High-level expression and characterization of the recombinant enzyme, and tissue distribution of human succinic semialdehyde dehydrogenase.
The succinic semialdehyde dehydrogenase gene (SSADH; EC 1.2.1.24) from human brain was cloned and overexpressed in Escherichia coli. Based on SDS-PAGE, the apparent molecular mass of subunit was 54 kDa, in good agreement with the theoretical size. The purified SSADH appears to be a tetramer of identical subunits. The specific activity of the recombinant protein was 1.82 micromol NADH formedmin(-1)mg(-1) and the optimal pH was found to be 8.5. The Michaelis constants K(m) for succinic semialdehyde and NAD(+) were 6.3 and 125 microM, respectively. Initial velocity studies show NADH to be a competitive inhibitor with respect to NAD(+), but to be non-competitive inhibitor with respect to succinic semialdehyde. The overexpression of SSADH in E. coli and one-step purification of the highly active SSADH will facilitate further biochemical studies on this enzyme. In addition, an mRNA master dot-blot for multiple human tissues provided a complete map of the tissue distribution for SSADH. The major sites of SSADH expression are liver, skeletal muscle, kidney, and brain. The data indicate that mRNA expression of SSADH is ubiquitous, but highly regulated at the level of transcription in a tissue-specific manner.
Kang
Jeong Han
JH
Department of Biochemistry, Kyungpook National University, Daegu 702-701, Republic of Korea.
Park
Yong Bok
YB
Huh
Tae-Lin
TL
Lee
Won-Ha
WH
Choi
Myung-Sook
MS
Kwon
Oh-Shin
OS
D001921:Brain / Q000201:enzymology*; D003001:Cloning, Molecular / Q000379:methods; D015870:Gene Expression / Q000235:genetics; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D007700:Kinetics; D009243:NAD / Q000737:chemistry / Q000378:metabolism; D009928:Organ Specificity / Q000502:physiology; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D050644:Succinate-Semialdehyde Dehydrogenase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D012333:RNA, Messenger; D011994:Recombinant Proteins; D009243:NAD; D050644:Succinate-Semialdehyde Dehydrogenase