Apparent ornithine decarboxylase activity, measured by 14CO2 trapping, after frozen storage of rat tissue and rat tissue supernatants

Ornithine decarboxylase (ODC) activity of rat tissues was measured by the standard 14CO2 trapping method after frozen storage (-60 or -70 degrees C) of the tissues or their 105,000g supernatants. True ODC activity was determined by two methods: (a) addition of the inhibitors alpha-difluoromethylornithine (DFMO), a specific irreversible inhibitor of ODC, or aminooxyacetate (AOA), an inhibitor that blocks the decarboxylation of ornithine by mitochondrial enzymes; and (b) chromatographic analysis of the reaction products. In the frozen supernatants of liver and spleen, ODC activity changed only slightly after 1 day but increased 29 and 14%, respectively, by 30 days; activity in kidney supernatant decreased 17% after 1 day and remained near that level at 30 days. Kidney and spleen ODC activity was inhibited 90-100% by DFMO, but apparent liver ODC activity was inhibited only 60-75%. In the supernatant prepared from tissue stored frozen for 1 day, apparent ODC activity in liver increased 500% over that activity in the freshly prepared supernatant; at 23 days, apparent activity increased 755% for liver and 121% for kidney. After 23 days, DFMO did not inhibit apparent ODC activity in supernatants from frozen liver and inhibited ODC in frozen kidney by only 49%. With AOA, the ODC activities of the fresh and frozen supernatants were similar, indicating that the large increase in apparent ODC activity in frozen tissue was due to artifacts from the metabolism of ornithine via the mitochondrial pathway. HPLC analysis of the reaction products resulting from the incubation of uniformly labeled [14C]ornithine with the fresh and frozen preparations indicated no increase in putrescine with the frozen preparation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulation of ornithine decarboxylase activity in rat tissues by growth hormone and by serum growth factor from rats infested with spargana of Spirometra mansonoides

Ornithine decarboxylase activity was studied in heart, kidney, liver, thymus, lung, spleen, skeletal muscle and fat of hypophysectomized rats after growth hormone treatment. A marked increase in enzyme activity was observed in kidney and liver, and a significant increase in heart and thymus at 4 h after injection of growth hormone. The kidney was the most responsive organ with an increase in activity of about 100 fold. The enzyme activity in kidney responded to a dose of 10 μg of growth hormone. Daily injection for 12 days raised activity only in the heart. Infestation for 6–13 days with spargana of Spirometra mansonoides, which also causes growth of hypophysectomized rats, increased enzyme activity in the heart and thymus. Intravenous injection of serum of hypophysectomized rats infested with spargana of Spirometra mansonoides caused a significant increase in the enzyme activity in liver and kidney after 4 h. Growth hormone and the serum growth factor of sparganosis seem to share the characteristic of causing an early increase in ornithine decarboxylase activity in rat tissues. The marked response in kidney and liver raises the possibility that these organs are the primary targets of both substances.     

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.     

Brain Polyamine Stress Response: Recurrence After Repetitive Stressor and Inhibition by Lithium

Abstract: We recently demonstrated that, unlike in peripheral tissues, the increase in activity of polyamine synthesizing enzymes observed in the brain after acute stress can be prevented by long-term, but not by short-term, treatment with lithium. In the present study we sought to examine the effects of chronic intermittent stress on two key polyamine synthesizing enzymes, ornithine decarboxylase and S-adenosylmethionine decarboxylase, and their modulation by lithium treatment. Adult male rats were subjected to 2 h of restraint stress once daily for 5 days and to an additional delayed stress episode 7 days later. Enzyme activities were assayed 6 h after the beginning of each stress episode. In contrast to the liver, where ornithine decarboxylase activity was increased (300% of the control) only after the first stress episode, the enzyme activity in the brain was increased after each stress episode (to ～170% of the control). Unlike ornithine decarboxylase activity, S-adenosylmethionine decarboxylase activity was slightly reduced after the first episode (86% of the control) but remained unchanged thereafter. After cessation of the intermittent stress period, an additional stress episode 7 days later led again to an increase in ornithine decarboxylase activity in the brain (225% of the control) but not in the liver, whereas S-adenosylmethionine decarboxylase activity remained unchanged. The latter increase in ornithine decarboxylase activity was blocked by lithium treatment during the intervening 7-day interval between stressors. The results warrant the following conclusions: (a) Repetitive application of stressors results in a recurrent increase in ornithine decarboxylase activity in the brain but to habituation of this response in the liver. (b) This brain polyamine stress response can be blocked by long-term (days) lithium treatment. (c) The study implicates an overreactive polyamine response as a component of the adaptive, or maladaptive, brain response to stressful events and as a novel molecular target for lithium action.     

