Irreversible inhibition of putrescine-stimulated S-adenosyl-L-methionine decarboxylase by berenil and pentamidine.

The putrescine-stimulated S-adenosyl-L-methionine decarboxylases from rat liver and yeast were strongly inhibited by Berenil and to a lesser extent by Pentamidine. Ten times greater drug concentrations were needed to achieve a similar level of inhibition of a Mg2+-stimulated bacterial enzyme. The inhibition was irreversible in that extensive dialyses or precipitation with (NH4)2SO4 did not restore enzyme activity. Putrescine did not protect the enzyme against Berenil, but adenosylmethionine either alone or with putrescine partially protected the irreversible action of Berenil. The compound 4,4'-diamidinodiphenylamine, which differs from Berenil only in lacking the azo group between benzene rings, was a weaker inhibitor than Berenil, and its inhibition was reversible. Berenil also inhibited the activity of adenosylmethionine decarboxylase in vivo, by depressing the activity of the enzyme in normal rat liver, for at least 24 h after a single injection (50 mg/kg body wt.) of the drug.     

Isolation and identification of S-adenosylmethionine from human urine

Procedures are described for the isolation and identification of adenosylmethionine from human urine. Previously described preliminary separative procedures using anion and cation exchange columns and an XAD-4 resin column have been extended to permit the separation of adenosylmethionine. The adenosylmethionine has been identified by conversion to methylthioadenosine followed by rechromatography of the latter compound with three different types of columns and elution systems. Mean adenosylmethionine values for urine were as follows: adults, 0.26; children, 0.36 nmole/mumole creatinine. Recovery of adenosylmethionine added to urine and determined by this separative procedure was 52%.     

Adenosylmethionine decarboxylase from various organisms: Relation of the putrescine activation of the enzyme to the ability of the organism to synthesize spermine

S-adenosyl-L-methionine decarboxylase (EC 4.1.1.50) from most eukaryotic organisms is activated by putrescine whereas the corresponding enzyme from bacterial sources shows a stringent requirement for magnesium ions. Adenosylmethionine decarboxylase from lower eukaryotes such as protozoa, however, is not influenced by diamines, neither are any metals needed for its maximal activity. A common characteristic of those organisms containing putrescine-insensitive adenosylmethionine decarboxylase appeared to be either a total absence or very low intracellular content of spermine. While extracts of all organisms containing putrescine-activated adenosylmethionine decarboxylase (animal tissues and yeast) exhibited easily measurable spermine synthase activity, no such activity was detected in cells of Tetrahymena pyriformis, Escherichia coli or Pseudomonas aeruginosa all containing adenosylmethionine decarboxylase insensitive to putrescine and other diamines.The activation of adenosylmethionine decarboxylase by putrescine, the immediate precursor of spermidine, may thus assure the availability of sufficient amounts of decarboxylated adenosylmethionine (S-methyladenosyl-cysteamine) for the synthesis of spermidine even in the presence of a spermine synthesizing system competing for the same precursor (decarboxylated adenosylmethionine).     

Specific inhibition of the synthesis of putrescine and spermidine by 1,3-diaminopropane in rat liver in vivo.

