Purification and characterization of gamma-glutamylcysteine synthetase from Ascaris suum.

Abstract:

:We have purified and characterized the Ascaris suum gamma-glutamylcysteine synthetase, the rate-limiting step in the glutathione biosynthesis. The purified enzyme exhibited a specific activity of 18 U (mg protein)-1. Estimation of the molecular mass of the native enzyme by FPLC on Superdex S-200 revealed the presence of two enzyme activity peaks corresponding to molecular masses of 100 and 70 kDa. The higher-molecular-mass component could be dissociated by repeated gel filtration into the 70-kDa protein which is the enzymatically active subunit. The apparent Km values of the A. suum enzyme for L-aminobutyrate, L-cysteine and L-glutamate were 0.31, 0.41 and 0.94 mM, respectively. D,L-Buthionine-S,R-sulfoximine and cystamine showed time-dependent irreversible inhibitory effects on the A. suum enzyme activity with Ki values of 0.05 and 1.11 microM, respectively. The Ki values for the corresponding enzyme from rat kidney with D,L-buthionine-S,R-sulfoximine and cystamine were 7.19 and 22.2 microM, respectively. The time of half-inactivation of the enzyme at infinite concentration of D,L-buthionine-S,R-sulfoximine, tau 50, was determined to be 3.1 and 1.34 min, for the parasite and mammalian enzymes respectively. For cystamine, a tau 50 value of 3.32 min for the A. suum gamma-glutamylcysteine synthetase was determined, while a value of 2 min in case of rat kidney enzyme was found. The A. suum enzyme activity was competitively inhibited by glutathione with a Ki value of 0.11 mM.(ABSTRACT TRUNCATED AT 250 WORDS)

journal_name

Mol Biochem Parasitol

authors

Hussein AS,Walter RD

doi

10.1016/0166-6851(94)00064-t

subject

Has Abstract

pub_date

1995-06-01 00:00:00

pages

57-64

issue

1-2

eissn

0166-6851

issn

1872-9428

pii

0166-6851(94)00064-T

journal_volume

72

pub_type

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