title: Alanine dehydrogenase from soybean nodule bacteroids: purification and properties.
abstract: Alanine dehydrogenase (ALADH) from soybean nodule bacteriods was purified 184-fold with 14% yield, using ammonium sulfate precipitation, hydroxylapatite, gel filtration, ion exchange, and dye affinity chromatography. The subunit molecular weight was 43,000 and the native molecular weight was approximately 190,000, suggesting that ALADH is a tetramer. ALADH was confined to the bacteroid cytosol fraction only. ALADH is specific for NAD(H) and does not use NADP(H) as a substrate, but it does use glyoxylate and hydroxypyruvate as substrates in lieu of pyruvate. The pH optimum was 8.5 for the amination reaction and 10.0 for the deamination reaction. The apparent Michaelis constants for NADH, NH4+, pyruvate, L-alanine, and NAD were 86 microM, 8.9 mM, 0.49 mM, 1 mM and 200 microM, respectively. High concentrations of pyruvate, L-alanine, or NH4+ caused inhibition of activity with Ki's of 8.6 mM, 6.5-15 mM, and 188 mM, respectively. The amination reaction of ALADH was 95-100% of the control at levels of NADH/NAD corresponding to those measured in isolated bacteroids. The deamination reaction, on the other hand, was only 35-40% of control. Thus, an aminating role for ALADH is possible.
authors.affiliation: Department of Biochemistry, University of Missouri, Columbia 65211.
authors.forename: M T
authors.initials: MT
authors.lastname: Smith
authors.forename: D W
authors.initials: DW
authors.lastname: Emerich
mesh_terms: D050788:Alanine Dehydrogenase; D000594:Amino Acid Oxidoreductases / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D000641:Ammonia / Q000378:metabolism; D002451:Cell Compartmentation; D003600:Cytosol / Q000201:enzymology; D009243:NAD / Q000378:metabolism; D011773:Pyruvates / Q000378:metabolism; D012230:Rhizobiaceae / Q000201:enzymology*; D012965:Sodium Chloride / Q000494:pharmacology; D013025:Glycine max / Q000382:microbiology; D013559:Symbiosis
chemical_list: D011773:Pyruvates; D009243:NAD; D012965:Sodium Chloride; D000641:Ammonia; D000594:Amino Acid Oxidoreductases; D050788:Alanine Dehydrogenase