The possible effects of reduction and/or alkylation on LH are shown schematically in Fig. 2a; treatment with DTT results in the reduction of -S-S- bond which can reoxidize and provide an active enzyme on DTT removal (Fig. 2b); alkylation has no effect on LH because no free thiols are present (Fig. 2c); alkylation of reduced LH after treatment with DTT results in inactivation, even on the removal of DTT. The significant drop of 91% activity of reduced/alkylated LH strongly suggests that the second disulphide bond plays an important role in the LH activity. Veliparib cell line Cadmium interacts readily with thiols and prevents disulphide bond formation
in proteins (Stafford et al., 1999). The activated LH was reduced with DTT, and varying concentrations of CdCl2 (0–25 mM) were added to the enzyme
assay and incubated for 1 h (Fig. 3a). Measurement of the LH activities showed increasing amounts of Cd2+ resulted in substantial loss of activity of up to fivefold, as in data obtained from LH inactivation by iodomethane, confirming that two Cys residues were most likely disulphide bonded. In another experiment, CdCl2 ranging Idelalisib price from 0 to 10 mM was added to the assay mixture containing pre-activated LH, and showed activity loss of up to 11-fold (Fig. 3b). Because Cd2+ would not be expected to break a disulphide bond (Tan et al., 2005), these BCKDHA results suggest that it may have been prereduced during catalysis. Such reduction would enable Cd2+ to block the thiol groups formed and thus inhibit activity. The effect of Cd2+ on the -S-S- bond containing protein is shown in Fig. 2d, where Cd2+ crosslinks with newly formed thiol groups and subsequently inhibits re-oxidation of the bond. To determine whether LH activity was inhibited by Cd2+ in a dose-dependent manner, a second experiment was performed where increasing amounts of Cd2+ were added to the same enzyme assay at two intervals over a total of 3 min (data not shown). The results showed increasing amounts of Cd2+-inhibited
enzymic activity in a dose-dependent manner. The experiments conducted above indicated that 124Cys and 143Cys of LH are putatively disulphide bonded and that this bond probably plays a crucial role in the structure and/or the generation of enzymic activity. Plasmids pGEM-LH-His4-Δ143CysSer and pBlue-LH-His4-Δ124,143CysSer were separately transformed into E. coli TB1 cells with plasmid pEC86, producing clones pEC86/pGEM-LH-His4-Δ143CysSer and pEC86/pBlue-LH-His4-Δ124,143CysSer, respectively. Expression of the luh gene harboured in pEC86/pGEM-LH-His4-Δ143CysSer, pEC86/pBlue-LH-His4-Δ124,143CysSer and pEC86/pINK-LH-His4 was undertaken in phosphate-limited MOPS medium at 22 °C for 18 h. The periplasmic extract of the control had a strong pink colour, whereas the mutant forms had fainter pink colorations.