Potato tuber succinate semialdehyde dehydrogenase: purification and characterization.
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 (Mr) of 145,000 for the native enzyme. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave a single polypeptide band of Mr 35,000. Thus the enzyme appears to be a tetramer of identical subunits. Chromatofocusing of the enzyme gave a pI of 8.7. The enzyme was maximally active at pH 9.0 in 100 mM sodium pyrophosphate buffer. In 100 mM 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 Km of 4.6 microM for SSA when assayed in pyrophosphate buffer and was inhibited by that substrate at concentrations greater than 120 microM. The Km for NAD+ was found to be 31 microM. 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-ethyl-maleimide, by heavy metal ions Hg2+, Cu2+, Cd2+, and Zn2+, and by arsenite. These results indicate a requirement of a SH group for catalytic activity.
Satya Narayan
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Food Technology and Enzyme Engineering Division, Bhabha Atomic Research Centre, Bombay, India.
Nair
P M
PM
D000445:Aldehyde Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D007202:Indicators and Reagents; D007700:Kinetics; D008970:Molecular Weight; D010944:Plants / Q000201:enzymology*; D011198:Solanum tuberosum / Q000201:enzymology; D050644:Succinate-Semialdehyde Dehydrogenase; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D007202:Indicators and Reagents; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D000445:Aldehyde Oxidoreductases; D050644:Succinate-Semialdehyde Dehydrogenase