Modulation of insulin induced ornithine decarboxylase by putrescine and methylputrescines in H-35 hepatoma cells

Summary The effect of several methylputrescines on the activity of insulin-induced ornithine decarboxylase (ODC) was examined in H-35 hepatoma cells. The induction involved both protein and m-RNA synthesis. Actinomycin D inhibited ODC activity when given up to 1 h after insulin treatment. When added to the medium 2 h or 3 h after the insulin, the activity was increased 100% and 80% respectively. Insulin-induced ODC from H-35 cells had a biphasic half-life, a shorter one of 46 min and a longer one of 90 min.1-Methylputrescine and 2-methylputrescine were found to be competitive inhibitors of the ODC from H-35 cells with K_i values of 2.8 and 0.1 mM respectively. Putrescine itself was found to have a K_i = 2.4 mM. N-Methylputrescine was a very poor inhibitor of the cell free ODC while 1,4-dimethylputrescine did not show any inhibitory effect. When cellular ODC activity was measured, the four methylputrescines assayed as well as putrescine entirely abolished its activity in the H-35 cells when given at a 1 mM concentration together with insulin. 1-Methylputrescine and 1,4-dimethylputrescine abolished 60% of the activity at a 0.1 µM concentration. All the methylputrescines given at 0.1 mM concentrations decreased the putrescine content of the stimulated cells to the levels found in quiescent cells, but only 1-methyl and 2-methylputrescines decreased spermidine and spermine content. 1,4-Dimethyl and 1-methylputrescines showed a strong inhibition of ODC synthesis, while the other diamines were less inhibitory. At concentrations that abolished ODC activity, 1,4-dimethylputrescine decreased 70% of the total immunoreactive ODC bands, while 1-methyl and 2-methylputrescine decreased them by 50%, and N-methylputrescine and putrescine decreased them by 20%. The lack of decrease in immuno-reactive ODC with the latter two compounds was mainly due to the appearance of immunoreactive degradation products of ODC of low molecular weight. Putrescine and N-methylputrescine affected protein synthesis to a small extent in stimulated cells, while 1-methylputrescine decreased it to the level of non-stimulated cells. Insulin (1 µM concentration) stimulated DNA synthesis in the cells, and this stimulation was doubled in the presence of 2-methylputrescine or putrescine. It can be concluded that, among the methylputrescines assayed, 2-methylputrescine was the best inhibitor of cell-free ODC activity, while 1,4-dimethylputrescine and 1-methylputrescine were the best inhibitors of cellular ODC activity.Abbreviations ODC Ornithine Decarboxylase - TLC Thin Layer Chromatography - DNEM Dulbecco's Modified Essential Medium - PBS Phosphate Buffered saline - PEG Polyethyleneglycol     

Interaction of alkylputrescines with ornithine decarboxylase from rat liver and Escherichia coli: an in vitro and in vivo study

The inhibitory effect of a series of 2-alkylputrescines on rat liver and Escherichia coli ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) was examined. At 2.5 mM concentrations, 2-methyl-, 2-propyl-, 2-butyl-, 2-pentyl- and 2-hexylputrescines were stronger inhibitors of the mammalian enzyme than putrescine. Only the higher homologues (from 2-propyl- to 2-hexylputrescine) were inhibitors of the E. coli enzyme. An analysis of the effect of increasing concentrations of the 2-alkylputrescines showed that the main difference in the behaviour of the mammalian and E. coli decarboxylases toward 2-alkylputrescines was that the former was strongly inhibited by 2-methylputrescine whereas the latter was not. 2-Alkylputrescines were found to be competitive inhibitors of both the bacterial and mammalian enzyme. The smallest Ki values (0.1 and 0.5 mM) were found for the 2-hexyl- and 2-pentylputresciens. N-Methyl-, N-ethyl-, N-propyl- and N-butylputrescines (50 mumol per 100 g body weight) were assayed as inhibitors of thioacetamide-induced rat liver ornithine decarboxylase. N-Propylputrescine was found to be the most inhibitory (66% inhibition) and although the N-alkylputrescines were taken up by the liver, they did not inhibit the liver polyamine pools. Both putrescine and N-methylputrescine were found to stabilize the thioacetamide-induced ornithine decarboxylase at the onset of the enzyme's degradation, while 2-alkylputrescines were inhibitory under similar conditions. N-Methylputrescine induced antizyme in thioacetamide-treated rats. In thioacetamide- or dexamethasone-treated rats, 2-methylputrescine was found to be the strongest in vivo inhibitor of the liver decarboxylase. Although 2-alkylputrescines were efficiently taken up by the liver, they did not noticeably inhibit its polyamine pools. 2-methylputrescine decreased the putrescine concentration of the liver, but not its spermidine and spermine content. No induction of ornithine decarboxylase antizyme by 2-methylputrescine could be detected. The intrahepatic concentration of the latter decreased with time, very likely due to its degradation by a diamine oxidase, since the decrease was inhibited by aminoguanidine.     

