In vitro dissociation and reassociation of human alcohol dehydrogenase class I isozymes.
Freezing (-78 degrees C) and thawing (25 degrees C) a heterodimeric human alcohol dehydrogenase class I isozyme in the presence of 0.1 M sodium phosphate/0.1 mM DTT, pH 7.0, and the subsequent separation of the scrambled isozymes by HPLC are used to prepare homodimers from heterodimers, with recovery of enzyme activity ranging from 80 to 95%. The ratio of the three isozymes obtained from a heterodimer follows the binomial distribution of 1:2:1, indicating random reassociation of the two subunits. The physical and enzymatic properties of the reassociated isozymes are the same as those obtained directly from human liver preparations. The nature of subunit-subunit interactions of human ADH class I isozymes is examined by optimizing the conditions required for the formation of the new dimers "in vitro". The effect of a number of reagents previously used in the reversible dissociation of dehydrogenases is investigated. The coenzyme NAD+ is a potent inhibitor of the dissociation of dimers during the freeze/thaw procedure. The presence of sodium phosphate in the enzyme solution is essential during the freezing and thawing experiment. No appreciable dissociation/reassociation occurs in TES, HEPES, or even potassium phosphate. The reversible dissociation is due primarily to the decrease in pH because of the low solubility of Na2HPO4 at low temperatures. The reassociation occurs after thawing in a temperature-dependent process. There is no reactivation if the enzyme is incubated at 0 degrees C after thawing, while at 25 degrees C high recovery in activity is achieved in a time period ranging from 15 to 90 min.(ABSTRACT TRUNCATED AT 250 WORDS)
Briganti
F
F
Center for Biochemical and Biophysical Sciences, Harvard Medical School, Boston, Massachusetts.
Fong
W P
WP
Auld
D S
DS
Vallee
B L
BL
D000426:Alcohol Dehydrogenase / Q000302:isolation & purification / Q000378:metabolism*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence
D007527:Isoenzymes; D046911:Macromolecular Substances; D000426:Alcohol Dehydrogenase