Nucleotide sequence of rat S-adenosylmethionine decarboxylase cDNA. Comparison with an intronless rat pseudogene.

Due to two different polyadenylation signals, two forms of S-adenosylmethionine decarboxylase (AdoMetDC) mRNA (2.1 and 3.4 kb) are present in human and rodent tissues. The nucleotide sequences of rat and human cDNAs corresponding to the shorter mRNA were published previously by us [Pajunen et al., J. Biol. Chem. 263 (1988) 17040-17049]. These sequences covered the coding regions but were incomplete at their 5' ends. Here we report the sequence of rat cDNA spanning the entire longer mRNA with a substantially extended leader region, and compare the sequence with that of a rat psi AdoMetDC pseudogene isolated from a rat genomic library. Relative to the mRNA, the pseudogene has multiple base changes as well as insertions, and deletions. Furthermore, it lacks introns, and is flanked by a short direct repeat. These are typical characteristics of a processed retrogene.

Structure and organization of the gene encoding rat S-adenosylmethionine decarboxylase.

The gene for S-adenosylmethionine decarboxylase (AdoMetDC) was isolated from a rat genomic library using AdoMetDC cDNA as a probe. Nucleotide sequence analysis shows that the rat AdoMetDC gene consists of 8 exons which encode a protein identical to that inferred by a rat AdoMetDC cDNA sequence. The exons range in length from 43 to 1964 base pairs spanning 15672 bases of chromosomal DNA. All of the exon/intron junctions were found to conform to the consensus splice donor and acceptor sequences. Exon 8 corresponds to the 3' noncoding region of the 2 species of AdoMetDC mRNA which are formed by alternative utilization of 2 polyadenylation signals separated from each other by 1272 nucleotides. The transcription initiation site was located by S1 nuclease protection and by primer extension analysis, -325 nucleotides upstream of the translation initiation codon. The promoter region of the rat AdoMetDC gene contains a TATA box at -29 base pairs. No typical GC or CAAT boxes are located in the promoter, but six GC boxes and several putative binding sites for both tissue-specific and non-specific transcription factors are found in the proximal part of intron 1. Southern blot analyses reveal a complex hybridization pattern suggesting that there are multiple copies of the AdoMetDC gene in the rat genome.

Expression of catalytically active rat S-adenosylmethionine decarboxylase in Escherichia coli.

The cDNA coding for rat S-adenosylmethionine decarboxylase (AdoMetDC, EC 4.1.1.50) has been cloned into a plasmid expression vector, pKK-223-3, and expressed in E. coli. The authenticity of the expressed protein has been demonstrated by reactivity with antibodies specific for rat AdoMetDC, by size analysis on SDS gels visualized with immunotransblots, and, finally, by catalytic activity. The expression of the enzyme results in a decrease in the activity of the bacterial enzyme suggesting the replacement of the bacterial enzyme by the rat AdoMetDC. Similarly, the addition of exogenous spermidine to the growth medium reduces bacterial enzyme activity affecting only marginally the expression of the recombinant protein.

The inverse changes of mouse brain ornithine and S-adenosylmethionine decarboxylase activities by chlorpromazine and imipramine. Dependence of ornithine decarboxylase induction on beta-adrenoceptors.

Intraperitoneal injection of chlorpromazine and imipramine increases mouse brain ornithine decarboxylase but decreases S-adenosyl-L-methionine decarboxylase activity. Maximal effect was obtained 6-8 hr after treatment at which time single dose of chlorpromazine (50 mg/kg) stimulated ornithine decarboxylase activity 7-fold and decreased S-adenosylmethionine decarboxylase activity to 50% from the control level. Correspondingly, ornithine decarboxylase activity was 5.5 times higher than the control value and S-adenosylmethionine decarboxylase activity about 40% from that after imipramine injection (80 mg/kg). The possible dependence of the enzyme responses on adrenergic receptors was studied using alpha-adrenoceptor antagonist, phentolamine, and beta-adrenoceptor antagonist, propranolol, concurrently with chlorpromazine and imipramine. The stimulation of ornithine decarboxylase but not the inhibition of S-adenosylmethionine decarboxylase could be abolished by propranolol (10 mg/kg), whereas phentolamine (10 mg/kg) slightly increased ornithine decarboxylase activity even when given alone. This suggests that beta- but not alpha-adrenergic mediation is involved in the stimulation of mouse brain ornithine decarboxylase activity and that brain ornithine and S-adenosylmethionine decarboxylase activities are independently regulated. When chlorpromazine and imipramine were tested in vitro, both of them turned out to have an inhibitory effect on S-adenosylmethionine decarboxylase. The former caused 50% inhibition at a concentration of 1 mM and the latter at 2 mM. Preliminary tests suggest that the type of inhibition is noncompetitive for both of them.

