A rat monoclonal antibody which interacts with mammalian ornithine decarboxylase at an epitope involved in phosphorylation

Ornithine decarboxylase was purified from androgen-treated mouse kidney to homogeneity and high specific activity. The purified enzyme was utilized for production and screening of rat monoclonal and polyclonal antibodies. A rat monoclonal antibody was isolated which was capable of immunoprecipitation of native mouse kidney ornithine decarboxylase activity or the [3H]difluoromethylornithine-inactivated enzyme. Phosphorylation of mouse ornithine decarboxylase by casein kinase-II prior to immunoprecipitation led to complete loss of the epitope recognized by the monoclonal antibody but did not alter recognition by polyclonal antibody. Mammalian ornithine decarboxylase activity obtained from several species, in crude or partially purified extracts, was subjected to quantitative immunoprecipitation with monoclonal and polyclonal antibody. Polyclonal antibody immunoprecipitated all of the ornithine decarboxylase activity from every extract tested, while monoclonal antibody was capable of only limited immunoprecipitation (60-80%). Due to the inability of the monoclonal antibody to recognize ornithine decarboxylase phosphorylated in vitro by casein kinase-II and the partial immunoprecipitation of ornithine decarboxylase activity from cell extracts, a portion of the ornithine decarboxylase molecule population must exist in a phosphorylated state. This immunological evidence further confirms existing data that the enzyme exists in at least two distinct forms.     

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.     

Investigation of structure and rate of synthesis of ornithine decarboxylase protein in mouse kidney

An immunoblotting technique was used to study the forms of ornithine decarboxylase present in androgen-induced mouse kidney. Two forms were detected which differed slightly in isoelectric point but not in subunit molecular weight (approximately 55 000). Both forms were enzymatically active and could be labeled by reaction with radioactive alpha-(difluoromethyl)-ornithine, an enzyme-activated irreversible inhibitor. On storage of crude kidney homogenates or partially purified preparations of ornithine decarboxylase, the enzyme protein was degraded to a smaller size (Mr approximately 53 000) without substantial loss of enzyme activity. The synthesis and degradation of ornithine decarboxylase protein were studied by labeling the protein by intraperitoneal injection of [35S]methionine and immunoprecipitation using both monoclonal and polyclonal antibodies. The fraction of total protein synthesis represented by renal ornithine decarboxylase was increased at least 25-fold by testosterone treatment of female mice and was found to be about 1.1% in the fully induced androgen-treated female. Both forms of the enzyme were rapidly labeled in vivo, and the immunoprecipitable ornithine decarboxylase protein was almost completely lost after 4-h exposure to cycloheximide, confirming directly the very rapid turnover of this enzyme. Treatment with 1,3-diaminopropane which is known to cause a great reduction in ornithine decarboxylase activity did not greatly selectively inhibit the synthesis of the enzyme. However, 1,3-diaminopropane did produce an increase in the rate of degradation of ornithine decarboxylase and a general reduction in protein synthesis. These two factors, therefore, appear to be responsible for the loss of ornithine decarboxylase activity and protein in response to 1,3-diaminopropane.     

Studies of mammalian ornithine decarboxylase using a monoclonal antibody.

A monoclonal antibody of the immunoglobulin M class was produced against mouse kidney ornithine decarboxylase. Screening for the antibody was carried out using alpha-difluoromethyl[5-3H]ornithine-labelled ornithine decarboxylase. The antibody reacted with this antigen and with native ornithine decarboxylase. The antibody attached to Sepharose could be used to form an immunoaffinity column that retained mammalian ornithine decarboxylase. The active enzyme could then be eluted in a highly purified form by 1.0M-sodium thiocyanate. The monoclonal antibody could also be used to precipitate labelled ornithine decarboxylase from homogenates of kidneys from androgen-treated mice given [35S]methionine. Only one band, corresponding to Mr of about 55000, was observed. The extensive labelling of this band is consistent with the rapid turnover of ornithine decarboxylase protein, since this enzyme represents only about 1 part in 10000 of the cytosolic protein.     

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.     

Phosphorylation of ornithine decarboxylase in intact erythroleukemia cells

32P-labeled ornithine decarboxylase was isolated by immunoprecipitation from murine erythroleukemia cells incubated in a medium containing [32P]ortophosphoric acid. Analysis of immunoprecipitate by SDS-polyacrylamide gel electrophoresis and autoradiography revealed a radiolabeled band, which corresponded to the position of mouse ornithine decarboxylase, phosphorylated in vitro by casein kinase-2. A preparation of casein kinase-2 purified from nuclei of erythroleukemia cells could also phosphorylate mouse ornithine decarboxylase.     

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.     

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.     

