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Approaching the Structures of Mammalian Propylamine Transferases and their Genes
Abstract
Propylamine transferases, spermidine synthase (EC 2.5.1.16) and spermine synthase (EC 2.5.1.22) catalyze the two final, irreversible steps in the conversion of arginine and methionine to spermidine and spermine, respectively. Both of the amino acid precursors are nutritionally essential for animals and are present in animal tissues at low, well-conserved concentrations. Arginine plays a central role in protein catabolism by being an essential intermediate in the conversion of toxic ammonia to excretable nontoxic urea. Methionine, on the other hand, is vitally important for protein synthesis by being the amino acid needed for peptide chain initiation and for the transmethylation reactions involved in the control of metabolism at several steps of transcriptional, translational and post-translational level. The fraction of the precursor amino acids consumed in polyamine synthesis varies a great deal depending on the cell type, the stage of differentiation and the proliferation rate. As to methionine consumption, the share of polyamine synthesis is less than 1% in rat liver (Eloranta and Kajander, 1984) but may exceed 20% in some cultured cells (Iizasa and Carson, 1985). Although detailed action mechanisms of polyamines in animal physiology are poorly known, their essentiality for cellular functions have been widely demonstrated (for references see Tabor and Tabor, 1984; Pegg, 1986).
The polyamines encountered in the parasites responsible for African trypanosomiasis include putrescine or 1,4-diaminopropane and its mono N-propylated form, spermidine [1, 2]. These amines not only play a role in the parasite’s growth and differentiation, but also contribute to the formation of a trypanosome-specific molecule, trypanothione, which represents for the trypanosome the equivalent of glutathione in eukaryotic cells. The enzymatic systems which participate in the synthesis of these polyamines are hence preferential targets for research of inhibitors which could be put to use as antiparasitic drug treatment. Perhaps it is such a potential clinical application that has encouraged the numerous general reviews of the metabolism of these polyamines [2–9]; see also Fig. 1).
A model of the active site of aminopropyltransferases was proposed based on the study of a number of monoamino and diamino compounds as potential inhibitors and substrates, respectively, of spermidine synthase purified from pig liver. The active site seems to have a relatively large hydrophobic cavity adjacent to a negatively charged site, to which a protonated amino group of putrescine binds, with another amino group of putrescine being situated in the hydrophobic cavity as a free form to be aminopropylated by decarboxylated S-adenosylmethionine. On the basis of the above-mentioned model, another modified one was proposed for spermine synthase, and several compounds mentioned model, another modified one was proposed for spermine synthase, and several compounds designed according to the modified model were found to potently inhibit spermine synthase, purified from rat brain, in competition with spermidine. The newly developed inhibitors were about two orders of magnitude more potent in vitro than a known inhibitor of spermine synthase, dimethyl(5'-adenosyl)sulfonium perchlorate.
Using a synthetic deoxyoligonucleotide mixture constructed for a tryptic peptide of the bovine enzyme as a probe, cDNA coding for the full-length subunit of spermidine synthase was isolated from a human decidual cDNA library constructed on phage lambda gt11. After subcloning into the Eco RI site of pBR322 and propagation, both strands of the insert were sequenced using a shotgun strategy. Starting from the first start codon, which was immediately preceded by a GC-rich region including four overlapping CCGCC consensus sequences, an open reading frame for a 302-amino-acid polypeptide was resolved. This peptide had an Mr of 33,827, started with methionine, and ended with serine. The identity of the isolated cDNA was confirmed by comparison of the deduced amino acid sequence with resolved sequences of the tryptic peptides of bovine spermidine synthase. The coding strand of the cDNA revealed no special regulatory or ribosome-binding signals within 82 nucleotides preceding the start codon and no polyadenylation signal within 247 nucleotides following the stop codon. The coding region, containing a 13-nucleotide repeat close to the 5' end, was longer than, and very different from, that of the bacterial counterpart. This region seems to be of retroviral origin and shows marked homology with sequences found in a variety of human, mammalian, avian, and viral genes and mRNAs. By computer analysis, the first 200 nucleotides of the 5' end of the coding strand appear able to form a very stable secondary structure with a free energy change of -157.6 kcal/mole.(ABSTRACT TRUNCATED AT 250 WORDS)
We have isolated and sequenced cDNA clones that encode human spermine synthase (EC 2.5.1.22). The total length of the sequenced cDNA was 1,612 nucleotides, containing an open reading frame encoding a polypeptide chain of 368 amino acids. All of the previously sequenced peptide fragments of human and bovine spermine synthase proteins could be located within the coding region derived from the cDNA. An unusual sequence of AATTAA apparently signaled the initiation of polyadenylation. Sequence comparisons between human spermine synthase and spermidine synthases from bacterial and mammalian sources revealed a nearly complete lack of similarity between the primary structures of these two enzymes catalyzing almost identical reactions. A modest similarity found was restricted to a relatively short peptide domain apparently involved in the binding of decarboxylated S-adenosylmethionine, the common substrate for both enzymes. The apparent lack of an overall similarity may indicate that spermine synthase, the enzyme found only in eukaryotes, and spermidine synthase with more universal distribution, although functionally closely related, have evolved separately.
