The formation of 1-dimethylaminonaphthalene-5-sulphonamide during the preparation of 1-dimethylaminonaphthalene-5-sulphonylamino acids

1. The amount of 1-dimethylaminonaphthalene-5-sulphonamide formed during the reaction of an amino acid with 1-dimethylaminonaphthalene-5-sulphonyl chloride depends on the structure of the amino acid and on the conditions used. 2. The reaction probably involves attack of a further molecule of 1-dimethylaminonaphthalene-5-sulphonyl chloride on the 1-dimethylaminonaphthalene-5-sulphonyl-amino acid and also gives the aldehyde (or ketone) with one carbon atom less than the parent amino acid.     

Identification of Functional Amino Acid Residues Involved in Polyamine and Agmatine Transport by Human Organic Cation Transporter 2

Polyamine (putrescine, spermidine and spermine) and agmatine uptake by the human organic cation transporter 2 (hOCT2) was studied using HEK293 cells transfected with pCMV6-XL4/hOCT2. The Km values for putrescine and spermidine were 7.50 and 6.76 mM, and the Vmax values were 4.71 and 2.34 nmol/min/mg protein, respectively. Spermine uptake by hOCT2 was not observed at pH 7.4, although it inhibited both putrescine and spermidine uptake. Agmatine was also taken up by hOCT2, with Km value: 3.27 mM and a Vmax value of 3.14 nmol/min/mg protein. Amino acid residues involved in putrescine, agmatine and spermidine uptake by hOCT2 were Asp⁴²⁷, Glu⁴⁴⁸, Glu⁴⁵⁶, Asp⁴⁷⁵, and Glu⁵¹⁶. In addition, Glu⁵²⁴ and Glu⁵³⁰ were involved in putrescine and spermidine uptake activity, and Glu⁵²⁸ and Glu⁵⁴⁰ were weakly involved in putrescine uptake activity. Furthermore, Asp⁵⁵¹ was also involved in the recognition of spermidine. These results indicate that the recognition sites for putrescine, agmatine and spermidine on hOCT2 strongly overlap, consistent with the observation that the three amines are transported with similar affinity and velocity. A model of spermidine binding to hOCT2 was constructed based on the functional amino acid residues.     

Thin-layer chromatography of 1-dimethylaminonaphthalene-5-sulphonyl derivatives of amino acids present in superfutases of cat cerebral cortex

1. Five new solvent systems are reported for the separation of 1-dimethylaminonaphthalene-5-sulphonylamino acids by thin-layer chromatography on silica gel. After two-dimensional chromatography with a suitable pair of these solvent systems, most of the 1-dimethylaminonaphthalene-5-sulphonyl derivatives were completely separated and could be located by their intense yellow fluorescence when viewed under u.v. light. 2. These techniques have been used to identify 21 amino acids present in superfusates of cat cerebral cortex, plasma and cerebrospinal fluid. 3. A method for the semiquantitative estimation of amino acids in biological fluids is described in which the fluorescent intensity of their separated 1-dimethylaminonaphthalene-5-sulphonyl derivatives was measured.     

Regulation of growth and macromolecular synthesis by putrescine and spermidine in Pseudomonas aeruginosa

Growth of P. aeruginosa, slowed by the addition of monofluoromethylornithine, difluoromethylarginine and dicyclohexylammonium sulfate, could be restored by addition of 0.1 mM putrescine plus 0.1 muM spermidine, or 0.1 mM spermidine or 5 mM putrescine by themselves. Lower concentrations of putrescine (0.1 mM - 1 mM) also partially reversed the growth inhibition. Conversion of putrescine to spermidine continued, although at a markedly reduced ratio, in the drug-inhibited cells, but intracellular spermidine concentrations remained depressed suggesting that reversal of inhibition by putrescine may be a direct effect. There was appreciable back-conversion of any added spermidine to putrescine with a demonstrable increase in total intracellular putrescine levels, making conclusions on the effects of spermidine ambiguous. Spermine (0.1 mM), a polyamine not present in bacteria, was also effective in reversing growth inhibition, probably because of its conversion into spermidine and putrescine. The effects of putrescine, spermidine and spermine were specific in that the non-physiological amines, 1,3-diaminopropane, 1,5-diaminopentane (cadaverine), 1,6-diaminohexane, or 1,7-diaminoheptane could not reverse the effects of the three drugs. Rates of total protein, RNA and DNA synthesis were all slowed to the same extent as growth rate and showed similar recovery with the addition of putrescine or spermidine. A role for putrescine in P. aeruginosa growth processes is suggested.     

