The Activator-Binding Site of Onchocerca volvulus S-Adenosylmethionine Decarboxylase, a Potential Drug Target

2003 ◽  
Vol 384 (8) ◽  
pp. 1195-1201 ◽  
Author(s):  
D. Ndjonka ◽  
Y. Zou ◽  
X. Bi ◽  
P. Woster ◽  
R. D. Walter ◽  
...  
Keyword(s):  
Specific Activity ◽  
Competitive Inhibitor ◽  
Onchocerca Volvulus ◽  
Polyamine Biosynthesis ◽  
Adenosylmethionine Decarboxylase ◽  
Polyamine Analogues ◽  
Human Enzyme ◽  
Competitive Inhibitors ◽  
New Strategy ◽  
Key Enzyme

Abstract S-Adenosylmethionine decarboxylase (AdoMetDC) is a key enzyme in polyamine biosynthesis. In many eukaryotes its activity is stimulated specifically by putrescine. The AdoMetDC of the filarial parasite Onchocerca volvulus, however, is not only stimulated by putrescine but also by the naturally occuring polyamines spermidine and spermine. Several diamines, acetylated polyamines and polyamine analogues were used to analyse what molecular prerequisites are needed to stimulate nematode AdoMetDC activity. In the absence of an activator, the O. volvulus enzyme exhibits an extremely low specific activity. This fact, together with the unspecificity of activator binding, was thought to be useful for a new strategy to inhibit nematode AdoMetDC activity. Therefore, different polyamine analogues were tested as competitive inhibitors towards the stimulatory effect putrescine has on the O. volvulus and, in comparison, on the Caenorhabditis elegans and human AdoMetDC. Bis(aralkyl)- and bis(alkyl)-substituted polyamine analogues with a 3-7-3 backbone were found to inhibit AdoMetDC activities, however, probably without interfering with the putrescine stimulation. The best inhibitor, BW-1, was about 10-fold more effective against O. volvulus AdoMetDC than against the human enzyme. Unexpectedly, BW-1 was determined to be a competitive inhibitor with respect to AdoMet, having a Ki value of 310 uM for the putrescine-stimulated human AdoMetDC. Furthermore, we show for the O. volvulus and the human enzyme that the degree of inhibition by BW-1 depends on the actual putrescine concentration.

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 The biosynthesis of granulose by Clostridium pasteurianum

1974 ◽  
Vol 144 (3) ◽  
pp. 503-511 ◽  
Author(s):  
R L Robson ◽  
R M Robson ◽  
J G Morris
Keyword(s):  
Specific Activity ◽  
Competitive Inhibitor ◽  
Partial Purification ◽  
Clostridium Pasteurianum ◽  
Saturation Curve ◽  
Wild Type ◽  
Competitive Inhibitors ◽  
Fine Control ◽  
Adp Glucose Pyrophosphorylase ◽  
High Concentrations

1. Mutant strains of Clostridium pasteurianum were obtained, which are unable to synthesize granulose (an intracellularly accumulated amylopectin-like α-polyglucan). 2. These mutants lacked either (a) ADP-glucose pyrophosphorylase (EC 2.7.7.27), or (b) granulose synthase (i.e. ADP-glucose–α-1,4-glucan glucosyltransferase, EC 2.4.1.21). 3. Although both of these enzymes were constitutively synthesized by the wild-type organism, massive deposition of granulose in a sporulating culture coincided with a threefold increase in the specific activity of ADP-glucose pyrophosphorylase. 4. The soluble ADP-glucose pyrophosphorylase was partially purified (33-fold). Its ATP-saturation curve was not sigmoidal and its activity was not enhanced by phosphorylated intermediates of glycolysis, pyruvate, NAD(P)H or pyridoxal 5′-phosphate. ADP at relatively high concentrations acted as a competitive inhibitor (Ki=19mm). 5. The dependence of granulose synthase on a suitable polyglucan primer was demonstrated by using enzyme obtained from a granulose-free mutant strain (lacking ADP-glucose pyrophosphorylase). 6. Partial purification of granulose synthase from wild-type strains was facilitated by its being bound to the native particles of granulose. No activator was discovered, but ADP, AMP and pyridoxal 5′-phosphate were competitive inhibitors, ADP being most effective (Ki about 0.2mm). 7. It would appear that the synthesis of granulose in Cl. pasteurianum is not subject to the positive, fine control that is a feature of glycogen biosynthesis in most bacteria.

