Insect extramitochondrial glycerophosphate dehydrogenase I. Crystallization and physical properties of the enzyme from honeybee (Apis mellifera) thoraces

Abstract

Extramitochondrial glycerophosphate dehydrogenase (L-glycerol-3-phosphate: DPN⁺ oxidoreductase, EC 1.1.1.8) was purified and crystallized from honeybee (Apis mellifera) thoraces. The isolation procedure is quite simple, since it consists merely of a series of (NH₄)₂SO₄ precipitations.

The crystalline enzyme is homogeneous according to the following criteria: recrystallization to constant specific activity, electrophoresis on cellulose acetate strips, absence of 3 other glycolytic enzyme activities, chromatography on Sephadex G-200, and immunodiffusion on cellulose acetate strips.

Sedimentation velocity studies indicate that the native protein readily associates at moderate protein concentrations. The minimum molecular weight as determined by Sephadex G-200 chromatography is around 67 000.

Absorbance and fluorescence spectra show that neither DPNH nor adenosine diphosphate ribose is bound to the enzyme.

The bee enzyme differs from the crystalline rabbit-muscle enzyme in electrophoretic, ultracentrifugal, and immunological properties.