Purification of ornithine decarboxylase from regenerating rat liver

Ornithine decarboxylase was purified 175-fold over the crude 100 000 × g supernatant from homogenates of regenerating rat liver. It exhibited a single major band on acrylamide gels and a minor contaminant which may represent partially degraded enzyme. Antibody prepared against this enzyme gave a single precipitin line on Ouchterlony plates. The enzyme was precipitated by the antibody and substantial activity could be recovered from the immune precipitate. Several properties of the enzyme are described including differential effects of mercaptoethanol and dithiothreitol on enzyme activity.     

Dexamethasone restores hormonal inducibility of ornithine decarboxylase in primary cultures of rat hepatocytes

Induction of ornithine decarboxylase by various hormones was studied in quiescent primary cultures of adult rat hepatocytes maintained in a chemically defined medium. The following results were obtained: Enzyme activity rose transiently during the first day of cultivation in hormone-untreated cells. During this phase, insulin increased ornithine decarboxylase activity. Inducibility by insulin was maintained for more than 40 h only after pretreatment with 0.1 microM dexamethasone. Enzyme activity could be induced by 1 nM insulin and peaked after 7 h. Inducibility by glucagon and growth hormone required pretreatment with the glucocorticoid hormone. Ornithine decarboxylase activity was maximal 5 h after glucagon addition. Concentrations down to 0.1 nM were effective. Pretreatment with dexamethasone was most effective, when the hormone was present during the first 20 h of cultivation. The effect of the glucocorticoid during the pretreatment phase was diminished by colchicine and to a lesser extent by cytochalasine B. We suggest that part of the permissive effect of dexamethasone could be mediated by changes in the cytoskeleton and the function of hormone receptors. The fact that induction of ornithine decarboxylase was exerted by several hormones despite the absence of cell proliferation and DNA synthesis may indicate that polyamine biosynthesis has an important role in the quiescent hepatocyte.     

Characterization of highly purified ornithine decarboxylase from rat heart

The fatty acid composition of cultured Friend erythroleukemia cells was modified by supplementation of the medium with oleic or linoleic acid. There was a 30% reduction in saturated and a 35% reduction in polyunsaturated fatty acids in microsomal phospholipids when the cells were grown in media supplemented with oleic acid, and a 3-fold increase in polyunsaturated fatty acids when the cells were grown in linoleic acid-supplemented media. Electron-spin resonance studies with the 5- nitroxystearate probe demonstrated that there was no appreciable change in microsomal lipid mobility as measured by the order parameters. In contrast, changes in lipid mobility were detected with the spin-label probe when microsomes were first isolated from Friend erythroleukemia cells and subsequently modified by incubation with liposomes composed of either dioleoyl- or dilinoleoylphosphatidylcholine plus bovine liver phospholipid-exchange protein. The fatty acid compositional changes produced in these microsomes were similar to those obtained when the intact cells were grown in media containing supplemental fatty acids. These findings indicate that the lipid mobility of Friend cell microsomes can be altered by phospholipid replacements in vitro, but that this does not occur when similar microsomal fatty acid modifications are produced during culture of the intact cell.     

Mechanisms of dramatic fluctuations of ornithine decarboxylase activity upon tonicity changes in primary cultured rat hepatocytes.

We investigated the mechanisms underlying the marked induction of ornithine decarboxylase (ODC) activity by hypotonic treatment and its rapid decay upon reversal to isotonicity in primary cultures of adult rat hepatocytes. Upon hypotonic treatment, ODC synthesis rate increased progressively whereas the amount of ODC mRNA increased only about twofold. In addition, ODC was stabilized severalfold. ODC activity rapidly decreased upon restoration of isotonicity, owing to immediate and nearly complete suppression of ODC synthesis and 3-6-fold stimulation of ODC decay. The stimulation of ODC decay caused by restoration of isotonicity was mostly independent of time and protein synthesis. ODC decay was also stimulated by putrescine, even under hypotonic conditions, depending on time and new protein synthesis. Restoration of isotonicity and putrescine treatment together caused a synergistic stimulation of ODC decay, confirming that these act by different mechanisms.     

