lambda-crystallin related to dehydroascorbate reductase in the rabbit lens.
OBJECTIVE
To evaluate the relationship of lambda-crystallin to reduced nicotinamide adenine dinucleotide (NADH)-dependent dehydroascorbate (DHA) reductase found specifically in the rabbit lens.


METHODS
DHA reductase Fractions I-IV were separated from the lambda/betaL1-crystallin fraction of rabbit lens soluble protein by diethylaminoethyl (DEAE)-cellulose ion-exchange column chromatography, and then the enzyme was partially purified from Fraction II by rechromatography on the same ion-exchange column. The isolated DHA reductase fractions were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, native isoelectric focusing and two-dimensional gel electrophoresis.


RESULTS
Using Western blot and a probe of antiserum to recombinant lambda-crystallin, the main 33-kDa protein band was strongly stained in all the rabbit lens DHA reductase fractions, and most of the additional protein bands of approximately 25-30 kDa were also detectable. In the partially purified enzyme, the 33-kDa subunit alone was identified as a distinct protein band by SDS-PAGE, and a main basic protein was found at pI 7.6 by native isoelectric focusing. In addition, many bands of more acidic proteins were separated from other enzyme fractions, and protein spots corresponding to the 33 and/or approximately 25-30-kDa subunits were detected in each of the more acidic proteins by two-dimensional gel electrophoresis.


CONCLUSIONS
These results suggest that lambda-crystallin is closely related to the DHA reductase in the rabbit lens. The above heterogeneity of the enzyme-crystallin may arise from posttranslational modifications.
Suzuki
Takahiro
T
Department of Ophthalmology, Tokai University School of Medicine, Isehara, Japan.
Bando
Masayasu
M
Oka
Mikako
M
Tsukamoto
Hideo
H
Akatsuka
Ichiko
I
Kawai
Kenji
K
Obazawa
Hajime
H
Kobayashi
Shizuko
S
Takehana
Makoto
M
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D003459:Crystallins / Q000737:chemistry / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D006168:Guinea Pigs; D007525:Isoelectric Focusing; D007908:Lens, Crystalline / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010088:Oxidoreductases / Q000378:metabolism*; D011817:Rabbits; D011892:Rana catesbeiana; D051381:Rats; D017208:Rats, Wistar
D003459:Crystallins; D010088:Oxidoreductases; C020666:glutathione dehydrogenase (ascorbate)