Enzyme purification by electrodecantation

1. Electrodecantation has been applied to the initial fractionation from crude material of pig kidney β-d-glucosidase and sheep testicular N-acetylglucosaminidase. 2. The isoelectric points determined by isoelectric focusing were pH4.9 for the β-d-glucosidase and pH6.3 for the N-acetylglucosaminidase. 3. Electrodecantation of pig kidney extract and sheep testicular extract was carried out at pH4.9 and 6.3 respectively. 4. A six- to ten-fold increase in specific activity could be obtained with good recoveries after a single cycle of electrodecantation. 5. The technique has also been used to purify further an extracellular Bacillus subtilis protease preparation. 6. Attempts to use electrodecantation for the concentration of very dilute enzyme solutions resulted in considerable loss of activity. 7. The limitations and potential use of the technique in laboratory-scale enzyme preparation are discussed.

PROTEOLYTIC MICRODISSECTION OF SMOOTH-SURFACED VESICLES OF LIVER MICROSOMES

Digestion of rabbit liver microsomal smooth vesicles with Bacillus subtilis protease released proteins and peptide fragments from the vesicles, without solubilizing phospholipids and cholesterol. The proteolysis was, however, limited when about 30% of the protein had been solubilized. The same limitation was observed when the vesicles were treated with trypsin, chymotrypsin, or their combinations with the bacterial protease. The limited proteolysis was accompanied by selective solubilization of cytochrome b5 and microsomal NADPH-specific flavoprotein, leaving the CO-binding hemoprotein and some other enzymes still attached to the vesicular membranes. Sucrose density gradient centrifugation of protease-treated vesicles indicated that all the vesicles had been attacked by the protease to similar extents. The behavior of intact and digested vesicles in dextran density gradient centrifugation suggested that the vesicles, even after proteolytic digestion, existed in the form of closed sacs which were impermeable to macromolecules such as dextran and proteases. It was concluded that only the outside surface of the vesicles is susceptible to the proteolytic action and that cytochrome b5 and the NADPH-specific flavoprotein are located in the susceptible area.