Ornithine decarboxylase, transglutaminase, diamine oxidase and total diamines and polyamines in maternal liver and kidney throughout rat pregnancy.

Ornithine decarboxylase (ODC; EC 4.1.1.17), transglutaminase (EC 2.3.2.13), diamine oxidase (DAO; EC 1.4.3.6) and total di- and poly-amines were studied in rat liver and kidney cortex throughout pregnancy. In liver, ODC activity exhibited two major peaks (4.5-5 times the control activities) on days 15 and 17. Also putrescine and spermidine increased biphasically (3-4-fold), but no variation in spermine content was observed. Transglutaminase activity showed slight variations only near the end of gestation. In kidney, ODC activity did not fluctuate significantly during pregnancy, whereas both transglutaminase activity and putrescine content showed three major increases, in very early, middle and late pregnancy. No significant variations in spermidine and spermine were observed. In both organs, DAO activity, very low or undetectable until day 10, dramatically increased (10- and 20-fold in kidney and liver respectively) in the second half of pregnancy, reaching maxima on days 16-17 and 19. The results obtained for transglutaminase, ODC and total di- and poly-amines are interpreted on the basis of hyperplastic and hypertrophic events in the liver and kidney respectively. The behaviour of DAO suggests that the enzyme plays an important role in the control of intracellular diamine concentration.     

Rat colon ornithine and arginine metabolism: coordinated effects after proliferative stimuli

Ornithine decarboxylase (ODC) catalyzes the first step in the polyamine biosynthetic pathway, a highly regulated pathway in which activity increases during rapid growth. Other enzymes also metabolize ornithine, and in hepatomas, rate of growth correlates with decreased activity of these other enzymes, which thus channels more ornithine to polyamine biosynthesis. Ornithine is produced from arginase cleavage of arginine, which also serves as the precursor for nitric oxide production. To study whether short-term coordination of ornithine and arginine metabolism exists in rat colon, ODC, ornithine aminotransferase (OAT), arginase, ornithine, arginine, and polyamine levels were measured after two stimuli (refeeding and/or deoxycholate exposure) known to synergistically induce ODC activity. Increased ODC activity was accompanied by increased putrescine levels, whereas OAT and arginase activity were reduced by either treatment, accompanied by an increase in both arginine and ornithine levels. These results indicate a rapid reciprocal change in ODC, OAT, and arginase activity in response to refeeding or deoxycholate. The accompanying increases in ornithine and arginine concentration are likely to contribute to increased flux through the polyamine and nitric oxide biosynthetic pathways in vivo.     

A human neuroblastoma cell line with a stable ornithine decarboxylase in vivo and in vitro

A human neuroblastoma cell line (Paju) grew in 10 mM difluoromethyl-ornithine, which at this concentration normally stops the growth of all mammalian cells. Ornithine decarboxylase from Paju was resistant to inhibition in vitro by difluoromethylornithine, and required 10 microM of the compound for 50% inhibition, whereas ornithine decarboxylase from SH-SY5Y cells (another human neuroblastoma) and from rat liver needed only 0.5 microM difluoromethylornithine. Paju ornithine decarboxylase also exhibited a long half-life (over eight hours) in vivo. The half-life of immunoreactive protein was significantly longer than that of the activity. The long half-life of ornithine decarboxylase in Paju cells leads to its accumulation to a specific activity of 2000 nmol/mg of protein per 30 min during rapid growth (the corresponding activity in SH-SY5Y cells was about 2.5). When partially purified ornithine decarboxylase from Paju cells was incubated with rat liver microsomes it was inactivated with a half-life of 75 min. This inactivation was accompanied by a fall in the amount of immunoreactive protein. In the same inactivating system partially purified SH-SY5Y ornithine decarboxylase had a half-life of 38 min and its half-life in vivo was 50 min. The corresponding values for rat liver ornithine decarboxylase were 45 min and 40 min, respectively. Rat liver microsomes also inactivated rat liver adenosylmethionine decarboxylase. These results suggest that Paju ornithine decarboxylase has an altered molecular conformation, rendering it resistant to (i) difluoromethylornithine and (ii) proteolytic degradation both in vivo and in vitro.     

