Incorporation of [214C]malonyl CoA into fatty acids by a cell-free extract of Catharanthus roseus suspension culture cells |
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Authors: | J. J. MacCarthy P. K. Stumpf |
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Affiliation: | (1) Department of Biochemistry and Biophysics, University of California, 95616 Davis, CA, USA |
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Abstract: | A cell-free extract containing the enzymes for de-novo synthesis, elongation and desaturation of fatty acids was prepared from cultured cells of Catharanthus roseus G. Don. 14C-Fatty acids synthesized by the extract from [2-14C]malonyl CoA substrate were palmitic (16:0), stearic (18:0) and oleic (18:1). Dialyzed extract was active and stable at room temperature and at 4° C, but was inactivated on boiling. There was an absolute requirement for NADPH for incorporation of [2-14C]malonyl CoA into total fatty acids. Escherichia coli acyl carrier protein stimulated total fatty-acid synthesis without affecting the relative ratio of individual fatty acids. Total fatty-acid synthesis at a rate of 45 nmol·mg-1 protein·h-1 occurred at a substrate level of 73 M malonyl CoA, cofactor levels of 500 M NADPH, 30 g·ml-1E. coli ACP, and 1.0 mg·ml-1 extract protein. Total fatty acid synthesis was also sensitive to cerulenin and CoA levels. Variations in the relative abundance of individual 14C-fatty acids were regulated by concentrations of [14C]malonyl CoA. NADPH and ferredoxin, as well as by pH, temperature and length of incubation. Fatty-acid synthetase enzymes responsible for [14C]palmitic acid were rapidly saturated at a low substrate level (0.3 M malonyl CoA). Increasing the level of [2-14C]malonyl CoA permitted further synthesis of [14C]stearate and [14C]oleate. Desaturation of [14C]stearate to [14C]oleate was stimulated by increasing the levels of NADPH and ferredoxin. The desaturase and elongase enzymes were sensitive to acidic pH. The desaturase was also unstable at 41° C, although fatty acid synthetase and elongase were unaffected by this temperature.Abbreviation ACP Acyl carrier protein |
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Keywords: | Catharanthus Cell culture (cell-free) Desaturase Fatty-acid synthesis |
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