PROPERTIES OF THE AMINOPEPTIDASE-N FROM THE SILKWORM MIDGUT (BOMBYX-MORI)

The properties of aminopeptidase N in Bombyx mori larval midgut are de scribed, The brush border membrane (BBM) aminopeptidase N was isolated from Triton-solubilized BBM by column chromatography at a specific ac tivity of 61.3 U/mg, The enzyme showed optimum activity with leucine-p -nitroanilide or alanine-p-nitroanilide as substrates and was inhibite d by aminopeptidase inhibitors and leucine analogues in the following order of potency: leucinethiol (reduced form)>bestatin>Zn++>>alanine>l eucine, histidine, alpha-methylleucine, Bestatin and leucinethiol were competitive inhibitors of the enzyme, whereas Zn++ was a non-competit ive inhibitor and its effect was reversed by the addition of ethylene diamine tetraacetic acid (EDTA), Phosphatidylinositol-specific phospho lipase C but not papain reduced aminopeptidase activity in BBM vesicle s to 55%, These treatments did not affect amino acid transport activit y, Some kinetic properties of aminopeptidase N and the K+/neutral amin o acid symporter are compared, Leucine uptake into BBM vesicles was in hibited according to the following rank: alpha-methylleucine, leucine> histidine>bestatin>alanine>leucinthiol. The spectrum of amino acid ana logue inhibition was very different between the two systems, This was demonstrated better by alpha-methylleucine, which was 370 times more p otent on leucine transport, and by leucinthiol, whose K-i was 2250 tim es higher on the symporter than that measured on aminopeptidase N, Mor eover, the symporter, but not aminopeptidase N, was readily able to re cognize histidine and its analogues, These data support the conclusion that in the silkworm midgut the specificities of recognition of amino acids by aminopeptidase N and the transporter are different, The impl ications of these results are discussed. (C) 1997 Elsevier Science Ltd .