Myelin basic protein inhibits histone-specific protein methylase I

Bovine brain myelin basic protein, free of associated proteolytic activity, was found to be a specific inhibitor of histone-specific protein methylase I (S-adenosyl-L-methionine:protein-L-arginine N-methyltransferase, EC 2.1.1.23) purified from bovine brain. 50% of the methyl group incorporation into the histone substrate catalyzed by the methylase I was inhibited by myelin basic protein at a concentration of 0.326 mM. However, neither of the peptide fragments (residues 1-116 and residues 117-170) generated by the chemical cleavage of myelin basic protein at the tryptophan residue retained the inhibitory activity for histone-specific protein methylase I. Proteins such as gamma-globulin, bovine serum albumin, bovine pancreatic ribonuclease and polyarginine did not exhibit significant inhibitory activity toward the enzyme. The Ki value for myelin basic protein was estimated to be 3.42 X 10(-5) M for histone-specific protein methylase I and the nature of the inhibition was uncompetitive toward histone substrate.     

Polyamines are essential for the synthesis of 2-ricinoleoyl phosphatidic acid in developing seeds of castor

The major fatty acid component of castor (Ricinus communis L.) oil is ricinoleic acid (12-hydroxy-cis-9-octadecenoic acid), and unsaturated hydroxy acid accounts for >85% of the total fatty acids in triacylglycerol (TAG). TAG had a higher ricinoleate content at position 2 than at positions 1 and 3. Although lysophosphatidic acid (LPA) acyltransferase (EC 2.3.1.51), which catalyzes acylation of LPA at position 2, was expected to utilize ricinoleoyl-CoA preferentially over other fatty acyl-CoAs, no activity was found for ricinoleoyl-CoA in vitro at concentrations at which other unsaturated acyl-CoAs were incorporated rapidly. However, activity for ricinoleoyl-CoA appeared with addition of polyamines (putrescine, spermidine, and spermine), while polyamines decreased the rates of incorporation of other acyl-CoAs into position 2. The order of effect of polyamines on LPA acyltransferase activity was spermine > spermidine >> putrescine. At concentrations of spermine and spermidine of >0.1 mM, ricinoleoyl-CoA served as an effective substrate for LPA acyltransferase reaction. The concentrations of spermine and spermidine in the developing seeds were estimated at ∼0.09 and ∼0.63 mM, respectively. These stimulatory effects for incorporation of ricinoleate were specific to polyamines, but basic amino acids were ineffective as cations. In contrast, in microsomes from safflower seeds that do not contain ricinoleic acid, spermine and spermidine stimulated the LPA acyltransferase reaction for all acyl-CoAs tested, including ricinoleoyl-CoA. Although the fatty acid composition of TAG depends on both acyl-CoA composition in the cell and substrate specificity of acyltransferases, castor bean polyamines are crucial for incorporation of ricinoleate into position 2 of LPA. Polyamines are essential for synthesis of 2-ricinoleoyl phosphatidic acid in developing castor seeds.     

S-adenosylmethionine: protein-arginine methyltransferase. Purification and mechanism of the enzyme.

