Kinetic Properties of Fungal Polyamine Oxidases and Their Application to Differential Determination of Spermine and Spermidine

Polyamine oxidase from Penicillium chrysogenum oxidized spermine rapidly and spermidine slightly at pH 7.5. The apparent Km values for spermine and spermidine were calculated to be 2.25 × 10^?5 m and 9.54 × 10^?6 m, respectively. The relative maximum velocities for spermine and spermidine were 3.37 × 10^?3 m (H₂O₂) per min per mg of protein and 2.08 × 10^?4 m (H₂O₂) per min per mg of protein, respectively. Spermine oxidation of the enzyme was competitively inhibited by spermidine and putrescine. The apparent Ki values by spermidine and putrescine were calculated to be 3.00 × 10^?5 m and 1.80 × 10^?8 m, respectively. On the other hand, polyamine oxidase from Aspergillus terreus rapidly oxidized both spermidine and spermine at pH 6.5. The apparent Km values for spermidine and spermine were 1.20 × 10^?8 m and 5.37 × 10^?7 m, respectively. The relative maximum velocities for spermidine and spermine were 1.55 × 10^?2 m (H₂O₂) per min per mg of protein and 6.20 × 10^?3 m (H₂O₂) per min per mg of protein, respectively.

Differential determination of spermine and spermidine was carried out using the two enzymes. The initial rate was assayed with Penicillium enzyme and the end point was measured afte addition of Aspergillus enzyme. Small amounts of polyamines (25 to 200 nmol of spermine and 25 to 250 nmol of spermidine) were assayed by solving two simultaneous equations obtained from the rate assay method and the end point assay method. The calculated values were in close agreement with those obtained by an amino-acid analyzer.     

A New Isolation Method of Plasmid Deoxyribonucleic Acid from Staphylococcus aureus Using a Lytic Enzyme of Achromobacter lyticus

An enzymatic procedure for the differential determination of polyamines, spermine and spermidine, has been established using beef plasma amine oxidase. This method was specific for these polyamines and required only one reaction system. Small amounts of polyamines (10µm to 80 µm of spermine and 10 µm to 100 µm of spermidine) were assayed by solving two simultaneous equations obtained from the rate assay method and the end point assay method. The calculated values were in good agreement with those obtained by other method.     

Change in the Regulation of Enzyme Synthesis under Catabolite Repression in Bacillus subtilis Pleiotropic Mutant Lacking Transketolase

The regulation of enzyme synthesis has changed in Bacillus subtilis pleiotropic mutant lacking transketolase (tkt). The tkt mutant is hypersensitive to d-glucose repression of the synthesis of d-mannitol catabolic enzymes, such as d-mannitol-1-phosphate dehydrogenase and d-mannitol transport system. d-Gluconate, d-xylose and l-arabinose are also effectors for repression in the tkt mutant. In contrast, the synthesis of sorbitol catabolic enzymes, such as sorbitol permease and sorbitol dehydrogenase, are almost insensitive to d-glucose repression. These changes in the regulation of enzyme synthesis seem to be related to some defect in the cell surface structure of the tkt mutant by which other pleiotropic properties are also generated.     

Effects of 1-Aminocyclopropane-1-carboxylic Acid Production on the Changes in the Polyamine Levels in Hiproly Barley Callus after Auxin Withdrawal

Contents of polyamines and 1-aminocyclopropane-1-carboxylic acid (ACC) in Hiproly barley callus were examined under different culture conditions. After auxin withdrawal, the contents of free polyamines changed conversely to the contents of ACC. In the absence of auxin, incorporation of l-[3,4–¹⁴C]methionine into polyamines and the activity of S-adenosylmethionine decarboxylase (SAMDCase) in the callus increased, then remained stable, but incorporation of l-[3,4- ¹⁴C]methionine into ACC, precursor of ethylene and ACC synthase activity once declined and increased again.

Aminooxyacetic acid (AOA) affected the increase in the levels of polyamines in the callus. 1- Aminoisobutyric acid (AIB) had a slight effect on the polyamine production. The incorporation of l-[3,4–¹⁴C]methionine into ACC and ACC synthase activity were inhibited by AOA, but not by « 4 AIB. AOA stimulated the activity of SAMDCase, and also enhanced the incorporation of l-[3,4- ¹⁴C]methionine into polyamines in the callus. Methylglyoxal-bis(guanylhydrazone) (MGBG) greatly enhanced the ACC production. The rate of incorporation of l-[3,4–¹⁴C]methionine into ACC and ACC synthase activity in the callus were significantly enhanced by MGBG. MGBG strongly inhibited SAMDCase activity and the incorporation of l-[3,4–¹⁴C]methionine into polyamines. Moreover, the synthesis of polyamines was inhibited by MGBG.

