Vol. 65, No. 4, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ACTIVATION OF HIGHLY PURIFIED LiPOPROTEIN LIPASE FROM BOVINE MILK A.-M. Ostlund-Lindqvist and P.-H. Iverius
Department of Medical and Physiological Chemistry, University of Uppsala, Biomedicum, Box 575, S-751 23
Uppsala, Sweden.
Received June 27,1975 SUMMARY The effects of apolipoproteins from very low density human lipoproteins on the activity of highly purified lipoprotein lipase from bovine milk was studied using a triglyceride phospholipid emulsion (Intralipid) as substrate. No activity was recorded with the substrate alone and apoC-ll was the only apolipoprotein that activated the enzyme. Maximal activity required a sufficient concentration of activator as well as an optimal ratio between activator and substrate. At substrate excess, the enzyme activity was reduced by about 50% or less depending on the concentration of activator employed. Under these conditions, however, appropriate additions of apoC-I or apoC-lll restored part of the lost activity. These findings may explain the contradictory results reported previously.
INTRODUCTION Lipoprotein lipase (EC 3. i. i. 3 ) has a key function in the metabolism of plasma triglyeerides.
The chylomicrons and the very
low density lipoproteins are the natural substrates of the enzyme (i). Artificial triglyceride emulsions are also hydrolyzed, provided that certain protein components of the plasma lipoproteins are present (2). For this purpose three proteins of low molecular weight, belonging to the group of apolipoproteins which are major constituents of the very low density lipoproteins, have been used. ApoC-I + was reported to activate lipoprotein lipase from postheparin plasma but not from adipose tissue or milk (4,5).
A weak
+The nomenclature suggested by Alaupovic (3) will be used throughout: apoC-I (apoLp-Ser, formerly apoLp-Val) ; apoC-ll (apoLp-Glu) ; apoC-lll (apoLp-Ala). The alternative abbreviations within brackets are based on the C00H-terminal amino acids.
Copyright © 1975 by Academic Press, Inc. All rights o f reproduction in any form reserved.
'1447
Vol. 65, No. 4, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
activation by the same apolipoprotein has been reported since then (6). Recently, however, a strong inhibiting capacity has also been ascribed to apoC-I (7). The activating property of apoC-ll was first observed by LaRosa et al. (8), using adipose tissue extracts as the source of enzyme.
This finding has later been confirmed further and now seems
well-established (6,7,9-11).
Activation of lipoprotein lipase by
apoC-lll has also been reported (4,6,8,9), but some of these findings have been ascribed to impurities in the apoprotein preparation (6,12). The experiments in the present work were all performed with components of very high purity.
The results obtained may clarify
previous contradictory results and also shed some light on the activation mechanism of lipoprotein lipase.
MATERIALS AND METHODS Lipoprotein lipase:
Highly purified lipoprotein lipase was isolated
from bovine milk by a procedure including affinity chromatography on heparin-Sepharose (13) followed by adsorption and desorption of the enzyme with C -alumina gel and finally rechromatography on heparinY Sepharose. All buffers used contained glycerol (15 or 30%, v/v). After the last chromatographic step, the enzyme was precipitated by dialysis against 3.6M a~nonium sulfate and the precipitate was then dissolved in 50% (v/v) glycerol containing I0 mM sodium-phosphate (pH 7.5).
The con-
centrated enzyme solution was stored at -20°C (unpublished results).
The
purity was monitored by polyacrylamide gel electrophoresis in sodium dodecyl sulfate (Fig. IA) according to Neville (14).
The stock solution
of enzyme, which contained 2 ,i00 U/ml ++ (specific activity 780 U/mg), was before use appropriately diluted with 0.17M Tris-HCl buffer (pH 8.5 ; I 0.05) containing bovine serum albumin (i mg/ml).
++One unit (U) is defined as the amount of enzyme which liberates i ~mole of fatty acid per minute under assay conditions described previously (15).
