scholarly journals Relative effects of S-adenosylmethionine depletion on nucleic acid methylation and polyamine biosynthesis

1987 ◽  
Vol 247 (2) ◽  
pp. 259-265 ◽  
Author(s):  
D L Kramer ◽  
J R Sufrin ◽  
C W Porter
Keyword(s):  
Nucleic Acid ◽  
Fold Increase ◽  
Synthetase Activity ◽  
Polyamine Biosynthesis ◽  
L1210 Cells ◽  
Treatment Conditions ◽  
Key Enzyme ◽  
The Media ◽  
Minimal Concentration ◽  
Adomet Synthetase

Treatment of cultured L1210 cells with 1 mM-L-2-amino-4-methoxy-cis-but-3-enoic acid (L-cisAMB), a methionine-analogue inhibitor of S-adenosylmethionine (AdoMet) synthetase (EC 2.5.1.6), produced a rapid and near-total depletion of AdoMet by 4 h. After this, the pools recovered to 60% of control by 48 h, apparently because of an increase in AdoMet synthetase activity. Both AdoMet depletion and the accompanying increase in synthetase activity were substantially enhanced by lowering methionine concentrations in the media from 100 microM to 30 microM, the minimal concentration that supports cell growth at control values. During a 4 h incubation in media containing 30 microM-methionine, 1-5 mM-L-cisAMB depleted cellular AdoMet to undetectable values, and inhibited nucleic acid methylation by 44-72% and RNA methylation by 60-87%. Under these same treatment conditions, putrescine pools increased by about 3-fold, whereas spermidine pools decreased by only 20% and spermine pools remained the same. Pool changes were accompanied by a 2-4-fold increase in ornithine decarboxylase activities and AdoMet activities. Thus the rapid depletion of AdoMet pools by L-cisAMB results immediately in a decrease in methyl-transfer reactions involving nucleic acids, whereas, by contrast, biosynthesis of higher polyamines appears to be minimally affected, owing to compensatory increases in key enzyme activities.

scholarly journals Modulation of polyamine-biosynthetic activity by S-adenosylmethionine depletion

1988 ◽  
Vol 249 (2) ◽  
pp. 581-586 ◽  
Author(s):  
D L Kramer ◽  
J R Sufrin ◽  
C W Porter
Keyword(s):  
Enzyme Activity ◽  
Enzyme Activities ◽  
Enzyme Inhibitors ◽  
Fold Increase ◽  
Negative Control ◽  
Biosynthetic Activity ◽  
Drug Induced ◽  
Polyamine Biosynthesis ◽  
Sequence Characterization ◽  
Adomet Synthetase

The methionine-analogue inhibitor of S-adenosylmethionine (AdoMet) synthetase, L-2-amino-4-methoxy-cis-but-3-enoic acid (L-cisAMB), was used to study the early effects of AdoMet depletion on polyamine biosynthesis. In the presence of decreased methionine (30 microM) in the medium, treatment of cultured L1210 cells with 1 mM-L-cisAMB resulted in a near-total (95%) depletion of cellular AdoMet pools by 4 h. This was accompanied by a 3-fold increase in ornithine decarboxylase (ODC) activity, a 2.5-fold increase in AdoMet decarboxylase (AdoMetDC) activity and a 20% decrease in spermidine and spermine pools. The increase in enzyme activities seemed to be partially due to prolongation of enzyme activity half-life, since that of ODC was extended from 30 to 50 min and that of AdoMetDC from 65 to 310 min. By temporal sequence characterization (0-4 h), the onset of elevations of enzyme activity (0.5-1 h) seemed to be causally related to an earlier (0-0.5 h) decline in AdoMet pools, as opposed to the 20% decrease in spermidine and spermine pools, which occurred much later (2-4 h); the latter are known to regulate decarboxylase activities negatively. Drug-induced elevations in ODC and, to a lesser extent, AdoMetDC activities were reversed by later treatment with exogenous AdoMet. However, because the latter also increased spermine pools (which could not be prevented with various enzyme inhibitors), the reversal of elevations in enzyme activities could not be directly linked to AdoMet. Although not definitive, the data raise the interesting possibility that, in addition to being negatively regulated by polyamines, ODC and AdoMetDC activities may also be subject to negative control by cellular AdoMet (or an AdoMet metabolite). The net effect of either or both of these influences would be to conserve polyamine-biosynthetic activity in the face of declining AdoMet supplies.