These results suggested that in Hiproly barley callus ACC production has an important effect on changes in the polyamine levels, and that polyamine and ethylene biosynthetic pathways are regulated by competition against each other.     

New Metabolites of Streptomyces alboniger

A mutant unable to grow under acidic conditions was isolated from the acidophilic heterotrophic bacterium Acidiphilium facilis 24R. The growth of the mutant could be fully restored by the addition of spermidine or lysine at the concentration of 100 μm. The HPLC analysis of polyamine composition showed that spermidine and putrescine were major polyamine components in the parental strain. In the mutant strain, putrescine was replaced by cadaverine. It was found that some polyamines in the cells were conjugated with the other cell components. The growth of the bacterium in the medium below pH 4.5 was inhibited in the presence of α-methylornithine or methylglyoxal-bis(guanylhydrazone), which are inhibitors of rate-limiting enzymes involved in the biosynthesis of polyamines. The growth of the bacterium that had been inhibited in the presence of inhibitors could be fully restored by the addition of putrescine or spermidine. On the basis of these results, it was concluded that polyamines have a significant role in the growth of Acidiphilium facilis 24R under acidic conditions.     

Polyamines and abiotic stress tolerance in plants

Environmental stresses including climate change, especially global warming, are severely affecting plant growth and productivity worldwide. It has been estimated that two-thirds of the yield potential of major crops are routinely lost due to the unfavorable environmental factors. On the other hand, the world population is estimated to reach about 10 billion by 2050, which will witness serious food shortages. Therefore, crops with enhanced vigour and high tolerance to various environmental factors should be developed to feed the increasing world population. Maintaining crop yields under adverse environmental stresses is probably the major challenge facing modern agriculture where polyamines can play important role. Polyamines (PAs)(putrescine, spermidine and spermine) are group of phytohormone-like aliphatic amine natural compounds with aliphatic nitrogen structure and present in almost all living organisms including plants. Evidences showed that polyamines are involved in many physiological processes, such as cell growth and development and respond to stress tolerance to various environmental factors. In many cases the relationship of plant stress tolerance was noted with the production of conjugated and bound polyamines as well as stimulation of polyamine oxidation. Therefore, genetic manipulation of crop plants with genes encoding enzymes of polyamine biosynthetic pathways may provide better stress tolerance to crop plants. Furthermore, the exogenous application of PAs is also another option for increasing the stress tolerance potential in plants. Here, we have described the synthesis and role of various polyamines in abiotic stress tolerance in plants.Key words: abiotic stress tolerance, putrescine, spermidine, spermine, polyamines     

A new pathway for the synthesis of oligosaccharides by the use of non-Leloir glycosyltransferases

The growing recognition of the roles of carbohydrates in fundamental biological processes and their potential application as functional foods and new therapeutics have generated a need for larger amounts of different carbohydrate structures. Leloir glycosyltransferases catalyze the synthesis of complex oligosaccharides. However they are difficult or expensive to obtain, and require expensive nucleotide activated sugars. In contrast non-Leloir pathway enzymes use sucrose, which is known to be a high energy donor of d-glucose for glucosyltransferases like dextransucrase, or a donor of d-fructose for fructosyltransferases like inulin- and levansucrases for the synthesis of polysaccharides. Here we present the synthesis and kinetic studies of oligosaccharides using non-Leloir glycosyltransferases and sucrose analogues as new substrates, like β-d-fructofuranosyl-α-d-galactopyranoside (Gal-Fru) by a fructosyltransferase (FTF) from B. subtilis NCIMB 11871. The sucrose analogues carry a high binding energy in the glycosidic bond similar to that of sucrose. Thus, β-d-Fructofuranosyl-α-d-galactopyranoside (Gal-Fru) and β-d-Fructofuranosyl-α-d-fucopyranoside (d-Fuc-Fru) have been shown to be substrates for fructosyltransferases, which produce oligo- or polysaccharides, also in the presence of acceptors.     

