S-Adenosylmethionine Decarboxylase and Spermidine Synthase from Chinese Cabbage

Abstract

The enzyme, S-adenosylmethionine (SAM) decarboxylase (EC 4.1.1.50), has been demonstrated in leaves of Chinese cabbage, (Brassica pekinensis var Pak Choy). All of the enzyme can be found in extracts of the protoplasts obtained from the leaves of growing healthy or virusinfected cabbage. The protein has been purified approximately 1500-fold in several steps involving ammonium sulfate precipitation, affinity chromatography, and Sephacryl S-300 filtration. The reaction catalyzed by the purified enzyme has been shown to lead to the equimolar production of CO2 and of decarboxylated S-adenosylmethionine (dSAM). The K, for SAM is 38 micromolar. The reaction is not stimulated by Mg' or putrescine, and is inhibited by dSAM competitively with SAM. It is also inhibited strongly by methylglyoxal bis(guanylhydrazone). The enzyme, spermidine synthase (EC 2.5. of Lathynrs sativus (19), a small amount of activity was concentrated and purified by affinity chromatography. The stoichiometry of the reaction was not determined nor was the amount of activity related to the amount of spermidine synthesized in the plant. This activity, as well as that found in mung bean sprouts (6), was shown to be Mge' dependent, as are some bacterial SAM decarboxylases. The levels ofbiosynthetic activity detected in extracts of these plant materials were extremely low and may have arisen from bacterial contamination. On the other hand, the activity of SAM decarboxylase isolated from corn seedlings (20) has been reported to be independent of Mge' or putrescine, as is the enzyme we have now isolated from Chinese cabbage. Nevertheless, the low level of activity of the corn enzyme, by comparison to the amount of spermidine found in plants, has similarly raised the problem of the significance of this activity.One product of the SAM decarboxylase reaction, dSAM, is difficultly available. Despite its recent synthesis, an extinction coefficient has not been published (15) and a direct measurement of the stoichiometry of the decrease of SAM and appearance of dSAM has not previously been attempted. For this reason the decarboxylation reaction catalyzed by several presumed SAM decarboxylases cannot be considered unequivocal. The compound, dSAM, was generated from SAM by the Escherichia coli enzyme in 87% yield, calculated on the assumption that dSAM had an extinction coefficient identical to that of SAM (21, 28). Low yields of dSAM via enzymatic decarboxylation of SAM, arise from the relatively high Km of SAM in the reaction, as well as feedback inhibition by dSAM. Thus, stoichiometry in the decarboxylation has been demonstrated only by coupling spermidine synthase and putrescine to the system to trap the aminopropyl of dSAM in spermidine and to compare the yield of this triamine to that of CO2 (12,14).A newly developed analytical system has been applied to the determination of picomole amounts of SAM and dSAM (9). We have determined the course of the SAM decarboxylase reaction and have established the equimolar production of dSAM and CO2 from SAM by t...

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).…”

A Polyamine Metabolon Involving Aminopropyl Transferase Complexes in Arabidopsis

“…Although the levels of spermidine seem to be regulated primarily through its recycling to putrescine by acetyltransferases in animals (Pietilä et al, 1997), spermidine production correlates with the tissue-specific regulation of SAMDC and SPDS in plants (Malmberg et al, 1998). A coordinated reduction of spermidine, SAMDC, and SPDS levels during stress suggests that the regulation of SPDS activity plays an important role in the proper adjustment of plant polyamine levels (Yamanoha and Cohen, 1985;Tiburcio et al, 1993). Duplicated genes that encode two closely related and differentially regulated SPDS isologs (SPDS1 and SPDS2) have been identified in pea, Datura , and Hyoscyamus (Hashimoto et al, 1998;Alabadí and Carbonell, 1999).…”

Putrescine Aminopropyltransferase Is Responsible for Biosynthesis of Spermidine, Spermine, and Multiple Uncommon Polyamines in Osmotic Stress-Tolerant Alfalfa

“…Apparently, two specific enzymes are required because the two consecutive propylamine transfers are made to opposing ends of the original putrescine molecule. However, PAPT has been partially purified from only two higher plant sources (Hirasawa and Suzuki, 1983;Yamanoha and Cohen, 1985) and appears to be a soluble cytoplasmic enzyme (Sindhu and Cohen, 1984a). SAPT has not been characterized in any plant, although detection of its activity has been reported in a rare case (Sindhu and Cohen, 198413).…”