Properties of the aminopepidase N from the silkworm midgut (Bombyx mori)
1997
Parenti, P. | Morandi, P. | McGivan, J.D. | Consonnic, P. | Leonardi, G. | Giordana, B.
The properties of aminopeptidase N in Bombyx mori larval midgut are described. The brush border membrane (BBM) aminopeptidase N was isolated from Triton-solubilized BBM by column chromatography at a specific activity of 61.3 U/mg. The enzyme showed optimum activity with leucine-p-nitroanilide or alanine-p-nitroanilide as substrates and was inhibited by aminopeptidase inhibitors and leucine analogues in the following order of potency: leucinethiol (reduced form)>bestatin>Zn++>>alanine>leucine, histidine, alpha-methylleucine. Bestatin and leucinethiol were competitive inhibitors of the enzyme, whereas Zn++ was a non-competitive inhibitor and its effect was reversed by the addition of ethylene diamine tetraacetic acid (EDTA). Phosphatidylinositol-specific phospholipase C but not papain reduced aminopeptidase activity in BBM vesicles to 55%. These treatments did not affect amino acid transport activity. Some kinetic properties of aminopeptidase N and the K+/neutral amino acid symporter are compared. Leucine uptake into BBM vesicles was inhibited according to the following rank: alpha-methylleucine, leucine>histidine>bestatin>alanine>leucinthiol. The spectrum of amino acid analogue inhibition was very different between the two systems. This was demonstrated better by alpha-methylleucine, which was 370 times more potent on leucine transport, and by leucinthiol, whose Ki was 2250 times higher on the symporter than that measured on aminopeptidase N. Moreover, the symporter, but not aminopeptidase N, was readily able to recognize 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 implications of these results are discussed.
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