Genetic Evidence for the Subunit Structure of Lactate Dehydrogenase Isozymes.

The lactate dehydrogenases commonly occur in from one to five molecular forms (isozymes) in the various tissues of an animal species.' By means of zone electrophoresis, characteristic patterns of these isozymes may be demonstrated in each tissue. In adult animals, most tissues contain all five isozymes but in different proportions. Usually, heart muscle exhibits principally the first, or most negatively-charged, isozyme (LDH-1), while skeletal muscle and liver contain chiefly the most positively-charged (LDH-5). Appella and Markert2 treated crystalline lactate dehydrogenase from beef heart with 5 M guanidine-HCl. This reagent dissociates the LDH molecule into four subunits of equal molecular weight, as determined by sedimentation studies. These subunits, or polypeptide chains, were shown to exist in two different electrophoretic varieties which Markert3 has designated as subunits A and B. Moreover, he theorized that the five isozymes are the five different tetramers that would be obtained by associating these two subunits in all possible combinations of four.2' 3 Cahn et al.,4 using chicken LDH, showed that the two "pure" tetramers (LDH-1 and -5) are immunologically distinct and, further, that the three intermediate isozymes (LDH-2, -3, and 4) are cross-reactive with both pure types. Thus, these intermediate isozymes presumably contain both kinds of subunits. Recently, Markert3 dissociated LDH-1 and LDH-5 into subunits by freezing in 1 M NaCl; after thawing, the subunits recombined at random in groups of four to yield all five isozymes in approximately the expected ratio of 1:4:6:4:1. The postulated subunit formulae for the LDH isozymes makes these relationships clear: LDH-1 = BBBB, LDH-2 = BBBA, LDH-3 = BBAA, LDH-4 = BAAA, LDH-5 = AAAA. It has been tacitly assumed that the two different polypeptides, A and B, are under separate genetic control. This study presents genetic data which support this assumption. It also reinforces the concept of a tetrameric structure of the LDH molecule. Materials and Methods.-The animals were obtained from a laboratory population of deer mice, Peromyscus maniculatus. All animals studied were at least 2 months old, and females were nonpregnant and nonlactating at the time of study. The mice were killed by decapitation, and tissues to be analyzed were removed immediately, washed in cold 0.9% saline solution, blotted, weighed, and homogenized in a glass homogenizer with the appropriate amount of cold distilled water, which was usually 3-10 times the volume of the tissue. Liver, kidney, brain, and testis were routinely studied. The homogenate was placed in a polypropylene centrifuge tube, frozen and thawed twice, and centrifuged at 25,000 X g for ten min at 4-80C. The clear supernatant was removed, and 40 microliters were added to the starch-gel insert. Vertical electrophoresis was carried out for 18 hr at a voltage