scholarly journals Antitrypanosomal effects of polyamine biosynthesis inhibitors correlate with increases in Trypanosoma brucei brucei S-adenosyl-l-methionine

1991 ◽  
Vol 274 (2) ◽  
pp. 527-533 ◽  
Author(s):  
T L Byers ◽  
T L Bush ◽  
P P McCann ◽  
A J Bitonti
Keyword(s):  
Trypanosoma Brucei ◽  
Mammalian Cells ◽  
Time Course ◽  
Fold Increase ◽  
Complete Disappearance ◽  
Trypanosoma Brucei Brucei ◽  
Irreversible Inhibitor ◽  
Polyamine Biosynthesis ◽  
Key Enzyme ◽  
Contrast Exposure

We reported recently that administration of ([(Z)-4-amino-2-butenyl]methylamino)-5′-deoxyadenosine (MDL 73811), an enzyme-activated irreversible inhibitor of S-adenosyl-L-methionine decarboxylase (AdoMetDC; EC 4.1.1.50), a key enzyme in the synthesis of spermidine, cures African trypanosome infections in mice. The precise mechanism of action of MDL 73811 was not clear because a rapid disappearance of trypanosomes from the bloodstream of treated rats occurred before significant depletion of spermidine. Administration of MDL 73811 to Trypanosoma brucei brucei-infected rats resulted in a 70% decrease in parasitaemia within 1 h and a complete disappearance of parasites by 5 h. The reduction in parasitaemia was accompanied by complete inhibition of AdoMetDC activity by 10 min after injection of MDL 73811; inhibition was sustained for at least 4 h. Polyamine levels in trypanosomes were unaffected during the first 1 h in which the marked decrease in parasitaemia was observed, but parasite AdoMet levels increased 20-fold within this time. In contrast, exposure of cultured mammalian cells to MDL 73811 resulted in only a 1.5-2-fold increase in AdoMet levels over a 6 h time course. Experiments with inhibitors of ornithine decarboxylase (ODC) also suggested that the increased AdoMet levels might be an important factor for antitrypanosomal efficacy. Trypanosomes taken from rats treated for 36 h with eflornithine, an inhibitor of ODC, were depleted of putrescine and had markedly decreased spermidine levels. These organisms also had less than 10% of control AdoMetDC activity, and had elevated decarboxy AdoMet (greater than 4000-fold) and AdoMet (up to 50-fold) levels. The methyl ester of alpha-monofluromethyl-3,4-dehydro-ornithine (delta-MFMO-CH3), which cures murine T. b. brucei infections, and the ethyl ester analogue of this compound (delta-MFMO-C2H5), which does not cure this infection, become ODC inhibitors upon hydrolysis and thus were tested for their effects on trypanosomal polyamines, AdoMet and decarboxy AdoMet levels. Although both esters of delta-MFMO depleted trypanosomal polyamines, AdoMet and decarboxy AdoMet levels were elevated in T. b. brucei from infected mice treated with delta-MFMO-CH3 but not in parasites from mice treated with the delta-MFMO-C2H5. These data suggest that inhibition of AdoMetDC, either directly with MDL 73811 or indirectly with inhibitors of ODC, apparently leads to a trypanosome-specific elevation of AdoMet. It is possible that major changes in AdoMet, rather than changes in polyamines, may be responsible for the antitrypanosomal effects of these drugs.