Chicken ornithine transcarbamylase: purification and some properties

Ornithine transcarbamylase [EC 2.1.3.3] has been purified from chick kidney to homogeneity. The molecular weight is 110,000 as determined by gel filtration. Sodium dodecylsulfate polyacrylamide gel electrophoresis of the enzyme showed that the enzyme exists as a trimer of identical subunits of 36,000 daltons like other mammalian species ornithine transcarbamylases. In 0.1 M triethanolamine/HCl, the apparent optimum pH of the purified enzyme was 7.5 in the presence of 5 mM ornithine. The curve shifted toward a more alkaline region with a decrease in ornithine concentration. The specific activity of the purified enzyme as 77 units at pH 7.5. The Km for carbamyl phosphate was 0.11 mM and the Km for ornithine was 1.21 mM. With an increase in pH, a decrease in Km values for ornithine and an increase in the extent of inhibition by ornithine were observed. On using antibody against bovine liver ornithine transcarbamylase, the precipitin lines for the chick and bovine enzymes showed a spur pattern. Even when excess amounts of the antibody were added, the chick enzyme did not lose the activity while the bovine enzyme activity was inhibited completely.     

Contragestational action of hyperthermia on rat pregnancy. Effect on ornithine decarboxylase.

In the rat, there are marked changes in ornithine decarboxylase activity in the fetuses and reproductive tissues during gestation. Exposure of pregnant rats to moderate hyperthermia (40 degrees C, 60 min) produced a marked decrease (about 80%) of ornithine decarboxylase activity in fetuses, uterus and ovaries, while this change was more moderate in placenta (about 20%). This effect was observed in different stages of pregnancy. Ornithine decarboxylase activity was returned to control values within a few hours after the end of the hyperthermic treatment. Hyperthermia produced marked contragestational effects if given sequentially on days 8, 9 and 10 of gestation, but only a decrease in the weight of viable fetuses was observed when given on days 11, 12 and 13. These results indicate that part of the harmful effects produced by hyperthermia on pregnant rats may be mediated by the sustained fall of ornithine decarboxylase activity during critical periods of gestation.     

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.     

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.     

A human neuroblastoma cell line with an altered ornithine decarboxylase

A human neuroblastoma cell line (Paju) was resistant to 10 mM difluoromethylornithine, a concentration at which the growth of all mammalian cells normally stops. Ornithine decarboxylase from Paju was very resistant to inhibition by difluoromethylornithine in vitro (Ki = 10 microM compared to 0.5 microM for mouse kidney ornithine decarboxylase). After purification, apparently homogeneous Paju ornithine decarboxylase was inactivated with [3H]difluoromethylornithine and analyzed by polyacrylamide gel electrophoresis. Under denaturing conditions it was found to have an altered molecular structure, i.e. two nonidentical subunits of Mr = 55,000 and 60,000. Another unusual feature of Paju ornithine decarboxylase was its long half-life in vivo (T 1/2 = 8 h compared with 36 min in human HL-60 promyelocytic leukemia cells). The disappearance of immunoreactive protein was only slightly slower than the loss of catalytic activity. The long half-life of Paju ornithine decarboxylase was not shared by adenosylmethionine decarboxylase. Despite the altered structure of Paju ornithine decarboxylase, it was recognized by a specific antisera raised in rabbit against mouse kidney ornithine decarboxylase. The Paju karyotype did not contain double minute chromosomes or any large homogeneously staining region such as that seen in a mouse lymphoma cell mutant that is resistant to difluoromethylornithine and overproduces ornithine decarboxylase (McConlogue, L., and Coffino, P. (1983) J. Biol. Chem. 258, 12083-12086).     

