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Korean Society for Biochemistry and Molecular Biology (생화학분자생물학회)
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Purification and Reaction Mechanism of Rat Brain Succinic Semialdehyde Dehydrogenase

  • Kim, Kyu-Tae (Department of Biochemistry, College of Science, Yonsei University) ;
  • Joo, Chung-No (Department of Biochemistry, College of Science, Yonsei University)
  • Received : 1994.10.21
  • Published : 1995.03.31
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Abstract

Rat brain succinic semialdehyde dehydrogenase (EC 1.2.1.24 SSADH) activity was detected in mitochondrial, cytosolic and microsomal fractions. Brain mitochondrial soluble SSADH was purified by ammonium sulfate precipitation, DEAE Sephacel, and 5'-AMP Sepharose 4B affinity chromatography. The purified enzyme was shown to consist of four identical subunits, and the molecular weight of a subunit was 55 kD. The $K_m$ for short chain aliphatic aldehydes and aromatic aldehydes were at the $10^{-3}M$ level but that for succinic semialdehyde was 2.2 ${\mu}M$. Either $NAD^+$ or $NADP^+$ can be used as a cofactor but the affinity for $NAD^+$ was 10 times higher than that for $NADP^+$. The brain cytosolic SSADH was also purified by ammonium sulfate precipitation, DEAE Sephacel, Blue Sepharose CL-6B and 5'-AMP Sepharose 4B affinity chromatography and its Km for short chain aliphatic aldehydes was at the $10^{-3}$ level but that for succinic semialdehyde was 3.3 ${\mu}M$. $NAD^+$ can be used as a cofactor for this enzyme. We suppose that both enzyme might participate in the oxidation of succinic semialdehyde, which is produced during GABA metabolism. The activity of both cytosolic and mitochondrial SSADH was markedly inhibited when the concentration of succinic semialdehyde was high. The reciprocal plot pattern of product inhibition and initial velocity indicated a sequential ordered mechanism for mitochondrial matrix SSADH. Chemical modification data suggested that amino acid residues such as cysteine, serine and lysine might participate in the SSADH reaction.

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