Partial purification and some properties of a liver alkaline ribonuclease from the frog Rana esculenta.

1. RNAases varying in pH optimum, activation with pCMB, sensitivity towards temperature and acid treatment, as well as electrophoretic mobility were found in Rana esculenta liver extract. 2. Of the three activity peaks of alkaline ribonuclease separated on CM-cellulose with 2000-fold purification, RNAase of peak C is thermo- and acid-stable and exhibits specificity for pyrimidine bases, preferring poly(U) over poly(C). 3. Differences in the specific "inhibitory effect" of frog liver supernatant on the frog liver alkaline RNAase were observed.     

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.     

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.     

Purification and some properties of rat intestinal ornithine decarboxylase

Ornithine decarboxylase (ODC) was induced in rat small intestine by treatment with hypotonic solution in vitro and purified by two procedures, a conventional procedure and an immunoaffinity procedure. SDS-polyacrylamide gel electrophoresis showed that the molecular weight of the preparation purified by the immunoaffinity procedure (Mr = 53,000) was slightly larger than that of the preparation obtained by the conventional procedure (Mr = 52,000). Values for the Km for L-ornithine (0.1 mM), the isoelectric point (5.4), and the final specific activity (5.1-5.5 x 10(5) nmol CO2/mg protein/30 min) of the two preparations were similar to those reported for the rat liver ODC. Addition of a protease inhibitor (limabean trypsin inhibitor) to the crude extract prevented the appearance of the smaller enzyme (Mr = 52,000) obtained by the conventional purification procedure. Our result indicates that the large enzyme is native ODC and the smaller one is a partial proteolysis product of native ODC.     

Hepatic cyclic AMP generation and ornithine decarboxylase induction by glucagon and beta adrenergic agonists

The relationship of hepatic ornithine decarboxylase (ODC) activity to cyclic AMP levels and nutritional status was studied in the pre-weanling rat. Previous studies demonstrated that 2 hr without food causes a loss of hepatic ODC induction after glucagon or catecholamine injection. Isoproterenol or glucagon administration produced increased hepatic cyclic AMP and tyrosine aminotransferase activity which were not prevented by nutritional deprivation. Blockade of hepatic beta 2 receptors by the selective antagonist ICI 118,551 prevented increased cAMP levels and ODC activity after isoproterenol administration. Blockade of beta 1 receptors by atenolol did not prevent increased cAMP levels or ODC induction by isoproterenol although it did block activation of cardiac ODC. The phosphodiesterase inhibitor RO20-1724 increased hepatic cAMP levels as well as ODC and TAT activities, although the increase in ODC activity was attenuated by nutritional deprivation. RO20-1724 also potentiated the induction of hepatic ODC after glucagon or isoproterenol administration. Administration of 8-bromo cAMP elevated hepatic ODC activity regardless of nutritional status but also elevated serum levels of growth hormone and corticosterone. Hepatic ODC induction by glucagon or beta 2 agonists can be dissociated from changes in cAMP levels during nutritional deprivation.     

Entamoeba histolytica: purification and characterization of ornithine decarboxylase

Ornithine decarboxylase, a rate-limiting enzyme in polyamine biosynthesis in eukaryotes, was stabilized and purified from trophozoites of the parasite protozoan E. histolytica. Analytical electrophoresis revealed the presence in the purified preparations of a major polypeptide of 45 kDa and barely detectable amounts of two other proteins of 70 and 120 kDa. Both the 45 and 70 kDa polypeptides were recognized by a mouse anti-ODC monoclonal antibody. The major polypeptide exhibited amino terminal sequence homology in the range of 40-73% with ODCs from other organisms. The immunoreactive polypeptide of 70 kDa was not identified. The molecular masses of 216 and 45 kDa determined for the native enzyme by gel filtration and for the major polypeptide by SDS-PAGE, respectively, suggest that the amoeba ODC is a homopentamer. Dialysis against hydroxylamine rendered the enzyme activity fully dependent on pyridoxal 5'-phosphate (PLP). As expected for an oligomeric enzyme, ODC activity exhibited sigmoidal kinetics when it was measured as a function of increasing concentrations of L-ornithine and PLP yielding S(0.5) values of 0.45 and 0.18 mM, respectively. Purified ODC was inhibited by 1,3-diaminopropane and 2,4-diamino-2-butanone but was largely insensitive to inhibition by alpha-difluoromethylornithine (DFMO), indicating that the enzyme may not be a suitable target for this anti-parasitic drug. Other features of the amoeba ODC were common with the enzyme from prokaryotes and eucaryotes.     

