Eye-specific lactate dehydrogenase (EC 1.1.1.27, LDH) C4 isozyme in the eyes of greenlings (Hexagrammos otakii) was successfully purified by affinity chromatography and continuous-elution electrophoresis. The molecular weight of the purified eye-specific LDH C4 isozyme was 154.8 kDa, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Optimal pH for enzymatic reaction of the eye-specific LDH C4 isozyme was pH 8.5. Km PYR value of the purified eye-specific LDH C4 isozyme was 1.88×10-5 M using pyruvate as a substrate. These results indicate that we must consider pH when measuring eye-specific LDH C4 isozyme activity. The eye-specific LDH C4 isozyme had a higher binding affinity for the substrate as a pyruvate than LDH A4 isozyme. Antibodies produced against the purified eye-specific LDH C4 isozyme may be used in the diagnosis of several human diseases and in comparative physiological studies of fishes.
The lactate dehydrogenase (EC 1.1.1.27, LDH) isozymes in tissues from Channa argus were purified and characterized by biochemical, immunochemical and kinetic methods. The activity of LDH in skeletal muscle was the highest at 380.4 units and those in heart, eye and brain tissues were 13.4, 3,5 and 5.4 units, respectively. Citrate synthase (EC 4.1.3.7, CS) activity in heart tissue was the highest at 20.7 units. LDH/CS in skeletal muscle, heart, eye and brain tissues were 172.9, 0.6, 0.32 and 0.47. Protein concentration in skeletal muscle tissue was 14.7 mg/g and specific activities of LDH in skeletal muscle, heart, eye and brain tissues were 25.88, 0.79, 0.31 and 1.38 units/mg, respectively. Therefore, skeletal muscle tissue was anaerobic and heart tissue was aerobic. The LDH isozymes in tissues were identified by polyacrylamide gel electrophoresis, immunoprecipitation and Western blot with antiserum against A4, B4, and eye-specific C4. LDH A4, A3B, A2B2. AB3 and B4 isozymes were detected in every tissue, C4, AC3, A2C2 and A3C were detected in eye tissue, and A3C was found in brain tissue. LDH A4, A3B, A2B2, AB3, B4, eye-specific C4 isozymes were purified by affinity chromatography and Preparative PAGE Cells. The LDH A4 isozyme was purified in the fraction from elution with NAD + containing buffer of affinity chromatography. Eye-specific C4 isozyme was eluted right after A4, after which B4 isozyme was eluted with plain buffer. As a result, one part of molecular structures in A4, B4 and eye-specific C4 were similar, but were different from each other in B4 and C4. Therefore the subunit A may be conservative in evolution, and the evolution of subunit B seems to be faster than that of subunit A. The activity of LDH A4, A2B2, B4, and eye-specific C4 isozymes remained at 39.98, 21.28, 19.67 and 16.87% as a result of the inhibition by 10 mM of pyruvate, so the degree of inhibition was very high. The Km PYR values were 0.17, 0.27 and 0.133 mM in A4, B4 and eye-specific C4 isozymes, respectively. The optimum pH of LDH A4, B4, eye-specific C4, A2B2, A3B, and AB3 were pH 6.5, pH 8.5, pH 5.5, pH 6.0-6.5, pH 5.0 and pH 7.5. The A4 and heterotetramer isozymes stabilized a broad range of pH. Especially, LDH activities in skeletal muscle tissue were high, resulting in a high degree of muscle activity.LDH metabolism in eye tissue seems to be converted faster from pyruvate to lactate by eye-specific C4 isozyme as eye-specific C4 have the highest affinity for pyruvate, and right after the conversion, oxidation of lactate was induced by A4 isozyme. It was found that expression of Ldh-C, affinity to substrate and reaction time of C4 isozyme were different according to the ecological environmental and feeding capturing patterns.
In this study, the properties and gene expression of the lactate dehydrogenase (EC 1.1.1.27, LDH) isozyme were studied in angelfish (Pterophyllum scalare) -known for their adaptation to the low oxygen environment of the tropics -which were acclimated to acute temperature change (27±0.5→18±0.5℃) and dissolved oxygen (DO) change (6±1→18 ppm) for 2 hours. The properties of the LDH isozymes were confirmed in the native-polyacrylamide gel electrophoresis, Western blot analysis and enzyme activity measurement. Liver-and eye-specific Ldh-C gene were expressed in liver, eye and brain tissues. Through Western blot analysis, the LDH A4 isozyme was shown to have a more cathodal mobility relative to the B4 isozyme. In the liver tissue, the LDH A4 isozyme increased with temperature drop while the B4 isozyme decreased. The LDH A4 and C4 isozymes increased with DO increment, while the B4 isozyme decreased. In the eye tissue, the LDH A4 and B4 isozymse increased with temperature drop while the C4 isozyme decreased. The LDH A4 and B4 isozymes increased with DO increment, but the C4 isozyme and isozymes including the subunit C decreased. In the heart tissue, LDH activity increased with DO increment, as well as the LDH B4 isozyme. In the brain tissue, the LDH A4 and B4 isozymes increased with temperature drop. The LDH B4 isozyme increased with DO increment. Accordingly, since the liver-and eye-specific Ldh-C are influenced by changes in DO and the LDH B4 and C4 isozymes are relatively controlled in the liver and eye tissues, the C4 isozyme can be considered to have a lactate oxidase function.
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.
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