Hydroxylamine derivatives for regulation of spermine and spermidine metabolism

The biogenic polyamines spermine, spermidine, and their precursor putrescine are present in micro-to-millimolar concentrations in all cell types and are vitally important for their normal growth. High intracellular content of spermine and spermidine determines the multiplicity of the cellular functions of the polyamines. Many of these functions are not well characterized at the molecular level, ensuring the ongoing development of this field of biochemistry. Tumor cells have elevated polyamine level if compared with normal cells, and this greatly stimulates the search for new opportunities to deplete the intracellular pool of spermine and spermidine resulting in decrease in cell growth and even cell death. O-Substituted hydroxylamines occupy their own place among chemical regulators of the activity of the enzymes of polyamine metabolism. Varying the structure of the alkyl substituent made it possible to obtain within one class of chemical compounds highly effective inhibitors and regulators of the activity of all the enzymes of putrescine, spermine and spermidine metabolism (with the exception of FAD-dependent spermine oxidase and acetylpolyamine oxidase), effectors of the polyamine transport system, and even actively transported in cells “proinhibitor” of ornithine decarboxylase. Some principles for the design of specific inhibitors of these enzymes as well as the peculiarities of cellular effects of corresponding O-substituted hydroxylamines are discussed.     

Effect of Oxidized Polyamines on the Infectivity of φX174 DNA

Oxidized spermine and oxidized spermidine inhibited markedly the infectivity of the 6 m-urea treated φX174 particle, whereas they did not inactivate the infectivity of the untreated phage particle. They also markedly inhibited the infectivity of φX174 DNA, while φX174 RF I DNA was less sensitive to these reagents. These facts suggested that oxidized polyamines could react with phage DNA.

The possible reasons of the insensitivity of phage φX174 particle and less sensitivity of φX174 RF I DNA to these reagents were discussed.     

Studies of non-metabolizable polyamines that support growth of SV-3T3 cells depleted of natural polyamines by exposure to alpha-difluoromethylornithine.

A number of synthetic polyamine derivatives that included five achiral gem-dimethylspermidines and two analogous tetramethylated spermines were tested for their abilities to serve as substrates for enzymes metabolizing polyamines and for their capacities to substitute for the natural polyamines in cell growth. It was found that none of the compounds were effective substrates for spermine synthase, and only one, namely 8,8-dimethylspermidine, was a substrate for spermidine/spermine N1-acetyltransferase. However, all of the spermidine derivatives and 1,1,12,12-tetramethylspermine were able to support the growth of SV-3T3 cells in which endogenous polyamine synthesis was prevented by the addition of alpha-difluoromethylornithine. These results suggest that either spermidine or spermine can support cell growth without the need for metabolic interconversion. In contrast with the result with 1,1,12,12-tetramethylspermine, 3,3,10,10-tetramethylspermine did not restore growth of polyamine-depleted SV-3T3 cells. Comparison of the properties of these derivatives may prove valuable in understanding the physiological role of polyamines.     

Hooked on α-d-galactosidases: from biomedicine to enzymatic synthesis

α-d-Galactosidases (EC 3.2.1.22) are enzymes employed in a number of useful bio-based applications. We have depicted a comprehensive general survey of α-d-galactosidases from different origin with special emphasis on marine example(s). The structures of natural α-galactosyl containing compounds are described. In addition to 3D structures and mechanisms of action of α-d-galactosidases, different sources, natural function and genetic regulation are also covered. Finally, hydrolytic and synthetic exploitations as free or immobilized biocatalysts are reviewed. Interest in the synthetic aspects during the next years is anticipated for access to important small molecules by green technology with an emphasis on alternative selectivity of this class of enzymes from different sources.     

Synthesis of β-D-Fructopyranosyl-(2→6)-D-glucopyranose from D-Glucose and D-Fructose by a Thermal Treatment

The synthesis is reported of β-D-fructopyranosyl-(2→6)-D-glucopyranose that had previously been isolated from a fermented plant extract as a new saccharide. A disaccharide was predominately formed from an equal amount of D-glucose and D-fructose under melting conditions at 140 °C for 60 to 90 min. This saccharide was isolated from the reaction mixture by carbon-Celite column chromatography and preparative HPLC, and was confirmed to be β-D-fructopyranosyl-(2→6)-D-glucopyranose by TOF-MS and NMR analyses.     

Regulatory Properties of Prephenate Dehydrogenase and Prephenate Dehydratase from Corynebacterium glutamicum

Regulatory properties of the enzymes in l-tyrosine and l-phenyalanine terminal pathway in Corynebacterium glutamicum were investigated. Prephenate dehydrogenase was partially feedback inhibited by l-tyrosine. Prephenate dehydratase was strongly inhibited by l-phenylalanine and l-tryptophan and 100% inhibition was attained at the concentrations of 5 × 10^?2mm and 10^?1mm, respectively. l-Tyrosine stimulated prephenate dehydratase activity (6-fold stimulation at 1 mm) and restored the enzyme activity inhibited by l-phenylalanine or l-tryptophan. These regulations seem to give the balanced synthesis of l-tyrosine and l-phenyl-alanine. Prephenate dehydratase from C. glutamicum was stimulated by l-methionine and l-leucine similarly to the enzyme in Bacillus subtilis and moreover by l-isoleucine and l-histidine. C. glutamicum mutant No. 66, an l-phenylalanine producer resistant to p-fluorophenyl-alanine, had a prephenate dehydratase completely resistant to the inhibition by l-phenylalanine and l-tryptophan.     

