Electrophoretic detection of Trypanosoma cruzi peptides

Greig, Stephen; Ashall, Frank

doi:10.1016/0166-6851(90)90005-7

Cited by 44 publications

(23 citation statements)

References 18 publications

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“…Using haemoglobin (substrate)-SDS-PAGE these intracellular proteases are detected as 5 enzymatic bands: 4 of them are cysteine proteases and the other is a metalloprotease (Zuo & Woo 1997a, present study). This protease zymogram is similar to that in Trypanosoma cruzi epimastigotes (Greig & Ashall 1990). The pathogenic C. salrnositica has lost its virulence (e.g.…”

Section: Discussionmentioning

confidence: 49%

“…Trypanosorna cruzi, a human pathogen in Central and South America, contains a metallo-protease which is mainly located in the cell membrane (Greig & Ashall 1990, Bonaldo et al 1991. T cruzi trypomastigotes penetrate host cells and a surface or secreted metalloprotease is presumed to be involved in penetration of cells (Piras et al 1985, Greig & Ashall 1990, Bonaldo et al 1991. The metallo-protease in trophozoite of Entarnoeba histolytica, a human pathogen, readily degrades host collagens, suggesting that it also plays an important role in the invasion of host tissue (Munoz et al 1982(Munoz et al , 1990.…”

Section: Discussionmentioning

confidence: 99%

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Purified metallo-protease from the pathogenic haemoflagellate Cryptobia salmositica and its in vitro proteolytic activities

Zuo¹,

Woo²

1997

Dis. Aquat. Org.

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The metallo-protease (200 kDa) from Cryptobia salmositica was punfied using ionexchange chromatography and gelfiltration. The purity of the enzyme was confirmed as there was only one homogeneous band using SDS-PAGE. Under in vitro conditions the purified metallo-protease completely degraded the extracellular matrix proteins (collagen types I. [V. V and laminin) and the membrane proteins isolated from rainbow trout erythrocytes. The results confirm that the C. salmositica metallo-protease is a histolytic enzyme and it contributes to salmonid cryptobiosis and to the direct transmission of the parasite between fish.

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Section: Discussionmentioning

confidence: 49%

Section: Discussionmentioning

confidence: 99%

Purified metallo-protease from the pathogenic haemoflagellate Cryptobia salmositica and its in vitro proteolytic activities

Zuo¹,

Woo²

1997

Dis. Aquat. Org.

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“…Parasite extracts were obtained by solubilising 7: cruzi in 0.75% (by vol.) Nonidet P-40 at approximately lo9 cells/ml (Greig and Ashall, 1990). 25-pg-protein aliquots (non-denatured) were fractionated through a 10% (masshol.)…”

Section: Isolation Of Cosmid Clonesmentioning

confidence: 99%

Overexpression of Cruzipain, the Major Cysteine Proteinase of Trypanosoma cruzi, is Associated with Enhanced Metacyclogenesis

Tomás

Miles

Kelly

1997

European Journal of Biochemistry

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Cruzipain, the major cysteine proteinase of Trypanosoma cruzi has been proposed as a target for chemotherapy against Chagas' disease. To investigate the role of cruzipain we transfected 7: cruzi epimastigotes with a recombinant cosmid containing approximately 20 tandenily repeated cruzipain genes. Transformed cells had multiple episomal copies of the vector and exhibited considerable overexpression of cruzipain activity. The upregulation was maintained throughout the parasite life-cycle, and electrophoretic detection techniques indicated that overexpression was correlated with correctly processed enzyme. Immunoelectron microscopy demonstrated that cruzipain had the same developmentally regulated subcellular localisation in transformed and non-transformed cells. In the insect epimastigote form, the enzyme was restricted to vesicles of the endosomal/lysosomal system, whereas in the intracellular forms it was also readily detectable on the cell surface. Phenotypic analysis of the transformed parasites showed that they had an enhanced ability to undergo metacyclogenesis and suggested an association between overexpression of cruzipain and increased resistance to the cysteiiie proteinase inhibitor Cbz-Phe-Phe-CHN, (where Cbz is benzoyloxycarbonyl). The increased resistance, however, was less than might be expected if cruzipain was the primary target of the inhibitor. Transgenic parasites did not exhibit increased infectivity.

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“…Various peptidases have been detected in Trypanosoma cruzi, the protozoan parasite that causes South American trypanosomiasis (Chagas' disease) in Man and other animals (Itow & Camargo, 1977;Bongertz & Hungerer, 1978;Rangel et al 1981;Bontempi et al 1984;Ashall, 1990;Greig & Ashall, 1990), although few of these enzymes have been well characterized. Bongertz & Hungerer (1978) purified a peptidase from T. cruzi epimastigotes that was reported to cleave peptide bonds on the carboxyl side of arginine, lysine and tyrptophan.…”

Section: Introductionmentioning

confidence: 99%

“…We characterized 5 major peptidases in T. cruzi epimastigotes using an electrophoretic system in which gelatin is hydrolysed in polyacrylamide gels (Greig & Ashall, 1990). Here we describe some other peptidase activities of epimastigotes and we present evidence that this parasite contains a lysosomal dipeptidyl aminopeptidase, one or more aminopeptidases, a neutral serine peptidase, and three enzymes that cleave carbobenzoxy-phenylalanyl-argininylaminomethylcoumarin (Z-Phe-Arg-MCA).…”

Section: Introductionmentioning

confidence: 99%

Detection of peptidases inTrypanosoma cruziepimastigotes using chromogenic and fluorogenic substrates

et al. 1992

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SUMMARYDetergent extracts of Trypanosoma cruzi epimastigotes catalysed the hydrolysis of a range of amino-acyl and peptidyl />-nitro-anilides and aminomethylcoumarins. At least three enzymes were detected that cleave Z-Phe-Arg-MCA. Two of these were optimally active at alkaline pH, the other at pH 4-0. Of the two enzymes with alkaline pH optima, one was a cysteine peptidase and was unable to cleave Bz-Arg-MCA readily, whilst the other cleaved Bz-Arg-MCA and was inhibited by diisopropyl fluorophosphate. The acidic enzyme was similar to cathespin L of other eukayrotes with respect to its pH profile, substrate-specificity and inhibitor-sensitivity. Evidence was presented that epimastigotes contain a cysteine-type dipeptidyl aminopeptidase, one or more aminopeptidases, and a serine peptidase that cleaves Boc-AlaAla-pNA. Digitonin solubilization of the activities from cells supports the hypothesis that the cathespin L-like enzyme and the dipeptidyl aminopeptidase are lysosomal, whilst the Bz-Arg-MCA hydrolase, the aminopeptidases and the Boc-Ala-Ala-pNA serine peptidase are cytosolic.

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Electrophoretic detection of Trypanosoma cruzi peptides

Cited by 44 publications

References 18 publications

Purified metallo-protease from the pathogenic haemoflagellate Cryptobia salmositica and its in vitro proteolytic activities

Purified metallo-protease from the pathogenic haemoflagellate Cryptobia salmositica and its in vitro proteolytic activities

Overexpression of Cruzipain, the Major Cysteine Proteinase of Trypanosoma cruzi, is Associated with Enhanced Metacyclogenesis

Detection of peptidases inTrypanosoma cruziepimastigotes using chromogenic and fluorogenic substrates

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