PURIFICATION AND CHARACTERIZATION OF ZETA-CRYSTALLIN FROM THE CAMEL LENS

zeta-crystallin a novel NADPH: quinone oxidoreductase was purified from the cortex of the camel (Camelus dromedarius) lens to homogeneity by Sepharose CL-6B gel filtration column and 2', 5' ADP-Sepharose 4B affinity column chromatography in the presence of dithiothreitol. The purified zeta-crystallin has a molecular weight of 140 kDa, as determined by Superose 12 gel filtration column. SDS-PAGE showed a single polypeptide band of molecular weight 35 kDa, suggesting that the native enzyme is composed of four identical subunits. The isoelectric point of the enzyme was 7.6 on native polyacrylamide gel. The enzyme was purified 20-fold over homogenate with a specific activity of 26.0 Units/mg protein, and an overall recovery of 53%. This enzyme was NADPH specific and followed Michaelis-Menten kinetics. K-m values for the reduction of 9,10-phenanthroquinone and oxidation of NADPH were 17 mu M and 6.9 mu M, respectively, at pH 7.8. The V-max values of the enzyme for 9,10 phenanthroquinone and NADPH were 32 mu mole min(-1), mg(-1) protein and 22.7 mu mole min(-1) mg(-1) protein, respectively. (C) 1995 Acadenic Press, Inc.