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

Abstract 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 p I 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 k cat value between these two subgroups. δa-Crystallins also have lower K m 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 M r 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 p I values were shown to be much more thermostable than those with lower p I values. Our system may represent the first in vivo information that oxidation does not always lead to inactivation of an enzyme.