Changes in L-ornithine decarboxylase activity during the cell cycle

The activity of L-ornithine decarboxylase (L-ornithine carboxy-lyase; EC 4.1.1.17), the enzyme that catalyzes the initial and rate-limiting step in polyamine biosynthesis, has been studied in Chinese hamster ovary fibroblasts synchronized by selective detachment of mitotic cells. At various times after plating the distribution of cells among the G1, S and G2+M phases of the cell cycle was calculated from DNA distributions obtained by high-speed flow cytometric analysis. At these same times determination of the cellular L-ornithine decarboxylase activity showed that polyamine (putrescine) synthesis was initiated in mid-G1, that the rate of synthesis was maximal prior to DNA synthesis, and that it decreased during the S phase. A second increase in enzyme activity occurred before mitosis.     

Identification of the amino acid residues conferring substrate specificity upon Selenomonas ruminantium lysine decarboxylase

Lysine decarboxylase (LDC, EC 4.1.1.18) from Selenomonas ruminantium has decarboxylating activities towards both L-lysine and L-ornithine with similar K(m) and Vmax. Here, we identified four amino acid residues that confer substrate specificity upon S. ruminantium LDC and that are located in its catalytic domain. We have succeeded in converting S. ruminantium LDC to an enzyme with a preference in decarboxylating activity for L-ornithine when the four-residue of LDC were replaced by the corresponding residues of mouse ornithine decarboxylase (EC 4.1.1.17).     

Decarboxylases for polyamine biosynthesis in Drosophila melanogaster larvae.

Ornithine decarboxylase (L-ornithine carboxy-lase, EC 4.1.1.17) and S-adenosyl-methionine decarboxylase (S-adenosyl-L-methionine carboxy-lase, EC 4.1.1.50) were assayed in Drosophilia melanogaster larvae. The highest enzyme activities were detected in 24 and 48 h larvae, with diminishing activities in subsequent larval stages. Stimulation of S-adenosylmethionine decarboxylase by putrescine was demonstrable in late but not in early stages of larval development.     

Apparent kinetic differences between brain and liver ornithine decarboxylase.

Kinetic studies of ornithine decarboxylase activity in homogenates of rat brain and liver indicate that rat brain ornithine decarboxylase has a higher affinity for substrate L-ornithine and competitive inhibitor putrescine. These data suggest different forms of ornithine decarboxylase may exist in these tissues.     

Effects of (2R, 5R)-6-heptyne-2,5-diamine, a potent inhibitor of L-ornithine decarboxylase, on rat hepatoma cells cultured in vitro

DL-alpha-Difluoromethylornithine (F2MeOrn), the most widely-used inhibitor of L-ornithine decarboxylase, has been a useful tool to demonstrate that polyamine biosynthesis is required to maintain maximum rates of cell proliferation. However, in most eukaryotic cell systems, F2MeOrn exerts cytostatic rather than cytotoxic effects. This may be due to the fact that this inhibitor creates only incomplete polyamine deficiency. In particular, F2MeOrn scarcely depletes intracellular spermine levels. We now demonstrate in rat hepatoma tissue culture (HTC) cells that (2R, 5R)-6-heptyne-2,5-diamine, a more potent irreversible inhibitor of L-ornithine decarboxylase than F2MeOrn, decreases the concentrations of all polyamines including spermine. In parallel with the depletion of these amines, there is a progressive decrease in the rate of cell proliferation and in cell viability. Restoration of the intracellular polyamine content, by addition to the medium of polyamines or a high concentration of L-ornithine, the substrate of L-ornithine decarboxylase, further demonstrates that the antiproliferative effects of (2R, 5R)-6-heptyne-2,5-diamine do result from polyamine deficiency. These findings support the concept that polyamines play an essential function in the cell division processes and emphasize the vital function of spermine in mammalian cells.     

