Kleinzeller A, McAvoy E M
Biochim Biophys Acta. 1976 Nov 11;455(1):109-25. doi: 10.1016/0005-2736(76)90157-7.
An improved analytical procedure for the extraction and determination of total, free and phosphorylated tissue sugar is described. This method, employing ZnSO4 plus Ba(OH)2 for the precipitation of sugar phosphates, yields values identical with those obtained by the more laborious separation of free and phosphorylated sugar by ion-exchange chromatography. Erroneous values for free sugar due to the action of a Zn2+ -activated phosphatase and/or the lability to acids of some sugar phosphates, are avoided. Using this technique for the sudy of transport and phosphorylation of D-galactose in rabbit renal cortical slices and tissue extracts, it was found: 1. The cellular uptake of D-galactose was associated with the appearance of both free and phosphorylated sugar whether or not external Na+ was present. At 1 mM sugar, galactose was accumulated in the cells against a modest concentration gradient of 1.445 +/- 0.097 (n = 17). Galactose phosphate appeared in the cells considerably faster than free sugar under conditions of net uptake as well as of steady-state exchange (pulse-labelling). 2. Increasing saline pH (6-8) increased the cellular levels of sugar phosphate without affecting the steady-state values of free sugar. With tissue extracts, increasing pH also stimulated the activity of galactokinase and the dephosphorylation of galactose 1-phosphate by a Zn2+ -activated phosphatase. 3. 0.5 mM phlorizin inhibited the tissue uptake of galactose and its subsequent oxidation to CO2 only to a minor degree (30 and 10%, respectively). The absence of external Na+ further depressed the phlorizin effect. Preincubation of the tissue with phlorizin and subsequent washing in part abolished the inhibitory effect. The data suggest that a major portion of the galactose uptake by the tissue proceeds by a mechanism with a low affinity for phlorizin. 4. Efflux studies showed that the wash-out of free galactose from slices was associated with a net decrease of both free and phosphorylated tissue sugar. 5. The above results suggest the possibility that phosphorylation may represent a step in the Na+ -independent, phloretin-sensitive transfer of D-galactose across the antiluminal cell membrane. The participation of intracellular galactokinase and a Zn2+ -activated alkaline phosphatase in the maintenance of the steady state of free and phosphorylated galactose in the cells has been demonstrated.
本文描述了一种改进的分析方法，用于提取和测定组织中的总糖、游离糖和磷酸化糖。该方法采用硫酸锌加氢氧化钡沉淀糖磷酸盐，所得结果与通过离子交换色谱法更繁琐地分离游离糖和磷酸化糖所获得的结果相同。避免了由于锌离子激活的磷酸酶的作用和/或某些糖磷酸盐对酸的不稳定性而导致的游离糖错误值。使用该技术研究兔肾皮质切片和组织提取物中D-半乳糖的转运和磷酸化，发现：1. 无论外部钠离子是否存在，D-半乳糖的细胞摄取都与游离糖和磷酸化糖的出现有关。在1 mM糖浓度下，半乳糖在细胞中逆着1.445±0.097（n = 17）的适度浓度梯度积累。在净摄取以及稳态交换（脉冲标记）条件下，磷酸半乳糖出现在细胞中的速度比游离糖快得多。2. 增加盐水pH值（6-8）会增加细胞中糖磷酸盐的水平，而不会影响游离糖的稳态值。对于组织提取物，增加pH值也会刺激半乳糖激酶的活性以及锌离子激活的磷酸酶对半乳糖1-磷酸的去磷酸化作用。3. 0.5 mM根皮苷仅在较小程度上抑制组织对半乳糖的摄取及其随后氧化为二氧化碳（分别为30%和10%）。外部钠离子的缺失进一步降低了根皮苷的作用。用根皮苷预孵育组织并随后洗涤部分消除了抑制作用。数据表明，组织对半乳糖的摄取主要通过对根皮苷亲和力低的机制进行。4. 流出研究表明，切片中游离半乳糖的洗脱与组织中游离糖和磷酸化糖的净减少有关。5. 上述结果表明，磷酸化可能代表D-半乳糖跨反腔细胞膜进行的不依赖钠离子、对根皮素敏感的转运过程中的一个步骤。已经证明细胞内半乳糖激酶和锌离子激活的碱性磷酸酶参与维持细胞中游离和磷酸化半乳糖的稳态。
