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KMID : 0613820120220020209
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Journal of Life Science
2012 Volume.22 No. 2 p.209 ~ p.219
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Biochemical Properties of Lactate Dehydrogenase Eye-Specific C4 Isozyme: Lepomis macrochirus and Micropterus salmoides
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Yum Jung-Joo
Ku Bo-Ra
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Abstract
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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 {K_m}^{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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KEYWORD
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Lactate dehydrogenase eye-specific C₄ isozyme, enzyme kinetics, pyruvate substrate inhibition, Lepomis macrochirus, Micropterus salmoides
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