Effect of amino acids on inhibition of lactate dehydrogenase-X by gossypol

Gossypol acetic acid (GAA) has been shown to have male antifertility effects, but there are pronounced differences among animal species. In the search of endogenous effector molecules, which interfere with the functions of GAA, we have studied the in vitro effect of various amino acids on the inhibition of the purified LDH-X by GAA. Histidine, cysteine and glycine were shown to block the effect of GAA. The effects of these amino acids were concentration dependent. Histidine and glycine protection was found to be complex type in which both the Km and Vmax were decreased compared to control. Arginine, glutamic acid, phenylalanine and valine were found to be ineffective against the inhibitory action of GAA.

Purification and characterization of lactate dehydrogenase from Varanus liver

Lactate dehydrogenase was purified 21-fold from liver of Varanus bengalensis using colchicine-sepharose column chromatography. The crude enzyme showed two isoenzymes (LDH-5 and LDH-4) by agarose gel electrophoresis (AGE). The purified enzyme showed a single band after SDS-PAGE corresponding to molecular mass of 35 kDa. The molecular mass of native enzyme was about 140 kDa. The optimum pH for the forward reaction was 7.5 while that for the reverse reaction was pH 9.5. The K-m values for pyruvate, NADH, lactate and NAD(+) were 0.17 +/- 0.037, 0.02 +/- 0.004, 12.4 +/- 3.05 and 0.38 +/- 0.032 mM, respectively. Pre-heating of enzyme showed that its t(50) was 40-50 degrees C. Oxalate and n-hexanediol were inhibitors for both forward and reverse reactions. Among divalent ions, Cu++ was shown to be more effective inhibitor for the forward reaction.

Estimation and separation of N-acetyl-beta-D-glucosaminidase isoenzymes in urine

N-acetyl Beta-D-glucosaminidase is a lysosomal enzyme made up of two isoenzymes [A and B]. It has been used extensively as a marker for kidney damage. However, its estimation in urine has not been standardized. We have established a method for the estimation and separation of NAG isoenzymes by ion-exchange chromatography. In 19 experiments done so far, this method has given reproducible results. The significance of this method is that with a single experiment, one can estimate total as well as individual isoenzyme activity. Furthermore, urine constituents do not appear to interfere in this assay

Effect of various metals and metabolites on the activity of lactete dehydrogenase

The effects of various chemicals were studied on the activity of purified lactate dehydrogenase [LDH] from buffalo's liver. Gossypol acetic acid, cupric and mercuric ions had completely inhibited the enzyme. Zinc showed inhibition only for reverse reaction. Lead, nickel and silver had shown more inhibition for forward reaction while cobalt produced inhibition only for forward reaction. The inhibition of reverse reaction by two metal chelators [hydroxyquinoline and dipyridyl] suggests that LDH from buffalo liver may require metal ions for its activity in reverse direction only. Oxalate also inhibited both sides of reaction and its Ki with respect to pyruvate was found to be 0.35 mM. Most of the nucleotides tested had almost no effect on the activity of LDH on either direction. Pyrophosphate and n - hexanediol also had shown no effect