Partition in dextran-poly(ethylene glycol) aqueous-phase systems can be used for both membrane subfractionation and gaining information on membrane surface properties [H. Walter (1977) in Methods of Cell Separation (Catsimpoolas, N., ed.), vol. 1, pp. 307-354, Plenum, New York]. Smooth, light rough and heavy rough rat liver microsome (obtained by sucrose-density-gradient centrifugation) were subjected to countercurrent distribution in such a system. Smooth microsomal membranes had the highest, heavy rough microsomal membranes the lowest and light rough microsomal membranes an intermediate partition coefficient. The separation is based primarily on hydrophobic differences in the membrane surfaces of the three preparations and is thus due to microsomal properties not previously utilized in their fractionation. The method permits additional subfractionations of microsomes.
1. Two different gels have been prepared suitable for the separation of a number of enzymes, in particular NAD + -dependent dehydrogenases, by affinity chromatography. For both the matrix used was Sepharose 4B. For preparation ( a ), NAD + –Sepharose, 6-aminohexanoic acid has been coupled to the gel by the cyanogen bromide method and then NAD + was attached by using dicyclohexylcarbodi-imide; for preparation ( b ), AMP–Sepharose, N 6 -(6-aminohexyl)-AMP has been coupled directly to cyanogen bromide-activated gel. 2. Affinity columns of both gels retain only the two enzymes when a mixture of bovine serum albumin, lactate dehydrogenase and glyceraldehyde 3-phosphate dehydrogenase is applied. Subsequent elution with the cofactor NAD + yields glyceraldehyde 3-phosphate dehydrogenase whereas lactate dehydrogenase is eluted by applying the same molarity of the reduced cofactor. 3. The binding of both glyceraldehyde 3-phosphate dehydrogenase and lactate dehydrogenase to the gel tested, AMP–Sepharose, is strong enough to resist elution by gradients of KCl of up to at least 0.5 m . A 0.0–0.15 m gradient of the competitive inhibitor salicylate, however, elutes both enzymes efficiently and separately. 4. The elution efficiency of lactate dehydrogenase from AMP–Sepharose has been examined by using a series of eluents under comparable conditions of concentration etc. The approximate relative efficiencies are: 0 (lactate); 0 (lactate+semicarbazide); 0 (0.5m m -NAD + ); 80 (lactate+NAD + ); 95 (lactate+semicarbazide+NAD + ); 100 (0.5m m -NADH). 5. All contaminating lactate dehydrogenase activity can be removed from commercially available crude pyruvate kinase in a single-step procedure by using AMP–Sepharose.