• Title, Summary, Keyword: PW-Plot

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Identification of Three Competitive Inhibitors for Membrane­Associated, $Mg^{2+}-Dependent$ and Neutral 60 kDa Sphingomyelinase Activity

  • Kim Seok Kyun;Jung Sang Mi;Ahn Kyong Hoon;Jeon Hyung Jun;Lee Dong Hun;Jung Kwang Mook;Jung Sung Yun;Kim Dae Kyong
    • Archives of Pharmacal Research
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    • v.28 no.8
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    • pp.923-929
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    • 2005
  • Methanol extracts of domestic plants of Korea were evaluated as a potential inhibitor of neutral pH optimum and membrane-associated 60 kDa sphingomyelinase (N-SMase) activity. In this study, we partially purified N-SMase from bovine brain membranes using ammonium sulfate. It was purified approximately 163-fold by the sequential use of DE52, Butyl-Toyopearl, DEAE-Cellulose, and Phenyl-5PW column chromatographies. The purified N-SMase activity was assayed in the presence of the plant extracts of three hundreds species. Based on the in vitro assay, three plant extracts significantly inhibited the N-SMase activity in a time- and concentration-dependent manner. To further examine the inhibitory pattern, a Dixon plot was constructed for each of the plant extracts. The extracts of Abies nephrolepis, Acer tegmentosum, and Ginkgo biloba revealed a competitive inhibition with the inhibition constant (Ki) of $11.9 {\mu}g/mL,\;9.4{\mu}g/mL,\;and\;12.9{\mu}g/mL$, respectively. These extracts also inhibited in a dose-dependent manner the production of ceramide induced by serum deprivation in human neuroblastoma cell line SH-SY5Y.

Inhibition of a Neutral Form of Sphingomyelinase by Alkylthioureido-l,3-propandiols, KY353X Series

  • Jung, Sang-Mi;Jeong, Eui-Man;Jo, Dong-Hwawn;Chin, Mi-Reyoung;Jun, Hyung-Jin;Kim, Yong-Hyun;Jeon, Hyung-Jun;Lee, Dong-Hun;Park, Mi-Ja;Oh, Mi-Jung;Yim, Chul-Bu;Kim, Dae-Kyong
    • Biomolecules & Therapeutics
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    • v.11 no.3
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    • pp.169-173
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    • 2003
  • Alkylthioureido-1,3-propandiols (KY353X series) were synthesized and evaluated as inhibitors for neutral sphingomyelinase (N-SMase). To examine whether KY353X series inhibit N-SMase, we purified the N-SMase from bovine brain. The N-SMase was partially purified by sequential chromatographies of DEAE-Cellulose anionic exchange and phenyl-5PW hydrophobic HPLC. These seqeuntial procedures for N-SMase resulted in a 67-fold purification and excluded other isoforms of SMase. Based on in vitro assay, KY353X series inhibited N-SMase activity in time, concentration-dependent manners and completely inactivated N-SMase at 50 $\mu$M. In particular, KY3535 and KY3536 inhibited more effectively than the others. To further determine the .inhibitory pattern, a Dixon plot was constructed, to showing that the inhibition by KY3535 and KY3536 were competitive. The inhibition constant (Ki) of KY3535 and KY3536 was 1.7 $\mu$M and 2.5$\mu$M in 100 mM Tris-HCl buffer, pH 7.0, respectively.