Ornithine decarboxylase from Saccharomyces cerevisiae. Purification, properties, and regulation of activity

Ornithine decarboxylase has been purified 1,500-fold to homogeneity from a spe2 mutant of Saccharomyces cerevisiae which lacks S-adenosylmethionine decarboxylase and is derepressed for ornithine decarboxylase. The ornithine decarboxylase is a single polypeptide (Mr = 68,000) and requires a thiol and pyridoxal phosphate for activity. Addition of 10(-4) M spermidine and 10(-4) M spermine to the growth medium reduces the activity of the enzyme by 90% in 4 h. However, immunoprecipitation studies showed that the extracts of polyamine-treated cells contain as much enzyme protein as normal cell extracts. This loss of ornithine decarboxylase activity is probably due to a post-translational modification of enzyme protein because we found no evidence for any inhibitor of activity in the polyamine-treated cells.     

Multiple ionic forms of ornithine decarboxylase differ in degree of phosphorylation

Two major ionic forms of ornithine decarboxylase were separated by column chromatography of extracts of kidneys from androgen-treated male CD-1 mice on DEAE-Sepharose CL-6B, and purified individually to apparent homogeneity. On SDS-PAGE, a single major protein band of Mr 50000 was present in each. When incubated with casein kinase II, purified from rat liver cytosol, only one form of the enzyme, which represented 20% of the total ornithine decarboxylase in the tissue, became phosphorylated. The major form, which was eluted later from the column, could be phosphorylated only after treatment with alkaline phosphatase, indicating that the phosphatase removed enzyme-bound phosphate already attached at the casein kinase II phosphorylation site. Evidence for the occurrence of a phosphorylated form of the enzyme in kidneys of dexamethasone-treated rats is also presented.     

Stabilization of ornithine decarboxylase and N1-spermidine acetyltransferase in rat liver by methylglyoxal bis(guanylhydrazone)

The activities of ornithine decarboxylase and spermidine N1-acetyltransferase started to rise in normal rat liver 4 h after the intraperitoneal injection of methylglyoxal bis(guanylhydrazone) (MGBG; 80 mg/kg). Ornithine decarboxylase had its greatest activity 24 h after a single injection of MGBG and the acetyltransferase peaked 8 h after the injection. Measurement of the apparent half-life of ornithine decarboxylase after MGBG treatment revealed a clear decrease in the decay rate of the enzyme in both normal and regenerating rat liver. MGBG slowed the decay of the transferase also in normal rat liver, as well as inhibiting its activity in vitro. The stabilization by MGBG of these two short-lived proteins involved in metabolism of polyamines should lead to their accumulation in liver, thus explaining their increased activities. In the case of ornithine decarboxylase, studies with a specific antibody against mouse kidney ornithine decarboxylase showed that the rise in ornithine decarboxylase activity after MGBG application was not due to the appearance of an immunologically different isozyme.     

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.     

Phosphorylation of ornithine decarboxylase by casein kinase II from RAW264 cells

Casein kinase II and ornithine decarboxylase were purified from a virally-transformed macrophage-like cell line, RAW264. The addition of casein kinase II to a reaction mixture containing [tau-32P]GTP, Mg++, and ornithine decarboxylase led to the phosphorylation of a 55,000 dalton protein band in the purified preparation of ornithine decarboxylase. Stoichiometric estimates indicated that casein kinase II incorporated 0.15 mole of phosphate per mole of ornithine decarboxylase, which was increased to 0.3 mole/per mole in the presence of spermine. The apparent Km and Vmax values for the casein kinase II-mediated phosphorylation of ornithine decarboxylase were 0.36 microM and 62.5 nmol/min./mg kinase. The addition of spermine to the reaction did not alter the Km but increased the Vmax to 100 nmol/min./mg kinase. The phosphorylation of ornithine decarboxylase by casein kinase II affected neither the rate of maximal ornithine decarboxylase activity nor the affinity of the enzyme for ornithine.     

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.     

Androgen-regulated ornithine decarboxylase mRNAs of mouse kidney

Ornithine decarboxylase, the first enzyme of the polyamine biosynthetic pathway, is induced by androgens in the mouse kidney. We have isolated from a kidney cDNA clone bank two plasmids, pODC1440 and pODC934 , that contain different cDNA inserts corresponding to ornithine decarboxylase mRNA. Identification was based upon the ability of plasmid-specific mRNAs to encode a 53,000-dalton polypeptide that reacts with antibody to purified mouse kidney ornithine decarboxylase. Plasmid pODC1440 hybridizes predominantly to a mRNA that is 2.1 kilobases (kb) long and is induced about 20-fold in the kidneys of female mice treated with testosterone. Plasmid pODC934 hybridizes to mRNAs of lengths 2.2 and 2.6 kb, which are induced about 8-fold by testosterone. There are probably no more than 1-2 copies of the pODC1440 -specific sequence in the mouse genome, while there may be as many as 12 copies of the pODC934 -specific sequence. That the two plasmids correspond to different ornithine decarboxylase mRNAs is suggested by two observations. First, several mouse strains express normal levels of pODC934 -specific RNA and little or no pODC1440 -specific RNA; furthermore, pODC934 -specific RNA is expressed in several tissues while pODC1440 -specific RNA is kidney-specific. Thus, androgen-mediated stimulation of kidney ornithine decarboxylase activity levels involves alterations in the concentrations of at least two distinct mRNAs.     