Purification of ornithine aminotransferase by immunoadsorption

Ornithine aminotransferase was purified by conventional biochemical methods from rat kidney, rat liver, and human liver. Affinity-purified antibodies raised to the rat kidney enzyme were used to produce an immunoadsorbent enabling a one-step purification of ornithine aminotransferase to be made from crude human liver extracts. The harsh chemical conditions often required to desorb immunoadsorbents were avoided by isolating antibodies with low functional affinity and employing an electrophoretic desorption method which allowed the enzyme activity to be retained. The close structural similarity between human and rat ornithine aminotransferase was demonstrated by immunodiffusion reactions. It was therefore possible to purify the enzyme from human liver using immobilized antibodies raised against rat kidney ornithine aminotransferase. Furthermore, desorption was more readily achieved due to the lower affinity for the human enzyme.     

Ornithine decarboxylase and thymidine kinase activity in tissues of prolactin-treated rats: effect of hypophysectomy

The activities of ornithine decarboxylase and thymidine kinase were determined in tissues of young intact and hypophysectomized rats at various times after treatment with prolactin. In both types of animals, ornithine decarboxylase activity increased in liver, kidney, spleen and adrenal of prolactin treated rats. Thymidine kinase activity increased only in liver and spleen of intact rats. Increase in the kinase activity was smaller, and occurred later than the change in ornithine decarboxylase. In hypophysectomized animals, thymidine kinase activity increased in spleen, but not in liver, following prolactin treatment.     

Hydrazine stress in the diabetic: ornithine decarboxylase activity

Streptozotocin-induced diabetes of 7 weeks duration increased male Sprague-Dawley rat kidney ornithine decarboxylase activity by 4.8-fold but did not affect the liver enzyme. Hydrazine treatment of 4 hr duration stimulated equally kidney ornithine decarboxylase activities of nondiabetic and diabetic rats. Hydrazine treatment increased liver ornithine decarboxylase activity in the nondiabetic rat but did not increase it in the diabetic rat. Since hydrazine stimulates ornithine decarboxylase activity prior to polyamine and protein syntheses, we speculate that the lack of hydrazine stimulation of ornithine decarboxylase in the diabetic liver may be related in part to the unrestrained gluconeogenesis and depressed Kreb's cycle activity: the latter being required for protein synthesis.     

Underestimated contribution of skeletal muscle in ornithine metabolism during mouse postnatal development

Ornithine aminotransferase (l-ornithine 2-oxoacid aminotransferase, OAT) is widely expressed in organs, but studies in mice have focused primarily on the intestine, kidney and liver because of the high OAT-specific activity in these tissues. This study aimed to investigate OAT activity in adult mouse tissues to assess the potential contribution to ornithine metabolism and to determine OAT control during postnatal development. OAT activity was widely distributed in mouse tissues. Sexual dimorphism was observed for most tissues in adults, with greater activity in females than in males. The contribution of skeletal muscles to total OAT activity (34 % in males and 27 % in females) was the greatest (50 %) of the investigated tissues in pre-weaned mice and was similar to that of the liver in adults. OAT activity was found to be regulated in a tissue-specific manner during postnatal development in parallel with large changes in the plasma testosterone and corticosterone levels. After weaning, OAT activity markedly increased in the liver but dropped in the skeletal muscle and adipose tissue. Anticipating weaning for 3 days led to an earlier reduction of OAT activity in skeletal muscles. Orchidectomy in adults decreased OAT activity in the liver but increased it in skeletal muscle and adipose tissue. We concluded that the contribution of skeletal muscle to mouse ornithine metabolism may have been underestimated. The regulation of OAT in skeletal muscles differs from that in the liver. The present findings suggest important and tissue-specific metabolic roles for OAT during postnatal development in mice.     

