162 METABOLISM AND PHYSIOLOGY 



Extracts were made by disruption of cell suspensions (200-250 mg, 

 wet weight, of washed cells per ml of 0,05 M glycylglycine buffer, 

 pH 8) at 0° in a French Pressure Cell (American Instrument Com- 

 pany, Silver Springs, Maryland) at 15,000 pounds (Carver Press, 

 Fisher Scientific Company), 



Intact cells and large debris were removed by centrifugation at 

 37,000 X g for 30 minutes. The supernatant fluid was centrifuged 

 again for 30 minutes at 37,000 x g. The final supernatant is referred 

 to as the crude extract. 



Fractionation of crude extract by centrifugation in sucrose density 

 gradients 



All operations were performed at 0-5 °C, Following the methods 

 of Britten and Roberts (2), SW39 rotor tubes were loaded with a linear 

 sucrose gradient made by emptying a Bock and Ling mixing device 

 containing 2,3 ml 5% sucrose and 2,3 ml 20% sucrose. All sucrose 

 solutions used also contained 0,05 M glycylglycine pH 8, This was 

 followed by the addition of 0.2 ml of crude extract and 0,2 ml 4% 

 sucrose to the gradient tubes with a smaller mixing device; accord- 

 ingly, the extract concentration attained its highest value at the mini- 

 mum sucrose concentration (at the top of the tube). The tubes were 

 centrifuged in the SW39 rotor, following the procedure of Martin and 

 Ames (3). The tubes then were punctured with a hollow needle and 

 nine to ten fractions, each containing 40 drops, collected. 



Assay of sucrose gradient fractions 



The protein contents of sucrose gradient fractions were estimated 

 routinely by measuring optical density at 280m/^with a Zeiss spectro- 

 photometer. In other instances, protein was measured by the method 

 of Lowry et al. (4), Chlorophyll was estimated on the basis of the 

 optical density of sucrose gradient fractions at 880 m/i. 



Enzymatic assays were facilitated by fitting a Leeds and Northrup 

 Speedomax Type G recorder to the spectrophotometer. With special 

 chart paper (No, TCI 1180 Chart paper, Technical Recording Chart 

 Division, Graphic Controls Corporation, Buffalo 10, N, Y.), the full 

 span of the recorder was equivalent to 0, 1 optical density unit. About 

 15 seconds afte. mixing, the time course of enzymatic reactions was 

 followed for one minute. All enzymatic assays were done at room 

 temperature (23°-25°C), unless otherwise stated. 



The assay of DPNH oxidase activity consisted of measuring the 

 rate of decrease in optical density at 340 m/i induced by the addition 

 of enzyme preparation to 0,5 mlof 0,1 mM DPNH in 0,1 M K phosphate 

 + 5 mM MgCl2 pH 7.0, 



In the succinic dehydrogenase assays, measurements were first 

 made of the decrease in optical density at 600 rafi induced by addition 

 of enzyme to 0,5 ml of 0.1 M K phosphate + 5 mM MgCl2 pH 7.0 



