Hydrophobic chromatography: use for purification of glycogen synthetase.
A homologous series of omega-aminoalkylagaroses [Sepharose-NH(CH(2))(n)NH(2)] that varied in the length of their hydrocarbon side chains was synthesized. This family of agaroses was used for a new type of chromatography, in which retention of proteins is achieved mainly through lipophilic interactions between the hydrocarbon side chains on the agarose and accessible hydrophobic pockets in the protein. When an extract of rabbit muscle was subjected to chromatography on these modified agaroses, the columns with short "arms" (n = 2 and n = 3) excluded glycogen synthetase (EC 2.4.1.11), but the enzyme was retained on delta-aminobutyl-agarose (n = 4), from which it could be eluted with a linear NaCl gradient. Higher members of this series (e.g., n = 6) bind the synthetase so tightly that it can be eluted only in a denatured form. A column of delta-aminobutyl-agarose, which retained the synthetase, excluded glycogen phosphorylase (EC 2.4.1.1), which in this column series and under the same conditions requires side chains 5-(or 6)-carbon-atoms long for retention. Therefore, it is possible to isolate glycogen synthetase by passage of muscle extract through delta-aminobutyl-agarose, then to extract phosphorylase by subjecting the excluded proteins to chromatography on omega-aminohexyl-agarose (n = 6). On a preparative scale, the synthetase (I form) was purified 25- to 50-fold in one step. This paper describes some basic features and potential uses of hydrophobic chromatography. The relevance of the results presented here to the design and use of affinity chromatography columns is discussed.
D000818:Animals; D002845:Chromatography; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D005964:Glucosyltransferases / Q000302:isolation & purification*; D006006:Glycogen Synthase / Q000302:isolation & purification / Q000378:metabolism; D008722:Methods; D009132:Muscles / Q000201:enzymology; D006005:Phosphorylases / Q000302:isolation & purification / Q000378:metabolism; D011134:Polysaccharides; D011817:Rabbits
D011134:Polysaccharides; D005964:Glucosyltransferases; D006005:Phosphorylases; D006006:Glycogen Synthase