Kramer W, Nicol S B, Girbig F, Gutjahr U, Kowalewski S, Fasold H
Hoechst Aktiengesellschaft, Frankfurt am Main, Germany.
Biochim Biophys Acta. 1992 Oct 19;1111(1):93-102. doi: 10.1016/0005-2736(92)90278-t.
The Na(+)-dependent uptake system for bile acids in the ileum from rabbit small intestine was characterized using brush-border membrane vesicles. The uptake of [3H]taurocholate into vesicles prepared from the terminal ileum showed an overshoot uptake in the presence of an inwardly-directed Na(+)-gradient ([Na+]out > [Na+]in), in contrast to vesicles prepared from the jejunum. The Na(+)-dependent [3H]taurocholate uptake was cis-inhibited by natural bile acid derivatives, whereas cholephilic organic compounds, such as phalloidin, bromosulphophthalein, bilirubin, indocyanine green or DIDS - all interfering with hepatic bile-acid uptake - did not show a significant inhibitory effect. Photoaffinity labeling of ileal membrane vesicles with 3,3-azo- and 7,7-azo-derivatives of taurocholate resulted in specific labeling of a membrane polypeptide with apparent molecular mass 90 kDa. Bile-acid derivatives inhibiting [3H]taurocholate uptake by ileal vesicles also inhibited labeling of the 90 kDa polypeptide, whereas compounds with no inhibitory effect on ileal bile-acid transport failed to show a significant effect on the labeling of the 90 kDa polypeptide. The involvement of functional amino-acid side-chains in Na(+)-dependent taurocholate uptake was investigated by chemical modification of ileal brush-border membrane vesicles with a variety of group-specific agents. It was found that (vicinal) thiol groups and amino groups are involved in active ileal bile-acid uptake, whereas carboxyl- and hydroxyl-containing amino acids, as well as tyrosine, histidine or arginine are not essential for Na(+)-dependent bile-acid transport activity. The irreversible inhibition of [3H]taurocholate transport by DTNB or NBD-chloride could be partially reversed by thiols like 2-mercaptoethanol or DTT. Furthermore, increasing concentrations of taurocholate during chemical modification with NBD-chloride were able to protect the ileal bile-acid transporter from inactivation. These findings suggest that a membrane polypeptide of apparent M(r) 90,000 is a component of the active Na(+)-dependent bile-acid reabsorption system in the terminal ileum from rabbit small intestine. Vicinal thiol groups and amino groups of the transport system are involved in Na(+)-dependent transport activity, whereas other functional amino acids are not essential for transport activity.
利用刷状缘膜囊泡对兔小肠回肠中胆汁酸的钠依赖性摄取系统进行了表征。与空肠制备的囊泡相比，从回肠末端制备的囊泡中，[3H]牛磺胆酸盐在存在内向性钠梯度（[Na+]外>[Na+]内）时表现出过冲摄取。天然胆汁酸衍生物对钠依赖性[3H]牛磺胆酸盐摄取具有顺式抑制作用，而亲胆有机化合物，如鬼笔环肽、溴磺酞、胆红素、吲哚菁绿或二异丙基氟磷酸（DIDS）——所有这些都会干扰肝脏对胆汁酸的摄取——均未显示出显著的抑制作用。用牛磺胆酸盐的3,3-偶氮和7,7-偶氮衍生物对回肠膜囊泡进行光亲和标记，导致一条表观分子量为90 kDa的膜多肽发生特异性标记。抑制回肠囊泡对[3H]牛磺胆酸盐摄取的胆汁酸衍生物也抑制了90 kDa多肽的标记，而对回肠胆汁酸转运无抑制作用的化合物对90 kDa多肽的标记未显示出显著影响。通过用多种基团特异性试剂对回肠刷状缘膜囊泡进行化学修饰，研究了功能性氨基酸侧链在钠依赖性牛磺胆酸盐摄取中的作用。发现（邻位）巯基和氨基参与了回肠胆汁酸的主动摄取，而含羧基和羟基的氨基酸以及酪氨酸、组氨酸或精氨酸对于钠依赖性胆汁酸转运活性并非必需。DTNB或NBD-氯化物对[3H]牛磺胆酸盐转运的不可逆抑制可被2-巯基乙醇或二硫苏糖醇等巯基部分逆转。此外，在用NBD-氯化物进行化学修饰期间，增加牛磺胆酸盐的浓度能够保护回肠胆汁酸转运体不被灭活。这些发现表明，一条表观分子量为90000的膜多肽是兔小肠回肠中钠依赖性胆汁酸主动重吸收系统的一个组成部分。转运系统的邻位巯基和氨基参与钠依赖性转运活性，而其他功能性氨基酸对于转运活性并非必需。