Transgenic mice aberrantly expressing human ornithine decarboxylase gene.

We have generated transgenic mice carrying human ornithine decarboxylase gene. Two different transgene constructs were used: (i) a 5'-truncated human ornithine decarboxylase gene and (ii) an intact human ornithine decarboxylase gene. Transgenic mice carrying the 5'-truncated gene did not express human ornithine decarboxylase-specific mRNA. Transgenic mice carrying the intact human ornithine decarboxylase gene expressed human-specific ornithine decarboxylase mRNA in all tissues studied. However, as indicated by actual enzyme assays, the expression pattern was highly unusual. In comparison with their wild-type littermates, the transgenic mice exhibited greatly elevated enzyme activity in almost every tissue studied. Ornithine decarboxylase activity was moderately elevated in parenchymal organs such as liver, kidney, and spleen. Tissues like heart, muscle, lung, thymus, testis, and brain displayed an enzyme activity that was 20 to 80 times higher than that in the respective tissues of nontransgenic animals. The offspring of the first transgenic male founder animal did not show any overt abnormalities, yet their reproductive performance was reduced. The second transgenic founder animal, showing similar aberrant expression of ornithine decarboxylase in all tissues studied, including an extremely high activity in testis, was found to be infertile. Histological examination of the tissues of the latter animal revealed marked changes in testicular morphology. The germinal epithelium was hypoplastic, and the spermatogenesis was virtually totally shut off. Similar examination of male members of the first transgenic mouse line revealed comparable, yet less severe, histological changes in testis.     

Changes in ornithine transcarbamylase activity and protein level during perinatal period in rat liver. Effects of actinomycin D

Ornithine transcarbamylase activity and immunoreactive enzyme level are compared during perinatal period and in adult rat. Ornithine transcarbamylase activity regularly rises during late fetal period and presents a marked increase 24 hours after birth. Immunoreactive enzyme level does not correlate with this developmental pattern. Ornithine transcarbamylase level increases from 0.06 mg on day 19.5 of pregnancy to 0.417 mg/g liver on day 21.5 and remains constant after birth (0.418 mg/g liver). These results suggest that inactive mitochondrial ornithine transcarbamylase accumulates before birth and that the postnatal increase in enzyme activity is mainly associated with an activation. Furthermore, the paradoxical effect of actinomycin D on ornithine transcarbamylase activity is associated with an increase in enzyme level (about 25%).     

Changes in antizyme-ornithine decarboxylase complexes in tissues of hormone-treated rats

The presence of antizyme-ornithine decarboxylase complex in thymus and kidney of rats was demonstrated using the method of Y Murakami et al. [(1985) Biochem. J. 225, 689-697]. A very small amount of complex was found in kidney of control rats, accounting for only 1-3% of total enzyme in the tissue, while in thymus, approximately one-third of the total ornithine decarboxylase in thymus occurred as an antizyme-enzyme complex. After treatment with dexamethasone, both free ornithine decarboxylase and antizyme-ornithine decarboxylase decreased in thymus, the free enzyme activity decreasing more rapidly. In kidney, the concentration of the antizyme-ornithine decarboxylase complex increased after dexamethasone treatment, but only after the induction of free enzyme activity had reached its peak and begun to decrease. The pattern of the changes in amount of antizyme-ornithine decarboxylase complex after prolactin treatment differed from those observed in the dexamethasone-treated animals. In both kidney and thymus, the concentration of antizyme-ornithine decarboxylase complex increased concurrently with the induction of free enzyme activity. Both free and complexed ornithine decarboxylase had increased at 2.5 h after prolactin treatment and continued to increase to maximum specific activities at similar rates. In thymus, the amount of ornithine decarboxylase present as a complex reached 70% of the total in the tissue. In both thymus and kidney, the concentration of antizyme-ornithine decarboxylase complex decreased more slowly than did free enzyme activity. Free antizyme was observed only in thymus of dexamethasone-treated animals. The amount of measurable inhibitor was decreased if cycloheximide was given with dexamethasone.