Stimulus-secretion coupling of arginine-induced insulin release. Functional response of islets to L-arginine and L-ornithine

L-Arginine and L-ornithine stimulate insulin release from pancreatic islets exposed to D-glucose. This coincides with an increased outflow of 86Rb and 45Ca from prelabelled islets and an increased net uptake of 45Ca by the islets. In the presence of D-glucose, L-lysine stimulates insulin secretion to the same extent as L-arginine or L-ornithine, but the hormonal release is not further enhanced by combinations of these cationic amino acids. L-Arginine or L-ornithine failed to enhance insulin release evoked by either L-leucine or 2-ketoisocaproate. The inhibitor of ornithine decarboxylase D,L-alpha-difluoromethyl ornithine failed to affect the metabolism and insulinotropic action of D-glucose in pancreatic islets, and only caused a partial inhibition of the secretory response to either L-arginine or L-ornithine. The latter amino acids inhibited modestly but significantly D-glucose utilization and oxidation by pancreatic islets. These and complementary findings suggest that the secretory response to L-arginine and L-ornithine is not attributable to any major change in the overall oxidative catabolism of nutrients, but involves mainly a biophysical component, such as the depolarization of the plasma membrane by these cationic amino acids.     

Characterization of ornithine decarboxylase of tobacco cells and tomato ovaries.

Some characteristics of L-ornithine decarboxylase of tomato ovaries and tobacco cells are described. The enzyme has a pH optimum of 8.0. It requires pyridoxal phosphate and thiol reagent (dithiothreitol) for activity. It is specific for L-ornithine and has an apparent Km of 1.4 X 10-4 M. It has an apparent molecular weight of 107000. Putrescine inhibited the activity in vitro. Spermidine and spermine also inhibit the enzyme, but less effectively. It is concluded that the enzyme is similar to that of mammalian origin and likewise fulfils a function related to cell proliferation.     

Purification and characterization of antizyme inhibitor of ornithine decarboxylase from rat liver

A protein inhibiting a protein inhibitor (antizyme) to ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) (ODC), antizyme inhibitor, was purified from the liver cytosol of thioacetamide-treated rats by procedures including antizyme affinity chromatography. Overall purification was roughly estimated to be about 17,000,000-fold and recovery was about 2.4%. The purified preparation showed one major protein band and a faint band corresponding in mobility to molecular weights of 51,000 and 53,500, respectively, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Judging from the ornithine decarboxylase activity of the final preparation, the faint band may be ornithine decarboxylase. The apparent molecular weight of antizyme inhibitor estimated by gel filtration on Sephacryl S-200 was approx. 62,000, indicating that antizyme inhibitor may be composed of a single polypeptide chain. In order to examine the question of whether antizyme inhibitor is a protein derived from ornithine decarboxylase, an inactive ornithine decarboxylase, in an immunotitration study and analysis of the binding to antizyme were investigated. The results indicate that antizyme inhibitor may be a protein distinct from ornithine decarboxylase.     

Ornithine decarboxylase activity in relation to DNA synthesis in mouse interfollicular epidermis and hair follicles.

The relationship between ornithine decarboxylase (L-ornithine carboxylyase, EC 4.1.1.17) activity and DNA synthetic activity was studied in mouse epidermis. Interfollicular epidermis and hair follicles were investigated separately. It was found that, in hair follicles, the variations of DNA replicative activity, which are reflected in the cyclic growth of hair, are paralleled by corresponding changes in ornithine decarboxylase activity. In both interfollicular epidermis and hair follicles, stimulation of DNA synthetic activity by plucking of hair induced a rapid and marked increase in ornithine decarboxylase activity. The relationship of steady-state and induced ornithine decarboxylase activity to DNA synthetic activity was compared in hair follicles and interfollicular epidermis. A correlation between the activity of this enzyme and DNA replication was found thereby in each of these tissues.     

Effect of DL-alpha-hydrazino-delta-aminovaleric acid, an inhibitor of ornithine decarboxylase, on polyamine metabolism in isoproterenol-stimulated mouse parotid glands.

The effects of DL-alpha-hydrazino-delta-aminovaleric acid (DL-HAVA) on polyamine metabolism in isoproterenol(IPR)-stimulated mouse parotid glands were investigated both in vitro and in vivo. Using partially enzyme preparations, it was found that DL-HAVA strongly inhibited ornithine decarboxylase (EC 4.1.1.17) by competing with L-ornithine. Other enzymes metabolizing ornithine and pyridoxal phosphate-dependent enzymes were at least 2-3 orders of magnitude less sensitive to DL-HAVA than ornithine decarboxylase. Administration of DL-HAVA greatly depressed the increases in both the putrescine level and putrescine formation from L-ornithine induced by IPR in the mouse parotid glands. Under the same conditions, the stimulation of DNA synthesis and subsequent cell proliferation in the glands were also suppressed. However, the IPR-dependent increases in S-adenosyl-L-methionine decarboxylase (EC 4.1.1.50) activity, synthesis and the tissue concentration of spermidine, and RNA synthesis in the parotid glands were not affected appreciably by DL-HAVA. The inhibition of DNA synthesis by DL-HAVA was effectively prevented by putrescine, but not by spermidine or 1,7-diaminoheptane, given at the same time when DL-HAVA inhibited stimulation of putrescine formation by IPR. From these results, it is proposed that putrescine is involved in cell proliferation besides being a precursor of spermidine. The effects of methylglyoxal bis(guanylhydrazone) (MGBG), an inhibitor of S-adenosyl-L-methionine decarboxylase, on the metabolism of polyamines and nucleic acids in growing parotid glands were also examined.     

