Propylamine Transferases in Chinese Cabbage Leaves

Abstract

We have found spermidine synthase and spermine synthase activities in extracts of leaves of Chinese cabbage (Brassica pekinensis var. Pak Choy) and have developed an assay of the former in crude extracts. The method is based on the transfer of the propylamine moiety of decarboxylated S-adenosylmethionine to labeled putrescine, followed by ion-exchange separation of the labeled amine substrate and product, which are then converted to the 5-dimethylamino-1-napthalene sulfonyl (dansyl) derivatives and further purified and identified by thin layer chromatography. The specific radioactivity of putrescine present in the reaction mixture is determined, as is the radioactivity present in dansyl spermidine. The enzyme is also present in extracts of spinach leaves.Spermidine synthase has been purified about 160-fold from Chinese cabbage leaves. After partial purification, a rapid coupled enzymic assay has been used to study various properties of the enzyme. The plant enzyme shows maximum activity at pH 8.8 in glycine-NaOH buffer and has a molecular weight of 81,000. The Km values for decarboxylated Sadenosylmethionine and putrescine are 6.7 and 32 micromolar, respectively. The enzyme activity is inhibited strongly by dicyclohexylamine, cyclohexylamine, and S-adenosyl-3-thio-1, 8-diaminoctane. Of these, dicyclohexylamine is the most potent inhibitor with an Iso at 0.24 micromolar.Propylamine transferases, catalyzing the synthesis of spermidine from putrescine (spermidine synthase, EC 2.5.1.16) and of spermine from spermidine (spermine spermidine, EC 2.5.1.-), utilize decarboxylated SAM2 as the propylamine donor. Both the polyamine and MTA are formed in equivalent amounts. Spermidine synthase was first discovered in Escherichia coli (23) and was later purified and characterized from this source (2). Spermidine synthase and spermine synthase have been reported in several mammalian tissues (reviewed in 26) and are separable from SAM decarboxylase. With the aid of affinity chromatography, these enzymes have now been purified to homogeneity from rat ventral prostate and bovine brain and characterized (16,17).In (25), and a significant fraction of the triamine is found in a nonexchangeable association with the virus (5). Despite the advances with the bacterial and mammalian enzymes, there have been few studies on the enzymes ofspermidine and spermine synthesis in plants. Decarboxylated SAM, the propylamine donor, is not commercially available and has to be prepared either enzymically or synthesized chemically. Furthermore, the crude plant extracts are complex, which limit the choice of a suitable assay of the enzymes. Methods dependent on the isolation of labeled MTA from labeled decarboxylated SAM (10) could not be applied to the Chinese cabbage extracts because MTA was rapidly degraded. Coupled enzymic assays (2,22,27) are affected adversely by the presence of degradative enzymes. Bypassing these difficulties, we have developed an assay for spermidine synthase which is useful for crude extracts. After partial purifica...

Subcellular Localization of Amines and Activities of Their Biosynthetic Enzymes in p-Fluorophenylalanine Resistant and Wild-Type Tobacco Cell Cultures

“…Nearly all the SDC activity was recovered from the supernatant fraction in both cell lines (Table II). Given the present and earlier findings on the subcellular location of ADC, ODC, and SDC, and of propylamine transferases in Chinese cabbage (27,28,35), it would appear that the entire pathway for the generation of polyamines is also found within the cytosolic system (Tables II, III) (2,3,22). This is not, however, necessarily an exclusive location since correction for organelle breakage would place small but significant levels of ADC and ODC in organelle-enriched fractions (Table II; 33).…”

S-Adenosylmethionine Decarboxylase and Spermidine Synthase from Chinese Cabbage

“…The estimation reported in the present paper was conducted on Sephacryl S-300, which yielded a value for spermidine synthase, i.e. 81,000, essentially identical to that previously observed on Sephadex G-100 (17). It must be noted that Sephacryl may have resolved a minor component of spermidine synthase of slightly greater mol wt (Fig.…”

“…It must be noted that Sephacryl may have resolved a minor component of spermidine synthase of slightly greater mol wt (Fig. 5); this had not been seen earlier (17). It may be asked if this new result relates to the observed relative instability of this enzyme.…”

“…This enzyme had been detected in Chinese cabbage and purified approximately 150-fold (4,17). The sample of ATPA in this laboratory's possession during those studies did not permit the preparation of a suitable affinity column.…”

“…The enzyme, spermidine synthase, is inhibited by dicyclohexylamine (17) and this inhibitor acts relatively specifically on this enzyme in the protoplasts (9). Nevertheless dSAM continued to be generated from SAM and was diverted to spermine synthesis (9 [1,4-14C]putrescine dihydrochloride (107.1 mCi/ mmol) were purchased from New England Nuclear Co. and unlabeled SAM was obtained from Sigma Chemical Co.…”