The activity of arginine decarboxylase (ADC), a key enzyme in plant polyamine biosynthesis, was manipulated in two generations of transgenic tobacco plants. Second-generation transgenic plants overexpressing an oat ADC cDNA contained high levels of oat ADC transcript relative to tobacco ADC, possessed elevated ADC enzyme activity and accumulated 10-20-fold more agmatine, the direct product of ADC. In the presence of high levels of the precursor agmatine, no increase in the levels of the polyamines putrescine, spermidine and spermine was detected in the transgenic plants. Similarly, the activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase were unchanged. No diversion of polyamine metabolism into the hydroxycinnamic acid-polyamine conjugate pool or into the tobacco alkaloid nicotine was detected. Activity of the catabolic enzyme diamine oxidase was the same in transgenic and control plants. The elevated ADC activity and agmatine production were subjected to a metabolic/physical block preventing increased, i.e. deregulated, polyamine accumulation. Overaccumulation of agmatine in the transgenic plants did not affect morphological development.
We have previously shown that the gene (speD) for S-adenosylmethionine decarboxylase is part of an operon that also contains the gene (speE) for spermidine synthase (Tabor, C. W., Tabor, H., and Xie, Q.-W. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 6040-6044). We have now determined the nucleotide sequence of this operon and have found that speD codes for a polypeptide of Mr = 30,400, which is considerably greater than the subunit size of the purified enzyme. Our studies show that S-adenosylmethionine decarboxylase is first formed as a Mr = 30,400 polypeptide and that this proenzyme is then cleaved at the Lys111-Ser112 peptide bond to form a Mr = 12,400 subunit and a Mr = 18,000 subunit. The latter subunit contains the pyruvoyl moiety that we previously showed is required for enzymatic activity. Both subunits are present in the purified enzyme. These conclusions are based on (i) pulse-chase experiments with a strain containing a speD+ plasmid which showed a precursor-product relationship between the proenzyme and the enzyme subunits, (ii) the amino acid sequence of the proenzyme form of S-adenosylmethionine decarboxylase (derived from the nucleotide sequence of the speD gene), and (iii) comparison of this sequence of the proenzyme with the N-terminal amino acid sequences of the two subunits of the purified enzyme reported by Anton and Kutny (Anton, D. L., and Kutny, R. (1987) J. Biol. Chem. 262, 2817-2822).
A method is presented for locating protein antigenic determinants by analyzing amino acid sequences in order to find the point of greatest local hydrophilicity. This is accomplished by assigning each amino acid a numerical value (hydrophilicity value) and then repetitively averaging these values along the peptide chain. The point of highest local average hydrophilicity is invariably located in, or immediately adjacent to, an antigenic determinant. It was found that the prediction success rate depended on averaging group length, with hexapeptide averages yielding optimal results. The method was developed using 12 proteins for which extensive immunochemical analysis has been carried out and subsequently was used to predict antigenic determinants for the following proteins: hepatitis B surface antigen, influenza hemagglutinins, fowl plague virus hemagglutinin, human histocompatibility antigen HLA-B7, human interferons, Escherichia coli and cholera enterotoxins, ragweed allergens Ra3 and Ra5, and streptococcal M protein. The hepatitis B surface antigen sequence was synthesized by chemical means and was shown to have antigenic activity by radioimmunoassay.
