A BACTERIA-DIGESTING MIDGUT-LYSOZYME FROM MUSCA-DOMESTICA (DIPTERA) LARVAE - PURIFICATION, PROPERTIES AND SECRETORY MECHANISM

Two lysozymes were purified to homogeneity from heated acid extracts o f Musca domestica larval midguts, using an S-Sepharose column, and a s emi-preparative polyacrylamide gel electrophoresis (PAGE). The final y ield was 60%. Lysozymes 1 and 2 display M(r) 22,000, determined by ult racentrifugation or electrophoresis in non-denaturing conditions, and M(r) 17,000, determined by SDS-polyacrylamide gradient gel electrophor esis. Isoelectric focusing showed the following pI values: lysozyme 1, 7.9; lysozyme 2, 8.2. Lysozyme 1 and 2 display identical kinetic prop erties, which include decrease in activity, displacement of the pH opt imum toward acidic values and a K(m) increase as the ionic strength of the medium becomes higher. The lysozymes are resistant to a cathepsin D-like proteinase present in M. domestica midgut, and display a chiti nase activity which is 6-fold higher than that of chicken lysozyme. Ly sozyme immunolabeling revealed that lysozyme mainly occurs in secretor y vesicles and at the outside surface of microvilli from M. domestica anterior midgut cells. The results showed that M. domestica lysozymes are similar to ruminant stomach lysozyme in being more active at acid pH values, when present in media with physiological ionic strengths, a nd in being resistant to an acid proteinase derived from the same anim al as the lysozyme. Furthermore, the data support the assertion that M . domestica midgut lysozyme is secreted by exocytosis, partly remainin g adsorbed to the cell glycocalyx.