Liquid-liquid partitioning of some enzymes, especially phosphofructokinase, from Saccharomyces cerevisiae at sub-zero temperature.
The effects of low temperature (-18 degrees C) on the stability and partitioning of some glycolytic enzymes within an aqueous two-phase system 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 phosphofructokinase, isolated from bakers' yeast, was also examined. The two-phase systems were composed of water, poly(ethylene glycol), dextran, and ethylene glycol and buffer. THe influence on the partitioning of the presence of ethylene glycol, phenylmethylsulfonyl fluoride and poly(ethylene glycol)-bound Cibacron Blue F3G-A was investigated at -18, 0 and (in some cases) 20 degrees C. The presence of ethylene glycol, phase polymers and low temperature stabilized all three enzyme activities. Cibacron Blue, an affinity ligand for phosphofructokinase, increased its partitioning 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 implications of the observed effects on the use of aqueous two-phase systems for the extraction and fractionation of proteins are discussed.
Grimonprez
B
B
Department of Biochemistry, University of Lund, Sweden.
Johansson
G
G
D000426:Alcohol Dehydrogenase / Q000737:chemistry / Q000378:metabolism; D003911:Dextrans / Q000737:chemistry*; D004791:Enzyme Inhibitors / Q000737:chemistry; D004795:Enzyme Stability; D004798:Enzymes / Q000737:chemistry* / Q000378:metabolism; D005026:Ethylene Glycols / Q000737:chemistry; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry / Q000378:metabolism; D010664:Phenylmethylsulfonyl Fluoride / Q000737:chemistry; D010732:Phosphofructokinase-1 / Q000737:chemistry* / Q000378:metabolism; D011092:Polyethylene Glycols / Q000737:chemistry*; D011108:Polymers / Q000737:chemistry; D011480:Protease Inhibitors / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013696:Temperature; D013997:Time Factors; D014227:Triazines / Q000737:chemistry; D014867:Water / Q000737:chemistry*
D003911:Dextrans; D004791:Enzyme Inhibitors; D004798:Enzymes; D005026:Ethylene Glycols; D011108:Polymers; D011480:Protease Inhibitors; D014227:Triazines; D014867:Water; D011092:Polyethylene Glycols; D010664:Phenylmethylsulfonyl Fluoride; C012842:Cibacron Blue F 3GA; D000426:Alcohol Dehydrogenase; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D010732:Phosphofructokinase-1