IDENTIFICATION OF A 44 KDA PROTEIN LOCALIZED WITHIN THE ENDOPLASMIC-RETICULUM OF TRYPANOSOMA-BRUCEI-BRUCEI

Immunoaffinity chromatography and gel electrophoresis were used to iso late a 44 kDa protein that was bound to a 72 kDa chaperone in Trypanos oma brucei brucei. A polyclonal antiserum to the 44 kDa protein was ra ised in rats and employed in conjunction with chromatography using DEA E-cellulose, Sephacryl S-300, and hydroxyapatite to purify the protein from membranes of bloodstream forms of the trypanosomes. Immunoblot a nalysis using this antiserum revealed a protein doublet of 44/45 kDa i n T. b. brucei and a single protein band of 53 kDa in almost equivalen t amounts throughout the life-cycle stages of T. congolense. Indirect immunofluorescence using affinity-purified antibodies specific for the 44 kDa protein showed labelling of the perinuclear area and reticular system extending throughout the parasites, suggesting that this prote in was located in the endoplasmic reticulum. Localization of the 44 kD a molecule in the endoplasmic reticulum was confirmed by immunoelectro n microscopy. Protease protection experiments demonstrated that the ep itopes bound by antibody were buried within the membrane or towards th e lumenal face of the endoplasmic reticulum. Ruthenium Red overlay of nitrocellulose blots containing the 44/45 kDa doublet suggested that t he molecules have the potential to bind calcium. The N-terminal amino acid sequence of the 44 kDa protein showed no sequence similarity to a ny proteins in the database.