Thiol/disulfide interconversion in bovine lens aldose reductase induced by intermediates of glutathione turnover.
The effectiveness of cysteine and cysteinylglycine to act as protein thiolating agents was investigated using bovine lens aldose reductase (ALR2) as the protein target. Disulfides of both thiol compounds appear to be very effective as ALR2 thiolating agents. Cysteine- and CysGly-modified ALR2 forms (Cys-ALR2 and CysGly-ALR2, respectively) are characterized by the presence of a mixed disulfide bond involving Cys298, as demonstrated by a combined electrospray mass spectrometry and Edman degradation approach. Both Cys-ALR2 and CysGly-ALR2 essentially retain the ability to reduce glyceraldehyde but lose the susceptibility to inhibition by Sorbinil and other ALR2 inhibitors. Cys-ALR2 and CysGly-ALR2 are easily reduced back to the native enzyme form by dithiothreitol and GSH treatment; on the contrary, Cys and 2-mercaptoethanol appear to act as protein trans-thiolating agents, rather than reducing agents. The treatment at 37 degrees C of both Cys-ALR2 and CysGly-ALR2, unlikely what observed for glutathionyl-modified ALR2 (GS-ALR2), promotes the generation of an intramolecular disulfide bond between Cys298 and Cys303 residues. A rationale for the special susceptibility of Cys-ALR2 and CysGly-ALR2, as compared to GS-ALR2, to the thermally induced intramolecular rearrangement is given on the basis of a molecular dynamic and energy minimization approach. A pathway of thiol/disulfide interconversion for bovine lens ALR2 induced, in oxidative conditions, by physiological thiol compounds is proposed.
Vilardo
P G
PG
Dipartimento di Fisiologia e Biochimica, Università di Pisa, Via S. Maria 55, 56100 Pisa, Italy.
Scaloni
A
A
Amodeo
P
P
Barsotti
C
C
Cecconi
I
I
Cappiello
M
M
Lopez Mendez
B
B
Rullo
R
R
Dal Monte
M
M
Del Corso
A
A
Mura
U
U
D000449:Aldehyde Reductase / Q000378:metabolism*; D000478:Alkylation; D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D004220:Disulfides / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism*; D006868:Hydrolysis; D007908:Lens, Crystalline / Q000201:enzymology*; D008958:Models, Molecular; D021241:Spectrometry, Mass, Electrospray Ionization; D013438:Sulfhydryl Compounds / Q000378:metabolism*
D004220:Disulfides; D013438:Sulfhydryl Compounds; D000449:Aldehyde Reductase; D005978:Glutathione