Hepatic tyrosine aminotransferase from the rainbow lizard Agama agama: purification and some properties

Tyrosine aminotransferase, induced by dexamethasone in the liver of the rainbow lizard, Agama agama, was extracted under optimal conditions which yield the native undegraded enzyme; purified by heat treatment at 65 degrees C, ammonium sulfate precipitation, chromatography on DEAE-Sephacel and Sephadex G-150-120 and then characterized. The enzyme was purified over 2000-fold to a specific activity of 2653 units/mg of protein. It had an optimum pH of 7.6 in potassium phosphate buffer, KmTyr: 1.0 mM; K alpha-KGm: 0.32 mM; Vmax: 1.33 nmol/min and a molecular weight of about 130,000. It was inhibited by L-glutamate (competitively, Ki, 2.5 mM), and by metal ions Ca2+, Mn2+, Zn2+, Hg2+ and Ag2+, but was unaffected by chelating agents and other divalent cations. Lizard hepatic cytosolic tyrosine aminotransferase was specific for L-tyrosine and alpha-ketoglutarate as substrates sensitive to sulfhydryl inactivation and to protection from thermal lability by alpha-ketoglutarate and pyridoxal phosphate.     

Plasmodium falciparum: purification, properties, and immunochemical study of ornithine decarboxylase, the key enzyme in polyamine biosynthesis

Ornithine decarboxylase, the rate-limiting enzyme in the polyamine biosynthetic pathway has been purified 7,600 fold from Plasmodium falciparum by affinity chromatography on a pyridoxamine phosphate column. The partially purified enzyme was specifically tagged with radioactive DL-alpha-difluoromethylornithine and subjected to polyacrylamide gel electrophoresis under denaturing conditions. A major protein band of 49 kilodalton was obtained while with the purified mouse enzyme, a typical 53 kilodalton band, was observed. The catalytic activity of parasite enzyme was dependent on pyridoxal 5'-phosphate and was optimal at pH 8.0. The apparent Michaelis constant for L-ornithine was 52 microM. DL-alpha-difluoromethylornithine efficiently and irreversibly inhibited ornithine decarboxylase activity from P. falciparum grown in vitro or Plasmodium berghei grown in vivo. The Ki of the human malarial enzyme for this inhibitor was 16 microM. Ornithine decarboxylase activity in P. falciparum cultures was rapidly lost upon exposure to the direct product, putrescine. Despite the profound inhibition of protein synthesis with cycloheximide in vitro, parasite enzyme activity was only slightly reduced by 75 min of treatment, suggesting a relatively long half-life for the malarial enzyme. Ornithine decarboxylase activity from P. falciparum and P. berghei was not eliminated by antiserum prepared against purified mouse enzyme. Furthermore, RNA or DNA extracted from P. falciparum failed to hybridize to a mouse ornithine decarboxylase cDNA probe. These results suggest that ODC from P. falciparum bears some structural differences as compared to the mammalian enzyme.     

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.     

Trypanosoma brucei ornithine decarboxylase: enzyme purification, characterization, and expression in Escherichia coli

Ornithine decarboxylase from the African trypanosome is an important target for antitrypanosomal chemotherapy. Despite this, the enzyme had not been previously purified or extensively characterized as it is a very low level protein. In this paper we describe the purification of Trypanosoma brucei brucei ornithine decarboxylase from bloodstream form trypomastigotes by 107,000-fold to a specific activity of 2.7 x 10(6) nmol CO2/h/mg of protein in the parasite. T. brucei ornithine decarboxylase had a native molecular weight of 90,000 and a subunit molecular weight of 45,000. The isoelectric point of the protein was 5.0. The Km for ornithine was 280 microM and the Ki for the irreversible inhibitor alpha-difluoromethylornithine (DFMO) was 220 microM with a half-time of inactivation at saturating DFMO concentration of 2.7 min. T. brucei ornithine decarboxylase appears similar to mouse ornithine decarboxylase, further supporting our previous suggestion that the selective toxicity of DFMO to the parasite is not due to catalytic differences between the two proteins. Although a small quantity of T. brucei ornithine decarboxylase was purified from T. brucei, extensive structural and kinetic studies will require a more ample source of the enzyme. We therefore expressed our previously cloned T. brucei ornithine decarboxylase gene in Escherichia coli using a vector that contains an inducible lambda promoter. T. brucei ornithine decarboxylase activity was induced in E. coli to levels that were 50 to 200 fold of that present in the long-slender bloodstream form of T. brucei. Ornithine decarboxylase activity in the crude E. coli lysate was 1500-6000 nmol of CO2/h/mg of protein and represented 0.05-0.2% of the total cell protein. The recombinant T. brucei ornithine decarboxylase was purified to apparent homogeneity from the transformed E. coli. The purified recombinant enzyme had kinetic and physical properties essentially identical to those of the native enzyme.     

