This chapter focuses on Aminoadipic Semialdehyde - Glutamate Reductase. An enzyme, aminoadipic semialdehyde-glutamate reductase, has been purified over 100-fold from bakers' yeast, which catalyzes the reversible reaction. Enzyme activity is conveniently assayed by measuring the saccharopine-dependent reduction of nicotinamide adenine dinucleotide phosphate (NADP) (or NAD) spectrophotometrically at 340 mμ, reverse reaction. The complete assay system contains 2 micromoles of saccharopine; 2 micromoles of NADP; 80 micromoles of glycine- sodium hydroxide (NaOH), pH 9.5, buffer; and enzyme protein in a volume of 2.0 ml. A unit is the amount of enzyme that gives an absorbance change of 0.001 per minute at 25 °. If crude extracts contain sufficient nicotinamide adenine dinucleotide phosphate (NADPH) oxidase to interfere seriously with this assay, an alternative possibility is to measure the aminoadipic semialdehyde formed as the o-aminobenzaldehyde adduct, that can also be measured spectrophotometrically. In view of the relative instability of aminoadipic semialdehyde coupled with the ease of studying reaction in the reverse direction, it is desirable rather to prepare saccharopine to permit this latter preferred method of assay. A description of the isolation of saccharopine from yeast is given in the last section of this chapter.
