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.     

Studies on the mechanisms of ornithine decarboxylase in vitro inactivation

Hydrocortisone-induced rat liver ornithine decarboxylase appears quite stable in the soluble fraction of the homogenate incubated at 37 degrees C. In contrast, the incubation of the whole homogenate causes a rapid loss of activity. The ornithine decarboxylase-inactivating capacity appears mainly bound to microsomes. Lysosomes seem to play a role only after the microsome-induced inactivation. Different reducing agents (dithiothreitol, NADPH, NADH, GSH) are effective both in preventing and in reversing ornithine decarboxylase inactivation. NADPH is peculiar in that it can reactivate the enzyme at very low concentrations. Oxidized glutathione potentiates the inactivating effect of microsomes. On the basis of present results it is suggested that ornithine decarboxylase may be reversibly inactivated through microsome-catalyzed formation of mixed or enzyme-enzyme disulfides and that NADPH plays a crucial role in ornithine decarboxylase reactivation, probably by cytosolic reductase(s).     

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.     

Circadian rhythm of ornithine decarboxylase and its endogenous high molecular weight inhibitor in rat pineal gland]

The biochemical mechanisms involved in circadian variations of the activity of ornithine decarboxylase (EC 4.1.1.17)--the rate-limiting enzyme of polyamine biosynthesis in rat pineal gland were studied. The enzyme was separated from its endogenous high molecular weight inhibitor by gel-filtration of the cytosol fraction from this organ through Sephadex G-100 in the presence of 250 mM NaCl. The inhibitor was similar in its molecular weight (30 000) and activity to ornithine decarboxylase inhibior from rat liver. The amount of the enzyme in the pineal gland undergoes much smaller circadian variations as compared to that of the inhibitor. It is concluded that the circadian variations of the ornithine decarboxylase activity in the pineal gland may be largely due to the changes in the enzyme/inhibitor ratio.     

Nuclear ornithine decarboxylase. Electron microscope autoradiographic identification of the active enzyme using alpha-[5-3H]difluoromethylornithine

alpha-Difluoromethylornithine (DFMO) is an enzyme-activated irreversible inhibitor of ornithine decarboxylase, that forms a covalent bond with the active enzyme. The highly selective binding of tritium-labeled DFMO to ornithine decarboxylase in vivo, as identified by electron microscope autoradiography, was used to determine the intracellular distribution of the enzyme in the germ cells of a polychaete (Ophryotrocha labronica). In mid-oogenesis ornithine decarboxylase was predominantly located in the nurse cells, which are actively supporting growth of the oocytes. On the basis of biochemical analyses ornithine decarboxylase has been considered mainly cytoplasmic in its distribution. However, in metabolically active polychaete cells (oocytes, nurse cells, intestinal and body wall cells), binding sites for tritiated DMFO, indicating the presence of active ornithine decarboxylase, were as abundant in the nucleus. The nucleolus was the most densely labeled organelle in nurse cells and oocytes.     

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.     

Activation of ornithine decarboxylase in monolayer cells treated with trypsin

When monolayer Chinese hamster cells are treated with trypsin for short periods of time, ornithine decarboxylase (ODCase) activity increases two- to fourfold. This increase can be blocked by aprotinin, a protease inhibitor, and is not observed when cultures are dislodged from substrate mechanically prior to contact with exogenous trypsin. The trypsin-induced increase in ornithine decarboxylase activity is not due to degradation of enzyme or inhibitor molecules or to new enzyme synthesis. Immunoprecipitable protein, radiolabeled with [3H]alpha-difluoromethylornithine in vitro, is the same molecular weight in cells harvested with or without trypsin. Protein-bound levels of this specific enzyme-activated irreversible inhibitor of ornithine decarboxylase are unchanged by trypsin treatments that increase enzyme activity. Trypsin treatment of rat embryonic fibroblasts, transformed by a temperature-sensitive mutant of Rous sarcoma virus, increases ODCase activity in cells growing at the nonpermissive, but not at the permissive, temperature for the transformed phenotype. These results suggest that ornithine decarboxylase can be activated by exogenous trypsin treatment in a manner that is dependent on cell adhesion properties, which are modified in transformed cells.     