1448
Vol. 65, No. 4, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Apolipoproteins:
Very low density lipoproteins were isolated from
eitrated plasma of hypertriglyceridemic patients as described elsewhere (16).
Delipidation was performed in chloroform-methanol (17).
Frac-
tionation of apolipoproteins by gel chromatography and ion exchange chromatography, as well as the checking of the purity by polyaerylamide gel electrophoresis in urea (Fig. IB, C and D), was carried out according to Brown et al. (18).
The apolipoprotein species was iden-
tified from its elution position in the ion exchange chromatogram.
®
i
m
®
A Fig. i.
B
C
O
Polyacrylamide ~el electrophoresis of lipoprotein lipase and apolipoproteins. A: enzyme (45 ~g). B: apoC-I (20 ~g). C: apoC-ll (20 ~g). D: apoC-lll:2 (20 ~g). The gels were stained with amido black.
1449
Vol. 65, No. 4, 1975
Enzyme assay:
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The assay procedure for lipoprotein lipase was a micro-
modification based on the method described previously (15). The basic medium was 0.11M NaCI-0.17M Tris-HCl buffer (pH 8.5;
I 0.16) containing
55 mg/ml of bovine serum albumin and various concentrations of a triglyceride-phospholipid emulsion (Intmalipid, Vitrum AB, Stockholm, Sweden).
Variations in the composition of the medium were achieved as
described under "Results". Incubations were performed in stoppered microcentrifuge tubes of glass (total assay volume 0.5 ml) at 37°C for 30 rain. Duplicate samples (0.I ml) were withdrawn at the beginning as well as at the end of the incubation period and analyzed for free fatty acid (19) against a standard of stearic acid.
All enzyme activities (Dmoles.min-l'ml -I) , which express
the velocity of fatty acid release per ml of assay medium, were within the linear range of the assay.
The experimental values in each diagram
have been obtained with the same amount of enzyme. Protein deternd_nation:
For the measurement of enzyme and activator
concentrations, the Lowry method (20) was used against a standard of bovine sert~n albumin. RESULTS
Activatin$ power of isolated apolipoproteins: the lipase (Fig. 2).
0nly apoC-ll activated
When the concentration of this activator was
increased, the enzyme activity increased to a maximum. higher concentrations the protein acted as an inhibitor.
At still ApoC-I
and apoC-lll:2 +++ were completely devoid of activating power. Concentration effects of substrate and activator:
The relationship
between enzyme activity and substrate concentration was influenced by the amount of added activator (Fig. 3).
At all triglyceride con-
centrations tested, virtually no activity was observed in the absence +++ApoC-Ill may contain various amounts of sialic acid. ApoC-IIl:2 designates the species containing two sialic acid residues per molecule (21).
1450
Vol. 65, No. 4, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
0.05
~4 ~ o.o~ LU "~.
OOl A
A
A
60 APOLI POPROTEIN
Fig. 2.
8~0
100
CONCN. (,,ug / m l )
Test for activating power of different apolipoprotein s. Enzyme was incubated with various concentrations of apoC-I (O O ), apoC-ll ( C ~ ) or apoC-ill :2 ( ~ ). The substrate concentration was 2.5 mg/ml.
of activator.
The maximum enzyme activity increased with increasing
concentration of activator.
At substrate excess, the enzyme activity
levelled out to a value corresponding to about 50% or more of the peak activity. Role of apoC-I and apoC-lll:
Although apoC-I and apoC-lll:2 did not
possess any activating power (Fig. 2), they could nevertheless influence the activation by apoC-ll (Pig. 4).
When either of them was added in
the presence of apoC-ll and the substrate concentration was varied, the activity peak seen with apoC-ll alone was displaced to higher substrate concentrations.
In addition, the height of the peak seemed to be reduced.
The interrelation between the curves in Fig. 4 means that apoC-I and apoC-lll:2 can inhibit as well as enhance the lipase activity under certain conditions.
When these apolipoproteins are added to combinations
of substrate and activator giving an activity maximum, mainly inhibition ensues.