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.     

The biochemistry, genetics, and regulation of polyamine biosynthesis in Saccharomyces cerevisiae

We have studied the enzymes and genes involved in the biosynthesis of putrescine, spermidine, and spermine in Saccharomyces cerevisiae. Mutants have been isolated with defects in the biosynthetic pathway as follows: spe10 mutants, deficient in ornithine decarboxylase, cannot make putrescine, spermidine, or spermine; spe2 mutants, lacking S-adenosylmethionine decarboxylase, cannot make spermidine or spermine; spe3 mutants, lacking putrescine aminopropyltransferase, cannot make spermidine or spermine; and spe4 and spe40 mutants, lacking spermidine aminopropyltransferase, contain no spermine and permit growth of spe10 mutants. Studies with these mutants have shown that in yeast: 1) polyamines are absolutely required for growth; 2) putrescine is formed only by decarboxylation or ornithine; 3) two separate aminopropyltransferases are required for spermidine and spermine synthesis; 4) spermine and spermidine are important in the regulation of ornithine decarboxylase and the amines exert this control by a posttranslational modification of the enzyme; and 5) spermidine or spermine is essential for sporulation of yeast and for the maintenance of the double-stranded RNA killer plasmid. Recent studies in amine-deficient mutants of Escherichia coli have shown an important role of the polyamines in protein synthesis in vivo.     

Fluorescence technique for comparative studies of substrate-binding subsites in serine proteinases. Application to subtilisins.

A fluorescence technique for comparative studies of substrate-binding subsites in serine proteinases is described. It consists of: selective labelling of the corresponding subsites with a fluorescent group by using N alpha-dansyl(5-dimethylaminonaphthalene-1-sulphonyl)ated peptide chloromethanes containing different numbers of amino acid residues, and probing the immediate environment of the subsites by quenching experiments using ionic and neutral quenchers. Intramolecular distances between the subsites and particular chromophores can be also determined. The technique is of general applicability to all serine proteinases. The above mentioned approach was applied to two proteinases: subtilisin Novo and mesentericopeptidase. It was concluded that the substrate-binding site of mesentericopeptidase is considerably more polar than that of subtilisin Novo. Intramolecular distances between the labelled subsites and tryptophan residues in the two proteinases were determined.     

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.     

Excretion of putrescine and spermidine by the protein encoded by YKL174c (TPO5) in Saccharomyces cerevisiae

The properties of the protein encoded by YKL174c (TPO5) were studied. It was found that TPO5 excretes putrescine effectively and spermidine less effectively. Gamma-aminobutyric acid slightly inhibited the excretion of putrescine, but basic amino acids did not affect excretion, suggesting that TPO5 preferentially recognizes polyamines. Accordingly, yeast cells transformed with the plasmid encoding YKL174c (TPO5) were resistant to toxicity caused by 120 mm putrescine or by 3 mm spermidine, and a mutant with a disrupted YKL174c (TPO5) gene was sensitive to toxicity by 90 mm putrescine. The growth of this mutant was faster than that of the wild-type strain. In parallel, there was an increase in putrescine and spermidine content of the YKL174c (TPO5) mutant compared with wild-type. It is noted that TPO5 functions as a suppressor of cell growth by excreting polyamines. The level of YKL174c (TPO5) mRNA was increased by the addition of polyamines to the medium. The degree of induction of the mRNA was spermine > spermidine > putrescine. The subcellular localization of TPO5 was determined by immunostaining of hemagglutinin-tagged TPO5, and it was found on Golgi or post-Golgi secretory vesicles. Excretion of putrescine and spermidine by TPO5 was reduced in cells that have mutations in the secretory or endocytic pathways, indicating that both processes are involved in the excretion of polyamines.     