Oxidation of N-alkyl and C-alkylputrescines by diamine oxidases

N-Methyl-, N-ethyl-, N-propyl- and N-butylputrescine were assayed as substrates of diamine oxidase from pea seedling and pig kidney. With the exception of N-methylputrescine they were found to be oxidized to the corresponding aminoaldehydes. 1-Methyl-, 2-methyl-, 1-ethyl- and 1-propylputrescine were oxidized by the oxidases at lower rates than the N-alkylderivatives. 1,3-Dimethylputrescine had negligible oxidation rates while 1,4-dimethylputrescine (2,5-diaminohexane) was not a substrate. The oxidation of putrescine by the kidney oxidase was inhibited by 1,4-dimethylputrescine, while the pea oxidase was strongly inhibited by the former as well as by 2-methylputrescine and 1,3-dimethylputrescine. Serum amine oxidase did not oxidize the substituted putrescines although several of the latter inhibited spermidine oxidation by this oxidase.     

Diamine-induced inhibition of liver ornithine decarboxylase

Repeated injections of 1,3-diaminopropane, a potent inhibitor of mammalian ornithine decarboxylase, induced protein-synthesis-dependent formation of macromolecular inhibitors or ;antienzymes' [Heller, Fong & Canellakis (1976) Proc. Natl. Acad. Sci. U.S.A.73, 1858-1862] to ornithine decarboxylase in normal rat liver. Addition of the macromolecular inhibitors, produced in response to repeated injections of diaminopropane, to active ornithine decarboxylase in vitro resulted in a profound loss of the enzyme activity, which, however, could be partly recovered after passage of the enzyme-inhibitor mixture through a Sephadex G-75 columin in the presence of 0.4m-NaCl. This treatment also resulted in the appearance of free inhibitor. In contrast with the separation of the enzyme and inhibitory activity after combination in vitro, it was not possible to re-activate, by using identical conditions of molecular sieving, any inhibited ornithine decarboxylase from cytosol fractions obtained from animals injected with diaminopropane. However, the idea that injection of various diamines, also in vivo, induces acute formation of macromolecular inhibitors, which reversibly combine with the enzyme, was supported by the finding that the ornithine decarboxylase activity remaining after diaminopropane injection appeared to be more stable to increased ionic strength than the enzyme activity obtained from somatotropin-treated rats. Incubation of the inhibitory cytosol fractions with antiserum to ornithine decarboxylase did not completely abolish the inhibitory action of either the cytosolic inhibitor or the antibody. A single injection of diaminopropane produced an extremely rapid decay of liver ornithine decarboxylase activity (half-life about 12min), which was comparable with, or swifter than, that induced by cycloheximide. However, although after cycloheximide treatment the amount of immunotitrable ornithine decarboxylase decreased only slightly more slowly than the enzyme activity, diaminopropane injection did not decrease the amount of the immunoreactive protein, but, on the contrary, invariably caused a marked increase in the apparent amount of antigen, after some lag period. The diamine-induced increase in the amount of the immunoreactive enzyme protein could be totally prevented by a simultaneous injection of cycloheximide. These results are in accord with the hypothesis that various diamines may result in rapid formation of macromolecular inhibitors to ornithine decarboxylase in vivo, which, after combination with the enzyme, abolish the catalytic activity but at the same time prevent the intracellular degradation of the enzyme protein.     

Effect of 1,3-diaminopropane on ornithine decarboxylase enzyme protein in thioacetamide-treated rat liver.

