Abstract

Lactate dehydrogenase-X from testes of several rodent species was purified to homogeneity by an 8-(6-aminohexyl)-amino-AMP-Sepharose affinity column. In the case of mouse, the testicle extracts was first heated to 60° for fifteen minutes before the passage through the affinity column. A biospecific elution with reduced NAD+-pyruvate adduct resulted in a homogeneous preparation of lactate dehydrogenase-X. A similar procedure was also employed for the purification of lactate dehydrogenase-X from hamster, guinea pig and rat. After purification by affinity chromatography, lactate dehydrogenase-X was separated from residual somatic lactate dehydrogenase isozymes by DEAF-Sephadex chromatography. Adenosine, AMP, ADP, and ADP-ribose were shown to be coenzyme-competitive inhibitors of lactate dehydrogenase-X. The effectiveness of binding of these compounds increased with the size of the adenosine derivatives employed. Multiple inhibition analysis suggested that these compounds are interacting with the same region of coenzyme-binding site as shown by the mutual exclusion of one another from binding to the enzyme. The data suggest that the binding of coenzyme to the enzyme occurs through interactions involving the adenosine moiety and pyrophosphate grouping. Fluorescence spectroscopy was employed for the study of the mechanism of action of mouse lactate dehydrogenase-X. Both oxidized and reduced coenzymes induced significant quenching of protein fluorescence. Significant enhancements of NADH fluorescence and protein energy transfer were observed upon the addition of lactate dehydrogenase-X to the coenzyme solution. In the presence of lactate dehydrogenase-X and NAD+, the addition of pyruvate or α-ketovalerate resulted in a time-dependent quenching of protein fluorescence and an increase in absorbance at 325 nm indicating the formation of a ternary complex. The results of this study suggest a similar molecular mechanism for different lactate dehydrogenase isozymes.

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