ABSTRACT The influence of glucagon and dibutyryl cyclic 3′,5′-AMP on ornithine decarboxylase (EC 4.1.1.17) and nuclear RNA polymerase (EC 2.7.7.6) activity of rat liver was studied. Intraperitoneal administration of 0.2 mg of glucagon per 100 g body weight resulted in a four- to six-fold increase in ornithine decarboxylase activity. This enzyme activity was also markedly increased by a single ip injection containing 2.5–5 mg of dibutyryl cyclic AMP. The time course of this effect demonstrated a maximum at 3–4 h. Both agents also stimulated nuclear RNA polymerase activity. The increase in hepatic ornithine decarboxylase activity by dibutyryl cyclic AMP was not abolished by adrenalectomy but was totally prevented by the administration of actinomycin D, α-amanitin and cycloheximide, which suggests that the increase in activity represents synthesis de novo of the enzyme. A sharp rise in liver ornithine decarboxylase activity was also observed after administration of theophylline, whereas no change in enzyme activity was found after the administration of adenosine or adenosine 5′-nucleotides.

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.

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.

IT has been reported that medium modified by contact with living cells (conditioned medium) has a favourable effect on cell growth, but the nature of the effector has usually remained obscure. We report here the properties of a growth stimulating factor produced in cultures of human fibroblasts and show that most of its effect is due to putrescine (diaminobutane).

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.

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 ° 10−6Ml-canaline.

FEBS LettersVolume 16, Issue 1 p. 1-4 Full-length articleFree Access Separation of enzyme activities catalysing spermidine and spermine synthesis in rat brain Aarne Raina, Aarne Raina Department of Medical Chemistry, University of Helsinki, Helsinki, FinlandSearch for more papers by this authorPekka Hannonen, Pekka Hannonen Department of Medical Chemistry, University of Helsinki, Helsinki, FinlandSearch for more papers by this author Aarne Raina, Aarne Raina Department of Medical Chemistry, University of Helsinki, Helsinki, FinlandSearch for more papers by this authorPekka Hannonen, Pekka Hannonen Department of Medical Chemistry, University of Helsinki, Helsinki, FinlandSearch for more papers by this author First published: July 15, 1971 https://doi.org/10.1016/0014-5793(71)80669-5Citations: 33AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL References 1 A.E. Pegg, H.G. Williams-Ashman, J. Biol. Chem., 244, (1969), 682– 2 A.E. Pegg, H.G. Williams-Ashman, Arch. Biochem., 137, (1970), 156– 3 A. Raina, P. Hannonen, Acta Chem. Scand., 24, (1970), 3061– 4 J. Jänne, H.G. Williams-Ashman, Biochem. Biophys. Res. Commun., 42, (1971), 222– 5 J. Jänne, A. Schenone, H.G. Williams-Ashman, Biochem. Biophys. Res. Commun., 42, (1971), 758– 6 A. Raina, J. Jänne, P. Hannonen, E. Hölttä, Ann. N.Y. Acad. Sci., 171, (1970), 697– 7 O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, J. Biol. Chem., 193, (1951), 265– 8 H.M. Kalckar, J. Biol. Chem., 167, (1947), 461– Citing Literature Volume16, Issue1July 15, 1971Pages 1-4 ReferencesRelatedInformation