A radioimmunoassay for ornithine decarboxylase was used to study the regulation of this enzyme in rat liver. The antiserum used reacts with ornithine decarboxylase from mouse, human or rat cells. Rat liver ornithine decarboxylase enzyme activity and enzyme protein (as determined by radioimmunoassay) were measured in thioacetamide-treated rats at various times after administration of 1,3-diaminopropane. Enzyme activity declined rapidly after 1,3-diaminopropane treatment as did the amount of enzyme protein, although the disappearance of enzyme activity slightly preceded the loss of immunoreactive protein. The loss of enzyme protein after cycloheximide treatment also occurred rapidly, but was significantly slower than that seen with 1,3-diaminopropane. When 1,3-diaminopropane and cycloheximide were injected simultaneously, the rate of disappearance of enzyme activity and enzyme protein was the same as that seen with cycloheximide alone. These results show that the rapid loss in enzyme activity after 1,3-diaminopropane treatment is primarily due to a loss in enzyme protein and that protein synthesis is needed in order for 1,3-diaminopropane to exert its full effect. A macromolecular inhibitor of ornithine decarboxylase that has been termed antizyme is induced in response to 1,3-diaminopropane, but our results indicate that the loss of enzyme activity is not due to the accumulation of inactive ornithine decarboxylase-antizyme complexes. It is possible that the antizyme enhances the degradation of the enzyme protein. Control experiments demonstrated that the antiserum used would have detected any inactive antizyme-ornithine decarboxylase complexes present in liver since addition of antizyme to ornithine decarboxylase in vitro did not affect the amount of ornithine decarboxylase detected in our radioimmunoassay. Anti-(ornithine decarboxylase) antibodies may be useful in the purification of antizyme since the antizyme-ornithine decarboxylase complex can be immunoprecipitated, and antizyme released from the precipitate with 0.3 M-NaCl.     

Inhibition of ornithine decarboxylase activity and spermidine accumulation in regenerating rat liver.

Injections of 1,3-diaminopropane, a close structural analogue of putrescine (1,4-diaminobutane), into partially hepatectomized rats powerfully inhibited ornithine decarboxylase (EC 4.1.1.17) activity in the regenerating liver in vivo. The compound did not have any effect on the enzyme activity in vitro (under assay conditions employed) but appeared to exert an inhibitory influence on the synthesis of ornithine decarboxylase itself.Repeated injections of diaminopropane into rats after partial hepatectomy, starting at the time of the operation and continued until 33 h postoperatively, markedly diminished the stimulation of ornithine decarboxylase activity in the regenerating liver remnant, and completely prevented the increases in hepatic spermidine concentration normally occurring in response to partial hepatectomy.Treatment of the rats with diaminopropane did not depress the activity of adenosylmethionine decarboxylase (EC 4.1.1.50) in the regenerating liver. Nor did the compound have any effect, whatsoever, on the activity of spermidine synthase (EC 2.5.1.16) in vitro, thus obiviously proving that the increased accumulation of liver spermidine after partial hepatectomy primarily depends upon a stimulation of ornithine decarboxylase activity and a concomitant accumulation of putrescine. The results also showed that 1,3-diamino-propane could not replace putrescine in the synthesis of higher polyamines in rat liver. The inhibition of ornithine decarboxylase by diaminopropane thus appears to represent “gratuitous” repression of polyamine biosynthesis and might conceivably be used for studies devoted to the elucidation of the physiological functions of natural polyamines.     

Regulation of l-ornithine decarboxylase and S-adenosyl-l-methionine decarboxylase in rat ventral prostate and seminal vesicle

1. The activities of l-ornithine decarboxylase (EC 4.1.1.17) and S-adenosyl-l-methionine decarboxylase (EC 4.1.1.50) were dramatically enhanced in both the ventral prostate and the seminal vesicle of castrated rats in response to androgenic stimulation. The time course of the stimulation of ornithine decarboxylase together with the quantitatively different response of adenosylmethionine decarboxylase to testosterone treatment in the prostate gland and seminal vesicle indicated that the enhancement in polyamine synthesis in the ventral prostate may reflect both cellular proliferation and the restoration of the secretory functions of the organ. In the seminal vesicle, however, the stimulation of the polyamine-biosynthetic pathway more closely resembled the pattern found in other rat tissues, such as regenerating liver, undergoing compensatory growth. 2. Ornithine decarboxylase activity in the ventral prostate and especially in the seminal vesicle of sexually mature rat was diminished in vivo by various short-chain diamines such as 1,2-diaminoethane, 1,3-diaminopropane and putrescine (1,4-diaminobutane). These diamines had no direct effect on the enzyme activity in vitro. 3. In contrast with the marginal decrease in ornithine decarboxylase activity produced by diaminoethane in the ventral prostate of non-castrated animals, repeated injections of the latter amine completely prevented the intense stimulation of the enzyme activity in the ventral prostate and seminal vesicle of castrated rats at 24h after the commencement of testosterone treatment. 4. The decrease in ornithine decarboxylase activity observed after injections of diamines (putrescine) in the ventral prostate was apparently associated with a similar decrease in the amount of immunoreactive protein as revealed by immunotitration of the enzyme with antiserum to rat ornithine decarboxylase.     