Purification and properties of ornithine decarboxylase from rat liver

Ornithine decarboxylase was purified to homogeneity, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and polyacrylamide gel electrofocusing, about 710,000-fold with a 35% yield from the liver cytosol of thioacetamide-treated rats. The final specific activity was approximately 24,400 nmol/min/mg of protein. The apparent molecular weight of the enzyme determined by gel filtration analyses on Sephacryl S-200 was 55,000 in the presence of 0.25 M NaCl and 145,000 in its absence. The minimum molecular weight of the enzyme was determined to be 54,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point of the enzyme was estimated as 5.7 in the presence of 8 M urea. Some catalytic properties of the enzyme were also studied.     

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.     

Effect of epidermal growth factor on ornithine decarboxylase activity and urea synthesis in isolated rat hepatocytes.

Ornithine decarboxylase (ODC) activity is induced by protein-synthesis independent mechanisms in freshly isolated rat hepatocytes, incubated either without or with a mixture of amino acids in the incubation medium. Urea synthesis rates were two- to three-fold higher in those hepatocytes incubated in the presence of amino acids that in those lacking amino acids in the medium. Epidermal growth factor (EGF) delayed ODC induction, but only in the presence of amino acids. EGF significantly decreased ureagenesis when hepatocytes were incubated in the presence of amino acids and only endogenous substrates were available. No evidence of any link between ODC induction and urea synthesis was found.     

Allylisopropylacetamide induces rat hepatic ornithine decarboxylase

In rat liver, allylisopropylacetamide (AIA) treatment strongly induced (25-fold) the activity of rat hepatic ornithine decarboxylase (ODC). By either the oral or the subcutaneous route, AIA produced a long-lasting induction (30 to 40 hours) of hepatic ODC activity. Three analogs of AIA, propylisopropylacetamide (PIA), allobarbital, and allylbenzene, were active ODC inducers while a fourth, allylacetate, was not. Although induction of hepatic aminolevulinic acid (ALA) synthetase activity and the accumulation of hepatic porphyrins depend on the allyl moiety of AIA, this is not the case with hepatic ODC induction. Allylisopropylacetamide did not elevate serum alanine aminotransferase (SGPT) nor did it cause DNA damage, as measured by the alkaline elution assay. Thus, hepatic cell death is not a likely explanation of AIA's long-lasting induction of ODC. As AIA does not belong to any of the common categories of ODC inducers, it may be the chemical prototype of a new class of hepatic ODC inducers.     

Purification and some properties of ornithine decarboxylase from rat liver

Ornithine decarboxylase (EC 4.1.1.17) was purified to near homogeniety from livers of thioacetamide- and dl-α-hydrazino-δ-aminovaleric acid-treated rats by using three types of affinity chromatography with pyridoxamine phosphate-Sepharose, pyridoxamine phosphate-dipropylenetriamine-Sepharose and heparin-Sepharose. This procedure gave a purification of about 3.5·10⁵-fold with an 8% yield; the specific activity of the final enzyme preparation was 1,1·10⁶ nmol CO₂/h per mg protein. The purified enzyme gave a single band of protein which coincided with activity peak on polyacrylamide gel electrophoresis and also gave a single major band on SDS-polyacrylamide gel electrophoresis. A single precipitin line was formed between the purified enzyme and an antiserum raised against a partially purified enzyme, on Ouchterlony immunodiffusion. The molecular weight of the enzyme was estimated to be 105 000 by polyacrylamide gel electrophoresis at several different gel concentrations; the dissociated subunits had molecular weights of 50 000 on SDS-polyacrylmide gels. The isoelectric point of the enzyme was pH 4.1.     

Characterization of arginine decarboxylase in rat brain and liver: distinction from ornithine decarboxylase

We compared the properties of mammalian arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) in rat liver and brain. Mammalian ADC is thermally unstable and associated with mitochondrial membranes. ADC decarboxylates both arginine (Km = 0.75 mM) and ornithine (Km = 0.25 mM), a reaction not inhibited by the specific ODC inhibitor, difluoromethylomithine. ADC activity is inhibited by Ca2+, Co2+, and polyamines, is present in many organs being highest in aorta and lowest in testis, and is not recognized by a specific monoclonal antibody to ODC. In contrast, ODC is thermally stable, cytosolic, and mitochondrial and is expressed at low levels in most organs except testis. Although ADC and ODC are expressed in cultured rat C6 glioma cells, the patterns of expression during growth and confluence are very different. We conclude that mammalian ADC differs from ADC isoforms expressed in plants, bacteria, or Caenorhabditis elegans and is distinct from ODC. ADC serves to synthesize agmatine in proximity to mitochondria, an organelle also harboring agmatine's degradative enzyme, agmatinase, and a class of imidazoline receptor (I2) to which agmatine binds with high affinity.     

