Aarne Raina

Stimulation of polyamine synthesis in relation to nucleic acids in regenerating rat liver

Abstract The effect of partial hepatectomy on the liver polyamines and nucleic acids was studied in the rat. Partial hepatectomy causes an early stimulation of spermidine synthesis, as indicated by the marked increase in the incorporation of [14C]methionine into this polyamine, as early as 4–8 h after operation. At 16 h the specific activity of spermidine was about 10-fold that of the sham-operated controls. The total spermidine content of the liver was already significantly elevated at 16 h, and at 64 h it was 3.6-fold that of the controls. The specific activity of spermine, on the other hand, did not increase until 16–20 h and the total amount of spermine per liver not until 64 h post-operatively. The total amount of liver ribonucleic acid was significantly increased at 32 h and that of deoxyribonucleic acid at 64 h. The ratio polyamine nitrogen to RNA phosphate remained quite constant during liver regeneration. The present observations are discussed on the basis of the hypothesis that tissue polyamines may be essential as physiological stabilizers of nucleic acids, especially RNA.

Mechanism of stimulation of polyamine synthesis by growth hormone in rat liver

Abstract The effect of growth hormone on the synthesis of polyamines, RNA and protein was studied in the normal rat liver. The hormone markedly stimulated the synthesis of spermidine from [14C]-methionine. The time-course of this stimulation was close to that observed in RNA and protein. In addition to stimulating spermidine synthesis, growth hormone also caused an early increase in the concentration of hepatic putrescine and markedly stimulated the incorporation of label from [14C]ornithine into putrescine. The formation of putrescine from [14C]ornithine was also demonstrated in vitro, using the 100 000 × g supernatant fraction of liver homogenate as the source of the enzyme (ornithine decarboxylase). A 4-fold increase in this enzyme activity was observed after growth-hormone treatment. The effects of growth hormone on the ornithine decarboxylase activity, as well as on the accumulation of putrescine and the synthesis of spermidine, could be partially reversed by actinomycin D or puromycin. No change was observed in the capacity of the liver to synthesize spermidine from [14C]putrescine after growth-hormone treatment. It is suggested that the stimulation of spermidine synthesis after growth-hormone treatment depends on a primary increase in the concentration of putrescine, a precursor of spermidine.

Polyamine synthesis in the regenerating rat liver: Stimulation of S-adenosylmethionine decarboxylase, and spermidine and spermine synthases after partial hepatectomy

The activities of the enzymes involved in the synthesis of spermidine and spermine, i.e. putrescine-activated S-adenosylmethionine decarboxylase, and spermidine and spermine synthases, increased in rat liver after partial hepatectomy. At early stages of regeneration all these three enzyme activities increased fairly parallelly and were above the control level as early as 12 h after the operation. The activity of S-adenosylmethionine decarboxylase reached a peak, about three times the control level, approx. at 48 h postoperatively and then declined fairly rapidly. Spermine synthase activity showed a time pattern roughly comparable to that of S-adenosylmethionine decarboxylase. Spermidine synthase activity increased up to 96 h and was still elevated at 8 days after the operation. The stimulation of ornithine decarboxylase activity (EC 4.1.1.17) preceded the increases in the spermidine and spermine synthesizing enzyme activities. When cycloheximide was given to partially hepatectomized animals the activity of liver S-adenosylmethionine decarboxylase declined very rapidly with an apparent half-life of about 35 min, whereas no change was observed in the activities of spermidine and spermine synthases within 2 h after the inhibitor. These results are in accord with the view that at least three different proteins are required for the synthesis of spermidine and spermine.

Inhibition of pyridoxal enzymes by l-canaline

Abstract The effect of l -canaline, a structural analogue of l -ornithine, was studied on several mammalian enzymes in vitro. The results obtained with three ornithine metabolizing enzymes indicated that canaline is not an effective competitor of ornithine in these reactions. However, canaline strongly inhibited the activity of all seven pyridoxal-dependent enzymes studied, including amino acid decarboxylases [ornithine decarboxylase (EC 4.1.1.17), 5-hydroxytryptophan decarboxylase (EC 4.1.1.28)], aminotransferases [ornithine-ketoacid aminotransferase (EC 2.6.1.13), tyrosine aminotransferase (EC 2.6.1.5)], ornithine transcarbamylase (EC 2.1.3.3) and plasma diamino-oxidase (EC 4.1.3.6). The reversibility of this inhibition by excess pyridoxal phosphate, as well as a strong interaction between canaline and pyridoxal phosphate in aqueous solution, support the view that canaline inhibition is due to complex formation between canaline and the pyridoxal coenzyme. l -canaline is one of the most potent inhibitors of pyridoxal enzymes. Ornithine-ketoacid amino-transferase, for example, was inhibited by 50% in the presence of 3 ° 10 −6 M l -canaline.

