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[42] Formation of 12-cis-dehydrosqualene catalyzed by squalene synthetase
FORMATION OF 12-ciS-DEHYDROSQUALENE
[42]
373
phatidylinositol have not been found to activate, and may inhibit the residual activity. It appears that permitting at least 45 min to pass between removing detergent and assaying the enzyme may result in improved stoichiometries of proton release and squalene synthesis, although this has not been systematically analyzed. The ability of the reducing cofactor to enhance the condensation reaction is also restored on reconstitution. This suggests that, if there are two enzyme proteins of -55,000 Da separately in solution, they may reassociate during or after reconstitution. Acknowledgments The author acknowledges being supported by N I H - N I N C D S Grant N S 17928 and a grant from the National Multiple Sclerosis Society during the writing of this article.
[42] F o r m a t i o n o f 1 2 - c i s - D e h y d r o s q u a l e n e C a t a l y z e d b y Squalene Synthetase B y TOKUZO N I S m N O a n d HIROHIKO KATSUKI
Upon incubation ofa microsomal fraction from Saccharomyces cerevisiae with [14C]famesyl pyrophosphate in the presence of Mn 2÷, radioactivity is incorporated into 12-cis-dehydrosqualene. L2 Dehydrosqualene formation is observed also with a microsomal fraction of Rhodotorula glutinis 3 or mammalian microsomes. 2 When the reaction is performed in the presence of NADPH, squalene is formed instead of dehydrosqualene. With a squalene synthetase preparation which is solubilized and partially purified from S. cerevisiae using a Sephacryl S-200 column by a modification 2 of the method of Agnew and Popj~ik, 4 the involvement of squalene synthetase in the dehydrosqualene formation was established. Dehydrosqualene is presumed to be formed through elimination of a proton from Ca of an allylic cation, an intermediate in squalene formation in the presence of NADPH. t T. Nishino, H. Takatsuji, S. Hata, and H. Katsuki, Biochem. Biophys. Res. Commun. 85, 867 (1978): 2 H. Takatsuji, T. Nishino, K. Izui, and H. Katsuki, J. Biochem. (Tokyo) 91, 911 (1982). 3 T. Nishino, N. Suzuki, H. Takatsuji, and H. Katsuki, Mem. Fac. Sci., Kyoto Univ. 36, 67 (1981). 4 W. S. Agnew and G. Popjfik, J. Biol. Chem. 253, 4578 (1978).
METHODS IN ENZYMOLOGY, VOL. 110
Copyright © 1985 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-182010-6
374
LINEARCONDENSATIONSOF ISOPRENOIDS
[42]
Assay Methods Principle. When crude microsomal preparations including squalene synthetase from which cofactors such as NADPH or NADP have been removed are incubated with farnesyl pyrophosphate and Mn 2÷ in the absence of NADPH, dehydrosqualene is formed instead of squalene. The formation of dehydrosqualene can be easily detected by reversed phase TLC in which dehydrosqualene is separated from squalene. Presqualene pyrophosphate which is formed under the above conditions 5 is not extracted in the petroleum ether used for extraction of dehydrosqualene. R e a g e n t s . [14C]Farnesyl pyrophosphate can be prepared by incubation of [1-14C]isopentenyl pyrophosphate (specific radioactivity, 56 Ci/mol, Amersham) with geranyl pyrophosphate and the 105,000 g supernatant of cell homogenates of R. glutinis 2 or S. cerevisiae 6 after preincubation with iodoacetamide for inactivation of isopentenyl pyrophosphate isomerase. Tris-HC1 buffer, 0.1 M, pH 7.4 MgCI2, 0.1 M MnCIE, 0.1 M [1-14C]Farnesyl pyrophosphate (sp. act., more than 50 Ci/mol), 0.1 mM KF, 1 M KOH, 6 M Petroleum ether Procedure. In a 15-ml screw-capped test tube are placed 0.1 ml of Tris buffer, 50/~1 of MgCI2,20/xl of MnCI2, I0 ttl of KF, l0/~1 of [14C]farnesyl pyrophosphate, the microsomal preparation, and water (final volume 1.0 ml). When the crude microsomal preparation is to be used instead of the partially purified one, it should be passed through a Sephadex G-50 column to remove cofactors such as NADPH, NADP and metal ions. Reaction is for 120 min at 30°. The reaction is stopped by the addition of 0.5 ml of KOH, and the mixture is heated for 60 min at 80°. Nonpolar lipids including dehydrosqualene are extracted with petroleum ether. Product analysis. The lipids are subjected to TLC analysis on kieselguhr plate (Merck) impregnated with liquid paraffin using 95% acetone saturated with liquid paraffin as solvent (Rf of dehydrosqualene, 0.39; Rf of squalene, 0.25). In radio-gas chromatography using a column of 2% Dexisi1300 GC or 1.5% SE-30, dehydrosqualene has a retention time relative to squalene of 1.40 or 1.23, respectively.
H. C. Rilling, J. Biol. Chem. 241, 3233 (1966). 6 T. Nishino, N. Suzuki, and H. Katsuki, J. Biochem. (Tokyo) 92, 1731(1982).
