Proteins of the kidney microvillar membrane. Aspartate aminopeptidase: purification by immunoadsorbent chromatography and properties of the detergent- and proteinase-solubilized forms

作者：E. Michael Danielsen, Ove Norén, Hans Sjöström, Jean C. Ingram, A J Kenny · 发表于：Biochemical Journal · 年份：1980 · DOI：10.1042/bj1890591 · 被引用次数：82 · 研究领域：Peptidase Inhibition and Analysis、Neuropeptides and Animal Physiology、Pancreatic function and diabetes

Aminopeptidase A (aspartate aminopeptidase, EC 3.4.11.7) was purified 2000-fold from pig kidney cortex. The essential step in the purification was chromatography on an immunoadsorbent column prepared from a rabbit antiserum raised against pig intestinal aminopeptidase A. Glutamyl and aspartyl substrate were attacked most rapidly and their hydrolyses were stimulated by Ca2+. The 2-naphthylamide derivatives of neutral and basic amino acids were also hydrolysed by aminopeptidase A, but at rates about two orders of magnitude lower, and Ca2+ was inhibitory. The possibility that these atypical substrates were hydrolysed by traces of aminopeptidase M (EC 3.4.11.2) contaminating the preparation could be excluded on several grounds. Aminopeptidase A was sensitive to inhibition by chelating agents and the inactive enzyme could be reactivated by Ca2+ or Mn2+. Atomic absorption spectrophotometry revealed 1 g-atom of Ca/143000 g of protein. Two forms of the enzyme were purified: an amphipathic form solubilized from the membrane by Triton X-100 (detergent form) and a hydrophilic form released by incubation with trypsin (proteinase form). The detergent form exhibited charge-shift in crossed immunoelectrophoresis when anionic or cationic detergents were present. On gel filtration, mol.wts. of 350000--400000 and 270000 were calculated for the detergent and proteinase forms. Electron microscopy after negative staining of the proteinase form revealed a dimeric structure. Electrophoresis of eith...