Purification and immunocytochemical localization of neuraminidase from Tritrichomonas foetus.
Parasitology
; 118 ( Pt 1): 17-25, 1999 Jan.
Article
em En
| MEDLINE
| ID: mdl-10070657
Lysis of Tritrichomonas foetus with a solution of the non-ionic detergent Triton X-114 at 0 degree C, followed by low-speed centrifugation, resulted in a detergent-insoluble pellet and a detergent-soluble supernatant. The supernatant was further fractionated by phase separation at 30 degrees C into a detergent-rich phase and an aqueous phase. Neuraminidase activity was mostly located in the detergent-insoluble pellet. When the parasites were incubated with bacterial phosphatidylinositol phospholipase C (PI-PLC) prior to detergent solubilization and phase separation neuraminidase activity was predominantly recovered in aqueous phase, rather than in the pellet and detergent phase. The molecular mass determined by gel permeation in high performance liquid chromatography (HPLC) and SDS-PAGE was 80,000 Da. Indirect immunofluorescence microscopy using polyclonal antibodies raised in rabbits against the purified neuraminidase, indicated that the enzyme is exposed on the cell surface. Previous treatment of the cells with PI-PLC significantly reduced antibody binding. Incubation of cryo-sections with the antibodies followed by detection using gold-labelled anti-rabbit IgG confirmed the presence of neuraminidase in the plasma membrane enclosing the cell body and flagella and in the membrane of vesicles preferentially located at the peripheral region of the protozoan.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Proteínas de Protozoários
/
Tritrichomonas foetus
/
Neuraminidase
Limite:
Animals
Idioma:
En
Revista:
Parasitology
Ano de publicação:
1999
Tipo de documento:
Article
País de afiliação:
Brasil
País de publicação:
Reino Unido