Synthesis and content of polyamines in bloodstream Trypanosma brucei

The sensitive dansyl procedure was used to detect putrescine and spermidine, but not spermine and cadaverine, in pleomorphic Trypanosoma brucei. The polyamines were synthesized in vitro from [3H]ornithine, [14C]arginine and [14C]methionine. Proline, agmatine, and citrulline, but not glutamine, glutamic or pyroglutamic acids, stimulated spermidine formation from [4C]methionine. Putrescine and sperimidine synthesis occurred rapidly from ornithine: putrescine synthesis peaked in 0.5 h, spermidine in 1 h. Trypanosoma brucei assimilated exogenous 14C-labeled putrescine, spermidine, and spermine; spermidine and spermine were taken up 5 times as rapidly as putrescine. Polyamine syntheses may therefore be a practical target for novel trypanocies.     

Uptake of putrescine and spermidine by Gap1p on the plasma membrane in Saccharomyces cerevisiae

It has been reported that Gap1p on the plasma membrane of Saccharomyces cerevisiae can catalyze the uptake of many kinds of amino acids. In the present study, we found that Gap1p also catalyzed the uptake of putrescine and spermidine, but not spermine. The Km and Vmax values for putrescine and spermidine were 390 and 21 microM, and 4.6 and 0.59 nmol/min/mg protein, respectively. The uptake of putrescine was strongly inhibited by basic amino acids, lysine, arginine, and histidine, whose Ki values were 25-35 microM. Thus, it is deduced that spermidine and basic amino acids have almost the same affinity for Gap1p. When the concentrations of amino acids in the medium were reduced to one-third and 0.5 mM putrescine or 0.1 mM spermidine was added to the medium, accumulation of putrescine or spermidine by Gap1p was observed. Furthermore, when yeast was transformed with the GAP1 gene and cultured in the presence of 60 mM putrescine, cell growth was inhibited through overaccumulation of putrescine. GAP1 mRNA was found to be induced by polyamines. This is the first report of the identification, at a molecular level, of a polyamine uptake protein on the plasma membrane in eukaryotes.     

Association of putrescine,spermidine, spermine,and GABA with structural elements of brain cells

The present experiments are the first survey of the association of endogenous and exogenous putrescine, spermidine, and spermine with subcellular structures of rat brain cortex. The differences of distribution in subfractions obtained from salt-free and salt-containing density gradients were studied, with the following results: (1) In contrast with liver preparation, putrescine and the polyamines spermidine and spermine are not distributed in parallel with RNA. (2) In salt-containing media, putrescine and the polyamines were preferentially associated with synaptosomes and with synaptosomal membranes. Significant association with myelin constituents was observed only in salt-free media. (3) Exogenous putrescine and the polyamines were less firmly attached to synaptosomes and to synaptosomal membrane fractions than the endogenous amines. There is good evidence for similar subcellular localizations of putrescine and GABA. Putrescine seems to be entrapped in the nerve endings. (4) Uptake studies with crude mitochondria under conditions of high-affinity uptake showed no temperature-sensitive component of polyamine accumulation in synaptosomes, in contrast with GABA, monoacetylputrescine, and ornithine. (5) Polyamines bound to myelin constituents or mitochondria could be displaced by a 200-fold concentration of nonradioactive amines; this was not the case with polyamines bound to synaptosomes. Mg²⁺ did not effectively compete with spermine for binding sites at synaptic regions. (6) Electrical stimulation and stimulation by mono- and bivalent cations did not change the concentrations of the polyamines and GABA in guinea pig cortex. (7) There is no evidence for a neurotransmitter role of putrescine, spermidine, or spermine, although these compounds might function as modulators of neurotransmission.     

Intramolecular distances between tryptophan residues and the active-site serine residue in alkaline bacterial proteinases as measured by fluorescence energy-transfer studies.

Singlet-singlet energy transfer from the tryptophan residues to an active-site-serine-bound 5-dimethylaminonaphthalene-1-sulphonyl group was investigated in four subtilisins. The transfer distances for subtilisin Novo and mesentericopeptidase are 1.93 +/- 0.20 nm (19.3 +/- 2.0 A) and 1.81 +/- 0.20 nm (18.1 +/- 2.0 A) respectively. The positions of the indole groups in the three-dimensional structures of the two pairs of proteinases, namely subtilisin Novo and mesentericopeptidase on the one hand and subtilisins Carlsberg and DY on the other, are essentially identical.     