scholarly journals Overexpression of arginine decarboxylase in transgenic plants

1997 ◽  
Vol 325 (2) ◽  
pp. 331-337 ◽  
Author(s):  
Daniel BURTIN ◽  
Anthony J. MICHAEL
Keyword(s):  
Transgenic Plants ◽  
Arginine Decarboxylase ◽  
Polyamine Metabolism ◽  
Morphological Development ◽  
Polyamine Biosynthesis ◽  
Adenosylmethionine Decarboxylase ◽  
Two Generations ◽  
Key Enzyme ◽  
Polyamine Conjugate ◽  
And Control

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.

scholarly journals Phosphoribosyl pyrophosphate and phosphoribosyl pyrophosphate synthetase in rat mammary gland. Changes in the lactation cycle and effects of diabetes, insulin and phenazine methosulphate

1986 ◽  
Vol 238 (2) ◽  
pp. 553-559 ◽  
Author(s):  
S Kunjara ◽  
M Sochor ◽  
N Salih ◽  
P McLean ◽  
A L Greenbaum
Keyword(s):  
Mammary Gland ◽  
Fold Increase ◽  
Pentose Phosphate ◽  
Diabetic Rat ◽  
Synthetase Activity ◽  
Phosphoribosyl Pyrophosphate ◽  
Rat Mammary Gland ◽  
Lactation Cycle ◽  
Phenazine Methosulphate

Changes in the tissue content of phosphoribosyl pyrophosphate (PPRibP), glucose 6-phosphate, ribose 5-phosphate (Rib5P), RNA and DNA, of the activity of PPRibP synthetase (EC 2.7.6.1) and the conversion of [1-14C]- and [6-14C]-glucose into 14CO2 were measured at mid-lactation in the normal and diabetic rat and in pregnancy, lactation and mammary involution in the normal rat. The PPRibP, glucose 6-phosphate and Rib5P contents increase during pregnancy and early lactation to reach a plateau value at mid-lactation, before falling sharply during weaning. The PPRibP content, PPRibP synthetase activity and flux of glucose through the oxidative pentose phosphate pathway (PPP) all change in parallel during the lactation cycle. Similarly, after 3 and 5 days duration of streptozotocin-induced diabetes, ending on day 10 of lactation, there were parallel declines in PPRibP content, PPRibP synthetase and PPP activity. The effect of streptozotocin was prevented by pretreatment with nicotinamide and partially reversed by insulin administration. Addition of insulin to lactating rat mammary-gland slices incubated in vitro significantly raised the PPRibP content (+47%) and the activity of the PPP (+40%); phenazine methosulphate, which gives a 2-fold increase in PPP activity, raised the PPRibP content of lactating mammary gland slices by approx. 3-fold. It is concluded that Rib5P, generated in the oxidative segment of the PPP, is an important determinant of PPRibP synthesis in the lactating rat mammary gland and that insulin plays a central role in the regulation of the bioavailability of this precursor of nucleotide and nucleic acid synthesis.

scholarly journals Role of the 5′-untranslated region of mRNA in the synthesis of S-adenosylmethionine decarboxylase and its regulation by spermine

1994 ◽  
Vol 302 (3) ◽  
pp. 765-772 ◽  
Author(s):  
L M Shantz ◽  
R Viswanath ◽  
A E Pegg
Keyword(s):  
Secondary Structure ◽  
Untranslated Region ◽  
Fold Increase ◽  
Negative Regulator ◽  
Mrna Levels ◽  
Coding Region ◽  
Polyamine Biosynthesis ◽  
Adenosylmethionine Decarboxylase ◽  
Spermine Synthase ◽  
Synthase Inhibitor

