53] Glycerol-3-phosphate dehydrogenase from the honey bee

Abstract

Extramitochondrial glycerol-3-phosphate dehydrogenase plays a central role in insect flight metabolism. It is one of the two enzymic members of the glycero-P cycle, which accounts for rapid reoxidation of cytoplasmic DPNH during glycolysis. The activity of this dehydrogenase is very high in the flight muscles of insects, such as honey bees, which utilize carbohydrate as fuel. This chapter examines the assay method, purification procedure, and properties of glycerol-3-phosphate dehydrogenase from the honey bee. The method is based upon the oxidation of NADH by dihydroxyacetone-P. The reverse reaction can also be used, but it often does not show reproducible kinetics. In the early stages of purification, protein is determined by the biuret reaction. The protein is first precipitated by addition of trichloroacetic acid to a final concentration of 10% (w/v), washed once with 10% (w/v) trichloroacetic acid, and once with ether. The precipitation of the protein eliminates interference of (NH 4 ) 2 SO 4 in the biuret reaction. Bovine serum albumin is used as protein standard. The enzyme preparation is homogeneous by the following criteria: (a) purification to a constant specific activity, (b) electrophoresis on cellulose acetate at several pH values, (c) disc gel electrophoresis, (d) gel electrophoresis in the presence of sodium dodecylsulfate, (e) sedimentation in the ultracentrifuge, (f) reaction of rabbit antibodies prepared against the enzyme with honey bee extracts. The honey bee enzyme binds to honey bee actin, as do some of the bumble bee isozymes. Glycerol-3-P dehydrogenase is localized in honey bee flight muscle with immunofluorescent techniques; it is located at or near the Z-band cross striations.