Correlation between the loss of the chaperone-like activity and the oxidation, isomerization and racemization of gamma-irradiated alpha-crystallin.
Alpha-crystallin possesses a molecular chaperone-like activity that prevents proteins from aggregating; however, the mechanism of this activity is not well known. Here we have taken gamma-irradiated alpha-crystallin and studied the relationship between the decrease in chaperone-like activity and the modifications such as oxidation, isomerization and racemization of amino acids in this molecule. We found that the chaperone-like activity of alpha-crystallin decreased with increasing gamma irradiation. After 4000 Gy gamma irradiation the activity of alpha-crystallin was reduced to 40% of the level of nonirradiated, native alpha-crystallin. The circular dichroism spectrum showed that the secondary structure of the irradiated alpha-crystallin had not changed. However, its tertiary structure appeared to change following more than 1000 Gy irradiation. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis also indicated that cross-linking of alpha-crystallin increased with increasing radiation doses. Irradiated and nonirradiated alpha-crystallin was subjected to trypsin digestion and peptide analysis by reverse-phase high-performance liquid chromatography and mass and sequence analysis. Depending on the radiation dose, Met-1 of alpha A-crystallin was oxidized to methionine sulfoxide. In addition, Asp-151 of alpha A-crystallin was isomerized to the beta-Asp form after irradiation, and racemization of Asp-151 decreased. Thus, the loss of the chaperone-like activity of alpha-crystallin is related to changes in its isomerization, oxidation and racemization.
Fujii
N
N
Research Reactor Institute, Kyoto University, Kumatori, Sennan, Osaka 590-0494, Japan. nfujii@hl.rri.kyoto-u.ac.jp
Hiroki
K
K
Matsumoto
S
S
Masuda
K
K
Inoue
M
M
Tanaka
Y
Y
Awakura
M
M
Akaboshi
M
M
D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry / Q000528:radiation effects; D002417:Cattle; D003459:Crystallins / Q000737:chemistry* / Q000528:radiation effects*; D005720:Gamma Rays / Q000009:adverse effects; D066298:In Vitro Techniques; D007536:Isomerism; D018832:Molecular Chaperones / Q000737:chemistry* / Q000528:radiation effects*; D010084:Oxidation-Reduction; D010777:Photochemistry; D017433:Protein Structure, Secondary / Q000528:radiation effects; D017434:Protein Structure, Tertiary / Q000528:radiation effects; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013237:Stereoisomerism
D003459:Crystallins; D018832:Molecular Chaperones; D001224:Aspartic Acid