S-Adenosylmethionine decarboxylase (AdoMetDC), a rate-limiting enzyme in polyamine biosynthesis, is regulated by polyamines at the levels of both transcription and translation. Two unusual features of AdoMetDC mRNA are a long (320 nt) 5′-untranslated region (5′UTR), which is thought to contain extensive secondary structure, and a short (15 nt) open reading frame (ORF) within the 5′UTR. We have studied the effects of altering these elements on both the expression of AdoMetDC and its regulation by n-butyl-1,3-diaminopropane (BDAP), a spermine synthase inhibitor. Human AdoMetDC cDNAs containing alterations in the 5′UTR, as well as chimaeric constructs in which the AdoMetDC 5′UTR was inserted ahead of the luciferase-coding region, were transfected into COS-7 cells. Construct pSAM320, which contains all of the 5′UTR, the AdoMetDC protein-coding region and the 3′UTR, was expressed poorly (2-fold over the endogenous activity). Deletion of virtually the entire 5′UTR, leaving nt -12 to -1, increased expression 59-fold, suggesting that 5′UTR acts as a negative regulator. The same effect was seen when the 27 nt at the extreme 5′ end were removed (pSAM293, 47-fold increase), or when the internal ORF which is present in this region was destroyed by changing the ATG to CGA (pSAM320-ATG, 38-fold increase). The expression and regulation of pSAM44 (made by deleting nt -288 to -12), which has very little predicted secondary strucutre, was very similar to that of pSAM320 indicating that the terminal 27 nt including the internal ORF rather than extensive secondary structure may be responsible for the low basal levels of AdoMetDC expression. These results, confirmed using luciferase constructs, suggest that the negative effect on expression is predominantly due to the internal ORF. Depletion of spermine by BDAP increased the expression from pSAM320 more than 5-fold without affecting AdoMetDC mRNA levels. Expression from pSAM293 was unchanged by spermine depletion, whereas that from pSAM320-ATG was increased 2.5-fold. These results indicate the presence of a spermine response element in the first 27 nt of the 5′UTR that may include but is not entirely due to the internal ORF.

scholarly journals Protein kinase C phosphorylation of rat liver S-adenosylmethionine synthetase: dissociation and production of an active monomer

1994 ◽  
Vol 303 (3) ◽  
pp. 949-955 ◽  
Author(s):  
M A Pajares ◽  
C Durán ◽  
F Corrales ◽  
J M Mato
Keyword(s):  
Alkaline Phosphatase ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Rat Liver ◽  
Oligomeric State ◽  
Kinase C ◽  
Phosphatase Treatment ◽  
Key Enzyme ◽  
Adenosylmethionine Synthetase ◽  
Adomet Synthetase

The regulation of rat liver S-adenosylmethionine synthetase (AdoMet synthetase), a key enzyme in methionine metabolism, by protein kinase C (PKC) phosphorylation has been studied. Both enzyme forms, tetramer and dimer, are phosphorylated by this kinase in the same residue, Thr-342, of the sequence. Phosphorylation of the dimer leads to its dissociation, with production of a fully-active monomer. The kinetics of the monomer have been studied, and a KmMet of 931.9 microM, a KmATP of 708 microM and a Vmax of 66.8 nmol/min/mg have been calculated. Alkaline phosphatase treatment of both enzyme forms (tetramer and dimer) produces a reduction in their activity with no change in the oligomeric state. On the other hand, PKC phosphorylation of the alkaline phosphatase-treated AdoMet synthetase forms leads to the dissociation of the dimer to produce a monomer. Rephosphorylation occurs again in the same residue, Thr-342, of the sequence. The significance of AdoMet synthetase regulation by PKC phosphorylation is further discussed.

Isolation and characterization of an interferon-resistant cell line deficient in the induction of (2'-5')oligoadenylate synthetase activity

1983 ◽  
Vol 3 (10) ◽  
pp. 1759-1765
Author(s):  
S Salzberg ◽  
D H Wreschner ◽  
F Oberman ◽  
A Panet ◽  
M Bakhanashvili
Keyword(s):  
Leukemia Virus ◽  
Fold Increase ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Synthetase Activity ◽  
Oligoadenylate Synthetase ◽  
Similar Reduction ◽  
Isolation And Characterization ◽  
Ifn Treatment ◽  
Nih 3T3