Putrescine biosynthesis in Tetrahymena thermophila.

The putrescine-biosynthesis pathway in Tetrahymena thermophila was delineated by studying crude extracts prepared from exponentially growing cultures. A pyridoxal phosphate-stimulated ornithine decarboxylase activity competitively inhibited by putrescine was detected. CO2 was also liberated from L-arginine, but analyses by t.l.c. and enzyme studies suggested that the activity was not due to arginine decarboxylase, nor could enzyme activities converting agmatine into putrescine be detected. We conclude that the decarboxylation of L-ornithine is probably the only major route for putrescine biosynthesis in this organism during exponential growth.     

Induction of ornithine decarboxylase in guinea-pig lymphocytes and its relation to phospholipid metabolism

Treatment of lymphocytes with exogenous phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3.) derived from Clostridium perfringens at concentrations similar to those which induced ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) activity produced diacylglycerol and phosphatidate. A divalent cation ionophore, A23187, and phytohemagglutinin induced not only diacylglycerol formation, but also ornithine decarboxylase activity. Dibutyryl cAMP inhibited both diacylglycerol formation and ornithine decarboxylase induction to a similar extent in phytohemagglutinin-stimulated lymphocytes, but stimulated them somewhat in ionophore A23187-activated lymphocytes. This suggests that the activation of intracellular phospholipase C and the formation of diacylglycerol is involved in ornithine decarboxylase induction in lymphocytes.     

The induction of ornithine decarboxylase by L-asparagine and intracellular alkalinization

The uptake of transport systems A and N amino acids, most noticeably L-asparagine, is essential for the induction of ornithine decarboxylase (L-ornithine carboxylase, EC 4.1.1.17) in cultured cells and we have proposed that the uptake-associated pH and ionic changes might constitute part of the cell activation signal (1). In the present study, it was shown that extracellular L-asparagine caused an immediate and transient increase in intracellular pH which was continuously monitored by the fluorescence probe BCECF (2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein). NH4Cl and NH4OH which caused intracellular alkalinization also caused ornithine decarboxylase activity to increase.     

L-ornithine-induced inactivation of mammalian ornithine decarboxylase in vitro

Partially purified ornithine decarboxylase, isolated from the liver of thioacetamide-treated rats, is stable in the absence of added low-molecular-mass thiols or other reducing agents. However, under these conditions, the enzyme is rapidly inactivated upon incubation with L-ornithine or L-2-methylornithine. The inactivation process follows first-order kinetics, and saturation kinetics are observed. Rapid recovery of activity is observed after subsequent addition of dithiothreitol. As distinct from L-ornithine, D-ornithine, putrescine, spermidine, or spermine do not produce inactivation of ornithine decarboxylase. Very similar results are obtained with pure ornithine decarboxylase isolated from androgen-stimulated mouse kidney, stabilized with a rat liver extract.     

Control of utilization of L-arginine, L-ornithine, agmatine, and putrescine as nitrogen sources in Escherichia coli K-12.

The regulation of the synthesis of the enzymes involved in the utilization of L-arginine, L-ornithine, agmatine, and putrescine as a sole nitrogen source in Escherichia coli K-12 was examined. The synthesis of agmatine ureohydrolase, putrescine aminotransferase, and pyrroline dehydrogenase is dually controlled by catabolite repression and nitrogen availability. Catabolite repression of agmatine ureohydrolase, but not that of putrescine aminotransferase or pyrroline dehydrogenase, is relieved by the addition of cAMP. Agmatine ureohydrolase synthesis in addition is subject to induction by L-arginine and agmatine. Arginine decarboxylase and ornithine decarboxylase synthesis is not sensitive to catabolite repression or to stimulation by nitrogen limitation or subject to substrate induction.     