Chronic administration of 1,3-diaminopropane, a compound inhibiting mammalian ornithine decarboxylase (EC 4.1.1.17) in vivo, effectively prevented the large increases in the concentration of putrescine that normally occur during rat liver regeneration. Furthermore, repeated injections of diaminopropane depressed by more than 85% ornithine decarboxylase activity in rat kidney. Administration of diaminopropane 60 min before partial hepatectomy only marginally inhibited ornithine decarboxylase activity at 4 h after the operation. However, when the compound was given at the time of the operation (4 h before death), or any time thereafter, it virtually abolished the enhancement in ornithine decarboxylase activity in regenerating rat liver remnant. An injection of diaminopropane given 30 to 60 min after operation, but not earlier or later, depressed S-adenosyl-L-methionine decarboxylase activity (EC 4.1.1.50) 4 h after partial hepatectomy. Diaminopropane likewise inhibited ornithine decarboxylase activity during later periods of liver regeneration. In contrast to early regeneration, a total inhibition of the enzyme activity was only achieved when the injection was given not earlier than 2 to 3 h before the death of the animals. Diaminopropane also exerted an acute inhibitory effect on adenosylmethionine decarboxylase activity in 28-h regenerating liver whereas it invariably enhanced the activity of tyrosine aminotransferase (EC 2.6.1.5), used as a standard enzyme of short half-life. Treatment of the rats with diaminopropane entirely abolished the stimulation of spermidien synthesis in vivo from [14C]methionine 4 h after partial hepatectomy or after administration of porcine growth hormone. Both partial hepatectomy and the treatment with growth hormone produced a clear stimulation of hepatic RNA synthesis, the extent of which was not altered by injections of diaminopropane in doses sufficient to prevent any enhancement of ornithine decarboxylase activity and spermidine synthesis.     

Studies on the metabolic effects of methylthioformycin

1. 5′-Methylthioformycin, a structural analog of 5′-methylthioadenosine in which the N-C glycosidic bond is substituted by a C-C bond, has been synthesized by a newly developed procedure. 2. Membrane permeability of the molecule has been compared to that of methylthioadenosine in intact human erythrocytes and Friend erythroleukemia cells. The formycinyl compound is taken up with a rate significantly lower than that of 5′-methylthioadenosine and is not metabolized by the cells. 3. 5′-Methylthioformycin inhibits Friend erythroleukemia cells' growth: the effect is dose-dependent, fully reversible and not caused by cytotoxicity. 4. Several enzymes related to methylthioadenosine metabolism are inhibited by methylthioformycin. Rat liver methylthioadenosine phosphorylase is competitively inhibited with a K_i value of 2 μM. Among the propylamine transferases tested only rat brain spermine synthase is significantly inhibited, while rat brain spermidine synthase is less sensitive. Rat liver S-adenosylhomocysteine hydrolase is irreversibly inactivated with 50% inhibition at 400 μM methylthioformycin. 5′-Methylthioformycin does not exert any significant effect on protein carboxyl-O-methyltransferase. Inferences about the mechanism of the antiproliferative effect of the drug have been drawn from the above results.     

A human neuroblastoma cell line with a stable ornithine decarboxylase in vivo and in vitro

A human neuroblastoma cell line (Paju) grew in 10 mM difluoromethyl-ornithine, which at this concentration normally stops the growth of all mammalian cells. Ornithine decarboxylase from Paju was resistant to inhibition in vitro by difluoromethylornithine, and required 10 microM of the compound for 50% inhibition, whereas ornithine decarboxylase from SH-SY5Y cells (another human neuroblastoma) and from rat liver needed only 0.5 microM difluoromethylornithine. Paju ornithine decarboxylase also exhibited a long half-life (over eight hours) in vivo. The half-life of immunoreactive protein was significantly longer than that of the activity. The long half-life of ornithine decarboxylase in Paju cells leads to its accumulation to a specific activity of 2000 nmol/mg of protein per 30 min during rapid growth (the corresponding activity in SH-SY5Y cells was about 2.5). When partially purified ornithine decarboxylase from Paju cells was incubated with rat liver microsomes it was inactivated with a half-life of 75 min. This inactivation was accompanied by a fall in the amount of immunoreactive protein. In the same inactivating system partially purified SH-SY5Y ornithine decarboxylase had a half-life of 38 min and its half-life in vivo was 50 min. The corresponding values for rat liver ornithine decarboxylase were 45 min and 40 min, respectively. Rat liver microsomes also inactivated rat liver adenosylmethionine decarboxylase. These results suggest that Paju ornithine decarboxylase has an altered molecular conformation, rendering it resistant to (i) difluoromethylornithine and (ii) proteolytic degradation both in vivo and in vitro.     

