Acid and Neutral Hydrolases in <i>Trypanosoma cruzi</i>. Characterization and Assay*

Avila, JoséLuis; Casanova, M A; Ávila, Andrea; Bretan̄a, Antonio

doi:10.1111/j.1550-7408.1979.tb02786.x

Cited by 42 publications

(14 citation statements)

References 2 publications

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“…The acid proteinase described here rather resembles the thiol proteinase reported from another protozoan, Tritrichomonas foetus [43], which like the T. brucei enzyme (Steiger R. F., unpublished) also acts on casein at more neutral pH values. The specific activity of a-mannosidase of T. brucei is considerably lower than that of T. cruzi [14,42] and mouse macrophages [44] ; moreover, we were unable to detect pronounced stimulation by Mg2+ or Mn2+ as has been reported for the T. cruzi enzyme [42].…”

Section: Acid Proteinase Acid Rnase and A-mannosidasecontrasting

confidence: 40%

“…Preliminary studies by us revealed that it was not cathepsin-D-like [14], as evidenced by the absence of sensitivity towards pepstatin and by total inhibition in the presence of 1 mM p-chloromercuribenzoate. It is probable that the existence of 'cathepsin D' in Trypanosoma cruzi [42] has to be discarded on the same grounds. The acid proteinase described here rather resembles the thiol proteinase reported from another protozoan, Tritrichomonas foetus [43], which like the T. brucei enzyme (Steiger R. F., unpublished) also acts on casein at more neutral pH values.…”

Section: Acid Proteinase Acid Rnase and A-mannosidasementioning

confidence: 99%

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Subcellular Fractionation of Trypanosoma brucei Bloodstream Forms with Special Reference to Hydrolases

1980

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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, a-glucosidase, inosine diphosphatase and a-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 a-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 a-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 a-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 a-glucosidase is mainly recovered in the nuclear and microsomal fraction; its particulate part equilibrates as a single band at e = 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 a-glucosidase and acid phosphatase appear to be highly accessible to their substrates.It is tentatively concluded that (a) acid proteinase and a-mannosidase are lysosomal enzymes, (b) acid phosphatase and acid phosphodiesterase are associated with the flagellar pocket and part of the former enzyme probably with the endoplasmic reticulum, (c) galactosyl transferase is a constituent of the Golgi apparatus, and (d) a-glucosidase may serve as a marker for the plasma membrane. Inosine diphosphatase may also be derived from the latter structure.

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Section: Acid Proteinase Acid Rnase and A-mannosidasecontrasting

confidence: 40%

Section: Acid Proteinase Acid Rnase and A-mannosidasementioning

confidence: 99%

Subcellular Fractionation of Trypanosoma brucei Bloodstream Forms with Special Reference to Hydrolases

1980

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“…The failure to detect a-L-fucosidase in epimastigotes or even the exceedingly low activities reported for blood trypomastigotes by different groups (12,33) is, most likely, a reflection of the enzymes lability under the assay conditions used. Nevertheless, despite the inherent difficulties in measuring the enzyme activity, the presence of L-fucosidase on the plasma membrane of T. cruzi raises the provocative question of a possible role of the enzyme in glycoconjugate function.…”

Section: Discussionmentioning

confidence: 99%

“…Glycopeptides isolated from Gp-72 by affinity chromatography contain rhamnose, fucose, xylose and galactose (1:1:2:3) linked to the peptide by a less usual phosphodiester linkage (11). Interestingly, no a-L-fucosidase was detected in one of the few studies concerning the degradation of these glycoconjugates in T. cruzi, although the existence of galactosidases, glucosidases and mannosidases has been described (12,13).…”

Section: Introductionmentioning

confidence: 99%

Immunocytochemical and biochemical detection of alpha-L-fucosidase in Trypanosoma cruzi

Miletti

Almeida-de-Faria

Colli

et al. 2003

Braz J Med Biol Res

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The aim of the present study was to demonstrate the presence of a-Lfucosidase in Trypanosoma cruzi. Immunocytochemical and biochemical techniques were used to localize and characterize a membrane-associated, neutral-pH-optimum, a-L-fucosidase from the parasite. Light and electron microscopy localized the a-L-fucosidase specifically on the surface of the parasite and on membranes in the posterior region of the epimastigote stage. Although much less intense, labeling was also detected on the surface of trypomastigotes. At least 50% of the a-L-fucosidase activity was associated with epimastigote membrane solubilized with 1 M NaCl or 1% Triton X-100, suggesting that a-L-fucosidase is peripherally associated with membranes. The enzyme from epimastigotes had a neutral pH optimum (near 7) but displayed low specific activity when p-nitrophenyl-a-Lfucoside was employed as substrate (0.028 U/mg protein for epimastigotes and 0.015 U/mg protein for tissue culture-derived trypomastigotes). Polyacrylamide gel electrophoresis and Western blotting analysis both showed an expected 50-kDa polypeptide which was immunoreactive with anti-a-L-fucosidase antibodies.

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“…Since the identification of a number of proteolytic activities in cell-free extracts of epimastigotes [2,3], several enzymes have been purified from the parasite and characterized. They include cysteine proteinases (CPs), serine proteinases (SPs), metalloproteinases (MPs), and the proteasome.…”

Section: Introductionmentioning

confidence: 99%

Proteinases of Trypanosoma cruzi: Potential Targets for the Chemotherapy of Chagas Disease

Cazzulo

2005

MCRO

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Trypanosoma cruzi, the agent of the American Trypanosomiasis, Chagas Disease, contains cysteine, serine, threonine and metallo proteinases. Aspartic proteinases have not been found so far. The most abundant among these enzymes is cruzipain, a cysteine proteinase expressed as a complex mixture of isoforms by the major developmental stages of the parasite, including some membrane-bound isoforms. The enzyme is an immunodominant antigen in human chronic Chagas disease and seems to be important in the host/parasite relationship. Inhibitors of cruzipain kill the parasite and cure infected mice, thus making the enzyme a very promising target for the development of new drugs against Chagas disease. In addition, 30 kDa cathepsin B-like enzymes have been described. Serine peptidases described in the parasite include oligopeptidase B, a member of the prolyl oligopeptidase family involved in Ca 2+ -signaling during mammalian cell invasion; a prolyl endopeptidase (Tc80), against which inhibitors are being developed, and a serine carboxypeptidase, belonging to the S10 family. Metalloproteinases homologous to the gp63 of Leishmania spp. are also present. The proteasome has properties similar to those of other eukaryotes, and its inhibition by lactacystin, blocks some differentiation steps in the life cycle of the parasite.

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Acid and Neutral Hydrolases in Trypanosoma cruzi. Characterization and Assay*

Cited by 42 publications

References 2 publications

Subcellular Fractionation of Trypanosoma brucei Bloodstream Forms with Special Reference to Hydrolases

Subcellular Fractionation of Trypanosoma brucei Bloodstream Forms with Special Reference to Hydrolases

Immunocytochemical and biochemical detection of alpha-L-fucosidase in Trypanosoma cruzi

Proteinases of Trypanosoma cruzi: Potential Targets for the Chemotherapy of Chagas Disease

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