[43]
SQUALENE EPOXIDASE
375
Properties
Presqualene pyrophosphate (also isopentenyl pyrophosphate when the 15,000 g supernatant is used) can be used as a substrate instead of farnesyl pyrophosphate. F o r the dehydrosqualene formation, Mn z+ is more effective than Mg 2÷. This is presumed to be due to the action of Mn 2÷ which makes the cleavage of the C - O bond of presqualene pyrophosphate easier. 6 The ratio o f squalene to dehydrosqualene formed under optimal conditions, in the presence or absence of N A D P H , is about 200 : I. Presqualene alcohol and several species of C30-alcohols 2 with unidentified structures, which are presumed to be derived from the cation intermediates postulated by Rilling, Poulter, and their collaborators,7,s are formed besides dehydrosqualene. 7 H. C. Pilling, C. D. PouRer, W. W. Epstein, and B. Larsen, J. Am. Chem. Soc. 93, 1783 (1971). s C. D. Poulter, O. J. Muscio, and R. J. Goodfellow, Biochemistry 13, 1530(1974).
[43] S q u a l e n e
Epoxidase
from Rat Liver Microsomes
B y TERUO ONO and YOH IMAI
Squalene + 02 + NADPH + H+
~ 2,3-Oxidosqualene + NADP+ + H20
The mixed-function oxidases of liver microsomes which catalyze the hydroxylation of a wide variety o f lipophilic substrates have been shown to consist o f a N A D P H - c y t o c h r o m e P-450 reductase, c y t o c h r o m e P-450 isozymes, and phosphatidylcholine, t The squalene epoxidase [EC 1.14.99.7, squalene m o n o o x y g e n a s e (2,3-epoxidizing)] system is comprised of the terminal oxidase which is distinct from hemoproteins such as c y t o c h r o m e P-450 isozymes, 2 and a flavoprotein identical with N A D P H c y t o c h r o m e P-450 reductase. 3 N A D P H - C y t o c h r o m e c (P-450) Reductase [EC 1.6.2.4] Assay Method
Assay o f N A D P H - c y t o c h r o m e c reductase activity is performed with an Hitachi 124 spectrophotometer. F o r determining activity the e n z y m e 1M. J. Coon, this series, Vol. 52, p. 200. 2 T. Ono, K. Nakazono, and H. Kosaka, Biochim. Biophys. Acta 709, 84 (1982). 3 T. Ono, S. Ozasa, F. Hasegawa, and Y. Imai, Biochim. Biophys. Acta 486, 401 (1977). METHODS IN ENZYMOLOGY, VOL. 110
Copyright © 1985 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-182010-6
[42]
373
phatidylinositol have not been found to activate, and may inhibit the residual activity. It appears that permitting at least 45 min to pass between removing detergent and assaying the enzyme may result in improved stoichiometries of proton release and squalene synthesis, although this has not been systematically analyzed. The ability of the reducing cofactor to enhance the condensation reaction is also restored on reconstitution. This suggests that, if there are two enzyme proteins of -55,000 Da separately in solution, they may reassociate during or after reconstitution. Acknowledgments The author acknowledges being supported by N I H - N I N C D S Grant N S 17928 and a grant from the National Multiple Sclerosis Society during the writing of this article.
[42] F o r m a t i o n o f 1 2 - c i s - D e h y d r o s q u a l e n e C a t a l y z e d b y Squalene Synthetase B y TOKUZO N I S m N O a n d HIROHIKO KATSUKI
Upon incubation ofa microsomal fraction from Saccharomyces cerevisiae with [14C]famesyl pyrophosphate in the presence of Mn 2÷, radioactivity is incorporated into 12-cis-dehydrosqualene. L2 Dehydrosqualene formation is observed also with a microsomal fraction of Rhodotorula glutinis 3 or mammalian microsomes. 2 When the reaction is performed in the presence of NADPH, squalene is formed instead of dehydrosqualene. With a squalene synthetase preparation which is solubilized and partially purified from S. cerevisiae using a Sephacryl S-200 column by a modification 2 of the method of Agnew and Popj~ik, 4 the involvement of squalene synthetase in the dehydrosqualene formation was established. Dehydrosqualene is presumed to be formed through elimination of a proton from Ca of an allylic cation, an intermediate in squalene formation in the presence of NADPH. t T. Nishino, H. Takatsuji, S. Hata, and H. Katsuki, Biochem. Biophys. Res. Commun. 85, 867 (1978): 2 H. Takatsuji, T. Nishino, K. Izui, and H. Katsuki, J. Biochem. (Tokyo) 91, 911 (1982). 3 T. Nishino, N. Suzuki, H. Takatsuji, and H. Katsuki, Mem. Fac. Sci., Kyoto Univ. 36, 67 (1981). 4 W. S. Agnew and G. Popjfik, J. Biol. Chem. 253, 4578 (1978).