Polyamine distribution and activity of their biosynthetic enzymes in the European sea bass (Dicentrarchus labrax L.) compared to the rat

1. In the liver, heart and brain of the European sea bass, putrescine concentrations are much higher than in the equivalent rat tissues; spermidine and spermine levels are smaller. 2. Ornithine decarboxylase in the bass liver is more active, but less stable than that in the rat; stability is acquired upon partial purification. Bass liver adenosylmethionine decarboxylase activity is less than that found in the rat. Both are activated and stabilized by putrescine. 4. The activating effect of putrescine decreases as the assay temperature is decreased. This may explain the high level of putrescine but low levels of spermidine and spermine in the bass liver.     

Characterization of a defect in the pathway for converting 5'-deoxy-5'-methylthioadenosine to methionine in a subline of a cultured heterogeneous human colon carcinoma

5'-Deoxy-5'-methylthioadenosine (methylthioadenosine) is cleaved to adenine and 5-methylthioribose-1-phosphate (methylthioribose-1-P). Methylthioribose-1-P is converted to 2-keto-4-methylthiobutyrate ( ketomethylthiobutyrate ) which is transaminated to methionine. We report that one subline of a heterogeneous human colon carcinoma, DLD-1 Clone D, only forms methylthioribose-1-P from methylthioadenosine or 5'-deoxy-5'-methylthioinosine (methylthioinosine), a deaminated derivative of methylthioadenosine, whereas Clone A converts methylthioadenosine and methylthioinosine to methionine, as shown by growth studies in culture of Clone A and Clone D cells and radioactive studies utilizing [methyl-14C]methylthioadenosine or [methyl-14C]methylthioinosine in the presence of extracts of these cells lines. To characterize this defect, we utilized three protein fractions isolated from rat liver which together convert methylthioribose-1-P to ketomethylthiobutyrate . Addition of only Fraction A to Clone D sonicates restores its ability to convert methylthioadenosine to methionine. This fraction is responsible for converting methylthioribose-1-P to 5- methylthioribulose -1-phosphate; radioactive studies confirm this observation. Thus, Clone D is deficient in an enzyme contained in Fraction A; this represents a qualitative biochemical difference between the two clones derived from a single human tumor.     

A novel reaction of S-adenosyl-L-methionine correlated with the activation of pyruvate formate-lyase

Conversion of the inactive form of pyruvate formate-lyase to the catalytically active enzyme is accomplished by the Fe-dependent ‘enzyme II’; reduced flavodoxin, S-adenosyl-L-methionine and the effector pyruvate are required. It was found that adenosylmethionine is reductively processed during activation of pyruvate formate-lyase to yield methionine, adenine and 5-deoxyribose. We suggest that transient adenosylation of enzyme II is required for its function as a converter enzyme.     

Rapid and convenient method for the assay of aminopropyltransferases.

A new method for the assay of aminopropyltransferase activity is described. The method measures the formation of [methyl-14C]methylthioadenosine from decarboxylated S-adenosyl[methyl-14C]methionine in the presence of an amine acceptor. When used with extracts from rat ventral prostate, kidney, liver or brain, and with putrescine or spermidine as amines, the method gave results in excellent agreement with those obtained by the much more time-consuming conventional method. It was found that 1,3-diamino-propane and 1,8-diamino-octane were not acceptors for the prostatic enzyme fraction, but 1,5-diaminopentane (cadaverine) was active and 1,9-diaminononane and 1,12-diaminododecane also lead to the production of [methyl-14C]methylthioadenosine.     

S-adenosylmethionine decarboxylase from baker''s yeast.