A monoclonal antibody to rat liver ornithine decarboxylase

A monoclonal antibody was obtained against rat liver ornithine decarboxylase by using hybridoma technology with a small amount of partially purified enzyme. The antibody, IgG1 of kappa-type, was affinity-purified to homogeneity from culture supernatants of hybridoma cells. While the antibody had no inhibitory effect on ornithine decarboxylase activity when tested alone, it precipitated up to 87 units (60 ng) of the enzyme per microgram in the presence of formalin-fixed Staphylococcus aureus Cowan I bacteria. Immunoadsorption on a column of the monoclonal antibody-Sepharose 4B was shown to be useful for the removal of ornithine decarboxylase from antizyme inhibitor preparations, an essential procedure for the accurate assay of either ornithine decarboxylase-antizyme complex or antizyme inhibitor. It was also shown that antizyme could be affinity-purified by using a column of the monoclonal antibody-Affi-Gel 10 to which ornithine decarboxylase had been bound.     

Comparison of ornithine decarboxylase from rat liver, rat hepatoma and mouse kidney.

Comparisons were made of ornithine decarboxylase isolated from Morris hepatoma 7777, thioacetamide-treated rat liver and androgen-stimulated mouse kidney. The enzymes from each source were purified in parallel and their size, isoelectric point, interaction with a monoclonal antibody or a monospecific rabbit antiserum to ornithine decarboxylase, and rates of inactivation in vitro, were studied. Mouse kidney, which is a particularly rich source of ornithine decarboxylase after androgen induction, contained two distinct forms of the enzyme which differed slightly in isoelectric point, but not in Mr. Both forms had a rapid rate of turnover, and virtually all immunoreactive ornithine decarboxylase protein was lost within 4h after protein synthesis was inhibited. Only one form of ornithine decarboxylase was found in thioacetamide-treated rat liver and Morris hepatoma 7777. No differences between the rat liver and hepatoma ornithine decarboxylase protein were found, but the rat ornithine decarboxylase could be separated from the mouse kidney ornithine decarboxylase by two-dimensional gel electrophoresis. The rat protein was slightly smaller and had a slightly more acid isoelectric point. Studies of the inactivation of ornithine decarboxylase in vitro in a microsomal system [Zuretti & Gravela (1983) Biochim. Biophys. Acta 742, 269-277] showed that the enzymes from rat liver and hepatoma 7777 and mouse kidney were inactivated at the same rate. This inactivation was not due to degradation of the enzyme protein, but was probably related to the formation of inactive forms owing to the absence of thiol-reducing agents. Treatment with 1,3-diaminopropane, which is known to cause an increase in the rate of degradation of ornithine decarboxylase in vivo [Seely & Pegg (1983) Biochem. J. 216, 701-717] did not stimulate inactivation by microsomal extracts, indicating that this system does not correspond to the rate-limiting step of enzyme breakdown in vivo.     

Specific inhibition of the synthesis of putrescine and spermidine by 1,3-diaminopropane in rat liver in vivo.

Chronic administration of 1,3-diaminopropane, a compound inhibiting mammalian ornithine decarboxylase (EC 4.1.1.17) in vivo, effectively prevented the large increases in the concentration of putrescine that normally occur during rat liver regeneration. Furthermore, repeated injections of diaminopropane depressed by more than 85% ornithine decarboxylase activity in rat kidney. Administration of diaminopropane 60 min before partial hepatectomy only marginally inhibited ornithine decarboxylase activity at 4 h after the operation. However, when the compound was given at the time of the operation (4 h before death), or any time thereafter, it virtually abolished the enhancement in ornithine decarboxylase activity in regenerating rat liver remnant. An injection of diaminopropane given 30 to 60 min after operation, but not earlier or later, depressed S-adenosyl-L-methionine decarboxylase activity (EC 4.1.1.50) 4 h after partial hepatectomy. Diaminopropane likewise inhibited ornithine decarboxylase activity during later periods of liver regeneration. In contrast to early regeneration, a total inhibition of the enzyme activity was only achieved when the injection was given not earlier than 2 to 3 h before the death of the animals. Diaminopropane also exerted an acute inhibitory effect on adenosylmethionine decarboxylase activity in 28-h regenerating liver whereas it invariably enhanced the activity of tyrosine aminotransferase (EC 2.6.1.5), used as a standard enzyme of short half-life. Treatment of the rats with diaminopropane entirely abolished the stimulation of spermidien synthesis in vivo from [14C]methionine 4 h after partial hepatectomy or after administration of porcine growth hormone. Both partial hepatectomy and the treatment with growth hormone produced a clear stimulation of hepatic RNA synthesis, the extent of which was not altered by injections of diaminopropane in doses sufficient to prevent any enhancement of ornithine decarboxylase activity and spermidine synthesis.     