A method is described for the affinity purification of antibodies using protein samples that have been electrophoretically transferred to diazotized paper. Using differentiated neuroblastoma cells as the protein sample and a heterogeneous anti-microtubule protein serum, antibodies were isolated that specifically bound only to tubulin on blots and that stained microtubule networks in cells. The general utility of this method for various types of applications is discussed.
The addition of [1,4-14C]putrescine into Zea mays seedlings yielded radioactive spermidine. The rate of incorporation of [14C]putrescine was depressed by pretreatment with methylglyoxal-bis-(guanylhydrazone) (MGBG). Spermidine synthetase has been purified 115-fold from etiolated maize shoots. The MW determined by Sephadex G-200 filtration was ca 43 000 and the optimum pH was 7.2. The enzyme was inhibited by sulfhydryl reagents. The purified enzyme had no spermine synthetase or polyamine oxidase activities.
Spermine synthase, a propylamine transferase, which catalyses the biosynthesis of spermine from S-methyladenosylhomocysteamine and spermidine has been purified to an apparent homogeneity (about 6000-fold) from bovine brain using spermine-Sepharose affinity chromatography. The enzyme preparation was free from S-adenosylmethionine decarboxylase and spermidine synthase activities. The molecular Stokes radius of the enzyme was calculated to be 4.16 nm. The enzyme has an apparent molecular weight of approximately 88000, composing of two subunits of equal size. The enzyme showed a broad pH optimum between 7.0 and 8.0 and an acidic isoelectric point at pH 5.10. The apparent Km value for S-methyladenosylhomocysteamine was 0.6 μM and about 60 μM for spermidine. The enzyme showed strict specificity to spermidine as the propylamine acceptor.
Both the reaction products, spermine and 5′-methylthioadenosine inhibited the enzyme activity, methylthioadenosine being a powerful competitive inhibitor with respect to S-methyladenosyl-homocysteamine (Ki value of about 0.3 μM). Putrescine also inhibited competitively with respect to spermidine (Ki value of about 1.7 mM). Spermine synthase had no requirements for metal or other cofactors.
A cDNA for chicken avidin was identified in a chicken oviduct cDNA library by screening with antibodies and synthetic oligodeoxyribonucleotides.
Four recombinant clones were characterized and each contained the sequence of the ollgonucleotide probes used in screening.
They were capable also of expressing an antigen recognizable by a polyclonal or a mixture of monoclonal antibodies raised
against avidin. The longest clone, λcAV4, contained the entire coding sequence of avidin along with a signal peptide of 24
amino acids. An avidin mRNA, approximately 700 nucleotides in length, was induced by a single injection of progesterone over
a period of twenty four hours. The avidin mRNA was distributed in a tissue-specific manner, since detectable concentration
of the mRNA appeared only in the oviduct after stimulation with progesterone alone or with a combination of progesterone and
estrogen. No avidin mRNA was detected in the liver or kidney under these conditions. Preliminary results on the genomic complexity
of avidin suggest a single copy gene. Isolation of the natural gene for avidin and studies on its regulation now can be initiated
using the cDNA probe.
The consumption of S-adenosylmethionine during polyamine synthesis and transmethylation reactions yields stoichiometric amounts of 5'-deoxy-5'-methylthioadenosine and S-adenosylhomocysteine, respectively. Information concerning the regulation of the two routes of S-adenosylmethionine metabolism in viable cells under changing growth conditions is limited. The present experiments have measured the time-dependent accumulation of 5'-deoxy-5'-methylthioadenosine and L-homocysteine in the medium of four malignant human and murine cell lines deficient in 5'-deoxy-5'-methylthioadenosine phosphorylase (5'-methylthioadenosine: orthophosphate methylthioribosyltransferase). Included in this group were anchorage-independent and anchorage-dependent cells. The enzyme-deficient cells did not detectably cleave 5'-deoxy-5'-methylthioadenosine, and did not appreciably metabolize homocysteine. A comparison of 5'-deoxy-5'-methylthioadenosine and homocysteine excretion therefore provided a noninvasive method for estimating the relative rates of polyamine synthesis and transmethylation. Early after the release of human CEM lymphoblasts from density dependent growth arrest, 5'-deoxy-5'-methylthioadenosine production increased, and exceeded homocysteine synthesis. 5'-Deoxy-5'-methylthioadenosine formation reached a maximum of 0.9 nmol/12 h per 10(6) cells prior to the onset of exponential growth. The kinetics of homocysteine synthesis were different. Homocysteine accumulation was proportional to the specific growth rate, and achieved a peak of 3.1 nmol/12 h per 10(6) cells during mid-exponential phase, at which time 5'-deoxy-5'-methylthioadenosine production was falling. Similar patterns of 5'-deoxy-5'-methylthioadenosine and homocysteine excretion were observed in other 5'-deoxy-5'-methylthioadenosine phosphorylase deficient cell lines. These data show that polyamine synthesis and transmethylation are differentially regulated during the growth cycle of mammalian cells.