Protein methylase I (S-adenosylmethionine: protein-arginine methyltransferase, EC 2.1.1.23) has been purified from calf brain approximately 120-fold with a 14% yield. The final preparation is completely free of any other protein-specific methyltransferases and endogenous substrate protein. The enzyme has an optimum pH of 7.2 and pI value of 5.1. The Km values for S-adenosyl-L-methionine, histone H4, and an ancephalitogenic basic protein are 7.6 X 10(-6), 2.5 X 10(-5), and 7.1 X 10(-5) M, respectively, and the Ki value for S-adenosyl-L-homocysteine is 2.62 X 10(-6) M. The enzyme is highly specific for the arginine residues of protein, and the end products after hydrolysis of the methylated protein are NG,NG-di(asymmetric), NG,N'G-di(symmetric), and NG-monomethylarginine. The ratio of [14C]methyl incorporation into these derivatives by enzyme preparation at varying stages of purification remains unchanged at 40:5:55, strongly indicating that a single enzyme is involved in the synthesis of the three arginine derivatives. The kinetic mechanism of the protein methylase I reaction was studied with the purified enzyme. Initial velocity patterns converging at a point on the extended axis of abscissas were obtained with either histone H4 or S-adenosyl-L-methionine as the varied substrate. Product inhibition by S-adenosyl-L-homocysteine with S-adenosyl-L-methionine as the varied substrate was competitive regardless of whether or not the enzyme was saturated with histone H4. On the other hand, when histone H4 is the variable substrate, noncompetitive inhibition was obtained with S-adenosyl-L-homocysteine under conditions where the enzyme is not saturated with the other substrate, S-adenosyl-L-methionine. These results suggest that the mechanism of the protein methylase I reaction is a Sequential Ordered Bi Bi mechanism with S-adenosyl-L-methionine as the first substrate, histone H4 as the second substrate, methylated histone H4 as the first product, and S-adenosyl-L-homocysteine as the second product released.     

Polyamines inhibit DNA methylation in vitro.

DNA methylase from rat liver differs from RNA methylase in response to polyamines, being inhibited rather than stimulated. With methyl-deficient DNA from rat liver as substrate, the DNA methylase is inhibited 97%, 59% and 42% respectively, by spermine, spermidine and putrescine at 1 mM concentration.     

NG-Methylarginines: Biosynthesis,biochemical function and metabolism

Summary N^G-Methylarginines (N^G-monomethylarginine, N^G, N^G-dimethylarginine and N^G, N^G-dimethylarginine) occur widely in nature in either proteinbound or in free states. They are posttranslationally synthesized by a group of enzymes called protein methylase I with S-adenosyl-L-methionine as the methyl donor. The enzymes are highly specific not only towards arginine residues but also towards the protein species. Since transmethylation reaction is energy-dependent in the form of S-adenosyl-L-methionine and is catalyzed a group of highly specific enzymes, it is quite logical to assume that the enzymatic methylation of protein-bound arginine residues play an important role in the regulation of the function and/or metabolism of the protein. When determined with histones asin vitro substrates, protein methylase I activity parallels closely the degree of cell proliferation, and the myelin basic protein (MBP)-specific protein methylase I activity decreases drastically in dysmyelinating mutant mouse brain during myelinating period, suggesting an important role played in the formation and/or maintenance of myelin. When the methylated proteins are degraded by intracellular proteolytic enzymes, free N^G-methylarginines are generated. Some of these free N^G-methylarginines, particularly N^G-monomethylarginine, are extensively metabolized by decarboxylation, hydrolysis, transfer of methylamidine and deimination reaction. Recent experiment demonstrates that some of the N^G-methylarginines may be involved in the neutralization of activity of nitric oxide (NO) which has attracted a great deal of attention as vascular smooth muscle relaxation factor.     

Substrate specificity for myelin basic protein-specific protein methylase I.

