Carol-Ann Manen

Proposed model of major sequential biochemical events of a trophic response.

Abstract It appears that the induction of ornithine decarboxylase regulates the rate of ribosomal RNA synthesis as well as regulating the rate of synthesis of polyamines. Further, ornithine decarboxylase, in most cases, is induced after a significant activation of cAMP-dependent protein kinase. We propose a model for the process of hypertrophy based on studies of a considerable number of mammalian growth systems. The mechanism of parallel regulation of polyamines and RNA appears to be initiated by the direct effect of ornithine decarboxylase on RNA polymerase I.

Relationship of ornithine decarboxylase to RNA polymerase I activity

Abstract Ornithine decarboxylase activity can be rapidly elevated 50- to 100-fold by the administration of methylxanthine derivatives such as methylisobutylxanthine. This elevation occurs just prior to the increase in RNA polymerase I activity. Inhibitors of RNA synthesis and of protein synthesis suggest that any alteration in the ornithine decarboxylase response results in a similar alteration in the level of α-amanitin-insensitive RNA polymerase. Addition of a partially purified ornithine decarboxylase preparation to the RNA polymerase assay increased both the initial rate of 3H-UTP incorporation and the length of time that the polymerase assay was linear. It is suggested that ornithine decarboxylase is the labile protein that modulates the level of RNA polymerase I.

In vivo activation of cAMP-dependent protein kinase by aminophylline and 1-methyl, 3-isobutylxanthine

Summary Aminophylline and 1-methyl, 3-isobutylxanthine, inhibitors of phosphodiesterases, caused an increase in the cyclic AMP level and an elevation in the activity of cyclic AMP-dependent protein kinase in the liver of the rat within 10–20 min of administration. The activation of cyclic AMP-dependent protein kinase appeared to be a more accurate measure of cyclic AMP-dependent events than cyclic AMP concentration and was statistically more reliable. After Sephadex chromatography, the protein kinase yielded the same activation pattern as measurable in the crude supernatant of liver.

Regulation of RNA polymerase I activity by ornithine decarboxylase

Abstract Ornithine decarboxylase activity was elevated from 5- to 80-fold in rat liver by the administration of various methylxanthine derivatives, i.e. theophylline, aminophylline, and 3-isobutyl,1-methylxanthine. The increased activity of ornithine decarboxylase in all instances was closely followed (within 0.5 hr) by an increase in the activity of RNA polymerase I. The increment in ornithine decarboxylase activity and the increase in RNA polymerase I activity after all three drugs showed a parallel relationship. That is, theophylline administration resulted in the least stimulation of both ornithine decarboxylase and RNA polymerase I activities, and 3-isobutyl,1-methylxanthine administration produced the greatest stimulation of both enzymes. The enhanced RNA polymerase I activity resulted in a significant increase in the total RNA content of the liver within 24 hr. Administration of inhibitors of protein synthesis and RNA synthesis indicated that attenuation of the increase in ornithine decarboxylase activity was closely paralleled by attenuation of the increase in RNA polymerase I activity. RNA polymerase I activity was not stimulated by putrescine concentrations which were physiological and above. We suggest, therefore, that ornithine decarboxylase may be involved in the regulation of RNA polymerase I activity.

Further evidence of cyclic amp-mediated hypertrophy as a prerequisite of drug-specific enzyme induction

