acid-phosphatase and Trypanosomiasis--African

A sensitive diagnostic assay for parasitic infections based on the detection of anti-enzyme antibodies is presented. All serum antibodies produced in response to parasite antigens are immobilized via their Fc domain on matrix-bound protein G. Incubation of the immobilized antibodies with saturating amounts of parasite extract results in the binding of all recognized antigens, including those directed against a specific and readily measurable enzyme. The amount of bound enzyme is proportional to the anti-enzyme antibody concentration in the serum. The application of this principle is demonstrated for the diagnosis of both human African trypanosomiasis and visceral leishmaniasis by the detection of antibodies against parasite acid phosphatases.

Topics: Acid Phosphatase; Animals; Antibodies, Protozoan; Humans; Leishmania mexicana; Leishmaniasis, Visceral; Trypanosoma brucei brucei; Trypanosomiasis, African

Bloodstream forms of the African trypanosome Trypanosoma rhodesiense were subjected to analytic cell fractionation procedures using isopycnic sucrose gradient centrifugation. The fractions obtained were analyzed using fused rocket immunoelectrophoresis, which revealed the most antigenic components to be associated with particles sedimenting between density increments of 1.10 to 1.15 and 1.20 and 1.22. From the distribution of various marker enzymes and the fluorogenic label fluorescamine, these particle populations were identified as derived from flagella pocket and surface membrane, respectively. Crossed immunoelectrophoresis of postulated flagella pocket and surface membrane fractions demonstrated at least seven and 10 distinct antigens, respectively, with only limited cross-reactivity. On the basis of crossed affini-immunoelectrophoresis four to five of each of these membrane antigens were glycoproteins, being precipitated in a concanavalin A-containing gel. Of the various detergent treatments used, it was found that optimum extraction of these glycoprotein antigens was achieved using the zwitterionic detergent Zwittergent 3-12 (0.1%) in the presence of 0.4% Triton X-100. Fused rocket immunoelectrophoresis also revealed a single prominent antigen associated with a particle population having an equilibrium density of approximately 1.180, which was identified as lysosomal from the distribution of marker enzymes. Other intracellular sites were not significantly antigenic.

Topics: Acid Phosphatase; Aminopeptidases; Animals; Antigens, Surface; Flagella; Immunoelectrophoresis, Two-Dimensional; Isoelectric Focusing; Leucyl Aminopeptidase; Rats; Subcellular Fractions; Trypanosoma; Trypanosomiasis, African

Bloodstream forms of Trypanosoma brucei have been screened for the presence of enzymes that could serve as markers for the plasma membrane, flagellar pocket, lysosomes, endoplasmic reticulum and Golgi apparatus in order to study the subcellular organization of the digestive system of the parasite. Acetylesterase, acid DNase, acid phosphatase, acid phosphodiesterase, acid proteinase, acid RNase, alanine aminotransferase, galactosyl transferase, alpha-glucosidase, inosine diphosphatase and alpha-mannosidase were partially characterized and their assays optimized for pH-dependent activity, linearity of reaction with respect to incubation time and enzyme concentration, and the effect of inhibitors and activators. The association of these enzymes with particulate material and the presence of structural latency were investigated. Acid proteinase and alpha-mannosidase are particle-bound and latent in cytoplasmic extracts; they can be activated and solubilized in part by Triton X-100. Similar results were obtained for acid phosphatase, acid phosphodiesterase and inosine diphosphatase. Neutral alpha-glucosidase, though partly sedimentable, does not show latency and is readily solubilized by the detergent. Galactosyl transferase is firmly membrane-bound even in the presence of 0.1% Triton X-100. Cell fractionation by differential centrifugation and density equilibration on sucrose gradients revealed that both alpha-mannosidase and acid proteinase are associated with organelles that band at a density of about 1.20 g/cm3. Inosine diphosphatase, galactosyl transferase, acid phosphatase and acid phosphodiesterase sediment predominantly as microsomal constituents equilibrating at densities between 1.13 and 1.15 g/cm3. In addition, inosine diphosphatase and galactosyl transferase exhibit considerable activity at higher densities (1.18-1.25 g/cm3). Neutral alpha-glucosidase is mainly recovered in the nuclear and microsomal fraction; its particulate part equilibrates as a single band at rho = 1.22 g/cm3. Acetylesterase and acid DNase are largely soluble, whereas acid RNase does not produce distinct sedimentation and banding profiles. In intact cells, neutral alpha-glucosidase and acid phosphatase appear to be highly accessible to their substrates. It is tentatively concluded that (a) acid proteinase and alpha-mannosidase are lysosomal enzymes, (b) acid phosphatase and acid phosphodiesterase are associated with the flagellar pocket and part of the former enzyme probably wit

Topics: Acid Phosphatase; Animals; Centrifugation, Density Gradient; Endopeptidases; Galactosyltransferases; Hydrogen-Ion Concentration; Hydrolases; Mannosidases; Peptide Hydrolases; Phosphoric Diester Hydrolases; Ribonucleases; Subcellular Fractions; Trypanosoma brucei brucei; Trypanosomiasis, African

Topics: Acid Phosphatase; Alkaline Phosphatase; Animals; Atrophy; Glomerulonephritis; Kidney; Kidney Glomerulus; Kidney Tubules, Proximal; Rabbits; Succinate Dehydrogenase; Trypanosoma brucei brucei; Trypanosomiasis, African

Topics: Acid Phosphatase; Animals; Cytoplasmic Granules; Electron Transport Complex IV; Endoplasmic Reticulum; Glucose-6-Phosphatase; Glycogen; Guinea Pigs; Histocytochemistry; Injections, Intraperitoneal; Kupffer Cells; Lipids; Liver; Lysosomes; Male; Microbodies; Microscopy, Electron; Mitochondria, Liver; Phagocytosis; Trypanosoma brucei gambiense; Trypanosomiasis, African

Topics: Acid Phosphatase; Africa; Africa, Eastern; Africa, Western; Histocytochemistry; Humans; Trypanosoma; Trypanosomiasis; Trypanosomiasis, African; Virulence