Lui A, Lumeng L, Li T K
J Biol Chem. 1982 Dec 25;257(24):14903-6.
The transport of [14C]pyridoxal-P and [14C]pyridoxine into isolated rat mitochondria was studied by centrifugal filtration. The incubation medium contained 20 mM 2-oxoglutarate and 10 mM inorganic phosphate to inhibit metabolism of pyridoxal-P by the mitochondria. The ratio of [14C]pyridoxine space to [3H]H2O space rapidly attained unity independent of the [14C]pyridoxine concentration in the medium and remained unchanged for up to 90 min of incubation. These data suggest simple passive diffusion for the transport of pyridoxine into the mitochondria. By contrast, the ratio of [14C]pyridoxal-P space to [3H]H2O space rose rapidly to exceed 1 in the first 15 min and continued to rise at a slower rate for as long as it was measured. The accumulation of [14C]pyridoxal-P was not decreased by inhibitors and uncouplers of oxidative phosphorylation. Fractionation of the mitochondria with digitonin revealed that 19 and 340 pmol of [14C]pyridoxal-P/mg of protein were taken up by the mitochondria at 15 and 60 min of incubation, respectively. Most of the uptake in the first 15 min occurred in the intermembrane space, whereas the largest increase of [14C]pyridoxal-P between 15 and 60 min of incubation appeared in the matrix fraction. Significant binding of the [14C]pyridoxal-P to proteins in the two compartments was demonstrated by gel filtration. These data indicate that pyridoxal-P can rapidly enter the intermembrane space of isolated mitochondria, but its penetration into the matrix occurs at a slower and more sustained rate (i.e. 9-16 pmol/h/mg of protein). It is concluded that the transport of pyridoxal-P into isolated rat liver mitochondria is energy-independent and is most consistent with passive diffusion facilitated by protein binding once this coenzyme enters the different compartments of the mitochondria.
通过离心过滤研究了[14C]磷酸吡哆醛和[14C]吡哆醇进入分离的大鼠线粒体的转运情况。孵育培养基含有20 mM 2-氧代戊二酸和10 mM无机磷酸盐，以抑制线粒体对磷酸吡哆醛的代谢。[14C]吡哆醇空间与[3H]H2O空间的比值迅速达到1，与培养基中[14C]吡哆醇的浓度无关，并且在长达90分钟的孵育过程中保持不变。这些数据表明吡哆醇进入线粒体的转运是简单的被动扩散。相比之下，[14C]磷酸吡哆醛空间与[3H]H2O空间的比值在最初15分钟内迅速上升超过1，并在测量期间持续以较慢的速率上升。氧化磷酸化的抑制剂和解偶联剂不会降低[14C]磷酸吡哆醛的积累。用洋地黄皂苷对线粒体进行分级分离显示，在孵育15分钟和60分钟时，线粒体分别摄取了19和340 pmol的[14C]磷酸吡哆醛/毫克蛋白质。最初15分钟内的大部分摄取发生在内膜间隙，而在孵育15至60分钟之间，[14C]磷酸吡哆醛的最大增加出现在基质部分。凝胶过滤证明了[14C]磷酸吡哆醛在这两个部分与蛋白质有显著结合。这些数据表明，磷酸吡哆醛可以迅速进入分离的线粒体的内膜间隙，但其进入基质的过程较慢且持续时间更长（即9 - 16 pmol/小时/毫克蛋白质）。结论是，磷酸吡哆醛进入分离的大鼠肝脏线粒体的转运不依赖能量，并且最符合这种辅酶一旦进入线粒体的不同部分后通过蛋白质结合促进的被动扩散。
