AEROBIC PHOSPHORYLATION IN R. RUBRUM 163 



containing 7 jug 2,6-dichlorophenolindophenol, The rate observed was 

 deducted from the rate seen upon the subsequent addition of 10 jul of 

 1 M sodium succinate. 



Aerobic phosphorylation was measured by counting the radioactive 

 mannose-6-P produced by incubation of an aliquot of enzyme prepara- 

 tion with radioactive Pi. The standard incubation mixture consisted of 

 the following, together with enzyme, in a total volume of 0.5 ml 

 (pH 7.4): 0.1 fimole DPNH, 0.2//mole MgCl2, l.Oyumole K phosphate, 

 lO'^ cpm p32_inorganic phosphate (Nuclear Consultants Corporation, 

 St. Louis 19, Mo.), 0.1 yumole ATP, 10/t/moles mannose, 5.0/imoles 

 glycylglycine, and 0.25 mg yeast hexokinase (Fraction IV, Sigma 

 Chemical Corporation, St. Louis, Mo.), The reaction was started by 

 addition of enzyme to the mixture contained in a 3 ml tube. After 10 

 minutes in a 30 °C water bath in darkness, the reaction was terminated 

 by adding 10 jul 100% TCA.2 Zero time controls were prepared by 

 addition of TCA prior to all of the components of the incubation mix- 

 ture, Mannose-6-P was separated from Pi by electrophoresis. To 

 each acidified incubation sample, 10 /ul aliquots of 1 M K2HPO4 and 



1 M K mannose-6-P were added. A 10 /il aliquot of the mixture was 

 electrophoresed on Whatman 3 MM paper in 0.04 M Na citrate pH 3.6 

 for 30 minutes at 80 V/cm. With the apparatus used, consisting of 

 lucite tank No. LT-36 fitted with auxiliary steel cooling coils and a 5 

 K.V, power supply (Savant Instruments, Inc., Hicksville, N, Y.), 

 4500 volts were applied, with the current ranging from 100 to 160 mA. 

 Thirty samples could be processed in one run. The paper then was 

 thoroughly dried at 50 °C for one hour. The spots were visualized by 

 several hours exposure to Kodak No-Screen Medical X-ray Film and 

 by aniline hydrogen phthalate spray (5), In routine assays (e.g. inhibi- 

 tion experiments) the X-ray film exposure was omitted. The sugar 

 phosphate spots were cut out (3/4 in, x 1 in, rectangles), placed in 

 vials containing 10 ml POPOP-PPO mixture (4 g PPO and 0.1 g 

 POPOP per liter of toluene), and counted in a liquid scintillation spec- 

 trometer (Packard Instrument Co., Inc., La Grange, Illinois). The 

 mannose- 6- P count was compared with total phosphate count (obtained 

 by counting a paper rectangle spotted with a 10 /il aliquot of one hundred 

 fold-diluted incubation mixture). In this system, mannose-6-P migrated 

 toward the anode at two-thirds the velocity of Pi. 



In studies of the distribution of the aerobic phosphorylation sys- 

 tem in sucrose gradient fractions, a correction had to be made for 

 activity which was presumably due to exchange reactions unrelated 

 to DPNH oxidation. This was done by determination of phosphorylation 

 in duplicate tubes containing PMS (0.1 mg/ml). The incorporation of 



2 The abbreviations used are: TCA, trichloroacetic acid; PPO, 2,5-Diphenyl- 

 oxazole; POPOP, l,4-bis-2-(5-phenyloxazolyl)-benzene; SDH, succinic dehy- 

 drogenase; M-6-P, mannose-6-phosphate; P-F3COCCP, phenylhydrazone of 

 p-trifluoromethoxyphenyl carbonyl cyanide. 