1. S-Adenosyl-L-methionine decarboxylase (S-adenosyl-L-methionine carboxy-lyase, EC 4.1.1.50) was purified more than 1100-fold from extracts of Saccharomyces cerevisiae by affinity chromatography on columns of Sepharose containing covalently bound methylglyoxal bis(guanylhydrazone) (1,1'[(methylethanediylidene)dinitrilo]diguanidine) [Pegg, (1974) Biochem J. 141, 581-583]. The final preparation appeared to be homogeneous on polyacrylamide-gel electrophoresis at pH 8.4. 2. S-Adenosylmethionine decarboxylase activity was completely separated from spermidine synthase activity [5'-deoxyadenosyl-(5'),3-aminopropyl-(1),methylsulphonium-salt-putrescine 3-aminopropyltransferase, EC 2.5.1.16] during the purification procedure. 3. Adenosylmethionine decarboxylase activity from crude extracts of baker's yeast was stimulated by putrescine, 1,3-diamino-propane, cadaverine (1,5-diaminopentane) and spermidine; however, the purified enzyme, although still stimulated by the diamines, was completely insensitive to spermidine. 4. Adenosylmethionine decarboxylase has an apparent Km value of 0.09 mM for adenosylmethionine in the presence of saturating concentrations of putrescine. The omission of putrescine resulted in a five-fold increase in the apparent Km value for adenosylmethionine. 5. The apparent Ka value for putrescine, as the activator of the reaction, was 0.012 mM. 6. Methylglyoxal bis(guanylhydrazone) and S-methyladenosylhomocysteamine (decarboxylated adenosylmethionine) were powerful inhibitors of the enzyme. 7. Adenosylmethionine decarboxylase from baker's yeast was inhibited by a number of conventional carbonyl reagents, but in no case could the inhibition be reversed with exogenous pyridoxal 5'-phosphate.     

Mechanism of substrate inactivation of Escherichia coli S-adenosylmethionine decarboxylase

S-Adenosylmethionine decarboxylase, a pyruvoyl-containing decarboxylase, is inactivated in a time-dependent process under turnover conditions. The inactivation is dependent on the presence of both substrate and Mg2+, which is also required for enzyme activity. The rate of inactivation is dependent on the concentration of substrate and appears to be saturable. Inactivation by [methionyl-3,4-14C]-adenosylmethionine results in stoichiometric labeling of the protein. In contrast, when either S-[methyl-3H]adenosylmethionine or [8-14C]adenosylmethionine is used, there is virtually no incorporation of radioactivity. Automated Edman degradation of the alpha (pyruvoyl-containing) subunit reveals that substrate inactivation results in the conversion of the pyruvoyl group to an alanyl residue. These data suggest a mechanism of inactivation which involves the transamination of the nascent product to the pyruvoyl group, followed by the elimination of methylthioadenosine and the generation of a 2-propenal equivalent which could undergo a Michael addition to the enzyme. This is the first evidence for a transamination mechanism for substrate inactivation of a pyruvoyl enzyme.     

High density lipoprotein and low density lipoprotein catabolism by human liver and parenchymal and non-parenchymal cells from rat liver.

The capacity of the homogenates from human liver, rat parenchymal cells, rat non-parenchymal cells and total rat liver for the breakdown of human and rat high density lipoprotein (HDL) and human low density lipoprotein (LDL) was determined. Human HDL was catabolized by human liver, in contrast to human LDL, the protein degradation of which was low or absent. Human and rat HDL were catabolized by both the rat parenchymal and non-parenchymal cell homogenates with, on protein base, a 10-times higher activity in the non-parenchymal liver cells. This implies that more than 50% of the total liver capacity for HDL protein degradation is localized in these cell types. Human LDL degradation in the rat could only be detected in the non-parenchymal cell homogenates. These findings are discussed in view of the function of HDL and LDL as carriers for cholesterol.     

