Substrate specificity of the lactate dehydrogenase isoenzyme C4 from human spermatozoa and a possible selective assay

Abstract

The activity of lactate dehydrogenase (EC 1.1.1.27) in normal human sperm lysates and in human heart and liver homogenates was determined by using a variety of 2-oxoacids as substrates. Sperm preparations were active with pyruvate, 2-oxobutanoate, 2-oxopentanoate and 2-oxohexanoate, while heart and liver extracts utilized only pyruvate and 2-oxobutanoate. Selective staining after gel electrophoresis indicated that the fraction corresponding to lactate dehydrogenase C4, the sperm-specific isoenzyme, was responsible for the utilization of substrates with a linear chain of 3 to 6 carbon atoms. The use of 5 mM 2-oxohexanoate allowed the selective determination of isoenzyme C4 in preparations containing different lactate dehydrogenase molecular forms.

Oxamic acid analogues as LDH-C4-specific competitive inhibitors

“…7,8,10,21 All LDH isozymes catalyse the reaction from pyruvate to lactate and from lactate to pyruvate. However, the LDH-C4 shows activity towards α-keto and α-hydroxy acid, of longer carbon chain than those of pyruvate and lactate (Figure 1).…”

LDH-X and HBDH/LDH Ratio in Human Semen

“…Therefore, the determination of LDH-X in the semen can represent a reliable index of sperm quality [2, 41. The assay of total LDH activity does not give a direct estimate of LDH-X values; an electrophoretic enzymogram is necessary to measure, by densitometry , the relative proportions of each isoenzyme. Some investigators [2] have viewed this procedure as unduly complex, and requiring excessive time for routine clinical usage. Furthermore, many clinical laboratories do not have easy access to the technology necessary for this assay.…”

“…Furthermore, many clinical laboratories do not have easy access to the technology necessary for this assay. The solution to this problem has been a selective assay for LDH-X using substrates for which it has higher affinity than the rest of LDH isoenzymes [2,8,9]. Within this concept, the strong affinity shown by LDH-X for a-ketobutyrate has allowed us to measure the levels of HBDH and establish the ratio HBDHILDH, whose estimation is simple, rapid, sensitive, available to any laboratory, and appears to be a more accessible technique for looking at LDH-X activity.…”

“…1.1.1.27), is an isoenzyme exclusive to the germinal cells, and is essential to glycolysis in these cells. It is identifiable electrophoretically in human semen between isoenzymes LDH-3 and LDH-4 [2,4,6,8]. The assay of lactate dehydrogenase (LDH) does not give a direct estimation of isoenzyme LDH-X, and it is necessary to rely upon electrophoretic separation of LDH, and later quantification, to determine the percentage of this specific isoenzyme present.…”

“…The assay of lactate dehydrogenase (LDH) does not give a direct estimation of isoenzyme LDH-X, and it is necessary to rely upon electrophoretic separation of LDH, and later quantification, to determine the percentage of this specific isoenzyme present. A direct analysis for LDH-X has been done using as substrates compounds such as the a-ketoacids with chains longer than pyruvic acid, for which LDH-X has great affinity [2,8,91.…”