By contrast, the lipase activity is enhanced by apolipoprotein
additions at substrate excess. DISCUSSION The activation of lipoprotein lipase by apoC-ll is influenced
1451
Vol. 65, No. 4, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
•~ "
0.03
.
•~
001
Lu
t-.l
A
tu
5 I0 I FRtGLYCERIOE 60NCN (rag~mr)
Fig. 3.
20
Effect of apoC-ll at varying triglyceride concentration. concentration of apoC-l! was 0 (~ ~ ), i ( ~ ) , 5 (C O ) , 15 (O-----4) or 5O ~g/ml (6 m).
by the triglyceride/activator ratio (Fig. 3).
The
At substrate excess, when
the ratio is high all activator molecules should be bound to the substrate particles according to the law of mass action, since Havel et al. (22) have shown that Intralipid adsorbs apolipoproteins.
The non-maximal
enzyme activity, unaffected by the concentration of the substrate, therefore seems to depend on the number of activator molecules bound to the substrate particles.
The activity maxima observed at lower triglyceride/
activator ratios may be ascribed to an additional activation step in which free activator molecules are involved.
Alternatively, this activation
step is related to an increased concentration of activator molecules on the substrate particles.
The inhibition which occurs at low triglyceride/
activator ratios (Fig. 2 and 3) may be due to an overcrowding of the substrate particles by apolipoprotein (ii) as well as to an excess of free aet ivator molecules. The two-step activation mechanism postulated above may also offer an explanation of the activity-promoting effects of apoC-I and apoC-lll: 2 (Fig. 4).
These apoproteins were only effective in the presence of apoC-ll
1452
Vol. 65, No. 4, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
0.03
.~ °°Il L
I
I
I
I
I
I
I
5 I0 15 FRIGLYCERIDE CONCN (mg/ml)
l
20
B
"F" "i" 003 E E
E~.O.02
0.01 Lu
i
i
i
5 I0 15 TRIGLYCERIDE CONCN.( m g / m l )
Fig. 4.
i
20
Effect of non-activating apolipoproteins. ApoC-I (A) or apoC-lli:2 (B) were added at concentrations of 0 (4~-----O), 2 (~ ~ ) , 5 (O O ) or i0 pg/ml (Ab------A). The concentration of apoC-l! was 15 pg/ml throughout.
and at substrate excess. than apoC-ll alone.
Further, they did not afford higher, activities
Presumably, apoC-I and apoC-lll:2 compete with the
apoC-ll, bound to the substrate, liberating the activator into the solution and thereby initiating the second activation step.
Such effects
can also be anticipated by any substance that competes with apoC-ll for sites on the substrate particles. In view of the present findings, previous work in this field also
1453
Vol. 65, No. 4, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
deserves a comment.
Experiments similar to those shown in Fig. 3 have
been performed by Krauss et al. (ii).
With no activator added, they
observed a considerable activity, while our enzyme was completely inactive.
It should be noted, however, that they employed a crude extract
from adipose tissue as enzyme source and also a different substrate.
The
assertion by Ganesan et al. (4,5) that apoC-I could activate lipoprotein lipase is only valid for enzyme from postheparin plasma but not from adipose tissue and milk.
Very probably, the purified plasma enzyme was still
contaminated with apoC-ll and the substrate/activator ratio high, resulting in further stimulation by apoC-l.
Similar arguments may be
applied to other reports of stimulatory effects by apoC-I (6) as well as apoC-lll (4,6,8,9).
ACKNOWLEDGEMENTS The excellent technical assistance of Miss K. Lilja is gratefully acknowledged.
Funds for this work were provided by the Swedish Medical
Research Council (13P-3594;
03X-3966;
13X-4), Vitrum AB, Greta och
Johan Kocks stiftelse, Ostermans fond, Gustav V:s 80-Arsfond and the University of Uppsala.
REFERENCES
i.
