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KMID : 0613820100200020260
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Journal of Life Science
2010 Volume.20 No. 2 p.260 ~ p.268
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Purification and Characterization of Lactate Dehydrogenase Isozymes in Channa argus
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Park Eun-Mi
Yum Jung-Joo
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Abstract
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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 ㎎/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/㎎, 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 KmPYR 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.
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KEYWORD
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Channa argus, lactate dehydrogenase isozyme, eye-specific C₄ isozyme, Km, Vmax
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