (197 o) 290. 14 J. K. HFTTUNEN, D. STEINBERG AND S. E. MAYER, Clin. Res., in t h e press.
Received July 6th, 197o Biochim. Biophys. Acta, 224 (197 o) 295-298
T I T L E S OF R E L A T E D P A P E R S IN O T H E R S E C T I O N S T h e following p a p e r s t h a t h a v e r e c e n t l y a p p e a r e d in o t h e r sections of BIOCHIMICA ET BIOPHYSICA ACTA m a y be i n t e r e s t to t h e r e a d e r s of t h i s specialized section:
BBA-ENZYMOLOGY Effect of dipicolinic acid on bacterial cyclic 3 ' , 5 ' - n u c l e o t i d e p h o s p h o d i e s t e r a s e (BBA 63471 ) b y T. OKABAYASm AND M. IDE (Osaka) . . . . . . . . . . . . . . . . . 22o(197o)124 T h e extracellular nuclease a c t i v i t y of Micrococcus sodonensis. III. K i n e t i c s t u d i e s a n d control of p r o d u c t i o n (BBA 6617,5) b y S. A. BERRY AND J. N. CAMPBELL ( E d m o n t o n ) . . . . . . . . . . . . 22o(197o)256 T h e e x t r a c e n u l a r nuclease a c t i v i t y of Micrococcus sodinensis. IV. P h y s i c a l studies, c h a r a c t e r i z a t i o n as a glycoprotein a n d i n v o l v e m e n t w i t h t h e cell wall (BBA 66176) b y S. A. BERRY, K. G. JOHNSON AND J. N. CAMPBELL ( E d m o n t o n ) . . . . . 22o(197o)269
BBA-BIOENERGETICS E l e c t r o n t r a n s p o r t in m a m m a l i a n nuclei. II. O x i d a t i v e e n z y m e s in a large scale p r e p a r a t i o n of bovine liver nuclei (BBA 46oo1) b y R. BEREZNEY, L. K. FUNK AND F, L. CRANE (Lafayette, Ind.) . . . . . 223(197o ) 61
PRELIMINARY NOTES
BBA 91293
A purified triglyceride lipase, lipoprotein in noture, from rot odipose tissue We have recently shown that a soluble and stable triglyceride lipase preparation is obtained when adipose tissue is homogenized in 0.25 M sucrose containing I. l O - 3 M EDTA z-3. Lipase activity in this soluble fraction was further separated into two subfractions by preparative ultracentrifugation at a medium density of 1.12. Previous exposure of the intact fat pads to epinephrine caused, preferentially, an enhancement of lipase activity in the floating (d < 1.12) fraction. The present communication describes further purification yielding a fraction homogeneous by analytical ultracentrifugation. Epididymal fat pads from fifty Sprague-Dawley rats (200-300 g) previously fasted for 24 h were incubated for 15 rain in Krebs-Ringer bicarbonate, pH 7.4, containing 5" lO-5 M epinephrine. Homogenization of the tissue and preparation of the supernatant fraction were carried out as described earlier 3. Triglyceride lipase was assayed using a [l*Cltriolein emulsion substrate stabilized with 5 % gum arabic 3. The released radioactive fatty acids were isolated and counted by a modification of the resin method described by KELLEY4. Protein 5, inorganic phosphorus 6, cholesterol7 and triglyceride s were assayed with micromodifications of the published techniques. Sedimentation velocity was measured in a Spinco Model E analytical ultracentrifuge using an An-H rotor. Sedimentation constants were calculated from the ultraviolet scanner patterns with appropriate corrections for viscosity and density. Molecular weights were determined by sedimentation equilibrium technique °. Rotor speed used was 4609 rev./min and pzotein concentrations of the solutions were 0.05-0.2 mg/ml.
TABLE I PURIFICATION OF TRIGLYCERIDE LIPASE FROM RAT ADIPOSE TISSUE F F A s t a n d s for free f a t t y acids.
