E.S. Canellakis

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.

The regulation and function of ornithine decarboxylase and of the polyamines.

Publisher Summary This chapter highlights the regulation and function of ornithine decarboxylase (ODC) and of the polyamines. ODC catalyzes the synthesis of putrescine. It is necessary to distinguish among several possible functions of polyamines, to define the state of the cell at the time of induction, and to differentiate among various modes of induction of ODC activity. The chapter discusses a few new aspects of the regulation and function of ODC and of the polyamines and some of the uncertainties in the field. It also presents areas that are closely related to the regulation and the interrelationships of the polyamines in a variety of physiological and pathological states. The chapter also explores the possible functions of the polyamines.

The in vivo stimulation of rat liver ornithine decarboxylase activity by dibutyryl cyclic adenosine 3′,5′-monophosphate, theophylline and dexamethasone

Abstract The hepatic ornithine decarboxylase (ODC) activity of normal rats was stimulated more than 7-fold 3 hours after a single intraperitoneal injection of dibutyryl cyclic adenosine 3′,5′-monophosphate (dibu-cAMP). The 3-hour ODC activity was also stimulated by single injections of either theophylline or dexamethasone (10- and 21-fold, respectively). The simultaneous administration of actinomycin D with either dibu-cAMP, theophylline or dexamethasone reduced the 3-hour ODC activity by 91, 62 and 58 percent, respectively. When actinomycin D was given one hour after dibu-cAMP, no inhibition of ODC activity was observed.

Studies on the incorporation of deoxyribonucleotides and ribonucleotides into deoxyribonucleic acid

Abstract The purification of an enzyme fraction from calf-thymus nuclei catalyzing a limited incorporation of deoxyribonucleotides into the terminal positions of DNA has been described. The enzyme studied is apparently distinct from DNA polymerase. The same enzyme will also catalyze the incorporation of single ribonucleotides into the terminal positions of DNA.

Diacridines: Bifunctional intercalators I. Chemistry, physical chemistry and growth inhibitory properties

The synthesis, as well as the rationale for synthesis of diacridines, double intercalators, as potential inhibitors of nucleic acid synthesis is presented. The syntheses of (9-acridyl)-putrescine and -spermine, and bis(-9-acridyl)-putrescine, -spermidine, -spermine diamines and of bis(6-chloro-2-methoxy-9-acridyl)-putrescine and -spermine diamines, all substituted on the terminal NH2 groups are described. In addition, the homologous series of diacridines connected by the amino groups of the diamines NH2(CH2)nNH2 (where n = 2,3,4,6,8,10,12,14,16,18) to the C-9 of the diacridines has been synthesized. The chemical properties of these compounds as well as their molecular relationship to DNA are presented. The effect of the double intercalators on the Tm of DNA and of (A)n - (U)n, (dA)n - (dT)n, (G)n - (C)n and on (dG)n - (dC)n have been determined. The double acridine intercalators produce a much greater increase of the Tm of these nucleic acids than do the single acridine intercalators. They also profoundly affect the Tm of DNA in physiological salt concentrations; under these latter conditions the single intercalators have no effect. The relationship between the length of the chain connecting the two acridine rings and the inhibition of the growth of P-388 cells in vitro and vivo is presented. Their growth inhibitory properties appear, in general, to parallel their intercalative abilities.

Studies on the regulation of ornithine decar☐ylase activity by the microtubules: The effect of colchicine and vinblastine

Abstract Colchicine and vinblastine in micromolar concentrations inhibit the activity of ornithine decar☐ylase (E.C.4.1.1.17) (ODC), of mouse leukemia L1210 cells, which has been stimulated by dilution of the cells with fresh medium and serum. The colchicine analogues, lumicolchicine and colchiceine, which do not affect microtubular strcuture, do not inhibit ODC activity even at 10 −4 M. However, it appears that disruption of the microtubular structure is not in itself enough to inhibit ODC activity but that one or more additional temperature dependent steps are involved. We propose that the microtubule system is one of a series of components which regulates ODC activity.

Iodination of cell membranes: I. Optimal conditions for the iodination of exposed membrane components

Abstract Iodination of red blood cells under optimal conditions by the Phillips-Morrison method leads to the iodination of two surface proteins. Modification of these conditions leads to the labeling of additional membrane proteins; labeling of hemoglobin can also occur. These results lead to the conclusion that, depending on the conditions of iodination, proteins located at various depths of the membrane can be labeled. This information was used in establishing an assay for the optimal iodination conditions of HeLa cells. Such iodinated HeLa cells grow at the same rate as control HeLa cells; most of these iodinated surface proteins can be removed by subsequent treatment with pronase.

Ornithine decarboxylase activity in synchronously growing Don C cells

Abstract Ornithine decarboxylase activity was assayed in intact Don C cells taken from exponentially growing or Colcemid-synchronized cultures. During a short incubation with [ 14 C]ornithine, equivalent amounts of 14 C-labeled (putrescine + spermine) and 14 CO 2 were produced; labeled spermidine was not detected. Enzyme activity varied during the cell cycle, increasing during late G 1 -early S phase and late S phase. The activity increase at late G 1 -early S phase was abolished by several inhibitors of RNA and protein synthesis. The basal enzyme level may consist partly of pre-existing enzyme.

Regulation of polyamine biosynthesis by antizyme and some recent developments relating the induction of polyamine biosynthesis to cell growth: Review

This review considers the role of antizyme, of amino acids and of protein synthesis in the regulation of polyamine biosynthesis.The ornithine decarboxylase of eukaryotic ceils and ofEscherichia coli coli can be non-competitively inhibited by proteins, termed antizymes, which are induced by di-and poly- amines. Some antizymes have been purified to homogeneity and have been shown to be structurally unique to the cell of origin. Yet, the E. c o l i antizyme and the rat liver antizyme cross react and inhibit each others biosynthetic decarboxylases. These results indicate that aspects of the control of polyamine biosynthesis have been highly conserved throughout evolution.Evidence for the physiological role of the antizyme in mammalian cells rests upon its identification in normal uninduced cells, upon the inverse relationship that exists between antizyme and ornithine decarboxylase as well as upon the existence of the complex of ornithine decarboxylase and antizyme in vivo. Furthermore, the antizyme has been shown to be highly specific; its Keq for ornithine decarboxylase is 1.4 x 1011 M-1. In addition, mammalian ceils contain an anti-antizyme, a protein that specifically binds to the antizyme of an ornithine decarboxylase-antizyme complex and liberates free ornithine decarboxylase from the complex. In B. coli , in which polyamine biosynthesis is mediated both by ornithine decarboxylase and by arginine decarboxylase, three proteins (one acidic and two basic) have been purified, each of which inhibits both these enzymes. They do not inhibit the biodegradative ornithine and arginine decarboxylases nor lysine decarboxylase. The two basic inhibitors have been shown to correspond to the ribosomal proteins S20/L26 and L34, respectively. The relationship of the acidic antizyme to other known B. coli proteins remains to be determined.

Purification of ornithine decarboxylase from regenerating rat liver.

Abstract 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.