Characterization of the Multiple Forms of Duck Lens δ-Crystallin with Endogenous Argininosuccinate Lyase Activity

Duck lens δ-crystallins were the translational products of two tandem arranged genes, δ1 and δ2. Two fractions of the δ-crystallin (δa and δb) were resolved when the duck eye ball lens extract was passed through a Pharmacia-LKB Q-Sepharose column. These fractions were further separated by chromatofocusing into multiple forms with pI value ranging from 5.05 to 5.45. The charge heterogeneity of δ-crystallins was also demonstrated on the polyacrylamide gel when electrophoresis was performed under nonreducing conditions. All of those multiple forms possessed endogenous argininosuccinate lyase activity with activation energy ∼12.5 ± 1.6 kcal/mol. δb-Crystallins showed higher enzyme activity than δa-crystallins. Slightly different kinetic parameters were observed for the multiple forms of δb-crystallins. On the other hand, δa-crystallins could be further divided into two subgroups according to their kinetic parameters. There is 12-fold difference in kcat value between these two subgroups. δa-Crystallins also have lower Km values than the δb-crystallins. When examined by polacrylamide gel electrophoresis under reducing conditions in the presence of sodium dodecyl sulfate, the multiple forms were shown to be composed of subunits with similar Mr of 55,000. All of these forms showed same antigenicity toward the rabbit anti-duck δ-crystallin antiserum; however, different carbonyl contents were observed for these forms, indicating that the origin of these multiple forms was due to post-translational oxidative modification of the protein molecules. Two δ-crystallin forms were demonstrated to be the N-truncated δ2-crystallin, lacking the first eight amino acid residues from N-terminus. Modification and/or truncation of the proteins resulted in changing of the intrinsic tryptophan and tyrosine fluorescence. Those forms with higher pI values were shown to be much more thermostable than those with lower pI values. Our system may represent the first in vivo information that oxidation does not always lead to inactivation of an enzyme.