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Peptides isolated from human liver with specific inhibitory effects on reassociation/reactivation of in vitro dissociated lactic dehydrogenase (LDH-M 4 and −H 4) isozymes
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Beschreibungen/Notizen
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-M
4 = muscle type; LDH-H
4 = 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-M
4 or LDH-H
4. From the amino acid analyses, gel-filtration and PAGE + SDS, molecular weight of 1800 for the M
4 and ≈2700 for the H
4 inhibitor were calculated. An apparent
K
i of ≈3 × 10
−5 mM for the H
4 and ≈7 × 10
−5 mM for the H
4 inhibitor was estimated. The interaction of the inhibitors with the enzyme system showed strong cooperativity with Hill coefficients of 2.9 (LDH-M
4-specific) and 2.4 (LDH-H
4-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 reacts with monomers, dimers or a transition state during the tetramerisation process.
k
1 for the dimerisation step of M
4 = 2.0 × 10
5 M
−1 · s
−1 and of H
4 = 8.2 × 10
4 M
−1 · s
−1;
k
2 for the tetramerisation step of M
4 = 2.8 × 10
5 M
−1 · s
−1 and of H
4 = 1.2 × 10
5 · M
−1 · s
−1, were calculated, the second step still being the faster one.