Robinson, D.S. (1970) Comprehensive Biochemistry Vol. XVIII, pp. 51-116, American Elsevier Publishing Co., New York. 2. Korn, E.D. (1955) J.Biol. Chem. 215, 15-26. 3. Alaupovic, P. (1971) A t h e r o s c l e ~ i s 13, 141-146. 4. Ganesan, D., Bradford, R.H., Alaupovic, P., and McConathy, W.J. (1971) FEBS Lett. 15, 205-208. 5. Ganesan, D., and Bass, H.B. (1975) FEBS Lett. 53, 1-4. 6. Havel, R.J., Fielding, C.J., Olivecrona, T., Shore, V.G., Fielding, F.E., and Egelrud, T. (1973) Biochemistry 12, 1828-1833. 7. Bensadoun, A., Enholm, C., Steinberg, D., and Brown, W.---V. (1974) J.Biol.Chem. 249, 2220-2227. 8. LaRosa, J.C., L e ~ , R.I., Herbert, P., Lux, S.E., and Fredriekson, D.S. (1970) Biochem.Biophys.Res.Conmun. 41, 57-62. 9. Havel, R.J., Shore, V.G., Shore, B., and Bier, D.M. (1970) Circulation Res. 27, 595-600.
1454
Vol. 65, No. 4, 1975
i0. ii. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
Miller, A.L., and Smith, L.C. (1973) J. Biol. Chem. 248, 3359-3362. Krauss, R.M., Herbert, P.N., Levy, R.I., and Fredrickson, D.S. (1973) Circulation Res. 33_, 403-411. Brown, W.V., and Baginsky, M.L. (1972) Biochem.Biophys.Res.Conmun. 46, 375-382. Olivecrona, T., Ege]rud, T., Iverius, P.-H., and Lindahl, U. (1971) Bioehem.Biophys.Res.Con~nun. 43, 524-529. Neville Jr., D.M. (1971) J.Biol.Chem. 246, 6328-6334. Iverius, P.-H., Lindahl, U., Egelrud, T., and Olivecrona, T. (1972) J.Biol.Chem. 247, 6610-6616. Iverius, P.-H, L(1972) J.Biol.Chem. 247, 2607-2613. Scanu, A.M., and Edelstein, C. (1971) Anal.Biochem. 44, 576-588. Brown, W.V., Levy, R.I., and Fredrickson, D.S. (1970) Bioehim.Biophys. Acta 200, 573-575. Ho, R.J. (1970) Anal.Bi0chem. 36, 105-113. Lowry, 0.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (1951) J.Biol.Chem. 193, 265-275. Herbert, P.N., Shulman, R.S., Levy, R.I., and Fredriekson, D.S. (1973) J.Biol.Chem. 248, 4941-4946. Havel, R.J., Kane, J.P. ~ and Kashyap, M.L. (1973) J.Clin.lnvest. 52, 32-38.
1455
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ACTIVATION OF HIGHLY PURIFIED LiPOPROTEIN LIPASE FROM BOVINE MILK A.-M. Ostlund-Lindqvist and P.-H. Iverius
Department of Medical and Physiological Chemistry, University of Uppsala, Biomedicum, Box 575, S-751 23
Uppsala, Sweden.
Received June 27,1975 SUMMARY The effects of apolipoproteins from very low density human lipoproteins on the activity of highly purified lipoprotein lipase from bovine milk was studied using a triglyceride phospholipid emulsion (Intralipid) as substrate. No activity was recorded with the substrate alone and apoC-ll was the only apolipoprotein that activated the enzyme. Maximal activity required a sufficient concentration of activator as well as an optimal ratio between activator and substrate. At substrate excess, the enzyme activity was reduced by about 50% or less depending on the concentration of activator employed. Under these conditions, however, appropriate additions of apoC-I or apoC-lll restored part of the lost activity. These findings may explain the contradictory results reported previously.
INTRODUCTION Lipoprotein lipase (EC 3. i. i. 3 ) has a key function in the metabolism of plasma triglyeerides.
