A Kinetic Analysis of the Endogenous Lactate Dehydrogenase Activity of Duck Lens ε-Crystallin in Reverse Micelles
✍ Scribed by Hwei-Jen Lee; Gu-Gang Chang
- Publisher
- Elsevier Science
- Year
- 1998
- Tongue
- English
- Weight
- 169 KB
- Volume
- 201
- Category
- Article
- ISSN
- 0021-9797
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✦ Synopsis
and optical clarity (1). In the past decade, some taxon-1-Crystallin is a structural protein in duck lenses with endogespecific crystallins were found to have structures related to nous lactate dehydrogenase (LDH) activity. When entrapped in cytosolic enzymes (1-3). Among them the 1-crystallin in an aerosol-OT (AOT)/isooctane/H 2 O reverse micellar system, 1duck lenses has been shown to have an amino acid sequence crystallin preserves this endogenous enzymatic activity. The catasimilar to the lactate dehydrogenase (S-lactate: NAD / oxilytic constant (k cat ) of 1-crystallin exhibited multiple peaks at doreductase; EC 1.1.1.27; LDH 2 ) (1-3). We have already varying degrees of system hydration ([H 2 O]/[AOT]), thereby sugcharacterized the kinetic mechanism of the endogenous LDH gesting that 1-crystallin exists as various oligomers in reverse miactivity of duck 1-crystallin in aqueous solution (4, 5). Howcelles and that each oligomer is enzymatically active. Substrate ever, since lenses contain closed, limited hydrated cells, a inhibition, similar to that found in aqueous solution, is also observed in reverse micelles, albeit with an inhibition constant lower reverse micellar system prepared by dissolving a surfactant than that in aqueous solution. Graphical analysis by the method in organic solvent with limited water content may mimic the of Wang and Srivastava [Anal. Biochem. 216, 15 (1994)] at low in vivo conditions. We thus proceeded to characterize the
, where 1-crystallin presumably existed as mono-kinetic properties of 1-crystallin in reverse micelles.
mers, suggests that there is only one pyruvate binding site per
Cytosolic LDH is found to be enzymatically active in monomer. A similar analysis of substrate inhibition data at high some reverse micellar systems, which also causes the en-
, where 1-crystallin might exist as tetramers, sug-zyme to dissociate or aggregate into various oligomers (6gests that monomeric 1-crystallin is enzymatically active, in accor-
8). To examine the endogenous LDH activity of duck lens dance with the multiple peaks in the k cat versus [H 2 O]/[AOT] plot.
1-crystallin in reverse micelles, we entrapped the 1-crystallin 1-Crystallin shows different pH dependencies on k cat in different in AOT/isooctane/H 2 O reverse micelles. We found that the solvent systems. In aqueous solution, only one amino acid residue endogenous LDH activity of 1-crystallin was preserved in with a pK a value of 8.11, which must be protonated, is found to be involved in the catalysis. However, two amino acid residues the reverse micellar solution. The possible oligomeric distriwith pK a values of 8.26 and 8.44, respectively, are obtained in bution and kinetic properties of 1-crystallin in reverse micelreverse micelles. The log ( k cat /K mPyr ) versus pH plots are similar lar solution with various hydration degrees were proposed.
in different solvent systems but the amino acid residue with p K a value 4.95 in aqueous solution raises its p K a value to 6.91 in reverse MATERIALS AND METHODS micelles. The pK a value of the other group is similar in the two Materials solvent systems (8.15 in aqueous and 7.69 in reverse micellar solution). The endogenous LDH activity of 1-crystallin is found to be AOT was purchased from Sigma-Aldrich (St. Louis, slightly sensitive to AOT concentration, thereby suggesting that MO). Isooctane (2,2,4-trimethylpentane) was purchased 1-crystallin has some affinity with the membranous structure. from E. Merck (Darmstadt, Germany). Other chemicals