Purification and comparison of two forms of S-adenosyl-L-methionine synthetase from rat liver

Only two S-adenosyl-L-methionine synthetase forms exist in rat liver: high-Mr S-adenosyl-L-methionine synthetase and low-Mr S-adenosyl-L-methionine synthetase, which have been purified to apparent homogeneity as judged by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. High-Mr S-adenosyl-L-methionine synthetase had an apparent molecular mass, determined by gel filtration, of 210 kDa and was a tetramer constituted by 48.5-kDa subunits, estimated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The apparent molecular mass of low-Mr S-adenosyl-L-methionine synthetase, as estimated by gel filtration, was 110 kDa and was constituted by two subunits of 47 kDa. An antiserum against low-Mr S-adenosyl-L-methionine synthetase cross-reacted with the two forms. Reverse-phase HPLC runs of tryptic digestions of high-Mr and low-Mr S-adenosyl-L-methionine synthetase showed that the peptide maps of the two forms were very similar, if not identical. High-Mr S-adenosyl-L-methionine synthetase activity was inhibited by S-adenosyl-L-methionine and pyrophosphate. Depending on the dose used, S-adenosyl-L-methionine activated or inhibited low-Mr S-adenosyl-L-methionine synthetase and pyrophosphate had no effect on this form. The two synthetases showed a different specific activity at the physiological concentration of methionine. This report shows that even though the two forms are constructed of the same polypeptide chains, they are regulated in a different manner by methionine and by the products of the reaction.     

Plasmodium falciparum: S-adenosyl-L-methionine decarboxylase

Putrescine-dependent S-adenosyl-L-methionine decarboxylase has been detected in the malaria parasite Plasmodium falciparum. Mg2+ did not affect the enzyme activity. The apparent Km value of the plasmodial enzyme for adenosyl-methionine was found to be 33 microM. Methylglyoxal bis(guanylhydrazone) competitively inhibited the enzyme activity with respect to adenosylmethionine. The inhibition constant for methylglyoxal bis(guanylhydrazone) was determined to be 0.46 microM. Spermidine was the main polyamine detected in the parasite. There was significant decrease in the S-adenosyl-L-methionine decarboxylase activity when the infected erythrocytes were incubated with chloroquine and mefloquine for 2 hr at 1 and 10 microM, respectively. Since at similar concentrations these drugs did not directly affect the plasmodial enzyme activity, the interaction of these drugs with the polyamine biosynthesis remains unclear.     

Biochemical genetic analysis of the role of methylthioadenosine phosphorylase in a murine lymphoid cell line

The enzyme methylthioadenosine phosphorylase functions in both purine and polyamine metabolism is dividing mammalian cells. To determine the effects of the loss of this enzyme on cell growth and metabolism, we selected two methylthioadenosine phosphorylase-deficient mutant clones of the transplantable murine T lymphoma cell line R1.1. The first had 3.5% of wild type methylthioadenosine phosphorylase activity. The second was completely enzyme-deficient. The loss of the enzyme did not alter the growth rate, cloning efficiency, or tumor-forming ability of the T lymphoma cells. The methylthioadenosine phosphorylase-deficient clones excreted substantial amounts of methylthioadenosine into the culture medium (0.13 and 0.32 nmol/h/mg of protein, respectively) and were unable to utilize the methylthioadenosine phosphorylase substrate 2',5'-dideoxyadenosine as a purine source when de novo purine synthesis was blocked. Spermine levels were 10-20% lower in the enzyme-deficient clones than in wild type cells. The loss of methylthioadenosine phosphorylase rendered the mutants exquisitely sensitive to the antiproliferative effects of methylthioadenosine. Methylthioadenosine at 3-6 microM inhibited their growth by 50%. The toxic effects of methylthioadenosine were not attributable to inhibition of purine, pyrimidine, or polyamine synthesis.     

Properties of catechol O-methyltransferases from brain and liver of rat and human.

Kinetic and electrophoretic properties of catechol O-methyltransferases (EC 2.1.1.6) from brain and liver were studied. The enzyme of either rat or human tissues exhibited a single molecular form when subjected to electrophoresis at pH7.9. At pH9 a second, apparently oxidized, form was detected. Isoelectric-focusing experiments also indicated only one enzyme form, which was identical from extracts of brain and liver of each species (pI = 5.2 for rat, 5.5 for human). Similarities between brain and liver catechol O-methyltransferase of a given species were also demonstrated by kinetic parameters, meta/para ratios of products, and inhibitor potencies. Human catechol O-methyltransferase exhibited lower Km values than did the rat enzyme for S-adenosyl-L-methionine, dopamine and dihydroxybenzoic acid. Adrenochrome inhibited both rat and human enzyme. It was concluded (1) that only a single enzyme form could be demonstrated in the physiological pH region; (2) that catechol O-methyltransferase of brain could not be distinguished from the liver enzyme of the same species; and (3) that species differences exist between the enzymes of rat and human tissues.     