Annals of the New York Academy of SciencesVolume 171, Issue 3 p. 697-708 SYNTHESIS AND ACCUMULATION OF POLY AMINES IN REGENERATING RAT LIVER* A. Raina M.D., A. Raina M.D. Department of Medical Chemistry University of Helsinki Helsinki, FinlandSearch for more papers by this authorJ. Jänne M.D., J. Jänne M.D. Department of Medical Chemistry University of Helsinki Helsinki, FinlandSearch for more papers by this authorP. Hannonen B.S., P. Hannonen B.S. Department of Medical Chemistry University of Helsinki Helsinki, FinlandSearch for more papers by this authorE. Hölttä B.S., E. Hölttä B.S. Department of Medical Chemistry University of Helsinki Helsinki, FinlandSearch for more papers by this author A. Raina M.D., A. Raina M.D. Department of Medical Chemistry University of Helsinki Helsinki, FinlandSearch for more papers by this authorJ. Jänne M.D., J. Jänne M.D. Department of Medical Chemistry University of Helsinki Helsinki, FinlandSearch for more papers by this authorP. Hannonen B.S., P. Hannonen B.S. Department of Medical Chemistry University of Helsinki Helsinki, FinlandSearch for more papers by this authorE. Hölttä B.S., E. Hölttä B.S. Department of Medical Chemistry University of Helsinki Helsinki, FinlandSearch for more papers by this author First published: October 1970 https://doi.org/10.1111/j.1749-6632.1970.tb39382.xCitations: 64 * This work was supported by grants from the National Research Council for Medical Sciences, Finland, and the Sigrid Jusélius Foundation. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Reference 1 Raina, A. 1963. Studies on the determination of spermidine and spermine and their metabolism in the developing chick embryo. Acta Physiol. Scand. 60 (Suppl. 218): 1. 2 Janne, J., A. Raina & M. Siimes. 1964. Spermidine and spermine in rat tissues at different ages. Acta Physiol. Scand. 62: 352. 3 Raina, A., J. Janne & M. Siimes. 1965. Polyamines and nucleic acids in re-generating rat liver. Abstracts, 2nd FEBS Meeting, Vienna. A 114. 4 Raina, A., J. Janne & M. Siimes 1966. Stimulation of polyamine synthesis in relation to nucleic acids in regenerating rat liver. Biochim. Biophys. Acta 123: 197. 5 Dykstra, W. G., Jr. & E. I. Herbst 1965. Spermidine in regenerating liver: Relation to rapid synthesis of ribonucleic acid. Science 149: 428. 6 Jänne, J. & A. Raina 1966. Further observations on the biosynthesis of polyamines in regenerating rat liver. Acta Chem. Scand. 20: 1174. 7 Jänne, J. 1967. Studies on the biosynthetic pathway of polyamines in rat liver. Acta Physiol. Scand. (Suppl. 300): 1. 8 Jänne, J. & A. Raina. 1968. Stimulation of spermidine synthesis in the regenerating rat liver: relation to increased ornithine decarboxylase activity. Acta Chem. Scand. 22: 1349. 9 Russell, D. & S. H. Snyder. 1968. Amine synthesis in rapidly growing tissues: ornithine decarboxylase activity in regenerating rat liver, chick embryo, and various tumors. Proc. Nat. Acad. Sci. U. S. A. 60: 1420. 10 Fausto, N. 1969. Studies on ornithine decarboxylase activity in normal and regenerating livers. Biochim. Biophys. Acta 190: 193. 11 Russell, D. H. & S. H. Snyder. 1969. Amine synthesis in regenerating rat liver: effect of hypophysectomy and growth hormone on omithine decarboxylase. Endocrinology 84: 223. 12 Schrock, T. R., N. J. Oakman & N. L. R. Bucher 1969. Ornithine decarboxylase in relation to liver growth. Fed. Proc. 28: 599. 13 Shapiro, S. K. & D. J. Ehninger. 1966. Methods for the analysis and preparation of adenosylmethionine and adenosylhomocysteine. Anal. Biochem. 15: 323. 14 Pegg, A. E. & H. G. Williams-Ashman. 1969. On the role of S-adenosyl-L-methionine in the biosynthesis of spermidine by rat prostate. J. Biol. Chem. 244: 682. 15 Cantoni, G. L. & J. Durell. 1957. Activation of methionine for transmethylation. II. The methionine-activating enzyme: Studies on the mechanism of the reaction. J. Biol. Chem. 225: 1033. 16 Higgins, G. H. & R. M. Anderson. 1931. Experimental pathology of the liver. Restoration of the liver in the white rat following partial surgical removal. Arch. Pathol. 12: 186. 17 Jänne, J. & A. Raina. 1969. On the stimulation of ornithine decarboxylase and RNA polymerase activity in rat liver after treatment with growth hormone. Biochim. Biophys. Acta 174: 769. 18 Raina, A. & J. Jänne. 1968. Biosynthesis of putrescine: characterization of ornithine decarboxylase from regenerating rat liver. Acta Chem. Scand. 22: 2375. 19 Raina, A., M. Jansen & S. S. Cohen 1967. Polyamines and the accumulation of ribonucleic acid in some polyauxotrophic strains of Escherichia coli.. J. Bacteriol. 94: 1684. 20 Ashwell, G. 1957. Colorirnetiic analysis of sugars. In Methods in S. P. Enzymology Colowick & N. O. Kaplan, Eds. 3: 73– 105. Academic Press. New York , N.Y . 21 Lowry, O. H., N. J. Rosebrough, A. L. Farr & R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265. 22 Raina, A. & J. Jänne. 1970. Polyamines and the accumulation of RNA in mammalian systems. Fed. Proc. 29. 23 Pegg, A. E. & H. G. Williams-Ashman 1968. Biosynthesis of putrescine in the prostate gland of the rat. Biochem. J. 108: 533. 24 Kekomäki, M., J. Jänne, E.-L. Rahiala, A. Raina & N. Räihä 1969. Canaline: a potent inhibitor of pyridoxal enzymes. Scand. J. Clin. Lab. Invest. 23 (Suppl. 108): 30. 25 Wickner, R. B., C. W. Tabor & H. Tabor. 1969. Purification of S-adenosyl-methionine decarboxylase: Evidence for a pyruvate prosthetic group. Fed. Proc. 28: 352. 26 Russell, D. H. & S. H. Snyder. 1969. Amine synthesis in regenerating rat liver: Extremely rapid turnover of ornithine decarboxylase. Molec. Pharmacol. 5: 253. 27 Siimes, M. 1967. Studies on the metabolism of 1,4-14C-spermidine and 1,4-14C-spermine in the rat. Acta Physiol. Scand. (Suppl. 298): 1. 28 Church, R. B. & B. J. McCarthy. 1967. Ribonucleic acid synthesis in regenerating and embryonic liver. I. The synthesis of new species of RNA during regeneration of mouse liver after partial hepatectomy. J. Molec. Biol. 23: 459. Citing Literature Volume171, Issue3Metabolism and Biological Functions of PolyminesOctober 1970Pages 697-708 ReferencesRelatedInformation

