Alternate putrescine metabolites: quantitative analysis of delta'-pyrroline oxidation to 2-pyrrolidone in tissue homogenates by high-pressure liquid chromatography.

A sensitive analytical procedure for following the oxidation of delta'-pyrroline to 2-pyrrolidone in tissue homogenates is described. Homogenates are extracted with chloroform/acetonitrile and fractionated by high-performance liquid chromatography, and 2-pyrrolidone is quantitated by monitoring the column effluent at 200 nm. The lower limit of 2-pyrrolidone that can be accurately (+/- 5%) quantitated is approximately 100 pmol. Phenazine methosulfate significantly enhances the rate of 2-pyrrolidone biosynthesis from delta'-pyrroline. Phenazine methosulfate and reduced glutathione are required to obtain proportionality between 2-pyrrolidone formation and incubation time. Formation of 2-pyrrolidone as a function of protein concentration is linear and 2-pyrrolidone biosynthesis as a function of delta'-pyrroline concentration is characterized by hyperbolic kinetics. Based on analysis of enzyme activity in different tissues, liver appears to play the dominant role in 2-pyrrolidone biosynthesis. The metabolic step from delta'-pyrroline to 2-pyrrolidone was localized in the cellular cytosol. These results demonstrate that the oxidation of delta'-pyrroline to 2-pyrrolidone is enzyme mediated and provide a useful method for further characterization of this metabolic step.

gamma-Glutamyl amino acids. Transport and conversion to 5-oxoproline in the kidney.

Transport of gamma-glutamyl amino acids, a step in the proposed glutathione-gamma-glutamyl transpeptidase-mediated amino acid transport pathway, was examined in mouse kidney. The transport of gamma-glutamyl amino acids was demonstrated in vitro in studies on kidney slices. Transport was followed by measuring uptake of 35S after incubation of the slices in media containing gamma-glutamyl methionine [35S]sulfone. The experimental complication associated with extracellular conversion of the gamma-glutamyl amino acid to amino acid and uptake of the latter by slices was overcome by using 5-oxoproline formation (catalyzed by intracellular gamma-glutamyl-cyclotransferase) as an indicator of gamma-glutamyl amino acid transport. This method was also successfully applied to studies on transport of gamma-glutamyl amino acids in vivo. Transport of gamma-glutamyl amino acids in vitro and in vivo is inhibited by several inhibitors of gamma-glutamyl transpeptidase and also by high extracellular levels of glutathione. This seems to explain urinary excretion of gamma-glutamylcystine by humans with gamma-glutamyl transpeptidase deficiency and by mice treated with inhibitors of this enzyme. Mice depleted of glutathione by treatment with buthionine sulfoximine (which inhibits glutathione synthesis) or by treatment with 2,6-dimethyl-2,5-heptadiene-4-one (which effectively interacts with tissue glutathione) exhibited significantly less transport of gamma-glutamyl amino acids than did untreated controls. The findings suggest that intracellular glutathione functions in transport of gamma-glutamyl amino acids. Evidence was also obtained for transport of gamma-glutamyl gamma-glutamylphenylalanine into kidney slices.

Pyrrolidone carboxylic acid synthesis in guinea pig epidermis.

To establish the in vivo mechanism of synthesis and accumulation of epidermal pyrrolidone carboxylic acid (PCA), enzymes potentially capable of PCA synthesis have been quantified and located within the guinea pig epidermis. Intermediates in the synthesis of [3H]PCA from a pulse of [3H]glutamine have been identified and quantified to determine which of the several possible metabolic routes occurs in vivo. PCA appears to be synthesized from substrate derived from the breakdown within the stratum corneum of protein synthesized several days earlier. The predominant route is probably via the nonenzymic cyclization of free glutamine liberated from this protein. In view of the high activity of gamma-glutamyl cyclotransferase in the stratum corneum, a minor contribution to PCA formation by the action of the enzyme on gamma-glutamyl peptides cannot be excluded.

N-(3-aminopropyl)pyrrolidin-2-one, a product of spermidine catabolism in vivo.

A high-pressure-liquid-chromatographic method suitable for the separation and sensitive detection of putreanine and isoputreanine is described. This method allowed us to study the formation of the metabolites of the oxidative deamination of spermidine and N1-acetylspermidine. Administration of spermidine trishydrochloride to mice causes a time-dependent accumulation of putreanine and N-(3-aminopropyl)pyrrolidin-2-one in various organs. The latter compound yields isoputreanine by hydrolysis. It can be assumed that the analogous lactam. N-(3-acetamidopropyl)pyrrolidin-2-one is formed from N1-acetylspermidine, since hydrolysis of tissue extracts of N1-acetylspermidine-treated mice produced isoputreanine. No putreanine is formed under these conditions. Pretreatment of the animals with 25 mg of aminoguanidine sulphate/kg body wt. completely inhibits the formation of putreanine and of the respective isoputreanine precursor from spermidine and N1-acetylspermidine. This suggests a role for a diamine oxidase-like enzyme in the oxidative deamination of spermidine and N1-acetylspermidine.

