Abstract
Succinate semialdehyde dehydrogenase (SSADH) has been purified from potato tubers with 39% yield, 832-fold purification, and a specific activity of 6.5 units/mg protein. The final preparation was homogeneous as judged from native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel filtration on Sepharose 6B gave a relative molecular mass ( M r) of 145,000 for the native enzyme. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave a single polypeptide band of M r 35,000. Thus the enzyme appears to be a tetramer of identical subunits. Chromatofocusing of the enzyme gave a p I of 8.7. The enzyme was maximally active at pH 9.0 in 100 m m sodium pyrophosphate buffer. In 100 m m Tris-HCl buffer, pH 9.0, the enzyme gave only 20% of the activity found in pyrophosphate buffer and had a shorter linear rate. The enzyme was specific for succinate semialdehyde (SSA) as substrate and could not utilize acetaldehyde, glyceraldehyde 3-phosphate, malonaldehyde, lactate, or ethanol as substrates. The enzyme was also specific for NAD + as cofactor and NADP + and 3-acetylpyridine adenine dinucleotide could not serve as cofactors. Potato SSADH had a K m of 4.6 μ m for SSA when assayed in pyrophosphate buffer and was inhibited by that substrate at concentrations greater than 120 μ m. The K m for NAD + was found to be 31 μ m. The enzyme required exogenous addition of a thiol compound for maximal activity and was inhibited by the thiol-directed reagents p-hydroxymercuribenzoate, dithionitrobenzoate, and N-ethylmaleimide, by heavy metal ions Hg 2+, Cu 2+, Cd 2+, and Zn 2+, and by arsenite. These results indicate a requirement of a SH group for catalytic activity.
Published Version
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