Abstract
An NAD-dependent alcohol dehydrogenase has been purified to apparent homogeneity from cell suspension cultures of Lithospermum erythrorhizon Sieb. et Zucc. (Boraginaceae), using protamine sulphate and ammonium sulphate precipitation and chromatography on DEAE-Sephacel, Superdex 200, hydroxyapatite and HiTrap blue. The enzyme is a homodimer with a Mr of ca. 77,000. Each subunit with a Mr of 40,000 contains two zinc atoms. Its isoelectric point was found at pH 5.0. The best alcohol substrate of the enzyme is ethanol. The pH optimum for ethanol oxidation is at pH 8.7 and for acetaldehyde reduction at pH 4.6. The Michaelis constants for ethanol and NAD are 2.49 and 0.05 (pH 8.7), and for acetaldehyde and NADH 2.2 and 0.078 mM (pH 4.6), respectively. Partial amino acid sequences of the purified enzyme showed high homology to alcohol dehydrogenases from other plants.
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Abbreviations
- ADH:
-
alcohol dehydrogenase
- DTT:
-
dithiothreitol
- PMSF:
-
dephenylmethylsulfonyl fluoride
- PVPP:
-
polyvinylpolypyrrolidone
- IAA:
-
indole-3-acetic acid
- TFA:
-
trifluoroacetic acid
References
Ashihara H, Horikosi T, Li XN, Sagishima K, Yamashita Y (1988) J. Plant Physiol. 133: 38–45
Bicsak TA, Kann LR, Reiter A, Chase T (1982) Arch. Biochem. Biophys. 216: 605–615
Bradford MM (1976) Anal. Biochem. 72: 248–254
Bruemmer J H, Roe B (1971) J. Agr. Food Chem. 19: 266–268
Danielsson O, Atrian S, Luque T, Hjelmqvist L, Gonzàlez-Duarte R, Jörnvall H (1994) Proc. Natl. Acad. Sci. USA 91: 4980–4984
Davies DD, Ugochukwu EN, Patil KD, Towers GHN (1973) Phytochemistry 12: 531–536
Dolferus R, Van den Bossche D, Jacobs M (1990) Mol. Gen. Genet. 224: 297–302
Felder MR, Scandalios JG, Liu EH (1973) Biochim. Biophys. Acta 318: 149–159
Freeling M, Bennett DC (1985) Ann. Rev. Genet. 19: 297–323
Fukui H, Yoshikawa N, Tabata M (1984) Phytochemistry 23: 301–305
Hatanaka A, Kajiwara T, Tomohiro S, Yamashita H (1974) Agr. Biol. Chem. 38: 1835–1844
Igaue I, Yagi M (1982) Plant Cell Physiol. 23: 213–225
Igaue I, Yagi M, Hayakawa T (1982) Niigata Daigaku Nogakubu Kenkyu Hokoku 45–53
Irigoyen JJ, Emerich DW, Sánchez-Díaz (1992) Physiol. Plant. 84: 61–64
Jarillo JA, Leyva A, Salinas J, Martínez-Zapater JM (1993) Plant Physiol. 101: 833–837
Jörnvall H, Danielsson O, Eklund H, Hjelmqvist L, Höög J-O, Parés X, Shafqat J (1993) In: Weiner H, Grabb DW, Flynn TG (eds) Enzymology and molecular biology of carbonyl compounds 4. Plenum Press, New York, pp 533–544
Laemmli UK (1970) Nature (London) 227: 680–685
Lineweaver H, Burk D (1934) J. Amer. Chem. Soc. 56: 658–660
Magonet E, Hayen P, Delforge D, Delaive E, Remacle J (1992) Biochem. J. 287: 361–365
Matsudaira PT (1989) A practical guide to protein and peptide purification for microsequencing. Academic Press, San Diego, pp 3–12
Nicolas M, Crouzet J (1980) Phytochemistry 19: 15–18
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. A laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, New York, Chapter 18
Tabata M, Fujita Y (1985) In: Day P, Zaitlin M, Hollaender A (eds) Biotechnology in Plant Science. Academic Press, Orlando, Florida, pp 207–218
Tabata M, Mizukami H, Hiraoka N, Konoshima M (1974) Phytochemistry 13: 927–932
Wastemack C, Gurannowski A, Glund K, Towers A, Walther, R. (1985) J. Plant Physiol. 120: 19–28
Yamashita I, Iino K, Yoshikawa S (1978) Agric. Biol. Chem. 42: 1125–1132
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Li, SM., Wang, ZX. & Heide, L. Purification and characterization of an alcohol dehydrogenase from Lithospermum erythrorhizon cell cultures. Plant Cell Reports 15, 786–790 (1996). https://doi.org/10.1007/BF00232230
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DOI: https://doi.org/10.1007/BF00232230