Effects of aliphatic diamines on rat liver ornithine decarboxylase activity.

Rat liver ornithine decarboxylase activity was decreased by administration of putrescine (1,4-diaminobutane) or other diamines, including 1,3-diaminopropane, 1,5-diaminopentane and 1,6-diaminohexane. This effect was seen in control rats and in rats in which hepatic ornithine decarboxylase activity had been increased by administration of growth hormone (somatotropin) or thioacetamide. Loss of activity was not dependent on the conversion of putrescine into polyamines and was short-lived. Within 6h after intraperitoneal administration of 0.8 mmol/kg body wt., ornithine decarboxylase activity had returned to normal values. This return correlated with the rapid loss of the diamines from the liver, and the decrease in activity could be slightly prolonged by treatment with aminoguanidine, a diamine oxidase inhibitor. A decrease in ornithine decarboxylase activity by these diamines was accompanied by the accumulation in the liver of a nondiffusible inhibitor that decreased the activity of a purified ornithine decarboxylase preparation. The possibility that administration of non-physiological diamines that are not converted into polyamines might be useful for the inhibition of polyamine synthesis is discussed.     

A simple enzymatic differential assay for diamines, spermidine, and spermine in urine and blood

This rapid, reliable enzymatic differential assay method for diamines (putrescine plus cadaverine), spermidine, and spermine in urine and blood is suitable for practical routine use and does not require special and expensive equipment. The method is based on a combination of the substrate specificities of two amine oxidases, i.e., polyamine oxidase from A. terreus and putrescine oxidase from M. rubens. Quinone dye, derived from hydrogen peroxide generated in each of three end-point reactions, is measured spectrophotometrically at 555 nm, and the amounts of the respective amines are simply calculated. Analytical recoveries and precisions are acceptable. The proposed method produces results which correlate with high-performance liquid chromatography. Twenty or more assays can be done within 3 hr by one technician.     

Determination of 5-dimethylaminonaphthalene-1-sulfonyl derivatives of urinary polyamines by ion-pair high-performance liquid chromatography

A sensitive and specific method for the determination of diamines and polyamines by ion-pair high-performance liquid chromatography is described. The 5-dimethylaminonaphthalene-1-sulfonyl derivatives of putrescine, 1,6-diaminohexane, spermidine and spermine are separated on a μBondapak C₁₅ reversed-phase column with 1-heptanesulfonic acid and acetonitrile as the mobile phase. All compounds are eluted within 30 min using a programmed solvent gradient system. The method has a lower detection limit of 1 pmole on column.Because of the simplicity of the method, its application provides a better means for closely monitoring patients undergoing treatment for various types of genito-urinary neoplastic diseases.     

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.     

A new method for isolation of polyamines from animal tissues

A new method for isolation of polyamines from tissues was developed and compared with the butanol extraction method which has been widely used for quantitative determination of polyamines. In the new method protein-free tissue extracts are applied to a small Dowex-50 column. The column is washed with appropriate buffers to remove ninhydrin-positive contaminants and the polyamines are eluted. With this method, the overall recovery of polyamines, after separation by paper electrophoresis and subsequent colorimetric determination with ninhydrin, is always over 90% (average 95%). This method is much better than the butanol method, which gives variable recoveries of 70–90%. The new method also has the advantages over the butanol method that the isolated polyamines are purer and the procedure is simpler.     

Fluorometric assay for polyamines in urine and tissues using electrophoresis on Titan III cellulose acetate

A simple, rapid assay method for polyamines (putrescine, spermidine, and spermine) in urine and tissues using electrophoresis on Titan III cellulose acetate was developed. In this procedure, polyamines are preliminarily extracted from a hydrolysate of urine or from supernatants of tissue homogenates by use of a Bio-Rex 70 minicolumn. After electrophoretic separation, polyamines are fluorometrically detected by the reaction with o-phthalaldehyde and 2-mercaptoethanol. Six extracts and two external standards of polyamines can be separated and detected in 11 min on a cellulose acetate strip. This method permits the determination of polyamines in a range of 0.1 mM (25 pmol) to 1.0 mM (250 pmol).     

