Purification of spermidine synthase from rat ventral prostate by affinity chromatography on immobilized S-adenosyl(5′)-3-thiopropylamine

Samejima, Keijiro; Yamanoha, Banri

doi:10.1016/0003-9861(82)90206-5

Cited by 48 publications

(33 citation statements)

References 29 publications

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“…1). The affinity of P. furiosus ACAPT for decarboxy-AdoMet (K m , 20 M) is similar to that measured for P. falciparum spermidine synthase (K m , 35 M) (18) but lower than that of corresponding enzymes from S. solfataricus (K m , 7.9 M) (8), E. coli (K m , 2.2 M) (4), and rat prostate (K m , 1.1 M) (41).…”

Section: Resultssupporting

confidence: 74%

“…The enzyme catalyzing this reaction was subsequently purified to homogeneity by Bowman et al (4). The development of rapid and sensitive methods of polyamine analysis, together with the utilization of affinity chromatography, allowed the purification to homogeneity and a complete characterization of spermine and spermidine synthases from bovine brain (36) and rat ventral prostate (41), respectively. Moreover, a propylamine transferase with unusual molecular and kinetic properties has been purified to homogeneity from Sulfolobus solfataricus (S. solfataricus aminopropyl transferase [APT]), an extreme thermophilic archaeon (8).…”

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confidence: 99%

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The First Agmatine/Cadaverine Aminopropyl Transferase: Biochemical and Structural Characterization of an Enzyme Involved in Polyamine Biosynthesis in the Hyperthermophilic Archaeon Pyrococcus furiosus

et al. 2007

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We report here the characterization of the first agmatine/cadaverine aminopropyl transferase (ACAPT), the enzyme responsible for polyamine biosynthesis from an archaeon. The gene PF0127 encoding ACAPT in the hyperthermophile Pyrococcus furiosus was cloned and expressed in Escherichia coli, and the recombinant protein was purified to homogeneity. P. furiosus ACAPT is a homodimer of 65 kDa. The broad substrate specificity of the enzyme toward the amine acceptors is unique, as agmatine, 1,3-diaminopropane, putrescine, cadaverine, and sym-nor-spermidine all serve as substrates. While maximal catalytic activity was observed with cadaverine, agmatine was the preferred substrate on the basis of the k cat /K m value. P. furiosus ACAPT is thermoactive and thermostable with an apparent melting temperature of 108°C that increases to 112°C in the presence of cadaverine. Limited proteolysis indicated that the only proteolytic cleavage site is localized in the C-terminal region and that the C-terminal peptide is not necessary for the integrity of the active site. The crystal structure of the enzyme determined to 1.8-Å resolution confirmed its dimeric nature and provided insight into the proteolytic analyses as well as into mechanisms of thermal stability. Analysis of the polyamine content of P. furiosus showed that spermidine, cadaverine, and sym-nor-spermidine are the major components, with small amounts of sym-nor-spermine and N-(3-aminopropyl)cadaverine (APC). This is the first report in Archaea of an unusual polyamine APC that is proposed to play a role in stress adaptation.Propylamine transferases represent a class of enzymes catalyzing the transfer of a propylamine group from S-adenosyl-(5Ј)-3-methylthiopropylamine (decarboxy-AdoMet) to various amine acceptors (7,39,48,60), according to the following reaction: decarboxy-AdoMet ϩ polyamine substrate 3 5Ј-methylthioadenosine ϩ polyamine product. The reaction involves a nucleophilic attack on the C-3 methylene of the propylamine moiety adjacent to the positively charged sulfur of decarboxy-AdoMet (7,39,48,60).In 1958, Tabor et al. (49) provided the first evidence that decarboxy-AdoMet is the donor of aminopropyl groups in the biosynthesis of spermidine in cell extracts of Escherichia coli. The enzyme catalyzing this reaction was subsequently purified to homogeneity by Bowman et al. (4). The development of rapid and sensitive methods of polyamine analysis, together with the utilization of affinity chromatography, allowed the purification to homogeneity and a complete characterization of spermine and spermidine synthases from bovine brain (36) and rat ventral prostate (41), respectively. Moreover, a propylamine transferase with unusual molecular and kinetic properties has been purified to homogeneity from Sulfolobus solfataricus (S. solfataricus aminopropyl transferase [APT]), an extreme thermophilic archaeon (8). Recently, recombinant forms of the spermidine synthases of the extremely thermophilic bacterium Thermotoga maritima (T. maritima PAPT) (28) and of the human malar...

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Section: Resultssupporting

confidence: 74%

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confidence: 99%

The First Agmatine/Cadaverine Aminopropyl Transferase: Biochemical and Structural Characterization of an Enzyme Involved in Polyamine Biosynthesis in the Hyperthermophilic Archaeon Pyrococcus furiosus

et al. 2007

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“…The sample of ATPA in this laboratory's possession during those studies did not permit the preparation of a suitable affinity column. In earlier studies such a column had previously increased the purification of animal enzymes (16). The column constructed in this laboratory earlier did not release the enzyme at all.…”

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confidence: 99%

“…The column constructed in this laboratory earlier did not release the enzyme at all. This experiment has not been repeated with newly synthesized ATPA; this substance has served quite well in preparing ATPA-Sepharose for affinity chromatography, the enzyme being eluted with dSAM as described previously (16).…”

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confidence: 99%

S-Adenosylmethionine Decarboxylase and Spermidine Synthase from Chinese Cabbage

Yamanoha

Cohen²

1985

Plant Physiol.

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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...

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“…Además de las posibles explicaciones avanzadas en los resultados de esta Tesis, en el apartado de "Características cinéticas de SPDS, tSPMS y PMT" (Página 73), también habría que considerar que en las reacciones de biosíntesis de poliaminas, además de la propia poliamina, se genera un producto secundario, el 5'-metiltioadenosina (MTA), que es la molécula resultante de eliminación del grupo aminopropilo del AdoMetdc. En condiciones fisiológicas, este MTA es degradado por MTA quinasas (Sauter et al 2004), pero en condiciones in vitro se acumula y actúa como un inhibidor tanto de las SPDS (Dufe et al 2005) como de las SPMS (Ikeguchi et al 2006), por lo que puede afectar a nuestras determinaciones de las constantes cinéticas.…”

Section: Mecanismos Bioquímicos De Las Aminopropil Transferasasunclassified

Bases moleculares de la síntesis de termoespermina y sus implicaciones en el desarrollo vascular de Arabidopsis thaliana

Sirera¹,

José²

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Purification of spermidine synthase from rat ventral prostate by affinity chromatography on immobilized S-adenosyl(5′)-3-thiopropylamine

Cited by 48 publications

References 29 publications

The First Agmatine/Cadaverine Aminopropyl Transferase: Biochemical and Structural Characterization of an Enzyme Involved in Polyamine Biosynthesis in the Hyperthermophilic Archaeon Pyrococcus furiosus

The First Agmatine/Cadaverine Aminopropyl Transferase: Biochemical and Structural Characterization of an Enzyme Involved in Polyamine Biosynthesis in the Hyperthermophilic Archaeon Pyrococcus furiosus

S-Adenosylmethionine Decarboxylase and Spermidine Synthase from Chinese Cabbage

Bases moleculares de la síntesis de termoespermina y sus implicaciones en el desarrollo vascular de Arabidopsis thaliana

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