The substrate specificity of bovine brain myelin basic protein (MBP)-specific protein methylase I (S-adenosyl-L-methionine:protein-L-arginine N-methyltransferase, EC 2.1.1.23), which methylates arginine residues of protein, has been studied using various MBPs, several synthetic peptides and heterogeneous nuclear ribonucleoprotein complex protein (hnRNP). (1) Among MBPs from different species of brain, the carp MBP was found to be the best substrate for MBP-specific protein methylase I. This high degree of methyl acceptability is most likely due to the fact that carp MBP is not in vivo methylated at the arginine residue (Deibler, G.E. and Martenson, R.E. (1973) J. Biol. Chem. 248, 2387-2391) and that the methylatable amino acid sequence is present in this protein. (2) In order to study the minimum chain length of MBP polypeptide which functions as the methyl acceptor, several synthetic polypeptides whose sequences are identical to the region surrounding the residue 107 of bovine MBP (the in vivo methylation site) were synthesized. It was found that the hexapeptide, Gly-Lys-Gly-Arg-Gly-Leu (corresponding to residues 104-109 of bovine MBP), was the shortest methyl accepting peptide, while the tetrapeptide, Gly-Arg-Gly-Leu (corresponding to residues 106-109) was inactive as a substrate. (3) hnRNP protein is known to contain methylarginine at residue 193 (Williams, K.R., Stone, K.L., LoPresti, M.B., Merrill, B.M. and Plank, S.R. (1985) Proc. Natl. Acad. Sci. USA 82, 5666-5670) which is post-translationally modified. Thus, the RNP protein overproduced in Escherichia coli and therefore did not contain methylarginine was examined for its methyl acceptability. It was found that neither MBP-specific nor histone-specific protein methylase I could methylate this methylarginine-less RNP protein. This suggests a possible existence of a distinct protein methylase I specific for this nuclear protein.     

Uptake of putrescine and spermidine by Gap1p on the plasma membrane in Saccharomyces cerevisiae

It has been reported that Gap1p on the plasma membrane of Saccharomyces cerevisiae can catalyze the uptake of many kinds of amino acids. In the present study, we found that Gap1p also catalyzed the uptake of putrescine and spermidine, but not spermine. The Km and Vmax values for putrescine and spermidine were 390 and 21 microM, and 4.6 and 0.59 nmol/min/mg protein, respectively. The uptake of putrescine was strongly inhibited by basic amino acids, lysine, arginine, and histidine, whose Ki values were 25-35 microM. Thus, it is deduced that spermidine and basic amino acids have almost the same affinity for Gap1p. When the concentrations of amino acids in the medium were reduced to one-third and 0.5 mM putrescine or 0.1 mM spermidine was added to the medium, accumulation of putrescine or spermidine by Gap1p was observed. Furthermore, when yeast was transformed with the GAP1 gene and cultured in the presence of 60 mM putrescine, cell growth was inhibited through overaccumulation of putrescine. GAP1 mRNA was found to be induced by polyamines. This is the first report of the identification, at a molecular level, of a polyamine uptake protein on the plasma membrane in eukaryotes.     

A rapid high-performance liquid chromatographic procedure for the simultaneous determination of methionine, ethionine, S-adenosylmethionine, S-adenosylethionine, and the natural polyamines in rat tissues

A method for the analysis of S-adenosyl-L-methionine (SAM) and S-adenosyl-L-ethionine (SAE) and their major metabolites by high-performance liquid chromatography is described. The procedure allows the simultaneous analysis of the natural polyamines, putrescine, spermidine, and spermine, and some of the major amino acids, methionine, tyrosine, and tryptophan. The uv absorbance at 254 nm is used for the determination of the SAM and SAE analogs, whereas the polyamines and amino acids are analyzed by fluorescence detection after postcolumn derivatization with o-phthalaldehyde. The method allows SAM and polyamine determinations by direct injection of the tissue extracts without prepurification. The procedure is applied to study the effects of DL-ethionine treatment on the SAM, SAE, methionine, and polyamine levels in various tissues of rats.     

Specific inhibition of ouabain sensitive and K+-dependent p-nitrophenylphosphatase by polyamines.

The ouabain sensitive and K⁺-dependent p-nitrophenyl-phosphatase was inhibited by polyamines. The order of effectiveness was spermine spermidine putrescine = cadaverine. The half maximum inhibition concentration of spermine was approximately 0.03 mM and 0.8 mM in the presence of 0.5 mM and 3.0 mM KCl in the reaction mixtures, respectively. Basic amino acids and hydroxylamine inhibited slightly. Other amines such as glycine and histamine were without effect. Spermine did not inhibit other membrane bound phosphatases, such as glucose-6-phosphatase, 5′-nucleotidase, alkaline phosphatase and ouabain insensitive p-nitrophenylphosphatase activity at pH 7.5     

