Abstract
Our principal goal is the complete resolution and reconstitution of the microsomal enzymes of cholesterol biosynthesis. Elucidation of the enzymology has been achieved primarily through dissection of the membrane-bound, 19-step multienzymic process. This report describes the dissection approach through both interruption of specific steps and reconstitution of enzymes that catalyze oxidation of the 14α-methyl group. In earlier work, 4-demethylation was resolved into 3 component reactions catalyzed by: 4-methyl sterol oxidase (NAD[P] H- and O2-dependnet); steroid 4α-carboxylic acid decarboxylase (NAD-dependent); and 3-ketosteroid reductase (NADPH-dependent). The 3-ketosteroid reductase and decarboxylase have been solubilized with Lubrol WX and deoxycholate, respectively, and characterized. The 4-methyl sterol oxidase (cytochrome b5-dependent) recently has been solubilized with Renex 690. This study represents successful elucidation of a microsomal enzyme sequence by interruption of the central 10-step segment of the multienzymic formation of cholesterol from lanosterol. The initial C-32 oxidative reaction of 14α-methyl group elimination is catalyzed by a from of cytochrome P-450 that is induced by isosafrole. The induced cytochrome P-450 has been solubilized with Emulgen 913 and purified to homogeneity (17 nmol of cytochrome/mg protein). 24,25-Dihydrolanosterol is oxidized by combination of cytochrome P-450 reductase, hematin, NADPH, glutathione, and the purified, isosafrole-induced cytochrome in an artificial liposome. Oxidation product identification is underway. This study represents successful elucidation of a microsomal multienzymic sequence by solubilization and reconstitution of a segment of the pathway. The remaining enzymes under study are the Δ8→Δ7 isomerase and 3 NADPH-dependent double bond reductases that catalyze reduction of: Δ7, Δ14- Δ24-sterol double bonds. Purification of these nonoxygenrequiring enzymes is in progress. Resolution of the enzymes has demonstrated unequivocally that cholesterol synthesis via this pathway could not have appeared biologically until membranes containedboth the cytochrome P-450- and cytochrome b5-electron transport enzymes. Chemically, all enzymic attacks in the formation of cholesterol from lanosterol appear to be initiated on the α-face of the relatively planar steroids. Thus, considerable genetic pressure must have been needed for the stereospecific clearing of the steroidal α-face to form the mature membrane component, cholesterol.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Gaylor, J.L. (1972) Adv. Lipid Res. 10, 89–141.
Gaylor, J.L. (1974) in MTP International Review of Science, Biochemistry Section (Lipids) (Goodwin, T.W., ed.) Vol. 1, pp. 1–42, Medical and Technical Publishing Co., Oxford, England.
Gaylor, J.L. (1981) in Polyisopropenoid Biosynthesis (Porter, J.W., ed.) Vol. 1, pp. 481–543, John Wiley and Sons, New York, NY.
Bloch, K.B. (1965) Science 150, 19–28.
Miller, W.L., Kalafer, M.E., Gaylor, J.L., and Delwiche, C.V. (1967) Biochemistry 6, 2673–2678.
Miller, W.L., and Gaylor, J.L. (1970) J. Biol. Chem. 245, 5369–5374.
Miller, W.L., and Gaylor, J.L. (1970) J. Biol. Chem. 245, 5375–5381.
Miller, W.L., Brady, D.R., and Gaylor, J.L. (1971) J. Biol. Chem. 246, 5147–5153.
Gaylor, J.L., Miyake, Y., and Yamano, T. (1975) J. Biol. Chem. 249, 1980–1987.
Rahimtula, A.D., and Gaylor, J.L. (1972) J. Biol. Chem. 247, 9–15.
Billheimer, J.T., Alcorn, M., and Gaylor, J.L. (1981) Arch. Biochem. Biophys. 211, 430–438.
Gibbons, G.F., and Mitropoulos, K.A. (1973) Eur. J. Biochem. 40, 267–273.
Gaylor, J.L., and Mason, H.S. (1968) J. Biol. Chem. 243, 4966–4972.
Aoyama, Y., Yoshida, Y., Sato, R., Susani, M., and Ruis, H. (1981) Biochim. Biophys. Acta 663, 194–202.
Fukushima, H., Grinstead, G.F., and Gaylor, J.L. (1981) J. Biol. Chem. 256, 4822–4826.
Gibbons, G.F., Bullinger, C.R., and Mitropoulos, K.A. (1979) Biochem. J. 183, 309–315.
Trocha, P.J., Jasne, S.J., and Sprinson, D.B. (1974) Biochem. Biophys. Res. Commun. 59, 666–671.
Aoyama, Y., and Yoshida, Y. (1978) Biochem. Biophys. Res. Commun. 85, 28–34.
Comai, K., and Gaylor, J.L. (1973) J. Biol. Chem. 248, 4947–4955.
Jefcoate, C.R.E., and Gaylor, J.L. (1969) Biochemistry 8, 3464–3472.
Gaylor, J.L., Moir, N.J., Seifried, H.E., and Jefcoate, C.R.E. (1970) J. Biol. Chem. 245, 5511–5513.
Gaylor, J.L., Hsu, S.T., Delwiche, C.V., Comai, K., and Seifried, H.E. (1973) in Oxidases and Related Redox Systems (King, T.E., Mason, H.S., and Morrison, M., eds.) Vol. 2, pp. 431–444, Univ. Park Press, Baltimore, MD.
Dickins, M., Bridges, J.W., Elcombe, C.R., and Netter, K.J. (1978) Biochem. Biophys. Res. Commun. 80, 89–96.
Fisher, G.J., Fukushima, H., and Gaylor, J.L. (1981) J. Biol. Chem. 256, 4388–4394.
Borchert, P., Miller, J.A., Miller, E.C., and Shires, T.K. (1973) Cancer Res. 33, 590–600.
Billheimer, J.T., and Gaylor, J.L. (1980) J. Biol. Chem. 255, 8128–8135.
Author information
Authors and Affiliations
About this article
Cite this article
Trazaskos, J.M., Bowen, W.D., Fisher, G.J. et al. Microsomal enzymes of cholesterol biosynthesis from lanosterol: A progress report. Lipids 17, 250–256 (1982). https://doi.org/10.1007/BF02535112
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF02535112