Tapani Hyvönen

Monitoring of the uptake and metabolism of aminooxy analogues of polyamines in cultured cells by high-performance liquid chromatography

A high-performance liquid chromatographic method for the determination of polyamines and their aminooxy analogues is described. Oxime derivatization with a ketone is used to protect the aminooxy group during post-column reaction with o-phthalaldehyde. The amount of the polyamines and of the oximes of their aminooxy analogues can be determined simultaneously in cultured cells and cell culture media. The limit of detection is 20-30 pmol, and the response of the fluorescence detection is linear up to 4 nmol. The separation of the aminooxy analogues from the naturally occurring polyamines can be varied by using different ketones for oxime formation. The method was used to measure the stability of aminooxy analogues of putrescine (1-aminooxy-3-aminopropane) and spermidine [N-(2-aminooxyethyl)-1,4-diaminobutane and 1-aminooxy-3-N-(3-aminopropyl)aminopropane] in cell culture media and the uptake into cultured baby hamster kidney (BHK21/C13) cells.

Polyamine-dependent alterations in the structure of microfilaments, Golgi apparatus, endoplasmic reticulum, and proteoglycan synthesis in BHK cells.

The activity of ornithine decarboxylase, the key enzyme in the synthesis of polyamines, is essential for proliferation and differentiation of all living cells. Two inhibitors of ornithine decarboxylase, α‐difluoromethylornithine (DFMO) and 1‐aminooxy‐3‐aminopropane (APA), caused swelling of endoplasmic reticulum (ER) and medial and trans Golgi cisternae, and the disappearance of stress fibers, as visualized by staining with fluorescent concanavalin A (ConA), C6‐NBD‐ceramide or wheat germ agglutinin (WGA), and phalloidin, respectively. In contrast, the pattern of microtubules, stained with a β‐tubulin antibody, was not affected. Rough ER seemed to be especially affected in polyamine deprivation forming whorls and involutions, which were observed by transmission electron microscopy. Since ER and Golgi apparatus are vital parts of the glycosylation and secretory machinery of the cell, we tested the ability of these structurally altered cell organelles to synthesize proteoglycans using [3H]glucosamine and [35S]sulfate as precursors. The total incorporation rate into proteoglycans and hyaluronan was not reduced in polyamine‐deprived cells, suggesting that the total glycosylation capacity of cells was not affected. However, the synthesis of a high molecular weight proteoglycan containing chondroitin and keratan sulfate was completely inhibited. The remodeling of cytoskeleton and rough endoplasmic reticulum in polyamine deprivation may perturb the synthesis and secretion of the components of membrane skeleton and of the extracellular matrix, e.g., proteoglycans. Rough ER and cytoskeleton may be the targets where polyamines affect cell proliferation and differentiation. J. Cell Biochem. 66:165‐174, 1997.

Synthesis of Hydroxylamine Analogues of Polyamines

Abstract Novel analogues of spermine and spermidine with terminal H2NCH2-group substituted by H2NO-group, were prepared starting the synthesis from EtO(Me)CNOH and subsequent extension of a polyamine backbone. To prepare their earlier unknown tritium labelled analoques, ω-[[(1′-ethoxyethylidene)amino]oxy]-poly-(iminomethylene) nitriles were reduced to amines by NaBT 4 CoCl 2 complex, which did not effect the N-O or CN bonds of ethoxyethylidene group, whereas aminooxy group deprotection was performed at the final step of synthesis by mild acidic hydrolysis. Novel monoacetylated (AcHN- or AcNHO-) analoques of spermidine were also synthesised.

