LIQUID-LIQUID PARTITIONING OF SOME ENZYMES, ESPECIALLY PHOSPHOFRUCTOKINASE, FROM SACCHAROMYCES-CEREVISIAE AT SUBZERO TEMPERATURE

The effects of low temperature (-18 degrees C) on the stability and pa rtitioning of some glycolytic enzymes within an aqueous two-phase syst em were studied. The enzymes were phosphofructokinase, glyceraldehyde- 3-phosphate dehydrogenase and alcohol dehydrogenase present in a crude extract of bakers' yeast. The partitioning of pure phosphofructokinas e, isolated from bakers' yeast, was also examined. The two-phase syste ms were composed of water, poly(ethylene glycol), dextran, and ethylen e glycol and buffer. The influence on the partitioning of the presence of ethylene glycol, phenylmethylsulfonyl fluoride and poly(ethylene g lycol)-bound Cibacron Blue F3G-A was investigated at -18, 0 and (in so me cases) 20 degrees C. The presence of ethylene glycol, phase polymer s and low temperature stabilized all three enzyme activities. Cibacron Blue, an affinity ligand for phosphofructokinase, increased its parti tioning into the upper phase with decreasing temperature. Depending on the conditions, various amounts of the enzymes were recovered at the interface, also in systems not containing ethylene glycol. The implica tions of the observed effects on the use of aqueous two-phase systems for the extraction and fractionation of proteins are discussed.