The appearance of an ornithine decarboxylase inhibitory protein upon the addition of putrescine to cell cultures.

Quiescent, contact inhibited H-35 rat hepatoma cell cultures maintained in minimal essential medium contain a very low level of ornithine decarboxylase activity. However, 2 h after the addition of 10% fetal calf serum to the culture medium, the enzyme activity increases by approx. 100-fold. This increase can be completely inhibited by the simultaneous addition of 10(-2) M putrescine. The presence of putrescine elicits the appearance of an intracellular inhibitor of ornithine decarboxylase. This inhibitor of ornithine decarboxylase has a molecular weight of 26500, is sensitive to the action of chymotrypsin and is noncompetitive with respect to ornithine. The intracellular appearance of this inhibitor is sensitive to cycloheximide but is only partially inhibited by actinomycin D.     

Induction of ornithine decarboxylase activity by growth and differentiation factors in FRTL-5 cells

Induction of ornithine decarboxylase has been correlated with the onset of cellular proliferation and cAMP production. Whether the resulting increases in polyamine levels are essential mediators of growth and/or differentiation or are merely incidental remains controversial. We have used FRTL-5 thyroid cells in culture to study the effects of three growth factors on ornithine decarboxylase activity. These factors [TSH, bovine calf serum, and 12-O-tetradecanoylphorbol-13-acetate (TPA)] are thought to act through different intracellular pathways. TSH stimulates cAMP production in thyroid cells, calf serum acts through ill-defined pathways to stimulate growth, and TPA is known to activate protein kinase C. Bovine calf serum and TSH acted synergistically to induce ornithine decarboxylase activity. Activity was maximal when the phosphodiesterase inhibitor, methyl isobutyl xanthine, was included. Individually, neither serum nor TSH was a potent stimulator of the enzyme. Ornithine decarboxylase mRNA was apparent on Northern blots as a doublet following one hour of exposure to these agents. TPA did not stimulate ornithine decarboxylase activity and had an inhibitory effect on enzyme induction by TSH and serum. Difluoromethylornithine, a specific inhibitor of ornithine decarboxylase, inhibited growth induced by both TPA and TSH in putrescine-free medium. This effect was not apparent in medium containing 10(-5) M putrescine. The data indicate that, although intracellular levels of cyclic AMP regulate ornithine decarboxylase activity, a component in serum is necessary for significant induction of this enzyme. Factors stimulating growth by non-cyclic AMP-dependent pathways may act without apparently stimulating this enzyme, although polyamines appear to be essential for their growth stimulatory effects.     

Studies of new intracellular proteases in various organs of rats. Participation of proteases in degradation of ornithine aminotransferase in vitro and in vivo.

Homogenates of the muscle layer of rat small intestine irreversibly inactivated endogenous ornithine aminotransferase at 37 degrees C. Addition to the homogenate of coenzymes and the various keto-acids which act as substrate inhibited conversion of the holoenzyme to the apoenzyme and its subsequent degradation. Addition of protease inhibitors including soybean trypsin inhibitor, chymostatin and phenylmethylsulfonyl fluoride almost completely prevented inactivation of he enzyme. Immunological activity decreased during inactivation of the enzyme, but its rate of decrease was much slower than that of loss of enzyme activity. Antigen-antibody precipitates from homogenates containing inactivated enzyme, were separated by electrophoresis on sodium dodecylsulfate-polyacrylamide gels. In this way breakdown products of the enzyme were found, indicating that the enzyme in homogenates was inactivated by limited proteolysis. These results obtained in vitro support our previous suggestion (1975) of a stepwise mechanism for degradation of pyridoxal enzymes.     

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.     

Distinct domains of antizyme required for binding and proteolysis of ornithine decarboxylase.

Selective degradation by proteasomes of ornithine decarboxylase, the initial enzyme in polyamine biosynthesis, is mediated by the polyamine-inducible protein antizyme. Antizyme binds to a region near the N terminus of ornithine decarboxylase (X. Li and P. Coffino, Mol. Cell. Biol. 12:3556-3562, 1992). This interaction induces a conformational change in ornithine decarboxylase that exposes its C terminus and inactivates the enzyme (X. Li and P. Coffino, Mol. Cell. Biol. 13:1487-1492, 1993). Here we show that the C-terminal half of antizyme alone can inactivate ornithine decarboxylase and alter its conformation, but it cannot direct degradation of the enzyme, either in vitro or in vivo. A portion of the N-terminal half of antizyme must be present to promote degradation.     