Translational regulation of the plant S-adenosylmethionine decarboxylase

2003 ◽  
Vol 31 (2) ◽  
pp. 424-427 ◽  
Author(s):  
C. Hanfrey ◽  
M. Franceschetti ◽  
M.J. Mayer ◽  
C. Illingworth ◽  
K. Elliott ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Rna Secondary Structure ◽  
Translational Regulation ◽  
Mrna Translation ◽  
Open Reading Frames ◽  
Polyamine Biosynthesis ◽  
Adenosylmethionine Decarboxylase ◽  
Key Enzyme ◽  
Upstream Open Reading Frames ◽  
Reading Frames

It is becoming apparent that control of protein synthesis by metabolites is more common than previously thought. Much of that control is exerted at the level of initiation of mRNA translation, orchestrated by upstream open reading frames (uORFs) and RNA secondary structure. S-Adenosylmethionine decarboxylase (AdoMetDC) is a key enzyme in polyamine biosynthesis and both mammalian and plant AdoMetDCs are translationally regulated by uORFs in response to polyamine levels by distinct mechanisms.

scholarly journals A novel trans-spliced mRNA from Onchocerca volvulus encodes a functional S-adenosylmethionine decarboxylase

1996 ◽  
Vol 320 (2) ◽  
pp. 519-530 ◽  
Author(s):  
Akram A. DA'DARA ◽  
Kimberly HENKLE-DÜHRSEN ◽  
Rolf D. WALTER
Keyword(s):  
Large Subunit ◽  
Deae Cellulose ◽  
Regulatory Elements ◽  
Leader Sequence ◽  
Onchocerca Volvulus ◽  
Genomic Sequences ◽  
Regulatory Function ◽  
Polyamine Biosynthesis ◽  
Spliced Leader ◽  
Adenosylmethionine Decarboxylase

Complete cDNA and genomic sequences encoding the Onchocerca volvulus S-adenosylmethionine decarboxylase (SAMDC), a key enzyme in polyamine biosynthesis, have been isolated and characterized. The deduced amino acid sequence encodes a 42 kDa proenzyme with a moderate level of sequence homology to eukaryotic SAMDCs. Enzymically active O. volvulusSAMDC was expressed at a high level in an Escherichia colimutant strain lacking endogenous SAMDC. The recombinant enzyme was purified to homogeneity using DEAE-cellulose, methylglyoxal bis(guanylhydrazone)–Sepharose and Superdex S-200 chromatography. It was determined that the recombinant proenzyme is cleaved to produce 32 and 10 kDa subunits. The sequence of the N-terminal portion of the large subunit was determined and comparison with the sequence of the proenzyme revealed that the precise cleavage site lies between Glu86 and Ser87. Gel-filtration experiments demonstrated that these two subunits combine to form an active heterotetramer. Comparison of the cDNA and genomic sequences revealed that the SAMDC mRNA undergoes both cis- and trans-splicing in its 5´-untranslated region (UTR). Anchored PCR on O. volvulusmRNA confirmed the cDNA sequence and identified two distinct trans-spliced products, a 22-nucleotide spliced-leader sequence and a 138 bp sequence containing the 22 nucleotide spliced-leader sequence. Genomic Southern-blot analysis suggests that the O. volvulusSAMDC is encoded by a single-copy gene. This gene spans 5.3 kb and is comprised of nine exons and eight introns. The first intron is located in the 5´-UTR and processing of this intron has a potential regulatory function. The 5´-flanking region of the gene contains potential transcriptional regulatory elements such as a TATA box, two CAAT boxes and AP-1-, C/EBP-, ELP-, H-APF-1-, HNF-5- and PEA3-binding sites.

