PROTEINS OF THE KIDNEY MICROVILLAR MEMBRANE - THE AMPHIPATHIC FORMS OF ENDOPEPTIDASE PURIFIED FROM PIG KIDNEYS
PROTEINS OF THE KIDNEY MICROVILLAR MEMBRANE - THE AMPHIPATHIC FORMS OF ENDOPEPTIDASE PURIFIED FROM PIG KIDNEYS
复制标题
DOI：
10.1042/bj2110743
复制
发表时间：
1983-01-01
影响因子：
4.1
通讯作者：
KENNY, AJ
中科院分区：
文献类型：
--
作者：
FULCHER, IS;KENNY, AJ
本文报道了用免疫吸附层析法纯化洗涤剂溶解的肾微绒毛内肽酶（EC 3.4.24.11）。与肾皮质匀浆相比，产物（d型）纯化了270倍，产率为5%。它是免费的其他肽酶活动和电泳分析均匀。它含有约15%的碳水化合物和1个锌原子/亚基。两种胰蛋白酶处理的形式也进行了表征。一种（dt型）是通过处理d型得到的。另一种（TT型）是通过用甲苯和胰蛋白酶处理溶解膜的结果。所有3种形式的表观亚基Mr [分子比]值均为apprx。89，000，但d型似乎略大于其他2。通过凝胶过滤法估计的Mr显示，TT型为216，000，而其他型为320，000。估计的去污剂（Triton X-100）结合的d-和dt-的形式占这种差异。 通过几个标准，包括电荷位移交叉免疫电泳和疏水色谱，d-和dt-形式被证明是两亲分子。tt型的性质是亲水性的。还注意到离子性质的差异，这与在dt-形式的情况下带正电荷的肽的损失一致。天然内肽酶可以是二聚体分子，每个亚基通过靠近一个或另一个末端的多肽的相对小的区域锚定在膜中。当通过125 I-胰岛素B-链的水解测定时，d-和dt-形式具有相似的酶活性。螯合剂和磷酰胺抑制内肽酶。以戊二酰甘氨酰甘氨酰苯丙氨酸2-萘酰胺为底物，氨基肽酶N（EC 3.4.11.2）水解苯丙氨酸2-萘酰胺，用新的两步荧光法测定了动力学常数。Km为68 μ M，Vmax为484 nmol/min/（mg蛋白质）。
The purification of detergent-solubilized kidney microvillar endopeptidase (EC 3.4.24.11) by immunoadsorbent chromatography is described. The product (the d-form) was 270-fold purified compared with the homogenate of kidney cortex and was obtained in a yield of 5%. It was free of other peptidase activities and homogeneous by electrophoretic analyses. It contained about 15% carbohydrate and 1 Zn atom/subunit. Two trypsin-treated forms were also characterized. One (dt-form) was obtained by treatment of the d-form. The other (tt-form) was the result of solubilizing the membrane by treatment with toluene and trypsin. All 3 forms had apparent subunit Mr [molecular ratio] values of .apprx. 89,000, but the d-form appeared to be slightly larger than the other 2. Estimates of Mr by gel filtration showed that of the tt-form to be 216,000 whereas those of the other forms were 320,000. An estimate of the detergent (Triton X-100) bound to the d- and dt-forms accounted for this difference. By several criteria, including charge-shift crossed immunoelectrophoresis and hydrophobic chromatography, the d- and dt-forms were shown to be amphipathic molecules. The tt-form was hydrophilic in its properties. Differences in ionic properties were also noted, consistent with the loss, in the case of the dt-form, of a positively charged peptide. The native endopeptidase may be a dimeric molecule, each subunit being anchored in the membrane by a relatively small region of the polypeptide close to one or other terminus. The d- and dt-forms had similar enzyme activity when assayed by the hydrolysis of 125I-insulin B-chain. Chelating agents and phosphoramidon inhibited the endopeptidase. The kinetic constants were determined by a new 2-stage fluorometric assay using glutarylglycylglycylphenylalanine 2-naphthylamide as substrate and aminopeptidase N (EC 3.4.11.2) to hydrolyse phenylalanine 2-naphthylamide. The Km was 68 .mu.M and Vmax 484 nmol/min per (mg of protein).