Iseki K, Yonemura K, Kikuchi T, Naasani I, Sugawara M, Kobayashi M, Kohri N, Miyazaki K
Department of Pharmacy, Hokkaido University Hospital, School of Medicine, Hokkaido University, Kita-14-jo, Nishi-5-chome, Sapporo 060, Japan.
Biochim Biophys Acta. 1998 Mar 6;1370(1):161-8. doi: 10.1016/s0005-2736(97)00258-7.
The transport activity of ceftibuten, a dianionic peptide-like compound, was extracted from rat intestinal brush-border membrane by n-octylglucoside and reconstituted into asolectin liposomes by dialysis. The proteoliposomes prepared from the membrane extract showed an inward H+-gradient-dependent uptake of ceftibuten and glycylsarcosine. Ceftibuten-immobilized affinity chromatography of the membrane extract permitted the isolation of two polypeptides (apparent molecular mass of 117 and 127 kDa) that can recognize the dianionic peptide structure of ceftibuten. Proteoliposomes prepared from reconstituting the isolated proteins into asolectin vesicles showed an overshooting uptake of ceftibuten in the presence of an inwardly directed H+ gradient, and this uptake could be inhibited by L-valyl-L-proline. N-glycanase digestion of the isolated proteins, 117 and 127 kDa, trimmed them into 78 and 120 kDa products, respectively. The protein core size of the smaller protein was in agreement with the calculated molecular mass of approximately 79 kDa for the rat PepT1 transporter obtained by other investigators.
头孢布烯是一种二阴离子肽样化合物，其转运活性通过正辛基葡萄糖苷从大鼠肠刷状缘膜中提取，并通过透析重组成大豆卵磷脂脂质体。由膜提取物制备的蛋白脂质体显示出对头孢布烯和甘氨酰肌氨酸的内向H⁺梯度依赖性摄取。对膜提取物进行头孢布烯固定化亲和层析可分离出两种多肽（表观分子量分别为117和127 kDa），它们能够识别头孢布烯的二阴离子肽结构。将分离的蛋白质重组成大豆卵磷脂囊泡制备的蛋白脂质体在存在内向H⁺梯度的情况下显示出对头孢布烯的过冲摄取，并且这种摄取可被L-缬氨酰-L-脯氨酸抑制。对分离的117 kDa和127 kDa蛋白质进行N-糖苷酶消化后，分别将它们修剪为78 kDa和120 kDa的产物。较小蛋白质的蛋白质核心大小与其他研究人员获得的大鼠PepT1转运蛋白约79 kDa的计算分子量一致。
