Ganapathy V, Burckhardt G, Leibach F H
J Biol Chem. 1984 Jul 25;259(14):8954-9.
Glycylsarcosine was found to be very resistant to hydrolysis by brush-border membrane vesicles from rabbit intestine. The dipeptide was transported intact into an osmotically responsive intravesicular space. The initial uptake rate of glycylsarcosine into these vesicles was greater in mannitol medium compared to that in the presence of an inward gradient of either Na+ or other monovalent cations. When vesicles preloaded with glycylsarcosine were incubated in a peptide-free medium, there was a rapid efflux of the dipeptide and the t1/2 for the process was less than 2 min. An inside-negative K+ diffusion potential generated by valinomycin stimulated glycylsarcosine uptake even in the absence of Na+. Experiments with the potential-sensitive dye DiS-C3 (5) showed that glycylsarcosine depolarized the brush-border membrane in the presence and absence of Na+. Imposition of an inward proton gradient stimulated the initial uptake rates of glycylsarcosine while the equilibrium uptake was not affected. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone decreased this proton gradient-induced stimulation. An inward proton gradient increased the Vmax of the transport system (10.8 +/- 0.8 nmol/min/mg of protein when [pH]o = [pH]i = 5.5; and 20.8 +/- 2.2 nmol/min/mg of protein when [pH]o = 5.5 and [pH]i = 7.8), without significantly affecting the apparent Kt (17.3 +/- 1.4 mM versus 19.5 +/- 2.0 mM). Glycyl-L-proline uptake was inhibited by glycylsarcosine and KI for the process was 20.8 +/- 3.0 mM. A relatively lower KI (2.8 +/- 1.2 mM) was obtained for the inhibition of glycylsarcosine uptake by glycyl-L-proline. The uptake of glycyl-L-proline and glycylsarcosine was strongly inhibited by L-carnosine, glycyl-L-leucine, and L-prolylglycine. With each inhibitory peptide, the KI values for the inhibition of glycyl-L-proline uptake and of glycylsarcosine uptake were comparable. Preloading the vesicles with unlabeled glycylsarcosine stimulated the uptake of labeled glycyl-L-proline. These data suggest that in rabbit intestinal brush-border membrane vesicles (i) glycylsarcosine and proton(s) are co-transported, (ii) this process results in a net transport of positive charge across the membrane and, (iii) a single transport system is involved in the translocation of glycyl-L-proline and glycylsarcosine.
发现甘氨酰肌氨酸对兔小肠刷状缘膜囊泡的水解作用具有很强的抗性。该二肽完整地转运到对渗透压有反应的囊泡内空间。与存在Na⁺或其他单价阳离子内向梯度时相比，在甘露醇培养基中甘氨酰肌氨酸进入这些囊泡的初始摄取速率更高。当预先装载有甘氨酰肌氨酸的囊泡在无肽培养基中孵育时，二肽会迅速流出，该过程的半衰期小于2分钟。缬氨霉素产生的内向负K⁺扩散电位即使在没有Na⁺的情况下也能刺激甘氨酰肌氨酸的摄取。使用电位敏感染料DiS-C3(5)进行的实验表明，无论有无Na⁺，甘氨酰肌氨酸都会使刷状缘膜去极化。内向质子梯度的施加刺激了甘氨酰肌氨酸的初始摄取速率，而平衡摄取不受影响。羰基氰化物对三氟甲氧基苯腙降低了这种质子梯度诱导的刺激作用。内向质子梯度增加了转运系统的Vmax（当[pH]o = [pH]i = 5.5时为10.8±0.8 nmol/min/mg蛋白质；当[pH]o = 5.5且[pH]i = 7.8时为20.8±2.2 nmol/min/mg蛋白质），而对表观Kt没有显著影响（分别为17.3±1.4 mM和19.5±2.0 mM）。甘氨酰-L-脯氨酸的摄取受到甘氨酰肌氨酸和KI的抑制，其抑制常数为20.8±3.0 mM。甘氨酰-L-脯氨酸对甘氨酰肌氨酸摄取的抑制作用获得的相对较低的KI为2.8±1.2 mM。甘氨酰-L-脯氨酸和甘氨酰肌氨酸的摄取受到L-肌肽、甘氨酰-L-亮氨酸和L-脯氨酰甘氨酸的强烈抑制。对于每种抑制性肽，抑制甘氨酰-L-脯氨酸摄取和甘氨酰肌氨酸摄取的KI值相当。用未标记的甘氨酰肌氨酸预先装载囊泡刺激了标记的甘氨酰-L-脯氨酸的摄取。这些数据表明，在兔小肠刷状缘膜囊泡中：(i) 甘氨酰肌氨酸和质子是共转运的；(ii) 该过程导致正电荷跨膜的净转运；以及(iii) 单个转运系统参与甘氨酰-L-脯氨酸和甘氨酰肌氨酸的转运。