Polyamines stimulate the phosphorylation of phosphatidylinositol in membranes from A431 cells

The phosphorylation of phosphatidylinositol in plasma membranes from A431 cells was investigated using [gamma-32P]ATP as the substrate. Phosphatidylinositol 4-phosphate was found to be the major product after an incubation time of 5-10 min. Little, if any, phosphatidylinositol 4,5-bisphosphate was found under these conditions. Epidermal growth factor (EGF) had no effect on the formation of phosphatidylinositol 4-phosphate or phosphatidylinositol 4,5-bisphosphate. On the other hand, the polyamines spermidine and spermine stimulated the phosphatidylinositol kinase activity about eightfold yielding almost exclusively phosphatidylinositol 4-phosphate as the reaction product. Half-maximum stimulation by spermidine occurred under near physiological conditions (1.5 mM). Furthermore various proteins and amino acid polymers containing clustered basic amino acid residues (e.g. histones and polylysine) stimulated the formation of phosphatidylinositol 4-phosphate to a similar extent. Half-maximal concentrations for the activation were considerably lower ranging from 1.5 microM to 80 microM. The ATP specificity of the phosphatidylinositol kinase(s) was investigated with a small set of selected ATP derivatives. In the presence of spermidine the specificity changed significantly indicating that (a) spermidine acts on a kinase and not on a phosphatase, (b) this activity is distinct from the EGF-receptor protein kinase activity. The results do not suggest an involvement of the EGF receptor in the growth-factor-dependent formation of phosphatidylinositol phosphates. It is proposed that the phosphorylation of phosphatidylinositol by polyamines might be a mechanism to replenish the pool of inositolphospholipids.     

Effects of polyamines on tyrosine hydroxylase activity in adrenals

The ability of polyamines (putrescine, spermidine, and spermine) to modify tyrosine hydroxylase (TH) activity was examined in crude or purified enzyme preparation and in adrenal tissue slices. Polyamines showed biphasic effects on TH activity in vitro at physiological pH 7.0, with an inhibitory effect at low concentrations (<1 mM) and a stimulatory effect at high concentrations. The degree of both inhibition and stimulation produced by polyamines at low and high concentrations, respectively, were proportional to the number of the amino group in the polyamines (putrescine < spermidine < spermine). The degree of inhibition by polyamines was much greater with purified enzyme than with crude enzyme preparations. Tyrosine hydroxylation in situ in adrenal tissue slices was stimulated by polyamines without inhibition at any concentrations tested. This evidence suggests that TH molecules in vivo could interact with polyamines or polyamine-like substances which inhibit the TH activity at physiological concentrations less than 1 mM.     

Activity of foot-and-mouth disease virus RNA-dependent RNA polymerase in vitro: inhibition by polyamines and poly(amino acid)s

Replication of foot-and-mouth disease virus RNA in vitro is inhibited by high concentrations of the following polyamines in decreasing order of effectiveness: putrescine, spermine, and cadaverine. The basic poly(amino acids), polylysine, polyornithine, and polyarginine, as well as the basic protein salmine, are several orders of magnitude more inhibitory than polyamines. The interaction between polyornithine and foot-and-mouth disease virus RNA and its inhibition of replicase activity are related to the ability of basic polypeptides to bind to the RNA template. The neutral polymer, polysarcosine, and the polyanions, polyglutamic acid and heparin sulfate, do not inhibit replication; however, both polyanions relieve the inhibition by polyamines and polyamino acids. The mode of inhibition by polyamines and poly(amino acids) and the antagonism by heparin is discussed.     