Aminooxy analogues of spermidine evidence the divergent roles of the charged amino nitrogens in the cellular physiology of spermidine

Two recently devised spermidine analogues, N-[2-aminooxyethyl]-1,4-diaminobutane (AOEPU) and 1-aminooxy-3-N-[3-aminopropyl]-aminopropane (APAPA), were used to elucidate the role of charge distribution in the functions of spermidine in cultured baby hamster kidney cells. The drugs did not affect cell proliferation nor did they relieve the growth-arrest but potentiated the metabolic disturbances caused by DL-alpha-difluoromethyl-ornithine (DFMO). Neither drug affected spermidine uptake but both competed with putrescine uptake. Neither drug could replace spermidine in the control of S-adenosylmethionine decarboxylase and accumulation of the reaction product. APAPA prevented spermine synthesis and showed that modest putrescine synthesis take place in the presence of DFMO. AOEPU, but not APAPA, interfered with cellular constituents resulting in enzymatic formation, accumulation and excretion to culture medium of UV-absorbing catabolites.

Effect of Dietary Methionine, Arginine and Ornithine on the Metabolism and Accumulation of Polyamines, S-Adenosylmethionine and Macromolecules in Rat Liver and Skeletal Muscle

The interrelationship and possible causality of polyamine synthesis and the transmethylation pathway in the growth-retarding effects of inadequate or excess dietary methionine was studied in young male rats. Feeding the rats for 2 weeks diets containing toxic concentrations of methionine had no effect on polyamine and S-adenosylmethionine metabolism in skeletal muscle, but resulted in markedly elevated concentrations of S-adenosylmethionine and S-adenosylhomocysteine and slightly decreased accumulation of spermine and RNA in the liver. These changes were accompanied by liver-specific stimulation of methionine adenosyltransferase and reduction of spermine synthase activities. Inadequate arginine feeding or supplementation of the diets with ornithine or excess arginine resulted in no apparent changes in tissue methionine or polyamine metabolism and did not alleviate the effects of varied dietary methionine supply. Inhibition of putrescine synthesis by supplementing the diets with 2-difluoromethylornithine did not modify the effects of toxic concentrations of dietary methionine. It is suggested that although hepatic spermine synthase is sensitive to excess methionine feeding, methionine toxicity is not mediated by defective polyamine metabolism.

Novel CoA-polyamine conjugates for effective inhibition of spermine/spermidine-N1-acetyltransferase.

New mimics of the transition state of spermine/spermidine-N1-acetyltransferase reaction were prepared starting from aminooxy analogues of spermidine or spermine and SH-CoA. The activity depended on the structure of polyamine fragment of the conjugate and best of the synthesized compounds were active at micromolar concentrations.

Approaching the Structures of Mammalian Propylamine Transferases and their Genes

Propylamine transferases, spermidine synthase (EC 2.5.1.16) and spermine synthase (EC 2.5.1.22) catalyze the two final, irreversible steps in the conversion of arginine and methionine to spermidine and spermine, respectively. Both of the amino acid precursors are nutritionally essential for animals and are present in animal tissues at low, well-conserved concentrations. Arginine plays a central role in protein catabolism by being an essential intermediate in the conversion of toxic ammonia to excretable nontoxic urea. Methionine, on the other hand, is vitally important for protein synthesis by being the amino acid needed for peptide chain initiation and for the transmethylation reactions involved in the control of metabolism at several steps of transcriptional, translational and post-translational level. The fraction of the precursor amino acids consumed in polyamine synthesis varies a great deal depending on the cell type, the stage of differentiation and the proliferation rate. As to methionine consumption, the share of polyamine synthesis is less than 1% in rat liver (Eloranta and Kajander, 1984) but may exceed 20% in some cultured cells (Iizasa and Carson, 1985). Although detailed action mechanisms of polyamines in animal physiology are poorly known, their essentiality for cellular functions have been widely demonstrated (for references see Tabor and Tabor, 1984; Pegg, 1986).

Stable analogues of coenzyme-substrate complex of spermidine/spermine-N 1-acetyltransferase reaction. Synthesis and interaction with the enzyme

A convenient two-step synthesis of conjugates of HS-CoA and D-pantetheine with aminooxy analogues of Spm and Spd was suggested. The use of acetone linker provided target conjugates with quantitative yields. The activity of CoA-derived “bisubstrate” inhibitors, being active at microM concentrations, was at least 100 times better than that of corresponding derivatives of D-pantetheine.