1. Studies were carried out with pure lactate dehydrogenase isoenzymes C4 (LDH isoenzyme X), B4, (LDH isoenzyme 1) and A4 (LDH isoenzyme 5) isolated from mouse testis, heart and muscle tissue respectively; with LDH isoenzyme X purified from pigeon testes and with crude lysates of spermatozoa from man, bull and rabbit. 2. LDH isoenzyme X from all species showed greater ability than the other isoenzymes to catalyse the NAD+-linked interconversions of 2-oxobutanoate into 2-hydroxybutanoate and of 2-oxopentanoate into 2-hydroxypentanoate. 3. Mouse LDH isoenzyme X presented the broadest spectrum of substrate specificity. It exhibited very similar Km values for a variety of 2-oxo acids: 2-oxopropanoate (pyruvate), 2-oxobutanoate, 2-oxo-3-methylbutanoate, 2-oxopentanoate, 2-oxo-3-methylpentanoate, 2-oxo-4-methylpentanoate, 2-oxohexanoate and 2-oxo-3-phenylpropanoate (phenylpyruvate). The corresponding 2-hydroxy acids were also readily utilized in the reverse reaction. A strong inhibition by substrate and product was demonstrated for the direct reaction. 4. Intracellular distribution of LDH isoenzyme X was investigated in mouse testes. LDH isoenzyme X activity was located in the fraction of “heavy mitochondria” and in the soluble phase. 5. A possible functional role for LDH isoenzyme X is proposed: the redox couple-2-oxo acid-2-hydroxy acid could integrate a shuttle system transferring reducing equivalents from cytoplasm to mitochondria.
Gossypol, a phenolic compound isolated from the cotton plant, is a powerful inhibitor of nicotinamide adenine dinucleotide-linked enzymes (alpha-hydroxyacid dehydrogenase and malate dehydrogenase) of Trypanosoma cruzi, the parasite that causes Chagas' disease. Parasites at the epimastigote stage that were incubated for 5 minutes with 100 micromolar gossypol were completely immobilized. Concentrations of gossypol as low as 0.01 micromolar markedly reduced the growth rate of T. cruzi in culture.
The proposed dual intracellular distribution of the sperm-specific lactate dehydrogenase (EC 1.1.1.27) isozyme C4 (LDH C4) has been based on indirect evidence. In order to obtain direct evidence on the localization of this LDH isozyme in mice, postembedding immunocytochemistry at ultrastructural level was performed on testes, epididymal spermatozoa, and isolated testicular mitochondria. The immunogold technique was applied to thin sections incubated first in partially purified specific anti-LDH C4 rabbit IgG, and immunoreactive sites were detected with colloidal gold adsorbed to anti-rabbit IgG. In the testis, immunostaining was found in the cytoplasm of spermatocytes and spermatids and in the principal and middle pieces of differentiating spermatozoa. Spermatozoa from epididymis also exhibited heavy labeling of colloidal gold in their middle and principal pieces, but the immunostaining was weak in the special type of mitochondria present in spermatocytes, spermatids, and spermatozoa (sperm-type mitochondria, STM). The isolation of STM produced several morphological changes in comparison with those in situ, including an enhancement of the LDH C4 labeling in the mitochondrial matrix. The other type of mitochondria (non-STM) from spermatocytes and nonspermatogenic cells were not immunostained and served as background control. The results presented here confirm previous findings, gathered by indirect methods, indicating a dual localization of LDH C4 in the cytosol of spermatocytes, spermatids, and spermatozoa, as well as in the matrix of sperm-type mitochondria.
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