Biochemical characteristics ofS-adenosylmethionine decarboxylase from carnation (Dianthus caryophyllus L.) petals

We have partially purified S-adenosylmethionine decarboxylase (EC 4.1.1.50, SAMDC) from carnation (Dianthus caryophyllus L.) petals and generated polyclonal antibodies against CSDC 16 protein (Leeet al., 1996) overexpressed inE. coli. The protein has been purified approximately 126.8 fold through the steps involving ammonium sulfate fractionation, DEAE-Sepharose column chromatography and Sephacryl S-300 gel filtration. Its molecular mass was 42 kDa in native form and we could also detect a band of 32 kDa molecular mass on SDS-PAGE in western blot analysis using the polyclonal antibodies. The Km value of this enzyme forS-adenosylmethionine was 26.3 μM. The optimum temperature and pH forS-adenosylmethionine decarboxylase activity were 35°C and pH 8.0, respectively. Putrescine and Mg²⁺ had no effects on the activation of the enzyme activity. Mg²⁺ did not have any significant effects on the enzyme activity. SAMDC activity was inhibited by putrescine, spermidine and spermine. Methylglyoxal bis-(guanylhydrazone) (MGBG), carbonyl reagents such as hydroxylamine and phenylhydrazine, and sulfhydryl reagent such as 5,5′dithio-bis (2-nitrobenzoic acid) (DTNB) were effective inhibitors of the enzyme. However, isonicotinic acid hydrazide known as an inhibitor of 5′-pyridoxal phosphate (PLP) dependent enzyme activity had no significant effect on the enzyme activity. These results and our previously reported results (Leeet al., 1997b) suggest thatS-adenosylmethionine decarboxylase is a heterodimer, αβ, and some carbonyl group and sulfhydryl group are involved in the catalytic activity.     

Polyamine synthesis in plants: isolation and characterization of spermidine synthase from soybean (Glycine max) axes

Spermidine synthase (EC 2.5.1.16) was purified to homogeneity for the cytosol of soybean (Glycine max) axes using ammonium sulfate fractionation and chromatography on DEAE-Sephacel, Sephacryl S-300, ω-aminooctyl-Sepharose and ATPA-Sepharose. The molecular mass of the enzyme estimated by gel filtration and SDS–PAGE is 74 kDa. Cadaverin and 1,6-diaminohexane could not replace putrescine as the aminopropyl acceptor. Kinetic behaviors of the substrate are consistent with a ping pong mechanism. The kinetic mechanism is further supported by direct evidence confirming the presence of an aminopropylated enzyme and identification of product, 5′-deoxy-5′-methylthioadenosine, prior to adding putrescine. The K_m values for decarboxylated S-adenosylmethionine and putrescine are 0.43 μM and 32.45 μM, respectively. Optimum pH and temperature for the enzyme reaction are 8.5 and 37°C, respectively. The enzyme activity is inhibited by N-ethylmaleimide and DTNB, but stimulated by Co²⁺, Cu²⁺ and Ca²⁺ significantly, suggesting that these metal ions could be the cellular regulators in polyamine biosynthesis.     

Propylamine transferases in chinese cabbage leaves

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 K_m values for decarboxylated S-adenosylmethionine 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 I₅₀ at 0.24 micromolar.

     

Isolation of a 110 000 molecular weight protein in the purification of the nuclear chick intestinal 1,25-dihydroxyvitamin D-3 receptor

A single protein band of molecular weight 110 000 has been obtained after sodium dodecyl sulfate polyacrylamide gel electrophoresis of purified 1,25-dihydroxyvitamin D-3 (1,25-(OH)₂D-3) receptor from crude nuclear extracts of chick intestinal mucosa, prepared in the presence of the protease inhibitors phenylmethylsulfonyl fluoride and ?-aminocaproic acid. The nuclear extract was subjected to a six-step purification scheme, involving polymin P and ammonium sulfate fractionation, DNA-cellulose affinity chromatography, Sephacryl S-200 gel filtration, blue dextran-Sepharose and a final DNA-cellulose chromatographic step. The receptor was obtained in about 1% yield and was purified approx. 3700-fold from the nuclear extract, as assessed by specific activity. Single peaks were observed with ³H-1,25-(OH)₂D-3-labeled crude nuclear extracts on Sephacryl S-200 gel filtration ($\text{Strokes′ radius}\text{= 35.5}\text{A}\text{?}$) and sucrose density gradient centrifugation (3.5 S). Although the identity of the M_r 110 000 protein will remain inconclusive until methods for further characterization are available, it may represent evidence for a higher molecular weight form of the 1,25-(OH)₂D-3 receptor than that observed previously.     