The chylomicrons and the very
low density lipoproteins are the natural substrates of the enzyme (i). Artificial triglyceride emulsions are also hydrolyzed, provided that certain protein components of the plasma lipoproteins are present (2). For this purpose three proteins of low molecular weight, belonging to the group of apolipoproteins which are major constituents of the very low density lipoproteins, have been used. ApoC-I + was reported to activate lipoprotein lipase from postheparin plasma but not from adipose tissue or milk (4,5).
A weak
+The nomenclature suggested by Alaupovic (3) will be used throughout: apoC-I (apoLp-Ser, formerly apoLp-Val) ; apoC-ll (apoLp-Glu) ; apoC-lll (apoLp-Ala). The alternative abbreviations within brackets are based on the C00H-terminal amino acids.
Copyright © 1975 by Academic Press, Inc. All rights o f reproduction in any form reserved.
'1447
Vol. 65, No. 4, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
activation by the same apolipoprotein has been reported since then (6). Recently, however, a strong inhibiting capacity has also been ascribed to apoC-I (7). The activating property of apoC-ll was first observed by LaRosa et al. (8), using adipose tissue extracts as the source of enzyme.
This finding has later been confirmed further and now seems
well-established (6,7,9-11).
Activation of lipoprotein lipase by
apoC-lll has also been reported (4,6,8,9), but some of these findings have been ascribed to impurities in the apoprotein preparation (6,12). The experiments in the present work were all performed with components of very high purity.
The results obtained may clarify
previous contradictory results and also shed some light on the activation mechanism of lipoprotein lipase.
MATERIALS AND METHODS Lipoprotein lipase:
Highly purified lipoprotein lipase was isolated
from bovine milk by a procedure including affinity chromatography on heparin-Sepharose (13) followed by adsorption and desorption of the enzyme with C -alumina gel and finally rechromatography on heparinY Sepharose. All buffers used contained glycerol (15 or 30%, v/v). After the last chromatographic step, the enzyme was precipitated by dialysis against 3.6M a~nonium sulfate and the precipitate was then dissolved in 50% (v/v) glycerol containing I0 mM sodium-phosphate (pH 7.5).
The con-
centrated enzyme solution was stored at -20°C (unpublished results).
The
purity was monitored by polyacrylamide gel electrophoresis in sodium dodecyl sulfate (Fig. IA) according to Neville (14).
The stock solution
of enzyme, which contained 2 ,i00 U/ml ++ (specific activity 780 U/mg), was before use appropriately diluted with 0.17M Tris-HCl buffer (pH 8.5 ; I 0.05) containing bovine serum albumin (i mg/ml).
++One unit (U) is defined as the amount of enzyme which liberates i ~mole of fatty acid per minute under assay conditions described previously (15).
1448
Vol. 65, No. 4, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Apolipoproteins:
Very low density lipoproteins were isolated from
eitrated plasma of hypertriglyceridemic patients as described elsewhere (16).
Delipidation was performed in chloroform-methanol (17).
Frac-
tionation of apolipoproteins by gel chromatography and ion exchange chromatography, as well as the checking of the purity by polyaerylamide gel electrophoresis in urea (Fig. IB, C and D), was carried out according to Brown et al. (18).
The apolipoprotein species was iden-
tified from its elution position in the ion exchange chromatogram.
®
i
m
®
A Fig. i.
B
C
O
Polyacrylamide ~el electrophoresis of lipoprotein lipase and apolipoproteins. A: enzyme (45 ~g). B: apoC-I (20 ~g). C: apoC-ll (20 ~g). D: apoC-lll:2 (20 ~g). The gels were stained with amido black.
1449
Vol. 65, No. 4, 1975
Enzyme assay:
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The assay procedure for lipoprotein lipase was a micro-
modification based on the method described previously (15). The basic medium was 0.11M NaCI-0.17M Tris-HCl buffer (pH 8.5;
I 0.16) containing
55 mg/ml of bovine serum albumin and various concentrations of a triglyceride-phospholipid emulsion (Intmalipid, Vitrum AB, Stockholm, Sweden).