Metabolism of putrescine to 5-hydroxy-2-pyrrolidone via 2-pyrrolidone.

Incubation of 2-[14C]pyrrolidone with sliced rat liver and analysis of the incubation medium by silica gel chromatography revealed that 2-[14C]pyrrolidone is metabolized to an unknown. It was previously shown by Lundgren and Hankins ((Lundgren, D.W, and Hankins, J. (1978) J. Biol. Chem. 253, 7130-7133) that slices of rat liver readily synthesized 2-pyrrolidone from putrescine. The unknown metabolite was partially purified by methanol/chloroform extraction, activated charcoal column chromatography, and two-dimensional thin layer chromatography on silica gel plates. The 2-pyrrolidone metabolite was derivatized with bis(trimethylsilyl)trifluroacetamide and analyzed by gas chromatography-mass spectrometry. The mass of the molecular ion (245) and fragment ions suggests that the 2-pyrrolidone metabolite is 5-hydroxy-2-pyrrolidone. The mass spectrum of synthetic 5-hydroxy-2-pyrrolidone was identical to that of the unknown metabolite. Synthetic 5-hydroxy-2-[3H]pyrrolidone co-chromatographed on silica gel sheets with the unknown 2-[14C]pyrrolidone metabolite obtained directly from incubation media. Under appropriate conditions (pH 7.5, no acid treatment of medium), putrescine is metabolized to 5-hydroxy-2-pyrrolidone via 2-pyrrolidone. Several effector compounds, but not necessarily the same ones, inhibit or enhance, or both, the conversion of putrescine to 2-pyrrolidone and of 2-pyrrolidone to 5-hydroxy-2-pyrrolidone. This is the first demonstration of the biosynthesis of 5-hydroxy-2-pyrrolidone.

Metabiolic products of microorganisms. Tirandamycin B(author's transl). / Stoffwechselprodukte von Mikroorganismen. 158. Mitteilung. Tirandamycin B.

Streptomyces flaveolus, strain Tü 1240 produces besides Tirandamycin A, a hitherto unknown antibiotic, which is closely related to Tirandamycin A. The new antibiotic Tirandamycin B contains one additional hydroxylgroup. Both antibiotics exhibit a similar antimicrobial spectrum and they seem to have the same mechanism of action. According to the data obtained from mass spectrometry, 13C-and 1H-NMR spectra formula II could be deduced for Tirandamycin B.

Microbial production of tenuazonic acid analogues.

The fungus Alternaria tenuis normally produces tenuazonic acid (3-acetyl-5-secbutyltetramic acid). On supplementation of the culture substrate with l-valine and l-leucine, the organism formed two new tetramic acids, 3-acetyl-5-isopropyltetramic acid and 3-acetyl-5-isobutyltetramic acid, respectively. l-Phenylalanine was not utilized by the organism as a tetramic acid precursor.

The formation of pyrrolid-2-one-5-carboxylic acid at the N-terminus of immunoglobulin G heavy chain.

We propose that pyrrolid-2-one-5-carboxyl-tRNA is not involved in the initiation of protein synthesis in eukaryotic cells and that the N-terminal pyrrolid-2-one-5-carboxylic acid group of an IgG (immunoglobulin G) (that secreted by the mouse plasmacytoma Adj PC5) is formed by the enzymic cyclization of the N-terminal glutamine of the heavy chain of the completed IgG molecule and that the cyclization takes place inside the cell. We base these conclusions on the following evidence. (1) Pyrrolidonecarboxyl-tRNA was not found in incorporation experiments with rat liver preparations and [U-(14)C]-pyrrolidonecarboxylic acid, glutamic acid and glutamine, even though an incorporation extent of less than 2% of the total products could have been detected. (2) Double-labelling experiments showed that less than 8% of the nascent peptides of heavy chains (those obtained by precipitation by the antibody to Fc fragment) began with pyrrolidonecarboxylic acid. (3) Further double-labelling experiments showed that 60-66% of the heavy chains of the completed intracellular IgG molecule began with pyrrolidonecarboxylic acid after both 1 and 5h of labelling. (4) The IgG, after secretion by plasmacytoma Adj PC5, was found to have the sequence [unk]Glu- Val-Gln-Leu- at the N-termini of the heavy chains.