Immunohistochemical localization of L-ornithine decarboxylase in developing rat brain.

L-Ornithine decarboxylase, the rate limiting enzyme of polyamine biosynthesis and a marker enzyme of tissue proliferation and maturation, was localized immunocytochemically in the developing rat central nervous system. It can be noted that the distribution of the enzyme protein underlies temporal alterations. Conclusions are drawn from the localization of the enzyme and possible functional roles played by ornithine decarboxylase in discrete brain areas.

Regulation of mammalian S-adenosylmethionine decarboxylase.

S-Adenosylmethionine decarboxylase is a key enzyme in the biosynthesis of polyamines that is the rate limiting step in the formation of spermidine and spermine. The activity of S-adenosylmethionine decarboxylase is known to be regulated negatively by these polyamines and positively by their precursor, putrescine. A specific antiserum to S-adenosylmethionine decarboxylase was raised by immunizing rabbits with the homogeneous enzyme purified from rat prostate and a specific radioimmunoassay for the protein was set up. Using this radioimmunoassay it was found that a number of inhibitors of other steps in the polyamine biosynthetic pathway lead to increases in the amount of S-adenosylmethionine decarboxylase protein. These changes were caused by both a decreased rate of degradation and an increased rate of synthesis of the protein. The increased synthesis was due to two factors; a rise in the amount of translatable mRNA and an enhanced translation efficiency. The mRNA content of the prostate was substantially increased by treatment for 3 days with alpha-difluoromethylornithine (2% in drinking water). The translation of mRNA for S-adenosylmethionine decarboxylase was studied using a polyamine-depleted reticulocyte lysate supplemented with mRNA from rat prostate and the antiserum to precipitate the proteins corresponding to S-adenosylmethionine decarboxylase. These studies indicated that the enzyme was synthesized as an inactive precursor of Mr 37,000 which was converted to the enzyme sub-unit of Mr 32,000. The conversion of the precursor to the active sub-unit in vitro was increased by putrescine. The precursor could also be detected by immunoblotting of extracts from prostates of rats depleted of putrescine by treatment with the ornithine decarboxylase inhibitor, alpha-difluoromethylornithine. The translation of the S-adenosylmethionine decarboxylase mRNA in the reticulocyte lysates was strongly inhibited by the addition of spermidine or spermine demonstrating that polyamines directly inhibit the synthesis of S-adenosylmethionine decarboxylase. cDNA clones corresponding to S-adenosylmethionine decarboxylase were isolated using prostatic mRNA from polysomes enriched in S-adenosylmethionine decarboxylase by immunopurification. The use of these probes showed that rat ventral prostate contains two S-adenosylmethionine decarboxylase mRNA species of approximately 3.4 and 2.1 kb which differ in the 3' non-translated sequence. The sequence of these cDNAs will enable the amino acid sequence of the precursor to be obtained. This will provide evidence on the origin of the pyruvate prosthetic group of S-adenosylmethionine decarboxylase.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of taurine, GABA and glutamate on the distribution of Na(+) and K(+) ions between the isolated synaptosomes and incubation medium.

Isolated synaptosomes of calf brain cortex lost 90% of their original Na(+) content and 70% of their K(+) content during 30 min incubation in Na(+)- and K(+)-free iso-osmotic medium. Electrical stimulation, and 5 mmol/l of taurine or GABA significantly reduced the outflow of Na(+) and K(+) ions during incubation, but 5 mmol/l of glutamate strongly increased their outflow. The effect of glutamate on ion outflow was slightly reduced by external electrical pulses.

