PURIFICATION AND CHARACTERIZATION OF ZETA-CRYSTALLIN FROM THE CAMEL LENS

zeta-crystallin a novel NADPH: quinone oxidoreductase was purified fro m the cortex of the camel (Camelus dromedarius) lens to homogeneity by Sepharose CL-6B gel filtration column and 2', 5' ADP-Sepharose 4B aff inity column chromatography in the presence of dithiothreitol. The pur ified zeta-crystallin has a molecular weight of 140 kDa, as determined by Superose 12 gel filtration column. SDS-PAGE showed a single polype ptide band of molecular weight 35 kDa, suggesting that the native enzy me is composed of four identical subunits. The isoelectric point of th e 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 pro tein, and an overall recovery of 53%. This enzyme was NADPH specific a nd 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 p henanthroquinone 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.