The appearance of an ornithine decarboxylase inhibitory protein upon the addition of putrescine to cell cultures

Quiescent, contact inhibited H-35 rat hepatoma cell cultures maintained in minimal essential medium contain a very low level of ornithine decarboxylase activity. However, 2 h after the addition of 10% fetal aclf serum to the culture medium, the enzyme activity increases by approx. 100-fold. This increase can be completely inhibited by the simultaneous additionof 10^?2 M putrescine. The presence of putrescine elicits the appearance of an intracellular inhibitor of ornithine decarboxylase. This inhibitor of ornithine decarboxylase has a molecular weight of 26500, is sensitive to the action of chymotrypsin and its noncompetitive with respect to ornithine. The intracellular appearance of this inhibitor is sensitive to cycloheximide but is only partially inhibited by actinomycin D.     

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.     

Affinity labeling of pig kidney 3,4-dihydroxyphenylalanine (Dopa) decarboxylase with N-(bromoacetyl)pyridoxamine 5'-phosphate. Modification of an active-site cysteine

Pig kidney 3,4-dihydroxyphenylalanine (Dopa) decarboxylase is inactivated by N-(bromoacetyl)pyridoxamine 5'-phosphate (BAPMP) in a reaction which follows first-order kinetics at pH 7.5 and 25 degrees C. The concentration dependence of inactivation reveals saturation kinetics with an apparent Ki of 0.16 mM and kinact of 0.086 min-1 at saturating inhibitor concentration. Enzyme can be protected from inactivation by pyridoxal 5'-phosphate. Inactivation of enzyme by [14C]BAPMP proceeds with the incorporation of a stoichiometric amount of labeled inhibitor. Proteolytic digestions of the radioactively labeled enzyme followed by high-performance liquid chromatography allow the isolation of the modified peptide corresponding to the sequence Ala-Ala-Ser-Pro-Ala-Cys-Thr-Glu-Leu in which cysteine (Cys111) is the modified residue. The conservation of this residue and also of an extended region around it in all Dopa decarboxylases so far sequenced is underlined. The overall conclusion of these findings is that Cys111 may be at, or near, the pyridoxal-5'-phosphate binding site of pig kidney Dopa decarboxylase and plays a critical role in the catalytic function of the enzyme. Furthermore, fluorescence studies of BAPMP-modified apoenzyme provide useful information on the microenvironment of the affinity label at its binding site.     

Mechanism of inactivation of ornithine decarboxylase by alpha-methylornithine

Ornithine decarboxylase from Lactobacillus 30a is gradually inactivated by treatment with alpha-methylornithine, but activity is restored by treatment of the inactivated enzyme with pyridoxal phosphate. Inactivation of the enzyme is associated with formation of pyridoxamine phosphate and 5-amino-2-pentanone, alpha-Methylornithine is decarboxylated by the enzyme about 6000 times more slowly than is ornithine under the same conditions. These observations provide an explanation for the previously observed inhibition of ornithine decarboxylase by alpha-methylornithine [M. M. Adbel-Monem, N. E. Newton, and C. E. Weeks (1974), J. Med. Chem. 17, 4447]: alpha-Methylornithine undergoes a decarboxylation-dependent transamination as a result of incorrect protonation of the quinoid intermediate which is formed by decarboxylation of the enzyme-bound pyridoxal phosphate-substrate Schiff base. This protonation produces inactive enzyme. Decarboxylation of ornithine by this enzyme produces a small amount of 4-aminobutanal, presumably also by decarboxylation-dependent transamination.     