Modulation of the induction of ornithine decarboxylase by some opioid receptor agonists in immune cells and cardiomyocytes

The ability of natural and synthetic opioids to modulate the induction of ornithine decarboxylase (ODC) was investigated in immune cells and cardiomyocytes in culture. In particular, Leu-enkephalin, which shows preference for -receptors, enhanced ODC activity in both thymocytes and cardiomyocytes, whereas the effect of U-50488H, a synthetic -selective agonist, was cell-specific. In thymocytes, U-50488H markedly inhibited the induction of the enzyme elicited by the mitogen concanavalin A (Con A) or by a combined treatment with PMA and A23187, and also reduced basal ODC activity. However the drug did not affect ODC induced by other stimuli. The inhibition of the induction of ODC activity was accompanied by a reduction of ODC mRNA level and an acceleration of ODC turnover. The action of U-50488H in thymocytes does not appear to be mediated by or other classical opioid receptors lacking both stereospecificity and antagonist sensitivity, but may involve a pertussis toxin-sensitive G protein. Splenocytes also showed the ODC inhibiting effect of U-50488H, although they were less sensitive compared to thymocytes. In contrast, U-50488H enhanced ODC activity in cardiomyocytes and this effect was blocked by a specific -antagonist. In conclusion, these results indicate that some opioid agonists can modulate ODC expression in non neural cells. In particular, -opioid receptors may be involved in the U-50488H action in cardiomyocytes, and a distinct site, linked to inhibition of cell proliferation, may operate in immune cells.     

The induction of ornithine decarboxylase by L-asparagine and intracellular alkalinization

The uptake of transport systems A and N amino acids, most noticeably L-asparagine, is essential for the induction of ornithine decarboxylase (L-ornithine carboxylase, EC 4.1.1.17) in cultured cells and we have proposed that the uptake-associated pH and ionic changes might constitute part of the cell activation signal (1). In the present study, it was shown that extracellular L-asparagine caused an immediate and transient increase in intracellular pH which was continuously monitored by the fluorescence probe BCECF (2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein). NH4Cl and NH4OH which caused intracellular alkalinization also caused ornithine decarboxylase activity to increase.     

The modulation of the induction of ornithine decarboxylase by spermine,spermidine and diamines

Extremely low concentrations of putrescine, spermidine and spermine added to the extracellular medium of cultures of mammalian cells inhibit the induction of ornithine decarboxylase activity despite 100- to 1,000-fold greater intracellular polyamine concentrations. The diamines, 1,2-diaminoethane, 1,3-diaminopropane, 1,5-diaminopentane, 1,7-diaminoheptane, 1,10-diaminodecane, 1,12-diaminododecane also inhibit ornithine decarboxylase at all concentrations tested (greater than 10^?6 M). In contrast, 10^?6 M to 10 ^?3 M 1,8-diaminooctane, the alkyl analog of spermidine, enhances ornithine decarboxylase activity. The concentraton of putrescine required to inhibit the activity of ornithine decarboxylase by 50% is a characteristic of each cell line; however, it varies by as much as 1,000-fold among the five cell lines we have tested (L1210 leukemic, H35 hepatoma, N18 neuroblastoma, W256 carcinosarcoma and 3T3 fibroblasts). The antizyme to ornithine decarboxylase can be induced in all these cells by high (di)(poly)amine concentrations. Based on these and other experiments we suggest a working hypothesis: that the polyamines regulate ornithine decarboxylase activity through two different sites that may be interrelated; a sensitive membrane-mediated site that responds to minute fluctuations of extracellular polyamine levels and a coarse site which may be intracellular or membrane associated that responds to larger fluctuations of intracellular polyamine levels. The consequences of such a control mechanism operating within the whole organism are discussed.     

Cardiac activity of some peptide hormones in the frog,Rana tigrina

1. The cardiac responses of isolated frog (Rana tigrina) atria to peptide hormones were studied.2. Calcitonin gene-related peptide (CGRP), arginine vasotocin (AVT), bovine parathyroid hormone fragment (bPTH-(1–34)) and oxytocin (OXY) produced dose-related positive chronotropic and inotropic responses; atrial natriuretic peptide (ANP) was negative chronotropic and inotropic; cholecystokinin (CCK), vasoactive intestinal peptide (VIP) were without effects.3. The dose-related responses under bPTH-(1–34) stimulation but not CGRP or AVT were attenuated in the presence of ANP (300 ng/ml, ≈0.98 × 10⁻⁷ M). As expected ANP decreased the basal AR and AT responses of the isolated atria and the inhibitory effects were dose-dependent.4. As shown previously, propranolol blocked the atrial tension stimulated by bPTH (1–34) but did not alter the cardiac responses to CGRP and AVT.5. In the presence of β-adrenergic blocker (propranolol 10⁻⁷M) or ANP (10⁻⁷M), the AR and AT changes under ISO stimulation in the frog were also decreased.6. These cardiac changes suggest the cardiac inhibitory effects of ANP are related to β-adrenoceptor activity and ANP might be a β antagonist.