Consecutive enzymatic modification of ornithine generates the hydroxamate moieties of the siderophore erythrochelin

Biosynthesis of the hydroxamate-type siderophore erythrochelin requires the generation of δ-N-acetyl-δ-N-hydroxy-L-ornithine (L-haOrn), which is incorporated into the tetrapeptide at positions 1 and 4. Bioinformatic analysis revealed the FAD-dependent monooxygenase EtcB and the bifunctional malonyl-CoA decarboxylase/acetyltransferase Mcd to be putatively involved in the generation of L-haOrn. To investigate if EtcB and Mcd constitute a two-enzyme pathway for the biosynthesis of L-haOrn, they were produced in a recombinant manner and subjected to biochemical studies in vitro. Hydroxylation assays employing recombinant EtcB gave rise to δ-N-hydroxy-L-ornithine (L-hOrn) and confirmed the enzyme to be involved in building block assembly. Acetylation assays were carried out by incubating L-hOrn with recombinant Mcd and malonyl-CoA as the acetyl group donor. Substrate turnover was increased by substituting malonyl-CoA with acetyl-CoA, bypassing the decarboxylation reaction which represents the rate-limiting step. Consecutive enzymatic synthesis of L-haOrn was accomplished in coupled assays employing both the L-ornithine hydroxylase and Mcd. In summary, a biosynthetic route for the generation of δ-N-acetyl-δ-N-hydroxy-L-ornithine starting from L-ornithine has been established in vitro by tandem action of the FAD-dependent monooxygenase EtcB and the bifunctional malonyl-CoA decarboxylase/acetyltransferase Mcd.     

Decarboxylation of ornithine and lysine in rat tissues.

The possibility that arginine and lysine might be decarboxylated by rat tissues was investigated. No evidence for decarboxylation of arginine could be found. Lysine decarbosylase (L-lysine carboxy-lyase, EC 4.1.1.18) activity producing CO2 and cadaverine was detected in extracts from rat ventral prostate, androgen-stimulated mouse kidney, regenerating rat liver and livers from rats pretreated with thioacetamide. These tissues all have high ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) activities. Lysine and ornithine decarboxylase activities were lost to similar extents on inhibition of protein synthesis by cycloheximide and on exposure to alpha-difluoromethylornithine. A highly purified ornithine decarboxylase preparation was able to decarboxylate lysine and the ratio of ornithine to lysine decarboxylase activities was constant throughout purification. Kinetic studies of the purified preparation showed that the V for ornithine was about 4-fold greater than for lysine, but the Km for lysine (9 mM) was 100-times greater than that for ornithine (0.09 mM). These experiments indicate that all of the detectable lysine decarboxylase activity in rat and mouse tissues was due to the action of ornithine decarboxylase and that significant cadaverine production in vivo would occur only when ornithine decarboxylase activity is high and lysine concentrations substantially exceed those of ornithine.     

Putrescine-sensitive (artifactual) and insensitive (biosynthetic) S-adenosyl-L-methionine decarboxylase activities of Lathyrus sativus seedlings.

The crude extracts of 3-day-old etiolated seedlings of Lathyrus sativus contained two S-adenosyl-L-methionine decarboxylase activities. The artifactual putrescine-dependent activity was due to the H2O2 generated by diamine oxidase (EC 1.4.3.6) of this plant system and was inhibited by catalase. This observation was confirmed by using an electrophoretically and immunologically homogeneous preparation of L. sativus diamine oxidase. In the presence of putrescine, diamine oxidase, in addition to S-adenosylmethionine, decarboxylated L-lysine, L-arginine, L-ornithine, L-methionine and L-glutamic acid to varying degrees. The decarboxylation was not metal-ion dependent. The biosynthetic S-adenosylmethionine decarboxylase (EC 4.1.1.21) was detected after removing diamine oxidase specifically from the crude extracts by employing an immunoaffinity column. This Mg2+-dependent decarboxylase was not stimulated by putrescine or inhibited by catalase. The enzyme activity was inhibited by semicarbazide, 4-bromo-3-hydroxybenzoylamine dihydrogen phosphate and methylglyoxal-bis (guanylhydrazone). It was largely localized in the shoots of the etiolated seedlings and was purified 40-fold by employing a p-hydroxymercuribenzoate/AH-Sepharose affinity column, which also separated the decarboxylase activity from spermidine synthase.