Fraction
Total Total activity protein (lzEq FFA/h) (rag)
Specific activity (l~Eq FFA/h/ mg protein)
Relative specific activity
Yield (%)
78 ooo × g supernatant
25o
435
o.58
i
p H 5.2 precipitate
235
91
2.6
4-5
94
d < 1.12 fraction
ioo
I o5
15.7
6.7
I 1.5
42
d 1.o6-1.12 fraction
58
8.3
7.o
12. I
23
Agarose fraction
48
3.0
16.o
27.6
19
8 7 ooo × g supernatant
38
o.61
62.0
lO6
15
Biochim. Biophys. Acta, 224 (197 o) 295-298
296
PRELIMINARY NOTES
The purification procedure is summarized in Table I. 9 ° °/o of the total triglyceride lipase activity in the 78 ooo X g supernatant fraction was precipitated at pH 5.2. The precipitate was taken up in 0.02 M tris, pH 7.4, containing lO _3 M EDTA and lO -3 M dithiothreitol, and the density of the solution was adjusted to 1.12 with sucrose. After 48 h ultracentrifugation in a 40.3 rotor (4° ooo rev./min, 5°), 45 % of the lipase activity was recovered in the floating (d < 1.12) fraction. This fraction was adjusted to d = 1.o6 and centrifuged at 40 ooo rev./min for 36 h. More than 5 ° ~o of the activity was found in the sedimenting (d 1.o6-1.12) fraction with only a slight increase in the specific activity. Large amounts of opalescent, lipid-containing material were, however, removed with the floating top fraction. The d 1.o6-1.12 fraction was further purified by chromatography on a 6 °/o agarose gel column. Lipase activity emerged from the column in the void volume, well separated from a second protein peak. Further purification was obtained by centrifugation at 35 ooo rev./min (40.3 rotor) for 30 rain. The lipase in the clear supernatant was used for subsequent characterization of the enzyme. A io6-fold purification over that in the original 78 ooo ×g supernatant fraction was obtained with 15-2o °/o overall recovery of triglyceride lipase activity. Equilibrium density gradient ultracentrifugation of the material in sucrose showed one peak of lipase activity in the d 1.o8-1.o 9 region with constant specific activity across the peak. The relative amounts of protein (bovine serum albumin standard), phospholipid and cholesterol in the purified material were 48 %, 45 %, and 6 °/o respectively. High speed sedimentation equilibrium analysis by the method of YPHANTIS9 was carried out on two independent preparations. The plot of logarithm of fringe displacement against r 2 yielded a single straight line within experimental error, as expected for a homogeneous preparation. Assuming a partial specific volume of 0.89 (the value for rat serum high-density lipoprotein, which also is approximately 50 9/o lipid by weight1°), the molecular weight was calculated to be 7.2. lO6. Additionally, three preparations were examined by the sedimentation velocity method using ultraviolet scanning. Symmetrical curves were observed on all scans (taken at 4 min intervals during runs of 3o to 4 ° min) and the calculated s20,~ values were 33.4, 35.2 and 34.o. Using tissue from fed rats, we have shown (unpublished results) that the bulk of lipoprotein lipase activity is removed during the first density ultracentrifugation step. Fig. I illustrates the pH-activity curve and the effect of dialysed serum on the activity of the purified lipase. At lower pH values a slight inhibition was observed, whereas at higher pH values there was activation. The effect of serum additions at pH 6.8 and 8.2 was followed during the purification of lipase from rats fasted for 36 hr. The shape of the pH-activity curves was similar throughout the purification. This suggests that the shift in the pH-activity curve in the presence of serum is characteristic for the isolated lipase and is not due to contamination by lipoprotein lipase. The lipase-stimulating effect of prior exposure of the fat pad to epinephrine has been demonstrated previously in the d < 1.12 fraction 3. In the present studies, yield of pure lipase from this fraction was about 4o %. Consequently, it seems most likely that the purified material represents the hormone-sensitive lipase of adipose tissue. The chemical composition of the pure enzyme and its behavior on sucrose-density centrifugation indicated that the enzyme is (or is associated with) a high-molecular Biochim. Biophys. Acta, 224 (197o) 295-298
297
PRELIMINARY NOTES
• t
II
_
SERUM
+
SERUM
1.o<
C-- o.5(.J
J
I
i
5.0
6.0
7.0
8.0
9.0
pH
Fig. i. The p H - a c t i v i t y curve for purified lipase in the presence and absence of dialysed serum. Triolein emulsion was incubated with s e r u m or equivalent a m o u n t of buffer for 3 ° min at 37 ° before the assay. Final concentration of s e r u m in the assay m i x t u r e was i o %. Circles indicate the values obtained with p h o s p h a t e buffer and squares the values obtained with Tris buffer, respectively. F F A s t a n d s for free f a t t y acids.
weight lipoprotein particle with a density of 1.o8-1.o 9. Whether the particle represents a multienzyme complex with several catalytic and possibly regulatory subunits remains to be established. Hormone-sensitive lipase is converted from an inactive to an active form by exposure of adipose tissue to any of a number of so-called lipolytic hormones and this conversion is related to increases in tissue levels of cyclic-3',5'adenosine monophosphate 11,1~. Recent studies in this laboratory show that activation can be effected in our d < 1.12 fraction using protein kinase from rabbit muscle in the presence of ATP and cyclic-3',5'-adenosine monophosphate TM. Thus, the inactive (or less active) form of the hormone-sensitive lipase fractionates with the active form at least through that stage of purification. Studies are in progress to determine whether the most highly purified lipase preparation also contains both "inactive" and active forms of the enzyme. We are indebted to Mr. Johannes Everse, Department of Chemistry, University of California, San Diego, for carrying out the ultracentrifuge analyses. This work was supported in part by a grant from the National Institutes of Health, USPHS, HE-I2373 and by a Public Health Service International Postdoctoral Research Fellowship, Fo5 TW 01386, to Jussi K. Huttunen.
