Mimosine, a non-protein amino acid, which occurs in two leguminous species, Mimosa pudica and Leucaena leucocephala, and its primary breakdown product, DHP, possess adverse biological properties and have been shown to produce toxicity symptoms in animals which ingest them.
The anti-metabolic effect of mimosine on insects has generated interest in attempting to transfer the genetic material responsible for mimosine synthesis from L. leucocephala to other agronomically important species. Such a process initially involves the elucidation of the biosynthetic pathway of mimosine inL.leucocephala necessitating in turn, a method of cell-free synthesis. A convenient and inexpensive assay technique for mimosine and/or DHP is also required.
Hylin (1964) proved that lysine is a specific precursor in mimosine biosynthesis by administering [2-14 C]-DL-lysine monohydrochloride to L.leucocephala plants, thereby demonstrating that the radioactive carbon atom is incorporated into the pyridone ring of mimosine.
Paper electrophoresis was investigated to assess its value, in conjunction with scintillation counting, as a suitable assay for radioactive mimosine and DHP in experiments involving administration of [6-14 C]-DL-lysine monohydrochloride toL.leucocephala plants and leaf homogenates.
Pure solutions of mimosine, DHP, lysine and other amino acids of the pyruvate family were used to study the migration of these molecular species under a variety of electrophoretic conditions. Satisfactory separation of DHP, lysine, alanine and a group comprising mimosine, leucine and valine was achieved, but the members within the group were inseparable.
The conditions thus determined were applied in the electrophoretic separation of a L. leucocephala leaf extract. A O.IM potassium phosphate buffer solution was used in the extraction and the crude extract obtained was passed through an ion exchange column to separate out the low molecular weight organic cations including mimosine and DHP. The combined concentration of mimosine/DHP in the purified extract was determined and adjusted to 5mg.ml-1. A sample loading of 1µl of concentrated extract (5µg of mimosine/DHP) proved satisfactory in combination with electrophoretic conditions of 12mA, ca 36 V.cm-1 and 45 minutes running time. Superior resolution was achieved when the extract was
clarified with polyvinylpolypyrrolidone (PVPP).
Experiments involving the addition of various quantities of PVPP and reducing agents to the extraction buffer solution were inconclusive.
The use of liquid nitrogen in the grinding of L. leucocephala tissue proved to be a superior method to that of maceration in cold buffer solution in an ice-bath.
Two attempts were made to incorporate 14C -lysine by the method described by Hylin. In the first attempt, using plants in the glasshouse, the administered labelled solution was taken up by the plant as evidenced by scintillation counting of segments of the electrophoretogram of leaf and floral bud extracts. However, no activity associated with any molecular species except lysine was discernible. A trail of residual activity in the migration path of lysine precluded the possibility of detecting any but very high levels of DHP activity. In a second attempt using Hylin's procedure, the 14C -lysine solution was not taken up successfully by the plant.
A series of two experiments in which 14C-lysine was added to homogenates of L.leucocephala with subsequent incubation at 30ºC produced no positive results. In the first experiment a slight peak of activity occurred in the putative mimosine region of the electrophoretogram of one of the incubation extracts. However this was not confirmed in the second experiment.