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Metabolism and Enzymology

Solubilization, Partial Purification, and Immunodetection of Squalene Synthetase from Tobacco Cell Suspension Cultures ¹

Plant Physiology/Biochemistry/Molecular Biology Program, Department of Agronomy, University of Kentucky, Lexington, Kentucky 40546-0091

Squalene synthetase, an integral membrane protein and the first committed enzyme for sterol biosynthesis, was solubilized and partially purified from tobacco (Nicotiana tabacum) cell suspension cultures. Tobacco microsomes were prepared and the enzyme was solubilized from the lipid bilayer using a two-step procedure. Microsomes were initially treated with concentrations of octyl--d-thioglucopyranoside and glycodeoxycholate below their critical micelle concentration, 4.5 and 1.1 millimolar, respectively, to remove loosely associated proteins. Complete solubilization of the squalene synthetase enzyme activity was achieved after a second treatment at detergent concentrations above or at their critical micelle concentration, 18 and 2.2 millimolar, respectively. The detergent-solubilized enzyme was further purified by a combination of ultrafiltration, gel permeation, and Fast Protein Liquid Chromatography anion exchange. A 60-fold purification and 20% recovery of the enzyme activity was achieved. The partially purified squalene synthetase protein was used to generate polyclonal antibodies from mice that efficiently inhibited synthetase activity in an in vitro assay. The apparent molecular mass of the squalene synthetase protein as determined by immunoblot analysis of the partially purified squalene synthetase protein separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 47 kilodaltons. The partially purified squalene synthetase activity was optimal at pH 6.0, exhibited a K_m for farnesyl diphosphate of 9.5 micromolar, and preferred NADPH as a reductant rather than NADH.

This article has been cited by other articles:

				T. P. Devarenne, A. Ghosh, and J. Chappell Regulation of Squalene Synthase, a Key Enzyme of Sterol Biosynthesis, in Tobacco Plant Physiology, July 1, 2002; 129(3): 1095 - 1106. [Abstract] [Full Text] [PDF]