Ornithine decarboxylase in difluoromethylornithine-resistant mouse lymphoma cells. Two-dimensional gel analysis of synthesis and turnover

Variant S49 mouse lymphoma cells with increased ornithine decarboxylase activity were obtained by selecting for resistance to alpha-difluoromethylornithine (DFMO), a specific inhibitor of the enzyme. Ornithine decarboxylase was identified as a specifically immunoprecipitable polypeptide that was made at an increased rate in the variant cells. Ornithine decarboxylase was also identified on a two-dimensional gel as a metabolically labeled polypeptide of Mr approximately 55,000 which was synthesized at an increased rate in two independently selected variants. Synthesis of this polypeptide was further augmented by treatment of cells with inhibitors of ornithine decarboxylase activity. The charge of the polypeptide was altered by treatment of either cells or cellular extracts with DFMO, a suicide substrate which binds covalently to the enzyme. This charge alteration and the inactivation of ornithine decarboxylase showed the same dependence on DFMO concentration and both effects were prevented by addition of either ornithine or putrescine. Pulse-chase experiments showed that the half-life of the ornithine decarboxylase polypeptide in these variant cells was 45 min. We conclude that ornithine decarboxylase is made at an increased rate in the resistant variants and that the polypeptide turns over rapidly.     

Stereospecific modulation of the calcium channel in human erythrocytes by cholesterol and its oxidized derivatives.

A marked decrease in activity of ornithine decarboxylase in thymus and spleen occurs soon after treatment of rats with a glucocorticoid. In the present study, evidence was obtained that extracts of these tissues prepared 5 h after administration of dexamethasone, when the enzyme activity is very low, contain an inhibitor of ornithine decarboxylase. The inhibitor is also present at 12 h after treatment and, in lesser amount, at 2.5 h, but was not evident at 24 h. The inhibitory activity was destroyed by treatment with heat or with trypsin, and was not lost on dialysis of the extract. Preliminary experiments indicate that the Mr of the inhibitor is greater than 50 000, which differentiates it from antizyme, an inhibitor of ornithine decarboxylase found in several other cell types. The inhibitor seems to act by a non-catalytic and non-competitive mechanism. The inhibition is dependent on the amount of inhibitor and does not change with time. Since inhibition is not changed by dialysis of the inhibitory extract, its activity apparently does not require small-Mr substances. This differentiates it from inhibitors which inactivate ornithine decarboxylase by covalent modification, such as the polyamine-dependent protein kinase or transglutaminase. The formation of this inhibitor is an early event in lymphoid tissues in response to dexamethasone and may be important in causing the inhibition of cell division which precedes the destruction of lymphocytes.     

The effect of 1,4-diaminobutanone on the stability of ornithine decarboxylase from Aspergillus nidulans.

1,4-Diaminobutanone, a competitive inhibitor of ornithine decarboxylase in Aspergillus nidulans, is able to increase the half-life of this enzyme and thus stimulate an increase in its activity in vivo. It also protects ornithine decarboxylase against proteolysis by chymotrypsin in vitro.     

Cloning and sequencing of the ornithine decarboxylase gene from Trypanosoma brucei. Implications for enzyme turnover and selective difluoromethylornithine inhibition

Ornithine decarboxylase of the African trypanosome Trypanosoma brucei brucei had an estimated native molecular weight of 100,000 by gel filtration and a subunit molecular weight of 45,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gene encoding this enzyme, present in a single copy in T. brucei, was identified by mouse ornithine decarboxylase cDNA under relatively stringent conditions of hybridization and subcloned in a 5.9-kilobase (kb) SstI fragment from a cosmid clone into the plasmid pUC 19. This clone encompassed a 2.8-kb SstII fragment that contained the entire T. brucei ornithine decarboxylase gene. The 2.8-kb SstII fragment hybridized to a 2.4-kb mRNA that presumably encodes the parasite enzyme. The 2.8-kb SstII fragment was partially sequenced and found to contain an open reading frame of 445 amino acids that has 61.5% homology with the corresponding sequence of the mouse enzyme. The only major discrepancies between the two enzymes are the addition of a 20-amino acid N-terminal peptide and the deletion of a 36-amino acid C-terminal peptide and the T. brucei ornithine decarboxylase. The C terminus has been postulated to be one of the structural factors associated with rapid in vivo turnover of mammalian ornithine decarboxylase. The absence of this C-terminal peptide in T. brucei ornithine decarboxylase predicts a slow turnover for the parasite enzyme in vivo, and this is supported by our experimental data. The lack of turnover of ornithine decarboxylase in trypanosomes may constitute the basis of selective antitrypanosomal action of the irreversible enzyme inhibitor DL-alpha-difluoromethylornithine.