Detection of multiple forms of rat liver ornithine decarboxylase.

Rat liver ornithine decarboxylase induced by injection of thioacetamide has been separated into at least two fractions by covalent chromatography on an activated thiol-Sepharose 4B column. The two major fractions could be distinguished by ion exchange chromatography and electrophoresis on acrylamide gels. In addition, the two forms displayed different Km values for ornithine. Although the two forms are separable, they display identical antigenic properties, pH optima, and they appear to be the same molecular size. The biological significance or the relationship between multiple forms of ornithine decarboxylase is not understood.     

Inhibition of rat heart ornithine decarboxylase by basic polypeptides.

Purified and partially purified ornithine decarboxylase (ODC) from rat heart was inhibited by basic polypeptides in vitro. Poly-L-arginine, the most effective, was inhibitory at a concentration as low as 0.1 microgram/ml; protamine and histone clearly inhibited ODC at concentrations higher than 2 micrograms/ml, but poly-L-lysine was less effective. The ability to inhibit ODC appeared to correlate with the arginine-residue content of basic polypeptides. The inhibition effect could be decreased by increasing substrate concentration and ionic strength.     

Induction of ornithine decarboxylase by biliverdin in rat liver.

The administration of biliverdin (0.1mg/g of body weight) into the peritoneal cavity of rats resulted in the induction of ornithine decarboxylase in the liver. When the temporal relationships between the changes in intracellular adenosine 3', 5'-cyclic monophosphate (cyclic AMP) level, cyclic AMP-dependent protein kinase activity and the induction of ornithine decarboxylase were investigated, the concentration of cyclic AMP increased significantly 2 h after the administration of biliverdin, while cyclic AMP-dependent protein kinase was activated after 2–4 h. The hepatic ornithine decarboxylase activity began to increase 4 h after biliverdin injection. These results suggest that there is some sequential relationship between the increase of cyclic AMP, the activation of cyclic AMP-dependent protein kinase and the induction of ornithine decarboxylase although the direct correlation of these three events remains to be elucidated.     

皮质酮对大鼠再生肝细胞鸟氨酸脱羧酶活性及其基因表达的影响

采用高效液相色谱和原位杂交技术研究了皮质酮对大鼠再生肝细胞鸟氨酸脱羧酶 (ODC)活性及ODCmRNA表达的影响。结果显示 ,大鼠完整肝脏中ODC水平较低 ,2 / 3肝切除 (PH)后 3h ,不同处理组ODC活性开始升高 ,6h达到最高值 ,其中 ,去肾上腺 NaCl组和糖皮质激素受体拮抗剂RU4 86处理组的酶活性高于对照组 (去肾上腺假手术组 ) ,而去肾上腺 皮质酮处理组的酶活性低于对照组 ,36h恢复到肝切除前水平 ;完整肝脏的ODCmRNA水平极低 ,PH后表达量迅速增加 ,5h达到最大值 ,不同处理组mRNA水平的高低顺序与酶活性一致 ,12h降至肝切除前水平 ;在PH前 12h给大鼠注射RU4 86 (10mg/kg体重 ) ,取得了与去肾上腺 NaCl处理鼠相似的结果。以上结果表明 ,在PH诱导的再生肝细胞中 ,ODCmRNA表达量的增加和 /或减少是造成ODC活性改变的原因之一 ,皮质酮对ODC活性及其mRNA的表达具有抑制作用 ,主要表现在肝再生的早期 ,该作用可能是通过受体实现的     

Prolactin-stimulated ornithine decarboxylase induction in rat hepatocytes: coupling to diacylglycerol generation and protein kinase C

The trophic effects of prolactin (PRL) in rat liver have been linked to activation of protein kinase C (PKC). Since alterations in PKC activity imply its activation by 1,2-diacylglycerol (DAG), we tested whether PRL treatment stimulated DAG generation coupled to induction of a growth response in primary hepatocytes. Addition of PRL to hepatocyte cultures significantly increased [3H]-glycerol incorporation into DAG within 5 minutes which was followed by a loss of cytosolic PKC activity by 10 minutes. Prolactin also significantly enhanced radiolabel incorporation into triacylglycerol and phospholipids within 10 minutes and induced ODC activity at 6 hours. Therefore, prolactin-stimulated alterations in PKC activity are preceded by enhanced DAG generation. Moreover, these events appear to be coupled to PRL-stimulated entry of hepatocytes into cell cycle.