Transfer ribonucleic acid methylase activity in adult and foetal rat liver.

Foetal rat liver extracts were found to have higher tRNA methylene activities than corresponding extracts of adult liver. When the specific activities were expressed per mg of liver or per mg of protein, the foetal tRNA methylating enzymes were respectively 2.5 and 6 times higher than those of adult livers. The presence of an inhibitor in adult liver can be excluded, since the same recoveries of total tRNA methylase activity were obtained after partial purification of both adult and foetal liver extracts: yields were close to 100%. The apparent Km's for the substrates in the methylating reactions were the same when tRNA methylases from either adult or foetal liver were used: values were 0.2 muM for Escherichia coli tRNA and 2.1 muM for S-adenosyl-L-methionine. After T1-T2 ribonuclease digestion of an in vitro methylated tRNA, similar methyl nucleotide patterns were observed in foetal and adult enzymatic extracts. It is concluded that the same tRNA methylase pool is present in adult and foetal liver. In addition, it is hypothesized that the different reaction rates exhibited by these enzymes might be due to the tRNA functional requirements rather than to the presence of a tRNA methylase inhibitor.     

Evidence that the decarboxylation reaction occurs before the first methylation in ubiquinone biosynthesis in rat liver mitochondria

Biosynthesis of ubiquinone-9 was studied by incubating rat liver mitochondria with p-hydroxy[U-14C]benzoate, solanesyl diphosphate and S-adenosyl-L-methionine. When methylation reactions were inhibited by replacing S-adenosyl-L-methionine with S-adenosyl-L-homocysteine, nonaprenyl p-hydroxybenzoate and three other labeled peaks, designated as P1, P2 and P3 according to their retention times on HPLC, were observed. No carboxyl group was present in P1, P2 or P3 because the radioactivities disappeared when p-hydroxy[U-14C]benzoate was replaced by p-hydroxy[carboxyl-14C]benzoate. Compound P2 seemed to be hydroxylated but not methylated because its radioactivity markedly diminished under anaerobic conditions and the radioactivity was not incorporated into the compound from S-adenosyl-L-[methyl-3H]methionine, suggesting that P2 is 6-hydroxynonaprenylphenol. The complete correspondence of the retention times of P2 and chemically synthesized 6-hydroxynonaprenylphenol on HPLC further confirmed this possibility. P2 was a precursor of ubiquinone-9 because the radioactivity of the compound was incorporated into ubiquinone when incubated with mitochondria. The results suggest that the decarboxylation may occur prior to the first methylation in the ubiquinone biosynthesis in rat liver mitochondria, though it has been generally considered that in eukaryotes the first methylation precedes the decarboxylation.     

GTP-insensitive ornithine decarboxylase in acetobacteria able to synthesize spermine

Several Acetobacteria contained large amounts of spermine in addition to the putrescine and spermidine, which are the polyamines normally found in prokaryotes. A spermine synthase present in cell extracts of these Acetobacteria is the first example of this enzyme in prokaryotes. Dicyclohexylammonium sulphate inhibited both spermidine synthase and spermine synthase activities in Acetobacteria. Their ornithine decarboxylase was not stimulated by GTP nor inhibited by ppGpp and pppGpp (magic spots I and II) in contrast to ornithine decarboxylase of nearly all bacteria studied so far. However, their S-adenosyl-L-methionine decarboxylase resembled other prokaryotic adenosylmethionine decarboxylases in requiring Mg2+ ions in vitro for full activity.