Construction of transgenic pea lines with modified expression of diamine oxidase and modified nodulation responses with exogenous putrescine

Diamine oxidase (DAO) might influence pea nodule development either by regulating the peroxide-driven cross-linking of glycoproteins in the infection thread matrix or by modifying the metabolism of diamines and polyamines in host cells. Transformed lines of pea (Pisum sativum) with the coding sequence for DAO (PSAO-1) in sense orientation behind a tissue-specific promoter (pENOD12A) showed strong co-suppression of DAO activity in extracts from nodules and epicotyls, whereas the antisense constructs were relatively unaffected. No difference in nodule number was observed between transformed lines and controls, suggesting that DAO does not normally have an essential role in nodule initiation. However, lines showing co-suppression of DAO were less sensitive to the inhibitory effects of exogenous putrescine and less active in the cross-linking of matrix glycoprotein, indicating that putrescine-derived products of DAO activity could retard nodule development. Inoculation of co-suppressed lines with Rhizobium strain B661 (a lipopolysaccharide-defective mutant) resulted in more extreme impairment of nodule development and nitrogen fixation capacity, relative to lines with normal levels of DAO, which suggests that DAO may serve to reduce the endogenous level of inhibitory diamines or polyamines in nodules under physiological stress. We conclude that the most critical role of DAO in pea nodule development is apparently in the regulation of diamine levels in host tissues.     

Inhibition of plant amine oxidases by a novel series of diamine derivatives

A series of N,N'-bis(2-pyridinylmethyl)diamines was synthesized and characterized for their inhibition effects towards plant copper-containing amine oxidase (EC 1.4.3.6) and polyamine oxidase (EC 1.5.3.11), which mediate the catabolic regulation of cellular polyamines. Even though these enzymes catalyze related reactions and, among others, act upon two common substrates (spermidine and spermine), their molecular and kinetic properties are different. They also show a different spectrum of inhibitors. It is therefore of interest to look for compounds providing a dual inhibition (i.e. inhibiting both enzymes with the same inhibition potency), which would be useful in physiological studies involving modulations of polyamine catabolism. The synthesized diamine derivatives comprised from two to eight carbon atoms in the alkyl spacer chain. Kinetic measurements with pea (Pisum sativum) diamine oxidase and oat (Avena sativa) polyamine oxidase demonstrated reversible binding of the compounds at the active sites of the enzymes as they were almost exclusively competitive inhibitors with K(i) values ranging from 10(-5) to 10(-3)M. In case of oat polyamine oxidase, the K(i) values were significantly influenced by the number of methylene groups in the inhibitor molecule. The measured inhibition data are discussed with respect to enzyme structure. For that reason, the oat enzyme was analyzed by de novo peptide sequencing using mass spectrometry and shown to be homologous to polyamine oxidases from barley (isoform 1) and maize. We conclude that some of the studied N,N'-bis(2-pyridinylmethyl)diamines might have a potential to be starting structures in design of metabolic modulators targeted to both types of amine oxidases.     

The interactions between nucleic acids and polyamines. II. Protonation constants and 13C-NMR chemical shift assignments of spermidine, spermine, and homologs

Macroscopic protonation constants have been determined by pulentiometric titration for spermidine, spermine and for the four pulyamines. 3,5-Spd, 4,4-Spd, 4,3,4-Spm and 4,4,4-Spm. which are homologs of spermidine and spermine. A method for calculation of microscopic protonation constants of polyamines based on data for mono- and diamines gives results for spermidine that agree well with the experimental macroscopic protonation constants and the protonation sequence of Kimberly and Goldstein. ¹³C-NMR spectra of spermidine, spermine and six homologs have been obtained and used to assign specific resonances, correcting some ambiguity in the assignments for spermidine and some errors in the assignments for spermine.     

