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MECHANISMS OF IRON-A CQUISITION FROM HEMOGLOBIN IN PORPHYROMONAS GINGIVALIS

从牙龈卟啉单胞菌血红蛋白中获取铁的机制

基本信息

批准号：
05454555
负责人：
SHIZUKUISHI Satoshi
金额：
$ 3.97万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

项目摘要

In this study, we investigated on the mechanisms of the acquisition of heme from hemoglobin in Porphyromonas gingivalis. The iron-depleted cells of P.gingivalis 381 were incubated in the basal medium plus test substrates such as hemoglobin, hemin, transferrin and various inorganic iron-compounds. The value of Ks, a parameter analogous to Michaelis-Menten constant, was estimated.P.gingivalis 381 showed a Ks value of 3.85,4.91 and 0.0017 mM for hemin, transferrin and hemoglobin, respectively. However, the inorganic iron compounds tested did not support P.gingvalis growth. These findings suggest that P.gingivalis utilizes hemoglobin as an iron source much more effectively than other iron-containing compounds under an iron -limited environment. Next, binding of human hemoglobin by P.gingivalis cells was determined by using [^3H]-labeled hemoglobin. The binding of hemoglobin occurred rapidly, reversibly and specifically. Scatchard analysis of the binding data revealed linear plot, indicating the presence of one population of binding protein. The apparent Kd was 1.0? B10.19x10^<-6>M and number of binding sites per cell was 3.2? B10.76x10^4. Hemoglobin binding was inhibited by unlabeled human hemoglobin but not hemin and protoporphyrin IX.The binding was only partially inhibited by serum albumin, transferrin, lactoferrin, catalase and cytochrome c. These results suggest that the ligand recognized by the binding protein may not be the heme moiety. The binding of hemoglobin much increased when the organisms were grown in hemin-limited conditions. These results suggest that P.gingivalis cells may interact with human hemoglobin through specific binding sites on their surfaces as a preliminary step in iron acquisition.

在本研究中，我们研究了牙龈卟啉单胞菌从血红蛋白中获取血红素的机制。将牙龈卟啉菌381的缺铁细胞在基础培养基和血红蛋白、血红蛋白、转铁蛋白及各种无机铁化合物等试验底物中培养。近似于Michaelis-Menten常数的Ks值进行了估计，p.g ingivalis 381对血红蛋白、转铁蛋白和血红蛋白的Ks值分别为3.85、4.91和0.0017 mM。然而，无机铁化合物测试不支持P.gingvalis生长。这些发现表明，在铁限制的环境下，牙龈假单胞菌比其他含铁化合物更有效地利用血红蛋白作为铁源。接下来，用[^3H]标记的血红蛋白测定牙龈卟啉菌细胞对人血红蛋白的结合。血红蛋白的结合发生迅速、可逆和特异性。结合数据的Scatchard分析显示线性图，表明存在一个结合蛋白群体。表观Kd为1.0？B10.19x10^<-6>M，每个细胞的结合位点数为3.2？B10.76x10 ^ 4。未标记的人血红蛋白抑制血红蛋白结合，而血红蛋白和原卟啉IX不受抑制。血清白蛋白、转铁蛋白、乳铁蛋白、过氧化氢酶和细胞色素c仅能部分抑制其结合。这些结果表明，结合蛋白识别的配体可能不是血红素部分。当生物在血红蛋白限制的条件下生长时，血红蛋白的结合大大增加。这些结果表明，牙龈卟啉菌细胞可能通过其表面的特定结合位点与人血红蛋白相互作用，作为铁获取的初步步骤。