Biosynthesis of spermidine, a direct precursor of pyrrolizidine alkaloids in root cultures of Senecio vulgaris L.

 The polyamine spermidine is an essential biosynthetic precursor of pyrrolizidine alkaloids. It provides its aminobutyl group which is transferred to putrescine yielding homospermidine, the specific building block of the necine base moiety of pyrrolizidine alkaloids. The enzymatic formation of spermidine was studied in relation to the unique role of this polyamine as an alkaloid precursor. S-adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.50) and spermidine synthase (SPDS, EC 2.5.1.16) from root cultures of Senecio vulgaris were partially purified and characterized. The SAMDC-catalyzed reaction showed a pH optimum of 7.5, that of SPDS an optimum of 7.7. The K _m value of SAMDC for its substrate S-adenosylmethionine (SAM) was 15 μM, while the apparent K _m values of SPDS for its substrates decarboxylated SAM (dSAM) and putrescine were 4 μM and 21 μM, respectively. The relative molecular masses of the two enzymes, determined by gel filtration, were 29 000 (SAMDC) and 37 000 (SPDS). Studies with various potential inhibitors revealed, for most inhibitors, profiles that were similar to those established with the respective enzymes from other plant sources. However, putrescine which is not known to be an inhibitor of plant SAMDC, strongly inhibited the enzyme from S. vulgaris roots. Spermidine synthase was sensitive to inhibition by its product spermidine. In the presence of the stationary tissue concentrations of the two polyamines (ca. 0.1 mM each) the activities of SAMDC and SPDS would be inhibited by >80%. The results are discussed in relation to the role of spermidine in primary and secondary metabolism of alkaloid-producing S. vulgaris root cultures. Received: 15 September 1999 / Accepted 10 December 1999     

Purification,properties and regulation of the level of bovine S-adenosylmethionine decarboxylase during lymphocyte mitogenesis

S-Adenosylmethionine decarboxylase was purified from the livers of calves treated with methylglyoxal bis (guanylhydrazone) to elevate the level of the enzyme. Purified bovine S-adenosylmethionine decarboxylase was similar in specific activity and subunit molecular weight (32 000) to the enzymes previously isolated from rat and mouse. The bovine liver enzyme immunologically crossreacted with S-adenosylmethionine decarboxylase from resting and mitogenically activated bovine lymphocytes. The rate of enzyme synthesis in activated lymphocytes was determined by labeling the cells with [³H]leucine and isolating the radioactive decarboxylase by affinity chromatography and sodium dodecyl sulfate gel electrophoresis. The rate of enzyme syntheis was increased 10-fold by 9 h after mitogen treatment, which accounts for the initial increase in cellular enzymatic. There was no further incraese in the rate of S-adenosylmethionine decarboxylase synthesis that correlated with a second elevation of activity occuring at approx. 24 h after mitogenic activation. It was concluded that the second increase in enzyme activity was due to lengthening the intracellular half-life of the enzyme by 2-fold.     

S-adenosylmethionine decarboxylase of corn seedlings

S-Adenosylmethionine decarboxylase (EC 4.1.1.50) has been purified 500-fold in 30% yield from the extract of etiolated corn seedlings (cv. Golden Crossbantam Bell). This preparation had a molecular weight of approximately 25,000. The K_m value was 5 micromolar for S-adenosylmethionine. Methylglyoxal bis(guanylhydrazone), hydroxylamine, and sulfhydryl reagents (such as p-hydroxymercuriphenylsulfonate and N-ethylmaleimide) were effective inhibitors of this enzyme. Germination of corn seed was accompanied by a rapid increase in enzyme activity and maximum activity occurred in 5-day-old seedlings.     

