Affinity chromatographic differentiation of lactate dehydrogenase isoenzymes on the basis of differential abortive complex formation

O'Carra, Pádraig; Barry, Standish; Corcoran, E.

doi:10.1016/0014-5793(74)80992-0

Cited by 38 publications

(14 citation statements)

References 12 publications

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“…Muscle-type lactate dehydrogenase (~ LDH) was purified from white skeletal muscle using an oxamate-Sepharose affinity column, following the techniques of Yancey and Somero (1978a) which are slightly modified from the procedures of O'Carra et al (1974). Purified~-LDH was concentrated by ultrafiltration, dialyzed against buffered saturated ammonium sulfate, and stored as a solution of 2-3 mg/ml protein.…”

Section: Methodsmentioning

confidence: 99%

Electrophoretic and Functional Enzymic Evolution in Four Species of Eastern Pacific Barracudas From Different Thermal Environments

Graves

Somero

1982

Evolution

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Section: Methodsmentioning

confidence: 99%

Electrophoretic and Functional Enzymic Evolution in Four Species of Eastern Pacific Barracudas From Different Thermal Environments

Graves

Somero

1982

Evolution

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“…In this respect isoenzyme LDH-X is comparable with isoenzyme LDH-5, which is also not retarded in the presence of NAD+, in contrast with isoenzymes LDH-1 and -2 (O'Carra et al, 1974). The retardation of isoenzyme LDH-1 on oxamate-Sepharose has been explained in terms of an abortive-complex formation (enzyme-NAD+-pyruvate), which is considered to be the cause of the inhibition of the enzyme activity at high pyruvate concentrations (O'Carra et al, 1974). But since human isoenzyme LDH-X (Wilkinson & Withycombe, 1965; A. H. J. Kolk, unpublished work) as well as mouse and rabbit isoenzyme LDH-X are inhibited by high pyruvate concentrations, the abortive-complex formation does not explain the separation of isoenzyme LDH-X from LDH-1 on the oxamate-Sepharose column.…”

Section: Resultsmentioning

confidence: 99%

Isolation of human lactate dehydrogenase isoenzyme X by affinity chromatography

Kolk¹,

Kuyk²,

Boettcher³

1978

Biochemical Journal

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Human isoenzyme LDH-X (lactate dehydrogenase isoenzyme X) was isolated from seminal fluid of frozen semen samples by affinity chromatography by using oxamateSepharose and AMP-Sepharose. In the presence of 1.6mm-NAD+, isoenzyme LDH-X does not bind to AMP-Sepharose, whereas the other lactate dehydrogenase isoenzymes do. This is the crucial point in the isolation of isoenzyme LDH-X from the other isoenzymes. The purified human isoenzyme LDH-X had a specific activity of 146 units/ mg of protein.

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“…골격근에 A4-항혈청을 사용하 여 Western blotting (Fig. 1b) [24,25]. 그리고 종에 따라 LDH 역학적 특성의 차이로 인해 정제 양상이 다르므로, 동위효소의 기능은 물론 진화상 의 유연관계를 고찰할 수 있다 [36].…”

Section: 서 론unclassified

Purification and Characterization of Lactate Dehydrogenase Isozymes in Channa argus

Park¹,

Yum²

2010

Journal of Life Science

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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 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.

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Affinity chromatographic differentiation of lactate dehydrogenase isoenzymes on the basis of differential abortive complex formation

Cited by 38 publications

References 12 publications

Electrophoretic and Functional Enzymic Evolution in Four Species of Eastern Pacific Barracudas From Different Thermal Environments

Electrophoretic and Functional Enzymic Evolution in Four Species of Eastern Pacific Barracudas From Different Thermal Environments

Isolation of human lactate dehydrogenase isoenzyme X by affinity chromatography

Purification and Characterization of Lactate Dehydrogenase Isozymes in Channa argus

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