Phyllostine,a New Phytotoxic Compound Produced by Phyllosticta sp.

Ethionine-resistant mutants derived from Corynebacterium glutamicum KY 9276 (Thr^?) were found to accumulate l-methionine in culture media. One of the mutants, ER-107-4, which produced 250 μg/ml of l-methionine was subjected to further mutagenesis to obtain better l-methionine producers. l-Methionine production increased stepwise by successive endowing such markers as selenomethionine, 1,2,4-triazole, trifluoromethionine and methionine hydroxamate resistance. Thus, a mutant multi-resistant to ethionine, selenomethionine and methionine hydroxamate, ESLMR-724, produced 2 mg/ml of l-methionine in a medium containing 10% glucose.

Increase of l-methionine production was accompanied by increased levels and reduced repressibility of methionine-forming enzymes. The levels of methionine enzymes in ESLMR-724 increased to 2.5~4.2 fold of those in KY9276, In addition, homoserine-O-trans-acetylase and cystathionine γ-synthase which were strongly repressed by l-methionine in KY 9276 were stimulated by exogenous l-methionine in ESLMR-724. Implications of these results were discussed in relation to the productivity of l-methionine and the regulation of l-methionine biosynthesis.     

Polyamines and environmental challenges: recent development

In this review, we will try to summarize some recent data concerning the changes in polyamine metabolism (biosynthesis, catabolism and regulation) in higher plants subjected to a wide array of environmental stress conditions and to describe and discuss some of the new advances concerning the different proposed mechanisms of polyamine action implicated in plant response to environmental challenges. All the data support the view that putrescine and derived polyamines (spermidine, spermine, long-chained polyamides) may have several functions during environmental challenges. In several systems (except during hypoxia, and chilling tolerance of wheat and rice) an induction of polyamines (spermidine, spermine) not putrescine accumulation, may confer a stress tolerance. In several cases stress tolerance is associated with the production of conjugated and bound polyamines and stimulation of polyamine oxidation. In several environmental challenges (osmotic-stress, salinity, hypoxia, environmental pollutants) recent results indicate that both arginine decarboxylase and ornithine decarboxylase are required for the synthesis of putrescine and polyamines (spermidine and spermine). Under osmotic and salt-stresses a production of cadaverine is observed in plants. A new study demonstrates that under salt-stress putrescine catabolism (via diamine oxidase) can contribute to proline (a compatible osmolyte) accumulation.     

Effect of Isopentenyladenine,a Cytokinin,on Proliferation and Protein Synthesis in Cultured Myoblasts

The effects of cytokinins, a class of plant hormones, on cell proliferation and protein synthesis were studied in rat-derived L₆ myoblasts cultured in a serum-free medium. Of the three cytokinins tested, isopentenyladenine, zeatin and ribosylzeatin, isopentenyladenine (5 μm) most stimulated the growth and DNA synthesis of the myoblasts, and it dose-dependently (0 ~ 10μm) enhanced the proliferation and DNA synthesis of the cells. Isopentenyladenine (5 and 10 μm) increased protein synthesis to twice that of control (0μm). These results suggest that isopentenyladenine, a trace component in plant food and a plant hormone, can affect the metabolism of an animal cell line of myoblasts.     

Spermidine level and protein synthesis are coregulated in nonproliferating hepatocytes

The relationship between polyamines and the rate of protein synthesis was investigated in non-proliferating cells: primary cultures of adult rat hepatocytes maintained in serum-free media, and treated with dexamethasone or dexamethasone + insulin. During the second day of culture, polyamine biosynthesis became induced along with the rate of protein synthesis. While the activity of ornithine decarboxylase and the intracellular concentration of putrescine increased only transiently and that of spermine declined, the rise of the protein synthetic rate was paralleled by that of the intracellular spermidine concentration. The polyamine analogue diamino-propanol specifically decreased spermidine content and the protein synthetic rate. The intracellular concentration of spermidine was found subject to tight homeostatic regulation, e.g. not being altered by the addition of up to 1 mM of this polyamine to the culture medium. In contrast, addition of putrescine or spermine led to an increase in their respective intracellular concentrations. These findings indicate that spermidine specifically of the polyamines is involved in protein synthesis in the intact hepatocyte. Moreover, spermidine may mediate part of the trophic action of dexamethasone and insulin upon cultured hepatocytes.