Regulation of ornithine decarboxylase activity by putrescine and spermidine in rat liver

The marked enhancement of the activity of ornithine decarboxylase (EC 4.1.1.17) in rat liver at 4 h following partial hepatectomy or the treatment with growth hormone could be almost completely prevented by intraperitoneal administration of putrescine. A single injection of putrescine to partially hepatectomized rats caused a remarkably rapid decline in the activity of liver ornithine decarboxylase with an apparent half-life of only 30 min, which is almost as rapid as the decay of the enzyme activity after the administration of inhibitors of protein synthesis. Under similar conditions putrescine did not have any inhibitory effect on the activity of adenosylmethionine decarboxylase (EC 4.1.1.50) or tyrosine aminotransferase (EC 2.6.1.5). Spermidine given at the time of partial hepatectomy or 2 h later also markedly inhibited ornithine decarboxylase activity at 4 h after the operation and, in addition, also caused a slight inhibition of the activity of adenosylmethionine decarboxylase.     

Effect of alterations in membrane lipid unsaturation on the properties of the insulin receptor of Ehrlich ascites cells

Rat heart ornithine decarboxylate activity from isoproterenol-treated rats was inactivated in vitro by reactive species of oxygen generated by the reaction xanthine/xanthine oxidase. Reduced glutathione, dithiothreitol and superoxide dismutase has a protective effect in homogenates and in partially purified ornithine decarboxylase exposed to the xanthine/xanthine oxidase reaction, while diethyldithiocarbamate, which is an inhibitor of superoxide dismutase, potentiated the damage induced by O2- on enzyme activity. Dithiothreitol at concentrations above 1.25 mM had an inhibitory effect upon supernatant ornithine decarboxylase activity, while at 2.5 mM it was most effective in the recovery of ornithine decarboxylase activity, after the purification of the enzyme by the ammonium sulphate precipitation procedure. The ornithine decarboxylase inactivated by the xanthine/xanthine oxidase reaction showed a higher value of Km and a reduction of Vmax with respect to control activity. The exposure of rats to 100% oxygen for 3 h reduced significantly the isoproterenol-induced heart ornithine decarboxylase activity. The injection with diethyldithiocarbamate 1 h before hyperoxic exposure further reduced heart ornithine decarboxylase activity.     

Changes in mouse kidney ornithine decarboxylase activity are brought about by changes in the amount of enzyme protein as measured by radioimmunoassay

Antibodies were produced in rabbits to homogeneous mouse kidney ornithine decarboxylase and used to determine the amount of this protein present in kidney extracts by a competitive radioimmunoassay procedure. The labeled ligand for this assay was prepared by reacting renal ornithine decarboxylase with [5-3H] alpha-difluoromethylornithine, an enzyme-activated irreversible inhibitor. The sensitivity of the assay was such that 1 ng of protein could be quantitated and the binding to ornithine decarboxylase of a macromolecular inhibitor (antizyme) or alpha-difluoromethylornithine did not affect the reaction. It was found that treatment of female mice with testosterone produced a 400-fold increase in ornithine decarboxylase protein in the kidney within 4-5 days. Exposure to cycloheximide or to 1,3-diaminopropane led to a rapid disappearance of the protein which paralleled the loss of enzyme activity. There was no sign of any immunoreactive but enzymatically inactive form of mouse kidney ornithine decarboxylase under any of the conditions investigated. The results indicate that fluctuations of the enzyme activity in this organ are mediated via changes in the amount of enzyme protein rather than by post-translational modifications or interaction with inhibitors or activators.     