Variations in the composition of the medium were achieved as
described under "Results". Incubations were performed in stoppered microcentrifuge tubes of glass (total assay volume 0.5 ml) at 37°C for 30 rain. Duplicate samples (0.I ml) were withdrawn at the beginning as well as at the end of the incubation period and analyzed for free fatty acid (19) against a standard of stearic acid.
All enzyme activities (Dmoles.min-l'ml -I) , which express
the velocity of fatty acid release per ml of assay medium, were within the linear range of the assay.
The experimental values in each diagram
have been obtained with the same amount of enzyme. Protein deternd_nation:
For the measurement of enzyme and activator
concentrations, the Lowry method (20) was used against a standard of bovine sert~n albumin. RESULTS
Activatin$ power of isolated apolipoproteins: the lipase (Fig. 2).
0nly apoC-ll activated
When the concentration of this activator was
increased, the enzyme activity increased to a maximum. higher concentrations the protein acted as an inhibitor.
At still ApoC-I
and apoC-lll:2 +++ were completely devoid of activating power. Concentration effects of substrate and activator:
The relationship
between enzyme activity and substrate concentration was influenced by the amount of added activator (Fig. 3).
At all triglyceride con-
centrations tested, virtually no activity was observed in the absence +++ApoC-Ill may contain various amounts of sialic acid. ApoC-IIl:2 designates the species containing two sialic acid residues per molecule (21).
1450
Vol. 65, No. 4, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
0.05
~4 ~ o.o~ LU "~.
OOl A
A
A
60 APOLI POPROTEIN
Fig. 2.
8~0
100
CONCN. (,,ug / m l )
Test for activating power of different apolipoprotein s. Enzyme was incubated with various concentrations of apoC-I (O O ), apoC-ll ( C ~ ) or apoC-ill :2 ( ~ ). The substrate concentration was 2.5 mg/ml.
of activator.
The maximum enzyme activity increased with increasing
concentration of activator.
At substrate excess, the enzyme activity
levelled out to a value corresponding to about 50% or more of the peak activity. Role of apoC-I and apoC-lll:
Although apoC-I and apoC-lll:2 did not
possess any activating power (Fig. 2), they could nevertheless influence the activation by apoC-ll (Pig. 4).
When either of them was added in
the presence of apoC-ll and the substrate concentration was varied, the activity peak seen with apoC-ll alone was displaced to higher substrate concentrations.
In addition, the height of the peak seemed to be reduced.
The interrelation between the curves in Fig. 4 means that apoC-I and apoC-lll:2 can inhibit as well as enhance the lipase activity under certain conditions.
When these apolipoproteins are added to combinations
of substrate and activator giving an activity maximum, mainly inhibition ensues.
By contrast, the lipase activity is enhanced by apolipoprotein
additions at substrate excess. DISCUSSION The activation of lipoprotein lipase by apoC-ll is influenced
1451
Vol. 65, No. 4, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
•~ "
0.03
.
•~
001
Lu
t-.l
A
tu
5 I0 I FRtGLYCERIOE 60NCN (rag~mr)
Fig. 3.
20
Effect of apoC-ll at varying triglyceride concentration. concentration of apoC-l! was 0 (~ ~ ), i ( ~ ) , 5 (C O ) , 15 (O-----4) or 5O ~g/ml (6 m).
by the triglyceride/activator ratio (Fig. 3).
The
At substrate excess, when
the ratio is high all activator molecules should be bound to the substrate particles according to the law of mass action, since Havel et al. (22) have shown that Intralipid adsorbs apolipoproteins.
The non-maximal
enzyme activity, unaffected by the concentration of the substrate, therefore seems to depend on the number of activator molecules bound to the substrate particles.
The activity maxima observed at lower triglyceride/
activator ratios may be ascribed to an additional activation step in which free activator molecules are involved.
Alternatively, this activation
step is related to an increased concentration of activator molecules on the substrate particles.