Reversed-phase ion-pair liquid chromatographic procedure for the simultaneous analysis of S-adenosylmethionine, its metabolites and the natural polyamines

A method using reversed-phase ion-pair liquid chromatography with dual detection was developed for the simultaneous determination of the S-adenosylmethionine (SAM) analogues and the natural polyamines. The separation is obtained with a gradient elution and by adjusting the concentration of octanesulfonic acid used as ion-pairing agent, the ionic strength of the eluent, the pH and the acetonitrile content of the eluents. The SAM analogues are analyzed by UV detection at 254 nm and the polyamines by fluorescence detection after post-column derivatization with o-phthalaldehyde. The method allows the determination of the SAM analogues and the polyamines in one single run by direct injection of tissue extracts. The procedure is applied to the study in rats and in hepatoma tissue culture cells of the biochemical effects of α-difluoromethylornithine, a potent enzyme-activated irreversible inhibitor of ornithine decarboxylase.     

Micro determination of polyamines with snake venom oxidase.

An accurate micromethod suitable for the assay of polyamines in concentrations of 2 to 30 nmol is described. It is based on the oxidation of polyamines by purified fractions of crude l-amino acid oxidase from Russell's viper venom. By a combination of two enzyme fractions, one which oxidizes polyamines and amino acids (AAP) and another which oxidizes only amino acids (AA), the technique can accurately determine polyamine concentrations in extracts of sera which may not be free of amino acids. Experiments described show 90 to 100% recovery of added polyamines in the presence of varying amounts of amino acids. Polyamines added to serum also showed recoveries ranging from 96 to 98%. The enzymes do not oxidize histamine and epinephrine and are very stable. The method does not require sophisticated equipment and is suitable for screening of large number of clinical samples to assess the importance of polyamines as a diagnostic test or their prognostic value in diseases like cancer.     

Microdetermination of d-amino acids and d-amino acid oxidase activity with 3-methyl-2-benzothiazolone hydrazone hydrochloride

A simple and rapid technique for the determination of the d-amino acids which are oxidized by d-amino acid oxidase has been presented. This method involves an oxidation of d-amino acids with d-amino acid oxidase in the presence of catalase, and the spectrophotometric determination of the resultant α-keto acids with MBTH. The additions of l-amino acids have no influence on the quantitative estimation of d-amino acids. The method is suitable for the assay of d-amino acids in the presence of the l isomers, and is also applicable for the determination of d-amino acid oxidase activity.     

Simultaneous liquid chromatography determination of polyamines and arylalkyl monoamines

The diamines putrescine (PUT) and diaminopropane (DAP), the polyamines spermidine (SPD) and spermine (SPM), and the arylalkyl amines phenethylamine (PEA), tyramine (TYR), dopamine (DA), and salsolinol (SAL) were dansylated and baseline separated by LC using a Waters ODS-2 column. The dansyl derivatives were detected by fluorescence (lambda(ex): 337 nm; lambda(em): 520 nm). Besides the amine function, the phenolic OH groups of TYR, DA, and SAL were also dansylated (LC-MS, formation of N,O-didansyl [TYR] and N,O,O'-tridansyl derivatives [DA and SAL]). Calibration curves revealed response factors being appreciably lower for (N,O-didansyl) aminophenol TYR and (N,O,O'-tridansyl) DA and SAL than for N-dansylamines. However, the method is suitable as a cheap alternative to LC-MS for the simultaneous determination of polyamines and arylalkyl amines of large quantities of samples.     

Novel copper amine oxidase activity from rat liver mitochondria matrix

Copper containing amine oxidases (Cu-AO) represent a heterogeneous class of enzymes classified as EC 1.4.3.6. The present study reports preliminary results on the presence of a novel amine oxidase activity in rat liver mitochondria lysates. Such enzymatic activity was found in the soluble mitochondrial fraction, obtained by simple osmotic shock. The mitochondrial amine oxidase was isolated by affinity chromatography on a newly synthesised spermine-Sepharose. SDS-PAGE showed a single band at about 60 kDa. Upon chromatographic purification, the enzymatic activity was very labile. The crude enzyme activity was tested by spectrophotometric measurements, determining hydrogen peroxide production following oxidative deamination of different substrates, such as polyamines (spermine, spermidine, putrescine and cadaverine) and monoamines (dopamine and benzylamine). The activity, observed on polyamines and not on monoamines, was inhibited by semicarbazide and azide, but not by pargyline, clorgyline and l-deprenil. Enzyme specificity was tested on several diamines characterized by different carbon atom chain length in the range 2-6 carbon atoms. The highest activity was found with 1,2-diamino-ethane and the highest affinity with 1,5-diamino-pentane. The above reported results suggest the presence of a novel copper-dependent amine oxidase in liver mitochondria matrix.     