A convenient technique for the partial purification of large quantities of functional, poly(adenylic acid)-rich mRNA is described. The method depends upon annealing poly(adenylic acid)-rich mRNA to oligothymidylic acid-cellulose columns and its elution with buffers of low ionic strength. Biologically active rabbit globin mRNA has been purified by this procedure and assayed for its ability to direct the synthesis of rabbit globin in a cell-free extract of ascites tumor. Inasmuch as various mammalian mRNAs appear to be rich in poly(adenylic acid) and can likely be translated in the ascites cell-free extract, this approach should prove generally useful as an initial step in the isolation of specific mRNAs.
Patients with seropositive rheumatoid arthritis (RA) have elevated titers of precipitating antibody toward an antigen designated RA nuclear antigen (RANA). Anti-RANA reactivity has been associated with prior Epstein-Barr virus (EBV) infection. Using the protein blot technique, we have identified, in extracts of WI-L2, an EBV+ nonproducer B-lymphoblast line, a Mr 80,000 polypeptide that is reactive with anti-RANA-containing sera. This same polypeptide can be recovered from RANA precipitin bands. The Mr 80,000 polypeptide appears to be EBV-associated, as a homologous antigen was detected in two other EBV+ cell lines, Daudi and Raji, but was not present in three EBV- human cell lines tested, HeLa, BJAB, and Ramos. Anti-RANA antibodies and antibodies reactive with the Mr 80,000 polypeptide also appear coincidently in the sera of individuals exhibiting an EBV infection (infectious mononucleosis). Further analysis of the RANA-associated Mr 80,000 polypeptide suggested its identity with the previously recognized EBV-associated nuclear antigen designated EBNA. The Mr 80,000 antigen shares with EBNA the properties of being a heat-stable, DNA binding protein. EBNA is traditionally assayed by a complement fixation reaction and anti-Mr 80,000 antibodies were shown to be reactive when a complement fixation assay was used in the immunoblot. Finally, when the Mr 80,000 antigen was used to absorb an anti-RANA/anti-EBNA serum, both antibodies were reduced.
To investigate the mechanisms by which androgens regulate ornithine decarboxylase (OrnDCase; L-ornithine carboxy-lyase, EC 4.1.1.17) in mouse kidney, a cDNA clone encoding OrnDCase mRNA was prepared. Purification of OrnDCase mRNA from kidneys of androgen-treated mice was accomplished by immunoadsorption of renal polysomes to a protein A-Sepharose column and enrichment for poly(A)-containing RNA by oligo(dT)-cellulose. Double-stranded cDNA synthesized from this mRNA was inserted into the Pst I site of plasmid pBR322 by using oligo(dG . dC)-tailing and was propagated in Escherichia coli. Plasmids containing cDNA sequences coding for OrnDCase were identified by differential colony hybridization, by radioimmunological detection of OrnDCase-like antigens in bacterial cultures, and by cell-free translation of hybrid-selected mRNA followed by immunoprecipitation with monospecific OrnDCase antiserum. A restriction endonuclease fragment of the selected plasmid DNA (pODC54) was labeled by nick-translation and used to study changes in OrnDCase mRNA concentration. After a single dose of testosterone, renal OrnDCase mRNA concentration increased as soon as 6 hr and peaked 24 hr after steroid injection, as measured by RNA blot hybridization. Continuous androgen treatment for 4 days resulted in a 10- to 20-fold increase in OrnDCase mRNA concentration in normal animals, but no induction of this mRNA was detected in mice that have an inherent defect of the androgen receptor (testicular feminization). These results indicate that androgens regulate OrnD-Case synthesis in mouse kidney, at least in part, by increasing OrnDCase mRNA accumulation.