The effect of amino acids,monoamines and polyamines on pyruvate dehydrogenase activity in mitochondria from rat adipocytes

Summary The ability of polyamines and other cationic compounds including monoamines, amino acids, poly-L-arginine, poly-D-lysine and poly-L-lysine, to alter pyruvate dehydrogenase (PDH) activity in mitochondria from rat epididymal adipocytes was determined. PDH was assayed with the substrate [1-¹⁴C] pyruvate in the presence of 0.05 mM Ca²⁺ and Mg²⁺. Nine of the fourteen compounds tested at 0.1 mM caused a significant increase (procaine, 3-(-morpholinopropionyl) benzo[b]thiophene [VII], spermine, spermidine, putrescine, lysine and tryptophan) or decrease (poly-L-arginine, 3-(-piperidinopropionyl) benzo[b]thiophene) in PDH activity. None of these compounds nonenzymatically decarboxylated [1-¹⁴C] pyruvate to release ¹⁴CO₂. NaF, a PDH phosphatase inhibitor, suppressed the stimulatory effects of those compounds tested: procaine, tryptophan, VII, spermine and spermidine. These results imply that these five compounds activate PDH activity through stimulation of the PDH phosphatase. When the Mg²⁺ concentration was increased from 0.05 to 4.5 mM, the stimulatory effect of spermine was increased, consistent with the finding by others that spermine lowers the Km of the enzyme for Mg²⁺. However, at Mg²⁺ concentrations greater than 0.3 mM, the stimulatory effect of VII was unaltered, procaine failed to alter PDH activity, lysine inhibited PDH activity, and poly-L-lysine stimulated PDH activity. Therefore, polyamines and other positively charged small molecules may be physiologic regulators of PDH activity.     

Polyamines secreted by cancer cells possibly account for the impairment of the human erythrocyte sodium pump activity.

Using an original microcalorimetric method, we previously showed that in erythrocytes from cancer patients, the sodium pump activity was decreased and returned to normal in patient in remission. In addition we suggested that a plasma-borne factor probably secreted by cancer cells accounted for this impairment of the sodium transporter. In the present study we sought to identify this factor as well as its mechanism of action. First we determined the effect of culture media from undifferentiated and differentiated colon cancer cell lines (Caco-2 and HT29-D4) on the sodium pump activity of normal human erythrocytes. The inhibitory powers of culture media from undifferentiated cells were higher than those of differentiated cells (38.6 +/- 3.5% vs 6.9 +/- 4.6%, p<0.05 for Caco-2 and 45.8 +/- 6.2% vs 9.0 +/- 5.0%, <0.05 for HT29-D4). The use of alpha difluoro-methylomithine (2 mM) to inhibit ornithine decarboxylase, the rate-limiting enzyme for polyamine biosynthesis, dramatically reduced the sodium pump inhibition induced by the two undifferentiated cell lines (75% for Caco-2 and 89% for HT29-D4). Polyamines secreted by undifferentiated cells and then taken up by human erythrocytes thus appeared as inhibitors of sodium pump of these red blood cells. Putrescine, spermidine, and spermine (the main polyamines) exerted a similar inhibitory effect (33 +/- 2%). Tested in vitro on Na,KATPase, these polyamines (3 mM) were inhibitors (putrescine = 23 +/- 2%; spermidine= 48 +/- 3%; spermine= 55 +/- 2%) when assay condition for the ATPase reaction was suboptimal (Na+ = 10 mM; K+ = 1 mM). The inhibitory effect appeared to be related to their charge and their aliphatic chain length. The effect of spermidine and spermine on the ionic substrates and ATP-Mg showed that molecules decreased the affinity (Km) of the Na,K-ATPase for Na+ (11.24 +/- 0.49 mM for control vs 23.51 +/- 1.53 mM for spermine and 18.86 +/- 0.98 mM for spermidine), indicating that polyamines exerted their inhibitory effect in a competitive manner.     

Human placental protein methylase--I. Purification and characterization.

