Purification and Characterization of Lactate Dehydrogenase Isozymes in Channa argus

Abstract

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.

Metabolism of Lactate Dehydrogenase in Tissues from Ldh-C Expressed Fish at Starved State

“…LDH 활성이 상대적으로 낮게 확인되어 서식 방법과 먹이 습 득 행동 방식에 따라서 활성이 다른 것으로 보고되었다 [32,33,50]. 또한 용존산소를 급격히 증가시킨 환경에 꺽지와 모래 무지를 순응시킨 경우 골격근에서 LDH A4가 증가되나, 심장 조직에서 B4가 증가되었고 [8], 온도를 증가 시켰을 경우 Ldh-C 가 발현된 간조직은 C4를 증가시켜 조절하였으므로 [21] …”

Biochemical Properties of Lactate Dehydrogenase Eye-Specific C4Isozyme: Lepomis macrochirus and Micropterus salmoides

“…또한 풀망둑과 얼룩동사리 눈조직의 LDH 활성이 높게 확인되어 농어목의 육식성어류에서 먹이 포획시 눈조직 의 LDH 활성이 기능을 할 것으로 예상하였다 [29,49]. 그러나 가물치, 쥐노래미에서는 LDH 활성이 비교적 낮고 Km PYR 이 낮 아 피루브산에 대한 친화성이 크게 나타났으므로, 에너지 대 사에서의 역할이 중요함이 인식되었고 [13,28] (Fig. 3).…”