VOL. 4 (1950) METABOLISM OF NUCLEATED RED CELLS 279 



of phosphorylation of glucose with the oxidative processes of the Krebs cycle. The 

 inhibitory effects of ions on the aerobic glycolysis suggested that we are here in presence 

 of an enzymatic system displaying this sensitivity towards ions which underlies the 

 mechanism of the metabohc response to cell stimulation. 



The possible general physiological significance of this phenomenon invites closer 

 investigation of its mechanism. The present report deals with experiments in this 

 direction. 



EXPERIMENTAL 



A . Preparation of the material 



Red blood cells of pigeons were used for the experiments. The animals were kept fasting for 

 at least 12 hours preceding the bleeding, which was carried out by cutting the throat on one side 

 after removal of feathers. The blood was caught in a dish containing 0.3 ml of 3.6% sodium citrate. 

 It was centrifuged and the upper stratum of the sediment, containing the white cells, was removed 

 as far as possible by pipetting. The remaining red cells were first washed twice with a fivefold volume 

 of a mixture of i part 3.6% sodium citrate and 9 parts of 0.9% NaCl and then 3 times with the NaCl 

 solution. The washed cells were hemolyzed by adding 1.5 parts of distilled water to i part of cells. 

 The pH of these hemolysates was found to vary between 7.25 and 7.15. As it was intended to investi- 

 gated the effect of salts on the metabolism of the hemolysate it was not possible to use buffers in 

 our experiments and we had to rely for the stabilization of pn during the experimental period on the 

 considerable buffering capacity of hemoglobin. Orienting experiments, however, showed that the 

 shift of PH due to acid formation during 4 hours at 25° did not exceed 0.2. The optimal pH for the 

 aerobic metabolism was found to be about 6.8. In most of our experiments the pn at time o was 

 therefore that of the original hemolysate or slightly lower, i.e., 6.9-7.0. The latter was obtained by 

 adding an appropriate amount of diluted HCl to the water used for hemolysis. 



B. Analytical methods 



In a certain number of experiments a complete balance of O2 uptake, COj production, and glu- 

 cose consumption was carried out. In these and most of the other experiments the total volume 

 of either water or of respective solutions added to the hemolysate was 0.2 ml per i ml of the original 

 hemolysate. The final dilution of the original cell suspension was therefore threefold. All experiments 

 were done at 25° and lasted as a rule four hours. The Oj uptake was measured on 2 ml of the hemo- 

 lysate in standard B.\rcroft-W.\rburg manometers with absorption of COj and NH3. This shifted 

 the PH of the hemolysate no more than o.i to the alkaline side. COj production was determined by 

 the direct method. To account for the retention of COj by the hemolysate the manometer in which 

 CO2 was not absorbed contained in a second sidearm 0.4 ml of diluted H2SO4. At the end of the ex- 

 periment the acid was tipped in from the sidearm into the hemolysate. The pH of the latter was then 

 shifted below 4. The hemolysate became very viscous at this pn but came into the equilibrium with 

 the gas phase after about 30 minutes. As the hemolysate contained from the beginning a certain 

 amount of bound COg the same procedure was carried out on a sample of the hemolysate at time o. 

 The difference of the increase in gas volume after addition of acid in the two samples gave the amount 

 of CO2 produced by oxidation and retained by the hemolysate. .\t the end of the experiment i ml 

 of the hemolysate was pipetted out of the manometer vessels, deproteinezed with 4 ml of 7.5% 

 trichloracetic acid. The centrifugate served for the determination of lactic acid and glucose and phos- 

 phate fractions. The lactic acid determination was carried out by the procedure of B.\rker and 

 SuMMERSON^^, glucose by the new spectrophotometric micromethod of Dische, Shettles and 

 OsNOS^^ based on a specific reaction of hexoses with cysteine in H2SO4. In this reaction fructose 

 gives only 12% more absorption than the equivalent of glucose, so that the phosphorylation of a 

 small amount of the latter to Harden-Young ester will not influence significantly the accuracy 

 of the determination. In some experiments we tested for this ester and triosephosphate by a new 

 highly sensitive reaction with carbazole, which allows the determination of fructose and triosephos- 

 phate in the same sample. Inorganic and the labile phosphate were determined by the Fiske- 

 SubbaRow method in the modification of King, ribose and adenosine-5-phosphate by the orcinol 

 reaction. 



C. Results 



In a first series of experiments the aerobic metabolism of the hemolysate was 

 examined to obtain information about the nature of enzyme reactions involved in this 

 References p. 2g2. 