1. Protein methylase I (S-adenosylmethionine[:]protein-arginine N-methyltransferase; EC 2.1.1.23) which methylates protein-bound arginine residues has been purified from human term placenta 400-fold with an approximate yield of 6%. 2. When histone was used as in vitro substrate, the methylation products were found to be NG-mono-, NG, NG-di- and NG, N'G-dimethylarginine. The enzyme was found to be sensitive toward Cu2+ with Ki value of 8 x 10(-5) M. The Km value for S-adenosyl-L-methionine was 5 x 10(-6) M. 3. When this partially purified protein methylase I was incubated with isolated human placental nuclei and S-adenosyl-L-[methyl-3H]methionine, the major endogenous [methyl-3H]-labeled proteins were protein species of 23, 38, 45 and 68 kDa, the 23 kDa species being the most predominant. 4. The endogenous enzyme activity during the pregnancy increased significantly, reaching more than 4 times the initial activity at the end of term.     

Characterization of antilipolytic action of polyamines in isolated rat adipocytes.

The interactions of polyamines with the lipolytic system were studied in isolated rat adipocytes. Spermine, spermidine and putrescine significantly inhibited adenosine deaminase-stimulated lipolysis. An antilipolytic effect of spermine was detectable at a concentration of 0.25 mM (P less than 0.05). At a concentration of 10 mM all three polyamines inhibited the stimulated lipolysis by 50-60% (P less than 0.001). In addition, spermine enhanced the antilipolytic sensitivity of insulin. Spermine (1 mM) decreased the half-maximal inhibitory concentration of insulin from 320 +/- 70 pM to 56 +/- 20 pM (P less than 0.01). The antilipolytic effects and the cyclic-AMP-lowering effects of the polyamines were almost completely prevented in the presence of different phosphodiesterase (PDE) inhibitors (3-isobutyl-1-methylxanthine and RO 20-1724) and, in addition, polyamines had no effect on lipolysis stimulated by dibutyryl cyclic AMP, indicating that polyamines may inhibit lipolysis by activating the PDE enzyme. This latter suggestion was confirmed by demonstrating that spermine (5 mM) significantly enhanced the low-Km PDE enzyme activity (P less than 0.01). Finally, the amounts of polyamines present in isolated adipocytes were measured, and the estimated cytoplasmic concentrations were 0.02 mM (putrescine), 0.86 mM (spermidine), and 1.0 mM (spermine). It is concluded that polyamines may possibly be involved in the physiological regulation of triacylglycerol mobilization in adipocytes.     

Spermidine and abscisic acid-mediated phosphorylation of a cytoplasmic protein from rice root in response to salinity stress

Importance of higher polyamines, spermidine, and spermine, in relation to the mechanism and adaptation to combat abiotic stress has been well established in cereals. Owing to their polycationic nature at physiological pH, polyamines bind strongly to negative charges in cellular components such as nucleic acids, various proteins, and phospholipids. To study the physiological role of polyamine during salinity stress, phosphorylation study was carried out in cytosolic soluble protein fraction isolated from the roots of salt tolerant (Nonabokra) and salt sensitive (M-1-48) rice cultivars treated with none (control), NaCl (150 mM, 16 h), spermidine (1 mM, 16 h) or with abscisic acid (100 μM, 16 h). A calcium independent auto regulatory 42 kDa protein kinase was found to phosphorylate myelin basic protein and casein but not histone. Interestingly, this was the only protein to be phosphorylated in root cytosolic fraction during NaCl/abscisic acid/spermidine treatment indicating its importance in salinity mediated signal transduction. This is the first report of polyamine as well as abscisic acid induced protein kinase activity in rice root in response to salinity stress.     

Activation of phosphoinositide-specific phospholipase C delta from rat liver by polyamines and basic proteins.

