Effects of β-citryl-L-glutamic acid and its metal ion-complex on the iron-dependent formation of free radicals

2006 Fiscal Year Final Research Report Summary

• Project/Area Number: 16591776
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Ophthalmology
• Research Institution: Kobe Gakuin University
• Principal Investigator: MIYAKE Masaharu Kobe Gakuin University, The Faculty of Pharmaceutical Sciences, Professor, 薬学部, 教授 (50093943)
• Co-Investigator(Kenkyū-buntansha): HAMADA Michiko Kobe Gakuin University, The Faculty of Pharmaceutical Sciences, Assistant Professor, 薬学部, 実験助手 (10248106)
• Project Period (FY): 2004 – 2006
• Keywords: β-citryl-L-glutamate, / iron / copper / competitive inhibitor, / xanthine oxidase / chelation

Research Abstract

We previously isolated a novel compound containing glutamate and citrate residue from a newborn rat brain and identified it as β-citryl-L-glutamate (β-CG). This compound appears at high concentrations (300〜600μM) during the period characterized by the growth and differentiation of neurons in developing rats, and then disappears with maturation,
Thin-layer chromatography results have shown that β-CG form a complex with ferrous ion and is also associated with calcium, magnesium, manganese, zinc, nickel, cobalt, and copper, where it has no affinity for ferric, potassium, or sodium ion. β-CG is able to solubilize iron more effectively from Fe(OH)_2 than Fe(OH)_3, and its Fe-solubilizing activities are inhibited by the presence of divalent metals in the following order, Co>Ca=Zn>Mp>Cu.
Both β-CG and deferoxamine inhibited the iron-dependent degradation of deoxyribose in a dose-dependent manner. In contrast, EDTA activated that degradation at a low concentration, and had scant effects at a hig her concentration. EDTA strongly inhibited bleomycin-iron-dependent damage to DNA, while both β-CG and deferoxamine showed slight stimulatory effects at low concentrations, whereas they inhibited the damage in a dose-dependent fashion at high concentrations.
Among various metal ion-complexes, only Cu-β-CG complex showed SOD activity in an assay system with xanthine-xanthine oxidase. Cu-EDTA complex also demonstrated SOD activity as did β-CG, while both Cu-and Mn-deferoxamine had strong SOD activities.
The Cu-complexes of β-CG and deferoxamine showed a concentration-dependent inhibition of xanthine oxidase, while those of glutamate and citrate also demonstrated inhibitory effects, in contrast to the Cu-complex of GHK, which showed only a weak inhibitory effect, and the complexes of EDTA and bathocuproine, which had no effect. Further, free Cu^<2+> alone was also found to inhibit xanthine oxidase. Using Lineweaver-Burk plots, the type of inhibition for Cu-β-CG and free Cu^<2+> were examined, and both compounds demonstrated competitive inhibition