PHYSIOLOGICAL AND BIOCHEMICAL TECHNICS 177 



Cell-free Enzyme Extracts 



For many types of studies, for example those involving reaction mecha- 

 nisms, cell-free or soluble enzyme extracts are desirable. There are 

 numerous technics available, none of which works in all cases. In prin- 

 ciple, all methods attempt to disrupt the cell structure, thus allowing the 

 liberation of intracellular material. The bacterial debris and any whole 

 cells left are removed by high-speed centrifugation, usually in the cold, 

 leaving the soluble elements in the supernatant liquid. If the desired 

 enzymatic activity is not located in the supernatant liquid, it is possible 

 that such activity may be found in the sedimented debris, and the latter 

 should be examined before the particular procedure employed to obtain 

 the cell-free extract is discarded as unworkable. 



Brief descriptions of these methods follow. Autolysis: cell suspension 

 made to pH 7.5-8.5 with phosphate buffer and kept at 37°C under toluene 

 for 12-24 hr. The degree of autolysis depends on the type of organisms. 

 Although autolysis increases with time, so does inactivation of enzymes. 

 Lysis : most commonly employed with Micrococcus lysodeikticus using the 

 enzyme lysozyme to disrupt the cells (see, for example, McManus, 1951). 

 Freezing and thawing: enzymes are often released by rupture of the cell 

 membrane with alternate freezing and thawing (see, for example, Koepsell 

 and Johnson, 1942). Wet grinding: (1) hand grinding of frozen cell paste 

 in a mortar with powdered glass, alumina, carborundum, or other suitable 

 abrasives (see, for example, Mcllwain, 1948) ; (2) semimechanical method 

 of Kalnitsky et at. (1945) which involves the passage of a paste of bacteria 

 and powdered glass between concentric cones of heavy glass, the inner 

 cone revolving by connection to a motor and the outer cone held firmly in 

 place; (3) a wet-crushing mill described by Booth and Green (1938) but 

 not generally available; (4) shearing action of minute glass beads in a 

 high-speed blender (Lamanna and Mallette, 1954). Dry grinding: con- 

 sists of slow rotation in vacuo for several hours of dried powders of bac- 

 teria mixed with glass beads. The shearing action of the beads appears 

 to be the important factor. Pressure: A large instantaneous pressure 

 forces the frozen cell material between two machined surfaces, with 

 resultant cell disruption. The apparatus has been referred to as the 

 ''Hughes press '^ (see Hughes, 1951, and a modification by Gest and 

 Nordstrom, 1956). Sonic vibration: this technic is becoming more 

 popular because of the ease of manipulations. The bacterial cells are 

 disrupted by the sonic energy (see, for example, Shropshire, 1947, and 

 Stumpf et at., 1946). The cells of various bacterial species differ in their 

 susceptibility to sonic disruption. Gram-positive cells are more resistant 

 than gram-negative cells; cocci are more resistant than rod forms; large 

 cells are more resistant than small ceUs. Thus, the time of exposure in 



