Rapid assay of spermidine synthase activity for high-performance liquid chromatography.

The final steps of spermidine and spermine biosynthesis involve the addition of an aminopropyl group from S-methyladenosylhomocysteamine (decarboxylated S-adenosyl-L -methionine) to putrescine and spermidine respectively 91-33 _ The reactions, forming 5’-methylthioadenosie as a secondary product, are catalyzed by two distinct synthases [4-7]_ Thus far, studies concerning polyamine metabolism have addressed primarily the decarboxyiations of omithine and S-adeno@nethionine (SAM), which are considered the rate-limiting steps in this metabolic process, Recent methodological improvements, including techniques for the preparation of decarboxylated SAM as well as for the separation of reactants from reaction products have provided important information regarding spermidine and spermine synthases [S-lo]. Our interest in polyamines derives from our findings that brain levels of SAM are decreased prior to seizures elicited by a single administration of the chemical convulsant L-methionine-d,Z-sulfoximin e (MSO) [ll] . This depletion of SAM is due to its increased utilization via transmethylation reactions 1121. Since the depletion of S-AM levels by MS0 could also limit the availability of decarboxylated SAM, the precursor of spermidine, we decided to investigate polyamine biosynthesis in the MS0 epileptogenic mouse brain. In this paper we describe a rapid and economic high-performance liquid chromatographic (HPLC) method for the separation and quantitation of spermidine formed upon incubation of putrescine with a crude extract of mouse brain. This method thus permits the rapid measurement of spermidine synthase (EC 2.5.1.16) activity_