A rapid assay method for spermidine and spermine synthases. Distribution of polyamine-synthesizing enzymes and methionine adenosyltransferase in rat tissues

Two enzymes involved in the biosynthesis of polyamines [1] which catalyse the transfer of the propylamine group of decarboxylated S-adenosylmethionine (S-methyladenosylhomocysteamine) to putrescine (spermidine synthase) or spermidine (spermine synthase) have not been extensively characterized from eucaryotic sources. The slow progress in this area is obviously due to the tedious and time-consuming methods used for the assay of these enzymes [2-5]. In this paper we describe a rapid and sensitive isotopic method for the assay of spermidine and spermine synthases. The method is based on the isolation of the radioactive polyamines formed from radioactive decarboxylated S-adenosylmethionine labelled in the propylamine moiety by using phosphocellulose ion exchange paper. Our results demonstrate marked differences between different tissues in the activities of spermidine and spermine synthases. No parallelism was found between the activities of these enzymes. Neither was there any correlation between the synthase activities and the activity of S-adenosylmethionine decarboxylase, which is in agreement with the view that these are three different enzymes.

Separation and partial purification of S-adenosylmethionine decarboxylase and spermidine and spermine synthases from rat liver

Summary Putrescine-activated S-adenosylmethionine decarboxylase and spermidine and spermine synthases, the enzymes catalyzing the synthesis of spermidine and spermine from S-adenosylmethionine and the appropriate amine, have been separated and partially purified from the soluble fraction of rat liver. During the purification of S-adenosylmethionine decarboxylase the stoichiometry between the decarboxylation of S-adenosylmethionine and the formation of spermidine in the presence of putrescine was lost.

Separation of enzyme activities catalysing spermidine and spermine synthesis in rat brain

Previous work from Williams-Ashmans laboratory [1,2] has demonstrated that the enzymic synthesis of spermidine and spermine in rat ventral prostate involves putrescine-activated decarboxylation of S-adenosyl-L-methionine (SAM) and the transfer of the propylamine group from decarboxylated SAM to putrescine or spermidine, yielding spermidine or spermine, respectively. Some evidence has also been provided which suggests that the same protein may catalyse both the conversion of putrescine to spermidine and the conversion of spermidine to spermine with SAM of decarboxylated SAM as the propylamine donor [2]. In disagreement with the latter notion, we observed that ammonium sulphate fractionation of soluble extracts from regenerating rat liver resulted in a partial separation of spermidine and spermine synthesizing enzyme activities [3]. The present paper describes separation and partial purification of enzymes from rat brain, which catalyse the synthesis of spermidine (spermidine synthase) or spermine (spermine synthase) from decarboxylated SAM and the appropriate amine. Both these enzyme preparations seem to be uncontaminated by the other and free from a significant amount of SAM decarboxylase activity. While this study was in progress, Jgnne et al. [4, 5] reported that a prostatic enzyme catalysing the synthesis of spermidine, but not spermine, from decarboxylated SAM can be separated from putrescine-activated SAM decarboxylase. 2. Materials and methods

A sensitive isotopic assay method for S-adenosylhomocysteine hydrolase: Some properties of the enzyme from rat liver

A rapid and sensitive isotopic method is presented for the assay of S-adenosylhomocysteine hydrolase (EC 3.3.1.1) activity, based on the formation of radioactive S-adenosylhomocysteine labelled in the adenosine portion. The radioactive product is separated either by low-voltage paper electrophoresis or by using phosphocellulose ion-exchange paper. Some kinetic properties of the enzyme from rat liver have shown to be clearly different from those reported earlier for this enzyme. The use of erythro-9-(2-hydroxy-3-nonyl)adenine, a potent inhibitor of adenosine deaminase, makes it possible to measure the S-adenosylhomocysteine hydrolase activity in tissues with a high adenosine deaminase activity, e.g. in intestinal mucosa.

Partial purification and characterization of spermine synthase from rat brain.

Abstract Spermine synthase, the enzyme catalyzing the formation of spermine from spermidine and 5′-ndeoxy-5′- S -(3-methylthiopropylamine)sulphonium adenosine (decarboxylated S -adenosylmethionine) has been purified more than 100-fold from rat brain cytosol fraction. Spermine synthase activity can be resolved from the other polyamine-synthesizing enzyme activities, i.e. from S -adenosylmethionine decarboxylase and spermidine synthase activities, by a single chromatography run on DEAE-cellulose. The purified spermine synthase, free of any S -adenosylmethionine decarboxylase or spermidine synthase activity, showed a broad pH optimum between 7.5 and 8.1 and an acidic isoelectric point at pH 5.0. Spermine synthase appeared to have a high affinity for decarboxylated S -adenosylmethionine, the apparent K m value being below 0.005 mM. The K m for spermidine was 0.07 mM. Putrescine was shown to be a competitive inhibitor with respect to spermidine, and spermine, the product of the reaction, was also inhibitory. No metal or other cofactor requirements for spermine synthase were found.

Effect of spermine and magnesium on the attachment of free ribosomes to endoplasmic reticulum membranes in vitro

Abstract A portion of the free ribosomes became attached to the isolated total endoplasmic reticulum membranes when incubated at 0°C for 60 min in buffers containing either 0.3–0.5 mM spermine or 5 mM Mg 2+ . This attachment was negligible if both spermine and Mg 2+ were omitted from the incubation medium. When smooth endoplasmic reticulum was tested alone, free ribosomes did not associate appreciably, whether or not spermine or Mg 2+ was present. These results suggest that spermine and Mg 2+ may function through forming ribosome-cation-membrane bridges. There is also an indication of the presence of ribosome binding sites on the endoplasmic reticulum membranes.