Division o/Metabolic Disease, Department o~ Medicine, University o/Cali/ornia, San Diego, La Jolla, Call/. 92037 (U.S.A,)
JossI K. HUTTUNEN ALEGRIA A. AOUINO DANIEL STEINBERG
Note added in proo]: Since this m a n u s c r i p t was s u b m i t t e d we h a v e s h o w n t h a t the p u r e p r e p a r a t i o n is a c t i v a t e d b y r a b b i t muscle p r o t e i n kinase. Activation is t o t a l l y d e p e n d e n t on addition of b o t h cyclic 3', 5 '-adenosine m o n o p h o s p h a t e a n d ATP. I n the presence of [ySZp-]ATP the increase in lipase a c t i v i t y as a f u n c t i o n of time is accompanied b y a parallel increase in lipase-bound r a d i o a c t i v i t y 14. Biochim. Biophys. Acta, 224 (197 o) 295-298
298
PRELIMINARY NOTES
I J. K. HUTTUNEN, J. ELLINGBOE, t{. C. PITTMAN AND D. STEINBERG, Federation Proc., 29 (197o) 267, Abs. 2 J. K. HUTTUNEN, J. ELLINGBOE, IR. C. PITTMAN AND D. STEINBERG, Clin. Res., 18 (197 o) 14o. 3 J. K. HUTTUNEN, J. ELLINGBOE, It. C. PITT.MAN AND D. STEINBERG, Biochim. Biophys. Acta, 218 (197 ° ) 333. 4 T. F. KELLEY, J. Lipid Res., 9 11968) 799. 5 0 . H. LOWRY, N. j. ROSEBROUGH, A. L. FARR AND R. j. I{ANDALL, J. Biol. Chem.,I93 (1951) 265 . 6 G. R. BARTLETT, J. Biol. Chem., 234 (1959) 466. 7 B. ZAK, Am. J. Clin. Path., 27 (1957) 1583. 8 E. VAN HANDEL AND D. B. ZILVERSMIT, J . Lab. Clin. Med., 5 ° (1957) 152. 9 D. A. YPHANTIS, Biochemistry, 3 (1964) 297. Io S. I{OGA, D. L. HORWlTZ AND A. M. SCANU, J. Lipid Res., IO (1969) 577. I I D. STEINBERG, in D. KRITCHEVSKY, R. PAOLETTI AND D. STEINBERG, Progress in Biochemical Pharmacology, Karger, Basel a n d N e w York, Vol. 3, 1967, PP. 139-15o. 12 I{. XV. BUTCHER AND E. W. SUTHERLAND, Ann. N . Y . Acad. Sci., 139 (1967) 849. 13 J. K. HUTTtlNEN, D. STEINBERG AND S. E. MAYER, Proc. Natl. Acad. Sci. U.S., 67 (197 o) 290. 14 J. K. HFTTUNEN, D. STEINBERG AND S. E. MAYER, Clin. Res., in t h e press.
Received July 6th, 197o Biochim. Biophys. Acta, 224 (197 o) 295-298
T I T L E S OF R E L A T E D P A P E R S IN O T H E R S E C T I O N S T h e following p a p e r s t h a t h a v e r e c e n t l y a p p e a r e d in o t h e r sections of BIOCHIMICA ET BIOPHYSICA ACTA m a y be i n t e r e s t to t h e r e a d e r s of t h i s specialized section:
BBA-ENZYMOLOGY Effect of dipicolinic acid on bacterial cyclic 3 ' , 5 ' - n u c l e o t i d e p h o s p h o d i e s t e r a s e (BBA 63471 ) b y T. OKABAYASm AND M. IDE (Osaka) . . . . . . . . . . . . . . . . . 22o(197o)124 T h e extracellular nuclease a c t i v i t y of Micrococcus sodonensis. III. K i n e t i c s t u d i e s a n d control of p r o d u c t i o n (BBA 6617,5) b y S. A. BERRY AND J. N. CAMPBELL ( E d m o n t o n ) . . . . . . . . . . . . 22o(197o)256 T h e e x t r a c e n u l a r nuclease a c t i v i t y of Micrococcus sodinensis. IV. P h y s i c a l studies, c h a r a c t e r i z a t i o n as a glycoprotein a n d i n v o l v e m e n t w i t h t h e cell wall (BBA 66176) b y S. A. BERRY, K. G. JOHNSON AND J. N. CAMPBELL ( E d m o n t o n ) . . . . . 22o(197o)269
BBA-BIOENERGETICS E l e c t r o n t r a n s p o r t in m a m m a l i a n nuclei. II. O x i d a t i v e e n z y m e s in a large scale p r e p a r a t i o n of bovine liver nuclei (BBA 46oo1) b y R. BEREZNEY, L. K. FUNK AND F, L. CRANE (Lafayette, Ind.) . . . . . 223(197o ) 61