Purification and immunocytochemical localization of neuraminidase from Trichomonas foetus

Lysis of Tritrichomonas foetus with a solution of the non-ionic detergent T riton X-114 at 0 degrees C, followed by low-speed centrifugation, resulted in a detergent-insoluble pellet and a detergent-soluble supernatant. The su pernatant was further fractionated by phase separation at 30 degrees C into a detergent-rich phase and an aqueous phase. Neuraminidase activity was mo stly located in the detergent-insoluble pellet. When the parasites were inc ubated with bacterial phosphatidylinositol phospholipase C (PI-PLC) prior t o detergent solubilization and phase separation neuraminidase activity was predominantly recovered in aqueous phase, rather than in the pellet and det ergent phase. The molecular mass determined by gel permeation in high perfo rmance liquid chromatograph! (HPLC) and SDS-PAGE was 80000 Da. Indirect imm unofluorescence microscopy using polyclonal antibodies raised in rabbits ag ainst the purified neuraminidase, indicated that the enzyme is exposed on t he cell surface. Previous treatment of the cells with PI-PLC significantly reduced antibody binding. Incubation of cryo-sections with the antibodies f ollowed by detection using gold-labelled anti-rabbit IgG confirmed the pres ence of neuraminidase in the plasma membrane enclosing the cell body and fl agella and in the membrane of vesicles preferentially located at the periph eral region of the protozoan.