Purified preparations of the pyridine nucleotide linked 17β-hydroxysteroid dehydrogenase of human placenta lose activity rapidly in buffered aqueous solutions at temperatures below 10-15°. The rate of the cold inactivation is reduced by the addition of low concentrations of 17β-estradiol and of certain pyridine nucleotides, and by high concentrations of some polyvalent anions. The addition of 20% or more of glycerol totally protects the enzyme against cold inactivation. The loss of enzyme activity which occurs at low temperatures may be largely restored by warming the enzyme solutions at 30° under carefully controlled conditions but is no longer reversible after prolonged cooling. Cold treatment of the enzyme preparations is accompanied by the formation of enzymatically inactive high molecular weight components which may be separated from the active enzyme by gel filtration on Sephadex or by electrophoresis on polyacrylamide gels. The process of cold inactivation appears to involve complex structural changes, including probable alterations in the configuration of the native protein as well as a series of aggregations resulting in the formation of multiple polymeric species. The properties of other cold-inactivated enzymes and the protective effect of glycerol are discussed.
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