208 E. Racker and R. Wu 



protein/ml.) phosphofructokinase and glyceraldehyde-3-phos- 

 phate (G-3-p) dehydrogenase were the major limiting enzymes 

 of glycolysis while hexokinase was the major pacemaker in 

 concentrated extracts. Addition of other glycolytic enzymes 

 had no effect. 



Table I 



Rate-limiting factors in tumour extracts 



Experiments were carried out in a final volume of 0-6 ml. containing 

 0-6 mg. or 2-4 mg. extract protein (12,000 g supernatant solution); 10 (xmoles 

 Tris buffer, pH 7-4; 4 [zmoles MgClg; 10 |j,moles potassium phosphate buffer, 

 pH 7-4; 1 (xmole DPN; 2 [xmoles ATP; 6 t^moles KCl and 8 ^xmoles glucose. 

 Incubated at 30°C for 20 minutes with shaking. 



[imoles Lactate 



Additions • 6 mg. protein 2 • 4 mg. protein 



— 0-31 2-5 



Hexokinase 0-30 3-8 



Phosphofructokinase 0-62 3-4 



Aldolase 0-34 2-7 



G-3-p dehydrogenase 0-63 3 1 



If we compare the glycolytic rate of these fortified extracts 

 with the glycolysis of intact cells (Table II) it becomes ap- 

 parent that there is a large excess of glycolytic potential in the 

 ascites cells. We can therefore draw our first conclusion, or 



Table II 

 Comparison of glycolytic rates of extracts 



AND intact tumour CELLS 



With extracts the experimental conditions were similar to those in Table I 

 with 2-4 mg. protein per 0-6 ml. With intact cells (10 mg. protein) the 

 reaction mixture contained in a final volume of • 6 ml : 20 [j.moles Tris buffer, 

 pH 7-4; 50 (xmoles NaCl; 12 (^.moles KCl; 3 [xmoles potassium phosphate buffer, 

 pH 7*4; and 6 (xmoles glucose. Incubated at 30° for 25 minutes. Results are 

 expressed as (xmoles per 140 mg. cell protein (or that amount of extract 

 obtained from 140 mg. cell protein) per hour. 



Lactate 

 Preparation Gas phase production 



