가물치(Channa argus) 젖산탈수소효소 동위효소들의 정제 및 특성 (Purification and Characterization of Lactate Dehydrogenase Isozymes in Channa argus)

서지정보

· 발행기관 : 한국생명과학회
· 수록지 정보 : 생명과학회지 / 20권 / 2호 / 260 ~ 268페이지
· 저자명 : 박은미, 염정주

초록

가물치(Channa argus) 조직의 젖산탈수소효소 동위효소(EC 1.1.1.27, lactate dehydrogenase, LDH)를 정제하고 생화학적, 면역학적 및 역학적 방법으로 특성을 연구하였다. LDH 활성은 골격근이 380.4 units로 가장 높고 심장 13.4, 눈 3.5, 뇌 조직 5.4 units이었으며, 심장의 CS 활성은 20.7 unit로 가장 높고, LDH/CS는 골격근 172.9, 심장 0.6, 눈 0.32, 뇌 0.47이고, 단백질 양은 골격근 14.7 mg/g이며, 특이활성(units/mg)은 골격근 25.88, 심장 0.79, 눈 0.31, 뇌 1.38 units/mg이었으므로 골격근은 혐기적이고, 심장은 호기적이었다. LDH A4, B4, eye-specific C4에 대한 항혈청을 사용한 Western blot, 면역침강반응 및 native- polyacrylamide gel electrophoresis에 의해 A4, A3B, A2B2, AB3 및 B4가 모든 조직에서 확인되었고, 눈 조직에서 C4와 AC3, A2C2, AC3, 뇌 조직에서 A3C도 확인되었다. LDH A4, A3B, A2B2, AB3, B4, eye-specific C4 동위효소는 affinity chromatography와 Preparative PAGE Cell에 의해 정제되었다. LDH A4 동위효소는 NAD+ 유입 후 정제되었고, eye-specific C4는 A4에 이어 용출되기 시작하였으며 B4는 buffer 유입 후 용출되었다. 정제한 결과 A4는 B4 및 eye-specific C4와 분자구조의 일부가 유사하였지만 B4와 C4는 서로 다른 것으로 나타났으므로, 하부단위체 A는 보존적이고, 하부단위체 B는 A보다 더 빠르게 진화된 것으로 보인다. 피루브산 10 mM에서 A4 동위효소 39.98%, A2B2 21.28%, B4 19.67% 및 eye-specific C4 16.87%의 활성이 남아있었고, 피루브산에 대한 KmPYR은 A4 0.17 mM, B4 0.27 mM, eye-specific C4 0.133 mM였다. A4, B4, eye-specific C4, A2B2, A3B 및 AB3의 최적 pH는 각각 pH 6.50, pH 8.5, pH 5.5, pH 6.0-6.5, 5.0 및 pH 7.5였고, 동질사량체 A4와 이질사량체 동위효소들은 넓은 pH 영역에서 안정하였다. 특히 골격근은 LDH 활성이 크므로 활동성이 크며, 눈조직에서 피루브산 친화력이 강한 eye-specific C4에 의해 피루브산 대사가 빠르게 일어나고, 이어서 A4에 의해 젖산이 산화되어지는 것으로 사료되므로, 종의 생태환경 및 먹이 획득 양식에 따라 LDH-C 발현, 기질에 대한 친화도 및 대사 시간이 다른 것으로 사료된다.

영어초록

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 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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