scholarly journals Glyoxal bis(guanylhydrazone) as an inhibitor of polyamine biosynthesis in tumour cells

1984 ◽  
Vol 221 (2) ◽  
pp. 483-488 ◽  
Author(s):  
P Seppänen ◽  
R Fagerström ◽  
L Alhonen-Hongisto ◽  
H Elo ◽  
P Lumme ◽  
...  
Keyword(s):  
Ornithine Decarboxylase ◽  
Parent Compound ◽  
Competitive Inhibitor ◽  
Inhibitory Effect ◽  
Dependent Manner ◽  
Experimental Conditions ◽  
Polyamine Biosynthesis ◽  
Adenosylmethionine Decarboxylase ◽  
L1210 Cells ◽  
In Cells

Glyoxal bis(guanylhydrazone), the parent compound of methylglyoxal bis(guanylhydrazone), was synthesized and tested for its ability to inhibit the biosynthesis of polyamines. It was found to be a powerful competitive inhibitor of adenosylmethionine decarboxylase (EC 4.1.1.50), yet the lack of the methyl group at the glyoxal portion increased the apparent Ki value for the enzyme by about 30-fold in comparison with methylglyoxal bis(guanylhydrazone). Glyoxal bis(guanylhydrazone) inhibited diamine oxidase (EC 1.4.3.6) activity as effectively as did methylglyoxal bis(guanylhydrazone). The cellular accumulation curves of glyoxal bis(guanylhydrazone) in L1210 cells were practically superimposable with those of methylglyoxal bis(guanylhydrazone), and the uptake of both compounds was distinctly stimulated by a prior treatment with 2-difluoromethylornithine. The drug decreased the concentration of spermidine in a dose-dependent manner and, in contrast with methylglyoxal bis(guanylhydrazone), without a concomitant accumulation of putrescine. The fact that putrescine concentrations were decreased in cells exposed to glyoxal bis(guanylhydrazone) was, at least in part, attributable to an inhibition of ornithine decarboxylase (EC 4.1.1.17) activity in cells treated with the compound. Under these experimental conditions equivalent concentrations of methylglyoxal bis(guanylhydrazone) [1,1′-[(methylethanediylidine)dinitrilo]diguanidine] elicited large increases in the enzyme activity. When combined with difluoromethylornithine, glyoxal bis(guanylhydrazone) potentiated the growth-inhibitory effect of that drug. Taking into consideration the proven anti-leukaemic activity of glyoxal bis(guanylhydrazone), its effectiveness to inhibit spermidine biosynthesis (without raising the concentration of putrescine) as well as its suitability for combined use with inhibitors of ornithine decarboxylase, this drug is apparently worthy of further testing in tumour-bearing animals, especially in combination with difluoromethylornithine or related inhibitors of ornithine decarboxylase.

A radiotracer method for the measurement of central nervous system catecholamines in vivo

1978 ◽  
Vol 56 (3) ◽  
pp. 535-538 ◽  
Author(s):  
S. W. Tang ◽  
H. C. Stancer ◽  
J. J. Warsh
Keyword(s):  
Central Nervous System ◽  
Animal Model ◽  
Nervous System ◽  
Specific Activity ◽  
Brain Catecholamines ◽  
Radiotracer Method ◽  
New Strategy ◽  
The Central Nervous System ◽  
6 Hydroxydopamine

A new strategy for measurement of brain catecholamines was tested in an animal model. [3H]Norepinephrine was infused intravenously in rabbits to label the peripheral norepinephrine pools. The specific activity of urinary 3-methoxy-4-hydroxymandelic acid was consistently higher than that for 3-methoxy-4-hydroxyphenylglycol (MHPG). Central sympathectomy with 6-hydroxydopamine abolished this difference. Using the formula we propose, it is estimated that 30–50% of urinary MHPG originates from the central nervous system.