Purification and partial characterization of rat kidney histamine-N-methyltransferase

Histamine-N-methyltransferase (EC 2.1.1.8) was purified 1700-fold with a yield of 9% from rat kidney. Purification included ammonium sulfate precipitation, linear gradient DEAE-cellulose chromotography and S-adenosylhomocysteine affinity chromotography. The purified enzyme preparation showed a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight of 35 000. The isoelectric point of the enzyme was at pH 5.2. The purified enzyme preparation did not contain detectable amounts of histamine. The purified enzyme was totally inhibited in 100 μM parahydroxymercuric benzoate and in 10 μM iodoacetamide, and it was found to be stabilized with dithiothreitol (1 mM), suggesting that the enzyme has an SH-group in the active center. The K_m values for histamine and S-adenosylmethionine were 6.0 and 7.1 μM, respectively. 50% inhibition of histamine-N-methyltransferase was obtained at 28 μM S-adenosylhomocysteine and 100 μM methylhistamine. The purified enzyme was slightly inhibited in 1 mM methylthioadenosine. Histamine in concentrations higher than 25 μM caused substrate inhibition.     

1-Aminocyclopropanecarboxylate synthase, a key enzyme in ethylene biosynthesis

1-Aminocyclopropanecarboxylate (ACC) synthase, which catalyzes the conversion of S-adenosylmethionine (SAM) to ACC and methylthioadenosine, was demonstrated in tomato extract. Methylthioadenosine was then rapidly hydrolyzed to methylthioribose by a nucleosidase present in the extract. ACC synthase had an optimum pH of 8.5, and a K_m of 20 μm with respect to SAM. S-Adenosylethionine also served as a substrate for ACC synthase, but at a lower efficiency than that of SAM. Since S-adenosylethionine had a higher affinity for the enzyme than SAM, it inhibited the reaction of SAM when both were present. S-Adenosylhomocysteine was, however, an inactive substrate. The enzyme was activated by pyridoxal phosphate at a concentration of 0.1 μm or higher, and competitively inhibited by aminoethoxyvinylglycine and aminooxyacetic acid, which are known to inhibit pyridoxal phosphate-mediated enzymic reactions. These results support the view that ACC synthase is a pyridoxal enzyme. The biochemical role of pyridoxal phosphate is catalyzing the formation of ACC by α,γ-elimination of SAM is discussed.     

Purification of DOPA decarboxylase from bovine striatum

Pyridoxal phosphate-dependent DOPA decarboxylase has been purified from bovine striatum to a specific activity of 1.6 U/mg protein. After ammonium sulfate precipitation (30–60%) it was purified by DEAE-Sephacel, Sephacryl S-200, and TSK Phenyl 5 PW chromatography. The purified enzyme showed a single silver staining band with polyacrylamide gel electrophoresis under both denaturing and non-denaturing conditions. The bovine striatal DOPA decarboxylase is a dimer (subunit M_r = 56000 by SDS-PAGE) with a native M_r of 106000 as judged by chromatography on Sephacryl S-200 and by sedimentation analysis. Similar to the DOPA decarboxylase purified from non-CNS tissues, the bovine striatal enzyme requires free sulfhydryl groups for activity, is strongly inhibited by heavy metal ions, and can decarboxylate 5-hydroxytryptophan as well. It should be noted, however, that the final enzyme preparation is enriched in DOPA decarboxylase activity. The distribution of the DOPA decarboxylase and 5-HTP decarboxylase activities also varies among several bovine brain regions. In addition, heat treatment of the enzyme preparation inactivated the two decarboxylation activities at different rates.Abbreviations AADC Aromatic L-amino Acid Decarboxylase - CNS Central Nervous System - DOPA 3,4-dihydroxyphenylalanine - DTT Dithiothreitol, 5-HTP - 5-hydroxytryptophan - Mr relative molecular weight - PLP pyridoxal 5-phosphate - SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis Part of this paper was presented at the 1987 Annual Pharmacology and Toxicology Conferences held at University of North Dakota School of Medicine, North Dakota, USA Res Commun Psychol Psychiat Behav 12: 227–228, 1987 (Abstr).     