A kinetic analysis including initial-velocity and product-inhibition studies were performed with spermine synthase purified from bovine brain. The enzyme activity was assayed in the presence of 5'-methylthioadenosine phosphorylase as an auxiliary enzyme to prevent the accumulation of the inhibitory product, 5'-methylthioadenosine, and thus to obtain linearity of the reaction with time. Initial-velocity studies gave intersecting or converging linear double-reciprocal plots. No substrate inhibition by decarboxylated S-adenosylmethionine was observed at concentrations up to 0.4 mM. Apparent Michaelis constants were 60 microM for spermidine and 0.1 microM for decarboxylated S-adenosylmethionine. Spermine was a competitive product inhibitor with respect to decarboxylated S-adenosylmethionine, but a mixed one with respect to the other substrate, spermidine. 5'-Methylthioadenosine showed a mixed inhibition with both substrates, predominantly competitive with respect to decarboxylated S-adenosylmethionine and predominantly uncompetitive with respect to spermidine. The observed kinetic and inhibition patterns are consistent with a compulsory-order mechanism, where both substrates add to the enzyme before products can be released.
The fate of S-adenosyl-L-methionine was studied in rat liver extracts by analysing the distribution of radioactivity from labelled adenosylmethionine in decomposition products, which were separated from each other by chromatographic and electrophoretic means. Marked non-enzymic degradation to adenine, pentosylmethionine, methylthioadenosine and homoserine was evident at pH 6.9-7.8. Enzymic cleavage to methylthioadenosine was stoichiometric with the accumulation of spermidine and could be totally prevented by inhibiting S-adenosyl-L-methionine decarboxylase. The results rule out the existence of adenosylmethionine cyclotransferase in rat liver and indicate that only two quantitatively significant enzymic processes are involved in hepatic adenosylmethionine degradation. Excluding nonenzymic decomposition, more than 99% of adenosylmethionine is demethylated and exclusively catabolized further by S-adenosyl-L-homocysteine hydrolase. Less than 1% of adenosylmethionine is decarboxylated and immediately utilized totally for polyamine biosynthesis.
Spermidine synthase was purified to homogeneity from rat and pig liver by a method modified from a previously reported one using DEAE-Sepharose, S-adenosyl(5')-3-thiopropylamine-Sepharose affinity chromatography, Sephacryl S-300 gel filtration and polyacrylamide gel electrophoresis. No apparent difference between the two enzymes was observed in specific activity, molecular weight (74,000), or subunit composition (two subunits). However, significant differences were observed in their pI values, which were 5.16 for the pig enzyme and 5.34 for the rat enzyme, and their peptide maps. Amino acid compositions of the two enzymes were closely related, but differed significantly in some amino acids. In addition, the rat enzyme was more sensitive to inhibition by S-adenosyl-1,8-diamino-3-thiooctane than the pig enzyme.
Spermidine synthase (EC 2.5.1.16) was purified to apparent homogeneity (about 11 000-fold) from bovine brain by affinity chromatography, with S-adenosyl-(5')-3-thiopropylamine linked to Sepharose as the adsorbent. The enzyme preparation was free from S-adenosylmethionine decarboxylase (EC 4.1.1.50) and spermine synthase (EC 2.5.1.22) activities. The native enzyme had an apparent Mr of 70 000, was composed of two subunits of equal size, and had an isoelectric point at pH 5.22. The apparent Km values for putrescine and decarboxylated adenosylmethionine [S-adenosyl-(5')-3-methylthiopropylamine] were 40 microM and 0.3 microM respectively. Cadaverine and 1,6-diaminohexane could replace putrescine as the aminopropyl acceptor, although the reaction rates were only 6% and 1% respectively of that obtained with putrescine. Ethyl, propyl and carboxymethyl analogues of decarboxy-S-adenosylmethionine could act as propylamine donors. Both the reaction products, spermidine and 5'-methylthioadenosine, were mixed-type inhibitors of the enzyme. On the basis of initial-velocity and product-inhibition studies, a ping-pong reaction mechanism for the spermidine synthase reaction was ruled out.