To screen for cells with different sensitivities to interferon (IFN), NIH 3T3 mouse fibroblasts were subcloned and examined for their response to IFN treatment. Of 30 clones tested, 2 appeared to be relatively resistant to IFN, since the replication of both vesicular stomatitis virus and mengovirus was not inhibited, even in the presence of 1,000 U of IFN per ml. One resistant (A10) and one sensitive (A5) clone were further analyzed. In both clones, murine leukemia virus replication was equally inhibited by IFN, indicating the presence of functional receptors for IFN in the resistant clone. Using the (2'-5')oligoadenylate (2-5A) radiobinding assay, we could demonstrate that both clones contained the RNase L protein. Furthermore, this enzyme appears to be active, since a similar reduction in the rate of protein synthesis was evident after the introduction of exogenous 2-5A to the cells. We also analyzed the activity of another enzyme in the 2-5A pathway, namely, 2-5A synthetase. In the sensitive cells (A5), the induction of enzyme activity was proportional to the IFN concentration used, reaching a maximum of more than a 10-fold increase over the background of untreated cells. However, little if any induction over the basal activity was observed in the resistant cells (A10) when similar doses of IFN were used. It is thus probable that the lack of induction of 2-5A synthetase activity by IFN in A10 cells is at least partly responsible for their relative resistance to IFN treatment.

scholarly journals Regulation of tropomyosin gene expression during myogenesis.

1981 ◽  
Vol 1 (3) ◽  
pp. 289-301 ◽  
Author(s):  
M Moss ◽  
R Schwartz
Keyword(s):  
Gene Expression ◽  
Nucleic Acid ◽  
Messenger Rna ◽  
Deoxyribonucleic Acid ◽  
Critical Role ◽  
Fold Increase ◽  
Nucleic Acid Content ◽  
Total Nucleic Acid ◽  
Synthetic Capacity ◽  
Rna Levels

In skeletal muscle, tropomyosin has a critical role in transduction of calcium-induced contraction. Presently, little is known about the regulation of tropomyosin gene expression during myogenesis. In the present study, qualitative and quantitative changes in the nucleic acid populations of differentiating chicken embryo muscle cells in culture have been examined. Total nucleic acid content per nucleus increased about fivefold in fully developed myotubes as compared to mononucleated myoblasts. The contribution of deoxyribonucleic acid to the total nucleic acid population decreased from 24% in myoblasts to 5% of total nucleic acid in myotubes. Concomitant with the decrement in deoxyribonucleic acid contribution to total nucleic acid was an increase in polyadenylated ribonucleic acid (RNA) content per cell which reached levels in myotubes that were 17-fold higher than those of myoblasts. Specific changes in the RNA population during myogenesis were further investigated by quantitation of the synthetic capacity (messenger RNA levels) per cell for alpha- and beta-tropomyosin. Cell-free translation and immunoprecipitation demonstrated an approximately 40-fold increase in messenger RNA levels per nucleus for alpha- and beta-tropomyosin after fusion in the terminally differentiated myotubes. Indirect immunofluorescence with affinity-purified tropomyosin antibodies demonstrated the presence of tropomyosin-containing filaments in cells throughout myogenesis. Thus, the tropomyosin genes are constitutively expressed during muscle differentiation through the production of tropomyosin messenger RNA and translation into tropomyosin protein.

scholarly journals Folate Insufficiency Due to MTHFR Deficiency Is Bypassed by 5-Methyltetrahydrofolate

2020 ◽  
Vol 9 (9) ◽  
pp. 2836
Author(s):  
Maša Vidmar Golja ◽  
Alenka Šmid ◽  
Nataša Karas Kuželički ◽  
Jurij Trontelj ◽  
Ksenija Geršak ◽  
...  
Keyword(s):  
Methylenetetrahydrofolate Reductase ◽  
Fold Increase ◽  
Mthfr Gene ◽  
Folate Supplementation ◽  
Disease States ◽  
Metabolic Defects ◽  
Key Enzyme ◽  
Cell Processes ◽  
Activity Groups ◽  
Mthfr Deficiency