S-Adenosyl-l-methionine decarboxylase activities in Mytilus edulis

• 1.1. The activities of S-adenosylmethionine decarboxylase (EC 4.1.1.50) were measured in cell extracts of mantle, hepatopancreas and foot from Mytilus edulis.
• 2.2. The apparent molecular weights of the enzymes estimated by gel filtration chromatography were 65,000 ± 10,000.
• 3.3. The enzymes do not require bivalent cations for catalysis and show optimum pH between 7.0–8.0 in phosphate buffer.
• 4.4. The hepatopancreas enzyme shows different behavior to the other two enzymes against temperature and its activity is strongly inhibited by NH₄⁺.
• 5.5. The apparent K_ms for S-adenosylmethionine were found to be 300, 200 and 250 μM for the hepatopancreas, mantle and foot enzymes, respectively.

     

Purification and some properties of an esterase from yeast

An esterase was isolated and purified from baker's yeast by ammonium sulfate precipitation and column chromatographies on Sephacryl S-200, DEAE-Sephacel, chromatofocusing, and DEAE-Sephacel again. The molecular weight of the enzyme was approximately 84,000 on Sephadex G-100 and 40,000 by sodium dodecyl sulfate-poly-acrylamide gel electrophoresis, suggesting a dimer for the activity. This enzyme hydrolyzed short-chain naphthyl esters and p-nitrophenyl esters, and its activity was strongly inhibited by mercuric compounds. The esterase appeared to be an arylesterase (EC 3.1.1.2) and its optimum pH was 8.0 at 30°C.     

Cottonseed malate synthase : purification and immunochemical characterization

Malate synthase (EC 4.1.3.2), an enzyme unique to the glyoxylate cycle, was purified to homogeneity from cotyledons of 72-hours, darkgrown cotton (Gossypium hirsutum L.) seedlings. Homogeneity of the enzyme was assessed by silver staining SDS-PAGE gels. Purification was accomplished by using a single buffer medium through six steps involving one ammonium sulfate fractionation and chromatography on three columns (Sephacryl S-300, DEAE Sephacel, Phenyl Sepharose). Large-scale preparation of glyoxysomes, a main step in all other published procedures, was not involved. The purified enzyme and that extracted from glyoxysomes appears to be a dodecamer with a native molecular weight of 750,000 (sedimentation coefficient of >20 Svedberg units [S] on sucrose gradients) composed of identical subunits (molecular weight approximately 63,000). The monomer (5S) occurs in the cytosol. Polyclonal antibodies raised in rabbits were judged to be monospecific for malate synthase by immunotitration, double immunodiffusion, and western blotting. Double immunodiffusion experiments revealed only partial immunological identity between the 5S (cytosolic) and 20S (glyoxysomal forms, although complete identity was observed between the 5S form in immature and germinated seeds, and the 20S form in immature and germinated seeds. Cross-reactivity of the cotton antimalate synthase serum was observed with extracts from five other oilseeds. Western blot analyses showed that malate synthase protein was not present in immature seeds prior to appearance of enzyme activity, but when present, subunit molecular weight was indistinguishable in immature, desiccated, and germinated seeds.     

