Abstract
The inactivation of bovine liver high- K m aldehyde reductase (ALR) by heat (47°C), 0.3 mM 2,4,6-trinitrobenzene sulfonate (TNBS) and 0.03 mM pyridoxal 5′-phosphate (PAL-P) followed pseudo-first-order kinetic as a function of incubation-time and concentration of TNBS. Nucleotides which have a 2′-phosphate group, especially β-NADPH and β-NADP +, showed effective protection on ALR-inactivation. However, typical substrates for ALR such as d,l-glyceraldehyde, d-erythrose, d-glucuronate and p-carboxybenzaldehyde could not protect the enzyme from inactivation. Completely inactivated enzyme was estimated to have 2.07 TNBS-modified lysine residues/mol enzyme from the determination of free amino group using fluorescamine (Ex = 390 nm, Em = 475 nm). Enzyme protected by β-NADP + (96.5% remaining activity) did not lose a significant number of lysine residues. Kd-values for β-NADPH and β-NADP + were estimated to be 0.48 μM and 4.7 μM, respectively and TNBS-treated enzyme lost its ability to bind to these nucleotides.
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