Subcellular distribution of ornithine decarboxylase in rat liver and accessibility of the enzyme to α-difluoromethylornithine,an irreversible inhibitor of ornithine decarboxylase

The subcellular localisation of ornithine decarboxylase and of its synthetic irreversible inhibitor, α-difluoromethylornithine, was investigated in control rat livers and in livers of animals in which the enzyme was induced by partial hepatectomy or by treatment with dexamethasone. Ornithine decarboxylase activity was distributed in normal rat liver between the nuclear (40%, mainly nucleolar) and the cytosolic (43%) fractions. Cytosolic liver ornithine decarboxylase was markedly induced after partial hepatectomy or treatment with dexamethasone, whereas the enzyme associated with the nuclear fraction was not induced by these procedures. The irreversible inhibitor was found only in the cytosol fraction and was totally absent from the nuclear fraction.     

Effect of oxygen radicals and hyperoxia on rat heart ornithine decarboxylase activity

Rat heart ornithine decarboxylase activity from isoproterenol-treated rats was inactivated in vitro by reactive species of oxygen generated by the reaction xanthine/xanthine oxidase. Reduced glutathione, dithiothreitol and superoxide dismutase had a protective effect in homogenates and in partially purified ornithine decarboxylase exposed to the xanthine/xanthine oxidase reaction, while diethyldithiocarbamate, which is an inhibitor of superoxide dismutase, potentiated the damage induced by O₂^? on enzyme activity. Dithiothreitol at concentrations above 1.25 mM had an inhibitory effect oupon supernatant ornithine decarboxylase activity, while at 2.5 mM it was most effective in the recovery of ornithine decarboxylase activity, after the purification of the enzyme by the ammonium sulphate precipitation procedure. The ornithine decarboxylase inactivated by the xanthine/xanthine oxidase reaction showed a higher value of K_m and a reduction of V_max with respect to control activity. The exposure of rates to 100% oxygen for 3 h reduced significantly the isoproterenol-induced heart ornithine decarboxylase activity. The injection with diethyldithiocarbamate 1 h before hyperoxic exposure further reduced heart ornithine decarboxylase activity.     

Measurement of the number of ornithine decarboxylase molecules in rat and mouse tissues under various physiological conditions by binding of radiolabelled α-difluoromethylornithine

The binding of alpha-difluoromethylornithine, an irreversible inhibitor, to ornithine decarboxylase was used to investigate the amount of enzyme present in rat liver under various conditions and in mouse kidney after treatment with androgens. Maximal binding of the drug occurred on incubation of the tissue extract for 60min with 3mum-difluoromethyl[5-(14)C]ornithine in the presence of pyridoxal phosphate. Under these conditions, only one protein became labelled, and this corresponded to ornithine decarboxylase, having M(r) about 100000 and subunit M(r) about 55000. Treatment of rats with thioacetamide or carbon tetrachloride or by partial hepatectomy produced substantial increases in ornithine decarboxylase activity and parallel increases in the amount of enzyme protein as determined by the extent of binding of difluoromethyl[5-(14)C]ornithine. Similarly, treatment with cycloheximide or 1,3-diaminopropane greatly decreased both the enzyme activity and the amount of difluoromethyl-[5-(14)C]ornithine bound to protein. In all cases, the ratio of drug bound to activity was 26fmol/unit, where 1 unit corresponds to 1nmol of substrate decarboxylated in 30min. These results indicate that even after maximal induction of the enzyme in rat liver there is only about 1ng of enzyme present per mg of protein. When mice were treated with androgens there was a substantial increase in renal ornithine decarboxylase activity, the magnitude of which depended on the strain. There was an excellent correspondence between the amount of activity present and the capacity to bind labelled alpha-difluoromethylornithine in the mouse kidney extracts, but in this case the ratio of drug bound to activity was 14fmol/unit, suggesting that the mouse enzyme has a higher catalytic-centre activity. After androgen induction, the mouse kidney extracts contain about 170ng of enzyme/mg of protein. These results indicate that titration with alpha-difluoromethylornithine provides a valuable method by which to quantify the amount of active ornithine decarboxylase present in mammalian tissues, and that the androgen-treated mouse kidney is a much better source for purification of the enzyme than is rat liver.     

Difluoromethylornithine irreversibly inactivates ornithine decarboxylase of Pseudomonas aeruginosa, but does not inhibit the enzymes of Escherichia coli.