Ornithine decarboxylase in the inner ear of the guinea pig.

L-Ornithine decarboxylase, the rate limiting enzyme of polyamine synthesis and a possible marker enzyme for tissue proliferation and maturation, has been found in the developing guinea pig cochlea using the unlabelled horseradish-peroxidase-antiperoxidase technique. Ornithine decarboxylase-like immunoreactive material was detected in the neurons of the Ganglion spirale and in their axonal and/or dendritic fibers. The location of the enzyme and the possible functional role of ornithine decarboxylase plays in the development and maturation of the auditory organ and of the hearing process are discussed.

Developmental expression of ornithine and S-adenosylmethionine decarboxylases in mouse brain.

The activities of the two key enzymes in mammalian polyamine synthesis, ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC) in mouse brain show distinct, but inverse, changes during ontogeny. The level of ODC activity is about 70 fold higher at the time of birth than in the adult mouse, whereas AdoMetDC activity is very low after birth and increases as the brain matures. The correlation between the changes in enzyme activities and in the levels of the corresponding mRNAs diminishes dramatically during development. The increase in AdoMetDC mRNA level exceeds the increase in enzyme activity by 100%. Whereas ODC mRNA level falls initially, in concert with decreasing enzyme activity, but then shows an abrupt rise to a very high level during the late period of brain maturation while the enzyme activity continues to decrease to an almost undetectable level. These data suggest the development-dependent appearance of post-transcriptional regulation mechanisms.

Inhibition of polyamine synthesis blocks urinary secretion of beta-glucuronidase from mouse kidney.

The effect of inhibition of polyamine synthesis on castrated male mouse kidney beta-glucuronidase induction and secretion by testosterone was studied. Inhibition of the activities of polyamine synthesis key-enzymes, L-ornithine and S-adenosyl-L-methionine decarboxylases, was performed with the combined treatment of 2-difluoromethylornithine and methylglyoxal' bis(guanylhydrazone). Blockage of polyamine synthesis did not affect testosterone-induced increase in renal beta-glucuronidase but blocked its secretion into the urine. After withdrawal of inhibitor-treatment beta-glucuronidase secretion normalized, and repeated testosterone administration produced undisturbed beta-glucuronidase secretion peak in urine suggesting that blockage of beta-glucuronidase secretion was not due to the tissue damage produced by inhibitors. These results indicate that the stimulation of renal polyamine synthesis by testosterone is not necessary for the induction of beta-glucuronidase but is required for the urinary secretion of this protein.

Increase in S-adenosylmethionine decarboxylase in SV-3T3 cells treated with S-methyl-5'-methylthioadenosine.

Treatment of SV-3T3 cells with the spermine synthase inhibitor S-methyl-5'-methylthioadenosine [AdoS+(CH3)2] led to a large increase in the activity of S-adenosylmethionine decarboxylase (AdoMetDC) without affecting ornithine decarboxylase. The elevation of AdoMetDC activity was due to an increased amount of enzyme protein, as demonstrated by radioimmunoassay and by immunoblotting. The increase in AdoMetDC protein was caused by at least three factors: an increase in the amount of translatable mRNA, an increased translation efficiency of the mRNA and an increase in the half-life of the protein. The depletion of spermine brought about by AdoS+(CH3)2 appeared to be responsible for the increased synthesis of AdoMetDC and for part of the decrease in its rate of degradation. An additional stabilization of the enzyme protein was probably due to the binding of AdoS+(CH3)2, which is also a weak inhibitor of AdoMetDC. These results demonstrate the importance of cellular spermine concentrations in regulating the activity of AdoMetDC, which is a key enzyme controlling polyamine synthesis.

Detection of proenzyme form of S-adenosylmethionine decarboxylase in extracts from rat prostate.

