The properties of lactate dehydrogenase (LDH, EC 1.1.1.27) eye-specific C4 isozyme were studied by polyacrylamide gel electrophoresis, Western blotting, immunoprecipitation, and enzyme kinetics. Furthermore, we proposed the optimal conditions for measuring the activity of LDH eye-specific C4 isozyme. The isozymes were detected in the cytosol of eye tissues from Lepomis macrochirus and Micropterus salmoides and were more similar to the A4 than the B4 isozyme. LDH/CS in the eye tissue of L. macrochirus was increased in September, so the ratio of anaerobic metabolism was high. The electrophoretic patterns of mitochondrial LDH were similar to those of cytosolic LDH in the eye tissues of L. macrochirus and Micropterus salmoides. LDH eye-specific C4 isozyme from eye tissue was purified by preparative native-PAGE. The activities of LDH eye-specific C4 isozymes in L. macrochirus and M. salmoides were reduced at concentrations greater than 0.2 mM and 0.1 mM of pyruvate, respectively. These concentrations remained at 5.2% and 15.8% as a result of the inhibition by 10 mM of pyruvate, so the degree of inhibition was very high. The LDH activities of eye tissues were reduced at concentrations greater than 22 mM and 24 mM of lactate, respectively, in L. macrochirus and M. salmoides. The Km PYR of eye-specific C4 was 0.088 mM in L. macrochirus and it was 0.033 mM in M. salmoides. The activities of cytosolic and mitochondrial eye-specific C4 isozymes were high in α-ketobutyric acid. Furthermore, the activities of eye tissue and eye-specific C4 isozyme had to be measured with 0.5 mM of pyruvate and a buffer solution of pH 7.5. As a conclusion, the eye-specific C4 isozyme in M. salmoides has a high affinity for pyruvate and exhibits maximum activity at a lower concentration of pyruvate and at higher concentration of lactate than that in L. macrochirus. Therefore, it seems that the energy produced by the LDH eye-specific C4 isozyme in M. salmoides was used at the first stage of predatory behavior.

The aim of this study was to examine the metabolic adjustment of lactate dehydrogenase (EC 1.1.1.27, LDH) isozymes to the environmental temperature in bluegill (Lepomis macrochirus). This study included three groups of bluegill collected in April (group Ⅰ), May (group Ⅱ), and September (group Ⅲ). The LDH activities of skeletal muscle, heart, and brain tissues were higher in group Ⅲ than in groups Ⅰ and Ⅱ. The citrate synthase (EC 4.1.3.7, CS) activity was higher in skeletal muscle but lower in heart and brain tissues of group Ⅱ as compared to group Ⅰ. In contrast, the CS activity was lower in skeletal muscle and higher in heart and brain tissues in group Ⅲ than in group Ⅱ. Furthermore, the LDH/CS activity ratio was higher in the skeletal muscle and brain in group Ⅲ than in groups Ⅰ and Ⅱ. Accordingly, anaerobic metabolism was increased in group Ⅲ. LDH A4, A2B2, and B4 isozymes were expressed in skeletal muscle, heart, liver, and brain tissues. The LDH C hybrid was detected in brain tissue. The LDH A4 isozyme was successfully purified by affinity chromatography. The molecular weight of the purified LDH A4 isozyme was 136 kDa and its optimal pH for enzymatic activity was 8.0. The Km PYR values of LDH in skeletal muscle were 0.161-0.227 mM using pyruvate as a substrate. These kinetic properties of LDH in skeletal muscle are consistent with the fact that bluegill is a cold-adapted species. These results may be useful for predicting the habitat use of this fish.

-The lactate dehydrogenase (EC 1.1.1.27, LDH) A4 isozyme in skeletal muscle of mandrin fish (Siniperca scherzeri) was successfully purified by affinity chromatography and ultrafiltration. The molecular weight of the purified LDH A4 isozyme was 140.4 kDa and its isoelectric point (pI) was 7.0. Optimal pH for enzymatic reaction was 7.5. Km PYR and Vmax value of the purified LDH A4 isozyme were 4.86×10-5 M and 13.31 mM/min using pyruvate as a substrate, respectively. These kinetic properties of the purified LDH A4 isozyme supported the fact that the mandrin fish was a warm-adapted species. The antibody against the purified LDH A4 isozyme may be used in the metabolic physiological studies of ectothermic vertebrates and in the diagnosis of several human diseases.