Plant Sterol Metabolism

A cell-free enzyme preparation obtained from bramble tissues (Rubus fruticosus) grown in vitro was found to be capable of opening the cyclopropane ring of cycloeucalenol, producing obtusifoliol. The identification of this substance was based on the use of radiochromatography, formation of functional derivatives, and crystallization to constant specific radioactivity. A boiled preparation failed to mediate the reaction, proving that the opening of the 9β,19β-cyclopropane ring is enzymatic. Enzymatic activity was bound to the microsomal fraction and was not dependent upon the addition of exogenous ATP or NADH. Microsomes from other higher plant species (germinating peas and tobacco tissue cultures) were found to be active in transforming cycloeucalenol into obtusifoliol, but microsomes from rabbit liver were unable to perform this reaction. 24-Methylene cycloartanol, cycloartenol, and cycloeucalenol, which are ubiquitous constituents of higher plants, were tested as substrates for the enzymatic reaction. The 4,4-dimethyl sterols are very poor substrates, whereas cycloeucalenol is efficiently converted. In a second group of experiments, three substrates differing only by the number of methyl substituents in position 4 were incubated with the microsomal fraction. Cycloeucalenol (a 4α-methyl sterol) and 24-methylene pollinastanol (a 4-desmethyl sterol) were converted to obtusifoliol and 4-desmethyl obtusifoliol, respectively. But 24-methylene cycloartanol (a 4,4-di-methyl sterol) was not transformed, which suggests that the 4β-methyl group hinders this reaction. On the basis of the results obtained, a tentative mechanism for the opening of the cyclopropane ring is proposed.