Proteins of the kidney microvillar membrane. Biosynthesis of endopeptidase-24.11, dipeptidylpeptidase IV and aminopeptidases N and A in pig kidney slices.
Proteins of the kidney microvillar membrane. Biosynthesis of endopeptidase-24.11, dipeptidylpeptidase IV and aminopeptidases N and A in pig kidney slices.
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肾微绒毛膜的蛋白质。
DOI：
10.1042/bj2240549
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发表时间：
1984
期刊：
影响因子：
--
通讯作者：
A. Kenny
中科院分区：
文献类型：
--
作者：
J. R. Stewart;A. Kenny
使用来自尤卡坦品系仔猪的肾皮质组织切片的器官培养物来研究四种微绒毛肽酶的生物发生：内肽酶-24.11（EC 3.4.24.11）、二肽基肽酶IV（EC 3.4.14.5）、氨肽酶N（EC 3.4.11.2）和氨肽酶A（EC 3.4.11.7）。通过保存超微结构的完整性和在实验期间细胞对[3 H]丙氨酸的摄取不变，证实了培养系统的活力。用[35 S]甲硫氨酸标记后，用Mg 2+处理产生两个部分，一个含有微绒毛，另一个含有细胞内膜和基底外侧膜的Mg 2+颗粒。标记形式的肽酶，通过免疫沉淀分离，分析了十二烷基硫酸钠/聚丙烯酰胺凝胶电泳和荧光。Mg 2+沉淀物含有最早可检测形式的酶。在每种情况下，比成熟形式更低的Mr并且对用内切-β-N-乙酰氨基葡糖苷酶H处理敏感的多肽是待检测的第一种形式。这些高甘露糖形式之后，约30分钟后的脉冲，由一个复杂的糖基化形式的高先生。只有后者的形式被观察到在微绒毛，然后只有在90分钟后的追逐期。二肽基肽酶IV的定量研究表明，在Mg 2+颗粒中观察到的形式是那些在微绒毛部分的前体。在细胞质中未观察到标记形式。因此，所有四种肽酶在膜隔室内合成，并在微绒毛中组装之前分两步糖基化。
An organ culture employing slices of renal-cortex tissue from piglets of the Yucatan strain was used to study the biogenesis of four microvillar peptidases: endopeptidase-24.11 (EC 3.4.24.11), dipeptidyl peptidase IV (EC 3.4.14.5), aminopeptidase N (EC 3.4.11.2) and aminopeptidase A (EC 3.4.11.7). The viability of the culture system was confirmed by the preservation of ultrastructural integrity and by an unchanged uptake of [3H]alanine into cells during the period of the experiments. After labelling with [35S]methionine, treatment with Mg2+ yielded two fractions, one containing microvilli and another, the Mg2+ pellet, containing intracellular and basolateral membranes. The labelled forms of the peptidases, isolated by immunoprecipitation, were analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and fluorography. The Mg2+ pellet contained the earliest detectable forms of the enzymes. In each case, a polypeptide of lower Mr than the mature form and sensitive to treatment with endo-beta-N-acetylglucosaminidase H was the first form to be detected. These high-mannose forms were followed, about 30 min after the pulse, by a complex glycosylated form of higher Mr. Only the latter form was observed in microvilli and then only after 90 min of the chase period. A quantitative study of dipeptidyl peptidase IV showed that the forms observed in the Mg2+ pellet were precursors of those in the microvillar fraction. No labelled forms were observed in the cytosol. All four peptidases were thus synthesized within membrane compartments and glycosylated in two steps before assembly in microvilli.