Receptor kinetics pertaining to blockade of nerve-released transmitter at synapses

1988 ◽  
Vol 233 (1273) ◽  
pp. 461-475 ◽  
Keyword(s):  
Channel Blocker ◽  
Competitive Inhibitor ◽  
Tissue Response ◽  
Receptor Density ◽  
Receptor Kinetics ◽  
Tissue Responses ◽  
Competitive Inhibitors ◽  
Transmitter Substance ◽  
Uncompetitive Inhibitor ◽  
Temporal Inhomogeneity

The question is raised as to whether competitive inhibitors should block responses of tissue to nerve-released neurotransmitter to the same extent as they block equivalent responses to exogenous agonist. From a simple dynamic model of synaptic events, which takes into account non-constancy of transmitter concentration in space and time, it is deduced that equal blockade of responses to nerve-released and exogenous transmitter substance will occur if: (i) there are locally many more receptor molecules than transmitter molecules; (ii) the active agonist–receptor complex, A n R, has n = 1 ; and (iii) tissue response is insensitive to spatial or temporal inhomogeneity of AR. In such a case there will also be equal sensitivity of responses to other modes of inhibition: irreversible competitive, uncompetitive, and non-competitive. Equal blockade of responses to equi-effective endogenous and exogenous agonist will also occur if nerve stimulation gives rise to a steady uniform concentration of agonist, so that equilibrium kinetics are applicable. When n > 1 and/or when tissue responses reflect local peak A n R, response to nerve-released transmitter will be relatively insensitive to receptor blockade by a competitive inhibitor. The same is true for irreversible competitive blockade or for modulation of receptor density. However, an uncompetitive inhibitor (e. g. a ‘channel blocker’) may be more effective against nerve-released agonist than against exogenous agonist.

Spermidine promotes adipogenesis of 3T3-L1 cells by preventing interaction of ANP32 with HuR and PP2A

2013 ◽  
Vol 453 (3) ◽  
pp. 467-474 ◽  
Author(s):  
Mervi T. Hyvönen ◽  
Taina Koponen ◽  
Janne Weisell ◽  
Marko Pietilä ◽  
Alex R. Khomutov ◽  
...  
Keyword(s):  
Protein Phosphatase ◽  
Rna Binding ◽  
Binding Protein ◽  
Cytoplasmic Localization ◽  
Polyamine Biosynthesis ◽  
Polyamine Analogues ◽  
Enhancer Binding Protein ◽  
Phosphatase 2A ◽  
Nuclear Phosphoprotein ◽  
Factor C

We have shown previously that the polyamine spermidine is indispensable for differentiation of 3T3-L1 preadipocytes. In the present study, we examined the mechanism of spermidine function by using the polyamine biosynthesis inhibitor α-difluoromethylornithine in combination with the metabolically stable polyamine analogues γ-methylspermidine or (R,R)-α,ω-bismethylspermine. At the early phase of differentiation, spermidine-depleted 3T3-L1 cells showed decreased translation of the transcription factor C/EBPβ (CCAAT/enhancer-binding protein β), decreased PP2A (protein phosphatase 2A) activity and increased cytoplasmic localization of the RNA-binding protein HuR (human antigen R). The amount of HuR bound to C/EBPβ mRNA was reduced, whereas the amount of bound CUGBP2, an inhibitor of C/EBPβ translation, was increased. ANP32 (acidic nuclear phosphoprotein 32) proteins, which are known PP2A inhibitors and HuR ligands, bound more PP2A and HuR in spermidine-depleted than in control cells, whereas immunodepletion of ANP32 proteins from the lysate of spermidine-depleted cells restored PP2A activity. Taken together, our data shows that spermidine promotes C/EBPβ translation in differentiating 3T3-L1 cells, and that this process is controlled by the interaction of ANP32 with HuR and PP2A.

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