METHODS IN ENZYMOLOGY, VOL. 110
Copyright © 1985 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-182010-6
374
LINEARCONDENSATIONSOF ISOPRENOIDS
[42]
Assay Methods Principle. When crude microsomal preparations including squalene synthetase from which cofactors such as NADPH or NADP have been removed are incubated with farnesyl pyrophosphate and Mn 2÷ in the absence of NADPH, dehydrosqualene is formed instead of squalene. The formation of dehydrosqualene can be easily detected by reversed phase TLC in which dehydrosqualene is separated from squalene. Presqualene pyrophosphate which is formed under the above conditions 5 is not extracted in the petroleum ether used for extraction of dehydrosqualene. R e a g e n t s . [14C]Farnesyl pyrophosphate can be prepared by incubation of [1-14C]isopentenyl pyrophosphate (specific radioactivity, 56 Ci/mol, Amersham) with geranyl pyrophosphate and the 105,000 g supernatant of cell homogenates of R. glutinis 2 or S. cerevisiae 6 after preincubation with iodoacetamide for inactivation of isopentenyl pyrophosphate isomerase. Tris-HC1 buffer, 0.1 M, pH 7.4 MgCI2, 0.1 M MnCIE, 0.1 M [1-14C]Farnesyl pyrophosphate (sp. act., more than 50 Ci/mol), 0.1 mM KF, 1 M KOH, 6 M Petroleum ether Procedure. In a 15-ml screw-capped test tube are placed 0.1 ml of Tris buffer, 50/~1 of MgCI2,20/xl of MnCI2, I0 ttl of KF, l0/~1 of [14C]farnesyl pyrophosphate, the microsomal preparation, and water (final volume 1.0 ml). When the crude microsomal preparation is to be used instead of the partially purified one, it should be passed through a Sephadex G-50 column to remove cofactors such as NADPH, NADP and metal ions. Reaction is for 120 min at 30°. The reaction is stopped by the addition of 0.5 ml of KOH, and the mixture is heated for 60 min at 80°. Nonpolar lipids including dehydrosqualene are extracted with petroleum ether. Product analysis. The lipids are subjected to TLC analysis on kieselguhr plate (Merck) impregnated with liquid paraffin using 95% acetone saturated with liquid paraffin as solvent (Rf of dehydrosqualene, 0.39; Rf of squalene, 0.25). In radio-gas chromatography using a column of 2% Dexisi1300 GC or 1.5% SE-30, dehydrosqualene has a retention time relative to squalene of 1.40 or 1.23, respectively.
H. C. Rilling, J. Biol. Chem. 241, 3233 (1966). 6 T. Nishino, N. Suzuki, and H. Katsuki, J. Biochem. (Tokyo) 92, 1731(1982).
[43]
SQUALENE EPOXIDASE
375
Properties
Presqualene pyrophosphate (also isopentenyl pyrophosphate when the 15,000 g supernatant is used) can be used as a substrate instead of farnesyl pyrophosphate. F o r the dehydrosqualene formation, Mn z+ is more effective than Mg 2÷. This is presumed to be due to the action of Mn 2÷ which makes the cleavage of the C - O bond of presqualene pyrophosphate easier. 6 The ratio o f squalene to dehydrosqualene formed under optimal conditions, in the presence or absence of N A D P H , is about 200 : I. Presqualene alcohol and several species of C30-alcohols 2 with unidentified structures, which are presumed to be derived from the cation intermediates postulated by Rilling, Poulter, and their collaborators,7,s are formed besides dehydrosqualene. 7 H. C. Pilling, C. D. PouRer, W. W. Epstein, and B. Larsen, J. Am. Chem. Soc. 93, 1783 (1971). s C. D. Poulter, O. J. Muscio, and R. J. Goodfellow, Biochemistry 13, 1530(1974).
[43] S q u a l e n e
Epoxidase
from Rat Liver Microsomes
B y TERUO ONO and YOH IMAI
Squalene + 02 + NADPH + H+
~ 2,3-Oxidosqualene + NADP+ + H20
The mixed-function oxidases of liver microsomes which catalyze the hydroxylation of a wide variety o f lipophilic substrates have been shown to consist o f a N A D P H - c y t o c h r o m e P-450 reductase, c y t o c h r o m e P-450 isozymes, and phosphatidylcholine, t The squalene epoxidase [EC 1.14.99.7, squalene m o n o o x y g e n a s e (2,3-epoxidizing)] system is comprised of the terminal oxidase which is distinct from hemoproteins such as c y t o c h r o m e P-450 isozymes, 2 and a flavoprotein identical with N A D P H c y t o c h r o m e P-450 reductase. 3 N A D P H - C y t o c h r o m e c (P-450) Reductase [EC 1.6.2.4] Assay Method
Assay o f N A D P H - c y t o c h r o m e c reductase activity is performed with an Hitachi 124 spectrophotometer. F o r determining activity the e n z y m e 1M. J. Coon, this series, Vol. 52, p. 200. 2 T. Ono, K. Nakazono, and H. Kosaka, Biochim. Biophys. Acta 709, 84 (1982). 3 T. Ono, S. Ozasa, F. Hasegawa, and Y. Imai, Biochim. Biophys. Acta 486, 401 (1977). METHODS IN ENZYMOLOGY, VOL. 110
Copyright © 1985 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-182010-6