Positions in human serum albumin which involve the indole binding site. Sequence of 107-residue fragment.

The first 107 residues of Fragment C of human serum albumin have been sequenced and two positions at which affinity labels block the indole site determined. Histidine 23 is the position of blockage by bromoacetyl-L-tryptophan and lysine 67 is the position of blockage by 5-dimethylaminonaphthalene-1-sulfonyl chloride and probably pyridoxal-5'-phosphate. The presence of an indole ligand at the binding site markedly reduces incorporation of the label into the above lysyl residue, and in the case of 5-dimethylaminonaphthalene-1-sulfonyl chloride, increases incorporation into three other positions, lysine residues 13, 39, and 84. It is concluded that binding of the indole ligand on the site brings about conformational changes in the albumin structure exposing new reactive positions for 5-dimethylaminonaphthalene-1-sulfonyl chloride. There is a large accumulation of basic and hydrophobic residues and no glycine, serine, threonine, valine, aspartate, or cysteine residues in the sequence 10 to 43. Lysine 71 has been identified by amino acid analyses and sequence studies as the position acetylated by acetylsalicylic acid (Hawkins, D. R., Pinckard, N., Crawford, C. P., and Farr, R. S. J. Clin. Invest. (1969) 48, 536), establishing the structural relationships of two major ligand binding sites on albumin. The lone tryptophan is at position 86. Evidence indicates that within residues 1 to 86 of Fragment C and within residues of the A-Phe fragment (Mr equals approximately 10,000), the latter known to be adjacent to Fragment C in the whole albumin structure, exists the major binding sites of all ligands for human serum albumin.     

Polyamines,floral induction and floral development of strawberry (Fragaria ananassa Duch.)

In the short-day plant, strawberry (Fragaria ananassa Duch.), polyamines (putrescine, spermidine and spermine), conjugated spermidine (water-insoluble compounds) and bound amines (putrescine, spermidine, phenylethylamine, 3-hydroxy, 4-methoxyphenylethylamine) accumulated in the shoot tips during floral induction and before floral emergence. Different associations of free amines and conjugated amines were observed during floral induction, as compared with the reproductive phase. During the whole period of floral development, phenylethylamine (an aromatic amine) was the predominant amine, representing 80 to 90% of the total free amine pool. Phenylethylamine conjugates (water-insoluble compounds) were the predominant amides observed prior to fertilization. These substances decreased drastically after fertilization. In vegetative shoot tips from plants grown continously under long days, free polyamines (putrescine, spermidine) and bound polyamines (putrescine, spermidine) were low and no change was observed. Free amines (spermine and phenylethylamine), bound aromatic amines (phenylethylamine, 3-hydroxy, 4-methoxyphenylethylamine), conjugated spermidine and phenylethylamine did not appear. Male-sterile flowers were distinguished by their lack of conjugated spermidine and phenylethyalamine and by a decrease in free phenylethylamine. In normal and sterile strawberry plants -DL-difluoromethylornithine (DFMO), a specific irreversible inhibitor of ornithine decarboxylase (ODC), caused inhibition of flowering and free and polyamine conjugates. When putrescine was added, polyamine titers and flowering were restored. A similar treatment with -DL-difluoromethylarginine (DFMA), a specific, irreversible inhibitor of arginine decarboxylase (ADC), did not affect flowering and polyamine titers. These results suggest that ornithine decarboxylase (ODC) and polyamines are involved in regulating floral initiation in strawberry. The relationship between polyamines, aromatic amines, conjugates, floral initiation and male sterility is discussed.Abbreviations ADC arginine decarboxylase - ODC ornithine decarboxylase - DFMA -DL-difluoromethylarginine - DFMO -DL-difluoromethylornithine - Put putrescine - Spd spermidine - Spm spermine - Phen phenylethylamine - 3H4M Phen 3-hydroxy, 4-methoxyphenylethylamine     

The occurrence, subcellular localization and partial purification of diamine acetyltransferase in the yeast Candida boidinii grown on spermidine or putrescine as sole nitrogen source

