Presence of bound substrate in lactate dehydrogenase from carp liver.

While attempting to purify UDP-galactose 4-epimerase from carp liver extract at pH 8.0, it was observed that the preparation even after dialysis could reduce NAD to NADH, interfering epimerase assay. The NAD reduction activity and the epimerase were co-eluted in a series of chromatographic steps. Mass spectrometric analysis of semi-purified fraction revealed that carp liver lactate dehydrogenase (LDH) contained bound lactate which was converted to pyruvate in the presence of NAD. The enzyme-bound lactate and the association with epimerase stabilized LDH from trypsin digestion and thermal inactivation at 45°C by factors of 2.7 and 4.2 respectively, as compared to substrate-free LDH. LDH and epimerase do not belong to any one pathway, but are the rate-limiting enzymes of two different pathways of carbohydrate metabolism. Typically, strongly associated enzymes work in combination, such as two enzymes of the same metabolic pathway. In that background, co-purification of LDH and epimerase as reloaded in this study was an unusual phenomenon.

LDH-C4-substrate binary complexes studied by intrinsic fluorescence method.

Lactate dehydrogenase-C4 (LDH-C4) has been studied in presence of substrates using intrinsic fluorescence measurements. Excitation maximum of LDH-C4 occurred at 282 nm whereas fluorescence emission maximum was obtained at 340 nm. Fluorescence intensities at 340 nm showed that ligands viz. NAD+, NADH, pyruvate and lactate quench the relative fluorescence intensities of LDH-C4 in a concentration dependent manner. NAD+ and NADH produced a maximum quenching between 92-93% while pyruvate and lactate quenched the fluorescence of LDH up to 29% and 21% respectively. Association constants (Ka) based on fluorescence measurements were 6.05 x 10(4)M-1, 20 x 10(4)M-1, 0.113 x 10(4)M-1 and 0.3 x 10(4)M-1, for NAD+, NADH, lactate and pyruvate respectively. Stern-Volmer constants (Ksv) show that NAD+ and NADH have single Ksv of 4.07 x 10(4)M-1 and 1.47 x 10(5)M-1, whereas lactate and pyruvate indicated quenching reaction to be made up of two components. Ksv at low and high concentration of lactate respectively were 0.645 x 10(2)M-1 and 0.05 x 10(2)M-1, whereas corresponding Ksv with pyruvate were 1.008 x 10(3)M-1 and 0.408 x 10(3)M-1. Low Ksv at higher concentrations suggested that the aromatic chromophores are located within a hydrophobic environment. Red shift in fluorescence maximum (lambda max) by 2nm with lactate and 6nm with pyruvate showed that interaction of these ligands with LDH-C4 exposes some buried chromophores of the enzyme to the surface.