Phosphorylation of rat heart ornithine decarboxylase by type-2 casein kinase

Highly purified preparations of rat heart ornithine decarboxylase are readily phosphorylated by rat liver type-2 casein kinase-TS at the same 54 KDa protein band which is also radiolabeled by 3H-DFMO. The reaction, which is stimulated by polylysine leads to the incorporation of up to 0.8 mol P/mol ornithine decarboxylase at seryl residue(s) included in a single 8.6 KDa CNBr fragment. Partially purified preparations of ornithine decarboxylase contain a type-2 casein kinase which promotes the phosphorylation of ornithine decarboxylase at the same CNBr fragment affected by rat liver casein kinase-TS.     

Two-Step Purification of Mouse Kidney Ornithine Decarboxylase

Abstract

We developed a simple two-step purification procedure for ornithine decarboxylase (ODC, EC 4. 1. 1. 17), consisting of DEAE-Cellulofine chromatography and affinity chromatography on a HO-101 monoclonal anti-rat liver ODC antibody-Affi-Gel 10 column. By this method, ODC was purified 1700-fold to homogeneity with about 80% yield from the kidney of ICR mice treated with testosterone enanthate. The final specific activity range between 1. 0 × 10⁶?1. 4 × 10⁶ nmol/h. mg protein. On SDS-polyacrylamide gel electrophoretic analysis, the final preparations gave a major protein band of Mr 54, 000 and a minor band of Mr 51, 000. Although relative staining intensity of the two bands varied depending on preparations, both bands could be stained by immunoblotting and labeled by a preincubation with [¹⁴C)difluoromethylornithine (DFMO). On Oudin double diffusion immunoanalysis, a single fused precipitin line was formed between purified anti-mouse kidney ODC IgG and both the purified enzyme and crude mouse kidney extract. In contradiction with earlier reports, no significant difference was observed between mouse kidney ODC and rat liver ODC in either final specific activity or specific binding of labeled DFMO.     

Two-step purification of mouse kidney ornithine decarboxylase

We developed a simple two-step purification procedure for ornithine decarboxylase (ODC, EC 4.1.1.17), consisting of DEAE-Cellulofine chromatography and affinity chromatography on a HO-101 monoclonal anti-rat liver ODC antibody-Affi-Gel 10 column. By this method, ODC was purified 1700-fold to homogeneity with about 80% yield from the kidney of ICR mice treated with testosterone enanthate. The final specific activity range between 1.0 x 10(6)-1.4 x 10(6) nmol/h.mg protein. On SDS-polyacrylamide gel electrophoretic analysis, the final preparations gave a major protein band of Mr 54,000 and a minor band of Mr 51,000. Although relative staining intensity of the two bands varied depending on preparations, both bands could be stained by immunoblotting and labeled by a preincubation with [14C]difluoromethylornithine (DFMO). On Oudin double diffusion immunoanalysis, a single fused precipitin line was formed between purified anti-mouse kidney ODC IgG and both the purified enzyme and crude mouse kidney extract. In contradiction with earlier reports, no significant difference was observed between mouse kidney ODC and rat liver ODC in either final specific activity or specific binding of labeled DFMO.     

Hepatic ornithine decarboxylase from the frog, Rana negromaculata: dietary induction, purification and some properties

1. In the liver of the frog, Rana negromaculata, the activity of ornithine decarboxylase (ODC) was induced by dietary stimuli and was rapidly lost upon intraperitoneal injection of cycloheximide or putrescine. 2. Frog liver ODC, purified by DEAE-Cellulofine and immunoaffinity column chromatographies, was used in a comparative study with mouse kidney ODC, also purified by the same method. 3. The purified frog ODC showed three bands on SDS-polyacrylamide gel electrophoretic analysis, as confirmed by [3H]alpha-difluoromethylornithine binding. 4. Frog ODC was found to be similar to mouse enzyme in some properties, for example molecular weight, immunoreactivity and inhibition by rat antizyme, except for a slightly higher Km value for ornithine.     

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.