Separation of rat lactate dehydrogenase isoenzyme C4 from other isoenzymes by affinity and ion-exchange chromatography

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Abstract
Lactate dehydrogenase [LDH] C, an isoenzyme composed of C polypeptide subunits and found only in mature testes and spermatozoa, differs kinetically, chemically and immunologically from the 5 common isoenzymes of LDH, each of which is a tetramer of A and/or B subunits. In the rat LDH C exists in 2 molecular forms, isoenzymes C4 and A1C3. In addition to these 2 forms of LDH C, rat testicular homogenate contains all the 5 isoenzymes of A and B type. Purification of isoenzyme C4 requires its separation from the other 6 isoenzymes, of which isoenzymes A1C3 and A3B1 are the most difficult ones to separate. Isoenzyme A3B1, along with other isoenzymes, was separated from isoenzyme C4 by AMP-Sepharose chromatography by using a gradient of increasing concentration of NAD+-pyruvate adduct. In the next step, isoenzyme A1C3 was separated from isoenzyme C4 by DEAE-cellulose chromatography, resulting in a pure LDH isoenzyme C4 preparation.