Phospholipase C from rat liver with a molecular weight of 87,000 (PLC delta) is stimulated by polyamines, basic proteins, and basic polyamino acids. The activation occurs in both the presence and the absence of detergents. Half-maximum activation by spermine is observed at 0.15 mM, with optimum effects between 0.2 and 0.5 mM. Spermine inhibits above 0.5 mM. Half-maximum activation by spermidine and putrescine is observed at 0.9 and 6 mM, respectively, with optimum effects at 2 and 5 mM, respectively. These polyamines also inhibit at higher concentrations. Neomycin activates the enzyme with an optimum concentration of 10 microM, but maximum activation is less than with polyamines. Half-maximum activation by histone 2B occurs at 0.5 micrograms/ml (36 nM), with maximum stimulation at 1.5 micrograms/ml. Other histones, protamine, melittin, poly-L-ornithine, poly-L-lysine, poly-D-lysine, and poly-L-arginine, activate optimally at 3-10 micrograms/ml. Myelin basic protein and lysozyme activate optimally at 50-100 micrograms/ml. Typical activations are three- to eightfold, but under some conditions the enzyme shows little or no activity in the absence of basic activators. The basic activators lower the salt concentration required for maximal activity. In the case of the detergent-micelle assay, histone shifts the optimum NaCl concentration from 350 to 200 mM for PIP2, from 260 to 100 mM for PIP, and from 150 to 0 mM for PI. Histone potentiates the activation by Ca2+, but does not shift the optimum Ca2+ concentration. The optimum salt and Ca2+ concentrations are linked, such that a decrease in the concentration of one decreases the optimum concentration of the other. Activation by histone is diminished by MgCl2 in a concentration-dependent manner.     

Protein methylases in Trypanosoma brucei brucei: activities and response to DL-alpha-difluoromethylornithine

Protein methylases I, II and III were detected in extracts of Trypanosoma brucei brucei, and characterized according to the specific amino substituent methylated. Only protein methylase II activity was elevated by difluoromethylornithine treatment of T. b. brucei, and hence this enzyme was characterized further. Protein methylase II transferred methyl groups from S-adenosyl-L-methionine (S-AdoMet) to the carboxyl residues of several protein substrates, exhibiting highest activity with histone VIII-S (arginine-rich subgroup f3). The crude enzyme had an apparent Km for histone VIII-S of 28 mg ml-1 (11.4 mM-aspartyl and 18.4 mM-glutamyl residues methylated), and an apparent Km for S-AdoMet of 8.4 microM. T. b. brucei protein methylase II was sensitive to inhibition by S-adenosyl-L-homocysteine and its analogue sinefungin with apparent Ki values of 12.9 and 1.6 microM, respectively. Using a partially purified preparation, analysis of kinetic data in the presence and absence of sinefungin indicated that this analogue acts as a competitive inhibitor of the S-AdoMet binding site, and as a non-competitive inhibitor of the (protein) histone VIII-S binding site. The possible role of the enzyme in morphological control and its potential as a chemotherapeutic target are discussed.     

Polyamine metabolism in enterocytes isolated from newborn pigs.

In the pig, the growth of intestinal mucosa is very intense after birth. Since the polyamines are key elements affecting cell proliferation and differentiation, the present work was undertaken in order to know whether this hypertrophy is associated with an adaptation of polyamine metabolism. Villus enterocytes isolated from pig immediately after birth or 2 days later were found to contain similar amounts of putrescine, spermidine and spermine, i.e., 0.23; 0.41 and 1.24 nmol/10(6) cells, respectively. At birth, despite a relatively high ODC activity, putrescine synthesis from 1 mM L-arginine or 2 mM L-glutamine was very low in isolated enterocytes (6.4 +/- 3.8 pmol/10(6) cells per 30 min), while spermidine and spermine production were not detectable. This could be explained by a very low L-ornithine generation from both amino acids and to an inhibitory effect of polyamines on ODC activity. Two days later, polyamine synthesis from L-arginine remained undetectable despite a higher L-ornithine generation. This was concomitant with a dramatic fall in ODC activity. At both stages, enterocytes were able to take up polyamines from the extracellular medium in a temperature-dependent manner. It is concluded that de-novo synthesis of polyamines from L-arginine or L-glutamine does not play a significant role in the control of polyamine content of pig enterocytes during the postnatal period. In contrast, polyamine uptake by enterocytes would contribute to maintain a steady-state polyamine content during this period.