PURIFICATION AND SUBSTRATE-SPECIFICITY OF 2 CYSTEINE PROTEINASES OF GIARDIA-LAMBLIA

Autor(en): WERRIES, E
FRANZ, A
HIPPE, H
ACIL, Y
Stichwörter: CYSTEINE PROTEINASES; ENTAMOEBA-HISTOLYTICA; GIARDIA-LAMBLIA; PEPTIDES; POLYACRYLAMIDE GELS; SPECIFICITY; SYNTHETIC PEPTIDES; TROPHOZOITES; Zoology
Erscheinungsdatum: 1991
Herausgeber: SOC PROTOZOOLOGISTS
Journal: JOURNAL OF PROTOZOOLOGY
Volumen: 38
Ausgabe: 4
Startseite: 378
Seitenende: 383
Zusammenfassung: 
The proteinase activity present in homogenates of trophozoites of Giardia lamblia, active on azocasein and urea-denaturated hemoglobin, was separated into two different enzymes by a series of purification procedures, These procedures included gel filtration on Fractogel TSK HW-55 (F), organomercurial agarose affinity chromatography, and ion exchange chromatography on DEAE-cellulose. By chromatography on Sephadex G-100, two purified enzymes exhibited relative molecular weights of M(r) = 95,000 and 35,000 /- 10%, respectively. On the basis of inhibition by thiol reagents and abrogation of this effect by dithiothreitol and cysteine, they were identified as cysteine proteinases. Proteinase I (M(r) = 95,000) and proteinase II (M(r) = 35,000) were active against the beta-chain of insulin releasing characteristic fragments. However, differences in substrate specificities of the two enzymes could be observed by using synthetic peptides that represent sequences 1-6, 8-18, and 20-30 of the insulin beta-chain. Furthermore, the synthetic tetrapeptides Arg-Gly-Phe-Phe, Arg-Gly-Leu-Hyp, and Arg-Arg-Phe-Phe were hydrolyzed by the two proteinases releasing Phe-Phe and Leu-Hyp, respectively. Compared with Arg-Gly-Phe-Phe, the rates of hydrolysis of Arg-Gly-Leu-Hyp and Arg-Arg-Phe-Phe at substrate concentrations of 1 mM were 91% and 63% (proteinase I) and 80% and 57% (proteinase II), respectively.
ISSN: 00223921
DOI: 10.1111/j.1550-7408.1991.tb01374.x

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