The yeast Candida boidinii when grown on spermidine, diaminopropane, putrescine or cadaverine as sole nitrogen source contains an N-acetyltransferase capable of acetylating the primary amino groups of spermine, spermidine, acetylspermidines, acetylputrescine and alpha, omega-diaminoalkanes. In the case of spermidine, the products were N1-acetylspermidine and N8-acetylspermidine in the ratio 50:45 with traces of other unidentified products. The enzyme was partially purified and the stoichiometry determined, together with apparent Km and V values for a number of substrates. The pH optimum was about 8.8 for putrescine and 9.3 for spermidine. The unstable enzyme was partially stabilized by 10% (v/v) glycerol or bovine serum albumin (5 mg/ml). The kinetic parameters were determined with putrescine as substrate and the mechanism shown to be of the sequential type. The enzyme was shown to be located in the mitochondria of C. boidinii, in contrast to mammalian N-acetyltransferases. The enzyme was found in a number of other yeast species when grown on spermidine or putrescine, but was only present in those species that had previously been found to contain polyamine oxidase. It is suggested that in C. boidinii, as in mammals, acetylation of spermidine and putrescine must precede their catabolism.     

Evidence for the existence of distinct transporters for the polyamines putrescine and spermidine in B16 melanoma cells

The uptake of intracellular putrescine and spermidine was examined in B16 melanoma cells. It was found that difluoromethylornithine preferentially induced putrescine transport (28-fold) compared to that for spermidine (3.5-fold). Putrescine uptake was partially Na+ dependent, whereas spermidine uptake was not. Inhibition studies with the two polyamines showed that putrescine was a poor competitive inhibitor of spermidine uptake, exhibiting a Ki of 69-75 microM, whereas the estimated Km for putrescine uptake was only 5.36 microM. By contrast, spermidine inhibition of putrescine transport produced a non-linear Eadie-Scatchard plot suggesting that putrescine was taken up by a spermidine-sensitive and a spermidine-insensitive process. The estimated spermidine Ki for inhibition of the spermidine-sensitive process was 0.125 microM. Using a series of polypyridinium quaternary salts to inhibit transport, no correlation between inhibition of putrescine uptake and inhibition of spermidine uptake was seen. Finally, the photoaffinity label, 1,12-di(N5-azido-2-nitrobenzoyl)spermine selectively inactivated the putrescine transporter(s) without affecting spermidine uptake. From these observations, it was concluded that multiple polyamine transporters are present on B16 melanoma cells and that separate, distinct transporter(s) account for the uptake of putrescine and spermidine in this cell-line following induction with difluoromethylornithine. The present of different transporters for the two polyamines indicates that expression of uptake activity for putrescine and spermidine may be under separate cellular control.     

Effect of Polyamines on Chlorophyll and Protein Contents, Photochemical Activity, and Energy Transfer in Detached Wheat Leaves during Dark Incubation

Spermine as compared to putrescine or spermidine retarded the loss of chlorophyll and protein contents to a greater extent in wheat primary leaves during dark incubation. Activities of whole chain electron transport, photosystem (PS) 1 and PS2, and absorbed excitation energy distribution in favour of PS 1 were protected by these amines in valency dependent manner during 72-h dark incubation.     

Chemical, photochemical and spectroscopic characterization of an alkaline proteinase from Bacillus subtilis variant DY

Circular-dichroism and fluorescence studies indicate that the 5-dimethylaminonaphthalene-1-sulphonyl and phenylmethanesulphonyl derivatives of subtilisin DY have three-dimensional structure closely similar to that of native enzyme. The single tryptophan residue is largely accessible to the aqueous solvent, and is not directly involved in the enzyme-substrate interactions, since its photochemical modification causes only a partial inhibition of the enzyme activity. It appears very likely that the location of the single tryptophan residue in the three-dimensional structure of subtilisin DY is similar to that of the single tryptophan residue in subtilisin Carlsberg. Fluorescence-quenching experiments further indicate that the 14 tyrosine residues are also largely accessible to the aqueous solvent, and probably interact with hydrated peptide carbonyl groups. The charge environment for tryptophan and tyrosine residues in subtilisin DY, as deduced by quenching experiments with ionic species, is also discussed. In general, subtilisin DY displays strong similarities to subtilisin Carlsberg, as suggested by a comparative analysis of the amino acid composition and fluorescence properties.