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 Mg2+. This attachment was negligible if both spermine and Mg2+ were omitted from the incubation medium. When smooth endoplasmic reticulum was tested alone, free ribosomes did not associate appreciably, whether or not spermine or Mg2+ was present. These results suggest that spermine and Mg2+ 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.

Some of the properties of the 80-S ribosome of Tetrahymena pyriformis have been examined. We estimate from sedimentation coefficient (79.5-S) and intrinsic viscosity (0.074 dl/g) a mol. wt. of 4.6·106 for the ribonucleoprotein particles. The RNA and protein ratio of the ribosomes is close to one. The high molecular weight ribosomal RNA (rRNA) components sediment at 26-S and 14-S. Disc-gel electrophoresis runs at pH 3.8 show 13 prominent bands while little, if any, protein migrated into the gel when the run was made at pH 9.4. The molar ratio of polyamine-nitrogen to RNA-phosphorus for these ribosomes is 0.24. Spermidine and putrescine are present at about the same level and account for 95 % of the polyamines bound to the ribosome.

The effect of growth hormone on the synthesis of polyamines, RNA and protein was studied in the normal rat liver.

Spermine and spermidine are present in rat liver mitochondria at the concentrations of 3.5 and 1.8 nanomoles/mg protein, respectively. Addition of 50 μM Ca2+and 1 mM Pi to respiring mitochondria induces, concomitantly with mitochondrial swelling and Mg2+efflux, a consistent release of polyamines, which is either prevented by cyclosporin A or Mg2+. Addition of 0.1 mM spermine to mitochondria deenergized by Ca2+and phosphate restores, like 1 mM Mg2+, transmembrane potential at the physiological level, while either cyclosporin A or ADP are ineffective. The presence of polyamines in the matrix space should be considered relevant, like Mg2+, in controlling the permeability transition of liver mitochondria.