60] Triosephosphate dehydrogenase from the honey bee

Abstract

This chapter discusses the triosephosphate dehydrogenase from the honey bee. The assay method is based upon the reduction of NAD + by glyceraldehyde-3-P in the presence of arsenate. In the early stages of purification, protein is determined by the biuret reaction. The protein is first precipitated by addition of trichloracetic acid to a final concentration of 10% (w/v). The centrifuged pellet is washed once with 10% trichloroacetic acid and once with ether. This protein precipitation eliminates interference of (NH 4 ) 2 S0 4 in the biuret reaction. Bovine serum albumin is used as protein standard. After the initial crystallization of triose-P dehydrogenase, concentrations are measued by absorbance at 280 nm using an extinction coefficient of 1.00 ml mg -1 cm -1 . Purification of triosephosphate dehydrogenase from honey bees is described. The preparation of thoraces and of the homogenate described in the chapter is identical to that of the isolation of glycerol-3-P dehydrogenase. The 2.2 M (NH 4 ) 2 SO 4 supernatant from the glycerol-3-P dehydrogenase isolation can be used to isolate triose-P dehydrogenase. Thus, four proteins can be isolated from one bee extract. The effect of glyceraldehyde-3-P and NAD + concentrations on the reaction velocity of the honey bee enzyme has been reported. In the presence of 4.3 m M DL-glyceraldehyde-3-P, a kinetic transition is observed when the NAD + concentration is raised from 0.16 to 0.18 m M . In the presence of 1 m M NAD + , a similar transition occurs when D-glyceraldehyde-3-P is increased from 0.62 to 0.72 m M . Half-maximal velocity of the honey bee enzyme is attained with about 40 μ M NAD + or 200 μ M D-glyceraldehyde-3-P. Iodoacetate, N- ethylmaleimide, ATP, ADP, AMP, and 3’,5’-AMP inhibit the honey bee enzyme. Other properties of honey bee enzyme are also discussed.