DL-alpha-Difluoromethylornithine, an enzyme-activated irreversible inhibitor of eukaryotic ornithine decarboxylase and consequently of putrescine biosynthesis, inhibited ornithine decarboxylase in enzyme extracts from Pseudomonas aeruginosa in a time-dependent manner t1/2 1 min, and also effectively blocked the enzyme activity in situ in the cell. Difluoromethylornithine, however, had no effect on the activity of ornithine decarboxylase assayed in enzyme extracts from either Escherichia coli or Klebsiella pneumoniae. However, the presence of the inhibitor in cell cultures did partially lower ornithine decarboxylase activity intracellularly in E. coli. Any decrease in the intracellular ornithine decarboxylase activity observed in E. coli and Pseudomonas was accompanied by a concomitant increase in arginine decarboxylase activity, arguing for a co-ordinated control of putrescine biosynthesis in these cells.     

Aminooxy analogues of spermidine as inhibitors of spermine synthase and substrates of hepatic polyamine acetylating activity

Aminooxy analogues of spermidine, 1-aminooxy-3-N-[3-aminopropyl]- aminopropane (AP-APA) and N-[2-aminooxyethyl]-1,4-diaminobutane (AOE-PU), were tested as substrates or inhibitors of the enzymes involved in methionine and polyamine metabolism. Both compounds were good competitive inhibitors and poor substrates of spermine synthase, good substrates of cytosolic polyamine acetyltransferase, inactivators of S-adenosylmethionine decarboxylase and inhibitors of ornithine decarboxylase. AP-APA and AOE-PU showed K1-values of 1.5 and 186 microM as inhibitors of purified spermine synthase, and Km-values of 1.4 and 2.1 mM as substrates of the crude hepatic polyamine acetyltransferase activity. AP-APA was more potent than AOE-PU in crude enzyme preparations. Neither drug had any significant effect at 1 mM concentration on the activities of spermidine synthase, methionine adenosyltransferase, S-adenosylhomocysteine hydrolase, and methylthioadenosine phosphorylase. The results suggest that compounds of this type are valuable tools in unraveling the physiology of polyamines.     

Immunochemical demonstration of increased accumulation of ornithine decarboxylase in rat liver after partial hepatectomy and growth hormone induction

Antiserum against ornithine decarboxylase (EC 4.1.1.17) was prepared in rabbits using purified ornithine decarboxylase from rat liver as the antigen. Immunoglobulins from the immune sera were covalently coupled to agarose by cyanogen bromide activation. With the aid of this immunoadsorbent against the enzyme it has been shown that following partial hepatectomy and growth hormone administration, the ornithine decarboxylase activity is elevated concomitantly with the increase in the immunoreactive enzyme protein. In addition, the rapid decay in ornithine decarboxylase activity in regenerating rat liver after cycloheximide injection is accompanied by a decrease in the immunoreactive protein. These results suggest that the activity of ornithine decarboxylase in rat liver is regulated through rapid changes in de novo synthesis and degradation of the enzyme protein.     

Regulation of S-adenosyl-L-methionine decarboxylase by 1-aminooxy-3-aminopropane: enzyme kinetics and effects on the enzyme activity in cultured cells

The kinetics of inactivation of adenosylmethionine decarboxylase of rat liver and of baby hamster kidney cells (BHK21/C31) by 1-aminooxy-3-aminopropane was studied. The apparent dissociation constants (Ki) for the hepatic and BHK21/C13 enzymes were 1.5 and 2.0 mM and the times of half-inactivation at infinite concentration of the inhibitor (tau 1/2) were 1.2 and 3.8 min, respectively. Treatment of BHK21/C13 with 0.5 mM 1-aminooxy-3-aminopropane prevented cell growth and depleted the cells of putrescine and spermidine within 1 day. The depletion of spermidine resulted in increased activity of S-adenosylmethionine decarboxylase which was due, at least partly, to the increase in the half-life of the enzyme activity. Because spermine levels were not significantly affected, it appears that spermidine is the principal feedback regulator of S-adenosylmethionine decarboxylase. So, 1-aminooxy-3-aminopropane is a very weak inhibitor of S-adenosylmethionine decarboxylase and the cellular effects can be correlated primarily with its inhibitory effects on ornithine decarboxylase and spermidine synthase. In cell-free systems, however, 1-aminooxy-3-aminopropane is likely to find use in unraveling the reaction mechanism of S-adenosylmethionine decarboxylase.