Properties and intracellular distribution of a cathepsin D-like proteinase active at the acid region of Musca domestica midgut

Abstract

Musca domestica larval midgut display in cells and luminal contents a proteolytic activity with a pH optimum of 3.0–3.5. This activity is abolished by pepstatin and is insensitive to soybean trypsin inhibitor and to sulfhydryl proteinase inhibitors. The acid proteinase occurs in multiple forms with M_r values in the range 40,000–80,000 and with pI values of about 5.5. The proteinase inactivates at 60°C according to apparent first-order kinetics and Lineweaver-Burk plots of its activity against albumin concentration are rectilinear, suggesting that the multiple forms have similar properties. The proteinase reacts slowly with diazoacetylnorleucine plus CuSO₄, is stable in alkaline media, is inhibited by dithiothreitol, hydrolyses hemoglobin better than albumin and is virtually not active upon synthetic substrates for pepsin. These properties are similar to those of cathepsin D. The specific activity of the acid proteinase determined by titration with pepstatin is 680 units/mg of proteinase and the K_D of the pepstatin-proteinase complex is 1.5 nM at 30°C. The acid proteinase occurs mainly in midgut subcellular fractions characterized by a high specific activity of molybdate-inhibited acid phosphatase and a large number of secretory-like vesicles. It is proposed that the M. domestica midgut acid proteinase is a cathepsin D-like proteinase evolved to function in luminal contents. The lack of ATP activation of the midgut enzyme supports this hypothesis, since ATP is thought to regulate cathepsin D-proteolysis inside lysosomes.