Masood H. Javed

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.

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.

GroEL protects the sarcoplasmic reticulum Ca ‐dependent ATPase from inactivation in vitro

The molecular chaperone, GroEL, facilitates correct protein folding and inhibits protein aggregation. The function of GroEL is often, though not invariably, dependent on the cochaperone, GroES, and ATP. In this study it is shown that GroEL alone substantially reduces the inactivation of purified Ca++‐ATPase from rabbit skeletal muscle sarcoplasmic reticulum. In the absence of GroEL, the enzyme became completely inactive in about 45‐60 hours when kept at 25°C, while in the presence of an equimolar amount of GroEL, the enzyme remained approximately 80% active even after 75 hours. Equimolar amounts of BSA or lysozyme were unable to protect the enzyme from inactivation under identical conditions. Analysis by SDS‐PAGE showed GroEL was acting by blocking the aggregation of ATPase at 25°C. GroEL was not as effective in protection at ‐20°C or 4°C. These results are discussed in the context of current models of the GroEL mechanism.