Alonso-Torre S R, Serrano M A, Medina J M, Alvarado F
Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Salamanca, Spain.
Biochem J. 1992 Nov 15;288 ( Pt 1)(Pt 1):47-53. doi: 10.1042/bj2880047.
The placental uptake of L-alanine was studied by using purified brush-border membrane vesicles from rat trophoblasts. Saturation curves were carried out at 37 degrees C in buffers containing 100 mM (zero-trans)-NaSCN, -NaCl, -KSCN, -KCl, or -N-methyl-D-glucamine gluconate. The uncorrected uptake results were fitted by non-linear regression analysis to an equation involving one diffusional component either one or two saturable Michaelian transport terms. In the presence of NaCl, two distinct L-alanine transport systems were distinguished, named respectively System 1 (S-1; Vm1 about 760 pmol/s per mg of protein; KT1 = 0.5 mM) and System 2 (S-2; Vm2 about 1700 pmol/s per mg; KT2 = 9 mM). In contrast, in the presence of K+ (KCl = KSCN) or in the absence of any alkali-metal ions (N-methyl-D-glucamine gluconate), only one saturable system was apparent, which we identify as S-2. When Na+ is present, S-1, but not S-2, appears to be rheogenic, since its maximal transport capacity significantly increases in the presence of an inside-negative membrane potential, created either by replacing Cl- with the permeant anion thiocyanate (NaSCN > NaCl) or by applying an appropriate K+ gradient and valinomycin. alpha-(Methylamino)isobutyrate (methyl-AIB) appears to be a substrate of S-1, but not of S-2. For reasons that remain to be explained, however, methyl-AIB inhibits S-2. We conclude that S-1 represents a truly Na(+)-dependent mechanism, where Na+ behaves as an obligatory activator, whereas S-2 cannot discriminate between Na+ and K+, although its activity is higher in the presence of alkali-metal ions than in their absence (Na+ = K+ > N-methyl-D-glucammonium ion). S-2 appears to be fully developed 2 days before birth, whereas S-1 undergoes a capacity-type activation between days 19.5 and 21.5 of gestation, i.e. its apparent Vmax. nearly doubles, whereas its KT remains constant.
利用从大鼠滋养层细胞纯化得到的刷状缘膜囊泡研究了L-丙氨酸的胎盘摄取情况。在含有100 mM（零转运）硫氰酸钠、氯化钠、硫氰酸钾、氯化钾或葡糖酸-N-甲基-D-葡糖胺的缓冲液中于37℃进行饱和曲线实验。未校正的摄取结果通过非线性回归分析拟合到一个包含一个扩散成分以及一个或两个可饱和米氏转运项的方程。在氯化钠存在的情况下，区分出了两个不同的L-丙氨酸转运系统，分别命名为系统1（S-1；每毫克蛋白质的Vm1约为760 pmol/s；KT1 = 0.5 mM）和系统2（S-2；每毫克的Vm2约为1700 pmol/s；KT2 = 9 mM）。相比之下，在钾离子存在的情况下（氯化钾 = 硫氰酸钾）或在不存在任何碱金属离子的情况下（葡糖酸-N-甲基-D-葡糖胺），仅出现一个可饱和系统，我们将其鉴定为S-2。当钠离子存在时，S-1似乎是生电的，因为在存在内向负膜电位时其最大转运能力显著增加，内向负膜电位可通过用渗透性阴离子硫氰酸盐替代氯离子（硫氰酸钠 > 氯化钠）或通过施加适当的钾离子梯度和缬氨霉素来产生。α-（甲基氨基）异丁酸（甲基-AIB）似乎是S-1的底物，而不是S-2的底物。然而，由于尚待解释的原因，甲基-AIB会抑制S-2。我们得出结论，S-1代表一种真正依赖钠离子的机制，其中钠离子作为必需的激活剂，而S-2无法区分钠离子和钾离子，尽管其在碱金属离子存在时的活性高于不存在时（钠离子 = 钾离子 > N-甲基-D-葡糖铵离子）。S-2在出生前2天似乎已完全发育，而S-1在妊娠第19.5天至21.5天之间经历容量型激活，即其表观Vmax几乎翻倍，而其KT保持不变。