Adequate levels of folates are essential for homeostasis of the organism, prevention of congenital malformations, and the salvage of predisposed disease states. They depend on genetic predisposition, and therefore, a pharmacogenetic approach to individualized supplementation or therapeutic intervention is necessary for an optimal outcome. The role of folates in vital cell processes was investigated by translational pharmacogenetics employing lymphoblastoid cell lines (LCLs). Depriving cells of folates led to reversible S-phase arrest. Since 5,10-methylenetetrahydrofolate reductase (MTHFR) is the key enzyme in the biosynthesis of an active folate form, we evaluated the relevance of polymorphisms in the MTHFR gene on intracellular levels of bioactive metabolite, the 5-methyltetrahydrofolate (5-Me-THF). LCLs (n = 35) were divided into low- and normal-MTHFR activity groups based on their genotype. They were cultured in the presence of folic acid (FA) or 5-Me-THF. Based on the cells’ metabolic activity and intracellular 5-Me-THF levels, we conclude supplementation of FA is sufficient to maintain adequate folate level in the normal MTHFR activity group, while low MTHFR activity cells require 5-Me-THF to overcome the metabolic defects caused by polymorphisms in their MTHFR genes. This finding was supported by the determination of intracellular levels of 5-Me-THF in cell lysates by LC-MS/MS. FA supplementation resulted in a 2.5-fold increase in 5-Me-THF in cells with normal MTHFR activity, but there was no increase after FA supplementation in low MTHFR activity cells. However, when LCLs were exposed to 5-Me-THF, a 10-fold increase in intracellular levels of this metabolite was determined. These findings indicate that patients undergoing folate supplementation to counteract anti-folate therapies, or patients with increased folate demand, would benefit from pharmacogenetics-based therapy choices.

scholarly journals Activation of 2',5'-oligoadenylate synthetase activity on induction of HL-60 leukemia cell differentiation.

1989 ◽  
Vol 9 (9) ◽  
pp. 3897-3903 ◽  
Author(s):  
E L Schwartz ◽  
L A Nilson
Keyword(s):  
Leukemia Cell ◽  
Fold Increase ◽  
Polyclonal Antiserum ◽  
Synthetase Activity ◽  
Human Leukemia ◽  
Type I ◽  
Mrna Levels ◽  
Oligoadenylate Synthetase ◽  
Granulocytic Differentiation ◽  
Ifn Alpha

A 27-fold increase in 2',5'-oligoadenylate synthetase activity, an enzyme associated with the antiproliferative actions of interferon (IFN), was observed after treatment of HL-60 human leukemia cells with dimethyl sulfoxide (DMSO), an inducer of granulocytic differentiation of the cells. Enzyme activity was elevated after 24 h of exposure to DMSO, was maximal at 48 hours, and declined thereafter. A comparable increase was observed after treatment with 1 U of alpha interferon (IFN-alpha) per ml or 8 U of beta interferon (IFN-beta) per ml. Elevated levels of expression of other IFN-inducible genes, including type I histocompatibility antigen (HLA-B) mRNA and 2',5'-oligoadenylate phosphodiesterase activity, were also observed with DMSO treatment. DMSO-treated HL-60 cells had an increased amount of a 1.8-kilobase mRNA for oligoadenylate [oligo(A)] synthetase when compared with that of control cells; both DMSO- and IFN-treated HL-60 cells also expressed 1.6-, 3.4-, and 4.3-kilobase mRNA. The increase in both oligo(A) synthetase activity and mRNA levels was inhibited by polyclonal antiserum to human IFN-alpha; however, no IFN-alpha mRNA could be detected in the cells. Antiserum to IFN-beta or gamma interferon (IFN-gamma) had no effect on oligo(A) synthetase expression or activity nor was there any detectable IFN-beta 1 or IFN-beta 2 mRNA in the cells. The anti-IFN-alpha serum did not block the elevation of HLA-B mRNA in DMSO-treated cells. These observations suggest that the increased expression of oligo(A) synthetase in DMSO-treated cells may be mediated by the release of an IFN-alpha-like factor; however, the levels of any IFN-alpha mRNA produced in the cells were extremely low.

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