从人肝脏中分离出的肽，对体外解离的乳酸脱氢酶（LDH-M4和-H4）同工酶的重新缔合/再激活具有特异性抑制作用。
Peptides isolated from human liver with specific inhibitory effects on reassociation/reactivation of in vitro dissociated lactic dehydrogenase (LDH-M4 and -H4) isozymes.
作者信息
Schoenenberger G A, Buser S, Cueni L, Döbeli H, Gillesen D, Lergier W, Schöttli G, Tobler H J, Wilson K
出版信息
Regul Pept. 1980 Dec;1(3):223-44. doi: 10.1016/0167-0115(80)90273-6.
Two different peptides have been purified from human liver, similar to those previously reported (Schoenenberger, G.A., and Wacker, W.E.C. (1966) Biochemistry 5, 1375--1379) to be present in human urine, which may serve as metabolic regulators of lactate dehydrogenase (EC 1.1.1.27) isoenzymes (LDH-M4 = muscle type; LDH-H4 = heart type). By trichloroacetic acid precipitation, ultrafiltration, Sephadex G-25 and Bio-Gel P-2 columns, affinity chromatography on immobilized LDH-isozymes and HPLC two peptides which differed with respect to molecular weight, retention on the affinity columns and amino acid composition were isolated. No effect was observed when native, tetrameric lactate dehydrogenase was incubated with these peptides. However, when lactate dehydrogenase was dissociated to monomers at low pH and allowed to reassociate by adjusting the pH to 7.5 complete inhibition of the reactivation occurred when the inhibitors were incubated together with respective reassociating monomeric isozymes. The two peptides showed no cross-specificity, i.e. each peptide exhibited inhibitory activity only on one of the two isozymes LDH-M4 or LDH-H4. From the amino acid analyses, gel filtrations and PAGE + SDS, molecular weights of 1800 for the M4 and approximately 2700 for the H4 inhibitor were calculated. An apparent Ki of approximately 3 X 10(-5) mM for the H4 and approximately 7 X 10(-5) mM for the H4 inhibitor was estimated. The interaction of the inhibitors with the enzyme system showed strong cooperativity with Hill coefficients of 2.9 (LDH-M4-specific) and 2.4 (LDH-H4-specific). Mathematical modelling of the reassociation and reactivation of lactate dehydrogenase and its specific inhibition by the peptides led to the conclusion that the peptides react with monomers, dimers or a transition state during the tetramerisation process. kappa 1 for the dimerisation step of M4 = 2.0 X 10(5) M-1 . S-1 and of H4 = 8.2 X 10(4) M-1 . S-1; kappa 2 for the tetramerisation step of M4 = 2.8 X 10(5) M-1 . S-1 and of H4 = 1.2 X 10(5) . M-1 S-1, were calculated, the second step still being the faster one (Rudolf, R. and Jaenicke, R. (1976) Eur. J Biochem. 63, 409--417).
从人肝脏中纯化出了两种不同的肽，类似于先前报道的（舍嫩贝格尔，G.A.，和瓦克尔，W.E.C.（1966年）《生物化学》5，1375 - 1379）存在于人尿液中的肽，它们可能作为乳酸脱氢酶（EC 1.1.1.27）同工酶（LDH - M4 = 肌肉型；LDH - H4 = 心脏型）的代谢调节剂。通过三氯乙酸沉淀、超滤、Sephadex G - 25和Bio - Gel P - 2柱、固定化LDH同工酶亲和色谱以及高效液相色谱，分离出了两种在分子量、在亲和柱上的保留情况和氨基酸组成方面存在差异的肽。当天然的四聚体乳酸脱氢酶与这些肽一起温育时，未观察到任何影响。然而，当乳酸脱氢酶在低pH下解离为单体，并通过将pH调至7.5使其重新缔合时，当抑制剂与各自重新缔合的单体同工酶一起温育时，再活化完全受到抑制。这两种肽没有交叉特异性，即每种肽仅对两种同工酶LDH - M4或LDH - H4中的一种表现出抑制活性。通过氨基酸分析、凝胶过滤以及PAGE + SDS，计算出M4抑制剂的分子量为1800，H4抑制剂的分子量约为2700。估计H4抑制剂的表观Ki约为3×10⁻⁵ mM，M4抑制剂的表观Ki约为7×10⁻⁵ mM。抑制剂与酶系统的相互作用表现出很强的协同性，Hill系数分别为2.9（LDH - M4特异性）和2.4（LDH - H4特异性）。对乳酸脱氢酶的重新缔合和再活化及其被肽特异性抑制进行数学建模得出结论，肽在四聚化过程中与单体、二聚体或过渡态发生反应。计算出M4二聚化步骤的κ1 = 2.0×10⁵ M⁻¹·s⁻¹，H4二聚化步骤的κ1 = 8.2×10⁴ M⁻¹·s⁻¹；M4四聚化步骤的κ2 = 2.8×10⁵ M⁻¹·s⁻¹，H4四聚化步骤的κ2 = 1.2×10⁵ M⁻¹·s⁻¹，第二步仍然是较快的一步（鲁道夫，R.和耶尼克，R.（1976年）《欧洲生物化学杂志》63，409 - 417）。
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