Abstract We have previously reported that the administration of phenobarbital, 3-methylcholanthrene or the polychlorinated biphenyl, Aroclor-1254, to rats resulted in an early and sequential increase in the activities of hepatic cAMP-dependent protein kinase(s), omithine decarboxylase and RNA polymerase I. It was suggested that this sequence of events was involved in both liver hypertrophy and the induction of microsomal mixed-function oxygenases. To further test this hypothesis, we investigated if mice unable to induce aryl hydrocarbon hydroxylase in response to 3-methylcholanthrene exhibited increases in these cAMP-mediated events. A single dose of 3-methylcholanthrene (150 mg/kg, i.p.) was administered to male C57B1/6J (aryl hydrocarbon responsive) and DBA/2J (aryl hydrocarbon nonresponsive) mice. In the C57B1/6J mice, the hepatic cAMP concentration increased to 165 per cent of control within 1 hr. Maximal increases in the activities of liver cAMP-dependent protein kinase(s) (160 per cent), ornithine decarboxylase (210 per cent) and RNA polymerase I (120 per cent) occurred at 2, 4 and 6 hr, respectively, in the responsive mice. There were no detectable increases in any of these parameters in the nonresponsive (DBA/2J) mice. Multiple doses of 3-methylcholanthrene (20 mg/kg, i.p., daily × 3) resulted in increases in hepatic aryl hydrocarbon hydroxylase (460 per cent) and liver weight/body weight ratios (118 per cent) in the responsive (C57B1/6J) mice killed at 5 days. There was no increase in either of these parameters in the nonresponsive (DBA/2J) mice. Both the responsive and nonresponsive mice responded similarly to a single parental dose of phenobarbital (100 mg/kg) with maximal increases in the activities of cAMP-dependent protein kinase(s) (150 per cent), ornithine decarboxylase (160 per cent) and RNA polymerase I (135 per cent) at 2, 4 and 8 hr respectively. Multiple doses of phenobarbital (100 mg/kg, i.p., daily × 3) resulted in increased ethylmorphine N -demethylase activity (160 per cent) and liver weight/body weight ratios (130 per cent) in both strains of mice at 5 days. These data provide further evidence of coupled cAMP-mediated hypertrophy and induction of mixed-function oxygenases in liver.

Posttranslationally modified ornithine decarboxylase may regulate RNA polymerase I activity

Purified ornithine decarboxylase (EC 4.1.1.17, ODC) transamidated with four putrescine moieties on four glutamine residues through the action of transglutaminase (EC 2.3.2.13, TGase) purified from guinea pig liver, when added to isolated rat liver nuclei, stoichiometrically increased the activity of RNA polymerase I (EC 2.7.7.6). The increase was relative to the pmoles of purified conjugated ODC added to the reaction and could be reinitiated after the reaction had plateaued by the further addition of ODC-putrescine conjugate. The kinetics of the reaction suggest that the ODC-putrescine conjugate was not reused but degraded after each initiation. Otherwise, the rapid plateau would not be observed. The repeated addition of 278 pmoles of purified ODC-putrescine conjugate to rat liver nuclear preparations containing 200 micrograms total protein consistently stimulated the incorporation of 600-700 pmoles UMP/mg protein. We suggest that ODC transamidated by its product putrescine may be the posttranslationally modified 65,000 Mr protein which has been reported by several laboratories to serve as a labile subunit of RNA polymerase I.

Polyamine biosynthesis and accumulation during the early development of the nudibranch Phestilla sibogae

Abstract Polyamine biosynthesis and accumulation were studied during the early development of the nudibranch Phestilla sibogae. Ornithine decarboxylase activity increases over 40-fold in the first 4 days of embryogenesis, with the maximum (400 pmole of 14CO2/30 min/mg of protein) occurring between Days 3 and 4, the time of most rapid growth. Putrescine-stimulated S-adenosyl- l -methionine decarboxylase activity is not detectable until the second day of development and attains maximal activity (100 pmole of 14CO2/30 min/mg of protein) at Day 4. The pattern of spermidine-stimulated S-adenosyl- l -methionine decarboxylase activity is similar. Putrescine and spermidine double in concentration between Days 0 and 6. Adults contain fairly high levels of putrescine and spermidine, similar to prokaryotes. The increase in the polyamine biosynthetic enzymes and the accumulation patterns of the polyamines correlate well with data from other early developmental systems and support the hypothesis of key roles for the polyamine pathway in the control of growth processes.

Rapid partial purification of S-adenosyl-L-methionine decarboxylase by affinity chromatography☆

Abstract A Sepharose-ethylenediamine-PCMB column can be used to obtain a rapid purification of S-adenosyl-L-methionine decarboxylase. PCMB-affinity fractions from both rat liver and sea urchin eggs have high specific activity, particularly the latter. The activity of the purified rat liver enzyme is stimulated by the addition of either putrescine or spermidine, whereas the purified enzyme fraction from sea urchin eggs has no measurable activity without the addition of either putrescine or spermidine. In both preparations there is a stoichiometric relationship between the release of 14 CO2 from S-adenosyl-L-carboxyl- 14 C-methionine and the formation of spermidine.