Purification and substrate specificity of two cysteine proteinases of Giardia lamblia.
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 Mr = 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 (Mr = 95,000) and proteinase II (Mr = 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.
Werries
E
E
Fachbereich Biologie/Chemie, Universität Osnabrück, Abteilung Biochemie, Germany.
Franz
A
A
Hippe
H
H
Acil
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003546:Cysteine Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005872:Giardia / Q000201:enzymology*; D007328:Insulin / Q000378:metabolism; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000378:metabolism; D010084:Oxidation-Reduction; D013379:Substrate Specificity
D007328:Insulin; D009842:Oligopeptides; D003546:Cysteine Endopeptidases