1. The specificity of rat prostatic spermidine synthase and spermine synthase with respect to the amine acceptor of the propylamine group was studied. 2. Spermidine synthase could use cadaverine (1,5-diaminopentane) instead of putrescine, but the Km for cadaverine was much greater and the rate with 1mM-cadaverine was only 10% of that with putrescine. 1,3-Diaminopropane was even less active (2% of the rate with putrescine) and no other compound tested (including longer alpha,omega-diamines, spermidine and its homologues and monoacetyl derivatives) was active. 3. Spermine synthase was equally specific. The only compounds tested that showed any activity were 1,8-diamino-octane, sym-homospermidine, sym-norspermidine and N-(3-aminopropyl)-cadaverine, which at 1mM gave rates 2, 17, 3 and 4% of the rate with spermidine respectively. 4. The formation of polyamine derivatives of cadaverine and to a very small extent of 1,3-diaminopropane was confirmed by exposing transformed mouse fibroblasts to these diamines when synthesis of putrescine was prevented by alpha-difluoromethylornithine. Under these conditions the cells accumulated significant amounts of N-(3-aminopropyl)cadaverine and NN'-bis(3-aminopropyl)cadaverine when exposed to cadaverine and small amounts of sym-norspermidine and sym-norspermine when exposed to 1,3-diaminopropane.
We isolated several strains of Saccharomyces cerevisiae containing mutations mapping at a single chromosomal gene (spe10); these strains are defective in the decarboxylation of L-ornithine to form putrescine and consequently do not synthesize spermidine and spermine. The growth of one of these mutants was completely eliminated in a polyamine-deficient medium; the growth rate was restored to normal if putrescine, spermidine, or spermine was added. spe10 is not linked to spe2 (adenosylmethionine decarboxylase) or spe3 (putrescine aminopropyltransferase [spermidine synthease]). spe 10 is probably a regulatory gene rather than the structural gene for ornithine decarboxylase, since we isolated two different mutations which bypassed spe10 mutants; these were spe4, an unliked recessive mutation, and spe40, a dominant mutation linked to spe10. Both spe4 and spe40 mutants exhibited a deficiency of spermidine aminopropyltransferase (spermine synthase), but not of putrescine aminopropyltransferase. This suggests that ornithine decarboxylase activity is negatively controlled by the presence of spermidine aminopropyltransferase.
A novel affinity chromatographic adsorbent was developed for purification of spermidine synthase from rat prostate. The adsorbent (S-adenosyl(5′)-3-thiopropylamine-Sepharose) possesses a ligand structurally similar to S-adenosyl(5′)-3-methylthiopropylamine (decarboxy AdoMet), a substrate of spermidine synthase. The S-adenosyl(5′)-3-thiopropylamine-Sepharose was prepared by an alkylation on sulfur of S-adenosyl-3-thiopropylamine by bromoacetamidohexyl-Sepharose under mild acidic conditions. The enzyme has been purified to homogeneity in 40% yield by using DEAE-cellulose, affinity chromatography employing S-adenosyl(5′)-3-thiopropylamine-Sepharose, and gel filtration. The enzyme had a molecular weight of approximately 73,000 and was composed of two subunits of equal size. The specificity of the reaction was rather strict, but cadaverine could replace putrescine as the aminopropyl acceptor, and the rate was 1/20th of the rate for spermidine formation. Apparent Km values for putrescine and decarboxy AdoMet were 0.1 mm and 1.1 μm, respectively. Inhibition by decarboxy AdoMet and 5′-deoxy-5′-methylthioadenosine was observed. The inhibition by 5′-deoxy-5′-methylthioadenosine was partially noncompetitive with respect to decarboxy AdoMet.
Polyamine synthesis in mammalian tissues. Isolation and characterization of spermine synthase from bovine brain
- R.-L Pajula
- A Raina
- T Eloranta
- R-L Pajula
Spermidine biosynthesis. Purification and properties of propylamine transferase from Escherichia coli
- W H Bowman
- C W Tabor
- WH Bowman