Succinic semialdehyde dehydrogenase from mammalian brain: Subunit analysis using polyclonal antiserum

1. 1. NAD⁺-dependent succinic semialdehyde dehydrogenase was purified to apparent homogeneity from rat brain and highly purified from human brain. 2. 2. Molecular exclusion chromatography of the purified enzymes on Sephadex G-150 and G-200 revealed M_r values of 203,000 and 191,000 for rat and human, respectively. 3. 3. Electrophoresis on sodium dodecylsulfate polyacrylamide gels revealed a single subunit of M_r 54,000 for rat and 58,000 for human. Isoelectric focusing of the purified rat enzyme yielded a pI of 6.1. 4. 4. For both proteins, K_m values for short-chain aldehydes acetaldehyde and propionaldehyde ranged from 0.33 to 2.5 mM; K_m values for succinic semialdehyde were in the 2-4 μM range. 5. 5. The subunit structure of both enzymes was investigated in brain extracts and purified preparations by immunoblotting, using a polyclonal rabbit antiserum against the purified rat brain enzyme. 6. 6. For rat and human extracts, single bands were detected at M_r, 54,000 and 58,000, comparable to findings in the purified preparations. Immunoblotting analyses in other species (guinea pig, hamster, mouse and rabbit) revealed single subunits of M_r, 54,000-56,500.