Reshkin S J, Lee S I, George J N, Turner R J
Clinical Investigations and Patient Care Branch, National Institute of Dental Research, Bethesda, Maryland 20892.
J Membr Biol. 1993 Nov;136(2):243-51. doi: 10.1007/BF02505766.
We demonstrate the presence of a 160 kD protein in rabbit parotid basolateral membranes that can be labeled with the irreversible sulfhydryl reagent [14C]-N-ethylmaleimide in a bumetanide-protectable fashion. The specificity of this labeling, and our previous evidence for the existence of an essential sulfhydryl group closely associated with the bumetanide-binding site on the parotid Na(+)-K(+)-Cl-cotransporter (J. Membrane Biol. 112:51-58, 1989), provide strong evidence that this protein is a part or all of the parotid bumetanide-binding site. When this protein is treated with endoglycosidase F/N-glycosidase F to remove N-linked oligosaccharides, its apparent molecular weight decreases to 135 kD. The pI of this deglycosylated protein is approximately 6.4. The bumetanide-binding protein was purified using two preparative electrophoresis steps. First, a Triton X-100 extract enriched in this protein was run on preparative electrophoresis to obtain fractions containing proteins in the 160 kD range. These were then deglycosylated with endoglycosidase F/N-glycosidase F and selected fractions were pooled and rerun on preparative electrophoresis to obtain a final 135 kD fraction. The enrichment of the bumetanide-binding protein in this final 135 kD fraction estimated from [14C]-N-ethylmaleimide labeling was approximately 48 times relative to the starting membrane extract. Since the bumetanide-binding site represents approximately 2% of the total protein in this starting extract, this enrichment indicates a high degree of purity of this protein in the 135 kD fraction.
我们证明在兔腮腺基底外侧膜中存在一种160 kD的蛋白质，该蛋白质能够以布美他尼可保护的方式被不可逆巯基试剂[14C]-N-乙基马来酰亚胺标记。这种标记的特异性，以及我们之前关于与腮腺钠-钾-氯共转运体上布美他尼结合位点紧密相关的必需巯基存在的证据（《膜生物学杂志》112:51 - 58，1989年），有力地证明了该蛋白质是腮腺布美他尼结合位点的一部分或全部。当用内切糖苷酶F/N-糖苷酶F处理该蛋白质以去除N-连接寡糖时，其表观分子量降至135 kD。这种去糖基化蛋白质的pI约为6.4。布美他尼结合蛋白通过两个制备性电泳步骤进行纯化。首先，将富含该蛋白质的Triton X-100提取物进行制备性电泳，以获得含有160 kD范围内蛋白质的组分。然后用内切糖苷酶F/N-糖苷酶F对这些组分进行去糖基化处理，将选定的组分合并并再次进行制备性电泳，以获得最终的135 kD组分。根据[14C]-N-乙基马来酰亚胺标记估计，在这个最终的135 kD组分中布美他尼结合蛋白的富集相对于起始膜提取物约为48倍。由于布美他尼结合位点约占该起始提取物中总蛋白质的2%，这种富集表明该135 kD组分中这种蛋白质具有高度纯度。
