Determination of isoenzyme contents of lactic dehydrogenase activity and 2-hydroxybutyric dehydrogenase activity in lactic dehydrogenase preparations.

In this inestigation, a determination of the isoenzyme contents of LDH and HBD activities in lactate dehydrogenase preparations and the differences in the interaction of these preparations with NAD analogues were examined. The results obtained were as follows. 1. The activity ratio between oxidation and reduction in LDH reaction is shown to be similar in both H4 and M4 preparations, whereas for HBD activity, the ratio seems to be lower in the M4 preparation than in H4(H4/M = 1/2). 2. NAD and its analogues (NXD, TNAD, and TNXD) are shown to be useful coenzymes for the LDH reaction, while 3-acetyl derivatives appear to be unsuitable for this purpose because of their lower activity. HBD activity with 3-acetyl NXD is shown to be higher than with TNAD or TNXD. among these NAD analogues, 3-acetyl NXD gives the highest HBD activity, especially in the M4 preparation. 3. The LDH activity of H4 relative to M4 preparations has been shown to be maximal when 450 mM lactic acid with NAD or 15 mM lactic acid with TNXD are used. Under these conditions, the contents of LDH subunits can be estimated with considerable reliability. As to HBD activity, the content of LDH subunit having HBD activity has been estimated by determing the enzyme activity under conditions in which either 300 mM 2-hydroxybutyrate with 3-acetyl NXD or 15 mM 2-hydroxybutyrate with NAD are employed.