A comparative physico‐chemical characterization of lactate dehydrogenase: Isozymes in brook trout, lake trout and their hybrid splake trout

Peptide maps and amino acid composition of LDH crystallized from skeletal muscle extracts of brook trout (Salvelinus fontinalis) and lake trout (S. namaycush) are similar to those of the hybrid splake trout. The molecular weights are identical to mammalian LDH on the basis of sucrose density gradient centrifugation. The five A′–A isozymes are restricted to LDH from trout muscle and migrate very slowly toward the anode. The B′–B subunit series of isozymes are ubiquitous in trout tissues, although cell specific proportions of isozymes occur. While the A′–A subunit genes must be present in all trout cells, they are active only in the muscle where their products are responsible for essentially all of the LDH activity in this tissue. Isozyme patterns reveal that B subunit‐containing tetramers (less anodal) predominate in LDH from liver, whereas B′ subunit molecules (more anodal) prevail in LDH from trout heart. Therefore, extracts from heart, muscle, and liver tissues were utilized to characterize and compare the duplicated subunit tetramers of LDH in terms of several physico‐chemical and physiological parameters. Substrate kinetics show that trout B′ subunit tetramers are very sensitive to increases in pyruvate and lactate. These isozymes attain maximum initial velocity at about 0.8 μmole of pyruvate, while substrate inhibition begins at 7 μmoles. The B and the A′–A tetramers reach maximum initial velocity at 1.3 and 3.5 μmoles of pyruvate, respectively, and inhibition does not begin until substrate concentrations of about 20 μmoles are reached. Results are similar with lactate as substrate. Only the B′ tetramers exhibit substrate inhibition with lactate. An analysis of competitive pyruvate and oxamate inhibition reveals that the LDH active site(s) binds one molecule of inhibitor (or substrate). The inactivating temperature for trout B′ subunit tetramers is 70–75°C; for B subunit tetramers, 65–70°C; and for A′–A tetramers, 46–54°. The A′–A and the B subunit‐containing tetramers possess the generally lower thermal stability characteristic associated with LDH‐5 from most other animals, but thermolability is much more pronounced in the A′–A tetramers. Kinetics of the thermal inactivation process show that it is a first‐order, unimolecular event consistent with protein denaturation. Homologies of trout LDH isozymes with those of higher vertebrates are discussed in relation to these data.