The inhibition which occurs at low triglyceride/
activator ratios (Fig. 2 and 3) may be due to an overcrowding of the substrate particles by apolipoprotein (ii) as well as to an excess of free aet ivator molecules. The two-step activation mechanism postulated above may also offer an explanation of the activity-promoting effects of apoC-I and apoC-lll: 2 (Fig. 4).
These apoproteins were only effective in the presence of apoC-ll
1452
Vol. 65, No. 4, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
0.03
.~ °°Il L
I
I
I
I
I
I
I
5 I0 15 FRIGLYCERIDE CONCN (mg/ml)
l
20
B
"F" "i" 003 E E
E~.O.02
0.01 Lu
i
i
i
5 I0 15 TRIGLYCERIDE CONCN.( m g / m l )
Fig. 4.
i
20
Effect of non-activating apolipoproteins. ApoC-I (A) or apoC-lli:2 (B) were added at concentrations of 0 (4~-----O), 2 (~ ~ ) , 5 (O O ) or i0 pg/ml (Ab------A). The concentration of apoC-l! was 15 pg/ml throughout.
and at substrate excess. than apoC-ll alone.
Further, they did not afford higher, activities
Presumably, apoC-I and apoC-lll:2 compete with the
apoC-ll, bound to the substrate, liberating the activator into the solution and thereby initiating the second activation step.
Such effects
can also be anticipated by any substance that competes with apoC-ll for sites on the substrate particles. In view of the present findings, previous work in this field also
1453
Vol. 65, No. 4, 1975
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
deserves a comment.
Experiments similar to those shown in Fig. 3 have
been performed by Krauss et al. (ii).
With no activator added, they
observed a considerable activity, while our enzyme was completely inactive.
It should be noted, however, that they employed a crude extract
from adipose tissue as enzyme source and also a different substrate.
The
assertion by Ganesan et al. (4,5) that apoC-I could activate lipoprotein lipase is only valid for enzyme from postheparin plasma but not from adipose tissue and milk.
Very probably, the purified plasma enzyme was still
contaminated with apoC-ll and the substrate/activator ratio high, resulting in further stimulation by apoC-l.
Similar arguments may be
applied to other reports of stimulatory effects by apoC-I (6) as well as apoC-lll (4,6,8,9).
ACKNOWLEDGEMENTS The excellent technical assistance of Miss K. Lilja is gratefully acknowledged.
Funds for this work were provided by the Swedish Medical
Research Council (13P-3594;
03X-3966;
13X-4), Vitrum AB, Greta och
Johan Kocks stiftelse, Ostermans fond, Gustav V:s 80-Arsfond and the University of Uppsala.
REFERENCES
i.
Robinson, D.S. (1970) Comprehensive Biochemistry Vol. XVIII, pp. 51-116, American Elsevier Publishing Co., New York. 2. Korn, E.D. (1955) J.Biol. Chem. 215, 15-26. 3. Alaupovic, P. (1971) A t h e r o s c l e ~ i s 13, 141-146. 4. Ganesan, D., Bradford, R.H., Alaupovic, P., and McConathy, W.J. (1971) FEBS Lett. 15, 205-208. 5. Ganesan, D., and Bass, H.B. (1975) FEBS Lett. 53, 1-4. 6. Havel, R.J., Fielding, C.J., Olivecrona, T., Shore, V.G., Fielding, F.E., and Egelrud, T. (1973) Biochemistry 12, 1828-1833. 7. Bensadoun, A., Enholm, C., Steinberg, D., and Brown, W.---V. (1974) J.Biol.Chem. 249, 2220-2227. 8. LaRosa, J.C., L e ~ , R.I., Herbert, P., Lux, S.E., and Fredriekson, D.S. (1970) Biochem.Biophys.Res.Conmun. 41, 57-62. 9. Havel, R.J., Shore, V.G., Shore, B., and Bier, D.M. (1970) Circulation Res. 27, 595-600.
1454
Vol. 65, No. 4, 1975
i0. ii. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
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