Purification and characterization of an alpha-glucosidase from germinating millet seeds

An α-glucosidase (α-d-glucoside glucohydrolase, EC 3.2.1.20) was isolated from germinating millet (Panicum miliaceum L.) seeds by a procedure that included ammonium sulfate fractionation, chromatography on CM-cellulofine/Fractogel EMD SO₃, Sephacryl S-200 HR and TSK gel Phenyl-5 PW, and preparative isoelectric focusing. The enzyme was homogenous by SDS-PAGE. The molecular weight of the enzyme was estimated to be 86,000 based on its mobility in SDS-PAGE and 80,000 based on gel filtration with TSKgel super SW 3000, which showed that it was composed of a single unit. The isoelectric point of the enzyme was 8.3. The enzyme readily hydrolyzed maltose, malto-oligosaccharides, and α-1,4-glucan, but hydrolyzed polysaccharides more rapidly than maltose. The K_m value decreased with an increase in the molecular weight of the substrate. The value for maltoheptaose was about 4-fold lower than that for maltose. The enzyme preferably hydrolyzed amylopectin in starch, but also readily hydrolyzed nigerose, which has an α-1,3-glucosidic linkage and exists as an abnormal linkage in the structure of starch. In particular, the enzyme readily hydrolyzed millet starch from germinating seeds that had been degraded to some extent.     

Isolation and characterization of Patnopecten mid-gut gland endo-beta-xylosidase active on peptidochondroitin sulfate

An endo-beta-xylosidase acting on the linkage region of peptidochondroitin sulfate was isolated from the mid-gut gland of the mollusc Patnopecten and purified about 375-fold, using a combination of ammonium sulfate fractionation, gel filtration on Sephacryl S-200, and DEAE-Sephacel chromatography. The pH optimum and the isoelectric point of this enzyme were 4.0 and 7.0, respectively. The molecular weight, estimated by gel filtration through Sephacryl S-200, was 78,000. The purified enzyme was completely free from protease, exoglycosidases, sulfatase, and phosphatase. This enzyme hydrolyzed the xylosyl serine linkage of the linkage region of various glycosaminoglycans, that is chondroitin sulfate, dermatan sulfate and heparan sulfate, all possessing a very small peptide segment, but not proteoglycans. It was concluded that this endo-beta-xylosidase was involved in the catabolism of proteoglycans.     

Putrescine N-methyltransferase – The start for alkaloids

Putrescine N-methyltransferase (PMT) catalyses S-adenosylmethionine (SAM) dependent methylation of the diamine putrescine. The product N-methylputrescine is the first specific metabolite on the route to nicotine, tropane, and nortropane alkaloids. PMT cDNA sequences were cloned from tobacco species and other Solanaceae, also from nortropane-forming Convolvulaceae and enzyme proteins were synthesised in Escherichia coli. PMT activity was measured by HPLC separation of polyamine derivatives and by an enzyme-coupled colorimetric assay using S-adenosylhomocysteine. PMT cDNA sequences resemble those of plant spermidine synthases (putrescine aminopropyltransferases) and display little similarity to other plant methyltransferases. PMT is likely to have evolved from the ubiquitous enzyme spermidine synthase. PMT and spermidine synthase proteins share the same overall protein structure; they bind the same substrate putrescine and similar co-substrates, SAM and decarboxylated S-adenosylmethionine. The active sites of both proteins, however, were shaped differentially in the course of evolution. Phylogenetic analysis of both enzyme groups from plants revealed a deep bifurcation and confirmed an early descent of PMT from spermidine synthase in the course of angiosperm development.     

Dicyclohexylamine-induced shift of biosynthesis from spermidine to spermine in plant protoplasts

An improved analytical method, based on high pressure liquid chromatography, has been developed for the simultaneous determination of the polyamines and S-adenosyl-containing compounds in extracts of plant protoplasts. The method involves simple procedures for sample preparation and permits quantification of 1 picomole or less for all the compounds. This method has been used to study the effects of dicyclohexylamine, an inhibitor of plant spermidine synthase (Sindhu, R. K., S. S. Cohen 1984 Plant Physiol 74: 645-649), on biosynthesis of polyamines and 1-aminocyclopropane-1-carboxylate in protoplasts derived from Chinese cabbage leaves. Dicyclohexylamine effectively inhibits spermidine synthase in vivo. Inhibition of the synthesis of spermidine by dicyclohexylamine resulted in a stimulation of spermine synthesis, without significant effect on the synthesis of 1-aminocyclopropane-1-carboxylate. Decarboxylated S-adenosylmethionine is present in control Chinese cabbage protoplasts at ~10⁻¹⁸ moles per cell, and dicyclohexylamine caused an increase of this metabolite of up to 10-fold in a 4-hour period. The increase in decarboxylated S-adenosylmethionine permitted an increased synthesis of spermine. These findings suggest that the availability of decarboxylated S-adenosylmethionine may be rate-limiting for the synthesis of spermine in plant protoplasts.     