A rapid high-performance liquid chromatographic procedure for the simultaneous determination of methionine, ethionine, S-adenosylmethionine, S-adenosylethionine, and the natural polyamines in rat tissues

A method for the analysis of S-adenosyl-L-methionine (SAM) and S-adenosyl-L-ethionine (SAE) and their major metabolites by high-performance liquid chromatography is described. The procedure allows the simultaneous analysis of the natural polyamines, putrescine, spermidine, and spermine, and some of the major amino acids, methionine, tyrosine, and tryptophan. The uv absorbance at 254 nm is used for the determination of the SAM and SAE analogs, whereas the polyamines and amino acids are analyzed by fluorescence detection after postcolumn derivatization with o-phthalaldehyde. The method allows SAM and polyamine determinations by direct injection of the tissue extracts without prepurification. The procedure is applied to study the effects of DL-ethionine treatment on the SAM, SAE, methionine, and polyamine levels in various tissues of rats.     

A Photometric Assay for Ethanol

A novel enzymatic photometric assay for ethanol determination using alcohol oxidase and peroxidase is described. The sensitivity of the method allows detecting ethanol in biological fluids (saliva and blood serum). Secondary alcohols and other organic compounds do not interfere with the assay. General-purpose spectrophotometers and photoelectric colorimeters can be used in the measurements. Methanol and propanol can also be determined by this technique.     

Photometric method for determining ethanol

A novel enzymatic photometric assay for ethanol determination using alcohol oxidase and peroxidase is described. The sensitivity of the method allows detecting ethanol in biological fluids (saliva and blood serum). Secondary alcohols and other organic compounds do not interfere with the assay. General-purpose spectrophotometers and photoelectric colorimeters can be used in the measurements. Methanol and propanol can also be determined by this technique.     

An NADH coupled assay system for galactose oxidase.

A galactose oxidase (EC 1.1.3.9); NADH-peroxidase (EC 1.11.1.1) coupled assay system is used for the estimation of galactose oxidase activity. Spectrophotometric measurement of NADH consumption yields direct quantitative value of enzymic activity or can be used for the end-point determination of the amount of galactose oxidase substrate present in test solutions. Use of similar coupled systems is suggested for the assay of other H₂O₂-producing enzymes and their substrates.     

Determination of the naturally occurring monoacetyl derivatives of di- and polyamines

A method is described for the determination of pmol quantities of monoacetylputrescine, N¹-acetylspermidine, N⁸-acetylspermidine and related compounds. The method is based on the derivatization of these compounds with 5-dimethylaminonaphthalene-1-sulphonyl-chloride, followed by thin-layer chromatographic separation. Cleanup steps allow the application of the method to urine analyses. From the repeated determination of acetylated polyamines in the urine of healthy individuals it can be concluded that these conjugates are the major excretory form of di- and polyamines.The cleanup steps used in this procedure and the method described for the stabilization of 5-dimethylaminonaphthalene-1-sulphonyl derivatives on thin-layer plates are advantageous also for the analyses of total polyamines in urine hydrolysates, and in related applications of the dansylation method.     

Retarded growth of an Escherichia coli mutant deficient in spermidine synthase can be unspecifically repaired by addition of various polyamines

The Escherichia coli mutant speE deficient in the gene encoding for spermidine synthase has no absolute requirement for spermidine but shows a retarded growth rate. This growth retardation could be unspecifically restored to the respective wild type level by exogenously supplied polyamines such as spermidine, spermine and homospermidine as well as the diamines putrescine and cadaverine. In comparison to the respective wild type, the mutant shows a two-fold increased level of endogenous putrescine but displays a reduced ability to accumulate the diamines putrescine and cadaverine. The ability to accumulate polyamines is not affected. The deleted spermidine synthase gene of the mutant was substituted by heterologous expression of the hss gene from Rhodopseudomonas viridis encoding homospermidine synthase.