Limited proteolysis of lactate dehydrogenase from porcine heart with trypsin: characterization and reactivation of the fragments.
The ternary complex formed by native lactate dehydrogenase (LDH) from porcine heart, NAD+ and sulfite, was digested with trypsin over a period of 12-16 h3. After removal of the ligands and residual native lactate dehydrogenase by ion exchange chromatography dimers were obtained which were almost inactive. The dimers were lacking a hexapeptide at the N-terminus; however, the secondary structure was the same as that of native lactate dehydrogenase. The circular dichroism spectra showed a dependence on temperature which suggested an equilibrium of two different structural states. The reaction of antibodies against native porcine heart LDH with the dimers restored the catalytic activity, and subsequently the dimers behaved similarly to the native enzyme. Addition of 1 M phosphate or NAD-sulfite to the dimers restored 80-90% of the catalytic activity. It could be demonstrated that the behavior of the reactivated dimers, in contrast to that of the inactive dimers, was similar to the behavior of native lactate dehydrogenase. For instance, ultracentrifugal analysis showed that dimers reactivated with NAD-SO3- were associated to give tetramers. The reaction of antibodies against native LDH with the dimers reactivated with NAD-SO3- demonstrated that the native LDH and the dimers have the same surface determinants.
Pfleiderer
G
G
Institut für organische Chemie, Biochemie and Isotopenforschung, Universität Stuttgart, Federal Republic of Germany.
Nagel
G
G
Bühler
H
H
D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology; D001665:Binding Sites; D004789:Enzyme Activation / Q000187:drug effects; D006321:Heart / Q000187:drug effects; D006868:Hydrolysis; D007527:Isoenzymes; D007770:L-Lactate Dehydrogenase / Q000276:immunology / Q000378:metabolism*; D008969:Molecular Sequence Data; D009206:Myocardium / Q000201:enzymology*; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D014357:Trypsin / Q000494:pharmacology
D000906:Antibodies; D007527:Isoenzymes; D007770:L-Lactate Dehydrogenase; D014357:Trypsin