Kinetic properties of phenylalanine ammonia-lyase from sunflower (Helianthus annuus L.) hypocotyls.

Phenylalanine ammonia-lyase (PAL) from sunflower hypocotyls has been partially purified by selective precipitation with ammonium sulfate and molecular gel filtration on Sephacryl S-300. Kinetic assays carried out with this partially purified PAL preparation revealed that the enzyme did not show a homogeneous kinetic behaviour. The observed kinetic pattern and parameters (Km and Vmax) depended on the assay conditions used and the protein concentration added to the assay mixture. PAL displayed Michaelian or negative cooperativity kinetics. Such behaviour can be explained by the existence of an association-dissociation process of PAL-protein subunits. The presence of mono-, tri- and tetrameric forms of PAL has been assessed by molecular gel filtration on Sephacryl S-200, using different elution conditions.     

A thermostable manganese-containing superoxide dismutase from the thermophilic fungus Thermomyces lanuginosus

A thermostable superoxide dismutase (SOD) from a Thermomyces lanuginosus strain (P134) was purified to homogeneity by fractional ammonium sulfate precipitation, ion-exchange chromatography on DEAE-Sepharose, Phenyl-Sepharose hydrophobic interaction chromatography, and gel filtration on Sephacryl S-100. The molecular mass of a single band of the enzyme was estimated to be 22.4 kDa, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using gel filtration on Sephacryl S-100, the molecular mass was estimated to be 89.1 kDa, indicating that this enzyme was composed of four identical subunits of 22.4 kDa each. The SOD was found to be inhibited by NaN₃, but not by KCN or H₂O₂, suggesting that the SOD in T. lanuginosus was of the manganese superoxide dismutase type. The SOD exhibited maximal activity at pH 7.5. The optimum temperature for the activity was 55°C. It was thermostable at 50 and 60°C and retained 55% activity after 60 min at 70°C. The half-life of the SOD at 80°C was approximately 28 min and even retained 20% activity after 20 min at 90°C.     

Purification of formaldehyde and formate dehydrogenases from pea seeds by affinity chromatography and S-formylglutathione as the intermediate of formaldehyde metabolism

Formaldehyde dehydrogenase (EC 1.2.1.1) and formate dehydrogenase (EC 1.2.1.2) have been isolated in pure form from pea seeds by a rapid procedure which employs column chromatographies on 5′-AMP-Sepharose, Sephacryl S-200, and DE32 cellulose. The apparent molecular weights of formaldehyde and formate dehydrogenases are, respectively, 82,300 and 80,300 by gel chromatography, and they both consist of two similar subunits. The isoelectric point of formaldehyde dehydrogenase is 5.8 and that of formate dehydrogenase is 6.2. The purified formate dehydrogenase gave three corresponding protein and activity bands in electrophoresis and isoelectric focusing on polyacrylamide gel whereas formaldehyde dehydrogenase gave only one band. Formaldehyde dehydrogenase catalyzes the formation of S-formylglutathione from formaldehyde, and glutathione. Formate dehydrogenase can, besides formate, also use S-formylglutathione and two other formate esters as substrates. S-Formylglutathione has a lower K_m value (0.45 mm) than formate (2.1 mm) but the maximum velocity of S-formylglutathione is only 5.5% of that of formate. Pea extracts also contain a highly active S-formylglutathione hydrolase which has been separated from glyoxalase II (EC 3.1.2.6) and partially purified. S-Formylglutathione hydrolase is apparently needed between formaldehyde and formate dehydrogenases in the metabolism of formaldehyde in pea seeds, in contrast to what was recently reported for Hansenula polymorpha, a yeast grown on methanol.