Previous work in which the synthesis of S-adenosylmethionine decarboxylase was studied by translation of its mRNA indicated that it was formed as a proenzyme having a M.W. of about 37,000 that was cleaved to form the enzyme sub-unit of M.W. 32,000 in a putrescine-stimulated reaction. The extent to which the proenzyme accumulates in vivo and is affected by the putrescine concentration was studied by subjecting prostate extracts to Western immunoblotting procedures. The proenzyme form was readily detectable in control prostates (about 4% of the total) and this proportion was increased to 25% when the rats were pretreated for 3 days with the ornithine decarboxylase inhibitor, alpha-difluoromethylornithine. Conversely, it was decreased to almost undetectable levels after treatment with methylglyoxal bis(guanylhydrazone). These results indicate that the processing of the proenzyme form of S-adenosylmethionine decarboxylase is regulated by the cellular putrescine concentration. This conversion provides another step at which polyamine biosynthesis may be controlled.

Changes in gene expression in response to polyamine depletion indicates selective stabilization of mRNAs.

We used differential display analysis to identify mRNAs responsive to changes in polyamine synthesis. As an overproducing model we used the kidneys of transgenic hybrid mice overexpressing ornithine decarboxylase and S-adenosylmethionine decarboxylase, two key enzymes in polyamine biosynthesis. To identify mRNAs that respond to polyamine starvation, we treated Rat-2 cells with alpha-difluoromethylornithine, a specific inhibitor of polyamine biosynthesis. We isolated 41 partial cDNA clones, representing 37 differentially expressed mRNAs. Of these, 15 have similarity with known genes, five appear to be similar to reported expressed sequence tags and seventeen clones were novel sequences. Of the 35 mRNAs expressed differentially after alpha-difluoromethylornithine treatment, 26 were up-regulated. The expression of only three mRNAs was altered in the transgenic animals and all three were down-regulated. Determination of mRNA half-life of three of the mRNAs up-regulated in response to polyamine depletion revealed that the accumulation results from stabilization of the messages. Because most of the transcripts identified from Rat-2 cells suffering polyamine starvation were accumulated, we conclude that polyamine depletion, while blocking cell growth, is stabilizing mRNAs. This may be due to the lack of spermidine for post-translational modification of the eukaryotic initiation factor 5A, which plays a major role in mRNA turnover. The coupling of mRNA stabilization with cell-growth arrest in response to polyamine starvation provides cells with an economical way to resume growth after recovery from polyamine deficiency.

The effect of testosterone on the half-life of ornithine decarboxylase-mRNA in mouse kidney.

The effect of testosterone on half-lives of ornithine decarboxylase and its mRNA in mouse kidney was studied. In addition to the prolongation of enzyme protein half-life by androgens, excess of testosterone increases in vivo the half-life of its mRNA to about 3-fold as manifested by the change of enzyme half-life in testosterone-treated animals after alpha-amanitin or actinomycin D. These results suggest that the accumulation of ornithine decarboxylase in mouse kidney by androgens is partly due to the stabilization of its mRNA.

Molecular cloning of the mouse S-adenosylmethionine decarboxylase cDNA: specific protein binding to the conserved region of the mRNA 5'-untranslated region.

The nucleotide sequence of a cDNA encoding the proenzyme of mouse S-adenosylmethionine decarboxylase (AdoMetDC) including 257 nucleotides of the 5' untranslated region has been determined. Comparison of the nucleotide sequence of the mouse 5' untranslated region with those of other mammals shows it to be highly conserved. The 52 nucleotides upstream from the translation initiation codon are identical in human, rat, bovine and mouse. The polyamines, spermidine and spermine, have been shown to inhibit AdoMetDC mRNA translation. An RNA gel retardation assay demonstrated that a cytoplasmic extract from mouse brain forms an RNA-protein complex with the completely conserved 5' untranslated sequence and that the complex formation is highly dependent on the presence of spermine. Crosslinking by UV irradiation shows that the complex contains a 39-kDa protein interacting with the 5' untranslated sequence. These data demonstrate spermine-dependent specific protein binding to a highly conserved 5' untranslated region of an mRNA translationally regulated by polyamines.