Determination of Covalently Bound Hypusine and Deoxyhypusine to Protein Using Submilligram of Protein Samples by HPLC.

Beppu, Takanobu; Shirahata, Akira; Samejima, Keijiro

doi:10.1248/bpb.19.1

Cited by 5 publications

(4 citation statements)

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“…Then, SDS\PAGE and fluorography were performed as described by Laemmli [34] and Laskey and Mills [35] respectively, using 100 µg of protein of the 100 000 g supernatant. Furthermore, hypusine in eIF5A was measured directly by reversed-phase HPLC with a fluorescence detection of o-phthalaldehyde using 1 mg of protein from the 100 000 g supernatant, as described previously [36].…”

Section: Measurement Of the Amount Of Eif5a And The Level Of Hypusinementioning

confidence: 99%

Inhibition of cell growth through inactivation of eukaryotic translation initiation factor 5A (eIF5A) by deoxyspergualin

et al. 2002

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The mechanism of inhibition of cell growth by deoxyspergualin was studied using mouse mammary carcinoma FM3A cells. Results of studies using deoxyspergualin analogues showed that both the guanidinoheptanate amide and glyoxyspermidine moieties of deoxyspergualin were necessary to cause inhibition of cell growth. When deoxyspergualin was added to the medium, there was a strong inhibition of cell growth and formation of active eukaryotic translation initiation factor 5A (eIF5A) at the third day of culture. There was also a marked decrease in cellular putrescine content and a small decrease in spermidine content. Accumulation of decapped mRNA, which is typically associated with eIF5A deficiency in yeast, was also observed. The inhibition of cell growth and the formation of active eIF5A was not reversed by addition of spermidine. The activity of deoxyhypusine synthase, the first enzyme in the formation of active eIF5A, was inhibited by deoxyspergualin in a cell-free system. These results, taken together, indicate that inhibition of active eIF5A formation is strongly involved in the inhibition of cell growth by deoxyspergualin.

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Section: Measurement Of the Amount Of Eif5a And The Level Of Hypusinementioning

confidence: 99%

Inhibition of cell growth through inactivation of eukaryotic translation initiation factor 5A (eIF5A) by deoxyspergualin

et al. 2002

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“…This analytical method can replace previous HPLC methods with retention times detecting the orthophthaldialdehyde derivatives of deoxyhypusine at 17.4 min and hypusine at 17.4 min (Beppu et al 1996;Beninati et al 1990). Moreover, reproduction of these experiments showed that a gradient performed with mobile phase A consisting of 20 mM sodium acetate and mobile phase B consisting of 40% methanol and 40% acetonitrile prolonged retention times of deoxyhypusine to 58.55 min and hypusine to 50 min (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…N-Epsilon-(5-aminopentyllysine) which is a structural analogue of deoxyhypusine was used as an internal standard. This separation resulted in retention times of deoxyhypusine with 17.2 min and for hypusine with 14.4 min, respectively (Beppu et al 1996) dependent on the gradient being used. However, reproduction of these experiments proved that the imine derivatives showed non-reproducible stabilities (Kaiser et al unpublished).…”

Section: Introductionmentioning

confidence: 99%

A rapid and robust assay for the determination of the amino acid hypusine as a possible biomarker for a high-throughput screening of antimalarials and for the diagnosis and therapy of different diseases

et al. 2011

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Eukaryotic initiation factor 5A (eIF5A) has recently been identified as a biomarker of prognostic significance and therapeutic potential for the treatment in hepatocellular carcinoma. This prompted us to establish a rapid and robust assay to determine deoxyhypusine and hypusine formed with the purified enzymes deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH) from Plasmodium to develop a rapid screening assay for antimalarial drugs. The peptide hydrolysate obtained from hypusinylated eIF5A was analyzed by ultra performance liquid chromatography (UPLC) with retention times for deoxyhypusine of 7.44 min and for hypusine of 7.30 min, respectively. The limit of detection for both compounds was 0.144 ng/μl. Determination of the specific activity of Plasmodium DOHH resulted in a twofold higher specific activity than its human counterpart. Following the iron-complexing strategy of the ferrous iron which is present in the active site of Plasmodium DOHH, a series of iron chelating compounds was tested. 2,2'-Dipyridyl and mimosine abolished DOHH activity completely while 4-oxo-piperidine-carboxylates i.e. the nitrophenylether JK8-2 and EHW 437, the oxime ether of the piperidine aldehyde, showed no inhibition although they were highly active in in vitro cultures of Plasmodium and in vivo in a rodent mouse model. The method allows a high-throughput screening (HPTS) of antimalarial drugs and the evaluation of eIF5A as a biomarker.

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“…16) HTC cells were grown as either a monolayer in culture dishes or a suspension in spinner flasks. HTC cells were maintained in Swim's S-77 medium containing 10% newborn bovine serum.…”

Section: )mentioning

confidence: 99%

Formation of Spermidine or Norspermidine from Synthetic Diacetylpolyamines by Acetylpolyamine Oxidase in Cultured Cells

Takao

Ozawa

Shibata

et al. 2007

Biological & Pharmaceutical Bulletin

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The polyamines spermidine and spermine, and their precursor putrescine, are widely distributed in eukaryotic cells, and are known to be involved in numerous cellular processes.1,2) Intracellular polyamine levels are regulated by the biosynthetic enzymes ornithine decarboxylase (ODC), Sadenosylmethionine decarboxylase, spermidine synthase and spermine synthase, the catabolic enzymes spermidine/spermine N 1 -acetyltransferase (SSAT), acetylpolyamine oxidase (PAO), spermine oxidase, and the cell membrane transport system. The critical role of polyamines in the regulation of cell growth has led to the development of polyamine biosynthesis inhibitors as an anti-neoplastic therapeutic strategy.

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Determination of Covalently Bound Hypusine and Deoxyhypusine to Protein Using Submilligram of Protein Samples by HPLC.

Cited by 5 publications

References 0 publications

Inhibition of cell growth through inactivation of eukaryotic translation initiation factor 5A (eIF5A) by deoxyspergualin

Inhibition of cell growth through inactivation of eukaryotic translation initiation factor 5A (eIF5A) by deoxyspergualin

A rapid and robust assay for the determination of the amino acid hypusine as a possible biomarker for a high-throughput screening of antimalarials and for the diagnosis and therapy of different diseases

Formation of Spermidine or Norspermidine from Synthetic Diacetylpolyamines by Acetylpolyamine Oxidase in Cultured Cells

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